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Abstract

Appropriate diagnosis is the key factor for treatment of viral diseases. Time is the most important factor in rapidly developing and epidemiologically dangerous diseases, such as influenza, Ebola and SARS. Chronic viral diseases such as HIV-1 or HCV are asymptomatic or oligosymptomatic and the therapeutic success mainly depends on early detection of the infective agent. Over the last years, aptamer technology has been used in a wide range of diagnostic and therapeutic applications and, concretely, several strategies are currently being explored using aptamers against virus proteins. From a diagnostics point of view, aptamers are being designed as a bio-recognition element in diagnostic systems to detect viral proteins either in the blood (serum or plasma) or into infected cells. Another potential use of aptamers is for therapeutics of viral infections, interfering in the interaction between the virus and the host using aptamers targeting host-cell matrix receptors, or attacking the virus intracellularly, targeting proteins implicated in the viral replication cycle. In this paper, we review how aptamers working against viral proteins are discovered, with a focus on recent advances that improve the aptamers' properties as a real tool for viral infection detection and treatment.

Aptamers HIV in Diagnostics

At present, the initial clinical testing for HIV in human is made with an antigen/antibody combination immunoassay that detects HIV-1 and HIV-2 antibodies and HIV-1 p24 antigen to screen for established infection with HIV-1 or HIV-2 and for acute HIV-1 infection [22] . Enzyme-linked immunosorbent assay (ELISA) and real time PCR are the methods used and accepted in clinical samples.

Aptamers to HIV as Antiviral Agents

At present, the treatment HIV-1/AIDS is by a combination of several antiretroviral drugs (cART), which can slow the progress of the disease and reduce the risk of death and disease complication, but it is not curative. Moreover, many patients do not tolerate cART because it has severe side effects, and it is too expensive for patients in developing countries. In this regard, aptamers have been considered an alternative or adjuvant to the chemical antiviral agents in cART to overcome these limitations. To date, highly specific, nucleic acid-based aptamers that target various parts of HIV-1 genomes, HIV-1 proteins (including HIV-1 protease (PR), reverse transcriptase (RT), nucleocapsid, gp120, and Gag) and cellular proteins (nucleolin, CD4 or CCR5) have been isolated and shown to effectively suppress viral replication to apply in HIV therapy [5, [25] [26] [27] (Figure 2 ). diamond field-effect transistor (FET) technique [24] . These "apta-biosensors" have high sensibility and specificity but the devices are complex and expensive.
At present, the treatment HIV-1/AIDS is by a combination of several antiretroviral drugs (cART), which can slow the progress of the disease and reduce the risk of death and disease complication, but it is not curative. Moreover, many patients do not tolerate cART because it has severe side effects, and it is too expensive for patients in developing countries. In this regard, aptamers have been considered an alternative or adjuvant to the chemical antiviral agents in cART to overcome these limitations. To date, highly specific, nucleic acid-based aptamers that target various parts of HIV-1 genomes, HIV-1 proteins (including HIV-1 protease (PR), reverse transcriptase (RT), nucleocapsid, gp120, and Gag) and cellular proteins (nucleolin, CD4 or CCR5) have been isolated and shown to effectively suppress viral replication to apply in HIV therapy [5, [25] [26] [27] (Figure 2 ). (1) The HIV viral particle has an inner capside containing ssRNA viral genome and integrase and retro-transcriptase proteins, mainly; (2) The outer envelope has gp120 and gp40 proteins involved in interaction with cellular receptors (CD4, CCR5, NLS) and fusion to cellular membrane; (3) In the cytoplasm the ssRNA viral genome is released and the retro-transcription step is produced; (4) The integrase protein binds to dsDNA viral genome by LTR ends sequences and other cellular proteins forming the pre-integration complex (PIC); (5) The PIC goes into nucleus through the nuclear pore and is integrated in the cellular genome by the integrase protein activity (provirus); (6) The viral RNAs are transcripted from proviral DNA and exported to cytoplasm to translate viral proteins as protease and a big pre-protein that are assembled to new RNA viral genomes and leave the cell with outer envelope from the cellular membrane; (7) After the budding viral particle, the proteases process the big pre-protein and get a mature viral particle.

Aptamers to HPV in Diagnostics

Several proteins from human papillomavirus, particularly E6 and E7, promote tumor growth and malignant transformation and are frequently associated with cervical cancer. Thus, these proteins represent ideal targets for diagnostic and therapeutic strategies. Belyaeva et al. reported two RNA aptamers to E6, named F2 and F4, which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. This aptamers were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor, while none of them inhibited E6-p53 interaction or p53 degradation [121] .

Aptamers to Rift Valley Fever Virus (RVFV)

The nucleocapsid protein (N) of RVFV is an RNA binding protein required for the production of viable virus because of its involvement in several stages of viral replication. This protein protects the viral genome from degradation and prevents the formation of double stranded RNA intermediates during replication and transcription by encapsidating viral genomic and antigenomic RNA [178] . Ellenbecker et al. isolated RNA aptamers that bound N with high affinity and identified GAUU and pyrimidine/guanine motifs in their sequences, which are also present within the coding region of the RVFV genome. Furthermore, the authors developed a truncated RNA aptamer labeled with fluorescein using a fluorescence polarization (FP) system. Titration of N with the 3 -FAM-labeled RNA aptamer gave an apparent Kd of 2.6 µM. Competitive binding experiments were conducted with four different aptamers and the apparent Ki values were all in the~200 nM range. These data demonstrate that these aptamers might be used to construct a sensitive fluorescence based sensor of N binding with potential applications for drug screening and imaging methodologies [179] .

Introduction

Viruses are infectious agents that enter and replicate only inside the living cells of other organisms. Once the virus replicates inside the cell, it may remain dormant for long periods of time or be released immediately and attach to other healthy cells to begin the infection process again. Many diseases are caused by viruses such as the influenza, hepatitis, human immunodeficiency virus (HIV) or emerging viral diseases. While they differ in symptoms such as fever and weakness, some present no symptoms at all. Rapid and secure diagnosis of viral infections is a key factor for treatment of these diseases avoiding new spread.

Aptamers: A Potential Diagnostic and Therapeutic Alternative

The SELEX process begins with the synthesis of an oligonucleotide library consisting of a central region with random sequence flanked by constant 5 and 3 ends that serve as primers ( Figure 1 ). Every member of the library is a linear oligonucleotide with a unique sequence that acquire three-dimensional structure depending on the experimental conditions (pH, ionic strength, temperature, etc.) or the presence of a ligand [9] . These highly structured aptamers are capable of binding to the target with high affinity and specificity. The diversity of an oligonucleotide library depends on the number of random nucleotides that contains each oligonucleotide molecule. Thus, an oligonucleotide library whose molecules contain a random sequence of 40 nucleotides (4 40 ) would be represented by 1.2 × 10 24 different sequences. However, in practical terms, the complexity of a combinatorial library of oligonucleotides is limited to 10 12 -10 18 different individual sequences [10] .

Aptamers to HIV Genome

Long terminal repeats (LTRs) are sequences necessary for proper expression of viral genes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. Srisawat and Engelke selected RNA aptamers that can bind to the LTRs of HIV-1 DNA [28] . They showed that conserved segments present in several of the aptamers could form duplexes via Watson-Crick base-pairing with preferred sequences in one strand of the DNA, assuming the aptamer invaded the duplex and, in consequence, these specific aptamers would inhibit the transcription process. Meanwhile, Sanchez-Luque et al. reported the in vitro selection of specific RNA aptamers against the 5 -untranslated region of HIV-1 genome. Those aptamers inhibited more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop (RNApt16; 5 -CCCCGGCAAGGAGGGG-3 ) that produced an HIV-1 inhibition close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication [29] . The reason to use RNA aptamer is to go into the cell as DNA plasmid and to get intracellular expression of RNA aptamer to block the target.

Aptamers to CCR5

HIV-1 commonly uses C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR-4) as co-receptors along with CD4 to enter target cells. Human CCR5 is an important co-receptor for macrophage-tropic virus expressed by T-cells and macrophages. Differences in CCR5 are associated with resistance or susceptibility to HIV-1. As an essential factor for viral entry, CCR5 has represented an attractive cellular target for the treatment of HIV-1. Thus, Zhou et al. have reported the selection of RNA aptamers against CCR5 using high throughput sequencing (HTS) to analyze the RNA pools from selection rounds 5 to 9. The individual sequences were classified into six major groups (Group 1-6). Group 2, 4 and 5 shared a conserved sequence, which is comprised of 10 nucleotides UUCGUCUG(U/G)G, named G3. The G3 activity was studied by a "prophylactic" HIV-1 experiment determining whether the aptamer would block HIV infectivity of R5 viruses in cell culture. The results showed that the G3 aptamer efficiently neutralized HIV-1 infectivity of R5 strains with IC 50 about 170~350 nM [25] .

Aptamers to Nonstructural Protein 3 (NS3)

Multifunctional enzyme NS3 is an essential protein for virus survival and it is considered a good target for the development of new antiviral-drugs. The protease activity of the protein is found in the N-terminal domain and the helicase activity is present in the C-terminal domain of the enzyme. During the last years, the group of Nishikawa has been working with different sets of RNA aptamers against the full-length or truncated NS3 protein to inhibit its dual-activity. Initially, they isolated NS3-specific aptamers that inhibited the protease activity (10-G1) or the dual activity, protease/helicase, of the enzyme NS3 (G6-16 and G6-19) [101, 102] . Afterward, new RNA aptamers against the helicase domain of NS3 protein, named G9-I, II and III, were able to inhibit protease activity in vitro [103] . Later on, the authors described the interaction between G9-I aptamer with Arg161/Arg130 residues in the truncated NS3 form as a putative target for protease activity inhibition [104] . To stabilize and protect G9 aptamers against exonuclease activity in vivo, Nishikawa et al. conjugated G9-II aptamer to the stem IV region of the Hepatitis delta virus (HDV) ribozyme. In addition, to allow nuclear export of the aptamer, the chimeric molecule HDV-G9-II (HA) was fused to a constitutive transport element (CTE) generating HAC molecule. Finally, the protease-inhibition capability of G9-II aptamer was checked, using HA and HAC expression vector in vivo [105, 106] . In order to inhibit the dual activity of NS3, a poly U tail in the 5 or 3 ends of G9-I aptamer (5 -14U-NEO-III or NEO-III-14U) was added. The two constructions were able to inhibit with high efficiency of the protease and helicase activities of NS3. Moreover, NEO-III-14U decreased the interaction between NS3 protein and the 3 end of the positive or negative sense HCV RNA and inhibited protease activity of NS3 in vivo [107] . Next, a new set of RNA aptamers were selected against the helicase domain using the truncated NS3 protein (NS3h) and the aptamer with greatest capability to deplete helicase activity in vitro was identified and named aptamer #5 [108] . Finally, a dual-functional aptamer named G925-s50 was designed using a truncated version of aptamer #5 plus G9-II aptamer linked by 50 mer poly(U) spacer. The designed molecule G925-s50 showed a significant inhibition of NS3 helicase-protease activity in vivo and is proposed by Nishikawa group as the best candidate for anti-HCV therapy [109] .

Aptamers to Rift Valley Fever Virus (RVFV)

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus (genus Phlebovirus) responsible for widespread outbreaks of severe disease such as hepatitis, encephalitis and hemorrhagic fever in humans [176] . The virus is endemic throughout much of the African continent. However, the emergence of RVFV in the Middle East, northern Egypt and the Comoros Archipelago has highlighted that the geographical range of RVFV may be increasing, and has led to the concern that an incursion into Europe may occur. At present, there is no licensed human vaccine [177] .

Aptamers to Dengue Virus (DENV)

The DENV capsid (C) protein functions as a structural component of the infectious virion but it may also have additional functions in the virus replicative cycle [183] . Balinsky et al. showed that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL) and that this interaction can be disrupted by the addition of a NCL binding aptamer (AS1411), developed as AGRO100 by Aptamera (Louisville, KY, USA). Treatment of cells with AS1411 produced a significant reduction of viral titers after DENV infection. Moreover, the authors showed that treatment with AS1411 affected the migration characteristics of the viral capsid and identified a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics [184] .

Introduction

Viruses cause different types of damage to the body which, if left untreated, can lead to death. There are many antiviral drugs that block the infection process at different stages. Some drugs prevent the virus from interacting with the healthy cell by blocking a receptor that helps internalize the virus into the cell. Other drugs inhibit the proliferation of the virus within the cell. The simultaneously use of several drugs affecting different processes increases the probability of recovery of the patient. Although some viral infections such as hepatitis or HIV remain latent for a long time current treatments control the virus and prevent further damage to the body. Viral infections usually produce an immune response in the host that eliminates the infecting virus. The same protective effect is produced by vaccines, which confer an artificially acquired immunity to the viral infection. However, some viruses including those that cause acquired immune deficiency syndrome (AIDS) and viral hepatitis evade these immune responses and result in chronic infections. Antibiotics have no effect on viruses, but several antiviral drugs have been developed. Because viruses use vital metabolic pathways within host cells to replicate, they are difficult to eliminate without using drugs that cause toxic effects to host cells in general. The most effective medical approaches to viral diseases are vaccinations to provide immunity to infection, and antiviral drugs that selectively interfere with viral replication.
Most of the antiviral drugs are nucleoside analogues which lack the hydroxyl groups. Viruses mistakenly incorporate these analogues into their genomes during replication and, in consequence, the newly synthesized DNA is inactive and the life-cycle of the virus is then halted. Some of the most frequently prescribed antiviral nucleoside analogues based-drugs are aciclovir for Herpes simplex virus infections and lamivudine for HIV and Hepatitis B virus infections [1] . During the last years, the nucleoside analogue drug ribavirin combined with interferon has been used for hepatitis C treatment [2] , although currently there is a more effective treatment that includes simeprevir available for patients with genotype 1 and genotype 4 [3] . The treatment of chronic carriers of the hepatitis B virus by means of a similar strategy using lamivudine has been developed [4] . Today, the first line treatment of choice includes one of three drugs: Peg-IFN, entecavir or tenofovir because of their greater power and because they produce a very low rate of resistance.

Aptamers against Human Immunodeficiency Virus (HIV)

Human immunodeficiency virus is a lentivirus (a subgroup of retrovirus) that causes HIV infection and over time AIDS [21] . HIV infects essential cells in the human immune system such as CD4 + T cells, macrophages, and dendritic cells. When CD4 + T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections. HIV enters cells by endocytosis through the interaction of gp120 viral surface protein (SU) with CD4 host cell. The subsequent interaction of this complex with chemokine coreceptors produces a conformational change in viral protein gp41 that promotes the fusion of virion and target cell membranes leading to the release of HIV particles into the cell. Once inside the cell, viral uncoating generates the viral reverse transcription complex, and the reverse transcription gives the HIV preintegration complex (PIC). The PIC gets into the nucleus and the HIV DNA (provirus) is integrated into the cellular chromosome. Integration can lead to either latent or transcriptionally active forms of infection. The latent form gives the viral latency in cells that can replicate in new infected cells; this provirus can remain hidden during years or replicate and form new viral particles in any moment. The transcriptional active forms are transcript and translated forming new viral particles that dead the host cell and goes to infect new cells. Great efforts are being made to get sensitive, fast and simple diagnostic methods and effective therapies.

Aptamers to Nucleocapsid Protein

Aptamers to Surface Glycoprotein (gp 120) Gp120 is essential for virus entry into cells as it plays a vital role in attachment to specific cell surface receptors mainly on helper T-cells. Several small aptamers containing G-quadruplex selected against gp120 have demonstrated antiviral activity [33] . The first of these molecules was the phosphorothioate 8-mer d(TTGGGGTT), named ISIS 5320, which forms a tetrameric G-quadruplex structure that binds the V3 loop of gp120 inhibiting virus entry [48] . Later on, Koizumi et al. synthesized a set of G-rich oligonucleotides and identified the hexadeosyribonucleotide d(TGGGAG), known as Hotoda's sequence, [49, 50] . Several authors have used the Hotoda's sequence as a lead sequence to make a series of modifications at the 5 and 3 ends of the molecule or mutations in the sequence that allowed to find molecules with high anti-HIV activity [50] [51] [52] [53] [54] [55] [56] .

Aptamers against HBV

Hepatitis B virus (HBV) is a partially double-stranded DNA virus of the Hepadnaviridae family classified into eight genotypes from A to H. The main element of the viral particle of HBV virus and also the most characterized component is the hepatitis B surface antigen (HBsAg) [72] .

Aptamers to 5′ and 3′ Untranslated Regions (5′ and 3′UTR)

Non-translated 5′ and 3′ regions have highly conserved sequences and structured regions closely related with transcription and replication of the HCV virus. Specifically, 5′ end contains the Internal

Aptamers to 5 and 3 Untranslated Regions (5 and 3 UTR)

Non-translated 5 and 3 regions have highly conserved sequences and structured regions closely related with transcription and replication of the HCV virus. Specifically, 5 end contains the Internal Ribosome Entry Site (IRES) domain responsible of transcription initiation by ribosome recognition of HCV viral genome. However, 3 UTR includes a region essential for viral replication named cis-acting replication element (CRE).

Aptamers against Human Papilloma Virus (HPV)

Human papillomavirus (HPV) is a DNA virus from the papillomavirus family. Most HPV infections cause no symptoms and resolve spontaneously, but some of them persist and result in warts or precancerous lesions which increase the risk of cancer of the cervix, vulva, vagina, penis, anus, mouth, or throat [119, 120] .

Aptamers to HPV in Diagnostics

Toscano-Garibay et al. isolated an aptamer (G5α3N.4) that exhibited specificity for E7 with a Kd comparable to aptamers directed to other small targets [122] that may be used for the detection of papillomavirus infection and cervical cancer. The same group characterized an RNA aptamer, named Sc5-c3, that recognized baculovirus-produced HPV-16 L1 virus-like particles (VLPs) with high specificity and affinity (Kd = 0.05 pM). This aptamer produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection [123] . Recently, Graham and Zarbl identified several DNA aptamers that have high affinity and specificity to the non-tumorigenic, revertant of HPV-transformed cervical cancer cells, which can be used to identify new biomarkers that are related to carcinogenesis produced by HPV [124] .

Aptamers to Herpes Simplex Virus (HSV)

Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are two members of the herpesvirus family, Herpesviridae, that infect epithelial tissues before invading the nervous system, where it becomes latent. Unfortunately until now, it has not found any treatment to eradicate the virus [128] . Table 5 shows information on the aptamers described against HPV and HSV. Aptamer technology has been used by Corbin-Lickfett et al. to identify RNA sequences capable of being recognized by HSV-1 ICP27 protein, an important regulator for viral gene expression. After SELEX procedure, GC-rich RNA sequences were isolated, which did not form stable secondary structures [129] . With a therapeutic purpose, Gopinath et al isolated two RNA aptamers (aptamer-1 and aptamer-5) against the ectodomain of the gD protein of HSV-1, which plays an important role in viral entry to the host cells. These aptamers specifically bind to gD protein of HSV-1 with high affinity but not the gD protein of HSV-2. Furthermore, aptamer-1 efficiently blocked the interaction between the gD protein and the HSV-1 target cell receptor (HVEM) in a dose-dependent manner with a EC 50 in the nanomolar range. Anti-HSV-1 activity of aptamer-1 was analyzed by using plaque assays and the results showed that this aptamer efficiently inhibited viral entry. A shorter variant of aptamer-1 named mini-1 aptamer (44-mer) had at least as high an affinity, specificity, and ability to interfere with gD-HVEM interactions [130] . In a similar way, Moore et al. have reported the isolation and characterization of one aptamer, G7a, that binds the gD protein of HSV-2 and neutralizes infection through the Nectin1 and HVEM entry receptors with IC 50 of 20 nM [131] . Interestingly, aptamers that prevent HSV-2 infection may also reduce the morbidity associated with HIV-1 as HSV-2 is a major risk factor for the acquisition of HIV-1.

