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Conflicts of interest:

No conflicts of interest reported. MicroRNAs (miRNA) are short (19-22 nucleotides), singlestranded, non-coding RNAs that specifically anneal with complementary sequences in multiple mRNA targets, and they silence mRNAs and suppress downstream protein translation. A miRNA can act as a fine-tuner of gene expression or an on/off switch. These features highlight the potential of miR-NAs as therapeutic targets. The role of miRNAs in myocardial fibrosis and hypertrophic cardiomyopathy has been widely studied in human patients. However, there is no data available for canine and human myxomatous mitral valve disease (MMVD). The aim of this study was to investigate miRNA transcriptomics in canine MMVD by using global transcriptional profiling, miRNA target prediction software (DIANA Tool, TargetScan 6.2) and network analysis software (BioLayout Express 3D ).
The most significantly down-regulated miRNA in MMVD was cfa-miR-218, which is an endothelial specific miRNA shown to regulate endothelial migration and vessel patterning. The top predicted target of cfa-miR-218is glucuronic acid epimerase (GLCE) which is the main enzyme controlling heparan sulphate biosynthesis. Other interesting findings were down-regulation of cfa-miR-29 and members of the cfa-miR-23 family. Cfa-miR-29 targets multiple extracellular matrix transcripts, such as collagens, elastin, integrin, laminin, MMP (matrix metalloproteinase) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), whereas cfa-miR-23 targets hyaluronic acid synthase 2 (Has2). Since the major pathology of MMVD is aberrant turnover of extracellular matrix proteins, this may be linked to miR-NA regulation. Dysregulation of valve miRNAs might be potential therapeutic targets in the treatment of canine MMVD.
No conflicts of interest reported. Sarcoplasmic reticulum (SR) Ca 2 + -ATPase and its regulatory proteins are pivotal determinants of myocardial active relaxation via calcium uptake against the SR-cytoplasmic gradient. The lowered density of the SR Ca 2 + -ATPase has been well demonstrated in many species during chronic hemodynamic overload. The genes linked to SR calcium uptake were reported not only being expressed in peripheral blood but serving as potential cardiac biomarkers in dogs with chronic mitral regurgitation, such as SR Ca 2+ adenosine triphosphatase isoform 2a (SER-CA2a), phospholamban (PLN), and HS-1 associated protein X-1 (HAX-1). The aim of this study is to determine whether the target genes expressed in the blood will be translatable to the myocardial setting as cardiac biomarkers.

ESVCN-O-3 EFFECTIVENESS OF A NEW DIETETIC WEIGHT MAN-AGEMENT FOOD TO ACHIEVE WEIGHT LOSS

Urinary d-aminolevulinic acid, porphobilinogen, uroporphyrin I, and coproporphyrin I concentrations were increased in the cat studied, suggesting an acute intermittent porphyria (AIP). The erythrocytic hydroxymethylbilane synthase (HMBS) activity in erythrocytes was approximately half normal suggesting a dominant enzymopathy, while the erythrocyte uroporphyrinogen IIIsynthase activity was normal. Sequencing the feline HMBS gene revealed a heterozygous intronic 12 base deletion (c.772-13_-2del) which results in an insertion in the mRNA and would predict a truncated protein.

Conflicts of interest:

CRP and Alb were by far the APPs most frequently correlated with clinical and laboratory abnormalities such as nutritional status, lethargy and skin ulcers (P < 0,05), as well as urinary protein to creatinine ratio (UPC), total serum protein, and urine specific gravity (P < 0,02). There were limited associations between Hp, Cp, SAA and clinicopathological parameters. A minor linear relationship was observed between CRP and clinical scoring. CRP and Alb were also correlated with parasitological scoring in bone marrow, but not lymph node cytology (P < 0.04). Dogs with Ehrlichia titers had higher CRP, Cp and lower Alb concentrations. Finally, CRP concentrations were higher in later compared to earlier stages of the infection, as defined by the Leishvet criteria.
The feline AIP gene was identified, encoding a 330 amino acid protein with 98% homology to the human AIP protein. A BLAST search revealed this gene contained 6 exons and exon specific primers were created to enable sequencing. A single nonconservative SNP was identified in exon 1 (AIP:c.9G>T), encoding for an amino acid change from aspartic acid to glutamic acid in 2/10 acromegalic patients and 0/10 control cats. Two additional conservative SNPs were also identified (AIP:c.826T>C and AIP:c.481T>C).
Exon 1 encodes for a region of the AIP protein considered essential for AIP-AIP receptor interaction. Although 70 different human AIP mutations have been identified to date, a human AIP:c9G>T mutation has not yet been identified. The AIP N-terminal is required for the stability of the AIP protein-AIP-receptor complex, and essential for the regulation of translocation into the nucleus, where it binds to aryl hydrocarbon receptor nuclear translocator leading to activation of genes thought to act as tumor suppressors. Loss of normal AIP activity is thought to promote somatotrophinoma development. It is therefore possible that the detected AIP:c.9G>T mutation predisposed to somatotrophinoma tumorigenesis in the two affected patients, and a study containing a larger number of cases is indicated.
In 53 of 100 dogs SBP exceeded 160 mmHg, none of the dogs had fundoscopic lesions secondary to hypertension. Body condition score was abnormal in 41 animals, 39 were overweight or obese. Physical examination revealed a heart murmur in 22, submandibular lymphadenopathy in 13, moderate to severe dental plaque in 51 and one or more (sub)cutaneous masses in 56 dogs. Twenty-three dogs were leukopenic, 29 had a decreased phosphorus, 32 an increased serum creatinine and one dog a decreased total thyroxine (with concurrent increased thyroid stimulating hormone). Crystalluria was commonly detected (62/96) and mostly due to low numbers (<1/high power field) of amorphous crystals (82%). Struvite crystals were present in 18% of the crystalluric dogs. Overt and borderline proteinuria were detected in 13 and 18 of 98 dogs, respectively. Four dogs had a positive urinary culture. SBP was not significantly different between the senior and geriatric group. There was no significant effect of obesity or gender on SBP. The platelet count (p = 0.014), total thyroxine concentration (p = 0.008) and the frequency of orthopedic problems (p = 0.007) and cutaneous masses (p = 0.002) were significantly higher in the geriatric compared to the senior dogs. Hematocrit (p = 0.007) and body temperature (p = 0.044) were significantly lower in the geriatric group.
No conflicts of interest reported. Feline immunodeficiency virus (FIV) infection has been associated with kidney disease, mainly characterised by an increased prevalence of proteinuria in FIV-infected cats. However, studies evaluating renal variables in FIV-positive cats are scarce. Recently, a higher systolic blood pressure (SBP) was reported in a small number of FIV-infected cats. Hypertension is an important cause of proteinuria and a frequent cause of renal disease in human immunodeficiency virus (HIV) positive patients. Therefore, our main objective was to describe SBP in clinically ill FIVpositive cats. Secondly we aimed to evaluate routine renal variables in this population.
The study included 99 cats, with a mean age of 8.0 AE 3.7 years and a mean body condition score of 4.6 AE 1.5 on a nine-point scale. The SBP ranged from 91 to 170 mmHg, with a mean of 116 AE 18 mmHg. Only two cats were hypertensive (SBP > 160 mmHg). Both had isosthenuric urine, were borderline proteinuric (UPC 0.2-0.4) and one of them was mildly azotemic. Mean sCreat was 101.7 AE 64.2 lmol/L (reference interval (RI) 44.2-141.4 lmol/L) and mean sUrea concentration 8.6 AE 5.2 mmol/L (RI 6.2-10.8 mmol/L). Thirteen cats showed increased sCreat levels, with decreased USG (< 1.035) in eight, proteinuria (UPC > 0.4) in seven and increased sUrea concentrations in ten of them. Five out of ten azotemic cats were proteinuric with a decreased USG. Mean UPC was 0.26 AE 1.06, with a wide range from 0.05 to 9.38. Borderline proteinuria was present in 40/99 (40.4%) and proteinuria in 28/99 (28.3%). Half of the proteinuric cats had a decreased USG. Mean USG was 1.040 AE 0.013. One third of all cats had a decreased USG, with isosthenuria in seven of them.
These results demonstrate that proteinuria and poorly concentrated urine are common in naturally infected clinically ill FIVpositive cats, confirming previous reports in cats and humans. However, longitudinal studies of (borderline) proteinuric patients are needed to elucidate the clinical relevance. The low number of hypertensive patients and low mean SBP in our study indicate that hypertension is uncommon and unlikely to be the cause of renal damage in clinically ill FIV-infected cats.
No conflicts of interest reported. Serum acute phase proteins (APPs) are considered biomarkers of the acute phase reaction, and are being increasingly used in human and veterinary medicine in diagnosis and monitoring of neoplastic diseases. In the cat, serum amyloid A (SAA) is considered a positive major APP, haptoglobin (Hp) a moderate APP, and albumin and insulin-like growth factor-1(IGF-1) negative APPs.

ESCG-P-3 A VERY LOW MOLECULAR WEIGHT POULTRY FEATHER HYDROLYZED-BASED EXTRUDED DIET

Nineteen dogs across France and Quebec entered the study. Dogs with food or antibiotic-responsive chronic enteropathy, hypoproteinemia, or treated with immunomodulating drugs were excluded from the study. Dogs were included in the study after complete clinical, ultrasonographic, endoscopic evaluation and histopathological evaluation of intestinal biopsies showing signs of intestinal inflammation. The owners were instructed to feed exclusively the study diet a .
The low molecular weight poultry feather hydrolyzed proteinbased dry extruded diet a appears to be effective in the management of idiopathic IBD without any concurrent immunosuppressive drug over the 10 week period of this pilot study. These preliminary findings should be confirmed by a prospective, randomized double blind study. Feline pancreatitis is the most common exocrine pancreatic disorder with varied mortality. However, there is no available and reliable method to evaluate the severity and prognosis of the disease. Ninety-two cats diagnosed as pancreatitis with acute onset of compatible clinical signs and a positive SNAP â fPL TM Test between October 2011 and September 2013 were enrolled in this study. All Cats were divided into survival (n = 48) and nonsurvival (n = 44) groups. Fifty-two parameters including signalments, clinical signs, physical examinations, clinicopathological examinations, diagnostic images, complications and concurrent diseases were analyzed and compared between the two groups. Parameters with P ≤ 0.05 were considered for further analyses. The mortality in this study was 47.8%. Hematocrit, albumin, BUN, creatinine, total bilirubin, calcium, phosphorous, body temperature, systolic blood pressure, the body cavity fluids, complications, e.g. systemic inflammatory response syndrome (SIRS) and acute renal failure (ARF) were found to be significantly associated with disease severity and prognosis, and were selected for constructing the scores. Continuous variables outside the reference interval were separated into quartiles to yield quartile-specific odds ratios (ORs) for survival. Based on the integer value of the OR, the scoring system was then developed by incorporating weighting factors assigned to each quartile. A predictive total score was calculated for each cat by summing all weighting factors. The total scores of each cat ranged from 12 to 83. The severity scores in this study achieved an area under receiver operating characteristic (AUROC) of 0.88. The optimal cut-off point for discriminating outcome was 32.5 with the sensitivity of 89.6% and specificity of 77.3%, respectively. The mortality was 87.2% with a score ≥ 33, whereas 18.9% with a score ≤ 32. There was significant difference (P < 0.001) between the two groups of the cut-off point. Furthermore, the mortality reached to 100% when the score more than 56. The severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in cats with pancreatitis.

Conflicts of interest:

No conflicts of interest reported. Alpha 1 -proteinase inhibitor (a 1 -PI) is a proteinase-resistent protein that can be quantified in fecal, urine, and serum samples from dogs. Recently, increased fecal and urinary canine a 1 -PI (ca 1 -PI) concentrations have been described in dogs with gastrointestinal diseases (e.g., inflammatory bowel disease [IBD] , but also in dogs with exocrine pancreatic insufficiency) and in dogs with chronic hepatitis or chronic kidney disease, respectively. Decreased serum ca 1 -PI concentrations have been reported in dogs with IBD, protein-losing enteropathy (PLE), and hypocobalaminemia. Treatment protocols for dogs with IBD and/or PLE commonly include corticosteroids, but the effect of corticosteroid therapy on serum ca 1 -PI concentrations have not yet been reported. The aim of this study was to evaluate the effect of hydro-cortisone on serum ca 1 -PI concentrations in healthy dogs.
The author works at Texas A&M University, whose GI Lab currently offer a commercial assay for faecal Alpha1-Proteinase Inhibitor. Canine chronic enteropathy (CE) is a common, but poorly understood syndrome, with variable response to therapy and prognosis. There is a need for novel biomarkers that are specific for intestinal disease and that provide objective measures of disease severity, progression, and prognosis. Serum citrulline is a useful biomarker in human intestinal disease as it is specific to the small intestine and indicates globally reduced enterocyte mass and absorptive function in various disease states. It is used to determine, quantitatively, intestinal integrity at the enterocyte level and is not influenced by nutritional or inflammatory status. The aim of this study was to determine whether serum citrulline can be used as a biomarker for CE in dogs.
No conflicts of interest reported. Matrix metalloproteinases (MMPs) 2 and 9 are zinc-dependent endopeptidases that contribute to the control of breakdown and reconstitution of extracellular matrix under both normal and pathological conditions. Intestinal mucosal levels of MMP-2 and -9 have been shown to be increased in animal models and human IBD. To our knowledge, the presense of MMP-2 and -9 has not been studied in the intestinal mucosal samples of healthy dogs as well as in canine spontaneous IBD. Thus, the main aim of this study was to identify the presence of MMP-2 and -9 in the mucosa of the small and large intestines of clinically healthy beagle dogs using gelatin zymography technique. For the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. The samples were taken and snap frozen in liquid nitrogen during necropsy from 12 healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota.
No conflicts of interest reported. There are only few laboratory markers being evaluated for diagnosing and/or monitoring canine chronic enteropathies, including inflammatory bowel disease (IBD). S100A12 belongs to the S100/calgranulin-protein family and has been proposed to play a central role in both innate and acquired immune responses. It has been reported to be increased in stool samples, serum and/or intestinal mucosa in human patients with IBD. Myeloperoxidase (MPO) is an enzyme found mostly in granulocytes. Intestinal mucosal levels of MPO have been shown to be increased in animal models and human IBD. To date, S100A12 and MPO levels in intestinal mucosal samples have been reported neither from healthy dogs nor from dogs suffering from IBD. To start investigating this aspect in dogs, the objective of this study was to evaluate mucosal S100A12 and MPO levels in the small and large intestines by using enzyme-linked immunoassay (ELISA) and spectrophotometric methods, respectively. For the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. The samples were taken and snap frozen in liquid nitrogen during necropsy from 12 healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota.
* Haptoglobin is a moderate acute phase protein in cats. As a part of the innate immune system its concentration rises within 24-48 hours after tissue damage.
No conflicts of interest reported. The total protein (TP) concentration and cell count of pleural and abdominal fluid is used to differentiate a transudate from an exudate. TP can be measured by automated wet chemistry analyser or more easily using a refractometer. The aim of this study was to assess if refractometer values of TP are useful for this purpose. Retrospectively samples from canine pleural and abdominal effusions in which TP concentration was measured both with a refractometer as well using Pentra 400 (ABX Horiba, Montpellier) were included. Samples were collected into heparinized tubes and analysed within 12 hours. Bland-Altman diagrams were created and correlation between both measurements was calculated by Spearman 0 s nonparametric correlation.
The refractometer is an acceptable, rapid and efficient method for determination of total protein concentration in pleural and abdominal effusions in dogs to differentiate transudates from exudates.
No conflicts of interest reported. Persistent renal proteinuria is considered an early marker of chronic kidney disease (CKD) and it is listed among the initiation factors and progression factors according to KDOQI guidelines. Nevertheless, few data are available about the prevalence of proteinuria in cats affected with CKD, in which it is assumed that nephropathy is mainly characterized by tubulointerstitial damage.
The aim of this study is to determine the prevalence of proteinuria in cats affected with CKD and to valuate the relations between urine protein to creatinine ratio (UPC) or IRIS substaging by proteinuria, towards purebred, sex, age, haematology, biochemistry and urinalysis. Wilcoxon test, linear regression and chi-square test were used for the statistical analysis.
Data from 251 cats were considered. Non-renal proteinuria was an exclusion criterion.
Proteinuric cats (UPC>0.4) were 15.4% in CKD cats, while 15.9% could be substaged as bordeline proteinuric (0.2) In cats, proteinuria tends to increase with aging (p < 0.0001) and with worsening of the nephropathy (p = 0.002). Proteinuria was related to the anaemic state in CKD cats: UPC significantly increases with RBC count, Hb, Ht and MCH decreasing (p < 0.0001 and p = 0.049 respectively). Proteinuria tends to increase with WBC count (p = 0.0001) and neutrophils increasing (p = 0.0001), while tends to decrease with lymphocytes increasing (p = 0.008). Furthermore, UPC significantly increases in presence of an inflammatory serum protein electrophoretic pattern.
Furthermore, proteinuria increases in presence of RBC in urinary sediment and in samples where casts were observed, in particularly when RBC casts (considered always pathological and indicative of glomerular damage) were present.
UPC values assessed in proteinuric cats and data analysis suggest the need of deepen the analytical variability of UPC and the opportunity to reconsider the intervals of substaging by proteinuria in cats.

Saccharomyces boulardii (Sb)is a non-pathogenic yeast used in the prevention and treatment of gastrointestinal disorders in human beings and horses. The aim of this study was to evaluate the effect of Sb in healthy dogs and dogs with chronic enteropathies (CE). Sb was formulated in 10x10 9 CFU capsules. Its concentration and viability within the capsules was controlled by yeast culture in subsequent steps until expiration date. Four healthy dogs (HD) and 18 dogs with CE (10 inflammatory bowel disease -IBD, 8 protein losing enteropathy -PLE) were included. In HD Sb was administered for 10 days (1x10 9 CFU/kg BID); daily clinical evaluation was performed to assess possible adverse effects and quantitative stool cultures for yeasts were performed before, during and after the administration. In dogs with CE a randomized double blind placebo-control study was performed, administering Sb (1x10 9 CFU/kg BID) or placebo (Pl). Sb or Pl administration was added to standard therapeutic protocols (diet, antibiotics and immunosuppressive drugs), to evaluate its efficacy for the treatment of IBD and PLE. Complete blood work, abdominal ultrasonography, gastro-duodenal and colon endoscopy and histopathological evaluation of intestinal samples were performed at diagnosis and after 60 days of treatment. Validated score system for the clinical signs (CECCAI), ultrasonography, endoscopy and histopathology were applied. Significance was set for P < 0.05. Results in HD showed the absence of Sb in the faeces before treatment, its presence after one day, its steady state (10x10 7 CFU/g) after 5 days and its complete elimination 4 days after withdrawal of treatment. No adverse effects were reported. In CE dogs the clinical score improved significantly in dogs receiving Sb compared to dogs receiving Pl (P = 0.009). In PLE dogs the albumin concentration increased significantly (P = 0.034) in the group receiving Sb with respect to Pl. The daily frequency of defecation in the Sb group was significantly lower with respect to Pl after 45 (P = 0.032) and 60 (P = 0.004) days of treatment. No statistical differences were found between dogs receiving Sb and Pl after treatment, based on the endoscopic evaluation of duodenum and colon. No statistical differences were found between the two groups on the duodenal ultrasonographic and histological evaluation after treatment. In conclusion, Sb can be safely used in dogs with CE, in addition to standard treatment, to achieve a better control of clinical signs and a significant increase in albumin concentration compared to the standard therapy alone.

Conflicts of interest:

No conflicts of interest reported. Canine inflammatory bowel disease (IBD) is an immune-mediated enteropathy likely triggered by environmental and immunoregulatory factors in genetically susceptible dogs. Previous studies suggest a pivotal role for gut bacteria in disease pathogenesis since luminal microbial composition is markedly altered (ie, dysbiosis) at diagnosis. Probiotic bacteria appear to be therapeutically effective in some forms of human IBD. Controlled studies evaluating the efficacy of probiotic therapy for canine IBD have not been previously reported. The aim of the present study was to characterize the mucosa-associated microbiota and determine the clinical, microbiological, and mucosal homeostatic effects of orally administered VSL#3 probiotics in dogs with IBD. Twenty dogs diagnosed with moderate-to-severe IBD (CIBDAI score > 5) were randomized to receive standard therapy (ie, elimination diet and glucocorticoids) with or without probiotic VSL#3. The mucosal microbiota from endoscopic intestinal biopsies of IBD dogs and controls was evaluated by fluorescence in situ hybridization (FISH) targeting the 16S rRNA genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human IBD. Epithelial tight junction protein (TJP) expression was studied using immunohistochemistry. Clinical signs and changes in mucosal microbiota and TJP expression were assessed before and after probiotic VSL#3 therapy. IBD dogs showed a reduction in GI signs following 8 weeks of probiotic therapy compared with baseline CIBDAI scores (P < 0.05). Adherent and sporadic invasive bacteria (EUB) were observed in the small intestines and colon of healthy dogs. The diseased canine duodenum was nearly bacteria-free. IBD dogs given probiotic VSL #3 had altered spatial redistribution of most bacterial groups in the mucus and adherent compartments of the colon. Subset analysis showed that Lactobacilli were significantly (P < 0.05) increased in the lumen and mucus post-VSL #3, while the number of mucus laden Bifidobacteria approached significance (P = 0.08). Expression of TJP showed that occludin was significantly lower in control intestines as compared to duodenal and colonic mucosa obtained from IBD dogs that received probiotic (P = 0.008 and P = 0.01, respectively). In contrast, claudin-2 expression in the colon was significantly higher (P < 0.002) in control dogs versus VSL #3 treated IBD dogs. Our data demonstrate that probiotic VSL#3 alters some of the mucosa-associated microbiota in dogs with IBD. These probiotic changes in bacterial composition are associated with up-regulated TJP expression indicative of enhanced epithelial barrier integrity, similar to VSL#3-induced disease protection seen in human IBD.
The probiotics used in the trial were supplied free of charge by the manufacturer. Canine Inflammatory bowel disease (IBD) is thought to be partially caused by an aberrant immune response towards the intestinal microbiome. In humans and mice, administration of probiotics can alleviate IBD severity and/or prevent relapse by induction of a more "tolerant"microenvironment. The aim of this study was to investigate the effect of probiotic Enterococcus faecium NCIMB 10415 E1707 (EF) on intestinal microbiome composition. Dogs were recruited to receive EF at 1x10e8 cfu in a double-blinded, placebo-controlled manner in addition to an exclusion diet (hydrolysed protein). Seven dogs were included in the probiotics group and 5 dogs in the placebo group. All dogs improved clinically after treatment, however, there was no obvious effect on clinical severity in those that received probiotics. Fresh naturally voided faecal samples were collected from all dogs before and after treatment, snap-frozen in liquid nitrogen and stored at -80°C until further analysis. Genomic DNA was extracted from each faecal sample using the Mobio Power soil DNA isolation Kit (MoBio Laboratories), as recommended by the manufacturer. Next generation sequencing was performed on the Ion-Torrent[TRADEMARK] (Life Technologies) platform based upon the V1-V3 region (E. coli position 27-519) of the 16S rRNA gene with the following primers: forward 28F: GAG-TTTGATCNTGGCTCAG and reverse 519R: GTNTTACN GCGGCKGCTG. Raw sequence data were screened, trimmed, filtered, and chimera depleted with default settings using the QIIME pipeline version 1.8 and UCHIME software, in which microbiome composition between treatment groups before and after treatment was compared.
At least in this group of patients fecal S100A12 concentration was more sensitive (but less specific) to detect dogs with a CCE-CAI ≥ 12 or histopathologic intestinal inflammation than fecal calprotectin concentration. Weight loss and malabsorption of fat, protein, cobalamin and tocopherol in the face of normal exocrine pancreatic function have been reported in up to 30-40% of cats older than 12 years of age fed a variety of nutritionally balanced dry and wet foods (Patil AP and Cupp CJ. Proc. Nestle-Purina Compan Anim Nutr Summit, Focus on Gastroenterology, [55] [56] [57] [58] [59] [60] [61] 2010) . The objectives of this study were to determine if serum cobalamin concentrations increased after oral administration of a cobalamin supplement to affected cats, and the duration of any positive response following cessation of supplementation.
No conflicts of interest reported. Centronuclear myopathy (CNM) is the most prevalent congenital inherited disorder affecting skeletal muscles in Labrador Retrievers. This disabling condition segregates worldwide and a recessive loss-of-function founder mutation was identified in the Protein Tyrosine Phosphatase-Like, member A gene (PTPLA/ HACD1).
No conflicts of interest reported. Feline hypertrophic cardiomyopathy (fHCM) is the most common heart disease in cats. HCM is considered an inherited disease of the sarcomere and fHCM has been linked to mutations in one sarcomere protein i.e. MYBPC3. However, the pathophysiologic mechanisms behind disease development and progression are largely unknown. In this study we investigate whether mitochondrial morphological changes in the myocardium accompany mitochondrial dysfunction and enhanced oxidative stress formation that we recently found in fHCM.
No conflicts of interest reported. During primary hyperfibrinogenolysis (PHF), FDPs production is increased but production of D-dimer is not. Therefore, elevated FDPs and normal D-dimer are considered an indicator of PHF. In humans and dogs, activation of coagulation and fibrinolysis develops in all type of ascites and it is associated with systemic PHF, suggesting that ascitis is inherently fibrinolytic. Preliminary data have shown that activation of coagulation followed by fibrinolysis occurs also in all type of pleural effusions (PE). The objective of this study was to determine if systemic PHF occurs also in dogs with PE. Thirty-three dogs referred to the San Marco Veterinary Clinic with PE, but without ascites, were studied (group 1). From the electronic data-base of the clinic dogs for inclusion in control groups 2 (healthy dogs) and 3 (sick dogs without PE or ascites) were randomly selected and individually matched to group 1 dogs for age, sex, and breed. Fibrinogen, FDPs, D-dimers, C-reactive protein (CRP), fibrinogen/CRP ratio, and prevalence of PHF (i.e., dogs with elevated plasma FDPs and normal D-dimer) were determined. Differences between the 3 groups were analyzed using ANOVA (fibrinogen), Chi-Square (FDPs and prevalence of PHF) and Kruskal-Wallis test (CRP, fibrinogen/CRP ratio, and D-dimer). Post-test analysis were performed by Tamhane and Mann-Whitney test. Fibrinogen concentration in group 1 was significantly increased compared to group 2 (p < 0.0001), but not compared to group 3 (p = 0.504). FDPs concentration in group 1 was significantly increased compared to groups 2 (p < 0.0001), but not compared to group 3 (p = 0.148). D-dimers concentration in group 1 was significantly increased compared to group 2 (p < 0.0001), but not compared to group 3 (p = 0.964). CRP was significantly increased in group 1 compared to group 2 and 3 (p < 0.0001 for both comparison). Fibrinogen/CRP ratio was significantly decreased in group 1 compared to group 2 and 3 (p < 0.0001 for both comparison). Prevalence of PHF was significantly higher in group 1 compared to groups 2 (p = 0.004), but not compared to group 3 (p = 0.186). These results support the hypothesis that PHF occurs significantly more often in dogs with PE compared to healthy dogs. Despite there was a trend of increased PHF also in dogs with PE compared to sick dogs, this difference did not reach significance. Nevertheless, the decreased in fibrinogen/CRP ratio in group 1 compared to group 3, in the face of a similar D-dimer concentration, would suggest that PHF is also more prevalent in dogs with PE compared to sick control dogs.
No conflicts of interest reported. The systemic inflammatory response syndrome (SIRS) refers to clinical signs of systemic inflammation in response to (non-) infectious insults. Current diagnosis of SIRS is based on clinical and basic laboratory data and is a sensitive screening to identify patients at risk. C-reactive protein (CRP) is a major canine acute phase protein with concentrations related to disease severity and underlying cause. CRP rises in response to proinflammatory cytokines, mainly interleukin (IL)-6 and tumor necrosis factor (TNF)-a, which are considered the main triggers of SIRS.
The assay used in the study was developed at the GI Laboratory, Texas A&M University. Most authors also work at the GI Laboratory, Texa A&M University. Canine leishmaniasis (CanL) is a multisystemic disease that is endemic in the Mediterranean region. In the past, concentrations of acute phase proteins (APPs), and specifically C-reactive protein (CRP), haptoglobin (Hp), ceruloplasmin (Cp), serum amyloid A (SAA) and albumin (Alb), have been reported to change in dogs with leishmaniasis, and revert to normal after successful treatment, highlighting the intrinsic inflammatory reaction of the host to the parasite.
The study was financially supported by the Swedish Juvenile Diabetes Foundation, the Fredrik and Ingrid Thuring Foundation, the Magnus Bergvall Foundation, the Lars Hierta Memorial Foundation, and the Foundation for Research, Agria Insurance Company. Feline acromegaly is an increasingly recognised endocrinopathy among diabetic cats, caused by chronic excessive growth hormone secretion by a functional somatotrophinoma in the pars distalis of the anterior pituitary gland. The majority of human somatotrophinomas are sporadic, however up to 20% of familial isolated pituitary adenomas are caused by germline mutations of the aryl-hydrocarbon-receptor interacting protein (AIP). Feline acromegaly has phenotypic and biochemical similarities to human familial acromegaly with AIP mutations, such as male predominance, somatotroph macroadenoma and resistance to octreotide therapy.
The affected Scottish Terriers (two male and one female) presented at 2 months of age with severe proliferative and ulcerative conjunctivitis and gingivitis/stomatitis; biopsy confirmed ligneous membranitis. Other clinical signs included increased upper respiratory tract noise, nasal discharge and lymphadenopathy. One male was cryptorchid. Clinical pathological findings included neutrophilia, proteinuria and hypoalbuinaemia. Serum plasminogen activity was measured in two dogs, and was low in one. The dam and sire of the affected dogs had normal serum plasminogen activity and no history or clinical signs consistent with ligneous membranitis.
Because life expectancy in dogs is related to body size, the inclusion of 100 dogs was based on a human/pet analogy chart to determine whether a dog was senior (n = 41) or geriatric (n = 59). To verify health status, owners were asked to complete an extensive questionnaire. Systolic blood pressure (SBP) was measured using the Doppler technique according to the ACVIM guidelines. Subsequently a thorough PE was performed, including body and muscle condition scoring, orthopedic examination, neurologic evaluation, indirect fundoscopy and bilateral Schirmer tear test. Complete blood count, serum biochemistry and urinalysis (including urinary sediment, urinary protein:creatinine ratio (UPC) and bacterial culture) were evaluated.
In total, 132 healthy cats between one and 16 years were included. Serum CysC was determined with a validated particleenhanced nephelometric immunoassay (PENIA). Serum Cr, urea, urine specific gravity (USG), urinary protein: creatinine ratio (UPC) and systolic blood pressure (SBP) were also measured. To test for difference between the groups, the F-test was used. The lower and upper value of the 95% reference interval were obtained as the 2.5% and 97.5% quantiles of the sCysC observations.
Blood and urine samples were obtained from cats at three UK first opinion practices as part of a geriatric screening programme. Haematology, serum biochemistry (including total thyroxine concentration (TT4)) and urinalysis (including urine protein:creati-nine ratio (UPC)) were performed. Dental disease score (calculus and gingivitis) and body condition score (BCS) were recorded. Cats with TT4 > 40 nmol/L, evidence of pyuria or bacteruria, or significant systemic disease were excluded. UAC and UCysC were determined in non-azotaemic cats (n = 50) and cats with azotaemic CKD (n = 10, defined as a serum creatinine concentration >153 lmol/L and concurrent urine specific gravity <1.035). Comparisons between the non-azotaemic and azotaemic CKD groups were made using the Mann Whitney U test. Correlations were assessed by Spearman's correlation coefficient. Data are presented as median [25 th , 75 th percentile] and statistical significance was defined as P < 0.05.
Naturally infected clinically ill FIV-positive cats were prospectively included. The Doppler ultrasonic technique was used to measure SBP according to ACVIM guidelines. Serum creatinine (sCreat) and urea (sUrea) concentrations, urine specific gravity (USG) and urinary protein:creatinine ratio (UPC) were determined.

