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Abstract

RNA viruses are known to replicate by low fidelity polymerases and have high mutation rates whereby the resulting virus population tends to exist as a distribution of mutants. In this review, we aim to explore how genetic events such as spontaneous mutations could alter the genomic organization of RNA viruses in such a way that they impact virus replications and plaque morphology. The phenomenon of quasispecies within a viral population is also discussed to reflect virulence and its implications for RNA viruses. An understanding of how such events occur will provide further evidence about whether there are molecular determinants for plaque morphology of RNA viruses or whether different plaque phenotypes arise due to the presence of quasispecies within a population. Ultimately this review gives an insight into whether the intrinsically high error rates due to the low fidelity of RNA polymerases is responsible for the variation in plaque morphology and diversity in virulence. This can be a useful tool in characterizing mechanisms that facilitate virus adaptation and evolution.

Poliovirus (PV)

The poliovirus population diversity was evaluated in the brain of the murine model during viral spread. It was observed that only a fraction of the original injected viral pool was able to move from the initial site of inoculation to the brain via the 'bottleneck effect.' To determine the maintenance of the quasispecies during infection in vivo, 6-10 weeks old mice were inoculated in the leg with individual viruses. Total RNA recovered from the brain tissues revealed that four viruses were shown to be capable of spreading to the brain with their introduced mutations unchanged. Therefore, it was postulated that the innate immune response reduced the viral pathogenicity by limiting the diversity of viruses during spread to vulnerable tissues [16] . Two mechanisms to explain the bottleneck effect have been speculated, namely-the "tough-transit" model and the "burned-bridge" model. The "tough-transit" model suggests that virus trafficking within the murine model has a low probability of success passing the blood-brain barrier. However, once in the CNS, it acts as a founder virus and re-establishes a population with initial limited diversity. On the other hand, the "burned bridge" model stipulates that it is not tough for the virus to physically reach the blood-brain barrier. Thus, when the first few viruses reach the gateway to the brain, the host innate immune response triggers an antiviral state [16] .
The poliovirus population diversity was evaluated in the brain of the murine model during viral spread. It was observed that only a fraction of the original injected viral pool was able to move from the initial site of inoculation to the brain via the 'bottleneck effect.' To determine the maintenance of the quasispecies during infection in vivo, 6-10 weeks old mice were inoculated in the leg with individual viruses. Total RNA recovered from the brain tissues revealed that four viruses were shown to be capable of spreading to the brain with their introduced mutations unchanged. Therefore, it was postulated that the innate immune response reduced the viral pathogenicity by limiting the diversity of viruses during spread to vulnerable tissues [16] . Two mechanisms to explain the bottleneck effect have been speculated, namely-the "tough-transit" model and the "burned-bridge" model. The "tough-transit" model suggests that virus trafficking within the murine model has a low probability of success passing the blood-brain barrier. However, once in the CNS, it acts as a founder virus and re-establishes a population with initial limited diversity. On the other hand, the "burned bridge" model stipulates that it is not tough for the virus to physically reach the blood-brain barrier. Thus, when the first few viruses reach the gateway to the brain, the host innate immune response triggers an antiviral state [16] .

West Nile Virus (WNV)

The genetic diversity of WNV in the avian host was also investigated using next-generation sequencing. The aim was to explore whether the genetically homogeneous cloned virus would go through genetic diversification after passages in young SPF chickens and wild juvenile carrion crows. Data collected revealed that the WNV population showed significant heterogeneity diverging from the quasispecies structure of the initial viral inoculum in both animal models. However, in-depth analysis enacted a comparison between the infection model (SPF chicken and wild juvenile carrion crows) to assess the variations in genetic diversity. It was demonstrated that the WNV genetic diversifications varied significantly from the inoculum in crows with 18 genetic variants but exhibited suboptimal levels of diversifications among the chickens with only 3 single nucleotide variants (SNV) being detected. Hence, natural WNV-susceptible avian hosts could provide a selective setting and contributed to genetic diversifications. NGS technologies have enabled the analysis of WNV quasispecies dynamics, leading to a better understanding of the virus and shed some light on its mechanism of pathogenicity [53] .