Aptamers to Influenza

Influenza is considered the most prevalent infectious disease in humans. Three emerging influenza viruses were responsible for major pandemics in the twentieth century: the 1918 Spanish influenza virus, the 1957 Asian influenza virus, and the 1968 Hong Kong influenza virus [132] . Indeed, the 1918 Spanish influenza virus was estimated to have killed 20-50 million people worldwide [133] . More recently, a highly pathogenic avian virus of the H5N1 subtype has produced sporadic infections in humans and, while it is associated with high rates of mortality, its poor transmission in humans prevented a more extensive spread among human populations. However, in 2009, a new influenza A virus of the H1N1 subtype emerged (pH1N1) that possessed high transmissibility but relatively low virulence, rapidly spreading across the entire globe and causing the first pandemic of the 21st century [134, 135] . Subsequently, 2013 witnessed the appearance of a new highly pathogenic avian virus of the H7N9 subtype in China [136] .
Influenza viruses are enveloped RNA virus of the family Orthomyxoviridae. The virion surface carries two membrane glycoprotein components, hemagglutinin (HA) and neuraminidase (NA) and, in the central core, the viral RNA (negative-sense) genome fragmented into eight single-stranded molecules and viral proteins that package and protects this RNA. Each segment contains one or two genes that code for the 15 viral proteins. Highly variable surface proteins, HA and NA, are used to classify influenza subtypes. The combination of hemagglutinin and neuraminidase mainly determines the host organism and the viral infectiousness. Currently, 18 HA and 11 NA types have been identified being the subtypes H1, H2 and H3, and N1 and N2 commonly found in humans.

Aptamers to Influenza Virus in Diagnostics

The detection rapid of influenza virus as well as the categorization of these viruses is particularly important due to the high risk of infection, the rapid propagation and the high frequency of mutation that often induces the arrival of new strains that can cause epidemics and even pandemics. An extensive review about the diagnostic strategies for influenza has been recently published [137] .
The recent advances in the development of rapid, automatic, point of care devices for the diagnosis and subtyping of influenza virus are sustained in two facts: (i) the rapid spread of influenza-associated H1N1 viruses that has caused serious concern in recent years; and (ii) H5N1 subtype of the avian influenza virus (AIV) caused the most lethal outbreaks of highly pathogenic avian influenza (HPAI) in poultry and fatal infections in human cases for over a decade. Thus, aptamers have been generated and found to be specific against these recent pandemic influenza viruses A/H1N1pdm [142] and H5N1 [143] .
Lee et al. developed an integrated microfluidic system that was used to screen a specific aptamer for the influenza A/H1N1 virus in an automated and highly efficient manner [144] . The selected aptamer showed a specific and sensitive detection of the influenza A/H1N1 virus, even in biological samples such as throat swabs. Later, they used a new approach for fluorescence-based detection of the influenza A H1N1 virus using a sandwich-based aptamer assay that is automatically performed on an integrated microfluidic system [145] . The entire detection process was shortened to 30 min using this chip-based system which is much faster than the conventional viral culture method. The limit of detection was significantly improved due to the high affinity and high specificity of the H1N1-specific aptamers. In addition, this two-aptamer microfluidic system had about 10 3 times higher sensitivity than the conventional serological diagnosis. The conformation of the aptamers changes in response to the solvent composition, including ion type and concentration, pH, and temperature. On the basis of this, Wang et al. have developed a microfluidic system that exploited the predictable change in conformation of the aptamer previously used in the group, exposed to different ion concentrations in order to detect multiple types of the influenza virus [146] . Thus, a single fluorescent-labelled aptamer is able to identify three different influenza viruses (influenza A H1N1, H3N2, and influenza B) at the same time, by modifying operating conditions, in 20 min. This chip-based aptamer-binding assay has several important advantages; it is rapid, accurate, and cheaper than multiple-aptamer screening.
Current methods for H5N1 AIV detection are virus isolation and RT-PCR that requires several days and expensive equipment and reagents. Rapid detection assays are also available (such as ELISA or immunochromatographic strips) but are less sensitive and specific. The alternative approach is biosensors technology, several biosensors have been developed to detect AIV among them biosensors using as probe aptamers (aptasensors) (reviewed in [147] . In the Li's lab, a highly specific DNA aptamer that can bind H5N1 virus with high affinity was selected. Using this aptamer, other authors have developed different aptasensors based on Surface Plasmon Resonance (SPR) [148] , a quartz crystal microbalance (QCM) aptasensor crosslinked polymer hydrogel [149] and several aptasensors based on impedance methods [150] [151] [152] . These aptasensors were able to detect H5N1 quickly and/or with more sensitivity than antibody-based biosensors.
The impedance-based aptasensor described Fu et al. has the lowest detection limit, however, it requires signal amplification with labels and a prolonged detection limit [150] . The impedance aptasensor with microfluidics chips has a lower detection limit than the SPR-based aptasensor [148] and the same sensitivity as the QCM aptasensor [149] , but the QCM-based aptasensors are not practical for in-field use due to the QCM's predisposition to environmental noise. The major advantage of the impedance aptasensor with gold nanoparticles for signal amplification described by Karash et al. is that it requires a small sample volume and is cheaper than the detection platforms based on QCM or that use interdigitated electrode microfluidic chips [152] . Recently, Nguyen et al. developed a sandwich-type SPR-based biosensor for the detection of H5Nx viruses using a pair of aptamers selected against a mixture of H5Nx whole viruses using Multi-GO SELEX [153] . The sensitivity of the dual aptamer-based system increased by more than 50-fold than for single-aptamers. In addition, the sensitivity was additionally enhanced when the secondary aptamer was conjugated with gold nanoparticles.

Aptamers to Influenza Virus as Antiviral Agents

Several aptamers against influenza virus have been developed for therapeutics purposes, mainly targeting hemagglutinin (reviewed in [5] ) (Figure 4) . These aptamers are able to inhibit the entry of the virus of the cells by blocking hemagglutinin activity. The common technique to measure the inhibitory activity of the aptamers in vitro is the hemagglutination inhibition assay. The model was more extensively used to test the effect of the aptamers on the viral infection involving the use of cell cultures, mainly Madin-Darby canine kidney (MDCK) cells. The cells are infected with the virus and incubated with the aptamers and the inhibition of viral infectivity is tested. Using these assays, DNA and RNA aptamers selected against HA from Influenza A virus [142, [154] [155] [156] [157] [158] or avian influenza virus [159] [160] [161] , able to significantly decrease the viral infection in cells, have been described. However, only a few studies have described aptamers capable of mediating a reduction in viral pathogenicity in mice models. Jeon et al. evaluated the effect of the administration intranasal of the A22 aptamer, a DNA aptamer selected against the HA-(91-261) peptide, in mice before, at the same time and after virus infection [154] . The aptamer-induced inhibition of viral infection was determined by prevention of weight loss, decrease of viral load in the lungs and restriction of the level of inflammation and cellular infiltration. A22 reduced up to 95% of infection in all the strains tested (H1N1, H2N2 and H3N2). A22 was most effective when administered concomitantly with the viral infection leading to 95% reduction in viral burden. The administration of A22 one day prior to infection (preventive treatment) was less effective, probably because the DNA is partially degraded. Interestingly, the treatment with A22 two days following the infection (therapeutic treatment) still leads to almost 95% reduction in viral titer in the lungs of the mice. In 2014, Musafia et al. used A22 aptamer as a starting point and the quantitative structure-activity relationship (QSAR) tool to produce aptamers with 10-15 times more potent antiviral activity in animal models than A22 aptamer. The binding of these aptamers to the virus (20 times higher than A22) may not necessarily be sequence-specific being the most important properties the aptamer length, 2D-loops and repeating sequences of C nucleotides [157] .
Another antiviral strategy is the inhibition of the enzymes involved in the viral replication, transcription and translation. The polymerase complex of Influenza virus catalyzes the viral replication and transcription. This heterotrimer is composed of three subunits named PA, PB1 and PB2 [164] [165] [166] . PA plays the role of an endonuclease, cleaving host mRNAs downstream of their mRNA cap structures, which are recognized and bound by PB2 [167] . The N-terminal of the PA subunit (PA N ), which holds the endonuclease activity site, is highly conserved among different subtypes of influenza virus, which suggests it is an attractive target in the development of anti-influenza agents. Yuan et al. selected DNA aptamers against both PA protein (three aptamers), and the PA N domain (six aptamers) of an H5N1 virus strain [168] . Four of the six PA N selected aptamers inhibited both endonuclease activity and H5N1 virus infection whereas the three PA-selected aptamers did not inhibit endonuclease activity and virus infection. Finally, one of the four effective aptamers, exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC 50 ) around 10 nM.
Pharmaceuticals 2016, 9, 78 19 of 33 aptamers, exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC50) around 10 nM. The association of the viral polymerase, bound to the cap, and eIF4GI may be involved in the preferential translation of viral mRNAs during influenza infection. In addition, the interaction of NS1, bound to a conserved 5untranslated region (UTR) element of the viral mRNA, with eIF4GI and PABP1 could promote the formation of a "closed loop" between the 5′ and 3′ ends of the viral mRNA; (4) RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/β production. Mitochondrial antiviral-signaling (MAVS) protein. Table 6 shows information on the aptamers described against influenza virus. Vaccination is a powerful approach to diminish the effects of influenza epidemics, but the use of antiviral drugs can also be very useful, particularly in delaying the spread of new pandemic viruses. Neuraminidase inhibitors like oseltamivir, laninamivir, zanamivir, and peramivir are commonly used as antiviral agents to treat influenza infection, especially in Japan. However, because of the rapid increases in drug-resistant influenza virus, it is essential to develop new antiviral drugs as an emerging strategy to block cellular factors important for the infective cycle. The advantage of blocking important cellular pathways for the virus inhibitory effect is that, in principle, it is not specific of influenza strain and the emergence of resistant virus is minimized. A limited number of aptamers targeting host cell factors have been described. Of these, the use of RIG-I as a target for aptamers to control viral infection should be emphasized [169] . RIG-I is a cytosolic receptor for nonself RNA that mediates immune responses against viral infections through IFNα/β production [170] . The use of a specific RIG-I aptamer that activates RIG-I efficiently blocks the replication of the Newcastle disease virus, vesicular stomatitis virus and influenza virus in infected cells, evidencing that aptamers targeting cellular factors can act as efficient antiviral agents [169] . The association of the viral polymerase, bound to the cap, and eIF4GI may be involved in the preferential translation of viral mRNAs during influenza infection. In addition, the interaction of NS1, bound to a conserved 5-untranslated region (UTR) element of the viral mRNA, with eIF4GI and PABP1 could promote the formation of a "closed loop" between the 5 and 3 ends of the viral mRNA; (4) RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/β production. Mitochondrial antiviral-signaling (MAVS) protein. Table 6 shows information on the aptamers described against influenza virus. Vaccination is a powerful approach to diminish the effects of influenza epidemics, but the use of antiviral drugs can also be very useful, particularly in delaying the spread of new pandemic viruses. Neuraminidase inhibitors like oseltamivir, laninamivir, zanamivir, and peramivir are commonly used as antiviral agents to treat influenza infection, especially in Japan. However, because of the rapid increases in drug-resistant influenza virus, it is essential to develop new antiviral drugs as an emerging strategy to block cellular factors important for the infective cycle. The advantage of blocking important cellular pathways for the virus inhibitory effect is that, in principle, it is not specific of influenza strain and the emergence of resistant virus is minimized. A limited number of aptamers targeting host cell factors have been described. Of these, the use of RIG-I as a target for aptamers to control viral infection should be emphasized [169] . RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/β production [170] . The use of a specific RIG-I aptamer that activates RIG-I efficiently blocks the replication of the Newcastle disease virus, vesicular stomatitis virus and influenza virus in infected cells, evidencing that aptamers targeting cellular factors can act as efficient antiviral agents [169] . However, aptamers directed against cellular factors that establish essential interactions with influenza virus proteins had not been reported before. The mRNAs of influenza virus possess a 5 cap structure and a 3 poly (A) tail that makes them structurally indistinguishable from cellular mRNAs. However, selective translation of viral mRNAs occurs in infected cells through a discriminatory mechanism, whereby viral polymerase and NS1 interact with components of the translation initiation complex, such as the eIF4GI and PABP1 proteins [171] [172] [173] . Thus, the inhibition of viral protein-translation factor interactions or their destabilization can be potentially used as an antiviral strategy. Recently, Rodriguez et al. studied whether two aptamers which bind hPABP1 with high affinity (ApPABP7 and ApPABP11) are able to act as anti-influenza drugs [174] . Both aptamers inhibit influenza virus replication of H1N1 or H3N2 subtypes at high and low multiplicity of infection and the viral polymerase-eIF4GI interaction. In addition, aptamer ApPABP11 inhibits the interactions between NS1 and eIF4GI or PABP1. These results indicate that aptamers targeting the host factors that interact with viral proteins may potentially have a broad therapeutic spectrum, reducing the appearance of escape mutants and resistant subtypes.

Aptamers against Other Emerging Viruses

An emergent virus is a virus that has adapted and emerged as a new disease/pathogenic strain, with attributes facilitating pathogenicity in a field not normally associated with that of virus. This includes viruses that are the cause of a disease which has notably increased in incidence; this is often a result of a wide variety of causes from both the influence of man and nature. Most emergent viruses can be categorized as zoonotic (an animal disease that can be transmitted to humans), and this has the advantage of possibly having several natural reservoirs for the disease.
Most of these viruses have newly appeared in a population or have existed but are rapidly increasing in incidence or geographic range and only recently aptamers against emergent viruses such as Rift Valley Fever, Tick-borne encephalitis, Dengue, Ebola viruses or other arboviruses have been developed [175] .

Aptamers to Tick-Borne Encephalitis Virus (TBEV)

Tick-borne encephalitis virus (TBEV) belongs to the family Flaviviridae, genus Flavivirus. This virus produces tick-borne encephalitis (TBE), an important emerging infectious disease that targets the central nervous system (CNS) [180] . There is currently no specific antiviral treatment for TBE because the specific immunoglobulin used in clinical practice has several disadvantages. The purpose of Kondratov et al. was to obtain an aptamer population against a fragment of the surface protein E of the TBEV, since it is available for aptamers outside of the host cell [181] . Authors showed that the treatment with the library of aptamers produced a TBEV neutralization index comparable with the results of neutralization of the commercial human immunoglobulin against tick-borne encephalitis (NPO Microgen, Russia). In addition, the enzyme immunoassay systems based on the immobilization of viral particles on antibodies are most commonly used for the TBEV diagnosis and the authors claim that protein E aptamers could substitute antibodies in these systems.

Aptamers to Dengue Virus (DENV)

Dengue viruses (DENVs) belong to the Flaviviridae family, and contain four serologically and genetically distinct viruses, termed DENV-1, DENV-2, DENV-3 and DENV-4. The envelope (E) protein plays an important role in viral infection but, however, there is no effective antibody for clinical treatment due to antibody dependent enhancement of infection. Chen et al. identified an aptamer (S15) that can bind to DENV-2 envelop protein domain III (ED3) with a high binding affinity. S15 aptamer was found to form a parallel quadruplex structure that together with the sequence on 5 -end were necessary for the binding activity to a highly conserved loop between βA and βB strands of ED3. Although S15 aptamer was selected against DENV-2, the authors demonstrated that this aptamer can neutralize the infections by all four serotypes of DENVs [182] .

Aptamers to Ebola Virus (EV)

Ebola virus belong to the genus Ebolavirus. Four of five known viruses in this genus cause a severe and often fatal hemorrhagic fever in humans and other mammals, known as Ebola virus disease (EVD). Ebola virus has caused the majority of human deaths from EVD, and is the cause of the 2013-2015 Ebola virus epidemic in West Africa.
In many cases, aptamers have been used as a technological and research tool to identify RNA sequences that are recognized by different virus proteins. The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of Moloney murine leukemia virus (MLV), Sindbis virus (SIN) and Ebola virus [187] , by targeting the viral mRNAs for degradation and preventing the accumulation in the cytoplasm. With the aim to identify RNA sequences that could be a target of ZAP, Huang et al. used aptamer technology identifying G-rich RNA aptamers that contained conserved "GGGUGG" and "GAGGG" motifs in the loop region. Interestingly, overexpression of the aptamers significantly reduced ZAP's antiviral activity and the substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity, suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA [188] .

Aptamers to Severe Acute Respiratory Syndrome (SARS)

Severe acute respiratory syndrome (SARS) is a disease caused by SARS coronavirus (SARS-CoV) that caused a pandemic pneumonia in 2002-2003, with a total of 8096 reported cases, including 774 deaths in 27 countries. SARS-CoV belongs to the Coronavirus genus in the Coronaviridae family and is an enveloped, positive-sense RNA virus with a genome of 27.9 kilobases. This RNA encodes two large polyproteins, pp1a and pp1ab, and four structural proteins including spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. Pp1a and pp1ab are proteolytically cleaved into 16 non-structural proteins (nsps) that form the viral replicase-transcriptase complex (reviewed in [189] ).
Only a few studies have been focused on the development of aptamers against SARS-CoV as antivirals, in spite of the fact that there is still no effective therapeutic treatment against the virus. Two studies have selected DNA and RNA aptamers against the non-structural nsp13 protein. This protein possesses NTPase, duplex RNA/DNA-unwinding and RNA-capping activities that are essential for viral replication and proliferation. These aptamers inhibited helicase activity with subnanomolar IC 50 , while the ATPase activity was not affected, suggesting that the aptamers may bind to the nucleic acid binding site of the helicase and block the unwinding activity [192, 193] . Table 7 shows information on the aptamers described against emerging viruses. [190, 191] Severe acute respiratory syndrome n.d. DNA/RNA non-structural nsp13 protein [192, 193] n.d. = not determined.