ISCAID-O-1 CLINICAL AND GENETIC CHARACTERIZATION AND VIRUS NEUTRALIZATION PATTERNS OF FELINE CAL-ICIVIRUS ISOLATES FROM FOUR VIRULENT-SYSTEMIC

Outbreak 1 occurred in a cattery in Liechtenstein and involved five non-vaccinated, 3-months old siblings with fever, edema, skin and tongue ulcerations. Outbreak 2 occurred in a small animal clinic in Zurich. A 10-year old cat presented with severe paw edema, fever, tongue and skin ulcerations, progressive hypoproteinemia and hyperbilirubinemia. Outbreaks 3 and 4 happened in a cattery in Lausanne five months apart and involved two litters of non-vaccinated, 2-to 3-months old kittens. The cats presented with fever, nasal discharge, edema and skin and oral ulcerations.

Conflicts of interest:

At initial presentation, the most common clinical signs were lethargy (96%), anorexia (88%), vomitus (85%), a painful abdomen (39%), diarrhea (38%), oliguria (27%), tachypnea (26%), delayed capillary refill time (18%), pale mucous membranes (17%), fever (15%), hypothermia (15%), and icteric mucous membranes (10%). Abnormal findings of the CBC included anemia (63%), thrombocytopenia (62%) and leukocytosis (57%). Biochemistry abnormalities included increased creatinine concentrations (82%), increased liver enzyme activities (80%), hyperbilirubinemia (70%), hyperphosphatemia (67%), hyponatremia (63%), and hypoalbuminemia (55%). Urinalysis often revealed glucosuria (77%) and an elevated urine-protein/creatinine-ratio (75%). Radiological pulmonary changes were detected in 57% of the dogs initially or during the course of disease. 32 dogs died or were euthanized, 24 of them due to "leptospiral pulmonary hemorrhage syndrome".
A total of 59 dogs were diagnosed with leptospirosis based on clinical signs and either microscopic agglutination test, blood/ urine polymerase chain reaction, and/or histopathology. At the time of admission and, in most patients, after an average of two weeks canine pancreatic lipase immunoreactivity (cPLI, as measured by Spec cPL â ), ultrasensitive cardiac Troponin I (cTnI), and C-Reactive Protein (CRP) were analyzed. Data were analyzed with non-parametric statistics. The level of significance was set at p < 0.05. Upon admission, common clinical signs reported included lethargy (n = 57), vomiting (n = 50), abdominal pain (n = 20), dyspnea (n = 16), pale mucous membranes (n = 13), oliguria (n = 11), hypothermia (n = 11), and fever (n = 10). Anemia (n = 39), thrombocytopenia (n = 41), leukocytosis (n = 38), were frequently reported hematology findings. Increased concentrations of creatinine (n = 48/59), phosphorus (n = 43/57), ALT (n = 31/ 58), SAP (n = 43/57) and bilirubin (n = 41/58) were also frequently recorded. CRP (median: 48.7 mg/L; range: 0.1-60.1 mg/L, reference interval (RI): 0.1-7.6 mg/L), cTnI (median: 0.137 ng/L; range: 0.005-24.063 ng/L, RI: 0-0.059 ng/L), and cPLI (median: 217 lg/L; range: 29-1001 lg/L, RI: 0-200 lg/L) concentrations were above the upper limit of the reference intervals in 52/59 (88%), 42/59 (71%), and 30/59 (51%) dogs, respectively and serum cPLI concentration was above the suggested cut-off value for a diagnosis of pancreatitis in 15/59 (25%) dogs. CRP and cTnI, but not cPLI were higher upon admission compared to the re-check measurement (p = 0.0001 and 0.0056, respectively). Dogs with increased serum cPLI concentrations also showed a higher proportion of dogs with increased serum cTnI concentrations (p = 0.001). There was no statistically significant correlation of cPLI concentrations with a history of abdominal pain and/or vomiting. Biochemical results were compatible with multiple organ impairment with involvement of kidneys, liver, heart, and exocrine pancreas where at least two organs were affected in 36/59 (61%) dogs. Forty (68%) of 59 dogs recovered, 10 (17%) died, and 9 (15%) were euthanized. cTnI and cPLI were higher in non-survivors, but these differences did not reach statistical significance. However, the number of organs affected and outcome were significantly correlated (p = 0.012).
Conflicts of interest: Financial support of Royal Canin. Canine chronic enteropathies (CCE) include diet-responsive, antibiotic-responsive, and immunosuppressive-responsive enteropathies (IRE). This prospective study was designed to evaluate a commercial hypoallergenic dry diet a containing oligopeptides as the only protein source for the management of dogs with IRE and as an alternative to immunosuppressive therapy over a 10 week period.

ESCG-P-8 SERUM CITRULLINE AS

Serum citrulline was measured in 49 dogs with CE and 69 controls. 17 dogs responded to dietary manipulation (food-responsive enteropathy, FRE) and 3 responded to antibacterials (antibiotic-responsive diarrhoea, ARD), with a further 2 having invasive mucosal bacteria, of which one responded to antibacterials and one was refractory. 27 dogs were diagnosed with idiopathic IBD (on the basis of exclusion of known causes and failure to respond to therapeutic dietary and antibiotic trials), of which 12 responded to immunosuppressive therapy, 13 were refractory, and 2 were lost to follow-up. Serum citrulline concentration did not differ between dogs with CE (median 8.5 lg/mL, range 1.4-20.6) and controls (median 8.2 lg/mL, range, 1.2-34.9, P = 0.96). There was also no difference in serum citrulline concentration amongst dogs with FRE, ARD, IBD, and controls (P = 0.48). Serum citrulline did not differ between dogs that responded well, or were refractory to treatment (P = 0.39), between 23 dogs with and 26 without protein-losing enteropathy (P = 0.67), or between 35 dogs that survived and 8 that were euthanased because of CE (P = 0.65). Serum citrulline did not correlate with CIBDAI (r 2 = 0.10).

Conflicts of interest:

The CU and CH draft genomes were~1.732 Mb and 1.844 Mb in size, and comprised on average 110 and 151 contigs, and on average 1782 and 1942 predicted genes, respectively. Of these CU had on average 497 and CH 622 hypothetical proteins. Using OrthoMCL, a core genome of 1459 and 1751 genes resulted for CU and CH, respectively. NeighborNet trees based on ribosomal MLST nucleotide sequences and the core genome confirmed the close phylogenetic relationship of CH and CU within the Campylobacter genus. PathogenFinder predicted all isolates as human pathogens with probabilities of 88.3-91.5%. Both PathogenFinder and VirulentPred identified many pathogenic proteins in CU and CH of different functions (e.g. chemotaxis, transporter and motility systems) but considerably fewer than in C. jejuni and C. coli.
Authors are affiliated with genetic disease screening test laboratory. Remarkably little has been published on haematological and serum biochemical phenotypes of the domestic dog. Information on the signalment and complete blood cell count of all dogs with normal red and white blood cell parameters judged by existing reference intervals was extracted from a veterinary database; similar information was collected from all dogs with normal serum biochemical profiles, considering all parameters other than glucose as inclusion criteria. Normal haematological profiles were available for 6046 dogs, 5447 of which also had machine platelet concentrations within the reference interval; normal serum biochemical profiles were available from 3045 dogs, 1495 of which also had accompanying normal serum glucose concentrations. For the haematological data, 75 pure breeds plus a mixed breed control group were represented by 10 or more dogs, while for the serum biochemical data, 60 pure breeds plus a mixed breed control group were represented by 10 or more individuals. All measured haematological parameters except mean corpuscular haemoglobin concentration (MCHC), and all serum biochemical analytes except sodium, chloride and glucose, varied with age. Concentrations of white blood cells (WBCs), neutrophils, monocytes, lymphocytes, eosinophils and platelets, but not red blood cell parameters, all varied with sex, as did total protein, globulin, potassium, chloride, creatinine, cholesterol, total bilirubin, and activities of alanine aminotransferase (ALT), creatine kinase (CK), amylase and lipase. Neutering status had an impact on haemoglobin concentration, mean corpuscular haemoglobin (MCH), MCHC, and concentrations of WBCs, neutrophils, monocytes, lymphocytes and platelets, as well as all serum biochemical analytes except albumin, sodium, calcium, urea and glucose. Principal component analysis (PCA) of haematological data revealed 37 pure breeds with distinctive phenotypes, while PCA of serum biochemical data revealed over 50 pure breeds with distinctive phenotypes. Furthermore, all haematological parameters except MCHC and all serum biochemical analytes except urea and glucose showed significant differences between specific individual breeds and the mixed breed group. Twenty-nine breeds had distinctive haematological phenotypes and 21 breeds had distinctive serum biochemical phenotypes when assessed in this way. Tentative breed-specific reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis. This study represents the first large-scale analysis of haematological and serum biochemical phenotypes in the dog and underlines the important potential of this species in the elucidation of genetic determinants of haematological and biochemical traits, triangulating phenotype, breed and genetic predisposition, as well as the urgent need for breed-specific reference intervals in clinical practice.
In human medicine, primary disorders of the heart often result in secondary dysfunction or injury to the kidneys. The coexistence of the two problems in the same patient is referred as cardiorenal syndrome (CRS). Just little information about CRS is available in veterinary medicine.

ESVCN-O-3 EFFECTIVENESS OF A NEW DIETETIC WEIGHT MAN-AGEMENT FOOD TO ACHIEVE WEIGHT LOSS

* Porphyrias are a group of inborn errors of metabolism resulting from accumulation of porphyrins due to deficient activities of specific enzymes in heme biosynthesis. In humans, they are clinically classified as either erythroid with cutaneous involvement or hepatic with acute neurovisceral attacks. Here we describe the clinical, biochemical, and molecular genetic studies in porphyric cats from New Brunswick, Canada.

Conflicts of interest:

The aim of this study was to adapt and validate test procedures and protocols previously developed for humans for use in dogs and cats.
No conflicts of interest reported. Feline diabetes mellitus shares many similarities with human type 2 diabetes mellitus (T2DM), including clinical, physiological and pathological features of the disease. Domestic cats spontaneously develop diabetes associated with insulin resistance in their middle age or later, with residual but declining insulin secretion. Humans and cats share the same environment and risk factors for diabetes, such as obesity and physical inactivity. Moreover, amyloid formation and loss of beta cells are found in the diabetic cat pancreas, as in humans. Subsequently, studying the molecular mechanisms in the failing beta cells may contribute to a better understanding of the pathophysiology of T2DM in both cats and humans.
No conflicts of interest reported. Hypersomatotrophism (HS) is an important cause of feline diabetes mellitus (DM). In humans surgical removal of the somatotrophinoma is generally recommended, though hypophysectomy programs have suffered from significant initial morbidity and mortality given a documented steep learning curve in newly established programs. Hypophysectomy as treatment for feline HS has thus far only been described in a handful of cases, all having been treated by one single experienced hypophysectomy team. This study's aim was to evaluate the learning curve of a de novo established hypophysectomy program, through analysis of peri-and post-operative morbidity and mortality, and endocrine outcomes in the first cohort of cats with HS treated.
Ligneous membranitis is well described in humans, where it is inherited in an autosomal recessive manner. Patients commonly present as infants. Ocular, oral and genital lesions are most common, but other organs are occasionally involved and congenital obstructive hydrocephalus is reported in some individuals. Numerous mutations and polymorphisms in the plasminogen gene have been identified in affected individuals.
The cost of examinations reported in this study were covered by Hill's Pet Nutrition Belgium. Systemic Lupus Erythematosus, SLE, is a chronic autoimmune disorder with varying clinical manifestations and diagnosis is based on both clinical signs and laboratory findings. Other systemic rheumatic diseases, referred to as SLE-related diseases or immune-mediated rheumatic disease (IMRD), are also described. The most common clinical signs in dogs are stiffness and pain from varying joints. One hallmark of SLE and SLE-related diseases in both dogs and humans is high titres of circulating antinuclear antibodies (ANA), which can be demonstrated by the indirect immunofluorescence (IIF) ANA test. Earlier studies have shown that canine IIF ANA positive samples may be divided into two main subgroups: homogenous (ANA H ) and speckled (ANA S ) IIF ANA fluorescence pattern. In humans, further determination of the specificity of ANA positive sera is frequently employed to characterize the ANA reactivity. Some of these ANA specificities have been demonstrated in man to strongly associate with different systemic autoimmune diseases and also with different IIF ANA staining patterns. Presence and character of antinuclear antibodies in canine SLE-related diseases are not well described.
Several specific ANA-reactivities earlier characterized in human patients were identified.
No conflicts of interest reported. Leptospirosis, a zoonotic bacterial disease with a worldwide distribution, is a re-emerging disease in humans and dogs. Acute renal and hepatic failure are the most frequently reported clinical manifestations of canine leptospirosis.
The results of this study suggest that C. perfringens type A is the most important C. perfingens genotype involved in the disease process of dogs with AHDS. Although C. perfringens enterotoxinhas been associated with intestinal diseases in humans, dogs, horses, pigs, and other animal species, this enterotoxin is most likely not responsible for the intestinal lesions in dogs with AHDS.
No conflicts of interest reported. In mitral valve disease, atrial remodeling is an indicator of evolution and prognosis, the duration of the P wave being considered suggestive of the dilatation of the left atrium. In humans' studies, neurological conditions have a significant impact on cardiac electrophysiology by altering the electrical impulse conductibility.
The study was partially supported by Laborat orio Segalab S.A. and Dechra Veterinary Products. Canine diabetes mellitus (cDM) has been proposed to be a spontaneous animal model of human autoimmune diabetes, and comparative research can be undertaken to investigate the interaction between genetic and environmental factors. Most epidemiological studies of cDM have been performed in northern European and North American populations.
No conflicts of interest reported. The cortisol-dehydroepiandrosterone (DHEA)-ratio is widely used in human medicine as a marker for stress however it is not clear whether it could also help in distinguishing hyperadrenocorticism (HAC) from other diseases which might have a negative impact on the outcome of a dexamethasone low dose test. Therefore the aim of the study was to evaluate the cortisol-DHEA-ratio as an additional diagnostic marker for HAC in dogs. To achieve this aim, a reference range of this ratio depending on the sex should be evaluated in healthy dogs and compared with dogs having a HAC. In 55 healthy dogs (age: 1 -11.4 years) and in 20 dogs with HAC (age: 7.1 -14.6 years) of different breeds the plasma concentration of cortisol (Immulite System, Siemens Healthcare Diagnostics) and DHEA (Beckman Coulter) was measured and the ratio was calculated. All dogs were patients of the Small Animal Clinic except five of the healthy dogs which were recruited from the Institute of Pharmacology, Toxicology and Pharmacy of the University. With these data the cortisol-DHEA-ratio was calculated for male dogs (healthy dogs n = 18; dogs with HAC n = 3), neutered males (healthy dogs n = 9; dogs with HAC n = 5), female dogs (healthy dogs n = 21; dogs with HAC n = 5) and spayed females (healthy dogs n = 7; dogs with HAC n = 7). The statistical analysis was performed with Sigma Stat. The plasma cortisol-DHEA-ratio of healthy male dogs was the lowest ratio of all sexual categories (mean average 84.8 AE 128) and it differed significantly to all other sexes (neutered males = 231 AE 138, P = 0.002; females = 244 AE 124, P < 0.001 and spayed females (183 AE 60.0, P = 0.006). The cortisol-DHEA-ratio showed no significant difference between male and female dogs with HAC. Spayed females with HAC had significantly higher cortisol-DHEA-ratios (501 AE 310) than healthy spayed females (P = 0.035) but no significant differences were found in other sexual categories. This preliminary data indicates that the cortisol-DHEA-ratio might not be a very promising tool for the diagnosis of HAC. In addition, the significant gender-dependency of this parameter has to be considered and may generally limit its clinical usefulness. This study is financially supported by the Bruns-Stiftung.
The study was partially funded by MSD Intervet. The main endocrinopathy affecting both humans and pet felines is diabetes mellitus. Accurate diagnosis is the most important aspect in the future outcome of the disease. A computer based Decision Support System (DSS) is targeted on assisting clinicians with one or more steps of the diagnostic process. The novelty of our DSS emerges from the possibility of assisting both clinical and paraclinical diagnosis stages of diabetes mellitus and all common combination of disorders associated with this endocrinopathy. The motivation behind the development of such system is the desire to maximize the reliability of clinical decisions.
The aim of this study was to characterise feline pancreatic neoplasms in more detail, based on the human classification system with a special view on cystic lesions.
The neoplasms showed a cystic (n = 8) or solid (n = 11) pattern. Cystic pancreatic tumors were up to 7 cm in diameter and were classified as benign variants in five and malignant variants in three cases. Based on the human classification system, they were classified as tubulopapillary (n = 2), acinar (n = 2) and mixed (n = 1) adenomas and mixed carcinomas (n = 3), respectively. Solid pancreatic nodules were diagnosed as carcinomas with a tubular (n = 5) or acinar (n = 6) differentiation pattern.
The author receives a salary as Editor of the BSAVA journal Companion, and has undertaken unrelated paid consultancies for Bayer and Merial. The author also receives a salary from Avacta Animal Health, and duties involved working directly on this project. Canine chronic enteropathy (CCE) can cause significant long-term morbidity. In some cases this is due to intestinal inflammation, resulting from idiopathic inflammatory bowel disease (IBD). Currently, the diagnosis of idiopathic IBD and assessment of disease severity relies on results of subjective clinical indices, laboratory data, diagnostic imaging and intestinal histopathology, whilst ruling out known causes of inflammation. In humans with IBD, a number of faecal biomarkers including lactoferrin, aid with diagnosis and determining disease activity. It may therefore be valuable to develop similar non-invasive objective methods to aid diagnosis and clinical assessment of disease severity in dogs with intestinal inflammation due to idiopathic IBD.
In conclusion, subclinical myocardial alterations were detected in both HET and CNM aging dogs from our French pedigree, suggesting a role for PTPLA in long-term cardiovascular homeostasis. These findings prompt globalized confirmation in additional PTPLA"'deficient dogs, which may thus be considered as a new large-size model for human left ventricular sub-clinical myocardial dysfunction.
Echocardiography represents the cornerstone of PDA diagnosis, but its role has been recently expanded to wider field of application: device sizing and intraoperative monitoring, as well as a tool to quantify cardiac morphology and function. Speckletracking echocardiography (STE) has been used to evaluate cardiac function in a wide variety of diseases in human and veterinary patients, however no study has evaluated its usefulness in dogs affected by PDA both before and after percutaneous closure of PDA.
Dr Bussadori receives royalties from ESAOTE (Florence, italy) related to an european patent (nr 071129712) he developed for Xstrain software. The study was not funded by a research grant. Cardiac cachexia which is characterized by progressive weight loss and depletion of lean body mass, is an independent predictor of survival in human patients with congestive heart failure. Chronic degenerative mitral valve disease (CDMD) is one of the most common cardiac diseases in dogs. The aims of this study were to evaluate the prevalence and the effects of cardiac cachexia in survival of dogs with CDMD.
The following conflicts of interest apply: The diet used in this study is manufactured by Royal Canin.whilst VB is employed by Royal Canin. VB and SS are employed by Royal Canin. AJG's Readership is funded by Royal Canin. Obesity and obesity-related metabolic dysfunctions are increasing in humans as well as in dogs. Obese dogs become affected by chronic diseases at young age, have a decreased quality of life and a shorter life-span. The aim of the study was to describe the metabolic and hormonal response to a feed-challenge test in lean and overweight dogs.

ESVCN-O-2 METABOLIC AND HORMONAL RESPONSE TO

The assigned BCS was supported by positive association with serum leptin concentrations. Postprandial triglyceride concentration was significantly higher in the overweight group. A tendency to higher cholesterol concentration was seen in the overweight group but cholesterol was not affected by food intake. Glucagon concentration rose after food intake and resembled the response seen in humans after a mixed meal. Glucose and insulin concentrations followed the same pattern while free fatty acids had declined one hour after the meal.