Togaviridae

Viruses from the Togaviridae family can be further classified into the genus Alphavirus and Rubivirus. Alphaviruses are anthropod-borne viruses [54] and they formed icosahedral particles of about 70 nm with a lipid envelope ( Figure 3A ) [55] . The spikes of the virion are made up of E1 and E2 glycoproteins organized in a T4 icosahedral lattice of 80 trimers. The alphavirus virion carries a positive single stranded RNA of approximately 11-12 kb as the genetic material [54] . The RNA has a 5 -methylated nucleotide cap and a polyadenylated 3 end. The viral genome is translated into three structural proteins (CP, E2 and E1) and four non-structural proteins (NSP1, NSP2, NSP3 and NSP4) ( Figure 3B ). The genetic diversity of WNV in the avian host was also investigated using next-generation sequencing. The aim was to explore whether the genetically homogeneous cloned virus would go through genetic diversification after passages in young SPF chickens and wild juvenile carrion crows. Data collected revealed that the WNV population showed significant heterogeneity diverging from the quasispecies structure of the initial viral inoculum in both animal models. However, in-depth analysis enacted a comparison between the infection model (SPF chicken and wild juvenile carrion crows) to assess the variations in genetic diversity. It was demonstrated that the WNV genetic diversifications varied significantly from the inoculum in crows with 18 genetic variants but exhibited suboptimal levels of diversifications among the chickens with only 3 single nucleotide variants (SNV) being detected. Hence, natural WNV-susceptible avian hosts could provide a selective setting and contributed to genetic diversifications. NGS technologies have enabled the analysis of WNV quasispecies dynamics, leading to a better understanding of the virus and shed some light on its mechanism of pathogenicity [53] .

Chikungunya Virus (CHIKV)

Similar to other RNA viruses with extensive mutation rates, CHIKV produces populations of genetically diverse genomes within a host. Up to date, the role of several of these mutations and the influence of disease severity in vertebrates and transmission by mosquitoes have been studied. Riemersma et al. investigated the intra-host genetic diversity of high and low-fidelity CHIKV variants using murine models. Both the high and low fidelity variants were expected to lower the virulence of CHIKV as compared to the wild-type (CHIKV-WT). However, the high-fidelity variant caused more acute levels of infection such that the onset of the swelling in the footpad exhibited earlier than the CHIKV-WT at 3-and 4-days post-infection (dpi). Moreover, the high-fidelity CHIKV (CHIKV-HiFi) infected mice also displayed higher peaks of disease severity when compared to the CHIKV-WT 7 dpi. This enhanced diversification was subsequently reproduced after serial in vitro passages. In high-fidelity variants, nsp2 G641D and nsp4 C483Y mutations increased CHIKV virulence in the adult mice. The NGS data showed that the CHIKV-HiFi variant produced more genetically diverse populations than the CHIKV-WT in mice. However, the low-fidelity variant gave rise to reduced rates of replication and disease [69] .
Plaque size is a common feature of viral characterization. Primary isolates of CHIKV containing variants with different plaque sizes were previously reported [70, 71] . Viral variants with different plaque morphology such as small and large plaques had been reported in the 2005 CHIKV outbreak isolates [72] . It is curious how small plaque variants with lower fitness were maintained as a natural viral quasispecies. Plausible explanations indicated that the plaque size might not represent the in vivo growth conditions and that cooperation among variants with different plaque sizes might be required for optimal in vivo replication and transmission fitness. Jaimipak et al. reasoned that if the plaque size did not represent the in vivo growth conditions and the small plaque variants had a similar fitness as the large plaque variants, they would be similarly virulent in a murine model. In order to explore the virulence of the small plaque CHIKV variant in vivo, the pathogenicity of the purified small plaque variant of the CHIKV virus isolated from the sera of the patient in Phang-nga, Thailand in 2009, was tested in neonatal mice [73] . The small plaque variant (CHK-S) showed stable homogenous small plaques after 4 plaque purifications. It also grew slower and produced lower titers when compared with the wild-type virus. After 21 days of infection in the suckling mice with the wild-type and CHK-S variants (injected 103 pfu/mouse), mice which received the CHK-S virus showed 98% survival rate while only 74% of mice survived after infection with the wild-type virus. The small plaque variant of CHIKV obtained by plaque purifications exhibited decreased virulence that makes it appropriate to serve as candidates for live-attenuated vaccine development. The CHIKV variant with the small plaque size formed a major subpopulation in the CHIKV primary isolate during multiple passages in C6/36 cells. This is in line with the reduction of virulence in the suckling mice and indicated that the small plaque variant had reduced in vivo fitness. This suggested that replication cycles in mosquito vectors might play an important role in maintaining the small plaque variant in natural infections. The persistence of the small plaque variant CHK-S clone after multiple passages in C6/36 cells showed that the CHK-S variant might be able to outcompete the large plaque variant when infecting the same cell by an unknown mechanism. Alternatively, small and large plaque variants might cooperate in a way that provided a selective advantage for maintaining the small plaque variant [73] .