Perspectives

The success of treatment in viral diseases depends on the early detection of the infective agent. The most probable use of aptamers in virus diagnostics involves the development of more simple, fast and cheap diagnostics devices. One of these simple detection systems can be the Lateral Flow Immunoassays (LFIAs) which are currently used for qualitative monitoring in resource-limited or non-laboratory environments. The LFIA biosensing platform mainly comprises the sample pad and test pad, which is generally composed of nitrocellulose membrane, and provides a platform for both reaction and detection where the capturing molecules are antibodies [197] . Lateral Flow biosensing platform has been developed using an aptamer against hepatitis C virus (HCV) core antigen [83] and could be applied for HIV or emerging virus detection using aptamers against specific proteins. This method allows detection of viruses in endemic or transit of human areas. Another interesting approach to obtaining cheaper diagnostics/genotyping devices is using only one aptamer to detect several targets. From this point of view, the strategy by Wang et al. [146] , in which they use the conformational change of one aptamer exposed to different ion concentrations to detect multiple types of the influenza virus could be used for genotyping of other viruses such as HBV or HCV.
From a therapeutic point of view, aptamers offer a hopeful solution in viral diseases because they can target elements of the virus or the infected host cell easier than the antibodies mainly due to their small size. The potential design of aptamers working against different targets might block the virion penetration into the cells or inhibit enzymes responsible for viral replication or other critical processes. In the case of HIV-1, despite efficient antiretroviral therapy, eradication of latent HIV-1 provirus is challenging and requires novel biological insights and therapeutic strategies. For this aim, novel target proteins should be chosen in HIV reservoir organs for the isolation of aptamers that could be applied to drug delivery or targeting of nanoparticles loaded with drugs to obtain HIV transcriptional activation.
As already mentioned above, RIG-I has been used as a target for aptamers to control viral infection [169] . Recently, Olagnier et al. investigated the inhibitory effect of a RIG-I agonist on the replication of Dengue and Chikungunya viruses [198] . The authors demonstrated that RIG-I stimulation generated a protective antiviral response against both pathogens. It would be motivating to study the use of RIG-I aptamers developed by Hwang for Dengue and Chikungunya therapy. Likewise, the effect of the aptamers developed by Guerra et al. against PABP in decreasing replication of influenza virus [174, 199] could be studied in other viruses that also use the PABP of the infected cell [200] .

Aptamers to HIV Genome

(2) The outer envelope has gp120 and gp40 proteins involved in interaction with cellular receptors (CD4, CCR5, NLS) and fusion to cellular membrane; (3) In the cytoplasm the ssRNA viral genome is released and the retro-transcription step is produced; (4) The integrase protein binds to dsDNA viral genome by LTR ends sequences and other cellular proteins forming the pre-integration complex (PIC); (5) The PIC goes into nucleus through the nuclear pore and is integrated in the cellular genome by the integrase protein activity (provirus); (6) The viral RNAs are transcripted from proviral DNA and exported to cytoplasm to translate viral proteins as protease and a big pre-protein that are assembled to new RNA viral genomes and leave the cell with outer envelope from the cellular membrane; (7) After the budding viral particle, the proteases process the big pre-protein and get a mature viral particle.

Aptamers to Reverse Transcriptase (RT)

Reverse transcriptase has two enzymatic activities, a DNA polymerase activity that can copy either a DNA or an RNA template, and an RNase H that cleaves RNA only if the RNA is part of an RNA/DNA duplex. The two enzymatic functions of RT, polymerase and RNase H, cooperate to convert the RNA into a double-stranded linear DNA [40] . DeStefano and Nair confirmed in vitro effectiveness of DNA aptamer, named 37NT, directed against the reverse transcriptase of HIV HXB2 strain. The aptamer competed with the natural template for the binding site in the enzyme, subsequently producing inhibition of the viral replication [41] . In parallel, Michalowski et al. identified three aptamers (RT5, RT6 and RT47) which contained a bimodular structure comprising a 5 -stem-loop module linked to a 3 -G-quadruplex. In addition, the authors demonstrated that this DNA aptamers inhibited RT from diverse primate lentiviruses with low nM IC 50 values [42] .

Aptamers HBV in Diagnostics

One of the current objectives in the diagnostic of HBV is to develop a daily screening assay with a short period of detection between infection and recognition. Therefore, Suh et al. have developed a fast and low cost detection test based on competitive binding assay combined with fluorescence resonance energy transfer (FRET) [73] . The assay was built with an aptamer selected against the hepatitis B virus surface antigen (HBsAg), the best characterize and most frequently used HBV marker [74] . The described aptasensor was approximately 40-fold more sensitive than the conventional method. In 2015, a new set of three different DNA aptamers was selected against HBsAg and applied to develop a chemiluminescence platform. The new aptasensor was designed with aptamers-conjugated to magnetic nanoparticles reaching a detection limit five-fold better than the current enzyme-linked immunosorbent assay (ELISA) kits used in hospitals [75] .

Aptamers HCV in Diagnostics

Aptamer-based biosensors are a promising diagnostic platform to allow HCV infection detection in early stages or in immunosuppressed patients. Thus, different groups have developed diverse aptasensors to improve diagnostic assay of HCV infection. First, Lee et al. developed a biosensor prototype that specifically recognizes the HCV core protein from sera of an infected patient using selected RNA aptamers against core antigen. The HCV viral particles were retained by the 2 -F aptamers immobilized in a 96-well plate and detected by sequential steps with anti-core and Cy3-labeled secondary antibodies [80] . Later on, Chen et al. developed an early diagnostic assay based on sandwich-ELISA to recognize HCV viral proteins using biotin-labelled DNA aptamers against HCV Envelope glycoprotein E2. The obtained results from infected patients showed a good correlation between viral genome quantification assay, HCV antibody detection and sandwich-ELISA aptasensor [81] . Afterwards, Shi et al. developed a similar platform for early detection, coating the bottom of the well with C7, a DNA aptamer against HCV core labelled with biotin, and HCV-core antibody conjugated with horseradish peroxidase (HRP) is applied over the surface. The platform was applied to the detection of the protein in sera from HCV-infected patients and showed a proportional relationship between amplified RNA copies and HCV core protein concentration [82] . Moreover, Wang et al. designed a rapid, easy-to-use diagnostic platform composed of lateral flow strips treated with thiol-DNA aptamers against HCV core antigen. HCV ELISA assay and core aptamer lateral flow strips showed positive coincidence rates when compared with HCV RNA amplification assay [83] . In an effort to develop a diagnostic test to monitor the infectivity of HCV samples, Park et al. have designed an ELISA-like assay replacing the capture and detection antibodies for DNA aptamers selected against HCV E2. The Enzyme Linked Apto-Sorbent Assay (ELASA) has been described to be used for qualitative and quantitative analysis of virus in infected samples [84] . Further, two laboratories have developed label-free aptasensor to eliminate the labelling step and simplify the HCV detection method. Hwang et al. have described a highly sensitive label-free aptasensor based on nanomechanical microcantilevers. The biosensors were able to measure the surface stress due to the interaction between immobilized RNA aptamers and the HCV helicase [85] . On the other hand, Roh et al. have developed a label-free diagnostic platform to detect and quantify the presence of HCV polymerase NS5B viral protein using conjugated streptavidin-biotin RNA aptamers on an Octet biosensor [86] .

Aptamers to HCV as Antiviral Agents

Eradication of HCV disease is one of the main objectives of global public health. Currently, HCV infected patients are treated combining protease inhibitors, as Telaprevir (TVR) and Boceprevir (BOC), with pegylated-interferon and Ribavirin. However, the new direct-acting antivirals (DAA), TVR and BOC, generate a high rate of side effects, are too expensive and are also susceptible to new resistant viruses [87] . Therefore, it is necessary to develop new DAA treatments that are more effective and with fewer side effects than current therapies. To this end, different advances have been made based on aptamers against HCV and host cell proteins as therapy [88] (Figure 3 ). amplification assay [83] . In an effort to develop a diagnostic test to monitor the infectivity of HCV samples, Park et al. have designed an ELISA-like assay replacing the capture and detection antibodies for DNA aptamers selected against HCV E2. The Enzyme Linked Apto-Sorbent Assay (ELASA) has been described to be used for qualitative and quantitative analysis of virus in infected samples [84] . Further, two laboratories have developed label-free aptasensor to eliminate the labelling step and simplify the HCV detection method. Hwang et al. have described a highly sensitive label-free aptasensor based on nanomechanical microcantilevers. The biosensors were able to measure the surface stress due to the interaction between immobilized RNA aptamers and the HCV helicase [85] .
Eradication of HCV disease is one of the main objectives of global public health. Currently, HCV infected patients are treated combining protease inhibitors, as Telaprevir (TVR) and Boceprevir (BOC), with pegylated-interferon and Ribavirin. However, the new direct-acting antivirals (DAA), TVR and BOC, generate a high rate of side effects, are too expensive and are also susceptible to new resistant viruses [87] . Therefore, it is necessary to develop new DAA treatments that are more effective and with fewer side effects than current therapies. To this end, different advances have been made based on aptamers against HCV and host cell proteins as therapy [88] (Figure 3) .

Aptamers to 5 and 3 Untranslated Regions (5 and 3 UTR)

In 2003, Toulmé et al. decided to use subdomain IIId of IRES element as an antiviral target. They selected RNA aptamer and verified that isolated aptamers inhibit HCV translation in vitro and in cell culture [89] . In the same year, Kikuchi et al. isolated RNA aptamers capable of binding to the domain II of HCV IRES and showed that IRES-mediated in vitro translation was reduced from 20% to 40% by using the 2-02 aptamer [90] . Later, they isolated a new RNA aptamer population against the HCV IRES domains III-IV and corroborated that 3-07 aptamer had a high inhibitory effect on IRES-mediated translation in vitro and in vivo [91] . To improve the inhibitory effect of selected aptamers, they constructed two new molecules, named 0207 and 0702, composed by 2-02 and 3-07 aptamers linked by their ends. The fused aptamers recognized two different subdomains of IRES element and are at least 10 times more efficient than the parental aptamers in the inhibition of mRNA IRES-dependent translation in vitro [92] . Following with IRES as an anti-viral target, Romero-López et al. described an innovative in vitro selection method to isolate aptamers fused to a hammerhead ribozyme with capacity to inhibit RNA translation mediated by IRES. Selected chimeric aptamer-ribozymes were able to recognize the IRES element and cleavage the 5 end at nucleotide position 363 [93] . The success of combining two functional elements in the same molecule was shown in the selected chimeric molecule HH363-50. Thus, the aptamer-ribozyme chimera did anchor to domain IV of the IRES element and inhibited in vitro and in vivo IRES-mediated translation [94] . Therefore, recruitment of ribosomal particles mediated by the IRES element was inhibited by the chimera HH363-24 that prevented both translation and replication in a hepatic cell line [95] . Moreover, to avoid HCV genome replication, Konno et al. isolated RNA aptamers against the 3 end of the negative strand of the virus genome [96, 97] . Interestingly, a RNA aptamer, named AP30, was able to recognize this minus-IRES region and reduce positive-strand genomic RNA synthesis [96] . To inhibit HCV replication, Marton et al. selected RNA aptamers against CRE element that were able to repress replication of HCV replicon in hepatic cells [98] . Subsequently, two selected aptamers, P58 and P78, interact with subdomain 5BSL3.2 of the CRE element and produce a structural reorganization of the 3 end HCV genome and a significant decrease of HCV replication in vivo [99] .

Aptamers to Nonstructural Protein 5A (NS5A)

It has been reported that NS5A protein is essential for HCV production and replication. Recently, Yu et al. have isolated and characterized DNA aptamers against HCV NS5A protein. Particularly, selected aptamer NS5A-5 was able to inhibit HCV virus infection by prevention of protein-protein interactions between NS5A and core protein [110] .
Aptamers to Nonstructural Protein 5B (NS5B) HCV nonstructural protein 5B (NS5B) is a RNA-dependent RNA polymerase protein (RdRp) responsible to the generation of positive-sense genomic HCV RNA and negative-sense RNA template. Reduction of HCV NS5B polymerase activity affects HCV viral life cycle and is one of the main objectives to isolate aptamers against NS5B. Thus, Biroccio et al. identified specific RNA aptamers against a truncated protein NS5B-∆55 without the C-terminal region. One of the selected aptamers, B.2, blocked RNA transcription but not competed with the complex RdRp-RNA, using different binding site than RNA template to the NS5B protein [111] . In the same way, Bellecave et al. selected DNA aptamers against the NS5B viral protein. One of the chosen aptamers, 27v, competed with positive and negative sense HCV viral RNA to bind RdRp polymerase and blocked initiation and elongation steps of RNA transcription [112] . However, 127v aptamer partially competes to dissociate RdRp-RNA complex formation and only inhibited initiation steps of HCV transcription [113] . Moreover, interference of viral production and transcription inhibition of HCV virus was confirmed in vivo using 27v aptamer. Table 4 shows information on the aptamers described against HCV. Five years later, another set of RNA aptamers against NS5B protein were obtained by Lee et al. [114] . To avoid aptamer degradation, oligonucleotides were modified with 2 hydroxyl (R-OH) or fluoropyrimidines (R-F). The R-OH aptamers blocked RNA synthesis of HCV replicon in cell culture without emergence of virus escape mutant or cellular toxicity. On the other hand, R-F oligonucleotides were truncated and conjugated with cholesterol-or galactose-PEG molecules to allow direct and specific liver delivery into cells or tissue. Cholesterol-and Gal-PEG-R-F t2 conjugated aptamer blocked RNA synthesis of HCV genome [115] . The above mentioned aptamers were non-genotype-specific; however, Jones et al. described for the first time aptamers against NS5 protein that exclusively recognized and inhibited RNA-polymerase activity of HCV virus subtype 3a [116] .

Aptamers to Structural Proteins E1, E2 and Core

Envelope E1 and E2 glycoproteins are putative targets in therapy due to its role in HCV viral recognition to enter into hepatic cells. Chen et al. have isolated DNA aptamer against E2 glycoprotein. The selected aptamers have higher affinity to genotype 1a, 1b and 2a than others, and strongly prevented HCV viral infection in Huh7 5.1 cells [81] . Afterwards, Yang et al. described the potential antiviral action of DNA aptamers selected against E1E2 protein by HCV infection suppression in HuH7.5 cells without innate immune response action [117] . In the case of core, an essential protein for HCV viral assembly, Shi et al. have applied for therapy the above-mentioned aptamers in diagnostics. In Huh7.5 cells, the aptamers against HCV core protein repressed viral production as a result of defective assembly of virus particle without stimulation of innate immune response [82] .

Aptamers to Influenza Virus in Diagnostics

The antibodies are the most common probe used to detect either the viral particles or host antibodies developed during the infection. However, although in most cases antibodies are able to distinguish between influenza A and B, only a few antibodies that differentiate subtypes of Influenza A or B have been reported. Alternative probes for subtyping are the aptamers. Thus, aptamers to hemagglutinin (HA) have been successfully and broadly used for the development on sensors for influenza detection. HA is expressed in high amounts in the viral surface and is required for binding and fusion with the host cell. Currently, more than 40 DNA and RNA aptamers to HA have been described since 2004, selected to recombinant hemagglutinins (H1, H3, H5, H9 and Ha from virus B) and to whole viruses (H5N1) (reviewed in [138] ). Misono and Kumar selected an RNA aptamer against to HA of A/Panama/ 2007/1999 (H3N2) using SPR-based SELEX [139] . Gopinath et al. generated two RNA aptamers against intact influenza virus A/Panama/2007/1999 and HA of B/Johannesburg/05/1999. These RNA aptamers are able to discriminate among both A and B influenza viruses [140, 141] .

Aptamers to Influenza Virus as Antiviral Agents

Other aptamers targeting NS1 or the PA polymerase subunit 1 have also been studied. NS1 is a nonstructural protein of small size, between amino acids 230 and 238 and with a molecular weight of 26 kDa. In view of its interaction with both RNAs and viral and cellular proteins, NS1 has been implicated in many of the alterations that occur during influenza virus infection. Moreover, NS1 has anti-interferon (IFN) properties leading to the inhibition of the host's innate immunity [162] . Thus, the importance of NS1 in viral infection makes it an attractive therapeutic target. Woo et al. selected a DNA aptamer specific to NS1 that induced IFN-β production by inhibiting NS1 function. In addition, the selected aptamer was able to inhibit the viral replication without affecting cell viability [163] .

Aptamers to Dengue Virus (DENV)

From a technological point of view, aptamers have been used for efficient isolation of endogenously assembled viral RNA-protein complexes. Hence, Dong et al. developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs) using the streptavidin-binding aptamer S1 sequence that was inserted into the 3 end of dengue virus (DENV) 5 -3 UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. This strategy led to identify several novel host DENV RBPs by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which were further implicated in DENV replication [185] .

Aptamers to Ebola Virus (EV)

Viral protein 35 (VP35) is a multifunctional dsRNA binding protein that plays important roles in viral replication, innate immune evasion and pathogenesis. These multifunctional proteins offer opportunities to develop molecules that target distinct functional regions. With this purpose, Binning et al. used a combination of structural and functional data to determine regions of Ebola virus (EBOV) VP35 (eVP35) to target aptamer selection. Two distinct classes of aptamers were characterized based on their interaction properties to eVP35. These results revealed that the aptamers bind to distinct regions of eVP35 with high affinity (10-50 nM) and specificity. In addition, the authors showed that these aptamers compete with dsRNA for binding to eVP35 and disturb the eVP35-nucleoprotein (NP) interaction. Consistent with the ability to antagonize eVP35-NP interaction, select aptamers can inhibit the function of the EBOV polymerase complex reconstituted by expression of select viral proteins [186] .

Use of Aptamers for Delivery of Therapeutic Molecules

Aptamers can also serve as elements that selectively recognize and bind to defined cell types or tissues. By attaching drug molecules, the aptamers can be used to deliver cargo molecules to or into specific cells or tissues of interest [64] . In order to reach efficient RNAi activity, aptamer-siRNA conjugates must be successfully internalized and released into the cytoplasm where they can meet the RNAi machinery [65] . To improve the Dicer entry and processing of the siRNA, one pair of complementary guanosine and cytosine (GC)-rich "sticky bridge" sequences can be chemically appended to the 3 end of the aptamer and one of the siRNA strands, respectively. Both the aptamer and siRNA portions are chemically synthesized and subsequently annealed via "sticky bridge" [66] .

Perspectives

In the field of viral diseases, the number of drugs for treating these infections is very small and most of the available therapeutics are not very effective [194] . In addition, current diagnostic tools for viral infections are expensive and time consuming. These important diagnostic and therapeutic limitations have favored the development of aptamer-based systems, mainly because these show several interesting advantages in relation to antibodies. Thus, aptamers can recognize and/or inhibit target activity through specific and strong interactions superior to other biologics and small molecule therapeutics, with lower toxicity and immunogenicity profiles. In this sense, during the last years, aptamer technology is being used in a wide range of diagnostic and therapeutic applications associated with viral pathologies [195, 196] . It is significant that aptamers selected as specific anti-viral molecules are effective in infected cells, however, none of the selected antiviral aptamers entered into clinical trials. In conclusion, it is necessary to continue research studies and successfully develop clinical trials to establish the use of aptamers as antivirals.
Nanoparticles have been considered for a wide range of applications, both in soluble and insoluble forms. An advantage of soluble forms of nanoparticles is that they can encapsulate antibiotics/drugs and then release them when they reach the cellular environment, making them highly applicable for drug delivery systems. Studies on aptamer-based enhanced drug delivery have been reported for prostate cancer and lymphoblastic leukemia cells [201] . With this purpose, for HIV therapy, nanoparticles successfully loaded with the antiretroviral (ARV) drugs efficiently inhibited HIV-1 infection [202, 203] . These results showed the benefit of the nanoparticles' application to delivery of antiviral drugs to improve its bioavailability.
In conclusion, the use of aptamers in the development of diagnostic platforms or as therapeutic drugs is a promising alternative for the treatment of viral diseases.
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Abstract

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horseassociated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.