Conflicts of interest:

Urs Giger and Raj Karthik are also part of the laboratory that offers DNA testing for this mutation. Fibrinogen decreases when coagulation is activated to form fibrin, while FDPs and D-dimers represent the products of fibrinolysis. In humans, activation of coagulation and fibrinolysis develops in all type of ascites and it is also associated with signs of systemic fibrinolysis.These results have lead to the suggestion that ascitic fluid is inherently fibrinolytic. Preliminary studies showed similar results also in dogs (JAVMA Nov. 2012 , ECVIM proceedings 2013 . In addition, in an old experimental study conducted in dogs, inoculation of blood or of a solution containing fibrinogen and thrombin into the pleural cavity resulted in the activation of the coagulation system followed by fibrinolysis. Therefore, the objective of the present study was to determine whether the activation of coagulation and fibrinolysis (i.e. low fibrinogen and elevated FDPs and Ddimer) occurs not only in the ascitic fluid, as alredy been demonstrated, but also in all type of pleural effusions in dogs. Thirty-three dogs referred to the San Marco Veterinary Clinic with pleural effusion, but without ascites, were studied. Fibrinogen, FDPs, and D-dimer concentrations were measured and then compared in both pleural fluid and venous blood via Wilcoxon signed ranks test. The dog's pleural effusions were categorized based on pathophysiology of fluid formation into 5 dogs with transudate (4 due to increased hydrostatic pressure and 1 due to decreased osmotic pressure), 23 with an exudate (of which 11 due to septic causes), 4 with a haemorrhagic pleural effusion, and 4 with a chylous effusions. The fibrinogen concentration in the pleural effusion (median: 59 mg/dL; range: 59-59) was significantly lower (p < 0.0001) than the plasma fibrinogen concentration (median: 419 mg/dL; range: 131-1406). In all dogs, the fibrinogen pleural fluid concentration was lower than the plasma concentration. The FDP concentration in the pleural effusion (median: 151 mg/dL; range: 0.69-151) was significantly (p < 0.0001) higher than plasma FDPs concentrations (median: 5.55 mg/dL; range: 0.83-108.47). In 1 case, the FDPs pleural fluid concentration was lower than the plasma concentration and in 32 cases the pleural fluid concentration was higher. The D-dimer concentrations were significantly(p < 0.0001) higher in the pleural effusion (median: 3.84 lg/mL; range: 0.05-9.61) than in the plasma (median: 0.07 lg/mL; range: 0.01-7.67). In one case, the D-dimer pleural fluid concentration was lower than the plasma concentration and in 32 cases was higher. These findings support the hypothesis that activation of coagulation followed by fibrinolysis occurs in all type of pleural effusions.
Residual samples of citrate anticoagulated blood were used from dogs and cats presented to a specialist referral centre for various reasons unrelated to clotting abnormalities. Initially the blood was stimulated using specific combinations of either arachidonic acid/epinephrine (AA/EPI) or ADP/U46619, designed to assess the effects of the anti-thrombotic agents aspirin and clopidogrel respectively. After 5 minutes stimulation, the blood samples were fixed using a patented platelet fixative solution developed for human platelets, which allows the delayed analysis of P-selectin an established marker of platelet activation.
Some of the authors are members of diagnostic laboratories (PennGen). Supported in part by the NIH OD #010939. Glucagon-like peptide-1 (GLP-1)is a gastrointestinal hormone released in response to food intake that increases insulin secretion, inhibits glucagon secretion, slows gastric emptying and induces satiation. It is also assumed to stimulate beta-cell proliferation. GLP-1 agonists are successfully used in humans with type 2 diabetes mellitus usually either in combination with insulin or other anti-diabetic drugs. In healthy cats twice daily (exenatide) as well as once weekly (exenatide extended-release (ER)) application of GLP-1 agonists induced pronounced insulin secretion. Benefits of exenatide ER are the regimen of once weekly injection and less side effects. The objective of the study was to assess whether administration of exenatide ER in addition to standard treatment leads to improved glycemic control and higher remission rates in cats with newly diagnosed diabetes.
The laser microdissection technique allows studies of islets without contamination of acinar cells, as shown in this study, and is of great advantage since it is difficult to get pure feline islets from collagenase-based isolation. Differences in gene expression in healthy and diabetic cats may reveal underlying mechanisms for beta cell dysfunction and decreased beta cell mass in human and feline type 2 diabetes.
The objective of this study was to identify the feline AIP gene, identify single nucleotide polymorphisms (SNPs) within this gene and compare any SNPs with reported human AIP SNPs. Stored pituitary tissue from an acromegalic cat was used to create feline AIP cDNA using feline specific AIP primers. Stored EDTA blood from 10 acromegalic cats (diagnosis of insulin resistant diabetes mellitus, serum IGF-1 > 1000 ng/ml and pituitary mass >4 mm identified using pituitary computed tomography or necropsy) and 10 control cats (no history of diabetes mellitus and greater than 15 years of age) were selected, DNA extracted and genotyped using PCR, agarose gel electrophoresis and Sanger sequencing.
Conflicts of interest: This study was supported by grants from the Swedish Research Council and the Foundation for Research, Agria Insurance Company. The concomitant occurrence of two or more endocrine tumors and/or hyperplasias, known as multiple endocrine neoplasia (MEN) is a well-known entity in humans. Multiple gene mutations have been identified. The two major forms are MEN1 and MEN2. In MEN1, the main affected organs are parathyroid, pancreas and pituitary gland. MEN2 occurs in 3 clinical variants: MEN2A, characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and primary hyperparathyroidism; MEN 2B, characterized by MTC, pheochromocytoma and additionally abnormalities; familial medullary thyroid carcinoma. In dogs and cats only a few cases have been reported and it is unknown whether hereditary MEN-like syndromes exist in these species. The aim of this study was to evaluate the prevalence of multiple endocrine tumors in dogs and cats at our institution, to identify possible breed and sex predispositions and to investigate similarities with the human MEN syndromes. Autopsy reports of dogs and cats from 2004 until 2014 were reviewed. Animals with at least two endocrine tumors/hyperplasias (ETH) were included. Autopsy reports of 951 dogs and 1155 cats were examined. 149 dogs had ETH affecting a single organ, 24 had multiple ETH; 123 cats had single ETH, 21 had multiple ETH. In dogs with multiple ETH, the most common breeds were West Highland White Terrier (WHWT, 3/24), Poodle, Golden Retriever, mixed-breed dogs (each 2/24). 14/24 were male (13 intact); 10/24 were female (10 neutered). Median age was 12 years (range 7-18). The most common combination was multiple testicle tumors of various types (4/24). The most common affected organs were the adrenals (18/24). Adrenal cortical adenomas/carcinomas/hyperplasias were mainly associated with pheochromocytomas (3/ 24), testicle tumors (3/24) and insulinomas (2/24). All 3 WHWTs had adrenal adenomas. Both Poodles had pheochromocytoma associated with pituitary adenoma or adrenal hyperplasia. 2 dogs showed tumor combinations similar to the human MEN1 syndrome: pituitary adenoma and insulinoma; pituitary adenoma and parathyroid hyperplasia. 19/21 cats were domestic short/long hair, 2/21 were Persians. 11/21 were male (7 castrated); 10/21 were female (9 neutered). The median age was 15.5 years (range 10-19). The most common affected organs were thyroid glands (18/21), combined mostly with lesions of parathyroid (10/21) and adrenal glands (7/21). None of the cats had combinations similar to the human MEN syndromes. The prevalence of multiple ETH in dogs and cats was 2.5% and 1.8%. MEN-like syndromes were extremely rare in dogs and non-existing in cats. No sex predisposition was observed. Possible breed predispositions need further investigations.
One of the authors, Erik Lattwein, is employed by EUROIMMUN where the analyses were performed. Chronic kidney disease (CKD) has a high prevalence in cats. Routine renal markers, serum creatinine (sCr) and urea are not sensitive or specific enough to detect early CKD. Serum Cystatin C (sCysC) has advantages over sCr for the detection of early kidney dysfunction, both in humans and dogs. A significant higher sCysC concentration in CKD cats has been demonstrated. The objective of this study was to determine the effect of age, gender and breed on feline sCysC and to establish a reference interval for feline sCysC.
Based on these findings, AMC and TMS would be the firstchoice antimicrobial agents for empiric treatment of bacterial The increasing rates of resistance exhibited by uropathogens represent a serious problem for the selection of an appropriate antibiotic. The aim of this study was to determine secular trends of companion animal urinary tract infection (UTI) that involve extended-spectrum b-lactamase (ESBL)-and carbapemenase-producing Gram negative bacteria (namely, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii), methicillin-resistant-staphylococci (MRS) and ampicillin and high-level-gentamicin-resistance (HLGR) enterococci. Nine hundred and twenty two uropathogenic bacteria were isolated from dogs and cats, between January 1999 and March 2014, at the Veterinary Teaching Hospital of the Faculty of Veterinary Medicine and at veterinary private practices in the Lisbon area. Isolates were identified using standard commercial systems. Susceptibility testing was performed using the disk diffusion and broth microdilution methods. CLSI breakpoints were applied. Extended-spectrum b-lactamases (ESBL) production was screened by double-disk synergy test. The ESBL, plasmid-mediated AmpC, carbapemenases, mecA and aac(6')-Ieaph(2'')-Ia genes were detected by PCR and gene enzymes were sequenced. Among Enterobacteriaceae 0.7% were DHAproducers, 2.7% were ESBL-producers and 3.6% were CMYproducers. All isolates were also multidrug-resistant. Cefalosporinases-producer Enterobacteriaceae were detected in 2000, the first being a CMY-2-producer E. coli. All the ESBL-producers were E. coli or K. pneumoniae producing CTX-M-group 1 enzymes. Ampicillin-resistance in enterococci was present throughout the years (15,4%, n = 8). HLGR appeared in enterococci in 2003 and was confirmed by the detection of the bifunctional enzyme that confers high level resistance to aminoglycosides (7 out of 8 isolates). In this study we showed that in the last decade the emergence of resistance to critically important antimicrobials among uropathogens from companion animals is a concerning fact. The multidrug-resistant Enterobacteriaceae may compromise effective therapeutic options, namely third and fourth generation cephalosporins, fluoroquinolones, trimethoprim/sulpha combinations. The emergence of MRSA/MRSP and HLGR among uropathogens is also a therapeutic challenge. The detection of uropathogens with antimicrobial resistance is not only an animal health issue but also a matter of public health, since companion animals may act as reservoirs of antimicrobial resistant bacteria or resistance genes for humans.
No conflicts of interest reported. In canines mastocytomas are among the most frequently diagnosed neoplasms of the skin. High grade mastocytomas (grade III, Patnaik classification) are characterized by an uncontrolled growth of neoplastic mast cells (MC) and a poor prognosis. Recently, the KIT-targeting tyrosine kinase inhibitors masitinib and toceranib have been approved for the treatment of canine MC tumors. These drugs are able to induce responses in mastocytoma patients. However, in many patients, relapses are seen. Therefore, research is focusing on new drug targets. Recently, the transcription factor STAT5 has been reported to play an important role in the proliferation and survival of human neoplastic MC. The aim of the present study was to evaluate the JAK2-STAT5 pathway in canine mastocytomas. To address this aim, the canine mastocytoma cell lines C2 and NI-1 as well as inhibitors directed against JAK2 or STAT5 were employed. As assessed by immunocytochemistry, C2 cells and NI-1 cells were found to express pSTAT5 in their cytoplasm and nuclei. Intracellular expression of pSTAT5 was confirmed by flow cytometry. Interestingly, C2 cells were found to express higher levels of pSTAT5 compared to NI-1 cells. Next, we treated C2 cells and NI-1 cells with various concentrations of the STAT5 inhibitors piceatannol and pimozide and the JAK2 inhibitors AZD1480 and TG101348. As assessed by 3 H-thymidine uptake, all 4 compounds were found to inhibit the proliferation of canine MC in a dose-dependent manner. Drug effects were found to vary in different cell lines, with the following rank-order of potency (IC 50 values): TG101348: 0.1-0.75 lM; pimozide: 0.1-2.5 lM; AZD1480: 1-2 lM; piceatannol: 5-50 lM. To further explore the mechanism of drug-induced inhibition of proliferation, we examined cell cycle progression and apoptosis in drug-exposed cells. Whereas all 4 drugs tested induced only moderate cell cycle arrests in the G1 phase, these drugs were found to induce substantial apoptosis in C2 cells and NI-1 cells as evidenced by microscopy and Annexin-V/PI staining. Together, our data show that JAK2-and STAT5-targeting drugs exert anti-proliferative and apoptosis-inducing effects in canine mastocytoma cells suggesting that this signaling pathway may be a promising new therapeutic target in canine mastocytomas. The clinical relevance of this observation remains to be determined.

ISCAID-O-5 IMMUNOHISTOCHEMICAL DETECTION OF IGG AND IGM IN LUNG TISSUE OF DOGS WITH LEPTOSPIRAL

PULMONARY HAEMORRHAGE SYNDROME (LPHS). S. Schuller 1 , S. Callanan 2 , S. Worrall 2 , T. Francey 1 , A. Schweighauser 1 , J.E. Nally 2 . 1 Bern University, Bern, Switzerland, 2 University College Dublin, Dublin, Ireland Leptospiral Pulmonary Haemorrhage Syndrome (LPHS) is a severe form of leptospirosis, which has been increasingly recognised in humans and many animal species in the past 20 years. Patients with LPHS may develop rapidly progressive intra-alveolar haemorrhage, leading to high mortality. The pathogenic mechanisms of LPHS are poorly understood hampering the application of effective treatment strategies. Studies in humans and experimentally infected guinea pigs have demonstrated deposition of immunoglobulin and complement C3 in LPHS lung tissue in the absence of significant numbers of leptospires, suggesting that LPHS is, in part, caused by autoimmunity. The aim of this project was to describe the histopathologic features of LPHS in dogs and to investigate whether IgG and IgM deposition is present in affected canine lung tissue.

Conflicts of interest:

Comparing spectra from the HE affected dogs with those from the control-group decreased values of Ins, Cho, NAA as well as High field MRI combined with brain MRS provide accurate and non-invasive diagnosis of canine HE. In accordance with human medicine publications, it could be state that MRS has a role in HE diagnosis and follow-up with particular mention monitoring.
This study demonstrates that the biopsies with a needle length of at least 10 mm brings satisfactory information for the evaluation of most of the inflammatory, vacuolar hepatopathies, fibrosis and diffuse tumoral infiltrations. Wedge biopsies allow to examine the largest number of portal triad, more contributory for certain forms of cholangitis affecting larger canals and for a single case, images of peri-hepatitis were counted at the level of the capsule. Fibrosis does not seem to be more important in the sub-capsular zone contrary to what is observed in human pathology.
No conflicts of interest reported. Gallbladder diseases like gallbladder mucocele and cholecystitis can reduce gallbladder motility and may lead to cholestasis. Since impaired gallbladder emptying contributes to sludge and gallstone formation, the evaluation of gallbladder motility requires accurate and appropriate methodology. Three-dimensional (3D) ultrasonography has been shown to be accurate and appropriate tool for measurement of gallbladder volume in humans. Therefore, we applied this novel technique for the first time to study preprandial and postprandial gallbladder volume in 10 healthy mixed-breed dogs and compared the results to twodimensional (2D) ultrasonography. The dogs were placed in dorsal recumbency to obtain ultrasonographic measurements of the gallbladder. Measurements by both 2D and 3D ultrasonography were recorded in preprandial state and after ingestion of full-fat milk. The preprandial and postprandial gallbladder volumes determined by 3D ultrasonography were significantly higher than corresponding volumes by 2D ultrasonography (1.11 AE 0.07 vs 0.77 AE 0.06 and 0.81 vs 0.61 ml/kg, respectively, P < 0.05). In 2D ultrasonography, most dogs (8/10 [80%]) had a preprandial gallbladder volume ≤ 1.00 ml/kg. However, in 3D ultrasonography, 6/10 (60%) of dogs had a preprandial gallbladder volume ≥ 1.00 ml/kg. Gallbladder contraction index was higher in 3D ultrasonography than 2D ultrasonography, however, it did not reach statistical significance (P = 0.25).
One of the co-authors (Marco Caldin) has a diagnostic laboratory offering citrulline assays. Chronic enteropathy (CE) is a multi-factorial disease, which involves aberrant immune responses to commensal bacteria or dietary antigens. Macrophages have an important role in human disease but little information is available in canine intestine. Data to date have relied solely on macrophage identification using MAC387, an antibody directed against calprotectin, which recognizes both macrophages and neutrophils. In this study an alternative antibody for macrophages, AM-3K, directed against a scavenger receptor (CD163) was used and distribution of both markers was compared. This antigen is of interest as positive cells accumulate in intestine of humans with CE.
No conflicts of interest reported. Campylobacter species are commonly isolated from faeces of dogs and cats with C. upsaliensis (CU) and C. helveticus (CH) being the most frequently isolated. These two species are usually not considered pathogenic in dogs and cats and are closely related to each other and to C. jejuni, the most common cause of bacterial gastroenteritis in humans in the developed world. Interestingly, despite their close genetic relationship, in humans CU is considered a pathogen while CH is not. This study aimed to describe whole genomes of CU and CH isolated from dogs and cats and to in silico investigate their pathogenic potential with comparison to several published genomes of C. jejuni and C. coli.
No conflicts of interest reported. Companion animals presenting to the emergency room in distress need to be assessed rapidly and accurately to implement life-saving therapies. Focused cardiac ultrasound (FOCUS) can be a useful adjunct to the physical examination in assessing dyspneic animals in the emergency room. Rapid bedside ultrasound evaluations performed by EC are commonly used in human medicine, however feasibility and utility of FOCUS by EC in veterinary medicine has not been fully evaluated. The purpose of this study is to determine the baseline accuracy of FOCUS performed by EC and whether or not a basic training session could improve accuracy compared to evaluation by a cardiology specialist. Fifteen EC including 6 boarded emergency-critical care specialists and 9 emergency res-idents performed FOCUS on four animals; a normal cat and dog, and a cat and dog with severe valvular and myocardial heart disease, respectively. EC semi-quantitatively assessed 6 thoracic and echocardiographic parameters including left atrial dimension, left ventricular systolic function and wall thickness, right heart dimension, and presence or absence of pleural or pericardial effusion before and after a structured didactic lecture and hands-on practical session. Primary outcome was the level of agreement with examination performed by a cardiologist. Level of agreement regarding EC assessment of all parameters improved from 0.70 to 0.78 after training (P < 0.01). Level of agreement concerning left atrial diameter improved from 0.52 to 0.75 (P < 0.01). EC confidence in their overall FOCUS evaluation and findings improved from 51% to 70% (P < 0.0001). In summary, EC accuracy and confidence in semi-quantitatively assessing basic cardiac parameters using FOCUS were improved following a simple structured training session. FOCUS might be a valuable tool to rapidly assess simple thoracic and cardiac parameters in the emergency setting.
The 3rd and 4th author (M. Hennies and C. Wienen) work for the company TECOmedical Group that developed the ELISA which was evaluated in the study. They provided the kits and they helped with performing the tests, but they did not have any influence on the results and the interpretation of the data. Canine idiopathic pulmonary fibrosis (CIPF) is a progressive interstitial lung disease that mainly occurs in the West Highland white terrier (WHWT) breed. The CIPF diagnosis commonly relies on thoracic high-resolution computed tomography (HRCT) findings and ultimately on histopathology. As those tests are not easily performed in practice, identification of measurable markers of fibrosis, that might help to diagnose and/or monitor the course of CIPF, is helpful. VEGF is an angiogenic regulator involved in a variety of physiological and pathological processes. In human IPF, serum VEGF concentration has been shown to be higher in IPF patients compared to healthy volunteers and may reflect the severity of the lung disease. The aims of the present study were (1) to investigate the potential role of VEGF as a peripheral blood biomarker in CIPF; and (2) to investigate possible breed-related differences in basal VEGF concentration, that might explain the high predisposition of the WHWT breed for CIPF.Therefore, VEGF was determined by ELISA (Canine VEGF Quantikine ELISA Kit, R&D systems) in the serum of 14 WHWT with CIPF confirmed by HRCT and/or histopathology (median age 11 years, range 5-14), 18 healthy WHWT (9, 3-17), and 85 healthy dogs of other breeds, including: 14 Scottish terrier (ST) (5, 1-10), 16 Jack Russell terrier (JRT) (7, 1-12), 15 Maltese (6, 1-13), 14 King Charles Spaniel (KCS) (6, 1-10), 12 Labrador Retriever (LR) (6, 2-12) and 14 Malinois Belgian Shepherd (6, 2-8). Health status was based on clinical examination, serum biochemistry and haematology in all healthy dogs and a thoracic HRCT was performed in 9/18 healthy WHWT. The Khi² test with the threshold 5% was used for the statistical analysis (XLStat â software). Eight CIPF WHWT (57%) have serum VEGF concentrations above the kit detection limit (39.1 pg/ml) compared to 1 WHWT (0.05%) in the group of healthy dogs (P = 0.001). Concerning inter-breed differences in healthy dogs, most values obtained were below the kit detection limit with only 3 KCS (21%), 3 JRT (19%), 3 LR (25%) and 1 ST (7%) having VEGF serum levels above 39.1 pg/ml (P = 0.147). Results of the present study show that (1) VEGF might be an interesting blood biomarker for CIPF; (2) canine VEGF Quantikine Elisa kit is not appropriate for measurement of serum VEGF levels in healthy canine populations.
In conclusion, we document here the presence of FVII deficiency in WSS based upon DNA and coagulation activity testing. The common Gly136Glu mutation must have arisen prior to the separation of the very different FVII deficient breeds. There is no knowledge of an advantage of the heterozygote state. While there is only a mild hemorrhagic tendency, bleeding dogs could be treated with fresh frozen or cryo-poor plasma or human recombinant FVIIa. This preliminary study indicates a high carrier frequency in WSS. Screening by new platform DNA methods for this and other ancestral defects is helpful to detect additional hereditary diseases and genetic predispositions in different breeds, while other mutations are new and restricted to one or related breeds.
Both authors are employees of MSD Animal Health. MSD Animal Health funded the study. MSD Animal Health has an approved veterinary medicinal product for the treatment of feline hyperthyroidism. This product is available commercially in some EU markets but not in Portugal. Diabetes mellitus is one of the most commonly encountered endocrinopathies in cats and its prevalence has increased in the past. Similar to human Type 2 Diabetes, feline Diabetes is associated with comparable lesions occurring in the pancreatic islets, namely islet amyloidosis and beta-cell loss. Studying the pathophysiology of feline diabetes and the molecular mechanisms through which glucose metabolism is disturbed is largely hampered by the lack of a method for the isolation of pure pancreatic islets. The aim of this project was to improve a previously established method for the isolation of pancreatic islets; in particular enhancing the purity of isolated islets in this species.
No conflicts of interest reported. Nesidioblastosis describes a syndrome of acquired hyperinsulinaemia and associated hypoglycaemia secondary to focal or diffuse (non-neoplastic) beta cell hyperplasia within the pancreas. Beta cell dysregulation is thought to occur secondary to pancreatic injury. This syndrome has been reported in humans with increasing frequency, but it has not previously been described in domestic pets.
While beta cell hyperplasia has been reported as an incidental histopathological finding in euglycaemic young Beagles, this is the first reported case of clinically significant hypoglycaemia secondary to nesidioblastosis in a domestic pet. While this condition is rare, nesidioblastosis is being increasingly recognised in the human field and it is an important differential to consider when investigating hypoglycaemia as it cannot be differentiated from insulinoma without histopathological evaluation. Age of onset may provide a clue to this non-neoplastic disease, as this cat was much younger than all previously reported cases of feline insulinoma (all > 12 years of age at diagnosis). While recurrence has been reported in humans, a favourable outcome is anticipated following partial pancreatectomy.
No conflicts of interest reported. Activins are cytokines belonging to the transforming growth factor (TGF)-b superfamily. It is thought that activins may be the key intermediary in TGF-b1 mediated fibrotic response. Activin A has been suggested to participate in the pathogenesis of human idiopathic pulmonary fibrosis (IPF), but studies regarding the role of activin B are still spares. Canine IPF (CIPF) is a chronic, incurable interstitial lung disease occurring particularly in West Highland White Terriers (WHWTs). During the disease course, acute exacerbations (AEs), with poor prognosis, can occur. Histopathologically AEs of CIPF are featured by diffuse alveolar damage, which is also a key feature in acute respiratory distress syndrome (ARDS). Our objective was to study the expression of activin A and B by immunohistochemistry in the lung tissue of CIPF WHWTs (n = 5), CIPF WHWTs with concurrent AE (n = 4), and dogs of various breeds with ARDS (n = 4), and to compare these findings to healthy WHWTs (n = 3). In addition, western blot analysis of activin B from bronchoalveolar lavage fluid (BALF) of CIPF WHWTs (n = 6) and healthy WHWTs (n = 6) was conducted. We demonstrated that activin B, but not activin A, is strongly expressed in the altered alveolar epithelium in lungs of diseased WHWTs as well as in ARDS lungs. Furthermore, activin B was detected in BALF of CIPF WHWTs, most notably in samples from dogs with AE, but not in BALF of healthy WHWTs. This novel finding suggests that activin B participates in the pathophysiology of CIPF and might act as a potential marker of alveolar epithelial damage.
No conflicts of interest reported. Canine idiopathic eosinophilic bronchopneumopathy (EBP) is a disease characterized by eosinophilic infiltration of the lung and bronchial mucosa in young adults. Aetiology remains unclear although immunologic hypersensitivity is clearly suspected, while inciting antigens are generally unidentified. In humans as in cats, infections with Mycoplasma spp. have been discussed as potential triggers in inflammatory bronchial disease 1,2 . Bordetella bronchiseptica (Bb) is a recognized pathogen agent of canine infectious tracheobronchitis. Detection of Bb and Mycoplasma spp, especially Mycoplasma cynos (M. cynos), and their potential role of in canine inflammatory bronchitis, have not been investigated.
In summary, the gross structure (solid versus cystic) seems to be of prognostic relevance. In contrast to solid tumors, cystic pancreatic lesions in the cat behave benign in a higher percentage of cases, resulting in a better prognosis. Therefore, surgical excision of these cystic masses can be recommended. With respect to the human classification system, three different subtypes of cystic pancreatic neoplasms were detected in the cat that have not been described before in veterinary medicine: tubulopapillary, acinar and mixed. To best of our knowledge, this is the first report of cystic adenocarcinomas in feline pancreas.
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Abstract

The threat of an influenza A virus pandemic stems from continual virus spillovers from reservoir species, a tiny fraction of which spark sustained transmission in humans. To date, no pandemic emergence of a new influenza strain has been preceded by detection of a closely related precursor in an animal or human. Nonetheless, influenza surveillance efforts are expanding, prompting a need for tools to assess the pandemic risk posed by a detected virus. The goal would be to use genetic sequence and/or biological assays of viral traits to identify those non-human influenza viruses with the greatest risk of evolving into pandemic threats, and/or to understand drivers of such evolution, to prioritize pandemic prevention or response measures. We describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin pH of activation, and polymerase complex efficiency) that contribute to pandemic risk.

Introduction

Multiple influenza A subtypes-defined by the patterns of antibody recognition of two surface proteins, hemagglutinin (HA) and neuraminidase (NA)-circulate in avian species and swine at any given time. Among these, a number are known to cause sporadic zoonotic infections in humans (Peiris, 2009 ). More than one thousand human infections with avian influenza viruses were detected in the last decade, for example H5N1 and H7N9 (Qin et al., 2015) as well as swine influenza viruses, e.g. an H3N2 variant that spilled over into humans attending agricultural shows in the early 2010s, H3N2v (Jhung et al., 2013) . In addition, zoonotic infections with other viruses from poultry or wild birds have occurred, including for example H7N7 (Fouchier et al., 2004) , H10N8 (Wohlbold et al., 2015) , H6N1 (Wei et al., 2013) , H9N2 (Butt et al., 2005) , and H5N6 ; for more examples and a fuller discussion see (Short et al., 2015) . The severity of zoonotic influenza A infections ranges from clinically inapparent (Gomaa et al., 2015; To et al., 2016) to fatal (de Jong et al., 2006; Gao et al., 2013) .