Ebola Virus (EboV)

Several studies of the Ebola virus glycoprotein showed that the two mutations at positions A82V and T544I might have caused an increase in viral infectivity in humans [88] [89] [90] [91] [92] [93] . These two mutations reduced the stability of the pre-fusion conformation of the EBOV glycoprotein. Kurosaki et al. investigated the viral pseudotyping of EBOV glycoprotein derivatives in 10 cell lines from nine mammalian species and the infectivity of each pseudotype. The data showed that isoleucine at position 544 mediated membrane fusion and increased the infectivity of the virus in all host species, whereas valine at position 82 modulated viral infectivity but was dependent on the virus and the host. Analysis via structural modeling revealed that the isoleucine 544 changed the viral fusion. However, the valine 82 residue influenced the interaction with the viral entry receptor, Niemann-Pick C1 [94] . The frequency of these two amino acid substitutions (A82V and T544I) varied between different Ebolavirus species.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

Scobey et al. reported the T1015N mutation in the spike glycoprotein during 9 passages of the virus was able to alter the growth kinetics and plaque morphology in vitro. The mutated MERS-CoV virus (MERS-CoV T1015N) replicated approximately 0.5 log more effectively and formed larger plaques compared to the wild type (MERS-CoV). The data suggested that the mutation T1015N was a tissue culture-adapted mutation that arose during serial in vitro passages [107] . The whole genome sequencing of MERS-CoV revealed the presence of sequence variants within the isolate from dromedary camels (DC) which indicated the existence of quasispecies present within the animal. A single amino acid (A520S) was located in the receptor-binding domain of the MERS-CoV variant. Strikingly, when detailed population analysis was performed on samples recovered from human cases, only clonal genomic sequences were reported. Therefore, the study speculated that a model of interspecies transmission of MERS-CoV whereby specific genotypes were able to overcome the bottleneck selection. While host susceptibility to infection is not taken into account in this setting, the findings provided insights into understanding the unique and rare cases of human of MERS-CoV [108] .

Mumps

The strain Urabe AM9 is one of the mumps virus strains that was widely used in vaccines but this strain was associated with meningitis and was withdrawn from the market. Sauder et al. performed serial passaging of the strain Urabe AM9 in cell cultures and compared the whole nucleotide sequences of the parental (Urabe P-AM9) and passaged viruses (Urabe P6-Vero or Urabe P6-CEF) to investigate the attenuation process and to identify the attenuation markers [123] . Passaging of the Urabe AM9 mumps virus in Vero or chicken embryo fibroblast (CEF) cell lines caused changes in the genetic heterogeneity at particular regions of the genome through either changing of one nucleotide at locations where the starting material showed nucleotide heterogeneity or the presentation of an additional nucleotide to produce a heterogenic site. Virulence of the passaged virus was dramatically decreased in the murine model. Moreover, similar growth kinetics of the virulent Urabe P-AM9 and passaged attenuated variants in the rat brain suggested that the impaired replication ability of the attenuated variants was not the main cause of the neuroattenuation. However, in the rat brain, the peak titer of the neuroattenuated variant was almost one log lower than that of the neurovirulent parental strain. For instance, identical but independent induction of heterogeneity at position 370 of the F-gene by substitution of threonine to alanine in passaged virus in Vero and CEF cells suggested a correlation of this mutation to the neuroattenuation phenotype. There was lack of ability to identify heterogeneity for those regions with differences of more than 10% between the detected nucleotides in the consensus sequence. The heterogeneity could be the result of new mutations at these positions or the selection of pre-existing sequences within the minority quasispecies. In addition, passaging of the parental strain in CEF and Vero cells led to the observation of several amino acid alterations in the NP, P, F, HN and L proteins that could affect the virulence of the virus. Thus, the modifications of genetic heterogeneity at particular genome sites could have important consequences on the neurovirulence phenotype. Therefore, extra caution should be exercised in order to evaluate genetic markers of virulence or attenuation of variants based on only a consensus sequence [123] .