Author Summary

The hepatitis C virus (HCV) is one of the most relevant causes of liver disease and cancer in humans. The lack of a small animal models represents an important hurdle on our way to understanding, treating, and preventing hepatitis C. The investigation of small mammals could identify virus infections similar to hepatitis C in animals that can be kept in laboratories, such as rodents, and can also yield insights into the evolution of those ancestral virus lineages out of which HCV developed. Here, we investigated a worldwide sample of 4,770 rodents, 2,939 bats, 210 horses and 858 cats and dogs for HCV-related viruses. New viruses were discovered in European bank voles (Myodes glareolus) and South African four-striped mice (Rhabdomys pumilio). The disease in bank voles was studied in more detail, suggesting that infection of the liver occurs with similar symptoms to those caused by HCV in humans. These rodents might thus enable the development of new laboratory models of hepatitis C. Moreover, the phylogenetic history of those viruses provides fascinating new ideas regarding the evolution of HCV ancestors.

Full genome characterization

The near full genomes of five representative hepaciviruses from all rodent clades were determined, including two viruses from R. pumilio, two from M. glareolus clade 1 and one from M. glareolus clade 2 (identified by red squares in Figure 3A ). The polyprotein genes were of different sizes including 2,781; 2,887; and 3,007 amino acid residues, respectively, compared to 3,008-3,033 in HCV. All genomes shared the typical hepacivirus polyprotein organization, encoding putative proteins in the sequence C-E1-E2-p7-NS2-NS3-NS4A/4B-NS5A-NS5B ( Figure 3B ). The putative structural C, E1, E2 and p7 proteins were predicted by signal peptidase cleavage site analysis (Supplementary Table S3 ) to be comparable in their sizes to that of known hepacivirus proteins. Placentalia (Eutheria) evolutionary lineages according to [76] . Major mammalian clades are identified at basal nodes of the Placentalia phylogeny: Afrotheria (e.g., elephants), Xenarthra, (e.g., anteaters) and Boreoeutheria, divided into the two superorders Euarchontoglires, (e.g., primates, rodents) and Laurasiatheria (e.g., dogs, bats). Sampled mammalian orders are shown in boldface type. Orders containing novel hepaciviruses identified in this study are shown in red and boldface. Orders with known hepaciviruses (perissodactyla, primates, carnivora) are given in red. Numbers of extant families and species per order adapted from [33] All rodent viruses had considerably fewer predicted glycosylation sites in their structural proteins, in particular their putative E2 proteins, as opposed to HCV. A detailed genome analysis is provided in Figure 3B . The 59-terminus of the core gene of the R. pumilio hepacivirus clade contained a putative adenosine-rich slippery sequence at codons 10-14 (AAAAAAAACAAAAA, Supplementary Figure 3B) . In HCV, a very similar sequence (AAAAAAAAAACAAA), located at nearly the same positions (codons 8-12) of the core gene induces production of a protein termed F in vitro due to ribosomal frameshift event [49] . Depending on the HCV genotype, the size of the F protein ranges from 126 to 162 amino acid residues which vary considerably in sequence composition [50] . The size of a putative F protein in SAR46 would be 65 amino acid residues and no homology to the HCV F proteins was observed.
The genome ends of representatives of all three rodent viruses were determined, including virus RMU10-3382 belonging to M. glareolus clade 1, NLR-AP-70 belonging to M. glareolus clade 2, and virus SAR-46 belonging to the R. pumilio hepacivirus clade. Figure 5 and Supplementary Figure S2A show that the 59genome terminus of RMU10-3382 contained structural elements typical of both pegi-and HCV-like internal ribosomal entry sites (IRESs). Predicted structural similarities with the HCV-like IRES included the first stem-loop element (termed Ia and highlighted in orange in Figure 5 ) and one of two sites involved in miRNA122 binding [51] , while most of the remaining stem-loop elements (termed 3, 4 and 5 and highlighted in blue in Figure 5 ) were more closely related to a pegivirus-like IRES. The 59-end of AP-70 was identical in structure to RMU10-3382 and contained only a few nucleotide exchanges. SAR-46 contained the typical HCV-like IRES structures including the characteristic stem-loop III ( Figure 5 and Supplementary Figure S2B) . The observed structural similarity between the first stem-loop of all rodent viruses described here and the prototype hepaciviruses HCV and GBV-B consisted of a hairpin with a six-nucleotide stem and fourfive nucleotide loop. The equine/canine hepaciviruses contained a similar structural element located as their second predicted IRES domain, instead of the most 59-position this domain occupied in all other hepaciviruses. The RMU10-3382 and NLR-365 translation initiation sites contained a cytosine immediately following the putative start codon at position +4, which is suboptimal in the original Kozak sequence context (ACCATGG) but should not block initiation [52] . The 39-ends of RMU10-3382 and SAR-46 contained three highly ordered stem-loop elements. In RMU10-3382, these RNA elements did not resemble any known 39noncoding sequence RNA structure. In SAR-46, the 39-terminal stem-loop structure, but not the preceding structures, resembled that of the HCV X-tail ( Figure 5 and Supplementary Figure S3) . A similar 39-terminal structure could be predicted for GBV-B, but not for the genetically related pegiviruses ( Figure 5 and Supplementary Figure S4) . The 39-end of NLR-AP-70 could not be determined. Contrary to HCV and GBV-B, no poly-uracil stretch was observed in the rodent hepaciviruses.

Discussion

In the canine/equine clade (also termed non-primate hepaciviruses or NPHV [23] ), it is striking that almost identical viruses have been found in horses and dogs. Additionally, horses but not dogs had antibodies against those viruses [24] . In the present study we augmented the number of studied dogs and horses considerably, and investigated cats in addition as these are related in the order of carnivores and have shared domestic habitats with dogs over a long history. The complete absence of viruses in cats and dogs, and the confirmation of highly similar viruses in other geographic regions, here and in another recent study [69] , suggest an actual equine association of the canine/equine clade. Whether acquisition of viruses might have occurred during the domestication of horses, or whether a more generic viral association with the equine stem lineage may exist, could be clarified by testing nondomestic equids such as wild asses or zebras. However, the overall phylogenetic position and monophyly of equine viruses suggest no role as ancestral hepacivirus hosts for horses. While the rodent hepaciviruses greatly extended the genetic diversity of the genus Hepacivirus, their role in the evolution of HCV precursors, if any, remains to be determined.

Introduction

Hepatitis C virus is one of the leading causes of human morbidity and mortality due to hepatitis, liver cirrhosis, and hepatocellular carcinoma [1, 2, 3] . It has become the main reason for liver transplantation in developed countries and represents an economic burden exceeding 1 billion US$ of direct health costs [4, 5] . New estimates of the burden of disease suggest at least 185 million individuals worldwide to have been seropositive in 2005, with a tendency to increase [6] . Treatment has considerably improved due to the optimization of antiviral regimens and the advent of new antiviral drugs [7, 8, 9] . However, treatment in resource-limited settings is hardly accessible [10] . The most effective instrument to prevent new infections with HCV would be a prophylactic vaccine. Unfortunately, chimpanzees are the only known animal species to adequately reflect human HCV infection [11] . Vaccine development is hampered by the lack of a small animal model accessible at early stages of vaccine development [12, 13] . Mice cannot be infected with HCV [14] , but rats and mice engrafted with human hepatoma cells or transgenic for human CD81 and other co-receptor molecules have been proposed [12, 15, 16, 17] . Mouse-adapted HCV has also been generated [16, 18] . Still, these models are highly demanding from a technical point of view and reflect only parts of the pathogenesis and lifecycle of HCV, precluding their wide application [12, 19] .

Full genome characterization

In a Bayesian phylogeny of the full polyprotein, the rodent hepaciviruses and GBV-B were monophyletic, forming a sister Figure 2 . Serological reactivity of bat and rodent sera with HCV antigens. A. Indirect immunofluorescence assay using bat serum. Typical reactivity of a positive E. helvum serum from Ghana (GH69) diluted 1:50 in sample buffer with HuH7 cells infected with HCV strain JHF1 is shown on the left. Arrows point at specific staining of cytoplasmatic antigen. On the right, lack of reactivity of GH69 with uninfected HuH7 cells is shown. IFA was done as described in the methods section. Cell nuclei were stained with DAPI. Scale bar represents 100 mm. B. HCV western blot reactivity patterns with bat sera. Representative reaction patterns of 11 bat sera with the HCV recomblot assay are shown. Sample 1, human positive control serum. Samples 2 to 12 correspond to the following bat species: 2-7, Eidolon helvum; 8-12, Rousettus aegyptiacus. C. HCV western blot reactivity patterns with rodent sera. Representative reaction patterns of 5 rodent sera with the HCV recomline are shown. Sample 1, human positive control serum. Samples 2 to 6 correspond to the following rodent species: 2, Rattus norvegicus; 3, Apodemus sylvaticus; 4, Myocastor coypus; 5, Rattus norvegicus; 6, Myodes glareolus. Blot antigens are indicated at the left of each row. Below each line in B and C, the result of a tentative evaluation is given following the manufacturer's criteria defined for human sera, as described below Table 1 clade to the canine/equine hepaciviruses and HCV ( Figure 5) . The rodent-associated clade had very long intermediary branches and originated close to the root of all viruses. The full genome tree had a better phylogenetic resolution compared to the partial NS3 phylogeny, but still contained topological uncertainties in some deep nodes leading to rodent-associated taxa (Supplementary Figure S1) .

Rattus norvegicus

Because of previous reports of canine/equine hepaciviruses, all RT-PCR assays used in this study were also applied on specimens from horses, cats and dogs. No HCV-related sequences were found in any of the 858 canine or feline specimens. In seven of 210 horse sera (3.3%), sequences closely related to those equine hepaciviruses described previously from the US and New Zealand [24] were detected (9.5-15.0% exchanges in the 978 nucleotide NS3 gene fragment). Most of those nucleotide differences represented synonymous mutations, resulting in low amino acid distances of 0-1.2%. The novel hepaciviruses from German horses clustered phylogenetically with the previously described equine viruses ( Figure 3A ).

Discussion

Including the novel rodent viruses congeneric with HCV and canine/equine viruses, the minimal distance between the genera Hepacivirus and Pegivirus would be 73.5%. While this is lower than the 85-88% between other pairs of genera, it is consistent with a separation threshold of 72.2% between all members of the genus Flavivirus and Tamana bat virus, for which a separate genus has been proposed [59] . This also corresponds to inter-generic distances within other well-studied families of plus-strand RNA viruses such as the Picornaviridae, whose twelve genera are mostly separated by 70-80% in the RdRp-encoding 3D gene [60] . The HCV 1a H77 (GenBank, NC_004102) . GenBank accession numbers of reference hepaciviruses are indicated to the right of taxon names. Tree topology was inferred using BEAST with a GTR nucleotide substitution model as described in the methods section. Rodent hepaciviruses from this study are shown in red and boldface, equine hepaciviruses from this study are shown in blue and boldface. Red squares indicate those viruses whose near full-length genomes were generated. Statistical support of grouping is shown as posterior probabilities at deep nodes. Scale bar corresponds to genetic distance. To the right, 5,000 tree replicates of the same analysis are rendered using Densitree (initial 5,000 trees discarded as burn-in). Green line color indicates low probability of all trees, line thickness corresponds to concordant topologies across tree replicates. B. Genome organization of the novel rodent hepaciviruses. Genes were annotated as described in the methods section. Black arrows on the top indicate predicted signal peptidase cleavage sites. Red arrows below indicate N-, blue arrows O-glycosylation sites. Putative gene starts and ends are numbered below polyprotein plots. HCV 1a strain H77 is depicted on top as a reference. RMU10-3382 (KC411777) also represents the highly similar virus NLR-365 ( For the Bayesian phylogeny shown to the left, the WAG amino acid substitution model was used in MrBayes as indicated in the methods section. Statistical support of grouping from Bayesian posterior probabilities is indicated at node points. Scale bar corresponds to genetic distance. The Pestivirus BVDV (NC_001461) was chosen as an outgroup and truncated for graphical reasons. Branches leading to the novel hepaciviruses from this study are in orange. GenBank accession numbers of analyzed hepaciviruses correspond to those indicated in Figure 3A . The 59-and 39-genome termini were re-drawn from published foldings for equine hepaciviruses [24] , HCV [63, 77] and GBV-B [78] and de novo for this study for the 59- The RNA-negative specimen RMU10-3187 from the same species was processed identically and is shown below as a control. Positive staining is visible as distinct red granules in the cytoplasm of hepatocytes. Magnification was 1006, the inserts shows details of single hepatocytes in 106 higher magnification. Scale bars are shown to the lower right. C. Histopathology of M. glareolus liver specimens. Liver sections were stained by Hematoxylin and Eosin (H&E) and Epson van Giesson (EvG) stains. In H&E stains, black arrows point to inflammatory lymphocytic infiltrate. In EvG stains, black arrows highlight potential signs of fibrosis. Specimen 3180 shows intermediate portal inflammatory lymphocytic activity with potential low-grade fibrosis in a case with high hepacivirus RNA concentrations (1.5610 8 copies/gram). Specimen 1602 shows low-grade portal inflammatory activity and low-grade fibrosis in a case with high hepacivirus RNA genomic organization of the novel viruses provides additional criteria for tentative classification. Like all hepaciviruses and in contrast to all members of the genus Pegivirus, the novel viruses have a discernible core gene [61] . In contrast to the genus Pestivirus [62] , their genomes contained no putative N pro and E rns genes in any reading frame. Finally, in contrast to the genus Flavivirus [63] , all rodent viruses showed IRES secondary structures in their 59genome termini. Some of the novel rodent IRES structures appeared to contain both elements related to type 3 IRES known from hepaci-and pestiviruses and type 4 IRES known from pegiviruses. Additionally, both M. glareolus rodent hepacivirus polyprotein clades were preceded by predominantly pegivirusrelated IRES structures, while the R. pumilio hepacivirus clade was preceded by a predominantly hepacivirus-related IRES. This may be compatible with ancient recombination events between Flaviviridae genera, a phenomenon known in the family Picornaviridae [64, 65] .
Within the genus, phylogeny suggests early divergence of ancestral rodent viruses from a lineage leading up to HCV and canine/equine hepaciviruses. Weakness of resolution in deep bifurcations of the NS3 gene phylogeny and lack of any highly significant preference for deep topological hypotheses in the NS5B gene phylogeny underline the ancestral origin of these viruses. Within the current dataset we can consider them equidistant from HCV and the canine/equine hepaciviruses, suggesting existence of independent taxonomic entities. HCV is one viral species whose genotypes are separated by more than 30% genomic nucleotide distance [67] , which corresponds to about 22-31% AA distance. Different species within the related sister genus Pegivirus, such as GBV-C and GBV-A, are separated from each other by about 45% AA distance [68] . Within the Genus Flavivirus, well-defined species such as dengue virus 1, West Nile virus, yellow fever virus and tickborne encephalitis virus are separated from each other by 48-60% AA distance. Comparing these values we could putatively assume that both Myodes-associated clades distant from each other by 70% AA sequence, as well as the Rhabdomys-associated clade separated from both of the aforementioned by 66-69% AA sequence, might form three distinct species. The canine/equine hepacivirus clade separated from HCV by 52-53% AA sequence would then also form a separate species. Furthermore, all rodent hepacivirus clades and specifically M. glareolus hepacivirus clade 2 were slightly more related to GBV-B than to HCV. GBV-B causes hepatitis in experimentally infected New World primates but not in humans and chimpanzees [61] . The true host of this virus is unknown, but our findings suggest that GBV-B might originate from rodents. Nevertheless, the genetic distance of GBV-B even to its closest relative, the Myodes hepacivirus clade 2 (63% AA sequence), suggests GBV-B to remain a solitary representative of a separate species of hepaciviruses.
Our serological evidence for hepaciviruses in bats is noteworthy even in absence of direct virus findings. Viruses from all Flaviviridae genera including Pegivirus, Pestivirus and Flavivirus have already been found in bats [48, 70, 71] . We could not exclude that the antibodies in bat sera reacting with HCV antigens were directed against viruses from other Flaviviridae genera, rather than bat hepaciviruses. However, there was no cross-reactivity between the NS3 proteins of the more closely related canine/equine hepaciviruses and HCV [24] . Similarly, the two bank vole hepacivirus clades from our study showed no serologic cross-reactivity. These data can therefore serve as very initial suggestions for the existence of bat hepaciviruses only. It should be noted that the degree of genomic similarity necessary for serologic cross-reactivity should have permitted RNA detection by the broadly reactive PCR assays used in this study. Whether bat hepaciviruses indeed exist will therefore require further evidence. A first step to this direction may be an analysis of an expanded bat sample by using the methods presented here.
Additional to phylogeny and genomic properties, the novel viruses resemble HCV in important traits of the natural history of infection. The detection of non-identical virus sequences in natural groups of animals, in combination with specific antiviral antibodies, proves continuous transmission of virus among animals. Induction of controlled infections in housed animals should thus be feasible. We found clear in-vivo evidence for hepatic tropism by demonstrating histopathological signs of liver inflammation, concentrations (3.4610 8 copies/gram). Specimen 3187 shows no significantly increased inflammatory activity and no signs of fibrosis in a case with no detectable hepacivirus RNA. Due to highest tissue quality, a terminal hepatic venule instead of a portal triad is shown. D. Recognition of rodent hepacivirus clade 1 antigens by M. glareolus serum. VeroFM cells expressing complete NS3 from M. glareolus hepacivirus RMU10-3382 (GenBank, KC411777) were incubated with 1:2000-diluted rabbit-anti-NS3 3382 antiserum (control) or 1:50-diluted rodent serum (picture shows exemplary results for animal NLR 3/C12), followed by goat-anti-rabbit-Cy2 (green) and goat-anti-mouse-Cy3 (red) secondary immunoglobulins. For colocalization analysis of fluorescence signals, the 6th of 12 1-mM Z-stags is shown for every channel. Cross-reactivity with HCV antigens was analyzed by incubation of HuH7 cells, transfected with HCV replicon JFH1, with a rabbit-anti-human-HCV-NS3-49 serum, diluted 1:400 (control) or rodent serum NLR 3/C12 diluted 1:50, followed by goat-anti-rabbit-Cy2 (green) and goat-anti-mouse-Cy3 (red) secondary antibodies. Counterstaining was performed using DAPI. Bar, 25 mm. doi:10.1371/journal.ppat.1003438.g006 excessive viral RNA concentrations in the liver, as well as in-situ hybridizations demonstrating intracellular genome replication in liver cells of bank voles. A somewhat lower degree of hepatic inflammation compared to that in some HCV-infected humans might be due to the shorter life span of bank voles rarely exceeding 1-2 years in the wild, or due to a higher capacity of tissue regeneration [72, 73] . Interestingly, our serological investigations suggested bank voles might be able to clear hepacivirus infections, as antibodies did not co-occur with RNA in most, but not all animals [1] . Bank voles may therefore be more capable of clearing hepacivirus infection than humans. This would be compatible with infection patterns also observed in other Flaviviridae members, exemplified by the flavivirus West Nile virus in rhesus macaques, the pestivirus BVDV1 in cattle and the hepacivirus GBV-B in experimentally infected tamarins [61, 74, 75] . However, it would differ from equine hepaciviruses, in which RNA and antibodies cooccurred [24] . Controlled infection experiments in bank voles might yield relevant scenarios for the study of HCV persistence. Bank voles can be kept in the laboratory with comparatively little effort and have been used for virus infection studies, e.g., with herpesviruses, bornaviruses, hantaviruses, and flaviviruses [53, 54, 55, 56] . Efforts to establish bank vole infection models may benefit from the discovery of two highly divergent clades in this species. Knowledge of three full genomes in total should enable efficient rescue of virus from cDNA. Notably, the Rhabdomys-associated virus clade has a host in even closer relationship (on subfamily level) to Mus musculus commonly kept in laboratories, for which powerful technologies such as gene knock out and in-vivo imaging exist. Also for this virus clade, two different full genomes have been determined. In the present study focusing on viral ecology, however, we have not conducted infection or virus rescue trials in cell cultures or animals. Due to the strict liver tropism those viruses can be expected to be as difficult to cultivate as HCV, and we currently lack any possibilities to generate primary Myodes hepatocytes. The housing of those animals is in preparation, as are attempts to rescue fully sequenced viruses by reverse genetics. Additionally, our finding of presence of hepaciviruses in the Murinae subfamily have triggered more targeted ecological investigations to potentially identify viruses from hosts in even closer relationship to Mus musculus. The availability of rodent surrogate models of HCV infection may obviate one of the most critical obstacles to HCV vaccine development by obviating the need for primate experiments in early stages of experimentation [12, 34] . Figure S1 BEAST polyprotein phylogeny including the novel rodent hepaciviruses. The complete polyprotein sequence of all hepaciviruses was analyzed in BEAST [37] using the FLU amino acid substitution matrix and a strict clock over 10,000,000 trees sampled every 1,000 generations. After exclusion of 2,500 trees as burn-in, all trees are depicted using Densitree [39] . Blue color corresponds to most probable topologies, red to second-best, green to third-best and dark green to remaining topologies. First round RT-PCR reactions were used a touchdown protocol with reverse transcription at 48u for 30 minutes, denaturation at 95u for 3 minutes, followed by PCR 10 cycles of 15 sec at 94uC, 20 sec at 60uC with a decrease of 1uC per cycle, and extension at 72uC for 45 seconds, followed by another 40 cycles at 50uC annealing temperature. Second round reactions used the same cycling protocol without the RT step. RNA quantification was performed in 25 mL reaction volumes using the SSIII One-Step RT-PCR system (Invitrogen) as described above with 300 nmol/L of respective forward and reverse primers and 200 nmol/L of respective probes. Amplification involved 15 min at 55uC; 3 min at 95uC; 45 cycles of 15 sec at 94uC, and 25 sec at 58uC. Fluorescence was measured at the 58uC annealing/extension step. Published assays from which oligonucleotide primers were used in this study included [35, 79, 80, 81, 82] . (DOC) Table S3 Putative cleavage sites for cellular signal peptidases within the N-terminal half of hepacivirus polyproteins. NN: neural networks; HMM: hidden Markov models (the values represent probabilities for putative SP cleavage sites). Only SP cleavage sites predicted by both NN and HMM were considered. All scores were re-calculated upon putting a suggested cleavage site at amino acid position 20 of a query polypeptide. *Y-scores were zero for these sites, however they were supported by uncorrected S-scores (not shown). Hepaciviruses included were SAR46 (KC411807) and SAR3 (KC411806) from Rhabdomys pumilio, RMU10-3382 (KC411777), NLR-365