B. Genetic and structural determinants of hemagglutinin-receptor interactions

The advent of inexpensive, large-scale sequencing, combined with improved computing power and novel algorithms to interpret nucleotide and protein sequences, have generated new approaches to characterizing the genotype-trait and genotype-transmission phenotype maps in influenza viruses. Some are well-established, while others are under active development. They include: Protein structural analysis to identify properties of individual amino acid residues and pairs of residues. A number of approaches have been devised to make use of databases of genome sequences and inferred protein sequences of influenza virus isolates, alone or in combination with metadata on the source (species), date of isolation and passage history of the isolates. Characterizing the predictors -at the level of individual amino acid residues within a proteinof variability or conservation can assist in identifying the major selection pressures on that protein. Evolutionary analysis of the predictors of high rates of nonsynonymous substitutions within HA showed solvent accessibility and proximity to the sialic acid receptor binding site are the strongest predictors of high nonsynonymous evolutionary rates (Meyer and Wilke, 2015) . Comparisons of residue-specific evolutionary rates in avian and human lineages can help to assess which sites are specifically involved in human adaptation and which may be evolving in avian reservoirs with potential consequences for human adaptation (Meyer et al., 2013) . Innovative use of metadata associated with sequences deposited in databases will be required to ensure that computational inferences from these databases are reliable. For example, methods that aim to identify sites under positive selection in the HA protein frequently find regions or sites that seem to contradict experimental evidence (Meyer and Wilke, 2015; Tusche et al., 2012; Kratsch et al., 2016) . Several of these apparent contradictions can be resolved by accounting for viral passaging. For example, passaging in regular MDCK cells produces a strong signal of positive adaptation underneath the sialic-acid binding site; this signal is entirely absent in unpassaged virus or virus passaged in SIAT1 MDCK cells (McWhite et al., 2016) . At the same time, passage bias mutations are assumed to increase fitness of the strain in the respective species and are often necessary to grow in culture at all. Therefore, sites associated with isolates passaged in mammalian cultures vs. those passaged in embryonated hen's eggs have the potential to further identify sites associated with mammalian or human adaptation. Metadata can also help to point to individual amino acids associated with human adaptation. For example, one proposed computational approach is to find potentially zoonotic human-isolated sequences when the majority of their database hits from preceding years were of animal origin. This serves, on one hand, as systematic survey to derive lists of times and places of likely zoonotic events and, on the other hand, provides close sequence pairs of zoonotic human and their putative animal precursors. In those pairs, common sites that repeatedly changed from the animal to the human zoonotic isolates could be reasoned as being involved in human adaptation. Combining these sites with those from passage changes, provides strong evidence for the involvement of a particular site in host adaptation. Network analysis of the level of sequence covariation of pairs of residues among protein sequences in the database has led to the identification of groups of mutually covarying sites, which have been used to define features of the HA protein that play a role in determining glycan receptor usage . Complementary to such covariation analysis is the analysis of predicted molecular interactions. Using X-ray co-crystal structures or modeled structural complexes of HA-glycan receptors, molecular features have been defined as distinct networks of inter-residue interactions involving key residues that make contacts with the different glycan receptor topologies. These features go beyond hallmark residue analyses and more accurately predict how amino acid variations in the receptor binding site impact the inter-residue interactions and glycan receptor binding specificity (Raman et al., 2014) . Similarly, network analysis of amino acid residues predicted to have significant interactions has shown that antigenic sites on the HA interact with residues controlling glycan receptor binding specificity, and that changes in these antigenic residues can then lead to changes in receptor-binding affinity (Soundararajan et al., 2011) . It seems likely that as these different lines of evidence -structural location, biophysical interaction, sequence covariation, sequence evolutionary rates, association with zoonotic or in vitro adaptation, etc.-begin to be understood at the resolution of individual amino acids within an influenza protein, such overlapping approaches will yield clearer understanding of the genetic and structural bases of host adaptation to human infection and transmission. A significant step toward such integration is the recent release of the FluSurver online tool which automatizes influenza sequence and structure analysis and highlights mutations that could alter the discussed traits based on extensive literature-derived genotype to phenotype lists, structural visualization of the mutation positions and their geographic and temporal frequency of occurrence and cooccurrence for epidemiological relevance (http://flusurver.bii.a-star.edu.sg and directly from within GISAID http://www.gisaid.org). In particular, the tool has been successful in picking up mutations affecting host receptor binding as well as pH dependency (Cotter et al., 2014; Maurer-Stroh et al., 2010) . However, also in this approach, annotations of the effects of mutations are based on inference from similarity to mutations studied in specific sequence contexts, which in most cases will not be identical to the investigated input sequences. Association studies. Understanding the genetic basis of adaptive phenotypic change is a central goal in biology, and influenza poses special challenges and advantages relative to other organisms. Association studies have begun mapping the genomes of Arabidopsis thaliana to over 107 quantitative traits and the genomes of humans to over 100,000 (Bergelson and Roux, 2010; Leslie et al., 2014) . These studies often investigate genetic variation at the scales of single nucleotide polymorphisms, alleles, and loci. Motif-based approaches have already proven useful in influenza (e.g., the insertion of multiple basic amino acids indicates highly pathogenic H5 and H7), and such simple, robust correlations simplify the prediction of phenotypic traits. Recent investigations of influenza (Thyagarajan and Bloom, 2014; Ashenberg et al., 2013; Pinilla et al., 2012) have shown that many mutations have roughly consistent impacts across diverse backgrounds. A complication of all association studies is confounding from genetic linkage and diverse environmental selective pressures. Although influenza's genes might be tightly linked over short time scales, the virus evolves quickly, and many traits can be assumed to be under stabilizing selection. Thus, association studies that appear statistically impractical now may be feasible with a few more years of expanded surveillance. As reviewed here, however, influenza often breaks simple genetic rules, perhaps due to epistasis (e.g., [Bloom et al., 2010] ). High-dimensional genotype-phenotype relationships obscure simple correlations from association studies. A relevant lesson comes from The Cancer Genome Atlas (TCGA), which amassed sequences from thousands of diverse tumors to investigate the mutations leading to different cancer types. Although metastatic cancers are typically conceptualized as possessing six main phenotypic traits (Hanahan and Weinberg, 2011) , TCGA revealed that the genetic commonality among tumors of any given type is shockingly low (Kandoth et al., 2013; Ledford, 2015) . Human genomes are much larger and more complex than influenza's, however, and so it is possible that an influenza atlas might reveal more patterns, which could inspire hypothesis-driven experiments (Weinberg, 2010) .

High-throughput, large-scale screens of mutational effects on hemagglutinin receptor binding.

In summary, genetic and protein sequence analysis, glycan arrays, and X-ray crystallography studies provide complementary data towards understanding the sialoglycan interactions of emerging viruses, with tradeoffs of equipment and reagent costs and throughput against level of precision and detail provided.

B. Functional, structural and genetic determinants of hemagglutinin pH of activation and its consequences

Based on surveillance studies, human-transmissible influenza isolates appear to have HA proteins that are more acid stable (have a lower activation pH) than avian influenza viruses (Russell, 2014). The HA activation pH values for H1N1, H2N2, and H3N2 seasonal viruses during Box 4. Ferret model: validity and limitations in pandemic risk assessment.

C. Experimental assays to measure hemagglutinin activation pH

A variety of experimental techniques have been developed to measure the activation pH of the HA protein, quantified as the highest pH at which the HA protein is activated to undergo the irreversible structural changes that mediate membrane fusion (Hamilton et al., 2012) , or alternatively the highest pH at which, in the absence of a membrane with which to fuse, the HA protein is inactivated (inactivation pH). Classical membrane fusion assays have measured the property in bulk (Hoekstra et al., 1984) . The pH of inactivation can be measured using aliquots of virions that are exposed to buffers of progressively lower pH and, after restoration to neutral pH, assayed for retention or loss of infectivity (Scholtissek, 1985) . In many classical fusion assays, fluorescent probes are used to label virions, HA-expressing cells, and/or target liposomes or cells. In these in vitro assays, HAbound target cells are typically exposed to buffers of various pH values and then lipid and/or contents mixing are measured by fluorescence (Loyter et al., 1988; Hoekstra and Klappe, 1993) . Alternatively, cell monolayers expressing cleaved HA proteins can be pulsed by low-pH buffers and then incubated to readout HAmediated cell-to-cell fusion either microscopically by syncytia formation or by reporter gene expression. If HA conformation-specific monoclonal antibodies are available for the subtype being studied, HA-expressing cells can be pulsed with low pH and then analyzed for conformational changes by flow cytometry (Reed et al., 2009 ). If such antibodies are lacking, HA-expressing cells can be assayed for trypsin susceptibility after low-pH exposure, with prefusion HA being resistant and postfusion HA susceptible to trypsin degradation (Steinhauer et al., 1996) . Recently, methods have been developed to study HA activation and membrane fusion by individual virions, including single virion fusion using total internal reflection fluorescence microscopy (Hamilton et al., 2012) .

A. Definition of the trait

The heterotrimer of influenza polymerase subunits (PA, PB1, PB2 gene products, together forming the RNA-dependent RNA polymerase) and the nucleoprotein (NP gene product) is required to transcribe and replicate the viral genome (Huang et al., 1990) . The polymerase genes of viruses isolated from avian hosts show a number of genetic differences from their counterparts in viruses isolated from humans (Chen et al., 2006) , and avian virus polymerase typically performs inefficiently in replicating the viral genome in human cells (Cauldwell et al., 2014; Naffakh et al., 2008) . Adaptation to efficient human-to-human transmission requires efficient activity of this complex of proteins, which we refer to as the polymerase complex, in human cells (Cauldwell et al., 2014; Naffakh et al., 2008) .

C. Experimental assays to measure polymerase complex efficiency in human cells

The original form of the in situ reconstituted polymerase assay requires expression of just the minimal set of four viral proteins to replicate the minigenome RNA: PB1, PB2, PA and NP. However, recent work showed an important additional role for another protein, the nuclear export protein (NEP), which is translated from a spliced mRNA derived from RNA segment 8 (that also encodes the major interferon antagonist NS2) (Robb et al., 2009) . In human H5N1 isolates that do not contain PB2 host-adapting mutations, the inefficient activity of these avian polymerases in human cells could also be compensated for by certain mutations in NEP (Mänz et al., 2012) . It appears that NEP is an important regulator of the balance between transcription and replication (Chua et al., 2013Paterson and , and can thus enhance fitness in viruses containing otherwise inefficient polymerases. The mechanism of this is as follows: the polymerase-enhancing domain of NEP is masked when NEP is folded in one conformation. However, mutations that increase the ability of NEP to rescue avian polymerase function allow more ready unfolding of the protein, unmasking the "activating" domain at the lower temperature of the mammalian respiratory tract. Interestingly, NEP overexpression in cells in which human-adapted polymerase is reconstituted is inhibitory because excess complementary RNA accumulates at the expense of messenger RNA and further viral RNA replication (Robb and Fodor, 2012) . Thus although a short-term adaptation of avian virus polymerase to mammalian cells can be achieved in this way, it may be that further compensatory changes rebalance NEP function in the face of polymerase adaptation during continued circulation in humans, although direct evidence for this selection is lacking. Indeed, although the rescue of low polymerase activity by NEP may explain the human infections by H5N1 viruses that lack other polymerase adaptations, it is not clear that such rescue is sufficient to create a level of transmissibility consistent with pandemic spread. Nonetheless, this finding shows that the minimal polymerase assay is not always sufficient to predict viruses that have functionally adapted polymerase activity to human cells and that a role for other viral proteins including at least NEP should also be considered in assessment of polymerase function.

Introduction

5. Finally, the infection must spread beyond the local area to infect members of distant populations, a process accelerated by modern global travel (Cooper et al., 2006) . This step and the one before are enhanced if the level of population susceptibility is high, as occurs when the surface proteins of the new strain are dissimilar to those on any currently or recently circulating human influenza A strains.
The appearance -by mutation or reassortment -and selection of genetic changes that encode human-adaptive viral traits may be seen as a process that can accelerate or increase the probability of one or more of these steps (though there is no guarantee that a given change that enhances one of these steps will enhance all of them. This is why the detection of phenotypes associated with human adaptation in avian or zoonotic isolates of novel influenza A viruses is thought to correlate with increased risk of pandemic emergence. As we describe throughout the paper, the process of human adaptation need not be complete to initiate a pandemic, so it may continue to occur at various stages throughout this progression. DOI: 10.7554/eLife.18491.002 general phenotype of 'transmissibility by respiratory droplets in mammals' such that experiments to select for such transmission in droplets in ferrets are important models of the process of adaptation to human transmission (Imai et al., 2012; Herfst et al., 2012) . This view is not universally shared . Starting from an zoonotic highly pathogenic avian influenza isolate from a human case of infection (or a reassortant of the HA from a different zoonotic H5N1 highly pathogenic avian influenza isolate, with the other segments from the 2009 pandemic H1N1 strain), it was shown that certain specific traits that had been previously associated with mammalian host adaptation were required to achieve respiratory droplet transmission. These ferret-transmission phenotypes in turn were associated with certain genetic changes relative to the original avian viruses (Imai et al., 2012; Herfst et al., 2012; Linster et al., 2014) . These specific changes occur both in HA and in polymerase-complex proteins.

High-throughput, large-scale screens of mutational effects on hemagglutinin receptor binding.

Greater precision and reproducibility has been achieved with the use of purified sialylated glycans to create solid-phase binding assays with fluorogenic or enzymatic detection (Gambaryan et al., 2006; Gambaryan and Matrosovich, 1992) . With these assays, it is possible to characterize the relative direct binding of whole virions or recombinant trimeric HA oligomers to glycans attached to a solid phase or the competition of such glycans with binding to a generic glycoprotein attached to the solid phase (Gambaryan and Matrosovich, 1992) . In recent work, biosensor interferometry and thermophoresis have been used to measure glycanbinding avidities and affinities in a more precise manner and to relate the two (Xiong et al., 2013a) . The development of glycan microarrays represented a turning point in the analysis of influenza virus receptor binding specificity, because it allowed simultaneous evaluation of virion or recombinant HA binding to a large repertoire of sialoglycans Blixt et al., 2004; Childs et al., 2009) . Several measures of preference for an HA molecule or whole virus have been defined, including the ratio of the number of a2,6 to a2,3 glycans bound (Stevens et al., 2006a (Stevens et al., , 2006b or the corresponding ratio of binding affinity or avidity (Imai et al., 2012; Xiong et al., 2013a) . A limitation to predictive power is that glycans tested on current arrays may not match those present in the human respiratory tract (Walther et al., 2013) . These arrays may also not present glycans in the same fashion as respiratory epithelial cells, so strategies such as measuring the binding of labelled viruses to human respiratory tissues (Chutinimitkul et al., 2010) or explant cultures ) may be promising alternatives, although challenges remain in standardization and quantification of such assays. Structural studies of wild-type and mutant HA in complex with representative sialoglycans provide the ultimate level of detail by characterizing interactions at the atomic level. X-ray to 4 simultaneous sites could be screened with substantial effort), and design of highly parallel screening, selection, and confirmatory assays. The mutagenesis and screening involved would be extremely large in scope: (before eliminating conserved residues, all 4-site mutnats~[550 residues x 20 amino acids] 4 = 1.4 x 10 16 variants for each subtype tested). However, some computational pre-screening to narrow the set of residues tested combined with contemporary mutagenesis and screening technologies such as deep scanning codon mutagenesis (Thyagarajan and Bloom, 2014; Bloom, 2015; Fowler and Fields, 2014 ) make such an endeavor feasible. DOI: 10.7554/eLife.18491.004 crystallography advances in recent years have accelerated structural determination, and similar progress in recombinant protein purification techniques combined with robotic crystal screening have reduced the amount of protein and labor required.

D. Receptor binding preference as a predictor of host adaptation of influenza viruses and pandemic risk

In summary, detection of a human receptor preference in a spillover virus may be an indication of increased risk, but exclusive human receptor preference is probably not necessary for an influenza A virus to initiate a pandemic. With several possible exceptions noted above, most viruses isolated to date fall within the shaded cells in Table 2 , which indicates concordance between the source of the isolate and the virus trait. Thus, prioritizing pandemic countermeasures against virus lineages with inferred or measured human receptor preference will likely lead to better targeting of such Figure 1 . Key phenotypic traits for the adaptation of avian influenza viruses to replicate efficiently in humans. (A) A switch in receptor binding preference from avian-like (a2,3-linked sialic acid) to human-like (a2,6-linked sialic acid) receptors. The human form on the left shows the typical distribution of human adapted influenza viruses determined by their receptor binding preference for a2,6, linked SA that is predominantly expressed in the upper respiratory tract but also in the lungs. The human form on the right shows that infection with avian influenza viruses is concentrated in the lungs where their preferred a2,3 linked SA receptor is expressed. (B) Lower HA pH of activation and increased polymerase complex efficiency. Free-floating viruses that enter the human respiratory tract (upper part of figure) encounter mucus and a mildly acidic extracellular environment that act as innate barriers to virus infection. If NA is able to desialylate decoy receptors on mucus and HA has a sufficiently low pH of activation, then the virus particle may reach the apical surface of the respiratory epithelium intact. There through a multiplicity of interactions between HA and cell-surface sialic acid, the virus enters the target cell. After the virus is internalized, it passes through the endosomal pathway where the pH is progressively decreased. The low pH of the endosomal environment triggers an irreversible conformational change in HA that fuses the viral and endosomal membranes and ultimately results in the release of virus genetic material in the form of the viral ribonucleoprotein complex (vRNP) into the cell cytoplasm. The eight vRNPs are subsequently imported into the cell nucleus by interactions between the vRNPs and cellular nuclear import machinery. Inside the nucleus the virus polymerase complex replicates the virus genome in conjunction with co-opted cell proteins. DOI: 10.7554/eLife.18491.005 Table 1 . Influenza virus adaptations that appear to be required for human-to-human transmission.

HA receptor binding specificity

Preference for a2,6-linked mammalian sialic acid receptors over a2,3-linked avian ones HA pH of activation HA avoids extracellular inactivation and undergoes conformational changes leading to membrane fusion at appropriate pH for human cells (5.0-5.4 or perhaps 5.5) (Russell, 2014) Polymerase complex efficiency Efficient replication in human cells (Cauldwell et al., 2014; Naffakh et al., 2008) Virus morphology Filamentous morphology associated with several adaptations to mammals (Seladi-Schulman et al., 2014; Seladi-Schulman et al., 2013; Campbell et al., 2014; Beale et al., 2014) Length of NA stalk Longer stalk of NA required to penetrate human mucus and deaggregate virions (Blumenkrantz et al., 2013) Antagonism of interferon production Species-specific binding of the NS1 protein to host factors (Rajsbaum et al., 2012) HA-NA "balance" Substrate selectivity and catalytic rate of NA are calibrated to "balance" avidity of HA for the cell-surface glycan receptor (Zanin et al., 2015; Baum and Paulson, 1991; Yen et al., 2011; Handel et al., 2014) DOI: 10.7554/eLife.18491.006 Glaser et al., 2005) ; most human H2 and H3 seasonal isolates (Connor et al., 1994; Matrosovich et al., 2000) *These anomalous results are speculated by the authors to be possibly, or even probably the result of laboratory adaptation to egg passage and may not reflect the properties of the primary isolate. A possible counter to this interpretation is that it is seen only in the earliest isolates from human pandemic viruses, while nearly all isolates from after the pandemic year, which should also have been passaged in eggs, show human-adapted phenotypes.

B. Functional, structural and genetic determinants of hemagglutinin pH of activation and its consequences

The HA is synthesized and folded such that the fusion peptide is buried and inactive until specific activation signals are provided. The structural changes that expose the fusion peptide and lead to fusion have been described in detail (Skehel and Wiley, 2000) . If the virion is exposed to sufficiently low pH outside of a host or host cell, the HA protein undergoes irreversible structural changes too early and is unable to mediate virus entry; such virions become inactivated. Thus the term acid stability is more broadly used to define the threshold for acidification that triggers membrane fusion (in the endosome) or inactivation (if triggered outside of the cell for an HA that is not sufficiently stable). During endocytosis, an influenza virion is exposed to sequentially lower pH values in early endosomes (pH 6.0-6.5), late endosomes (pH 5.0-5.5), and lysosomes (pH 4.6-5.0) (Mellman et al., 1986) . If the HA is too stable, and fusion is not triggered in the acidic endosome of the host cell, further traffic into lysosomes results in virus inactivation by lysosomal proteases (Skehel and Wiley, 2000) .
highly pathogenic avian influenza virus, an increase in HA activation pH within the range of 5.2-6.0 has been associated with increased replication and pathogenicity in chickens (DuBois et al., 2011) . Conversely, a mutation that decreased the HA activation pH of A/ chicken/Vietnam/C58/2004 (H5N1) from 5.9 to 5.4 has been shown to attenuate virus growth and prevent transmission in mallard ducks (Reed et al., 2009 ) but increase virus growth in the upper respiratory tracts of mice and ferrets (Zaraket et al., 2013a (Zaraket et al., , 2013b . Therefore, for H5N1 viruses, a higher HA activation pH (5.6-6.0) has been associated with a component of fitness in birds, and a lower HA activation pH (pH 5.0-5.4) has been linked to greater replication in the mammalian upper respiratory tract. Two H5N1 viruses were adapted to transmit by the airborne route between ferrets (Imai et al., 2012; Herfst et al., 2012) . After a switch in receptor-binding specificity from avian to human receptors (as described above) and deletion of a glycosylation site, in both studies a final mutation that decreased the HA activation pH was shown to be necessary for airborne transmissibility in ferrets. However, these and other studies have shown that this acid stability change is not sufficient in the absence of human receptor-binding specificity (Zaraket et al., 2013a; Shelton et al., 2013) . Recently, an HA protein whose activation pH was 5.5 or lower was shown to be required for the pandemic potential of 2009 pH1N1 influenza virus .
Nearly 100 mutations have been described to alter the HA activation pH values of various influenza A virus subtypes (Russell, 2014; Mair et al., 2014) . These acid stabilizing/destabilizing residues are located throughout the HA1 and HA2 subunits and tend to be positioned in regions of the molecule that undergo large-scale changes in structure during pH-activated protein refolding (Russell, 2014; Bullough et al., 1994; Wilson et al., 1981) . Mutations that modify the activation pH do not appear to alter the prefusion HA protein Box 5-figure 1. Transmission genomics of non-human transmission (top), spillover transmission (middle) and sustained human transmission (bottom). Haemagglutinin and neuraminidase gene segments have been color-coded to show an example shared infection history in humans who are current spillover hosts for H7N9 and H9N2. These shared evolutionary histories make it challenging to interpret serological studies of human spillover infections. Humans infected by H2N2 or H3N2 will likely have cross-reactive antibodies to H9N2, because of the similarity between the neuraminidase in those viruses. Because incidence of spill-over infection is likely to be low, even low-levels of cross-reactivity can make the interpretation of serological studies of the general population challenging. DOI: 10.7554/eLife.18491.014 DOI: 10.7554/eLife.18491.013 backbone in X-ray crystal structures (DuBois et al., 2011; Weis et al., 1990; de Vries et al., 2014) . Therefore, an experimental determination or modeling of intermediate structures may be required in order to reliably predict HA pH of activation. Further complicating genetic prediction of HA activation pH values are observations that the NA and M proteins can also modulate HA acid stability in some cases (Huang et al., 1980; Su et al., 2009; Reed et al., 2010; O'Donnell et al., 2014) .

C. Experimental assays to measure hemagglutinin activation pH

Although the biological trigger for HA's conformational change is a drop in pH, HA refolding can also be triggered by other destabilizing agents such as heat and urea (Scholtissek, 1985; Ruigrok et al., 1986; Carr et al., 1997) . Stability at a lower pH is associated with stability at higher temperatures and higher urea concentrations, permitting the use of these agents instead of, or in addition to, pH in assays of stability. Thermal stability has been determined by measuring the threshold temperature at which denatured HA protein loses its ability to bind erythrocytes and cause hemagglutination (Linster et al., 2014) .

D. Role of hemagglutinin activation pH in pandemic risk prediction

Many questions remain regarding whether HA activation pH plays a similar role in all influenza subtypes isolated from a wide variety of avian species. For early isolates of the H1N1pdm lineage in 2009, the HA protein has an activation pH of 5.5, which appears intermediate between the canonical human (lower) and avian (higher) ranges. Subsequent H1N1pdm isolates have HA activation pH values ranging from 5.2-5.4, suggesting pH 5.5 may be the upper limit for human pandemic potential and a lower value may be preferred. Indeed, a destabilizing HA mutation in the background of H1N1pdm results in a lossof-function of airborne transmissibility in ferrets and has been reported to be followed by regain-of-function by mutations that lower the HA activation pH to 5.3, a value representative of human-adapted H1N1pdm viruses . For the moment, it appears that while HA pH of activation that is shown experimentally to be suitable for human infection is highly typical of isolates from human pandemic and seasonal influenza ( Table 3 , bottom right) (Galloway et al., 2013) , it is possible for humans to have symptomatic infection with (though not extensively transmit) viruses with activation pH closer to the range associated with terrestrial birds ( Table 3 , bottom left). Conversely ( Table 3 , top right), there are avian H9, H10, H14, and H15 isolates that display activation pH typical of human viruses (Galloway et al., 2013) . The existence of these human-like avian viruses is perhaps unsurprising, as they may lack other essential adaptations for human transmission. As in the case of receptor binding, reliance on this trait to prioritize pandemic prevention measures should consider this property in conjunction with other properties associated with pandemic potential and will likely enrich the coverage of truly high-risk strains on average.