Pneumoviridae

The virions of the pneumoviruses are enveloped with a spherical shape and a diameter of about 150 nm. They have a negative-sense RNA genome of 13 to 15 kb ( Figure 8A ). The RNA-dependent RNA polymerase (L) binds to the genome at the leader region and sequentially transcribes each gene. The cellular translation machinery translates the capped and poly-adenylated messenger RNA of the virus in the cytoplasm. Members of the genus Orthopneumovirus possess 10 genes including NS1 and NS2 which are promoter proximal to the N gene. The gene order is NS1-NS2-N-P-M-SH-G-F-M2-L ( Figure 8B ). Alignment of the L proteins showed moderate conservation of the sequences between the human and bovine viruses. Bovine respiratory syncytial virus (BRSV) differs from HRSV in host range and the two viruses bear substantially similar sequences as well as antigenic relatedness [132] . Figure 8B ). Alignment of the L proteins showed moderate conservation of the sequences between the human and bovine viruses. Bovine respiratory syncytial virus (BRSV) differs from HRSV in host range and the two viruses bear substantially similar sequences as well as antigenic relatedness [132] .

Dengue Virus (DENV)

From another perspective, differences in the envelope (E) gene sequence was investigated using the plasma samples of six DENV infected patients. The first account of viral quasispecies of DENV in vivo was reported using clonal sequencing analysis whereby the simultaneous occurrence of diverse variant genomes was observed. The degree of genetic diversity was revealed to fluctuate among patients with the mean proportion being 1.67%. Moreover, out of 10 clones derived from dengue infected plasma, 33 nucleotide substitutions were detected, of which 30 were non-synonymous mutations. Of particular interest, mutations at amino acid residues 290 and 301 resulted in the presence of two stop codons which indicated that genome-defective dengue viruses (5.8%) were also present within the quasispecies population. It was hypothesized that this might have significant impact on the pathogenesis of the dengue virus [39] . Recently, Parameswaran et al. profiled the intra-host viral diversity of samples from 77 patients via whole-genome amplifications of the entire coding region of the DENV-3 genome. A significant difference in the viral makeup between naïve subjects and patients with DENV-3 immunity revealed that the immune repertoire of the host is responsible for the degree of diversity exhibited by the viral population. Subsequently, identification of the hotspots responsible for the intra-host diversity revealed that few spots were crucial for intra-host diversity. The major hotspots for diversity were revealed in more than 59% of the samples at three codon coordinates-amino acid residues 100 and 101 in the M protein and residue 315 in the AB loop of the E Domain III. The residue E 315 was speculated to have arisen as an immune escape variant in response to the pressure exerted by the immune defense mechanism. These findings highlighted the importance of host-specific selection pressures in the evolution of DENV-3 viral population within the host and this could eventually lead to the intelligent design of a vaccine candidate identified from the prevalent escape variants such as those bearing the E 315 [40] . It was reported that within the quasispecies population, amino acid substitutions occurred on the surface of the E protein which was involved in interactions with other oligomers, antibodies and host cell receptors. In particular, two amino acid substitutions at positions E452 and E455 were mapped to the E protein transmembrane domain, E450 to E472, which functioned as the membrane anchor for E protein. Intra-host quasispecies analysis using the E gene sequences also identified several amino acids on the surface of the E protein which altered the properties of the virus. The conformational rearrangements that led to the fusion of the virus and the host cell membrane was altered. The amino acids detected in the quasispecies consensus sequence were observed to be less frequent in the E proteins from patients suffering from mild disease than from patients with severe onset of dengue infection. Thus, the quasispecies might harbor specific variants that are crucial for the pathogenesis of the disease [41] . Understanding the significant molecular determinant of pathogenesis through the analysis of quasispecies could lead to the rational design of a DENV vaccine.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