Introduction

A HCV-related hepacivirus of unknown origin, termed GBV-B, has been used as a surrogate model for HCV infection involving New World monkeys, where it causes hepatitis upon experimental inoculation [20, 21] . The use of a surrogate model based on a related virus indicates a way to study HCV pathogenesis and immunity, even though neither monkeys nor apes are acceptable laboratory models in terms of accessibility and ethics [12, 13, 22] . Non-Primate hepaciviruses related to HCV have also been detected in dogs and horses [23, 24] . While horses cannot be considered as laboratory models, dogs at least have compatible body sizes. However, additional to ethical controversies, infected dogs showed grossly deviating pathology in that they appeared to have higher virus concentrations in respiratory specimens than in the liver [23] . So far there is no evidence of antibodies against the virus in dogs, limiting their utility as a vaccination challenge model [23, 24] . No hepaciviruses have been detected in other animals that could be kept in laboratories with reasonable effort, and under ethically acceptable conditions.
The targeted identification of animal hepaciviruses might help elucidating the obscure origins of HCV and yield more accessible HCV surrogate models. We have recently demonstrated that the systematic investigation of small mammal reservoirs can yield novel viruses that are genetically closely related to human pathogenic viruses, such as the paramyxoviruses mumps and Nipah virus [25] . Biological and ecological considerations direct research interests to animals with properties supportive of virus maintenance. The close social interaction of certain bat species forming large and dense social groups favors virus maintenance [25, 26] . Virus spreading by bats may be facilitated by their migratory lifestyle, but also by human activities such as hunting of bats as bushmeat and human invasion of remote habitats [27, 28, 29] . Several rodent species are also in focus as potential virus reservoirs, as they constitute habitat generalists and follow human civilization, providing opportunities for virus transmission [30, 31] . Even though rodents form smaller social groups than bats, some rodent species have a high population turnover, which should enable efficient maintenance of viruses through the continuous replenishment of susceptible individuals [26, 32] . Among terrestrial mammals, rodents and bats together constitute about two thirds of the 5,487 known mammalian species [33] . Screening of wild mammals with a view on laboratory models should be oriented by criteria such as small body size and the ability to adapt to laboratory conditions, which applies to rodents, but not bats [12, 34] . Here we have investigated 7,709 bats and rodents pertaining to 92 species sampled globally in ten tropical and temperate countries. The investigation was complemented by a comparison of virus diversity in 1,068 horses, cats and dogs.

Hepacivirus detection and quantification

Six nested PCR assays for amplification of hepacivirus RNA and two assays targeting the Flaviviridae sister-genera Flavivirus and Pestivirus were used to ensure broad detection. Highly sensitive HCV-specific assays targeting the X-tail, NS5B and 59-untranslated genomic regions were used in addition (see Supplementary Table S2 for oligonucleotide sequences and reaction conditions). RNA quantification relied on strain-specific real-time RT-PCR assays and photometrically quantified in vitro RNA transcripts generated as described previously [35] .

Full genome sequencing

No isolation attempts were made due to the small available specimen quantities and notorious difficulty of hepacivirus isolation. Instead, those rodent specimens with highest RNA concentrations were selected for full genome sequencing. Genomespanning islets were amplified by PCR using degenerate broadly reactive oligonucleotides (Supplementary Table S2 ). Bridging strain-specific oligonucleotide primers (available upon request) were then designed to perform long range PCR using the Expand High Fidelity kit (Roche) on cDNA templates generated with the SuperScriptIII kit (Invitrogen). Some cDNA templates were enriched using a Phi29-based hexamer-driven amplification using a modified protocol of the Qiagen Whole Transcriptome Amplification kit (Qiagen) as described previously [25] . Amplicons were Sanger sequenced using a primer walking strategy. The 59genome ends were determined using the Roche rapid amplifica-tion of cDNA ends (RACE) kit (Roche) generating contiguous PCR amplicons encompassing the complete 59-untranslated region (59-UTR) and the 59-terminus of the core gene. 454 junior next generation sequencing was used for confirmation of 59-UTR sequences. For determination of the 39-genome end, viral RNA was adenylated using a poly-A-polymerase (Clontech, Paris, France) followed by 39-RACE using the Invitrogen GeneRacer Kit (Invitrogen).

Statistics

Comparison of mean virus concentrations was done using an ANOVA analysis with Scheffé post-hoc tests in the SPSS V20 software package (IBM, Ehningen, Germany). Cross-tables were done using EpiInfo7 (www.cdc.gov/epiinfo).

Serology

Rodent hepacivirus immunofluorescence assay. VeroFM cells were transfected in suspension using FuGENE HD (Promega, Mannheim, Germany) with 0.75 mg plasmid expressing the complete His-tagged NS3 proteins of the rodent hepaciviruses RMU10-3382 (rNS3RMU10-3382) and NLR-AP70 (rNS3AP70) and fixed 24 hours later with acetone/methanol (80%/20%). Myodes glareolus sera were tested at screening dilutions of 1:10 and 1:40. For secondary detection, a goat-anti-mouse Ig (Dianova, 1:2000) and a donkey-anti-goat cyanine 3-labelled Ig (Dianova, 1:200) were applied. Recombinant rNS3RMU10-3382 protein including a cleavable Thioredoxin/His 6 tag was expressed in bacteria and purified under non-denaturing conditions following a standard protocol [47] . The untagged purified protein was used to produce specific rabbit polyclonal antisera at Thermo Scientific Pierce custom antibody service. Rabbit antiserum against rNS3RMU10-3382 (1:2000) was used in parallel to an rNS3RMU10-3382-reactive rodent serum (1:50) for a co-localization study by confocal laser scanning microscopy. Here, secondary detection was performed using a cyanine 2-labelled goat-anti rabbit Ig (Dianova, 1:200) and a cyanin 3-conjugated goat-anti-mouse Ig (Dianova, 1:200).

In-situ hybridization

RNAScope RNA probes targeting a 978 nucleotide NS3 gene fragment of the M. glareolus clade 1 hepacivirus detected in specimen RMU10-3379 were custom designed by Advanced Cell Diagnostics (Hayward, CA, USA). RMU10-3379 was selected due to best tissue quality and high virus concentration. In-situ hybridization was performed as described by the manufacturer.

Accession numbers

All virus sequences reported in this study were submitted to GenBank under accession numbers KC411776-KC411814.

Hepacivirus detection

For the molecular analysis of bats, 2,939 sera from Gabon, Ghana, Papua-New Guinea, Australia, Thailand, Panama and Germany were tested for Hepacivirus RNA using several broadly reactive and highly sensitive RT-PCR assays, as detailed in Supplementary Table 2 . Despite the apparent relatedness of putative bat hepaciviruses with HCV suggested by the serologic analyses, no hepacivirus RNA was detected in any of the specimens, whereas several PCR fragments from the NS3 gene were obtained which upon sequencing were identified as pegiviruses related to GBV-D [48] .
A Bayesian phylogeny of the partial NS3 gene shown in Figure 3A suggested that the M. glareolus hepacivirus clade 1 was monophyletic with HCV and the canine/equine hepaciviruses. M. glareolus hepacivirus clade 2 was most closely related to GBV-B while the R. pumilio-associated clade formed a sister taxon to all other hepaciviruses. An analysis of all replicate trees indicated that the deep phylogenetic nodes were not resolved ( Figure 3A ). The monophyly of HCV and the M. glareolus clade 1 hepaciviruses was maintained in 68.6% of tree replicates (3,430 of 5,000). In another 28.9% of trees (1,446/5,000), the two M. glareolus hepacivirus clades clustered together. Monophyly of all three rodent hepacivirus clades and GBV-B was indicated in only 15 of 5,000 tree replicates (0.3%).

Full genome characterization

The total amino acid diversity of all homologous genes within the polyproteins of the three rodent hepacivirus clades was larger than that of all HCV genotypes (Supplementary Table S4 ). Similar to HCV, the most variable genomic regions in rodent hepaciviruses were located in the Envelope E2 gene differing in up to 84.4% of encoded amino acids between the rodent virus clades; the NS2 gene differing in up to 79.8%; and the NS5A gene differing by up to 84.6%.

Natural history of hepacivirus infection in bank voles

Strain-specific real-time RT-PCR assays were used to determine viral RNA concentrations in tissues of 22 bank voles infected with clade 1 and 2 hepaciviruses. Mean RNA concentrations were highest in liver tissue (1.8610 8 copies/gram; range, 1.5610 6 -4.4610 9 ). These concentrations were significantly higher than those in other organs or serum (ANOVA, F = 7.592, p,0.0001; Figure 6A and Supplementary Figure S5 ). Figure 6B shows M. glareolus clade 1 hepacivirus RNA stained by in-situ hybridization (ISH) in liver tissue. Foci of viral RNA were located in the cytoplasm of M. glareolus hepatocytes, while no staining was observed in RT-PCR-negative M. glareolus liver specimens (Supplementary Figure S6 shows additional ISH details). Spleen, kidney, heart and lung tissues yielded no evidence of virus infection by ISH. Histopathological examination of eight RNApositive and two RNA-negative animals revealed low-grade focal lymphocytic invasion compatible with liver inflammation, such as shown in Figure 6C for two exemplary RNA-positive animals.
Serological investigations in wild rodents were complicated by the fact that the vast majority of animals from virus-positive species were not live-trapped, therefore yielding no blood samples. Only post mortem peritoneal lavage fluids were collected from carcasses, but these were not qualified for serology. However, a subset of 97 live-trapped M. glareolus with appropriate blood samples were

Rattus norvegicus

available. These were analyzed for antibodies against the Myodes hepacivirus clades 1 and 2 in an IFA using cells expressing the NS3 antigens of these viruses. Antibodies against the Myodes hepacivirus clade 1 NS3 antigen were found in eight animals (8.3%) at a median end-point titer of 1:200 (range, 1:100-1:1600). Antibodies against the Myodes hepacivirus clade 2 NS3 antigen were detected in 12 animals (12.4%) at a median end-point titer of 1:600 (range, 1:100-1:12800). The difference in antibody detection rates against clades 1 and 2 was not statistically significant (X2 = 0.5, p = 0.5).
Myodes hepacivirus clade 1 antigen specificity was proven by counterstaining with a high-titered rabbit serum raised against the same recombinant NS3 antigen down to dilutions of .1:20,000.
Myodes hepacivirus clade 2 antigen did not cross-react with this rabbit control serum even at high concentrations of 1:100, compatible with low NS3 amino acid sequence identity between the NS3 proteins of the two Myodes hepacivirus clades (42.4%, Supplementary Table S4 ). Neither hepacivirus clade 1, nor clade 2 antibody-positive sera cross-reacted with HCV by immunofluorescence and by immunoblot, indicating specific immune reactions against the viruses studied (exemplary results in Figure 6D ). This was compatible with low NS3 amino acid sequence identities between both Myodes hepacivirus clades and HCV, ranging from 37.9-42.2% (Supplementary Table S4 ).
Only one of the eight sera positive against M. glareolus clade 1 hepaciviruses also contained antibodies against M. glareolus clade 2 hepaciviruses (titers against clade 1 and clade 2 hepaciviruses were 1:200 and 1:3200, respectively). Additional highly sensitive real-time RT-PCR assays were designed specifically for the M. glareolus clade 1 and 2 hepaciviruses and used to analyze the association of viral RNA and antibody status in the 97 M. glareolus sera. No hepacivirus RNA was detected in any of the IFA-positive sera, neither with the broadly reactive screening assays, nor with the additional real-time RT-PCR assay. Therefore, another 239 RNA eluates still containing sufficient volumes to permit screening for M. glareolus clade 1 and 2 hepaciviruses were re-tested with the strain-specific real time RT-PCR assays. Another 57 specimens positive for clade 1 hepaciviruses (23.9%), but no additional clade 2 hepaciviruses were detected. Sera from these PCR-positive animals were obtained and tested for antibodies. Three of the 57 clade 1 RNA-positive sera contained antibodies against clade 1 hepaciviruses (5.3%).

Discussion

Here we found molecular evidence for viruses related to HCV in rodents. Rodent hepaciviruses were detected in four-striped grass mice from South Africa, as well as in bank voles from Central Europe. The latter have already been successfully bred under laboratory conditions, indicating an approach to establish surrogate models for hepacivirus infection [53, 54, 55, 56] .
All discovered viruses originated from deep nodes close to the bifurcations separating genera within the flavivirus tree. In phylogenies on whole genome and individual gene alignments, the novel viruses clustered in a monophyletic clade with previously known hepaciviruses and GBV-B. The clade is highly diversified with NS5b amino acid sequence distances between taxa ranging up to 66.1%, exceeding that in the well-studied genus Flavivirus (55.8%). Maximal distances within the genera Pegivirus (52.9%) and Pestivirus (42.0%) are even lower, suggesting a particularly high diversity to exist in a tentative genus defined by the novel clade. Whereas this indicates that some or all of the novel rodent viruses together with GBV-B might alternatively form an independent genus, recent descriptions of novel pegi-and pestiviruses in bats and swine suggest the diversity also within these genera to be understudied [48, 57, 58] .
The genetic elements potentially homologous to HCV detected in rodent viruses also included microRNA-122 binding sites in the 59-ncr, an X-tail-like element in the 39-terminus and a putative F gene in an alternative open reading frame (ORF) of the R. pumilioassociated virus. The F protein appears to be unessential for HCV replication, but the evolutionary conservation of its ORF suggests that it may play a critical regulatory role in virus propagation and survival [50] . In this regard, the F protein may be considered a counter-defensive security protein that evolved to overcome mechanisms of host resistance [66] . The absence of paramount features typical of other genera and the presence of hepaciviruslike features suggest a tentative classification of the novel rodent viruses within the genus Hepacivirus, rather than a novel genus.

Ethics statement

All animals were handled according to national and European legislation, namely the EU council directive 86/609/EEC for the protection of animals. For all individual sampling sites, study protocols including trapping, sampling and testing of animals were approved by the responsible animal ethics committees as detailed below. All efforts were made leave animals unharmed or to minimize suffering of animals. Any surgical procedure was performed under sodium pentobarbital/ketamine anesthesia. Trapping of rodents in Germany was conducted in the framework of hantavirus monitoring activities and was coordinated by the Friedrich-Loeffler-Institut, the Federal Research Institute for Animal Health. Rodent

Phylogeny

Bayesian tree topologies were assessed with MrBayes V3.1 [36] using the WAG amino acid substitution matrix and BEAST V1.7.4 [37] using the GTR model for nucleotide sequences and the FLU model for amino acid sequences. For MrBayes, two million MCMC iterations were sampled every 100 steps, resulting in 20,000 trees. For BEAST, 10,000,000 generations run under a strict clock were sampled every 1,000 steps, resulting in 10,000 trees. Burn-in was generally 25% of tree replicates.. A human pegivirus (previously termed GBV-C1; GenBank, U36380) was used as an outgroup. Maximum Likelihood analyses were used to confirm Bayesian tree topologies using the WAG amino acid substitution model and 1,000 bootstrap replicates in PhyML [38] . Trees were visualized in FigTree from the BEAST package and Densitree [39] .
Folding RNA secondary structures in viral 59-and 39-genome ends were inferred manually basing on covariant base pairing and thermodynamic predictions using mfold [40] in an alignment of rodent, primate and canine/equine hepaciviruses generated with MAFFT [41] .

Genome comparison

Putative genes were annotated based on predicted signal peptidase (SP) cleavage sites (where applicable) and sequence homology to HCV, GBV-B and canine/equine hepaciviruses. Alignments were generated using MAFFT [41] . Amino acid percentage identity matrices were calculated using MEGA5 [45] with the pairwise deletion option.

Results

Specimens from 8,777 individual animals from the orders Chiroptera, Rodentia, Carnivora and Perissodactyla were included in this study. The geographical origins of samples are summarized in Figure 1AB . The sample contained sera and liver tissue from 4,770 rodents (Rodentia, 41 species), sera from 2,939 bats (Chiroptera, 51 species), sera from 210 horses (Perissodactyla) and sera from 167 dogs (Carnivora). Due to the reported respiratory tropism of canine hepaciviruses, snout swabs were additionally obtained from 239 dogs and 452 cats. The detailed composition of the sample is listed in Supplementary Table 1.