Discussion

While predictions of virus phenotypes from sequence data can be informative, they are not infallible, for several reasons, notably the large number of sites involved in determining such traits (Raman et al., 2014; Russell, 2014; Cauldwell et al., 2014; Mehle and Doudna, 2009; Mänz et al., 2012; Herfst et al., 2010) , the important role of epistasis (dependence of a mutation's effect on the genetic background in which it appears) in determining these traits Kryazhimskiy et al., 2011; Gong and Bloom, 2014; Bloom et al., 2010; Wu et al., 2016; Raman et al., 2014; Tharakaraman et al., 2013; Gong et al., 2013) , and the consequent imperfections in our ability to map single sequence polymorphisms to a trait value. For example, the hallmark HA amino acid residues 190, 226 and 228 are important to human adaptation, but "human-adapting" mutations at these residues do not always change receptor-binding specificity; it depends on the genetic background. Similarly, amino acid residues 627 and 701 of the PB2 protein are often involved in human adaptation, but both carried the "avian-adapted" residue in the 2009 H1N1 pandemic strain. When these changes were introduced individually or together in the laboratory, the resulting polymerase showed greater activity in a minigenome assay, but replication was unchanged or attenuated in vitro, in mice, and in ferrets (Herfst et al., 2010; Jagger et al., 2010) . After the pandemic strain was identified and its anomalous residues at these sites noted, other sites within PB2 were identified and found to be responsible for human adaptation (Mehle and Doudna, 2009; Yamada et al., 2010) .
There are three complementary approaches to address these limitations: improving genetic prediction of biological traits, improving assays of these traits, and improving animal models of human transmission; all approaches are currently progressing in parallel Simon et al., 2011) . The first approach aims to further refine our understanding of sequence-totrait relationships by continued studies of diverse, naturally or artificially produced mutations and their effects on the traits of interest. Such research could use all of the approaches described above and higher-throughput assays that could be developed with improved technology, for example as described in Box 3. This will include generating mutations not found in known strains in nature to probe for those that could be involved in human adaptation in the future Thyagarajan and Bloom, 2014) . The goal would be to identify classes of functionally equivalent substitutions, sufficient individually or in defined combinations to confer a trait of interest when introduced into a defined, avian-adapted genetic background. Use of in vitro approaches with noninfectious viruses or viral components, or infectious viruses containing surface proteins to which there was already population-wide immunity would reduce the possible biosafety and biosecurity risks of such studies (Lipsitch and Inglesby, 2014) .

Introduction

1. The avian-adapted strain must become sufficiently widespread in wild or domestic birds, swine or other reservoir species to expose at least one human to infection.
2. One or more humans must acquire infection from the reservoir species.
4. The infection must be transmitted to additional humans, avoiding an "early" termination of the transmission chain due to chance. Such early termination is a significant risk given the relatively low infectiousness of influenza and the moderate degree of overdispersion in the number of secondary cases infected by each case, both of which contribute to the probability that a transmission chain will terminate by chance (Lloyd-Smith et al., 2005; Lipsitch et al., 2003) . It must also avoid extinction due to deliberate control efforts put in place by public health authorities (Ferguson et al., 2006; Merler et al., 2013) .
We know from serologic studies and human infections that several different influenza A viruses have achieved steps 1 and 2 at any given time (Gomaa et al., 2015; To et al., 2016) . Steps 3 and/or 4 appear to be the rare, rate-limiting steps; that is, the conditional probability of achieving step 3 and 4 given the previous steps is low, so that sustained human-to-human transmission of a novel strain occurs a few times per century while zoonotic infections must occur thousands or more times per year. No case is known in which an influenza A strain has reached stage 4 but failed to reach stage 5, although it may have happened undetected.
The rationale for these experiments was that, because the ferret model recapitulates many features of human infection, changes identified in adaptation to ferret transmission would also be important for adaptation to sustained transmission in humans Schultz-Cherry et al., 2014) , though this can never be known with certainty . Notably, viruses isolated from humans who were infected by contact with birds show some of these changes (Russell et al., 2012; Bi et al., 2015) , particularly the change at amino acid 627 of the PB2 gene (Jonges et al., 2014; Fonville et al., 2013) , which often affects polymerase complex efficiency (see below). This indicates that even the first generation of human infection from nonhuman hosts can initiate a process of host adaptation. It also indicates that not all the human-adaptive changes must be in place in the avian reservoir to initiate this process. Some human infections, including some zoonotic cases (de Jong et al., 2006; Chen et al., , 2006 Sha et al., 2016) and some cases early in a pandemic (Rogers and D'Souza, 1989; Connor et al., 1994; Glaser et al., 2005; Pappas et al., 2010) , involve viruses that are not yet fully human-adapted (see below and Tables 2-4). The interpretation of some of these isolates is complicated by uncertainty about whether they were passaged in hen's eggs at some point in their history.
. limited surveillance of nonhuman influenza viruses, such that high-risk viruses may not be detected and hence cannot be assessed (Butler, 2012) . Limitations include both the number, geographic and species diversity of hosts sampled, and the difficulty in sampling all genetic variants present in a given infection (Poon et al., 2016; Varble et al., 2014) ;
. practical, ethical and cost limitations of animal transmission experiments, as well as some exceptions to the correlation between human transmissibility and droplet transmissibility in nonhuman animal models (Buhnerkempe et al., 2015) ;
. the role of stochastic events in the ecology and evolution of influenza viruses during and after host-switching to humans (Gog et al., 2015; Lloyd-Smith et al., 2015) , including the potential for transmission bottlenecks to either promote or inhibit emergence of human-adapted viruses (Varble et al., 2014; Moncla et al., 2016; Wilker et al., 2013; Zaraket et al., 2015) .
Determining the appropriate taxonomic level for influenza virus risk assessment is a challenging endeavor. Influenza virus subtype is a convenient classification but there can be substantial variation in estimable risk within subtype. For example, H5N1 viruses can be roughly segregated into high pathogenicity and low pathogenicity phenotypes with the high pathogenicity variants generating substantially greater concern for both human and animal populations. Even within the high pathogenicity H5N1 variants, risk to animal populations and potential for adaptation to humans is likely to vary by phylogenetic lineages or clades of viruses. Much of the difficulty for predicting the threat posed by subtypes or coarse grained concepts of virus variants stems from two factors: first, a lack of understanding of how genetic context affects the ability of a virus to adapt for efficient spread in humans; and second, the critical, and geographically variable, role of ecology in determining likelihood of cross species transmission. Phylogenetic clade is a practical unit for risk prediction. However, in species where reassortment is frequent, phylogenetic clade must be considered on a gene by gene basis. The definition of phylogenetic clades can be challenging and arbitrary, but recent efforts to develop a unified nomenclature for highly pathogenic H5N1 viruses may offer a transferrable framework for the classification of other viruses (Smith and Donis, 2015 ; WHO/OIE/FAO H5N1 Evolution Working Group, 2008) . Clades of viruses circulating in poultry, swine or other domestic animals with extensive human interactions should be prioritized for surveillance and further study. Foundational efforts are required to assess the diversity of viruses present in these animal populations, particularly for low pathogenic avian influenza viruses. Further study will then be required to assess the abundance and prevalence of different virus subtypes and clades, along with geographic spread and overlap with ecological risk factors (Hill et al., 2015; Gilbert et al., 2014) , e.g. live animal markets, cohabitation of humans and animals, and biosecurity in animal processing facilities. Antigenic characterization of animal influenza viruses should form part of a comprehensive risk assessment, particularly of viruses from swine and possibly dogs. Swine influenza virus diversity is driven in large part by introductions of viruses from humans to swine (Nelson et al., 2015 Lewis et al., 2016) . The substantial antigenic diversity of viruses circulating in swine and antigenic differences with viruses circulating in humans poses an ever increasing risk for re-introduction into humans. Much of the antigenic variation in swine has a strong relationship to phylogenetic clade (Lewis et al., 2016) . Similarly, the high contact rates between humans and dogs, combined with increased circulation of H3N2 canine influenza viruses, may present increasing opportunities for reassortment (Na et al., 2015) and for zoonotic infections (Flanagan et al., 2012) . DOI: 10.7554/eLife.18491.003 viruses isolated from humans after a pandemic starts that retain some degree of avian-like traits, and we discuss these in more detail in the text -these represent the greatest challenge to use of genotypic or phenotypic information for pandemic prediction because they run the risk of false negatives. The other off-diagonal cell, which represents avian isolates with some human-like traits, simply shows that some circulation of viruses in birds is possible without the classical 'avian' phenotypes. How this happens is a phenomenon worthy of further study. We conclude with some recommendations for future research and for the practice of pandemic risk assessment.

A. Definition of the trait

Attachment of an influenza virus to a host cell requires binding of the viral HA to a sialylated glycan receptor (sialic acid) on the surface of the host cell. Cells of the avian gut and a minority of cells in deep lung in mammals predominantly express receptors terminated with an a2,3linked sialic acid: hereafter, a2,3 glycans or avian receptors Gambaryan et al., 1997; van Riel et al., 2006; Shinya et al., 2006) . By contrast, in humans and other mammals, upper respiratory epithelial cells express mainly glycan receptors terminated by a2,6-linked sialic acid: a2,6 glycans or human receptors Shinya et al., 2006; Chandrasekaran et al., 2008) . The human upper respiratory epithelium is the primary target site for infection of human-adapted viruses, and infection at this site is thought to be a prerequisite for efficient human-to-human transmission via respiratory droplets. Thus, it appears that human adaptation of an HA is associated with a switch in its binding preference from avian to human receptors. Receptor binding is not either-or; human-adapted influenza virus HA may show some binding to avian receptors, and vice versa.

B. Genetic and structural determinants of hemagglutinin-receptor interactions

Preference for binding human or avian glycan receptors is determined by the structure of the viral HA. Except for a few conserved amino acids in the sialic acid receptor binding pocket, the influenza HA has considerable structural plasticity to evolve variation at the rim of the pocket to engage different sialic acid linkages. Importantly, antigenic regions of the HA are located nearby regions that determine receptor-binding preference, meaning that selection for antigenic escape may be constrained by the need to maintain receptor preference (Koel et al., 2013) . More speculatively, selection for changes in receptor preference might also alter recognition of the HA by host antibodies.
Conformation of hemagglutinin as a determinant of receptor binding preference. Although the co-crystal structures of HA and sialylated glycans have not been solved for all pairs, there is evidence that avian-or human-adapted HA bind to different conformations of the avian and human receptors: the cis conformation of human receptors and the trans conformation of avian receptors Ha et al., 2001 Ha et al., , 2003 Gamblin et al., 2004; Liu et al., 2009; Xu et al., 2010; Yang et al., 2010; Lin et al., 2012; Yang et al., 2012; Zhang et al., 2013) . This finding has led to the concept of "hallmark" residues within the receptor-binding site of avianand human-adapted HAs. Avian-adapted HAs typically carry Glu at position 190, Gln at position 226, and Gly and position 228 (H3 numbering), and the Gln226->Leu, Gly228->Ser substitutions have been associated with a switch to human receptor preference in HAs of H2, H3 (Matrosovich et al., 2000) , and H5 (Chutinimitkul et al., 2010) viruses. In H1 HA, Glu190fiAsp and Gly225fiAsp have been considered as hallmark amino acid changes to switch receptor specificity leading to greater human Box 3. Novel approaches to identifying genomic predictors of traits and transmission phenotypes.

High-throughput, large-scale screens of mutational effects on hemagglutinin receptor binding.

Binding of upper-respiratory-tract glycans by the influenza virus hemagglutinin is one of the best-understood ingredients in making a virus capable of efficient human transmission. Yet the viral sequence determinants of this trait have been mapped only for a limited number of variants. A systematic screening strategy to scan the genetic "landscape" for sequences with a preference for human glycan receptors might include four components: (1) selection of viral genetic background, (2) large-scale mutagenesis, (3) screening and selection, and (4) confirmatory assays. Because both mutations near and far from the sialic-acid-binding site on hemagglutinin have been shown to alter glycan specificity, this should be based on a minimally biased approach to mutagenesis: screening combinations of all possible substitutions at all hemagglutinin residues that are not absolutely conserved across known subtypes. Critical considerations include choice of viral genetic background (both subtype and strain identity), extent of combinatorial screening (if conserved sites are omitted, every mutant containing changes at up adaptation (Glaser et al., 2005; Tumpey et al., 2007) . The determinants of specificity are reviewed in much more detail in (Paulson and de Vries, 2013) .
Additional structural features involved in receptor binding preference. The cis and trans definition of glycan conformation does not fully describe HA binding to a range of structurally diverse glycans displayed on human respiratory cells and tissues (Chandrasekaran et al., 2008) . This limitation motivated studies that revisited the definition of glycan conformation, extending the conformational analysis beyond the terminal sialic acid linkage to describe overall topology and dynamics of the glycan receptor upon binding to the receptor-binding site of avian and human-adapted HAs (Chandrasekaran et al., 2008; Xu et al., 2009) . HA sequence determinants of preference for the "cone"-like topology of avian receptors, versus the "umbrella"-like topology of human receptors, are still being defined (Raman et al., 2014). C. Experimental assays to measure hemagglutinin receptor binding specificity Experimental evidence on differential binding of avian and human viruses to sialic acid receptors in avian and human conformations, respectively, was first obtained by hemagglutination assays with erythrocytes whose surfaces had been chemically modified to display glycans terminating with either homogeneous a2,3 or homogeneous a2,6-linked sialic acids (Paulson and Rogers, 1987) . Subsequent analysis of the repertoire of glycan structures in erythrocytes of various animal species informed the use of cells from different species as probes of HA receptor binding preference in hemagglutination assays (Ito et al., 1997) .

D. Receptor binding preference as a predictor of host adaptation of influenza viruses and pandemic risk

As noted above, hallmark residues have substantial predictive power. These distinct sets of hallmark residues in the H1, H2 and H3 subtype (Paulson and de Vries, 2013) correlate with human-adaptation in known sequences collected from birds or humans (Connor et al., 1994; Paulson and de Vries, 2013) ; they induce changes in receptor-binding specificity when introduced experimentally (Chen et al., 2012; Leigh et al., 1995) ; and experimental selection for receptor binding in vitro (Chen et al., 2012) or in ferrets (Imai et al., 2012) cause these changes to appear.
However, hallmark residue predictions of receptor-binding specificity are imperfect, as evidenced by a failure to switch in vitro receptor-binding preference from avian to human when changes observed in H5N1 strains after selection in ferret gain-of-function experiments were introduced to other H5N1 viruses (Tharakaraman et al., 2013) . The involvement of other features in human adaptation, such as the topology of the bound HA-receptor complex, further complicate the genetic prediction of human adaptation, as the residues involved in these features are less well characterized (Shriver et al., 2009) .
In the case of early 2009 pandemic viruses, findings are mixed (Childs et al., 2009; Chen et al., 2011) . Some of the findings of dual or avian specificity may reflect artifacts introduced when human isolates were passaged in eggs before receptor specificity was assayed; alternatively, they may genuinely reflect a transitional stage in the evolution of HA genes in human populations after transmission from other species (Connor et al., 1994; Glaser et al., 2005; Stevens et al., 2010) , ( Table 2) . Consistent with this latter possibility, an H5N1 virus isolated from a human zoonotic case in Vietnam displayed strong avian receptor preference . This preference changed in the course of experiments to adapt it to respiratory droplet transmission in ferrets (Imai et al., 2012) . Taken together, these findings confirm that there is a strong correlation between measured receptor preference and the host from which a virus is isolated. However, they raise questions about the predictive value of human receptor binding preference. Indeed, the examples of mixed receptor preference in human isolates from the early phase of the H1 or H2 pandemics suggest that the ability to evolve human receptor specificity over a chain of human infections, which may be present in many avian-receptor-adapted viruses, may be sufficient for pandemic emergence.

Found in avian isolates

Avian H1-H4, H11 isolates (Galloway et al., 2013; Russier et al., 2016; DuBois et al., 2011; Reed et al., 2010) Avian H5, H8, H9,H10,H14,H15 isolates (Galloway et al., 2013) Found in human isolates H5N1 (Imai et al., 2012; Linster et al., 2014) and H7N9 (Schrauwen et al., 2016) human zoonotic isolates with pH 5.6. One human H1N1 (2008) * the role of amino acids 590 and 591 in adaptation was not recognized until after the 2009 strain had already emerged (Mehle and Doudna, 2009) ; it has the residues associated with avian adaptation at sites 627 and 701
Greater acid stability (lower pH of activation) is associated with greater human adaptation.

B. Functional, structural and genetic determinants of hemagglutinin pH of activation and its consequences

Box 4-figure 1. Ferret respiratory droplet transmission experiments predict the potential for sustained human-to-human transmission of influenza viruses. The solid line shows the weighted logistic regression relationship predicting the probability that a given strain is supercritical (i.e. capable of sustained spread among humans), and the dashed lines show the 95% confidence interval for the prediction. Filled circles show the measured secondary attack rates (SAR) in ferrets for influenza subtypes that are known to be subcritical (blue) or supercritical (red) in humans. The filled pink area shows the range of SAR for which the virus is significantly likely to be supercritical. Reprinted from (Buhnerkempe et al., 2015) . Box 5. Role of seroepidemiology in pandemic risk assessment.
Pandemic threat assessment can also be enhanced by immunological surveys of human populations in geographical area where strains of concern are known to be circulating (Van Kerkhove et al., 2011) . Serological surveys can help to estimate the frequency of spillover infections from non-human to human hosts and also to assess the degree of cross reactivity arising from endemic human strains that share recent genomic ancestors with non-human strains of concern (Figure for Box 4). Attempts have been made to use serological surveys to estimate the rate of spillover infections to humans for recent strains of concern Van Kerkhove et al., 2012) . Sometimes blood samples are obtained from the general population (Chao et al., 2011) and other times only high risk individuals are tested. Inherent measurement error and cross-reactivity between human and non-human strains make the measurement of low rates of incidence problematic (Van Kerkhove et al., 2012) . Confidence that serologic responses truly reflect zoonotic transmission, rather than cross-reactivity with antibodies generated in response to human influenza infection, may be enhanced by comparison of high-risk persons to those without known exposure to zoonotic sources (Huang et al., 2015; Gomaa et al., 2015) . Although there is evidence of exposure of poultry workers to H5N1 influenza viruses in China, rates are much lower than for other endemic non-human influenza viruses (Kim et al., 2011) , such as H9N2 (Blair et al., 2013) . More recent studies of exposure of high risk workers to the H7N9 lineage suggest even higher rates of exposure to this new strain than has been observed in similar studies of H5N1 or H9N2 . Even when rates of spillover can be estimated accurately, the use of such information in pandemic threat assessment is not obvious. Clearly, the first detected presence of human infections for a given strain is of concern because the degree of transmissibility among humans is unknown. Should the emergent strain fail to achieve sustained transmission, it is not immediately clear how best to use further information on the frequency of human spillover infections. For example, should we interpret high sustained levels of human spillover as evidence of increased risk because of the number of human infections, or as evidence of decreasing risk because of the number of times the strain has failed to achieve sustained transmission? Cross reactivity between non-human and human influenza strains has implications beyond the measurement of spillover infections. Often levels of cross reactivity in humans may indicate some degree of reduced population susceptibility (Worobey et al., 2014) . All else equal, such evidence of lower population susceptibility should reduce our level of concern about a pandemic threat from a particular virus, because even if it gains efficient human-to-human transmissibility, its effective reproductive number and the proportion of the population at risk will be less than for a virus to which there is no cross-reaction in the population. For example, older individuals are thought to have been far less susceptible to pandemic H1N1 than were younger individuals, because they had previously been exposed to similar strains early in life (Yu et al., 2008) . The low average age of infection with a swine variant form of H3N2 (H3N2v) in North America (Jhung et al., 2013) is likely driven by reduced susceptibility in adults because of early exposure to similar strains. Such immunological overlaps are likely to be a general feature of influenza emergence because human strains frequently emerge into swine populations (Nelson et al., 2015) . Data on reduced human susceptibility due to cross-reactivity must be synthesized with other data used for threat assessment. In some cases, the aging of the part of the population with prior exposure to a closely related strain could be the most important known factor increasing the risk of an emergence event. Mechanistic models could be used to estimate the degree of increased risk of emergence due to the aging of partially immune cohorts.

C. Experimental assays to measure polymerase complex efficiency in human cells

Polymerase complex efficiency in human cells can be measured by an in situ assay in which the influenza polymerase is reconstituted from cloned cDNAs in plasmids and then coexpressed with "minigenome," a viral-like RNA encoding a reporter, such as luciferase. By measuring the rate of reporter accumulation in the transfected human cell line, specific combinations of RNA sequences for the polymerase-complex viral genes can thereby be screened directly for their efficiency in producing the mRNA encoding the reporter gene product, providing a measure of human adaptation of the polymerase complex (Moncorgé et al., 2010) .
Ultimately, it would be valuable to develop a simple screen to assess the ability of a viral polymerase to support replication and transmission in humans. This phenotype is influenced by at least 4 different viral genes and involves interactions with several different human host factors. If all the relevant host factors were enumerated, one could imagine quickly converting sequence information into an assay that tested for interactions that should support activity. Along these lines the recent description of a host factor, ANP32A that differs between flighted birds and mammals and explains the poor activity of avian polymerase in mammalian cells is a step forward (Long et al., 2016) .

D. Role of polymerase complex efficiency in pandemic risk prediction

In summary, no single polymerase mutation appears to be predictive of pandemic risk for all viruses, but the concept that the polymerase must adapt to human cells before it can cause extensive human-to-human transmission appears consistent with the four pandemic jumps that have occurred in modern times.

Discussion

There has been tremendous progress in understanding the traits involved in the adaptation of avian influenza viruses for efficient human-tohuman transmission and the genetic and structural basis of each of these traits. While the ability to use virus sequence data to inform risk assessment of pandemic potential is improving, it remains essential to consider these data alongside other experimental and epidemiological data. For example, in 2013 there was a substantial increase in the number of human infections with A/H5N1 viruses in Cambodia. The increase in infections was cause for substantial concern by itself. Enhancing the level of concern was the finding that some of the viruses collected from infected humans contained previously identified genetic mutations suggestive of human adaptation . These findings led to extensive epidemiological and experimental investigations and then to the decision to produce a candidate vaccine virus from a virus representative of the 2013 Cambodian outbreaks.
The third approach is to improve animal models to more precisely study phenotypes that are important for human adaptation, and to clarify whether the notion of 'mammalian adaptation' is in fact a valid category. Ferrets are the closest known model for human transmission (see Box 4). Respiratory-droplet transmission studies in ferrets, and potentially in other animal models, have shown a remarkably strong correlation with human transmissibility of influenza A strains (Buhnerkempe et al., 2015) . While these assays are not perfectly predictive, they may be the most reliable way at present to assess the transmission potential of a virus in human populations. Here a partial counterexample to their overall strong predictive value is H7N9 avian influenza isolates from human zoonotic cases. These viruses transmit in ferrets, albeit less efficiently than human seasonal strains, yet humanto-human transmission has been extremely rare in the hundreds of human zoonotic cases caused by H7N9 (Buhnerkempe et al., 2015) . A challenge is the expense and practical challenge of using large enough numbers of ferrets (Buhnerkempe et al., 2015; Belser et al., 2013; Nishiura et al., 2013) to assess transmissibility; this will remain a technique of limited throughput for the foreseeable future. Nonetheless, the value of ferret testing for risk assessment can be enhanced in at least two ways: first, by standardizing the conditions for ferret transmission experiments, so these can be more confidently compared between laboratories; and second, by continuing to combine ferret studies with studies of viral traits and sequence/structural studies to further identify correlates of transmissibility in ferrets.
Evolutionary factors also play a role in pandemic risk evaluations. Even with excellent surveillance, we may never isolate exactly the virus that is destined to cause a pandemic from an animal reservoir or a zoonotic human case; rather, we may isolate its evolutionary precursor. Understanding the potential of a strain to produce pandemic-capable progeny is yet a further scientific challenge. Perhaps the most startling finding of the gain-of-transmissibility experiments with H5N1 avian influenza viruses was that so few mutations were required to convert a strain circulating in birds to mammalian transmission. This concern was reinforced by a finding that many of these mutations, including combinations of some of them, were already present in strains isolated during surveillance (Russell et al., 2012) . The interpretation of the latter finding, however, is complicated by the problem of epistasis: the effect of these mutations in the genetic background of field strains may or may not be the same as in the strain studied in the laboratory.
As we seek to improve our understanding of genetic and phenotypic bases of efficient human transmission of influenza viruses, there are multiple possible approaches. One approach that has received considerable attention recently is to perform gain-of-transmissibility studies in highly pathogenic avian viruses; this has been controversial because of concerns about the unusual biosafety and biosecurity risks entailed in such studies (for contrasting perspectives on these risks, see the exchange in 2014-5 between Lipsitch and Inglesby, and Fouchier Inglesby, 2014, 2015; Fouchier, 2015] ).

Introduction

Although secondary transmission can occur following some of these spillover events (Kucharski et al., 2014) , only a very small proportion of them-four in the last hundred years, which seems to be close to the historical average (Patterson, 1986 )-led to sustained person-to-person transmission with global spread (Box 1). There are 18 known HA types and 11 known NA types (Tong et al., 2013) , which could theoretically be found in any combination. So far, sustained spread in humans has been limited to the H1N1, H2N2, and H3N2 subtypes , though it is possible that other subtypes circulated prior to 1918, the year of the first pandemic from which viruses are available for study (Worobey et al., 2014) . Multiple virus-host interactions are necessary for replication and onward transmission; the differences in the genetic requirements to accomplish each of these interactions in humans versus other animals provide a barrier to sustained transmission following spillover .
Experiments in ferrets have been used to measure viral transmissibility via respiratory droplets (in this review we use this term to refer to any transmission through the air between ferrets that are not in direct or indirect physical contact). Droplet transmission in ferrets is a useful, albeit imperfect, correlate of the potential of influenza strains to transmit efficiently in human populations (Buhnerkempe et al., 2015) . For this reason, some have argued that there is a Box 1. Steps in pandemic emergence.
Certain types of countermeasures against an influenza pandemic are effective only against one lineage of viruses -for example, creating stockpiles or seed stocks of vaccines against a particular subtype, or culling poultry infected with that subtype. It is not currently feasible to invest in such countermeasures against all viruses circulating in avian or other reservoirs, or even against all those causing known zoonotic cases. Therefore, there would be value in an accurate system to assess the relative pandemic risks posed by each virus and prioritize them for the development of such strain-specific countermeasures, while directing fewer resources to strains of lower concern (Kaplan et al., 2016) . This consideration has motivated calls for comprehensive analysis of all available data to assess the threat to public health posed by these strains. One response is the CDC's Influenza Risk Assessment Tool (IRAT) (Trock et al., 2015) , which incorporates elements including properties of the virus, field and epidemiological findings, and attributes of the human population to provide a framework to differentiate among novel influenza viruses thought to possess pandemic potential. Such risk assessments can help focus pandemic prevention and response efforts on the viruses thought to pose the highest risk of pandemic spread , in the most worrisome cases providing a rationale for costly measures such as poultry culling or vaccine seed stock development, or even stockpiling of large quantities of vaccine. A guiding question of this article is to examine the degree to which it is justified to rely on measurements and predictions of viral genetic and phenotypic traits in prioritizing responses to particular viral subtypes and within-subtype lineages.
These difficulties are exacerbated by the fact that influenza pandemics are rare events, and that risk assessments are not yet made with enough quantitative precision to formally evaluate their practical application. Even perfect information about the viral determinants of pandemic risk might only be enough to distinguish between strains with a low risk of causing a pandemic (say, 0.1% per year) and those with an extremely low risk (say, less than 0.01% per year), with unpredictable ecological or evolutionary contingencies determining which of these low-probability events will actually occur. One such contingency is that an avian influenza virus could acquire one or more of the determinants of pandemic potential by reassorting with a human seasonal influenza virus.
With only one pandemic every few decades, the data set for testing the prediction of such rare events is inadequate, a problem that challenges predictions in many fields beyond infectious diseases (King and Zeng, 2001; Hansson, 2006) . Evolutionary events in which a strain increases human-to-human transmissibility, but not enough to spark a pandemic, are extremely hard to observe, but if we could do so it would increase our ability to characterize the process of adaptation (Kucharski et al., 2015) .
Despite these challenges, there has been tremendous interest and investment in making and using such predictions, and a number of new ideas to improve predictions are in various stages of development (Box 3). Building on the findings of a previous workshop , we considered in detail the present state of knowledge concerning three phenotypic traits: HA receptor binding specificity, and HA pH of activation, and polymerase complex activity, (Figure 1 ). These were chosen from a longer list of candidate traits (Table 1 ) because they span the viral life cycle ( Figure 1 ) and their role in host adaptation has been extensively studied. All three are believed to be required for an influenza virus to cause a pandemic; consistent with this assumption, all three traits have been present to some degree in the earliest viruses isolated in pandemics since the 20th century, though some have been enhanced by subsequent evolution during seasonal transmission in humans. Moreover, for each of these three traits, viruses isolated from avian hosts typically do not show the mammalian-adapted phenotype, reflecting divergent selection pressures in the two classes of hosts (Tables 2, 3, and 4). All three traits changed in the adaptation of zoonotic H5 influenza viruses to droplet transmission in ferrets (Imai et al., 2012; Linster et al., 2014) . We emphasize that each of these traits is quantitative, and that human-adaptation is not a threshold criterion but a continuum; in this review when we speak of human adaptation we mean a tendency to be better adapted to humans, rather than an absolute yes-or-no property.
This review starts with a summary of our knowledge about the role of each of the three functional traits in conferring pandemic potential on a virus strain. Following these case studies, we draw some generalizations about the prospects of predicting pandemic risk from virus genotype or from assays of particular viral traits. For each trait we present a table showing the degree to which the sequence changes or phenotypic properties associated with avian or human adaptation are present in isolates from birds and humans, respectively. If the avian traits were always found in avian isolates and human traits always in human isolates, only the shaded cells on the main diagonal would be filled. In such a case, however, it is hard to see how viruses would ever make the jump from birds to humans, since so many traits would have to change simultaneously, and indeed the off-diagonal cells are not empty. Finding avian-adapted traits in viruses isolated from humans most often occurs in zoonotic cases, showing that not all human-adapted traits are required for the first human infection. In some cases there are also Box 2. Granularity of pandemic risk prediction -for what taxonomic level does it make sense?