Alterations in the coronavirus spike glycoprotein by means of natural and experimentally induced mutations changed cell and organ tropism and virus pathogenicity. The wild-type MERS-CoV spike glycoprotein precursor contains 1353 amino acids arranged into two subunits-an aminoterminal subunit (S1) carrying the receptor binding domain (RBD) and a carboxy-terminal subunit (S2) containing the putative fusion peptide (FP/IFP), two heptad repeat domains (HR1/HR2) and the transmembrane (TM) and intracellular domains ( Figure 6 ). Lu et al. isolated a diverse population comprising the wild-type and a variant carrying a deletion of 530 nucleotides in the spike glycoprotein gene from the serum of a 75-year-old patient in Taif, Saudi Arabia. The patient subsequently died. Analysis of the MERS-CoV sequence showed an out of frame deletion which led to the loss a large part of the S2 subunit. It contained all the major structures of the membrane fusion in the S2 subunit preceding the early stop codon [103] and this also included the proposed fusion peptide (949-970 aa) [104] . The deletion resulted in the production of a shortened protein bearing only 801 amino acids. In the cell-free serum sample of the patient, mutant genomes with the S530∆ were abundant with an estimated ratio of 4:1 deleted to intact sequence reads. The spike gene deletion would cause the production of a defective virus which was incapable of causing infections or with a lowered rate of infection. Losing the S2 subunit caused a disruption in the membrane holding the spike protein and halted the fusion of the virus to the host. However, in the case of the mutant bearing the S530∆, the mutation helped to sustain the wild-type MERS-CoV infection by producing a free S1 subunit with a "sticky" hydrophobic tail and the additional disulfide bonds caused the aggregation and mis-folding of proteins. In addition, the mutated S530∆ could form steady trimer complexes that retained biding affinity for the dipeptidyl peptidase 4 (DPP4) and acted as a decoy such that the spike-specific MERS-CoV neutralizing antibodies were blocked.

Newcastle Disease Virus (NDV)

On the other hand, in vitro studies showed that the quasispecies distribution of the avirulent isolate harbored 10% of variants bearing the virulent F0 region (RRQRRF). Gene sequence analysis of Australian NDV isolates showed the existence of a novel clade of NDV viruses with the F0 cleavage site sequence of 112 -RKQGRL-117 and the HN region bearing seven additional amino acids. Four field isolates (NG2, NG4, Q2-88 and Q4-88) belonging to the novel clade were propagated for a longer time period in CEF cells prior to sequencing. Analysis revealed the existence of 1-2% of virulent strains with the F0 cleavage site of 112 -RKGRRF-117 in the population [131] .