Full genome characterization

The high degree of sequence homology of the RNA-dependent RNA polymerase (RdRp) genes between all members of the family Flaviviridae enabled a more comprehensive comparison of the novel viruses. In a Bayesian phylogeny of these genes across the flavivirus family, the rodent viruses formed a monophyletic sister-clade to HCV ( Figure 4A ). Topological robustness was assessed by the fixation, in parallel Bayesian phylogenies, of two alternative topological hypotheses, the first involving monophyly of HCV with the canine/ equine viruses and M. glareolus clade 1, and the second assuming a separation of HCV and the canine/equine viruses from all rodent viruses and GBV-B. A Bayes factor test comparing the total model likelihood traces of these analyses indicated borderline-significant preference of the second hypothesis over the first (Log10 Bayes factor = 2.94). Figure 4B provides a comparison of RdRp-based amino acid distances within and between Flaviviridae genera.
35 section matches

Abstract

Viruses exhibit rapid mutational capacity to trick and infect host cells, sometimes assisted through virus-coded peptides that counteract host cellular immune defense. Although a large number of compounds have been identified as inhibiting various viral infections and disease progression, it is urgent to achieve the discovery of more effective agents. Furthermore, proportionally to the great variety of diseases caused by viruses, very few viral vaccines are available, and not all are efficient. Thus, new antiviral substances obtained from natural products have been prospected, including those derived from venomous animals. Venoms are complex mixtures of hundreds of molecules, mostly peptides, that present a large array of biological activities and evolved to putatively target the biochemical machinery of different pathogens or host cellular structures. In addition, non-venomous compounds, such as some body fluids of invertebrate organisms, exhibit antiviral activity. This review provides a panorama of peptides described from animal venoms that present antiviral activity, thereby reinforcing them as important tools for the development of new therapeutic drugs.

Background

More than 200 viruses are known to cause human diseases [1, 2] . Some of them present high public health importance, such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B and C viruses (HBV and HCV, respectively), herpes simplex virus (HSV), human immunodeficiency virus (HIV), rabies virus and Ebola virus. The most recent worldwide estimates presented by the World Health Organization (WHO) reported 1.5 million deaths caused by HIV in 2012, 400 million people living with hepatitis B or C, 80% of liver cancer deaths caused by hepatitis viruses, 500 thousand cases of cervical cancer caused by HPV infection, and over 250 thousand cervical cancer deaths each year [3] .

Mechanisms of viral resistance to drugs

The viral DNA integration in the host cell chromosome represents the major problem to be overcome in a retroviral infection. Until now, there is no available drug capable of completely clearing the virus from the host [18] . Furthermore, silent retroviral infection is hidden at anatomical sites that are difficult to reach by drugs, such as the gut-associated lymphoid tissues, lymph nodes and central nervous system. Infected cells, including macrophages, are quiescent in these tissues and it is not known when they will activate and release new viral progenies. Another challenge for an antiviral candidate is posed by the mutation rate of viral genes, mainly among RNA virus, due to the polymerase synthesis error. This is much more intriguing among retroviruses, as the initial virion genome, maintained in quiescent cells in "sanctuary niche", are distinct, mutated from each round of cell infection. Thus, in each cycle of viral infection, the hijacked cell produces a growing number of recombinant new virions [19] .

Scorpion venoms

Both charybdotoxin (ChTx) and scyllatoxin, isolated from Leiurus quinquestriatus hebraeus venom, present the CS-α/β motif and are capable of blocking K + channels [29] [30] [31] [32] . These toxins have been used effectively as molecular scaffolds for gp120-CD4 interaction assays [28, 33, 34] . Since the amino acid residues Phe 43 and Arg 59 of CD4 were shown to be critical for CD4 binding to gp120, equivalent amino acid residues were added to the new compounds.
Recently, the antiviral activities of Scorpio maurus palmatus and Androctonus australis crude venoms were shown against HCV. They presented IC 50 values of 6.3 ± 1.6 and 88.3 ± 5.8 μg/mL, respectively. S. maurus palmatus venom was considered a good natural source for characterizing new anti-HCV agents targeting the entry step, since it impaired HCV infectivity in cell culture, but not intracellularly, through a virucidal effect. This effect was not inhibited by a metalloprotease inhibitor or heating at 60°C [44] .

Insect venoms

Mastoparan is a tetradecapeptide present in wasp (Vespula lewisii) venom [71] that forms amphipathic helical structures that insert into lipid bilayers of bacteria, erythrocytes, mast cells and others, forming pores [72, 73] . Mastoparan-7, a mastoparan analogue, displayed a wide spectrum of antiviral activity against enveloped viruses of five different families (Rhabdoviridae, Poxviridae, Flaviridae, Paramyxoviridae and Herpesviridae) in in vitro assays ( Table 2 ). Structural studies have indicated pore formation by the insertion of the mastoporan amphiphilic α helix into the viral lipidic envelope, causing its disruption [74] .
AMPs isolated from invertebrate organisms presented augmented antiviral activity in human diseases. Such peptides enclose melittin, cecropin and alloferon molecules [77] (Table 2) . Melittin, isolated from honey bee (Apis mellifera) venom, is an amphipathic peptide composed of 26 amino acid residues, arranged in two α helical segments. Inserted in nanoparticles, melittin exhibited virucidal activity against HIV-1 in the VK2 cell line, an epithelial vaginal cell line, and also inhibited HIV infection in TZM-bl reporter cells (HeLa cell line expressing HIV receptors) [78] [79] [80] . Among other antiretroviral mechanisms, melittin complemented the azidovudin reverse transcription inhibition [81, 82] . Hecate, an analogue of melittin, selectively reduced the protein biosynthesis of virus-specified glycoproteins B, C, D, and H of the HSV type 1 [83] . The mechanism is similar to the one detected among HIV-1 infected lymphoblastic cells, previously treated with melittin, by the intervention in the processing of the gag/pol protein precursor. Therefore, specific intracellular events are targeted by melittin and its derivatives [82, 84] .

Peptides from marine organisms

Sea organisms are also promising sources of antiviral cationic peptides. They present a broad spectrum of antiviral activity, while one single peptide may present activity against different viruses and other pathogens. The promiscuous antifreeze Pa-MAP peptide, which consists of an α-helix composed of 11 amino acid residues, was isolated from the polar fish Pleuronectes americanus ( Table 2 ). The Pa-MAP exerted antimicrobial activity against bacteria, fungi, neoplastic cells, and also interacted with the viral envelope of the HSV types 1 and 2, inhibiting the infection of susceptible cells [77, [90] [91] [92] .

Possible action mechanism of antiviral compounds

Other candidates may act intracellularly by interacting with the virion capsid to prevent its decapsidation; therefore, the viral nucleic acid would not be freed and transcribed. Concerning retroviruses, the antiviral candidates can act by inhibiting (i) the viral reverse transcriptase activity; (ii) the pre-integration complex, thus avoiding the transport of circular viral DNA to the nucleus; (iii) and also by inhibiting the action of the viral integrase, which would not allow the viral DNA to integrate into the cellular chromosome. The proviral DNA, after transcription, is transduced into a polyprotein that requires the viral protease in order to generate small proteins to assemble the viral capsid. In this manner, an antiviral compound could inhibit the viral protease by blocking the retroviral morphogenesis ( Fig. 1) [14] . Some retroviral proteins play a major role in the pathogenesis, by down regulation of CD4 and MHC molecules of the host cell, driving them to the proteasome for degradation. If supposed antiviral candidates target these viral proteins, HIV-1 Nef, Tat and Vpr, their actions can be restrained. All the mentioned mechanisms are directly performed by retroviral molecules [15] , but other mechanisms could also be triggered, such as those involved in the innate immune system, e.g. (i) the induction of toll-like receptor expression, that interacts Fig. 1 Action mechanism of animal venom peptides or derivatives at different retrovirus replication cycle phases. (1) The ChTx and Scyllatoxin-based mimetics, such as CD4M33, inhibit the attachment of the viral glycoprotein (gp120) to the host cell receptor CD4. (1a) The peptides cecropin A, magainin 2, papuamide A, dermaseptin DS4, caerins 1.1 and 1.9 and maculation 1.1 disintegrate the viral envelope. (1b and 1c) The peptides CD4M33, BmKn2, Kn2-7, polyphemusin, tachyplesin, immunokine and p3bv obstruct the interaction of the viral gp 120 to the CXCR4 and CCR5 co-receptors. ( 2) The peptides miramides A-H inhibit the fusion of the viral envelope to the host cell membrane. (3) The peptides melittin, didemnis A, B and C interfere with the reverse transcription process, aborting the synthesis of double-stranded viral DNA. (6) The peptides hecate and TVS-LAO act in the post-translation process, in the cleavage of the GAG/POL protein precursor thus interfering in the assembly of the viral capsid and in the organization of the polymerase complex with viral nucleic acid, or (ii) production of cytokines that stimulate the action of T cytotoxic cells, and NK cells, and even host cell expression of the major histocompatibility complex molecules, in order to present viral peptides to the other cells of the immune system [16] . Furthermore, antiviral compounds may activate innate restriction factors coded by the host cell [17] .

Scorpion venoms

As to scyllatoxin scaffold-based mimetics, a 27-amino acid residue miniprotein named CD4M3 was constructed, which inhibited CD4 binding to gp120 with an IC 50 value of 40 μM [34] . Structural and functional analysis performed with CD4M3 suggested additional mutations that, once incorporated in the new compound (CD4M9), caused an increased affinity for gp120, with IC 50 values of 0.1-1.0 μM, depending on the viral strains. Additionally, CD4M9 inhibited infection of CD4 + cells by different HIV-1 strains [34] . Its β-turn sequence ( 20 AGSF 23 ) is similar to that of TXM1. After that, based on CD4M9 structural analysis, a potent mimetic with bona fide CD4-like properties was synthesized [36] . Denominated CD4M33, it inhibited CD4-gp120 binding in different viral strains with 4.0-7.5 nM IC 50 , with these values being comparable to those obtained with CD4. CDM33 also inhibited HIV-1 cell-cell fusion and infection of cells expressing CD4 and either the CCR5 or CXCR4 co-receptors at similar concentrations to CD4 [36] . Its three dimensional structure was further analyzed in complex with gp120 [37] . Then, another analog was designed, denominated F23, which differs from CD4M33 due to the presence of Phe 23 in replacement by biphenylalanine in position 23 (Bip 23 ). The authors showed that F23 had higher mimicry of CD4 than CD4M33. In addition, F23 presented increased neutralization against isolates of phylogenetically related primate lentiviruses [37] .
Mucroporin is a cationic 17-amino-acid residue AMP isolated from Lychas mucronatus venom. One of its derivatives, named mucroporin-M1, has an enhanced net positive charge, and besides having antibacterial activity, presented antiviral activity against Measles, SARS-CoV and Influenza H5N1 viruses (Table 1) , possibly through a direct interaction with the virus envelope [39] . Additionally, it has been shown to reduce the production of HBV antigens and viral DNA in cell culture microenvironment and also to hinder HBV infection in mouse models [40] . The molecular mechanism implicated reveals the specific activation of mitogen-activated protein kinases (MAPKs) leading to down-regulation of HNF4α expression and consequently less binding to the HBV pre-core/core promoter region [40] . Mucroporin-M1 also presented anti-HIV-1 activity [38] .

Snake venoms

This homology occurs between the 30-40 highly conserved amino acid residues of snake venom neurotoxins long loop and the sequence 164-174 of short segment HIV-1 gp120. As a result, both may compete for the same receptor or binding site and present anti-HIV activity [50] . The sequence homology between HIV gp120 and snake neurotoxins, such as cobratoxin and bungarotoxin, had generated some antiretroviral patents [53] [54] [55] . Linking the gp120 fragment to the HIV peptide fusion inhibitors (fragments of gp41 ectodomains) was shown to improve their anti-HIV efficacy [56] . Besides structural homology, other action mechanisms of snake venoms against HIV are also discussed in the literature, such as catalytic/inhibitory activity through enzymes, binding interference (receptor/enzyme), and induction/interaction at the membrane level [50] .
The L-amino acid oxidases (LAAOs or LAOs, EC1.4.3.2), which constitute one of the most studied main components of snake venoms, are oxidoreductase flavoenzymes with molecular masses around 110 to 150 kDa and are usually non-covalently linked homodimeric glycoproteins [57, 58] . These compounds are widely distributed in other organisms and play an important role in biological activities such as apoptosis induction, cytotoxicity, inhibition or induction of platelet aggregation, hemorrhaging, hemolysis and edema, as well as anti-HIV, antimicrobial and antiparasitic activities [59] . TSV-LAO, characterized from Trimeresurus stejnegeri snake venom, seems to be the first snake venom LAO reported to present antiviral activity ( Table 2 ) [60] .
Other compounds found in snake venoms that exhibit antiviral activity are the phospholipases A 2 (PLA 2 ). Among their biological effects, they seem to interact with the host cells and prevent the intracellular release of virus capsid protein, suggesting that they block viral entry into the cells before virion uncoating [7, 49, 62] . The PLA 2 isolated from Crotalus durissus terrificus venom (PLA 2 -Cdt, HIV human immunodeficiency virus, HSV herpes simplex virus, IAV influenza virus, VSV vesicular stomatitis virus, DENV dengue virus. Adapted from Jenssen et al. [9] and Mulder et al. [77] inhibited both DENV and YFV in Vero E6 cells [48] . This PLA 2 is part of crotoxin, a heterodimeric protein composed of two different subunits non-covalently linked: the basic PLA 2 (~16.4 kDa) and the acidic protein crotapotin (~9.0 kDa) [48] . The mechanism proposed for PLA 2 -Cdt antiviral activity involves the cleavage of the glycerophospholipid virus envelope and protein destabilization on the virion surface, which partially exposes the genomic RNA and culminates with viral inactivation, making it unable to access the cell receptor [63] . PLA 2 -Cdt also showed in vitro activity against HIV (Table 2 ) [62, 64] , as well as the snake venom PLA 2 s NmmCM III from Naja mossambica mossambica, taipoxin from Oxyuranus scutellatus, and nigexine from Naja nigricollis [49] . Additionally, the PLA 2 variants, Lys49 and Asp49, denominated BlK-PLA 2 and BlD-PLA 2 , from Bothrops leucurus venom (Table 2) , reduced dengue viral RNA in cells treated with these compounds, and presented cytotoxic activity against DENV-infected cells in vitro [65] . BlK-PLA 2 and BlD-PLA 2 have 121 and 122 amino acid residues, respectively, including seven disulfide bonds.

Anuran skin peptides

The dermaseptin family of antimicrobial peptides comprise 24-34 amino acids, exhibiting a linear polycationic molecule disposed as an amphiphilic α-helical structure when associated with a lipid cell bilayer. Bergaoui et al. [69] described the dermaseptin S 4 , a chemically synthesized 28-amino-acid drug derived from an amphibian skin antimicrobial peptide, exhibiting anti-herpetic activity (HSV type 2), with reduced cytotoxic effects after biochemical modifications of the original peptide. It also reduced in vitro HIV-1 infection of an established cell line, P4-CCR5, expressing CD4, CCR5, and CXCR4 HIV-1 cell receptors and, primary T lymphocytes, being capable of acting on both R5 and X4 tropic HIV-1 virions. Upon insertion in the viral envelope, the dermaseptin S 4 disrupts the virion [69] . Caerin 1.1, caerin 1.9 and maculatin 1.1, peptides also derived from the skin secretions of the amphibians Litoria caerulea, Litoria chloris and Litoria genimaculata, respectively, completely abolished HIV infection of T cells, after a few minutes of virion exposure to these modified peptides, which disintegrates the viral envelope, preventing viral fusion to the cell membrane. Furthermore, these molecules obstructed viral transfection from dendritic cells to T cells. Caerin peptides are composed of 25 amino acid residues in their structure, including four central amino acid residues not present in maculatin peptides. In lipid bilayer membranes, these peptides are adjusted to two α-helices, interlinked by a flexible hinge region limited by Pro 15 and Pro 19 , which determine the disruption of viral envelope and cell membrane [70] .

Peptides from marine organisms

Some sponge species contain linear or cyclic bioactive peptides composed of atypical amino acid residues, generating unique structures that are rarely found in terrestrial organisms [90, 93] . These compounds, particularly the cyclic depsipeptides mirabamides A-H, isolated from Siliquaria spongia mirabilis and Stelletta clavosa, obstruct the HIV-1 virion entry into TZM-bl cells, thus neutralizing the viral glycoprotein fusion for expressing CD4 and CCR5 HIV cell receptors [94, 95] (Table 2 ). Peptide concentrations between 40 and 140 nM were sufficient to inhibit infection by 50% (IC 50 ). Another cyclodepsipeptide, homophymine A, obtained from Homophymia sp., conferred 50% cell protection at 75 nM concentration against HIV-1 infection in vitro [96] (Table 2) . Discovered in the early 1980s, didemnins A, B and C from the Caribbean tunicate Trididemnum solidum were the first antiviral marine depsipeptides described. Didemnins were effective against vaccinia virus, HSV type 1 and 2, coxsackie virus A-21 and equine rhinovirus, presenting strong activity at low doses [97] . Furthermore, these peptides were active in in vivo assays in a rat model infected with herpes simplex virus, reducing the skin lesions after topical administration [98] . Didemnins inhibit protein, DNA and RNA synthesis in cells [99, 100] . The protein synthesis inhibition mechanism may be related to the binding of didemnins to the elongation factor 1 alpha (EF-1 alpha) [101] . Didemnin B underwent phases I and II of clinical trials in the 1980s, but presented low selectivity and therapeutic index, as well as toxic side effects [102] . Dehydrodidemnin B (Aplidin®, Pharma Mar SA, Spain) is currently under phase III of clinical trials as an anticancer drug against multiple myeloma and T-cell lymphoma [103] .
Several antiviral peptides and depsipeptides have been described in marine sponges from the genus Theonella sp.: koshikamides F and H isolated from T. swinhoei and T. cupola [104] ; papuamides A and B, and theopapuamide A from Theonella sp. and T. swinhoei, respectively [105] [106] [107] . All of them inhibited HIV entry into T cells. Theopapuamide B was isolated from an Indonesian sponge, Siliquariaspongia mirabilis, and was also able to inhibit HIV-1 entry into host cells [108] . Papuamide A presented antiviral activity not only against HIV-1, but also against vesicular stomatitis virus and amphotropic murine leukemia virus. Due to its tyrosine residue and the presence of a hydrophobic tail, the peptide may insert into the viral membrane, causing its rupture [105] .