A. Definition of the trait

Receptor binding preference is defined as the ratio of affinity (or avidity) of an HA molecule for an a2,6 glycan relative to that for an a2,3 glycan, with higher values associated with greater human adaptation. The evolution of receptor binding specificity is driven by the host environment, with selection for specificity during the infection process within a host and during the process of transmission. The error-prone replication of influenza genomes can facilitate rapid emergence of viruses with amino acid substitutions that alter the receptor binding characteristics of the HA (Lakdawala et al., 2015) . Increased transmissibility may result from mammalian receptor adaptation, either because the virus shedding form the infected donor host is increased, or because the ability of virus to infect the recipient host at a low dose is enhanced, or for both of these reasons. Recent experimental evidence in ferrets implicates the soft palate as an important site of selection for a2,6 specificity (Lakdawala et al., 2015) .

D. Receptor binding preference as a predictor of host adaptation of influenza viruses and pandemic risk

At present, estimating the contribution of receptor specificity to the pandemic risk posed by a novel virus relies primarily on the similarity between the receptor binding characteristics of the emerging virus and that of the most closely related HA with known transmissibility among humans or a surrogate animal model.
In principle, phenotypic assays that directly measure the receptor-binding preference of HA -if performed under realistic conditions that capture the interaction of the HA trimer with the receptor (Gambaryan et al., 1997; Takemoto et al., 1996; Collins and Paulson, 2004 )-may better capture the trait of interest than genetic predictions of this preference. However, even here, a simple equivalence between binding preference for a2,6-linked glycans and pandemic risk could be misleading. Several viruses circulating in humans during the early phase of previous pandemics were found to show either a preference for avian receptors (Rogers and D'Souza, 1989; Connor et al., 1994) or a mixed preference for both human and avian receptors (Rogers and D'Souza, 1989; Glaser et al., 2005; Childs et al., 2009 ).

B. Functional, structural and genetic determinants of hemagglutinin pH of activation and its consequences

The use of small mammalian models in influenza virus pathogenesis and transmission has proven invaluable for the study of these complex, polygenic traits. The ferret model is particularly valuable, as ferrets are highly susceptible to most influenza A viruses without the need for prior host adaptation. However, even this gold-standard model is not a true substitute for humans. Below, we summarize the benefits, drawbacks, and alternatives to the ferret model for the study of influenza. Validity. Influenza is a respiratory pathogen in humans, and employing mammalian models that possess comparable lung physiology permits a greater extrapolation of results from the laboratory. Importantly, the linkage types and distribution of sialic acids throughout the ferret respiratory tract are generally comparable to humans (Jayaraman et al., 2012; Jia et al., 2014) : like humans, ferrets express the sialic acid N-acetylneuraminic acid ( Neu5Ac), but not the sialic acid N-glycolylneuraminic acid ( Neu5Gc), on respiratory epithelia. As a result, ferrets are uniquely suited for the study of influenza viruses compared with other small mammalian models which express Neu5Gc (Ng et al., 2014) . Furthermore, human and avian influenza viruses exhibit comparable binding to upper and lower respiratory tract tissues in ferrets and humans (van Riel et al., 2006; Shinya et al., 2006) . Secondly, ferrets infected with influenza viruses demonstrate numerous clinical signs and symptoms of infection associated with human disease. Ferrets infected with human influenza viruses often exhibit transient weight loss, transient fever, and sneezing, whereas infection with selected HPAI viruses in this species can lead to pronounced weight loss, sustained fever, lethargy, dyspnea, and neurological complications (Belser et al., 2009 ). Thus, ferrets represent a preclinical model to assess the ability of novel vaccine and antiviral treatments to mitigate influenza virus. As ferrets are a suitable model for the coincident study of pathogenesis and transmission, this model allows for a greater understanding of virus-host interactions and the interplay between both of these parameters. Finally, the ferret model can yield valuable insights about the potential human-to-human transmissibility of influenza viruses -the critical determinant of pandemic risk. A recent meta-analysis showed that estimates of transmissibility derived from ferret respiratory droplet transmission studies could explain 66% of measured variation in human transmissibility, for influenza subtypes that have been detected in humans (Buhnerkempe et al., 2015) . Furthermore, there is a strong statistical relationship between the attack rates measured in particular ferret experiments and the probability that the influenza strain in question is capable of sustained transmission among humans: if two-thirds or more of contact ferrets become infected via respiratory droplets, then the strain is likely to have pandemic potential (see figure) . However, extrapolation of this relationship to novel strains is inherently risky, and variable outcomes observed for H7N9 influenza transmission in ferrets highlight the potential for false alarms. Further analysis of ferret transmission experiments, ideally in concert with molecular and virological research, could raise their sensitivity and specificity for identifying pandemic threats. Limitations. There is no 'perfect' small mammalian model for influenza. A longstanding challenge of the ferret model has been limited availability of ferret-specific commercial reagents compared with other models, though recent sequencing of the ferret genome should improve this situation . Ethical considerations, and the size and cost of ferrets, necessitate generally small sample sizes in ferret experiments, limiting statistical power (Belser et al., 2013) . Like other vertebrate models, the ferret is not appropriate for high-throughput screens, so research in the ferret model is most potent when complemented with in vitro and computational approaches. Finally, ferrets are not well suited to model the multiple influenza exposures over several years that may be experienced by humans and may mold their immune responses in ways that affect the infection risk with subsequent viruses (Andrews et al., 2015) . Studies of first influenza infection in ferrets may thus overestimate infection and/or transmission risk relative to that in populations with a history of prior infection with related viruses (Belser et al., 2016) . the 20th Century range from pH 5.0 to 5.4 (Galloway et al., 2013) . In 2009, emerging pandemic H1N1 viruses had HA activation pH values of approximately 5.5, but numerous subsequent isolates have acquired mutations that lower the activation pH to the range of the 20th Century human influenza viruses (Cotter et al., 2014; Maurer-Stroh et al., 2010; Russier et al., 2016) . Broad surveys of avian and swine influenza isolates have shown that HA activation pH can vary substantially with a range from pH 4.6-6.0 (Galloway et al., 2013; Scholtissek, 1985) . Among avian viruses, low-pathogenic duck viruses appear to range in acid stability from pH 5.3-6.0 and highly pathogenic avian viruses range from 5.6-6.0 (Galloway et al., 2013) .
Consistent with observed patterns in natural isolates, some experimental evidence indicates that within the range of natural variation, lower activation pH is adaptive for mammalian replication while higher activation pH is adaptive for replication in avian hosts. For isolates of H5N1 Alternatives. The ferret is but one of several well-characterized mammalian models for influenza virus. Mice are widely used in the field as they offer a greater availability of commercially available species-specific reagents, permit studies with greater statistical power due to larger sample sizes, and offer the advantage of transgenic animals. However, not all human influenza viruses replicate well in mice without prior adaptation due to a predominance of avian-like receptors in the murine respiratory tract; also mice do not display clinical signs and symptoms of influenza that mimic humans, and are not a reliable model for virus transmission studies. The guinea pig is another model, and offers several comparable advantages to ferrets, including generally similar lung physiology to humans and potential for transmission studies. Experiments in guinea pigs are often less expensive than in ferrets, because of lower husbandry costs and reduced drug costs when dosing is based on body weight (Lowen and Palese, 2007) . However, guinea pigs do not exhibit clinical signs and symptoms of infection similar to humans, and do not exhibit severe disease following infection with HPAI or pandemic influenza viruses, limiting their utility for viral pathogenesis studies.

B. Genetic basis of polymerase complex efficiency

Some mutations in PB2 are consistently associated with efficient function of the polymerase complex in mammalian cells ( Figure 2 ). As long ago as 1977, it was shown that an avian influenza virus could achieve efficient replication in mammalian cells by acquiring mutations solely in the PB2 subunit of the viral polymerase (Spooner and Barry, 1977) . The most famous of these mutations was later described as PB2 residue 627 (Subbarao et al., 1993) , which is a glutamic acid (Glu) in avian influenza viruses but a lysine (Lys) in human-adapted viruses, including those that emerged in the pandemics of 1918, 1957 and 1968 , and their seasonal descendants. An important exception is the virus that sparked the pandemic of 2009. In this virus, the PB2 segment had been introduced from an avian precursor into swine viruses in the 1990s, and mammalian adaptation had been achieved by a different set of PB2 mutations including changes at residues at 271, 590 and 591 (Mehle and Doudna, 2009) . Now that the 3-dimensional structure of the viral polymerase has been elucidated, we can see that residue 627, 271, 590 and 591 lie on the same external surface. Mammalian-adapting mutations increase the positive charge of this domain, suggesting that they either adapt the virus for interaction with an enhancing host factor or enhance its ability to repel a restriction factor (Mehle and Doudna, 2009) . Recently a host factor, ANP32A, that differs between mammals and flighted birds was shown to be a cofactor of the influenza polymerase, and the species specific difference could explain the inefficient function of avian virus polymerase and the stringent selection for the 627Glu->Lys adaptive mutation in mammals (Long et al., 2016) .
The adaptive value of these mutations is shown by experimental or observational data in which a mammalian host is infected with a virus whose PB2 is not adapted for efficient mammalian replication, but such a mutation becomes common in the virus population over the course of infection. Such evolution has been observed in a fatal human case of influenza A/H7N7 (Jonges et al., 2014) and in mouse experiments following serial lung passage using an isolate from this outbreak . Lys at position 627 has also been associated with greater severity in zoonotic H7N9 (Sha et al., 2016) and H5N1 (de Jong et al., 2006) cases However, reverse genetics experiments show that certain strains of avian influenza may be less able to accept these mutations than others (Long et al., 2013) .

C. Experimental assays to measure polymerase complex efficiency in human cells

Alternatively, polymerase activity could be measured in the context of viral infections (although this will require proper containment). This could be achieved by measuring intracellular levels of viral transcripts using transcriptomics or qRT-PCR. Such experiments would provide important information if they are performed using appropriate cell lines (or respiratory explants) at the temperature of the human airway (33˚C). It has been suggested that plaque size at 33˚C can be used as a surrogate measure of polymerase function but plaque size is a mutligenic trait. The predictive value of such assays for transmissibility is limited.

D. Role of polymerase complex efficiency in pandemic risk prediction

Many H5N1 viruses that circulate today in the avian reservoir already have mutations in PB2 at 627 (Long et al., 2013) or 701 (de Jong et al., 2006) , likely resulting from the reintroduction of mammalian-adapted strains back into the wild bird reservoir. These have been associated in human infections with more severe cases (de Jong et al., 2006) . The fact that these strains have not achieved sustained human-tohuman transmission demonstrates that while polymerase adaptations to humans are likely necessary, they are not sufficient for a strain to spark a pandemic. Moreover, the absence of the signature PB2 627K mutation in the 2009 H1N1 pandemic strain demonstrates the limitations of relying on any single mutation for risk prediction (Herfst et al., 2010) ; viruses with the avian-like residue have also been isolated from zoonotic human cases of H5N1, H7N9, and H9N2 infections ( Table 4 , bottom left). On the other hand, the concept that adaptation of the polymerase is necessary for sustained human transmission is validated by findings that the 2009 pandemic strain had adapted to replication in human cells by changes other than E627K within the polymerase (Mehle and Doudna, 2009) . Identification of biophysical mechanisms common to mammalian-adaptive mutations may in the future provide the basis for new biological or biophysical assays of polymerase function to inform risk predictions.

Discussion

Based on the evidence to date, it seems clear that all three of the traits considered in this review, and possibly others in Table 1 , must be simultaneously present at least to some degree for a strain to cause a pandemic. Yet with only a few instances of pandemic strains emerging per century, it should not be surprising that a new pandemic strain would violate an apparent rule of human adaptation that applied perfectly to previous pandemic strains, as in the case of the PB2 residues associated with human adaptation in 2009, or as in the case of activation pH of HA in early 2009 isolates , which had a value outside the range previously seen in human influenza viruses. It is unclear whether the list of sites and phenotypic traits associated with human adaptation is nearly complete or will continue to grow as we experience additional pandemics. At least for the traits of receptor binding (Childs et al., 2009) (Table 2) and acid stability (Table 3) , full human adaptation may not be required to initiate a pandemic in a virus that is otherwise well-adapted for humans. Thus, whether or not the list of traits required for pandemic is now complete, our understanding of where the threshold lies for being sufficiently humanadapted continues to change.
While the biological properties of a virus certainly play a large role in determining the pandemic risk posed by a strain, it is possible that even a virus perfectly adapted for human-tohuman transmission might fail to transmit extensively, due to ecological factors, chance, or both. Initiation of a pandemic requires not only a well-adapted virus but ecological opportunity to spill over into humans (perhaps multiple times if the first introduction is not "successful" [Mills et al., 2006] ), as well as a human population that is immunologically susceptible and sufficiently connected to establish ongoing transmission (Box 5). Additional complexity arises from the fact that selection pressures for within-host proliferation and competition may diverge from those needed for efficient transmission (Park et al., 2013) . Genetic bottlenecks at the time of transmission (Poon et al., 2016; Varble et al., 2014; Zaraket et al., 2015) may further enhance the role of chance, as a highly adaptive variant arising in a host may not get transmitted in a particular event. However, selective bottlenecks, which have been observed in experimental transmission of H5N1 and avianlike H1N1 viruses in ferrets, could lower the barrier to emergence of human-adapted viruses (Moncla et al., 2016; Wilker et al., 2013) . Both ecological and host factors are considered in the CDC's IRAT (Trock et al., 2015) .
It seems clear that a pandemic risk assessment informed by genetic sequence data is better than one uninformed by such sequence data, but the thought experiment of considering the 2009 swine-origin virus, had it been seen prior to initiating the pandemic, shows that such efforts may fail to identify the risk posed by strains that subsequently cause a pandemic. According to the knowledge at the time, early human isolates of 2009 (and presumably their swine precursors) would have had an HA intermediate between human and avian adaptation in terms of receptor binding. They had an activation pH outside the range previously seen in human viruses and more typical of avian viruses. Moreover, these viruses lacked the amino acid residues then thought to confer human adaptation for the polymerase complex. We must imagine that had this strain been detected in swine surveillance prior to the pandemic emergence, genetic as well as phenotypic considerations would have marked it as low risk, creating a false-negative risk assessment. Given that this virus did in fact create a pandemic, it is evident that failure of a nonhuman influenza virus to fully meet the three criteria discussed in this review does not disqualify it from posing a significant risk of a pandemic.
There are alternative approaches to ferret gain-of-transmission experiments in highly pathogenic avian influenza viruses, though disagreement remains about the level of evidence such alternatives provide. One alternative is to perform similar experiments starting from avian viruses that are not highly pathogenic in mammals, and/or are related to currently circulating human seasonal viruses, so that immunity would already be present in the population. Such experiments can provide the same degree of causal rigor as gain-of-function in highly pathogenic avian viruses with novel surface antigens and can elucidate general principles of mammalian adaptation, but they cannot confirm that the same changes would be observed in other strains that are not used in the experiment. A recent report shows a related way forward: the recreation of the steps of mammalian adaptation using viruses whose HA and NA are already circulating in humans (Lakdawala et al., 2015) . Such loss+gain-of-transmissibility experiments reconstruct the properties of naturally occurring seasonal human strains, from laboratory-generated, avian-adapted (or at least human-deadapted) precursors. Reconstructing such seasonal strains should pose a risk similar to that of working with the seasonal strains themselves, less than that of a novel subtype. A 2011 report employed a similar strategy, demonstrating the importance of HA activation pH in mouse adaptation by selection experiments on a live attenuated H5N1 vaccine strain lacking the NS1 gene (Krenn et al., 2011) . More recently, a 2009 H1N1 pandemic virus was modified to express a mutation that increased pH of HA activation, then selected in ferrets for droplet transmission, and it was found that a second site mutation partially restored the lower pH of activation of the selected virus . One limitation of de-adaptation strategies is that the acquisition of transmissibility is perhaps most likely to evolve by reversion of the de-adaptation changes. As with all gain-of-function and loss-offunction studies, epistatic effects of other loci in the genetic background of the viruses used in such studies set limits to the generalizability of such experiments. Another kind of alternative is simple characterization of ferret transmissibility of naturally occurring highly pathogenic strains without selection for airborne transmission. This approach can provide correlative evidence for the importance of genetic differences but cannot prove the mechanistic role of any particular change.
Even if strain-based assessment methods were much better, surveillance would be a key rate-limiting step for pandemic risk assessment to direct countermeasure development. If a virus about to cause a pandemic is not found in surveillance it cannot be assessed. The fact that we have yet to identify a pandemic strain in nonhuman hosts or in human spillover cases before the pandemic starts indicates there is much work to be done. Although surveillance has expanded since the 2009 pandemic, it has not been designed to optimize the chances of detecting a pandemic strain before it becomes pandemic; indeed, how to do so is not clear at present. Some possible considerations would be to maximize the diversity of isolates collected, to preferentially sample strains that are known to cause human infections, and to feed back information from risk assessments to inform choice of sampling. Rapid sequencing and phenotypic characterization of strains and dissemination of this information, along with interpretations of the risk profiles implied, is also important to maximizing the value of surveillance. Further thought should be given to the possibility of using high-throughput sequencing as a screen for which viruses should be subjected to phenotypic testing, which for the moment is typically more costly, slower, and lower-throughput than sequencing. More deliberate approaches to the design of surveillance systems would also depend on answering the question addressed in Box 2: how different must a virus be from previously characterized viruses to merit separate evaluation of its pandemic risk? The uncertainties noted above about the phenotypic characteristics of viruses isolated from previous pandemics (which may have been present in the primary isolate or may have arisen during egg passage in the laboratory) underline the need for careful attention to passage histories of surveillance isolates to avoid altering their genotype and phenotype post-isolation (Bush et al., 2000) . Expanding and rationalizing surveillance in this way would require overcoming political, logistical and financial constraints that vary between countries and regions.

Acknowledgements

We thank Jesse Bloom, Ruben Donis, Judith Fonville, Scott Hensley, and Benjamin TenOever for contributing valuably to the workshop on which this paper is based and Ruben Donis for significant assistance in improving the manuscript. We thank James Hay for the Figure for Box 5. This work and the workshop from which it originated were supported in part by the Research and Policy for Infectious Disease Dynamics (RAPIDD) program of the Science and Technology Directorate, U.S Department of Homeland Security, and the Fogarty International Center, NIH. The findings and conclusions in this report are those of the authors and do not necessarily reflect the views of the Centers for Disease Control and Prevention, the Department of Health and Human Services or its Components, or the NIH or its institutes. This paper is dedicated to the memory of Ellis McKenzie, cofounder of RAPIDD and Senior Scientist at the Fogarty International Center of NIH, who was a valued friend and colleague and who enthusiastically supported the workshop that produced his paper.
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Abstract

Plant-made or "biofarmed" viral vaccines are some of the earliest products of the technology of plant molecular farming, and remain some of the brightest prospects for the success of this field. Proofs of principle and of efficacy exist for many candidate viral veterinary vaccines; the use of plant-made viral antigens and of monoclonal antibodies for therapy of animal and even human viral disease is also well established. This review explores some of the more prominent recent advances in the biofarming of viral vaccines and therapies, including the recent use of ZMapp for Ebolavirus infection, and explores some possible future applications of the technology.

Introduction

The science of "molecular farming", or the use of plants and plant cell cultures to produce high-value recombinant proteins, started with the production via transgenic tobacco and sunflower of chimaeric human growth hormone in 1986 [1] , then of monoclonal antibodies in transgenic tobacco in 1989 [2] , and human serum albumin in transgenic tobacco and cell cultures [3] . This was followed from 1992 onwards with the expression of the first candidate virus vaccine antigens: these were Hepatitis B virus (HBV) surface antigen (HBsAg) in 1992 [4] , and a VP1 epitope of foot-and-mouth disease virus (FMDV) expressed on the surface of particles of recombinant Cowpea mosaic virus (CPMV) in 1993 ( [5] see Table 1 ).
Use of rTMV to express R9 epitope of E2 protein as CTB fusion [30] Expression of chimaeric plant virus coat protein molecules containing R9 [31] [32] [33] R9-CMV CP VLPs orally immunogenic via feeding of lettuce leaves in rabbits [34] Mixed Th1/Th2 response in mice to rodlike PMV-E2 epitope chimaeric VLPs [36] Co-expression of whole E2 and calnexin and calreticulin increases E2 accumulation in plants [37] Influenza viruses

Hepatitis C vaccines

Another interesting recent development was the filing of a USPTO patent application on plant production of the whole E2 protein, as apparently this has not been particularly successful because of misfolding. The patent covers agroinfecting a N. benthamiana cell expressing the recombinant E2 protein with one or both of the molecular chaperones calnexin and calreticulin, as this leads to an improvement in the yield of recombinant E2 protein [37] . This adds to what appears to be a successful expression, albeit at low level, of HCV E1 protein in plants [38] .

Influenza virus vaccines

Perhaps fortunately, then, influenza vaccines have been the major success story for plant-expressed antigens, largely due to two major factors: first, the haemagglutinin (HA) protein is the main determinant of neutralisation, and the only essential component of a vaccine; second, it appears to be well expressed in plants, and to fold properly. Perhaps the most significant factor of plant expression of HA, however, is the fact that expression of this alone is sufficient for the efficient formation at high yield of highly immunogenic VLPs, which bud from the plant cell outer membrane [44] . This is especially relevant to influenza vaccine development in light of the fact that HA-containing VLPs produced in insect cells elicit broader cross-neutralising immune responses than whole virion inactivated influenza virus or recombinant HA [45] . These properties have been exploited by a number of groups in work reported on in detail elsewhere [46] [47] [48] ; accordingly, this review will be limited to recent developments of specific interest to pandemic and seasonal human vaccine development.
In 2012, accounts of Phase 1 trials of both H1N1pdm HA-derived (A/California/04/09) and HPAI H5N1 (A/ Indonesia/05/05) HA-derived products were published by researchers from what is now the Fraunhofer Center for Molecular Biotechnology in Delaware, USA (http://www. fraunhofer.org/MolecularBiotechnology), following proof that they could use a TMV-based transient expression system to produce both proteins [48] . Recombinant HA sequences both included KDEL ER retention and 6xHis affinity purification sequences at their C-termini and were purified using detergent-containing buffers, Ni-Sepharose and hydrophobic interaction and anion exchange chromatography columns, at scales up to 50 kg of N benthamiana biomass under cGMP. Mouse immunogenicity studies demonstrated that intramuscular (IM) injection of 1 μg of H1 HA or 5 μg of H5 HA protein adsorbed to 0.3% Alhydrogel as adjuvant elicited serum HAI antibody responses with titres ≥1:40 in at least 67% of vaccinated mice, with significant enhancement of immunogenicity and consequent dose-sparing afforded by use of the adjuvant. In rabbits, two IM doses of 45 μg of H1 HA plus Alhydrogel elicited HAI titres of ≥1:40 in 100% of animals, while two doses of 90 μg of H5 HA were required for the same titres in 80% of rabbits. Results in ferrets were similar, with lower responses to the H5 HA. Conclusions from this study were that both antigens were safe and immunogenic, and suited to human trial.
A number of other different plant-produced influenza vaccine candidates are at earlier stages of development, but show promise: these include HA protein attached to a number of different partner molecules, as well as candidate "universal vaccines" based on the highly conserved M2e or M2 ion channel protein ectopic domain.
TMV potentiated the response to HA. This group was able to produce 500 g of monomeric HA protein at pilot scale in less than a montha good advertisement for the potential of this technology for an emergency response vaccine capability.
Production of other influenza virus antigens has been limited, with the only other protein investigated to any extent being the M2e peptide, or ectopic domain of the M2 ion channel protein: this has considerable potential as a universal vaccine candidate, as it is highly conserved between all influenza A viruses. The insertion of a M2e 2-24 peptide in two different locations in the Human papillomavirus type 16 VLP-forming L1 protein has been reported [62] , as has the use of surface display on a filamentous VLP-forming potexvirus CP [63] , albeit without preclinical evaluation. However, a more recent investigation that used surface display on TMV virions was more noteworthy in that synthetic M2e peptides derived from a H1N1 virus inserted in between TMV U1 CP residues 155 and 156 were well displayed on virions. The candidate vaccines elicited high antibody titres in IM immunised mice, and were 100% protective against 5 LD50s of homologous challenge virus (A/PR/8/34), and 70% of heterologous (A/California/04/2009) challenge [64] .
While plant-made influenza viruses in general, and HA-based vaccines in particular seem to be products with significant potential, there are still some avenues to exploresuch as the expression and testing of neuraminidase (NA), matrix protein (M1) and nucleoprotein (NP), all of which can play a role in immunity to and recovery from influenza virus infections, and which could constitute valuable additions to the plant-made influenza vaccine armoury.