Introduction

With their diverse differences in size, structure, genome organization and replication strategies, RNA viruses are recognized as being highly mutatable [1] . Their high mutation rates make it very difficult for therapeutic interventions to work effectively and very often they develop resistance to antiviral drugs and antibodies elicited by vaccines [2, 3] . This poses a real threat to how emerging infectious agents could be prevented or treated [4] . The success of the evolution of RNA viruses arises from their capacity to utilize varying replication approaches and to adapt to a wide range of biological niches faced during viral spread in the host. One of the factors affecting the emergence or re-emergence of infectious diseases is the genetics of the infectious agents [1] .
Only about 15 viral diseases can be effectively prevented through FDA approved vaccinations [5] . This attests to the urgency to understand the mechanisms by which viruses can overcome the different pressures applied to restrict their replications. In the last four decades, breakthroughs in molecular biology have favored in-depth analysis of virus isolates. Findings from other studies have suggested that populations of RNA viruses are divergent and favor an active evolution of RNA genomes. The quick evolution of RNA genomes could lead to variant sequences that differ by one or two nucleotides from the wild-type sequence in the population. It is further suggested that each viral RNA population of 10 9 or more infectious particles was always a mixture of various variants despite being isolated from a single clone [6] .
Such heterogeneity within the virus population could be explained by the existence of not a single genotype within the species but rather an ensemble of related sequences known as the quasispecies [7] . Developed by Manfred Eigen and Peter Schuster, the concept of quasispecies was defined as a mutant distribution dominated by a primary sequence with the highest rates of replication between the components of the mutant distribution. The phenomenon of quasispecies was further supported by the "hypercycle" theory as a self-organization principle to include different quasispecies in a higher-order organization that eases evolution into more complicated forms such that the coding capacity and catalytic activities of proteins are taken into account [8] [9] [10] .
Mutants with varying levels of infectivity generated from a mutated gene occurred regularly in a virus population due to the high mutation rates [6] . The error-prone replication ability of RNA viruses and the shorter generation times can be used to explain the variations in evolution rates between DNA and RNA viruses. While mutation rates for DNA genomes have been estimated to be between 10 −7 and 10 −11 per base pair per replication [11] , the RNA dependent RNA polymerase (RdRp) showed typically low fidelity whereby the mutation rate is of roughly 10 −4 mutations per nucleotide copied, which is greater than that of almost all DNA viruses [7, 12, 13] . This characteristic of the RNA polymerase in RNA viruses led to the generation of diverse offspring with different genotypes in shorter generation times.
There is some general consensus regarding quasispecies that have been established. For instance, the presence of diverse mutants in a population of viruses is a reality which can affect the biological behavior of the virus in vivo due to the complexity and amplitude of the mutant spectra [14] . Moreover, interactions amongst variants of a quasispecies population was classified into three types namely-cooperation, interference and complementation. Cooperative interactions arise from those variants exhibiting advantageous phenotypes compared to the wild-type while interfering interactions are those exemplified in variants with detrimental effects on the replication of the virus. However, complementation interactions have no positive or negative effects on the virus population [15] .
Considerably less is known of the relationships between the evolution of RNA viruses with respect to virulence. The dynamics of quasispecies has explained the failure of monotherapy and synthetic antiviral vaccine but opened up new avenues for exploration [14] . Specifically, unanswered questions pertaining to quasispecies remain-What are the underlying mutations responsible for long term tenacity compared to those of extinction? Are there any molecular determinants which are the root cause of higher virulence in a quasispecies population?
Frequent occasional outbreaks of emerging and re-emerging viral diseases such as Dengue fever, West Nile Fever, Zika virus disease, Chikungunya disease, Middle east respiratory syndrome, Ebola virus disease and many others have been targets for therapeutic interventions. Long lasting protection against viral infections is best achieved via vaccinations through live attenuated viruses (LAVs). In order to generate stable vaccine strains, the evolution of these viruses must be properly understood. This review is centered on the examination of the evidence for the heterogeneous nature of RNA genomes (quasispecies), the factors leading to quasispecies formation and its implications on virulence.
The phenomenon of quasispecies has been well reported for many viruses belonging to different families and genera [16] [17] [18] . There is no clear idea whether emerging viruses such as Zika virus, Ebola virus, West Nile virus, Dengue virus and many others owe part of their evolution to higher virulence conferred by the presence of specific quasispecies within the viral population. Exploring the underlying mechanism of virulence stemming from a quasispecies population remains of interest. In this review, we examine reported cases of quasispecies and their implications on virulence.

Picornaviridae

Viruses of the family Picornaviridae can be classified into genera such as Enterovirus, Parechovirus, Aphotovirus and others. The virion is made up of a non-enveloped capsid of 30 nm surrounding a core positive stranded ssRNA genome ( Figure 1A ) [19] . The genome is approximately 7 kb in size and possesses a single long ORF flanked on both ends by the 5 -non-translated region (5 -NTR) and the 3 -non-translated region [20] . The 5 -NTR has an internal ribosome entry site (IRES) which controls cap-independent translation. The ORF comprising 6579 nucleotides can be classified into three polyprotein regions, namely, P1, P2 and P3. They encode for structural proteins (VP1 to VP4) in the P1 region and non-structural proteins in the P2 (2A-2C) and P3 regions (3A-3D) following proteolytic cleavage ( Figure 1B) . The viral capsid proteins VP1, VP2 and VP3 are displayed on the external structures of the EV-A71 viral particle whereas VP4 is found within the internal structures of the capsid [21] . (7.4 Kb) . The Open Reading Frame (ORF) contains the structural viral protein P1 which is cleaved to yield VP1, VP2, VP3 and VP4 and non-structural viral proteins P2 (cleaved to yield 2A, 2B and 2C) and P3 (cleaved to yield 3A, 3B, 3C and 3D). The 3′-NTR end of the genome contains the poly (A) tail.