Conclusions

As a consequence of the scarcity of new families of antiviral drugs, pharmaceutical companies have strengthened their efforts to increase developments of known current drugs, resulting in little or even no improvement to the existing therapies. These new patent protections guarantee the rights to the same stakeholders who are charging high consumer prices due to the lack of competition [114] . At the same time, the growing demand for new drugs and natural therapeutic products is a matter of extreme necessity to face the emergency of multiresistant viral pathogens. More than 45 compounds obtained from vertebrate and invertebrate organisms presented in vitro or in vivo antiviral activity. Although none of those has yet been launched on the market as an antiviral drug, they present chemical structures completely different from the current drugs used in therapy, despite acting on similar targets. Those compounds may lead to new classes of therapeutic drugs after additional chemical and pharmacological studies.
Emerging and reemerging viruses of medical relevance challenge health authorities all around the planet. Some viral vaccines have taken too long to be designed and approved for human and animal utilization, and even in some cases could not be developed. Preventive and curative measures should always be in the hands of health authorities to ensure control of epidemics, such as the recent Ebola virus in Africa or arboviruses, particularly in Brazilrepresented by the dengue, chikungunya and Zika virusesor worldwide pandemics, such as influenza and HIV. Therefore, prospection, screening and all other phases of biological activity, validation, clinical development of animal peptides represent an essential scientific investment for protecting and perpetuating humankind.

Possible action mechanism of antiviral compounds

Some peptides exhibit direct virucidal activity; others disturb attachment of virus particles to the cell membrane surface or interfere with the virus replication. Because of the limited efficiency of commonly used drugs and emerging resistance of viruses, antiviral peptides may have the potential for development as putative therapeutic agents [11] . In addition to their reduced market availability, the collateral effects and toxicity of the synthetic antiviral drugs have triggered an expanded search for natural compounds displaying antiviral activities [12, 13] . Any compound to be utilized as an antiviral should comply with the virus pathways during the cellular infectious cycle. Initially, any RNA or DNA virus, enveloped or not, expresses glycoproteins that are responsible for the interaction with surface molecules, receptors, usually glycosylated proteins, integrated in the host cell membrane. At this step, any potential antiviral candidate must compete for the cell receptor by inhibiting the virus attachment to the cell membrane, thereby aborting the viral infection.

Scorpion venoms

In relation to the activity of scorpion venom compounds against retroviruses, such as HIV/SIV, it has been reported that some DBPs can bind to HIV gp120 glycoprotein due to molecular mimicry of lentiviruses host cell CD4 + receptor. As a result, they abolish the gp120-CD4 interaction, which is essential to initiate the conformational changes in the viral envelope that trigger viral entry into host cells [28] . These CD4 mimetic scorpion toxins contain about 30 amino acid residues, with three or four disulfide bridges, characterized by the cysteine-stabilized α/β motif (CS-α/β), in which a β-turn between the two β-strands in these peptides resembles the CDR 2 loop of CD4.

Snake venoms

Additionally, another LAO, isolated from Bothrops jararaca venom and denominated BjarLAAO-I (Table 2) , reduced the viral load in cells infected with dengue virus type 3 strain exposed to the toxin in comparison to controls [61] . Its cDNA-deduced sequence has 484 amino acid residues and is similar to other snake venom LAOs. These flavoenzymes also produce hydrogen peroxide (H 2 O 2 ) as a free radical, which appears to enhance their antiviral activity [60] .

Insect venoms

HIV virions usually infect the host cells in the genital mucosae, by infecting macrophages, being denominated M-tropic virus; after migrating to the lymph nodes, they infect T lymphocytes, changing into T-tropic virus [75] . Based on the HIV tropism, a phospholipase A 2 from bee venom, bvPLA 2 , blocked the replication of both M and T-tropic HIV virions [65] , while a small peptide derived from bvPLA 2 , the p3bv, exclusively inhibited the replication of T-tropic virus, behaving as a ligand for the HIV-1 co-receptor CXCR4 [49, 76] (Table 2) .

Background

The very few antiviral drugs commercially available can induce severe and considerable adverse effects, especially to those patients receiving lifelong treatment for diseases such as HIV. Furthermore, viruses possess rapid mutational capacity to trick and infect host cells. All these facts together have propelled the prospection for new antiviral drugs, particularly from natural products, as they constitute more than 25% of the new drug prototypes approved in the last decades [4] . Among sources of natural products, animal venoms have revealed a great potential for drug discovery [5] [6] [7] , and despite the harmful action mechanism of animal venoms, most of them have components holding potential medicinal properties to cure diseases.
It is widely reported in the literature that animal venoms are rich sources of antimicrobial substances, and contain a vast array of active biological compounds with distinct chemical structures [8] . Thus, antimicrobial peptides (AMPs)a diversified group of peptides that exert essential function in the innate immune host response, when invaded by pathogenic organisms, such as bacteria, fungi and virusare considered the first line of defense of many organisms, including plants, insects, bacteria and vertebrates [9, 10] .

Scorpion venoms

The scorpion venom AMPs belong to NDBPs; many of them and their analogs exert strong antiviral activity, as shown in Table 1 . Some of these compounds act by direct rupture of the viral envelope, thereby decreasing viral infectivity [8] . AMPs could also prevent or block the virion from entering into the cell by occupying cell receptors utilized by the viral glycoproteins [38] . Other AMPs do not compete with viral glycoproteins to get attached to cell receptors. Instead, they can cross the cell lipoprotein membrane and internalize themselves in the cytoplasm and organelles, yielding alterations in the profile of host cells that can enhance the defense against the virus or may also block the expression of viral genes in the host cell, halting viral dissemination to other cells [9] .
An amphipathic α-helical peptide, Hp1090, was screened from the cDNA library of Heterometrus petersii venomous gland. This 13-amino-acid residue NDBP inhibited the HCV infection (Table 1) , acting as a viricide against HCV particles and preventing the initiation of HCV infection by permeabilizing the viral envelope and decreasing virus infectivity [41] . Also from H. petersii venom gland cDNA library, other α-helical NDBPs were synthesized. Two of them, Hp1036 and Hp1239, exhibited potent virucidal activity against HSV-1 (Table 1 ) [42] . They showed inhibitory effects on multiple steps of the virus replication cycle, caused the destruction of the viral morphology and also entered the infected cells where they reduced viral infectivity.
From the cDNA library of Mesobuthus martensii venom gland, a compound denominated BmKn2with 13 amino acid residueswas cloned and synthesized. Based on its sequence, Kn2-7 was designed by making Ctry2459-H2 HCV 1.08 μg/mL [43] Ctry2459-H3 HCV 0.85 μg/mL [43] the substitutions G3K, A4R and S10R, enhancing its net positive charge and α-helix structure [38] . Both compounds exerted anti-HIV-1 activity through inhibition of chemokine receptors CCR5-and CXCR4-mediated activities and replication of the viruses, of which Kn2-7 was the most potent (Table 1 ) [38] . Another NDBP, screened from Chaerilus tryznai scorpion venom gland, Ctry2459, was able to inhibit initial HCV infection in Huh7.5.1 cells by inactivating infectious viral particles (Table 1 ) [43] . However, due to the low bioavailability of this 13-amino-acid residue peptide, Ctry2459 could not suppress an established infection. Thus, in order to enhance the helicity, amphiphilicity and endosomal escape of peptides, the authors designed histidine-rich peptides based on a Ctry2459 template. Denominated Ctry2459-H2 and Ctry2459-H3, they were more effective against HCV than Ctry2459 (Table 1) , significantly reducing intracellular viral production. Unlike Ctry2459, these analogs reduced the viral RNA by 40 and 70%, respectively; however, Ctry2459 diminished viral infectivity in a manner similar to that of wild-type peptide [43] .

Snake venoms

Snake venoms are composed of a mixture of proteins, peptides (90-95%), free amino acids, nucleotides, lipids, carbohydrates and metallic elements coupled to proteins (5%) [45] . Some studies have reported the antiviral activity of snake venoms and their components against measles virus, Sendai virus, dengue virus (DENV), yellow fever virus (YFV) and HIV [46] [47] [48] [49] [50] . Thus, snake venoms are sources of promising candidates for new antiviral drugs ( Table 2 ). In relation to antiretroviral activity, the benefits of treating a patient with multidrug-resistant HIV with a snake venom preparation in addition to the antiretroviral therapy were demonstrated in clinical practice [51] . The response was a decreased viral load and elevated T CD4 + cell count. The authors suggest that this activity may be related to the presence of some snake venom molecules that are homologous to HIV-1 glycoprotein or proteases [51, 52] .

Insect venoms

Cecropins, isolated mostly from the hemolymph of infected pupae of the silk moth Hyalophora cecropia, but also from other insects, tunicates and Ascaris nematodes, are a family of AMPs, containing 35-37 amino acid residues arranged in two amphiphilic α-helices linked by a Gly-Pro hinge. Synthetic hybrid peptides, namely cecropin A (1-8)-magainin 2 (1-12), exhibited potent antiviral activity by a mechanism mainly based on the compound hydrophobicity and α-helical content, inhibiting the virushost cell fusion [85] (Table 2) .
Alloferon 1 and 2 are peptides constituted of 12-13 amino acid residues, isolated from the hemolymph of the blowfly Calliphora vicina. Alloferons exert immunomodulatory activities to control infection by the human influenza virus in mice model of lethal pulmonary infection [75] , whereas their derivatives also inhibited in vitro HSV replication in Vero cells [86, 87] (Table 2 ). These peptides also displayed a relevant role in the innate immunity, being considered prospective peptides for the pharmaceutical industry [88, 89] .

Peptides from marine organisms

Other peptides from marine sponges that inhibit HIV-1 entry into host cells are: callipeltin A, isolated from sponges of the genus Callipelta, which displayed antiviral activity with a high selectivity index (29) between the virus and host cells (SI ratio 50% cytotoxic dose [CD 50 ]/ED 50 ) [109] ; celebesides A-C from Siliquariaspongia mirabilis [108] ; neamphamide A, from Neamphius huxleyi, a compound with structural similarities to callipeptins and papuamides that exhibited low toxicity to host cells and a selectivity index above 10 [110] ; and microspinosamide, isolated from Sidonops microspinosa [111] .

Scorpion venoms

With the rapid increase in the number of characterized scorpion venom compounds, many new drug candidates have been identified as potential medicines to deal with emerging medical global threats [8, 20] . In scorpions the biologically active peptides are classified as disulfidebridged peptides (DBPs) and non-disulfide-bridged peptides (NDBPs) [26, 27] , with the former being the main components of scorpion venoms, responsible for the neurotoxic symptoms and signs observed during scorpionism. Usually these DBPs target the ion channels of excitable and non-excitable cell membranes. These properties make these molecules interesting prototypes of drugs for the treatment of diverse diseases, particularly those affecting the neural system [8] .

Background

Considering the most common pathologies in humans and other animals, cardiovascular and infectious diseases and cancer are among the leading causes of deaths. The cultural and educational background of affected people largely influences the prevention and treatment of human diseases; nevertheless, the availability of new drugs contributes greatly to mitigating diseases.

Scorpion venoms

The arachnid venoms, utilized as a tool for defense and attack, by killing or immobilizing their prey for feeding or their possible competitors and predators, are composed of a rich molecular diversity and complex mixture, with an intricate protein and peptide expression by mechanisms of gene regulation still under investigation [20, 21] . Scorpion venoms have been exhaustively studied, mainly due to the clinical effects after envenomation in humans, which sometimes lead to death [22] . Paradoxically, biotechnological applications are devised by the increased understanding of the action mechanisms of venom components, and therefore, many research works deal with the generation of new drugs based on the structure and function of molecules found in these venoms [23] [24] [25] .
40 section matches

Abstract

Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines.

Alphavirus Vectors

The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity.

Introduction

Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] .

Modified vaccinia virus Ankara (MVA) Vectors

Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine.

Universal Vaccines

Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here.

Introduction

Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] .

Virus-Vectored Vaccines

Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here.

Adenovirus Vectors

There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines.
Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains.
Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] .
One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] .

Adeno-Associated Virus Vectors

Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] .
AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] .

Alphavirus Vectors

Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material.
SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] .

Baculovirus Vectors

Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors.
Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response.
Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] .
Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] .
While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time.

Newcastle Disease Virus Vectors

Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] .

Parainfluenza Virus 5 Vectors

Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] .

Poxvirus Vectors

Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] .
Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] .

NYVAC Vectors

The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] .
While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] .

Veterinary Pox Vectors

While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use.

Vesicular Stomatitis Virus Vectors

Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines.
VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination.

Universal Vaccines

The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] .

Conclusions

The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches.
Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection.
Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

Introduction

The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] .

Adeno-Associated Virus Vectors

There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] .

Newcastle Disease Virus Vectors

Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection.

Parainfluenza Virus 5 Vectors

Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] .

Vesicular Stomatitis Virus Vectors

Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] .

Universal Vaccines

Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications.
The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] .
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Abstract

Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for their spread constitute potential targets for intervention, but are incompletely defined. The viral glycoprotein (GP) mediates filovirus entry into host cells. Recent studies revealed important insights into the host cell molecules engaged by GP for cellular entry. The binding of GP to cellular lectins was found to concentrate virions onto susceptible cells and might contribute to the early and sustained infection of macrophages and dendritic cells, important viral targets. Tyrosine kinase receptors were shown to promote macropinocytic uptake of filoviruses into a subset of susceptible cells without binding to GP, while interactions between GP and human T cell Ig mucin 1 (TIM-1) might contribute to filovirus infection of mucosal epithelial cells. Moreover, GP engagement of the cholesterol transporter Niemann-Pick C1 was demonstrated to be essential for GP-mediated fusion of the viral envelope with a host cell membrane. Finally, mutagenic and structural analyses defined GP domains which interact with these host cell factors. Here, we will review the recent progress in elucidating the molecular interactions underlying filovirus entry and discuss their implications for our understanding of the viral cell tropism.

The Filovirus Glycoprotein Is a Class I Membrane Fusion Protein

Mutagenic analyses and resolution of the structure of GP 1,2 at the atomic level identified features also present in other viral glycoproteins, termed class I membrane fusion proteins (Figure 1 ), including the human immunodeficiency viruses (HIV) envelope protein and the influenza viruses hemagglutinin [37, 42, 43] . Thus, class I membrane fusion proteins form metastable trimers which are oriented perpendicular to the viral membrane. Each monomer consist of an N-terminal surface subunit, which contains a receptor-binding domain, and a C-terminal transmembrane unit inserted in the viral membrane, which harbors domains integral to the membrane fusion reaction, a hydrophobic fusion peptide or fusion loop and two heptad repeats (HR). The surface and transmembrane units are separated by a proteolytic cleavage site, and processing of this site by a host cell protease primes the proteins for membrane fusion. The membrane fusion involves a major conformation rearrangement of the transmembrane unit, resulting in the formation of a characteristic, highly stable trimer-of-hairpins with a central α-helical coiled-coil [44] .

Figure 2.

Host cell surface proteins involved in filovirus uptake. Cellular lectins bind glycans present on the filoviral GP 1,2 and thereby promote attachment to target cells. The surface molecule TIM-1 interacts with GP 1,2 inserted in the viral membrane. TAM proteins do not bind to GP 1,2 but elicit signals which promote viral uptake by macropinocytosis. Integrins might interact with GP 1,2 and are important for intracellular processing of GP 1,2 .

Cathepsins B and L

Cathepsins comprise serine, aspartic and cysteine proteases and carry out diverse biological functions, including antigen processing for MHCII presentation [168] [169] [170] . Cysteine proteases of the papain family, some of which are localized in endosomes, are expressed as preproenzymes and are activated by proteolysis in the endoplasmatic reticulum and the late endosome/lysosome [168] [169] [170] . The low pH environment present in the latter compartment is essential for cathepsin enzymatic activity.

Introduction

Filovirus infection causes severe hemorrhagic fever in humans and non-human primates. Outbreaks of filovirus hemorrhagic fever occur in equatorial Africa and are associated with high case-fatality rates [1] . At present, neither vaccines nor antiviral drugs have been approved for combating filovirus infection. The filoviridae family comprises two genera, Ebolavirus (the ebolaviruses) and Marburgvirus (the marburgviruses). The genus Marburgvirus includes a single species, Marburg marburgvirus, which has two members, Marburg virus (MARV) and Ravn virus (RAVV). The genus Ebolavirus includes five species, each of which has a single member: Zaire ebolavirus (Ebola virus, EBOV), Sudan ebolavirus (Sudan virus, SUDV), Taï Forest ebolavirus (Taï Forest virus, TAFV), Bundibugyo ebolavirus (Bundibugyo virus, BDBV) and Reston ebolavirus (Reston virus, RESTV) [2, 3] . Filoviruses exhibit different virulence in humans: EBOV and MARV infection is associated with case-fatality rates of up to 90% [4, 5] while RESTV seems to be apathogenic [6] [7] [8] . Nevertheless, infection of non-human primates with RESTV can induce hemorrhagic fever [9] . Evidence is emerging that African [10] [11] [12] [13] , Asian [14] and possibly also European [15] bats are natural reservoirs of filoviruses and these animals could transmit the virus directly to humans or via intermediate hosts, including gorillas [16, 17] and swine [6, 14, 18] . Thus, filoviruses pose a threat to human and animal health in different continents, but virulence factors and pathogenesis are incompletely understood.

Attachment factors

By introduction of a cDNA library derived from permissive VeroE6 cells into non-susceptible Jurkat lymphocytes, Axl was identified as an EBOV entry factor expressed at the cell surface [117] . The ectopic expression of Axl and the TAM family members DTK and MER on lymphoid cells allowed for transduction of GP 1,2 -harboring pseudotypes, which could be blocked by Axl-specific antibodies, confirming that TAM-proteins could promote filovirus entry [117] , although the mechanism underlying the antibody-mediated blockade of viral entry is currently unclear. Mutational analysis revealed that both the extracellular ligand binding domain and the cytoplasmic tail of Axl were required for efficient GP 1,2 -mediated entry [116, 117] . A role of TAM family proteins in filovirus entry was confirmed by an independent study, which showed that expression of an mRNA encoding for Axl correlates with susceptibility of cell lines to EBOV infection [114] . The use of TAM protein-specific antibodies and siRNA knockdown indeed identified several cell lines, in which EBOV-GP 1,2 -faciliated entry was dependent on Axl expression. In contrast, down-regulation of Axl-expression in other cell lines did not compromise GP 1,2 -driven entry [114, 115, 117] . So far, all studies failed to detect a direct interaction between EBOV-GP 1 and Axl. However, Axl expression was shown to augment internalization of EBOV-GP 1,2 -bearing pseudovirions and virus-cell fusion [114] , and this activity correlated with enhanced macropinocytosis in Axl-expressing cells [115] . Thus, Axl might promote filovirus uptake by macropinocytosis, which was previously shown to be a pathway exploited by filoviruses for cellular uptake [136, 137] . Indeed, inhibition of Axl and blockade of PIK3, which is important for macropinocytosis and Axl-dependent signaling, both inhibit filovirus entry in a cell type-dependent fashion [21, 125] . Finally, it is noteworthy that Gas6 can promote Sindbis virus entry by bridging phosphatidylserine present in the viral envelope to Axl localized on target cells [138] . Whether a similar mechanism operates in the context of filovirus entry remains to be investigated. The observation that recombinant Gas6 inhibits Axl-and DTK-dependent GP 1,2 -mediated entry [117] might argue against this hypothesis.

Endo-/Lysosomal Host Cell Factors

Many viral GPs are synthesized as inactive precursor proteins which transit into a membrane fusion-competent state only upon proteolytic cleavage by host cell proteases, a process termed priming. A prominent example is the influenza virus, which depends on cleavage of its hemagglutinin by host cell proteases for acquisition of infectivity, and the nature of the cleavage sequences in HA determines the virulence of avian influenza viruses [166] . The subtilisin-like proprotein convertase furin is responsible for priming of several viral GPs in the Golgi apparatus of infected cells, and furin consensus sites are present in GP 0 of all filoviruses excluding RESTV, which harbors an incomplete furin recognition site [32] . Despite of its conservation, several studies indicate that this motive is dispensable for filoviral spread in cell culture and infected animals [46, 47] , and the reason for its presence is unknown.