Papillomavirus vaccines

Human papillomavirus (HPV) vaccines based on VLPs made by recombinant expression and assembly of the major capsid protein L1 are the latest blockbuster viral vaccines, with annual sales of both Merck's yeast-made Gardasil and GSK's insect cell-made Cervarix VLP-based vaccines close to or higher than US$1 billion as of 2012 [65] . Gardasil targets the two most prevalent HPVs found in 70% of cervical cancer cases -HPV-16 and HPV-18as well as the two most common genital wart-causing viruses, HPVs −6 and −11, while Cervarix contains only HPVs −16 and −18. While the global impact of the target disease is in the developing worldin 2001 women in developing countries accounted for~85% of both annual cases of cervical cancer (~500 000 cases worldwide) and annual deaths from cervical cancer (~300 000 worldwide) [66] the main market for the vaccines is in the developed world, largely due to their expense. While this is changing to some extent, with a number of governments negotiating favourable tenders for supply of HPV vaccines for EPI programmes, it remains a problemand the imminent release of nextgeneration products with wider coveragemixtures of more types of HPV L1, or chimaeric L1 vaccines including all or part of the minor capsid protein L2, or L2-based vaccines -will do nothing to fix this problem [67, 68] .
The only documented expression of the minor papillomavirus capsid protein L2 was from our laboratory (reported in [81] ): this was HPV-16 L2, human codon optimised, and expressed to levels of~30 mg/kgwhich we noted at the time could be useful, as the protein is present in virions at a maximum ratio of 1 L2 molecule per L1 capsomere, and given L1 yields that are~10-20fold higher, co-expression of the proteins could result in efficient L1 + L2 VLP formation.
Candidate therapeutic vaccines based on the HPV-16 oncoprotein E7 have been investigated in some detail too: the first expression of the protein in plantstransient expression in N benthamiana via a recombinant Potato virus X-derived vector -was accompanied by a proof of efficacy in a mouse model, with protection from tumour development caused by the HPV-16 E7-expressing C3 cell line accompanied by strong cytotoxic T-cell responses [82] . The same group went on to show 5-fold enhanced expression of E7 by targeting of the protein to the secretory pathway [83] . The possibly immune-enhancing properties of N benthamiana extracts indicated in previous work were further explored, with proof of immunomodulatory activity on human monocyte derived dendritic cells (MDDCs) [84] . Possibly in response to concerns about using an unmodified oncoprotein as a vaccine, they engineered mutations that abolished interaction with cell cycle governing proteins (E7GGG), and showed that a plantproduced fusion product of this with the beta-1,3-1,4-glucanase (LicKM) of Clostridium thermocellum elicited the same protective response [85, 86] . Experimental scale-up of production of this fusion protein as a therapeutic vaccine was achieved with 50% final yield (at 100 mg/kg biomass) of protein at 99% purity, by means of metal-ion affinity chromatography and gel filtration [87] .
Another investigation of the potential of the L2 108-120 epitope was undertaken using a fusion of the peptide to the N-terminus of the Potato virus X coat Protein (PVX CP) for surface display, expressed via a recombinant PVX vector in N benthamiana: expression levels of up to 170 mg/kg biomass were obtained, and the chimaeric protein elicited anti-L2 108-120 -reactive antibodies after SC injection or tattoo administration [97] .
In another use of PVX CP fusions, this time with the E7GGG protein mentioned previously [99] , it was shown that while both N-and C-terminal fusions with PVX CP formed long filamentous VLPs when expressed in E coli, and expression in N benthamiana was at high levels, the latter did not apparently form VLPs. Another production modality for E7GGG was investigated in the form of transplastomic Chlamydomonas reinhardtii, a well-characterised unicellular alga [100] . E7GGG was expressed alone or as a His6 or FLAG fusion protein, to levels of 0.12% of TSP. Affinity purification was performed for both tagged forms, and C57BL/6 mice were vaccinated SC with C reinhardtii extract or purified protein mixed with QuilA adjuvant. Both specific anti-E7 IgGs and E7-specific T-cell proliferation were shown in mice vaccinated with either inoculum, and tumour protection was shown after challenge with TC-1 tumour cell line expressing E7. The authors note that this was the first successful expression of a soluble E7 derivative in plants, as previously the protein had to be expressed as a fusion product in order to get a usable yield.
Another recent paper that explored the expression of E7 in transplastomic tobacco determined that targeting the protein by means of a transit peptide to the thylakoid lumen -a chloroplast inner compartment with different redox potential from the stroma, among other characteristicsincreased production by more than 80-fold [101] . However, these authors did not inactivate the oncogenic potential of the protein, which means it presently has value only as an illustration of a useful yield enhancement technique.

Bluetongue virus vaccine

It has been established that recombinant expression of BTV proteins in insect and other animal cell culture systems results in a variety of structures being formed: expression of VP3 alone produces subcore-like particles (SCLPs); VP3 and VP7 together produce core-like particles (CLPs); expression of these plus VP5 and VP2 produces authentic VLPs [133, 134] . Building on evidence that these VLPs are protective in sheep challenged with live virus [135] , the PlaProVa group investigated whether it was possible to do the same using plant-produced antigens. Gene constructs were based on the Netherlands NET2006/04 strain of BTV-8, and sequences for VP2, VP3, VP5 and VP7 genes were Nicotiana codon-use optimised. The genes were cloned for expression into pEAQ-HT, an expression vector containing the Cowpea mosaic virus (CPMV)-derived HT or HyperTrans translational enhancer sequence from RNA2 [136] . Interestingly, infiltration of N benthamiana plants with either VP3 and VP5 alone produced necrotic symptoms; if these were combined with the other proteins, no necrosis was observed. Simple screening of clarified extracts by density gradient ultracentrifugation showed that expression of VP3 alone, or of VP3 + VP7, or of all four together, resulted in virion proteins co-sedimenting as high-MW aggregates. Some problems with stoichiometry resulting from relative overexpression of VP3an overaccumulation of SCLPs -were addressed by use of vectors expressing more than one protein: VP2 and VP5 were expressed via a dual HT vector, with VP3 and VP7 together on another vector with VP3 expression being detuned by use of a non-HT-containing 5′ UTR. This had the result of down-regulating the formation of CLPs, and a significant shift in the equilibrium towards VLP formation. Total BTV-8 protein yield by use of these vectors was >200 mg per kg biomass; the final yield of gradient-purified VLPs was~70 mg per kggratifyingly high, given a vaccine dose (see below) of 50 μg VLPs/dose.
The results of this investigation are significant for biofarming and vaccinology in particular for a number of reasons. First, as a general finding, it showed it is possible to reasonably easily produce a multi-protein complex with varying stoichiometry: such an approach could also be applied to other complex viruses, such as the related African horsesickness virus, human rotavirus, or picornaviruses such as poliovirus or foot and mouth disease virus. Second, and for BTV in particular, the ability to produce high levels of properly-assembled VLPs that are protective in sheep is a valuable proof of concept and of efficacy for a vaccine against an important emerging disease. Additionally, the timescale of mere days for production following preparation of the agroinfiltration inoculum means that it is possible to respond quickly and potentially locally to outbreaks of emerging disease.

Dual-use or "One Health" vaccines

The development of an effective oral vaccine against rabies has also been reported, with single doses of 50 μg Vnukovo strain rabies glycoprotein G in transgenic maize seed containing the antigen at 1% of TSP, protecting all vaccinated mice from lethal vampire bat rabies virus challenge [140] . Further testing of this vaccine was done in sheep: maize kernels containing different doses of G protein (0.5, 1, 1.5 and 2 mg) were given in a single dose by the oral route, and 2 mg doses elicited a degree of protection comparable to that conferred by the injected commercial vaccine [141] . The authors state that "…this is the first study in which an orally administered edible vaccine showed efficacy in a polygastric model", which is an important landmark in veterinary and potentially One Health vaccinology.
A recent study investigated the expression of the two neutralizing epitope-rich CCHFV envelope glycoproteins Gc and Gn in hairy root cultures and in leaves derived from transgenic tobacco plants [144] . Proteins accumulated to levels of 1.8 mg/kg biomass in hairy roots and 1.4 mg/kg in leaves. Separate groups of mice were fed transgenic leaves or roots, or fed the plant material and injected SC with the plant-made proteins, or vaccinated with an attenuated CCHFV vaccine as a positive control. Mice in all the immunised groups had a consistent rise in anti-Gc and Gn IgG and IgA antibodies in serum and faeces, respectively. Mice in the group that was fed and parenterally boosted, however, exhibited a significant rise in anti-CCHFV IgG (titre of 1/32 000 compared to 1/256 for oral-only) after a single boost. Additionally, the plant-purified Gc and Gn proteins reacted with human immunoglobulins in serum from a patient who had recovered from CCHFV infection. The potential for recombinant protein production in plants as a CCHFV vaccine appears obvious, although yields seem low for routine production.

Anti-viral therapeutic antibodies

As background, use of a high-yielding geminivirusbased transient expression system in N benthamiana that is particularly suited to simultaneous expression of several proteins had previously allowed expression of a MAb (6DB) known to protect animals from Ebola virus infection, at levels of 0.5 g/kg biomass [149] . The same group also used the same vector system (described in detail here [24] ) in lettuce to produce potentially therapeutic MAbs against both Ebola and West Nile viruses [150] .
A novel application of the same technology was also used to produce an Ebola immune complex (EIC) in N benthamiana, consisting of the Ebola envelope glycoprotein GP1 fused to the C-terminus of the heavy chain of the humanised 6D8 MAb, which binds a linear epitope on GP1. Geminivirus vector-mediated co-expression of the GP1-HC fusion and the 6D8 light chain produced assembled immunoglobulin, which was purified by protein G affinity chromatography. The resultant molecules bound the complement factor C1q, indicating immune complex formation. Subcutaneous immunisation of mice with purified EIC elicited high level anti-GP1 antibody production, comparable to use of GP1 VLPs [158] . This is the first published account of an Ebola virus candidate vaccine to be produced in plants.

Introduction

Initially, the prevailing idea for the use of plantproduced vaccines was that these should be delivered in plant material, as edible vaccinessomething that was already being questioned as early as 1996 [6] . However, this concept has now largely fallen out of favour, with the realisation that administration of vaccines to humans requires standardisation of dose and some measure of quality control, and the necessity for purification and formulation has largely been accepted [7] . Over the years since 1989, then, many proteins have been expressed as candidate vaccines or therapeutics, and many different plant-based expression systems have been tried, with a growing trend towards transient expression systems based on infiltration of whole plants with recombinant Agrobacterium tumefaciens (agroinfiltration) and the use of "deconstructed" plant viral vectors (reviewed in [8] ).
Evidence of high-yield expression of H5 haemagglutinin-derived VLPs via transient expression [44] 10 million vaccine dose "rapid fire" milestone by Medicago Inc. in DARPA Blue Angel programme [50] Human clinical trial of plant-made H5N1 vaccine candidate [47] HA-only VLPs produced for H7N9 outbreak virus [54] Phase 1 trials of H1N1pdm and HPAI H5N1 HA-derived plant-made products [48] Testing of plant-made engineered soluble trimeric HA (H1N1pdm) in mice [58] Adjuvanting of monomeric H1N1pdm HA with SiO 2 and bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) [59] Emergency response influenza vaccine candidates made in South Africa [43] Conjugation of plant-made HA to TMV particles and successful testing in mice [60] Elicitation of neutralising Ab with elastin-like polypeptide fused with stabilised soluble trimer-forming H5N1 HA [61] Presentation of M2e epitope on surface of rTMV virions elicits protective immunity to homologous and heterologous challenge in mice [64] Papillomaviruses Proof of efficacy of a plant-made Cottontail rabbit and Rabbit oral papillomavirus vaccines [74, 75] High yields of HPV-16 L1 and VLPs via agroinfiltration-mediated transient expression or via transplastomic expression [76, 77] Transplastomic expression of capsomere-forming HPV-16 L1 fused with Escherichia coli LTB as a built-in adjuvant [78] Successful expression of HPV-8 and Bovine papillomavirus L1 VLPs in plants [79, 80] Co-expression of HPV-16 L1 with E coli LTB and oral immunisation elicits increased intestinal mucosal IgA responses to L1 [90] High-yield plant transient expression of chimaeric L1::L2 VLPs and proof of increased breadth of immune response [91] rPVX CP fusion with L2 108-120 epitope yields well and elicits high-titre anti-L2 protein sera in mice [97] Plant production, scale-up and protective efficacy in mouse model of therapeutic E7GGG-LicKM fusion protein vaccine [85] [86] [87] Plant expressed HPV-16 L1 with C-terminal string of E6 and E7 T-cell epitopes is viable prophylactic/therapeutic vaccine candidate [98] Production and proof of efficacy in mice of soluble E7GGG therapeutic vaccine in transplastomic Chlamydomonas reinhardtii [100] Proof of yield increase and efficacy in a mouse tumour model of shuffled E7 protein fused to Zera® peptide [102] Human immunodeficiency virus HIV-1 p24 capsid protein expressed successfully in transgenic tobacco [107] Transgenic maize as production platform for oral vaccine delivery tested using SIV major surface glycoprotein gp130 [108] Transiently-expressed gp41-derived molecule fused to CTB elicits anti-membrane proximal region (MPR) antibodies in mice [109, 110] Gag-derived antigens expressed transiently and transgenically as CTL-inducing booster immunogens [113] High-yield transplastomic expression of Gag VLPs [117] Phase I clinical trial of anti-HIV MAbs produced in transgenic tobacco [116] 20 years for a subunit vaccine to get to market. However, this was in the form of 22 nm subviral particles purified from the serum of human carriers of HBV, and although highly effective, was expensive to produce and of limited supplyto say nothing of the ever-present risk associated with a blood product isolated from HBV carriers who may carry any number of other, as yet undetected viruses. It was a triumph of modern molecular biology, therefore, when a very similar virus-like particle (VLP) vaccine derived from expression of the HBV small surface antigen (S-HBsAg) was developed in 1984 [12] . While this was initially still expensive -US$40/dose, with three intramuscular doses being necessaryprices have come down very significantly, to the point that more than 110 countries now routinely immunise infants as part of the Extended Programme of Immunisation (EPI) [13] . The recombinant vaccines are highly effective and safe, and have helped set the standard for later introductions, such as of the recombinant VLP-based Human papillomavirus (HPV) vaccines. However, there is still space for improvements in HBV vaccines, both in terms of cost of goods, and in specific antigen content. An increasing desire worldwide for "needle-free" vaccine delivery, for example, would require cheaper production of larger amounts of antigen for oral delivery, which current production modalities would not be able to meet. Problems with non-response of certain groups of people to the current vaccines have also necessitated the development of third generation products, containing the middle (M-HBsAg) and/or large (L-HBsAg) surface antigens, which contain the strongly immunogenic preS1 and/or preS2 domains. However, these vaccines are more expensive and less readily available [11] . Accordingly, plant production of HBsAgbased HBV vaccines has gone on for over 20 years, with a variety of products being made; pre-clinical testing of oral delivery of transgenic potato-delivered products for almost as long [14] , with a preclinical trial of an orallydelivered product in 2001 [15] , and human clinical trial in 2005 [16] .
The highest plant yield of conventional (=S, or small) HBsAg was achieved via use of deconstructed Tobacco mosaic virus-based cDNA MagnICON vectors (Icon Genetics, Halle, Germany): this was around 300 mg/kg wet weight in Nicotiana benthamiana, and the recombinant protein was full-length, formed disulphide-linked dimers, displayed the conformationally-determined 'a' antigenic determinant, and assembled correctly into VLPs. Table 1 Highlights of historical and recent activity in virus vaccine biofarming (Continued) rTMV-based expression of Gag VLP-based deconstructed multiantigens incorporating gp 41 MPER region [124] First successful published expression of HIV Env in plants: 89.6 .P gp140ΔCFI envelope protein expressed at high yield [125] Transient expression of HIV Env-H5 HA fusion molecule results in VLP formation [126] Veterinary and "One Health" vaccines Production in plants and proof of efficacy in sheep of intact VLPs of Bluetongue virus [128] Rabies virus vaccines produced in plants and shown to be protective including after oral administration [138] [139] [140] [141] Crimean-Congo haemorrhagic fever virus Gc and Gn glycoproteins immunogenic in mice via oral or parenteral administration [144] Plant-produced Rift Valley fever virus Gn and N proteins immunogenic after feeding mice transgenic Arabidopsis [145] Expression via rBeYDV and successful immunogenicity trial in mice of Ebola GP1/anti-GP1 MAb HC [158] Anti-viral therapeutic antibodies
Interestingly, vacuum-mediated agroinfiltration of whole plants led to a better product, as determined by presence of the 'a' determinant [20] . Plant expression of HBV antigens other than the standard S HBsAg has been attempted in recent years by a number of groups. The Arizona Biodesign Institute group, who pioneered much of the biofarming of HBV vaccines, showed in 2004 that the S form of HBsAg could be used via transient agroinfiltration-mediated expression as a useful fusion partner, with an N-terminal fusions of up to 239 amino acids being tolerated without alteration of its particle-forming ability or major antigenic properties [21] . They followed this with the demonstration that the HBsAg middle protein (M protein) -with the highly immunogenic 55 amino acid pre-S2 region fused at N-terminus of the S proteincould be successfully produced in plants, and elicited stronger humoral immune responses than the S protein when injected into mice [22] . The same group used MagnICON vectors to express over 2 g/kg wet weight of plants of VLP-forming and highly immunogenic HBcAg, the "core" antigen [23] : HBc has considerable potential both as a therapeutic vaccine and as a display vehicle for other peptides, including the HBV preS region [11] . A subsequent paper from the Biodesign Institute group detailed the use of a ssDNA Bean yellow dwarf mastrevirus-based vector system (reviewed here [24] ) to produce 800 mg/kg of HBcAg in N benthamiana [25] .

Hepatitis C vaccines

Plant production of candidate HCV vaccines has a reasonably long history, given that the virus was only described in 1989 [28, 29] . It is interesting that the first products have all been intended as therapeutic vaccines: in 2000, Nemchinov and colleagues described the use of a Tobacco mosaic virus (TMV)-derived vector in N benthamiana to express a synthetic hypervariable region 1 (HVR1)-derived peptide called R9, a potential neutralising epitope of HCV derived from the envelope protein E2, fused to the C-terminal of the B subunit of cholera toxin (CTB). The plant-derived HVR1/CTB reacted with immune sera from individuals infected with four of the major genotypes of HCV, and mice immunised intranasally with crude plant extract produced anti-HVR1 antibodies which specifically bound HCV VLPs [30] . This was followed by a succession of reports on plant expression of chimaeric plant virus coat protein molecules containing the same (R9) epitope: these included the use of Cucumber mosaic virus (CMV) CP, with immunoreactivity of sera from chronically infected patients with the recombinant CP [31] leading to use of purified R9-CMV to elicit in vivo responses in rabbits, and in vitro cellular responses as measured by interferon gamma production for lymphocytes from human patients [32, 33] . Subsequently, the same group showed that purified R9-CMV particles were stable under simulated gastric and intestinal conditions, and could elicit a humoral immune response in rabbits fed with R9-CMV infected lettuce plants [34] .
It appears as though plant production of candidate therapeutic vaccines for HCV is quite well covered; however, the prophylactic vaccine space is far less well investigated: hopefully, this will change with optimisation of plant production of full-length E2 protein as detailed above.

Influenza virus vaccines

While biofarming is often touted as being ideal for vaccine and other pharmaceutical production for the developing world, there is very little actually published from developing countries on use of the technology for their own purposes. One object example comes from my own lab in South Africa, where we explored the prospects of making emergency response pandemic influenza vaccines [43] . We were successful in making high-yield candidate subunit vaccines via transient agroinfiltrationmediated expression in N benthamiana from both a full-length (up to 130 mg/kg FW) and a soluble version (up to 675 mg/kg FW) of the H5N1 A/Viet Nam/1194/ 2004 HA, via a human codon-use optimization of the HA gene, and showed that while both proteins elicited usable titres of antibodies in mice and chickens by HAI assays, the full length protein was significantly better. Subsequent work showed that the full-length H5 HA could be extracted as semi-pure VLPs from the apoplastic space of unmacerated N benthamiana leaves via buffer infiltration and gentle centrifugation of cut leaves rolled into strips (H Inderthal, II Hitzeroth and EP Rybicki, unpublished) ( Figure 1 ).

Papillomavirus vaccines

The plant-based production of Human papillomavirus vaccines has had quite a long history, that has been well reviewed up to 2010 for all HPV vaccines [10] , and 2013 specifically for VLPs [9] . Briefly, the important early landmarks in farming of prophylactic papillomavirus vaccines are the proof that transgenically-expressed HPV-16 or HPV-11 L1 can assemble into immunogenic VLPS, albeit at low yield [70] [71] [72] [73] ; proofs of efficacy of a plant-made Cottontail rabbit papillomavirus (CRPV) L1based vaccine [74] and Rabbit oral papillomavirus or CRPV L2 peptides surface-displayed on rTMV vaccines [75] ; very high yields of HPV-16 L1 and VLPs via agroinfiltration-mediated transient expression [76] or via transplastomic (chloroplast) expression [77] . An interesting approach was shown in a study that used transplastomic plants to express a HPV-16 L1 mutated so as to form only pentameric capsomers rather than particles, fused to the Escherichia coli heat-labile enterotoxin subunit B (LTB) as a built-in adjuvant [78] . The resultant protein accumulated to 2% of total soluble protein (TSP), and had all the correct epitopes and biochemical activity of both parent molecules, indicating that it could be a viable vaccine candidate.
One of the few investigations to look at non-genital HPV L1s was a study of plant expression of the L1 of HPV-8, a high-risk cutaneous papillomavirus associated with epidermodysplasia verruciformis and non-melanoma skin cancer in immunocompromised people [79] . The expressed protein formed VLPs in planta, albeit at relatively low yield -and only if the 22aa C-terminal nuclear localisation signal was removed. This is similar to what was found for HPV-11 L1 [73] , and in contrast to results for HPV-16, where removal lowered yield [76] . In only the third investigation of expression of the L1 of a non-human papillomavirus, reasonably high levels of protein (183 mg/ml biomass) were achieved for transient agroinfiltration-mediated expression of Bovine papillomavirus type 1 L1 [80] . The protein formed VLPs, albeit only T = 1 30 nm particles rather than the 50 nm T = 7 native structure, which were nevertheless highly immunogenic in rabbits.
An interesting fusion of prophylactic and potentially therapeutic HPV vaccines was an investigation of the feasibility of producing chimaeric HPV-16 VLPs with L1 fused to a string of cytotoxic T-lymphocyte epitopes from HPV 16 E6 and E7 proteins, in transgenic tomato plants (Lycopersicon esculentum) [88] . While expression levels were low, VLPs were formed in planta and elicited both anti-L1 neutralising antibody and anti-E6/E7 CTL responses. A development from this work was the use of the chimaeric VLPs made in tomato plants for detection of HPV-16 specific antibodies in patients with grade 1 cervical intraepithelial lesions (CIN 1) [89] , pointing up the potential use of plant-made products as inexpensive reagents.
A study of chimaeric HPV-16 L1 molecules from our group [91] investigated neutralising antibody epitopes derived from HPV-16 L2 comprising amino acid residues 108-120, 56-81 or 17-36 substituted into the C-terminal helix 4 (h4) region of L1, from amino acid 414, following evidence that these were the most effective in terms of eliciting a wider spectrum of cross-neutralising antibodies to high-risk HPVs [92] [93] [94] [95] . Following previous experience with L1, we used a human codon-use optimised L1 gene with similarly-optimised inserts, transiently expressed in N benthamiana via agroinfiltration, and targeted for import into chloroplasts. All chimaeras were very highly expressed, with yields of up to 1.2 g/kg plant tissue. The L1 chimaera containing L2 amino acids 108-120 (L1:L2 108-120 ) was the most successful candidate in that it assembled into small VLPs (~30 nm), and elicited anti-L1 and anti-L2 responses in IM immunised mice, and immune sera neutralised homologous HPV-16 and heterologous HPV-52 pseudovirions. The other chimaeric L1s did not form distinct particles, and elicited significantly weaker humoral immune responses for the same dose of protein. The L1:L2 108-120 particle formation was in contrast to repeated previous expression of this and the other chimaeras in insect cells, where only loose aggregates of capsomers were formed for L1:L2 108-120 [95, 96] .
Our group has recently explored the potential of a novel HPV-16 E7-derived gene constructa synthetic shuffled HPV-16 E7 (16E7SH) that has lost its transforming properties, but retains all naturally-occurring CTL epitopesfor expression in plants as a candidate therapeutic vaccine [102] . The E7SH gene has previously successfully been used as a DNA vaccine in a mouse tumour model, eliciting potent cellular and humoral immune responses, including tumour protection and regression [103] . We fused the gene translationally to one encoding the Zera® peptide, a self-assembly domain of the maize gamma-zein seed storage protein that induces the accumulation of recombinant proteins into protein bodies (PBs) within the endoplasmic reticulum in a variety of eukaryotic expression systems [104] . This generally allows stabilisation of recombinant proteins, as well as enhanced accumulation and far easier purification. While E7SH alone was expressed at only a low level by agroinfiltration-mediated transient expression in N benthamiana, high-level expression of E7SH-Zera was achieved, with a maximum of 1.1 g/kg biomass, and the resultant protein bodies could be easily purified. Immune responses comparable to the E7SH DNA vaccine were demonstrated in mice, with specific humoral as well as cell-mediated immune responses, and significant tumour regression in vaccinated mice with pre-existing tumours. Interestingly, simply mixing Zera-only PBs and 16E7SH also enhanced immune responses, indicating an independent adjuvant activity for the Zera® component. Moreover, use of the E7SH-Zera gene as a DNA vaccine also resulted in increases in IFN-γ levels of mouse splenocytes compared to mice inoculated with the 16E7SH gene, a trend that was also observed for Granzyme B ELISPOT assays as well as in chromium release assays. We feel we have demonstrated proof of efficacy in a mouse tumour model of a novel HPV therapeutic vaccine candidate, which should be easy and cheap to produce and purify. For further development of this vaccine, a DNA vaccine prime followed by matched protein boost might be ideal in order to achieve further enhancements in immunogenicity.