Cathepsins B and L

Chandran and colleagues demonstrated that two lysosomal cathepsins, cathepsin B and L, cleave filovirus GP 1,2 and that cathepsin activity is essential for GP 1,2 -driven host cell entry [57] . This report showed that cathepsins B and L prime the filovirus GP 1,2 for membrane fusion and proposed that cathepsin cleavage of virion-associated GP 1,2 occurs in a sequential fashion: First, cathepsin L and/or B cleave GP 1,2 into an 18 kDa form, which is fully infectious but still requires cathepsin B activity for infectious entry. Subsequently, the 18 kDa form is processed by cathepsin B and cleavage might be sufficient to trigger membrane fusion [57] . An alternative model for GP 1,2 activation has been proposed by subsequent studies. Thus, Schornberg and colleagues demonstrated that processing of virion-inserted GP 1,2 by recombinant cathepsin B and L or the bacterial protease thermolysin yielded a 19 kDa form of GP 1 and was associated with a notable increase in infectivity [56], a finding confirmed by others [126] . Processing of GP 1 into the 19 kDa from proceeded via 50 kDa and 20 kDa intermediates and its was speculated that the 20 kDa form might differ from the 18 kDa form observed by Chandran and colleagues only in the presence of a N-linked glycan [56] . Virions bearing the 19 kDa form were largely resistant to cathepsin B but not L inhibitors but remained sensitive to a lysosomotropic agent and a cysteine protease inhibitor. On the basis of these findings, a two-step model was proposed, suggesting that GP 1,2 must first be processed by cathepsins B and L before the activity of a third lysosomal factor, potentially a thiol reductase, triggers GP 1,2 -dependent membrane fusion [56, 171] . Indeed, subsequent studies provided evidence that the 19 kDa form represents a metastable conformation in which the fusion machinery is not yet exposed [37, [172] [173] [174] and which can be triggered for membrane fusion by low pH and reduction [173] . In addition, it was demonstrated that cleavage of GP 1,2 removes a glycan cap and the MLR, while the N-terminal RBR and GP 2 remain in the molecule [172, 175, 176] . In sum, proteolytic processing by cathepsins B and L primes GP 1,2 for membrane fusion and exposes the RBR. Subsequently, an incompletely understood stimulus triggers membrane fusion and these final steps of the lysosomal escape of filoviruses critically depend on GP 1,2 binding to NPC1 (Figure 3) , as discussed below. . Infectious entry of filoviruses into target cells. After interaction between GP 1,2 and cellular surface molecules, virions are internalized via macropinocytosis into the endosomal compartment. Subsequently, the endosomal cysteine proteases cathepsin B and L proteolytically process GP 1,2 , thereby removing the glycan cap (indicated by grey caps) and allowing primed GP 1 binding to Niemann-Pick C1 (NPC1), which is essential for the following virus-host membrane fusion process. Finally, a so far incompletely understood stimulus triggers the membrane fusion activity in GP 2 .

NPC1

As the NPC1 protein is localized on the endosomal and lysosomal membranes, it was proposed to act downstream of filovirus GP 1,2 engagement of attachment and signaling factors at the cell surface. Indeed, GP 1,2 -mediated viral uptake was readily detectable in NPC1-deficient cells, where the virions accumulated in early endosomes, indicating that membrane fusion was not triggered [129] . Proteolytic processing of GP 1,2 and thus exposure of the RBR was a prerequisite of NPC1 binding, as only the cleaved 19 kDa form was able to physically interact with NPC1 [20, 130] . Mapping studies revealed that the 19 kDa form binds to the second luminal domain of NPC1 [130] and cell surface presentation of this domain in the context of an artificial receptor molecule was sufficient to allow entry of pseudotypes carrying thermolysin-primed filovirus GP 1,2 [130] . It can be speculated that the interaction between the 19 kDa form of GP 1,2 and NPC1 might expose the GP 2 residues involved in membrane fusion. However, fusion of pseudotypes bearing the primed 19 kDa GP 1,2 with the plasma membrane of target cells expressing the second loop of NPC1 at their surface could not be induced by low pH treatment. Thus, binding of primed GP 1,2 to NPC1 is not sufficient to trigger membrane fusion [130] .

Biosynthesis and Posttranslational Modification of the Filovirus Glycoprotein

The filoviral GP is synthesized as a precursor protein, GP 0 , in the secretory pathway of infected cells. Upon transport of GP 0 into the Golgi apparatus, subtilisin-like proprotein convertases, in particular furin, process the precursor protein into two subunits, the surface unit GP 1 and the transmembrane unit GP 2 [32] . Both subunits remain covalently linked by an intermolecular disulfide bond and trimers of GP 1 -GP 2 heterodimers (GP 1 , 2 ) are inserted into the cellular and the viral membrane [33] . The filoviral GP 1,2 is heavily glycosylated and harbors numerous consensus sites for N-linked glycosylation and GP 1 glycosylation has been determined on the molecular level [34, 35] . In addition, a mucin-like region (MLR) at the C-terminus of GP 1 , which is highly variable among filovirus species [28, 33, [36] [37] [38] , is extensively modified by O-linked glycans. The MLR is dispensable for entry into cell lines, as lentiviral vectors (pseudotypes) bearing GP 1,2 mutants with a deleted MLR show comparable or even enhanced capacity to transduce certain cell lines (for instance Vero cells) compared to particles carrying wild type GP 1,2 [28, 36, [39] [40] [41] . However, the MLR seems to be important for association with several host cell lectins, as discussed below, and might have an important role in immune evasion and filovirus pathogenesis.

α5β1-Integrin

Expression of EBOV-GP 1,2 was initially shown to interfere with surface expression of various cellular membrane proteins, including α3 and ß1 integrins [40, 124] . However, a more recent study suggested that GP 1,2 expression does not reduce cell surface levels of integrins but rather sterically occludes epitopes in these proteins otherwise recognized by antibodies [157] . Experiments with EBOV-GP 1,2 -bearing pseudotypes demonstrated that soluble recombinant ß1 integrin or ß1-reactive antibodies diminish GP 1,2 driven entry, suggesting that GP 1,2 might need to engage ß1 integrins for infectious entry [124] . However, a direct interaction between EBOV-GP 1,2 and integrins remains to be demonstrated. Work by Schornberg and colleagues provided evidence that α5β1-integrin is required for expression of the double chain forms of cathepsin B and L and for full cathepsin L activity [123] , an endosomal protease involved in priming of GP 1,2 for membrane fusion, as discussed below. In contrast, α5β1-integrin was dispensable for GP 1,2 -mediated binding and uptake into target cells [123] . These observations suggest that α5β1-integrin indirectly promotes GP 1,2 -driven entry by ensuring activity of GP 1,2 -priming cysteine proteases or by stimulating the protease maturation pathway, which might be required for viral entry. Whether physical interactions of GP 1,2 with α5β1-integrin also contribute to filovirus entry remains to be determined.

Cathepsins B and L

Despite the importance of cathepsin B and L in priming EBOV-GP 1,2 for membrane fusion in several cell lines, the dependence on these particular proteases for viral entry is not universal among filoviruses. A requirement for cathepsin B activity during entry of EBOV-, TAFV-and BDBV-but not SUDV-, RESTV and MARV-GP-bearing pseudotypes has been described, and the same group showed that particles harboring the GP 1,2 of EBOV, SUDV and MARV exhibited enhanced transduction efficiency when cathepsin L was active in concert with cathepsin B. In contrast, entry of RESTV was dependent on a cysteine protease distinct from cathepsins B and L [125] . Furthermore, it has been reported that cathepsin L activity is dispensable for ebolavirus GP 1,2 -driven entry into Vero cells and mouse embryonic fibroblasts [57, 125] as well as human monocyte-derived dendritic cells [128] . Moreover, the observation of the failure of EBOV-GP 1,2 -bearing pseudotypes to transduce CatB − / − CatL − / − mouse embryonic fibroblasts can be overcome by ectopic expression of CatB, suggests that a protease other than CatL is required for a post-CatB cleavage step necessary for membrane fusion [125] . In addition, transduction of primary human macrophages by EBOV-GP 1,2 -carrying pseudotypes was shown to be dependent on both cathepsin B and L, whereas MARV-GP 1,2 -facilitated entry was not blocked efficiently by cathepsin B/L inhibitors, suggesting that MARV-GP 1,2 might employ a so far unknown protease for priming in macrophages [177] . It is also noteworthy that many studies investigating the role of cathepsins in filovirus host cell entry were performed with GP 1,2 -bearing vectors and not with authentic filoviruses. It would thus be interesting to examine the effect of cathepsin B and L knock-out on filoviral spread and pathogenicity, particularly in the light of efforts to develop cathepsin inhibitors as treatment for SARS-coronavirus [178] and filovirus infection.

Conclusions

Host cell entry is the first essential step in filovirus infection. Cellular lectins can concentrate vectors bearing GP 1,2 at the cellular surface and can thereby promote infectious entry. However, lectin expression usually does not render cells susceptible to GP 1,2 -driven entry and a role of lectins in the cell tropism of filoviruses in the infected host remains to be demonstrated. TAM tyrosine kinase receptors and TIM-1 can augment entry of filoviruses into a subset of susceptible cell lines and into some primary cells but are not universally required for filovirus infection. In fact, neither TIM-1 nor the TAM family member Axl are expressed to appreciable amounts in human macrophages, key viral targets, and the factors regulating viral uptake into these cells remain to be elucidated. The respective studies might reveal that filoviruses can use diverse cell surface factors for uptake into TIM-1-, Axl-negative cells, which might account for the broad cell tropism of filoviruses. In contrast to Axl and TIM-1, the broadly expressed endosomal/lysosomal protein NPC1 is required for filovirus entry into all cellular systems tested so far and seems to play a key role in filovirus entry. Further experiments will clarify whether NPC1 alone is sufficient for triggering membrane fusion or whether another host cell factor is involved.

Introduction

Deciphering filovirus pathogenesis requires the elucidation of filovirus interactions with host cells. The entry of filoviruses into target cells is the first essential step in the viral life cycle, and the viral and cellular factors involved in this process are potential targets for antiviral strategies. Infectious filovirus entry is mediated by the viral glycoprotein (GP), which is the only viral envelope protein and thus constitutes the sole target for the neutralizing antibody response. Consequently, defining which domains in GP are essential for cellular entry and can be targeted by the humoral immune response is pivotal to the design of effective vaccines. In addition, insights into the multiple, sequential interactions of GP with host cell factors required for cellular entry can define novel targets for therapeutic inhibition. Key components of the filovirus entry cascade have been identified in the recent years, and some of these discoveries have already been translated into novel antiviral approaches in cell culture and small animal models [19] [20] [21] [22] [23] [24] . Here, we will review the current knowledge of the host cell factors involved in the cellular entry of filoviruses and we will discuss the implications of key findings in this area for our understanding of viral pathogenesis.

The Filoviral Glycoprotein: Structure and Function

The filovirus particle is predominantly of filamentous nature and consists of seven structural proteins and a non-segmented, single-stranded RNA genome of negative polarity. In the viral envelope, a single type I transmembrane glycoprotein is incorporated, which is a virulence factor [25] and the sole viral determinant of host cell entry. Thus, GP mediates attachment of virions to host cells and fusion of the viral envelope with a host cell membrane, which is essential for delivery of viral proteins and nucleic acid into the host cell cytoplasm [26] [27] [28] [29] [30] . To facilitate virus entry, GP interacts with host cell factors, and the expression pattern of these factors is believed to be a central determinant of filovirus cell tropism. In addition, the various soluble forms of GP produced in the context of ebolavirus infection might modulate viral pathogenicity, as reviewed elsewhere [31] .

Cell and Organ Tropism of Filoviruses

The cellular molecules involved in filovirus entry govern the spectrum of cells susceptible to filovirus infection. Before we discuss GP 1,2 interactions with these factors, we will therefore provide a brief overview of the filovirus cell tropism. Filoviruses exhibit a broad cell and organ tropism in infected humans and non-human primates. However, at the early stages of the infection, cells of the mononuclear phagocyte system are mainly targeted, in particular macrophages and dendritic cells in the spleen, lymph nodes and liver [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] . Infection of these cells not only amplifies the virus [71] and ensures its rapid dissemination [72] , but also triggers the uncontrolled release of pro-inflammatory cytokines [60, 66, 70] , a hallmark of filovirus pathogenesis.
Secondary targets of filovirus infection are mainly fibroblasts and endothelial cells located in many different organs, including liver, kidney and testis, and these cells are permissive to robust, lytic viral replication [64, 73] . It has been proposed that ebolavirus infection of the vascular endothelium and the ensuing GP 1,2 -induced cell rounding might result in loss of vascular integrity and hemorrhage [28] . However, the vascular endothelium is a late target in EBOV infected non-human primates, and no obvious cytotoxic effects are associated with its infection [64] , suggesting that hemorrhage is not a direct consequence of ebolavirus infection of vascular endothelial cells. Apart from fibroblasts and endothelial cells, virtually all cell types are susceptible to filovirus infection, and infectious EBOV and MARV could be isolated from all tissues tested [64, 67, [74] [75] [76] [77] [78] . The only cells refractory to the otherwise pantropic filoviruses are lymphocytes [65, 67] , and experiments with pseudotypes showed that these cells are not susceptible to GP 1,2 -driven host cell entry, potentially because of lack of host cell factors required for appropriate uptake and intracellular trafficking [26, 27, 27, [79] [80] [81] [82] [83] [84] .

Host Cell Factors Promoting Infectious Filovirus Entry

The entry of enveloped viruses into host cells commences with the attachment of the virus to the cell surface, which is frequently promoted by relatively nonspecific interactions between the viral GP and cellular attachment-promoting factors. Subsequently, highly specific engagement of cellular molecules by the viral GP is essential to trigger uptake of virions into target cells and/or fusion of the viral with a host cell membrane. A virus entry receptor is usually defined as a cellular binding partner of a viral GP which is essential for infectious viral entry into host cells. Given the complexity of filovirus entry, which involves both cell surface molecules and intracellular proteins, only some of which physically interact with GP, we will not employ the classical receptor definition in our description of filovirus entry. Instead, discriminate between attachment factors, which interact with GP 1,2 at the cellular membrane and promote viral attachment to cells, signaling factors, which induce filovirus uptake through activating signaling cascades (Figure 2 ), and endo-/lysosomal host factors, which prime and activate GP 1,2 for membrane fusion ( Figure 3 ).

Attachment factors

Signaling factors immunosuppressive cytokines [99] , which is believed to promote viral spread. Whether a similar mechanism is operative during filovirus infection is unknown. Finally, it is noteworthy that secreted lectins can modulate filovirus infection. Thus, recombinant mannose-binding C-type lectin (MBL) was shown to protect mice from a lethal EBOV infection potentially by targeting the virus for phagocytosis and complement-directed lysis [100] . The human T cell Ig mucin 1 (TIM-1) surface molecule was initially found to be a cellular receptor for hepatitis A virus [104] . By a bioinformatics-based correlation analysis between gene expression profiles and susceptibility of cell lines to EBOV-GP 1,2 -driven infection, TIM-1 was recently also identified as an entry factor for filoviruses [105] . TIM-1 is a type I membrane glycoprotein with an extracellular IgV domain and a mucin-like domain predicted to be heavily O-glycosylated [106] . The IgV domain allows highly specific recognition of phosphatidylserine exposed on the surface of apoptotic cells, and TIM-1 was shown to be involved in the clearance of apoptotic cells [107, 108] . TIM-1 is expressed on activated T-cells, epithelial cells, conjunctiva and renal tissue [105, 109, 110] as well as certain cell lines including the liver cell line Huh7 [105] (Table 2) , which is permissive for filovirus replication [111] .

Endo-/Lysosomal Host Cell Factors

The membrane fusion reaction driven by the GPs of enveloped viruses can be triggered by several stimuli. For some viruses, engagement of a receptor at the cell surface activates fusion with the plasma membrane at neutral pH [158] . Alternatively, receptor binding resulting in virus internalization, and membrane fusion is stimulated by protonation of the viral GP in the acidic environment of the endosome [158] . The mild pH (6.5-6) of early endosomes is sufficient to trigger membrane fusion facilitated by the Nipah and Hendra virus GPs [159, 160] , while the low pH (5.5-4) environment of late endosomes and/or lysosomes activates the membrane fusion proteins of influenza, bunya and dengue viruses [161] [162] [163] . In addition, some viruses require both low pH and receptor engagement as triggers for membrane fusion [164] , while others are triggered upon receptor engagement at the cell surface, but ultimately fuse with the endosomal membrane [165] , indicating that complex determinants govern the nature of the trigger and the subcellular location of membrane fusion reaction.
It has long been noticed that filoviruses depend on low pH for infectious cellular entry [26] . However, it has also been demonstrated that low pH does not trigger the fusion activity of GP 1,2 [167] . This conundrum has been resolved by a study demonstrating that filoviruses are activated by endo-/lysosomal cysteine proteases, which require a low pH environment for their enzymatic activity [57].

NPC1

An endosomal factor required for filovirus entry after GP 1,2 priming by cathepsins has recently been discovered by two independent studies as the Niemann-Pick C1 (NPC1) protein. Cote and colleagues discovered that filoviruses depend on NPC1 for cellular entry by screening a library of chemical compounds for entry inhibitors [20] , while Carette and coworkers found in a screen of haploid human cells that mutations in NPC1 are not compatible with filovirus GP 1,2 -driven entry [129] . Finally, a recent study found that CHO cells selected for resistance to EBOV-GP 1,2 -dependent entry harbored a defect in the NPC1 gene [183] . The NPC1 protein is highly conserved among species and is ubiquitously expressed in human tissues, with the highest expression in the liver [184, 185] (Table 2) . The protein is an integral membrane protein of late endosomes and lysosomes and exhibits a polytopic orientation, forming several luminal and cytoplasmatic loops. NPC1 is a cholesterol transporter and mutations in the NPC1 gene result in fatal, progressive neurodegenerative disorder, Niemann-Pick C1 disease, due to a defect in the export of cholesterol from lysosomes [186] . The abnormal accumulation of cholesterol in turn leads to altered protein and lipid trafficking [187, 188] .Which lines of evidence suggest that NPC1 facilitates filovirus entry? NPC1 interacts with primed GP 1 [20, 130] and the contribution of NPC1 to infectious entry of filoviruses can be separated from its cholesterol transport activity [20, 129] , indicating that the protein directly facilitates entry. NPC1-deficient cells, including primary fibroblasts derived from NPC1 patients, were resistant to filovirus infection, but still allowed for efficient cellular entry of several other viruses [20, 129] . Furthermore, transduction of the wild type NPC1 gene into NPC1-defective, patient-derived cells or NPC1-negative CHO cells fully restored infection [20, 129] and directed expression of NPC1 in non-susceptible reptilian cells or haploid hamster CHO-K1 cell clones was sufficient to render these cells susceptible to GP 1,2 -mediated infection [130] . Additionally, siRNA knockdown of NPC-1 in HeLa cells resulted in reduced v