HIV vaccines

There is little need to either reiterate the need for vaccines for Human immunodeficiency viruses (HIV) in general, or of the predominant HIV-1 in particular; there is also little need, in the face of a plethora of literature, to detail the approaches to, or problems inherent in, the development of said vaccines [105, 106] . As could be expected, HIV vaccines were an early target for plant expression studies, with a variety of targets: indeed, HIV-1 p24 capsid protein was expressed successfully in transgenic tobacco, albeit at low yield (0.35% of TSP), as long ago as 2002 [107] ; the suitability of transgenic maize as a production platform for oral delivery of HIV vaccines in seed extracts was tested using SIV major surface glycoprotein gp130 [108] ; a novel gp41-derived molecule incorporating a CTB fusion as polymerising agent and adjuvant was produced via agroinfiltration-mediated expression in N benthamiana [109] and shown to produce mucosal and serum antimembrane proximal region (MPR) antibodies in mice after mucosal prime-systemic boost immunisation [110] ; a Tat monomer was produced in spinach via rTMV as a potential oral vaccine [111] ; epitopes derived from HIV-1 Gag and Env were fused to HBsAg and expressed as VLPs in transgenic tomato fruit [112] ; various Gag-derived antigens (p24, p41, p55) were produced in transgenic tobacco and via rTMV in N benthamiana for investigation of their utility as CTL-inducing immunogens by our group [113] ; HIV-1 Nef was produced in N benthamiana using an agroinfiltration-mediated transient expression system [114] . The field was comprehensively reviewed recently [115] , so this review will be limited discussion of major advances, and more recent work.
While many and varied antigens have been produced in various types of plant using a variety of expression systems, there have been few systematic investigations of antigens regarded as central to mainstream HIV vaccine productionthat is, full-length Gag and Envand no clinical trials of any candidate vaccine products, although plant-produced anti-HIV MAbs have gone to Phase 1 trial [116] . The most successful expression of full-length Pr 55 Gag precursor polyprotein to date was done with subtype B HIV-1 Gag in transplastomic tobacco, with yields of enveloped~100 nm diameter VLPs of up to 400 mg/kg biomass [117] . This was close to 10 000-fold more than a previous best by our group, with VLPs produced in transgenic tobacco [81] , and represents a viable yield for a product with serious vaccine potential: recent evidence that lack of progression to AIDS is linked to strong CTL responses to Gag [118] [119] [120] reinforces the need for vaccines that can target such responses, and Pr 55 Gag VLPs are potent elicitors of CTL, especially when used as a protein boost to a heterologous prime in primate models [121, 122] . A recent paper details how HIV-1 p24 antigen expressed transgenically in either Arabidopsis thaliana or Daucus carota showed a priming effect in mice fed whole plant material, eliciting humoral immune responses detected as serum anti-p24-specific IgG after an intramuscular purified p24 protein boost [123] . It is interesting that dose-dependent antigen analyses using transgenic A. thaliana showed that low p24 antigen doses were superior to high doses.
A new plant-made HIV multiantigen that makes use of VLPs is "…enveloped particles… consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41)" [124] . This combines the proven qualities of Gag VLPs as self-adjuvanting potent humoral and cellular response-inducing immunogen with an envelope protein comprising the membrane proximal external region (MPER) of HIV gp41, which is important in infection processes and in eliciting broadly neutralising anti-HIV antibodies. Both the gag and gp41 genes were extensively deconstructed in terms of removal of potential methylation sites, and cryptic splice and polyadenylation sites, and plant codon-use optimised. Gag was expressed constitutively in transgenic N benthamiana plants under control of the CaMV 35S promoter, to levels of~22 mg/kg biomass. Transgenic plants were then infiltrated with Agrobacterium transformed with a deconstructed MagnICON replicating vector expressing the dgp41 envelope protein that was targeted to the apoplast by means of a barley alpha-amylase signal peptide. Coexpression allowed >2fold greater accumulation of both proteins, with dgp41 reaching~9 mg/kg. Iodixanol density gradient centrifugation indicated that the coexpressed proteins cosedimented in a denser fraction than for pgp41 when expressed alone, and similarly to Gag-only fractions that were known to contain VLPsand indeed, characteristic~100 nm diameter enveloped VLPs were seen in extracts and in situ in plant leaf sections, and could be shown to bud into the medium from protoplasted co-transfected cells. I believe these authors are not exaggerating in their claim that "These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV-1", given that their vaccine candidate includes both the whole of Gag, and a popular Env-derived target antigen.
One other Env-related production modality in plants that could be of commercial interest for HIV as well as for other viruses of humans and animals is revealed in a patent filed by Medicago Inc.: this details the use of chimaeric constructs of "ectodomains" from enveloped virus trimeric surface proteinssuch as retroviruses, rhabdoviruses, herpes-, corona-, paramyxo-, pox-and filoviruses -fused to an influenza virus HA protein transmembrane domain and cytoplasmic tail, in order to produce VLPs similar to the HA-only VLPs previously mentioned [126] . Their HIV Env construct was a fusion of various portions of the ConS ΔCFI gp145 -an engineered Env lacking the gp120-gp41 cleavage site, the fusion peptide, an immunodominant region in gp41 and the cytoplasmic tail (CT) domain [127] and the transmembrane (TM) and CT domains of the HA2 portion of either the H3 or the H5 HA molecule. In the words of the patent, "Although native HIV Env protein poorly accumulates in plants, a chimeric HIV Env protein, fused to a transmembrane (TM) and cytoplasmic tail (CT) domains from influenza HA accumulates at high level, and buds into HIV VLPs in absence of core or matrix protein, in plants". This could provide a genuinely novel source of HIV Env antigens of enhanced immunogenicity, especially for use as a boost vaccine for heterologous prime-boost vaccination regimes.

Bluetongue virus vaccine

One of the more exciting success stories in veterinary biofarming in recent years is undoubtedly the proof of efficacy of a plant-made VLP-based vaccine for Bluetongue virus (BTV), a muticomponent dsRNA-containing orbivirus in the family Reoviridae [128] . This project was a part of the EU FP7 Plant Production of Vaccines (PlaProVa) initiative (http://www.plaprova.eu/), whose activities to do with VLP-based vaccines are partly reported on here [129] . Bluetongue disease is a relatively newly emerged problem in sheep and goats in northern Europe [130] , and is almost certainly a result of climate change affecting the distribution of the Culicoides midges that transmit it [131] . Vaccines are seen as an essential part of disease control: however, unlike the case in endemic areas such as South Africa where attenuated live vaccines are used routinely, concerns about vaccine safety and the possible emergence of new strains of virus because of genomic reassortment with live vaccine strains, have resulted in a push for the development of recombinant proteinonly vaccines [132] .
Testing of the plant-made antigens was done in sheep: immunogenicity was assessed by injecting two sheep with 20 μg of VLPs mixed 1:1 (v/v) with Freund's incomplete adjuvant, and boosting at 21 and 42 days. Serum collected 18 days after first boost was positive for BTV antibodies using a commercial ELISA test kit, and serum from day 56 final bleed reacted with all four structural proteins in western blots, with strongest reactivity toward the major immunogenicity determinant (VP2) and the most abundant structural protein (VP7). Efficacy was tested by injecting four groups of five sheep with either 50 μg VLP or 200 μg CLP mixed 1:1 (v/v) with Montanide ISA70 VG adjuvant, or 5 × 10 4 TCID 50 /mL commercial live attenuated BTV-8, and boosting on day 28. Animals were challenged with 1 mL infected sheep blood containing live BTV-8 on day 63, and clinical reactions monitored for 2 weeks. Plant-produced VLPs had an identical protective efficacy profile as assessed by clinical reaction index (CRI) as the live attenuated, BTV-8 vaccine, while plant-produced CLPs were poorly protective. Sera from both the VLP and the live attenuated vaccine group showed high serum neutralization titres after day 28; however, VLPs induced high antibody levels only after booster injection, whereas neutralising antibodies were elicited by the attenuated vaccine as soon as 7 days after vaccination. Plant-produced CLPs offered partial protection against live virus challenge, similar to insect cell-produced CLPs.

Dual-use or "One Health" vaccines

The "One Health Initiative" (http://www.onehealthinitiative. com/) is a "…worldwide strategy for expanding interdisciplinary collaborations and communications in all aspects of health care for humans, animals and the environment", which it hopes to achieve by, inter alia, "Joint efforts in the development and evaluation of new diagnostic methods, medicines and vaccines for the prevention and control of diseases across species". There are a number of obvious viral vaccine targets for this initiative: these are all of the zoonotic viruses that affect domestic and farmed livestock for which there are either unsatisfactory human vaccines or therapeutics, or no human vaccines at all, and high-impact recently emerging viruses with no vaccines for animals or humans. A good example in the former category would be rabies virus; examples for the second would be agents such as Severe acute respiratory syndrome (SARS) and Middle Eastern respiratory syndrome (MERS) coronaviruses (CoVs), West Nile virus (WNV), and Ebola and Marburg filoviruses. Thomas Monath has also recently defined a "one health paradigm" [137] which specifies three frameworks for development and use of vaccines to control zoonoses: Given the obviousness of the targets, it is surprising that very few of them have been explored for their potential as plant-made vaccines. Rabies is one of these, and relatively early on: a chimaera of Alfalfa mosaic virus CP and epitopes derived from glycoprotein G and the nucleoprotein was successfully expressed in plants via two different plant virus-based systems. Parenterallyinjected extracts protected mice from challenge, and human volunteers who had ingested rCP-containing plant material produced rabies virus-neutralising antibodies [138] . A full-length synthetic G protein genewith a plant secretion signal peptide, and ER retention signalwas found to express reasonably well (0.4% TSP) in transgenic tobacco, and purified protein elicited complete protection against virulent intracerebral challenge in intraperitoneallyimmunised mice [139] .
The production of Rift Valley fever virus antigens in plants is much less well studied: there is one PhD thesis that reports expression of a truncated Gn or soluble ectodomain construct and of the nucleoprotein (N) gene in transgenic Arabidopsis thaliana [145] . This study chose these two antigens because Gn is the more effective at eliciting neutralising antibodies of the two envelope glycoproteins and the recombinant Gn ectodomain (tGn) is known to be protective, and the N antigen elicits non-neutralising antibodies but also a strong cellular immune response that is partially protective. The N protein accumulated to high levels while truncated Gn protein did not accumulate to levels detectable by western blot. Feeding groups of mice transgenic plant material three times (0, 2 and 4 weeks) containing tGn or~70 μg of N protein per 4 mice resulted in seroconversion after the second feeding for both antigens, with higher titres (10 3 -10 4 ) for N compared to tGn (10 2 -10 3 ). While these results are very preliminary as far as a vaccine goes, they indicate the feasibility of first, using transgenic or transientlyproduced antigen in plant tissue as an oral vaccine in animals, and second, of producing antigen for complete or partial purification processes that could result in a human vaccine.

Anti-viral therapeutic antibodies

A useful recent review details how plant-based antibody products may "…provide lower upfront cost, shorter time to clinical and market supply, and lower cost of goods (COGs)…[and] improvements in pharmacokinetics, safety and efficacy" [146] . As an object example, an important aspect of rabies disease prevention is therapy, given the many people in developing countries who get bitten by suspected rabid animals annuallyand a major development in this area is the production of potent rabies-neutralising antibodies in plants, given the prevailing situation of mainly equine-produced sera being in short supply and of variable quality. A consortium of researchers that includes South Africans have recently described the engineering and production in transgenic N tabacum plants of both a humanised IgG version of the broadly neutralising murine MAb E559, and the murine version [147] . Purification via agarose protein A/G affinity chromatography yielded 1.8 mg/kg biomass (0.04% TSP) for the chimaeric Mab, and 1.2 mg/kg biomass (0.03% TSP) for the murine Ab. Both antibodies assembled properly, and were equivalent to hybridomaproduced MAbs in neutralising a panel of lyssaviruses that included all the phylogroup I viruses classical RABV, Duvenhage virus, European bat lyssavirus types 1 and 2, and Australian bat lyssavirus, although no neutralisation was seen for the phylogroup II viruses Lagos bat virus and Mokola virus. The efficacy in post-exposure prophylaxis of the humanised Ab was tested in hamsters injected with a lethal dose of CVS-11 strain of virus: the plantproduced antibody was apparently more effective than the commercial human rabies immunoglobulin (HRIG; Rabigam), in that survival for both treatment groups was >50% after 14 days, and zero and 11% for HRIG and plant Ab groups, respectively, after 28 days.

Introduction

Virus vaccines have been a large and exciting part of this field almost from its beginning, for disease agents ranging from Hepatitis B to C to Foot and mouth disease viruses, from Human papillomavirus and Human rotavirus to ovine Bluetongue and Rabbit haemorrhagic disease viruses, to mention just a few. Aspects of this history have been covered recently, and in particular for virus-like particle based vaccines including rotaviruses and Norwalk virus [9] , Human papillomaviruses [10] and Hepatitis B virus [11] , and so these will not be discussed in detail here except where there is new material to be covered. This review will cover the relevant recent history of virus-specific candidate vaccines and virus-specific therapeutic antibodies made in plants, with a view to providing object examples of successful approaches and especially of dual human/animal use or "One Health" examples (http:// www.onehealthinitiative.com/), in order to help inform future work. First expression of HBV surface antigen in plants [4] Human clinical trial of plant-produced HBsAg [16] First production of HBsAg in transgenic banana [17] Use of rTMV-mediated transient expression to produce~300 mg/kg HBsAg [20] Use of rBeYDV-mediated transient expression to produce 800 mg/kg HBc Ag [24] Tabletised lyophilised transgenic lettuce containing HBsAg VLPs is orally immunogenic in mice [19] Hepatitis C virus

Influenza virus vaccines

Human seasonal influenza is currently mainly caused by viruses from two distinct genera of Influenzavirus: these are three Influenza A viruses (H1N1, H1N1pdm09, and H3N2), and two lineages of Influenza B virus (Yamagata and Victoria). Annual attack rates globally are estimated at between 5-10% in adults and 20-30% in children, with about 3-5 million cases of severe illness, and about 250 000-500 000 deaths [39, 40] . The conventional chicken egg-based inactivated whole-virus split vaccine technology has a production capacity as of 2011 of 1.42 billion doses of trivalent vaccine, and a production level of 620 million doses -albeit with a six-month lead-in period every year [41] . This is manifestly obviously not capable of dealing with pandemics, when "…the potential vaccine supply would fall several billion doses short of the amount needed to provide protection to the global population" [42] .
Latest developments from contenders in this challenge include preclinical and human clinical trials of a number of HA-based products. The first was of Medicago's H5N1 A/Indonesia/5/05 HA VLP vaccine candidate, which constituted "…the first ever report of administration of a plant-made VLP vaccine to humans" [47] . In preclinical work reported in this paper, two low doses of Alhydrogel® alum-adjuvanted plant-made VLPs (1.8 μg) in ferrets prevented pathology and reduced viral loads following heterotypic (A/Vietnam/1203/04 H5N1 clade 1 virus) lethal challenge. Interestingly, this protection occurred despite the fact that HAI titres to the A/Vietnam/ 1203/04 challenge virus were detectable in only 75-87.5% of the challenged ferrets: this prompted the comment that the correlates of protection for influenza virus infection are not fully understood, and that the VLPs are probably stimulating innate immune responses not seen for conventional vaccines.
The human trial of the H5 HA VLPs was performed in healthy adults 18-60 years of age who received 2 doses 21 days apart of 5, 10 or 20 mg of alum-adjuvanted H5 VLP vaccine or placebo (alum). Immunogenicity was evaluated using Haemagglutination-Inhibition (HAI), Single Radial Hemolysis (SRH) and MicroNeutralisation (MN) assays: results from all three assays were highly correlated, with clear dose-responses with all measures of immunogenicity. Almost 96% of those in the 2×10 or 2×20 μg dose groups mounted detectable MN responses, indicating promising immunogenicity. In their words, "These data are particularly encouraging in light of the fact that traditional, egg-based split H5N1 vaccines showed only modest immunogenicity in humans at doses as high as 45 mg with or without alum as an adjuvant".
In contrast to the preclinical results, however, testing of the H5 HA vaccine in human volunteers showed no enhancement of immunogenicity by Alhydrogel: 15 and 45 μg doses with Alhydrogel adjuvant, and at 90 μg dose with and without Alhydrogel, were administered in a twodose regimen three weeks apart; the highest responses were in the 90 μg unadjuvanted group [55] .
The H1 HA vaccine antigen was tested with sera derived from human volunteers vaccinated with a conventional AS03 adjuvanted pdmH1N1 vaccine in order to assess its vaccine potential [56] . It was recognized strongly by serum antibodies and antibody-secreting cells from vaccines. Additionally, there was good correlation between results obtained using the plant-made antigen and the conventional vaccine antigen both by ELISPOT and by intracellular cytokine staining assays. The conclusion was that the candidate H1 HA had good vaccine potential, but needed a good adjuvant for use in a clinical trial. This has been done recently [57] , in a first-in-human Phase 1 study with H1 HA given twice at dose levels of 15, 45 and 90 μg with and without Alhydrogel, in healthy adults 18-50 years of age. The highest seroconversion rates, measured by HAI (78%) and virus MN assays (100%), were in the 90 μg nonadjuvanted vaccine group after the second vaccine dose.

Future prospects for plant-produced vaccines

However, while the prospects for animal vaccines in general and possibly human therapeutic vaccines also seem favourable, given an increasing number of proofs of efficacy in this sphere, for prophylactic human vaccines specifically they are not as brightin the short term, at least. The length and rigour of the human prophylactic vaccine developmental and clinical testing path compared to animal and human therapeutic vaccines are a huge obstacle to commercial production of novel biofarmed vaccines, as is the entrenched investment in conventional technology by Big Pharma. It may be that the technology will find a niche on the fringes of conventional vaccinology, where there is a need for small-scale production of vaccines for orphan diseases or for rapid responses to bioterror-related or emerging viral disease outbreaks. One area where biofarmed vaccines could break through soon could be rapid-response vaccines to novel influenza virus outbreaks: the capacity for such a response has already been demonstrated (see above); it remains to put it to the test in a real-life scenario. Once this happens, there is an increased likelihood of the technology being employed for other agents, such as MERS-CoV, RVFV and West Nile and Chikungunya viruses, where "One Health" principles are important.

Conclusions

The widespread application of biofarmingor more properly, plant molecular farmingthat was anticipated in the 1990s has been long in coming, and there are still cynics that doubt that it will ever happen. However, it is almost certainly the case that there is now enough accumulated evidence of proofs of principle in humans and animals, and of efficacy mainly in animals but in humans too, that its great potential for the cheaper and more rapid and more widely scalable manufacture of highvalue biologics and pharmaceuticals can no longer reasonably be denied. In the case of viral vaccines and therapeutics specifically, it has proved possible to routinely make fully-assembled complex VLPs and immunoglobulins that can either elicit potent protective immune responses, or disease-reducing therapeutic effects, as shown in animal models.

Influenza virus vaccines

An illustration of the speed and scalability of transient expression in N benthamiana for emergency response to novel influenza virus outbreaks was shown recently by Medicago Inc., who produced grams of cGMP-grade plant-made H7N9 vaccine, as HA-only VLPs, in response to the outbreak in humans of that very severe influenza virus in China in 2013. The first vaccine lots were available only 19 days after the company accessed the H7 HA gene cDNA sequence, and as little as 3 μg in one dose of the H7 VLP vaccine administered with or without GLA (glucopyranosyl lipid A) adjuvant elicited high antibody titres in mice [54] .

Dual-use or "One Health" vaccines

Two important disease agents that are prime candidates for consideration as One Health vaccines and occur almost exclusively in developing countries, are the tickborne Crimean-Congo haemorrhagic fever virus (CCHFV) and mosquito-borne Rift Valley fever virus (RVFV), both familial bunyaviruses. CCHFV has the wider distribution, including Africa, Asia, southern and eastern Europe and the Middle East [142] , while RVFV is largely restricted to sub-Saharan Africa. However, both viruses are regarded as having emerging potential, with RVFV in particular regarded as having the potential to spread to Europe, Asia, and the Americas [143] . While attenuated live vaccines are available for RVFV, these are regarded as having significant side effects; there are no accepted vaccines for either virus for general use in humans.

Anti-viral therapeutic antibodies

A more comprehensive investigation was reported recently, of both plant production of Mabs and postexposure prophylaxis of Ebola virus infection in rhesus macaques [151] . Three Ebola-specific mouse-human chimaeric MAbs (h-13 F6, c13C6, and c6D8; the latter two both neutralising) were produced in whole N benthamiana plants via agroinfilration of magnICON TMV-derived viral vectors. A mixture of the three MAbscalled MB-003given as a single dose of 16.7 mg/kg per Mab 1 hour post-infection followed by doses on days 4 and 8, protected 3 of 3 macaques from lethal challenge with 1 000 pfu of Ebola virus. The researchers subsequently showed significant protection with MB-003 treatment given 24 or 48 hours post-infection, with four of six monkeys testing surviving, compared to none in two controls. All surviving animals treated with MB-003 experienced insignificant if any viraemia, and negligible clinical symptoms compared to the control animals. A significant finding was that the plant-produced MAbs were three times as potent as the CHO cell-produced equivalentsa clear case of plant production leading to "biobetters". A followup of this work investigated efficacy of treatment with MB-003 after confirmation of infection in rhesus macaques, "according to a diagnostic protocol for U.S. Food and Drug Administration Emergency Use Authorization" [152] . In this experiment 43% of treated animals survived, whereas all controls tested here and previously with the same challenge protocol died from the infection.
In news received during consideration of this article that may vindicate this view, a report quoted as coming from the National Institute of Allergy and Infectious Diseases states that two US healthcare workers who contracted Ebola in Liberia were treated with ZMapp [154] . Despite being given up to nine days post-infection in one case, it appears to have been effective [155] . In later developments, the therapy was also given to another five people, two of whom died. The US government was negotiating at time of writing to produce large amounts of ZMapp as a first-line therapy [156, 157] . As an illustration of the scale-up problem facing the manufacturers, in the macaque trial referred to above [151] , animals were given three doses of ZMapp at 50 mg/kg intravenously at 3-day intervals. For an adult 70 kg human given the same dose, this would mean a total of 10.5 (3 × 3.5) grams of ZMapp: assuming optimal purified yield of each of the three MAbs individually at 100 mg/kg plant biomass, this would mean 105 kg of N benthamiana would be needed to dose just one person optimally. While biofarming may be the most scalable technology for producing MAbs and other therapeutics, this sort of scale requires resources far larger than currently exist.

Conclusions

Thus, it seems a reasonably safe prediction that biofarmed viral vaccines will be approved more widely for at least animal use in the near future, and possibly also for use as therapy for existing infections and emergency response therapeutics and vaccines in humans in the slightly longer termsuch as for rabies, and possibly for Ebola. Hopefully, approval for prophylactic vaccines for humanssuch as for seasonal influenzawill follow in the slightly longer term.
56 section matches

INTRODUCTION

Recombinant proteins expressed in plants have emerged as a novel branch of the biopharmaceutical industry and hold great potential to produce different types of therapeutic proteins at low cost and with reduced risks of contamination with human and animal pathogens (Moustafa et al., 2015; Paul et al., 2015; Sack et al., 2015) . Transient expression of target proteins can be easily achieved by plant viruses or by agroinfiltration (Gleba et al., 2007) , saving the time spent in the generation of transgenic plants, often allowing higher protein yield due to the absence of chromosomal integration and consequently of position effects (Komarova et al., 2010) . Transient expression can also be used as a means for preliminary evaluation of correct expression before starting the generation of transgenic plants, or related platforms, such as plant cell cultures or microalgae (Franconi et al., 2010) .
The structural N protein is the most abundant protein in the SARS-CoV virion. It is a highly basic protein of 422 amino acids (46 kDa) of the helical nucleocapsid, playing an important role in viral pathogenesis, replication, RNA binding, cell cytokinesis and proliferation (Surjit and Lal, 2008) . N protein has been recognized as the preferred target for detection of SARS-CoV infection by reverse transcription-polymerase chain reaction (RT-PCR; Suresh et al., 2008) . In addition, the WHO guidelines for SARS diagnosis, developed during the outbreak in 2003, suggested the use of N-based ELISA for specific IgG detection as confirmatory test of SARS-CoV infection (World Health Organization [WHO] , 2003 SARS: Laboratory diagnostic tests) due to the ability of the host to mount an early antibody response against the N protein (Che et al., 2004) . Furthermore, since the N protein is able to induce a long-term cell-mediated immune response in animal models, it represents a potential vaccine candidate as well (Roper and Rehm, 2009) . To date, the production of recombinant N protein has been achieved in a variety of heterologous expression systems, including plants, (Zheng et al., 2009) , providing proofs of concept for its use in vaccine formulations (Roper and Rehm, 2009 ). However, the immune response in animal models (both natural and nonnatural SARS-CoV hosts) might be not useful to predict the human immune response.
The M protein is the most abundant protein in the SARS-CoV viral envelope. It is functionally involved in the assembly and budding of virions from the cell. M protein forms homo-oligomers and interacts with S, E, and N proteins (Hogue and Machamer, 2008) . It consists of 221 amino acids (25 kDa), with a short glycosylated N-terminal domain, three membrane-spanning domains and a long immunogenic C-terminal cytoplasmic domain. It has been reported that rabbit antiserum raised against recombinant M protein produced in yeast has a potent neutralizing activity in vitro (Pang et al., 2004) . Antibodies to the M protein were detectable in convalescent SARS patients and B-cell epitopes of the M protein have been identified (He et al., 2005) . It has also been shown that M acts as a dominant immunogen for CTL response in humans (Liu et al., 2010) . Moreover, it has been demonstrated that SARS-CoV M-specific memory CD4+ and CD8+ T cells were persistent in the peripheral blood of recovered SARS patients more than 1 year after infection . In a study, where different DNA vaccines were used, M generated the strongest T-cell response in an animal model, and recovered SARS patients had a long-lasting CD4+ and CD8+ memory for the M antigen (Roper and Rehm, 2009) . These data suggest that further research should be directed toward evaluating the potential efficacy of the M antigen for vaccine and diagnostic tools development.
In this study, we demonstrate the feasibility of using plant transient expression systems (Potato Virus X [PVX]-mediated infection and agroinfiltration) to produce two SARS-CoV antigens, the N and M proteins, as useful tools to face SARS-CoV infection.
In particular, we demonstrated that the SARS-CoV N protein produced in Nicotiana benthamiana is recognized by the specific antibodies of convalescent SARS patients. Moreover, the expression of the SARS-CoV M protein was achieved for the first time in plant.