Poliovirus (PV)

Poliovirus is found within the Human Enterovirus C species of the Picornaviridae family and can be classified into three distinct serotypes (1, 2 and 3). Most poliovirus infections cause an asymptomatic incubation period followed by a minor illness characterized by fever, headache and sore throat which mainly affects children. However, PV infections can lead to paralytic poliomyelitis which can result in death. Following the WHO 1988 polio eradication program, the number of poliomyelitis has been reduced by 99% worldwide but a small number of countries still have sporadic outbreaks of polio [22] .
In order to verify whether limiting the genomic diversity of a viral population has any effect on its evolution, a study was conducted on a strain of poliovirus with a substitution of Glycine 64 to Serine (G64S) in the RNA polymerase of the virus. The outcome of one-step growth curves and
Poliovirus is found within the Human Enterovirus C species of the Picornaviridae family and can be classified into three distinct serotypes (1, 2 and 3). Most poliovirus infections cause an asymptomatic incubation period followed by a minor illness characterized by fever, headache and sore throat which mainly affects children. However, PV infections can lead to paralytic poliomyelitis which can result in death. Following the WHO 1988 polio eradication program, the number of poliomyelitis has been reduced by 99% worldwide but a small number of countries still have sporadic outbreaks of polio [22] .
In order to verify whether limiting the genomic diversity of a viral population has any effect on its evolution, a study was conducted on a strain of poliovirus with a substitution of Glycine 64 to Serine (G64S) in the RNA polymerase of the virus. The outcome of one-step growth curves and northern blot analysis of genomic RNA synthesis confirmed that the G64S mutation showed greater fidelity without a considerable reduction in the overall efficiency of RNA replication. The study hypothesized that having greater heterogeneity within a viral population allows it to adapt better to changing environments encountered during an infection and indeed the finding showed that boosting the fidelity of poliovirus replication had a noticeable effect on viral adaptation and pathogenicity. The poliovirus strain with a mutated RNA polymerase carrying an altered amino acid residue (G64S) was observed to replicate similarly to the wild-type counterpart but produced lower genomic diversity and subsequently was incapable of adapting well under detrimental growth situations. This study showed that the diversity of the quasispecies was associated with increased virulence rather than selection of single adaptive mutations. Alongside previous observations, these findings indicated a rise in the error rate over the tolerable error threshold which induced viral extinction, suggesting that the rate of viral mutation was precisely modulated and most likely had been finely tuned during the evolution of the virus [7] . It was further revealed that curbing the diversity of a RNA viral population by raising the fidelity of the RNA polymerase had a direct effect on its pathogenicity and capacity of the viruses to escape antiviral immunity [23] . These findings support the fact that RNA viruses have developed minimal viral polymerase fidelity to facilitate quick evolution and adaptation to novel situations [24] .

Enterovirus 71 (EV-A71)

EV-A71 belongs to the genus Enterovirus within the family of Picornaviridae. It was first characterized in 1969 in California, USA [25] and is one of the main etiological agents of hand, foot and mouth disease (HFMD) [26] . Some cases of EV-A71 infections have been associated with neurological complications such as aseptic meningitis, brainstem encephalitis and acute flaccid paralysis [27] . In China, enteroviruses such as EV-A71 and CV-A16 have caused 7,200,092 cases of HFMD between 2008 to 2012. The mortality rate was highest among children below the age of five. It was further reported that 82,486 patients developed neurological complications and 1617 deaths were confirmed by the laboratory to be caused by EV-A71 [28, 29] .

Flaviviridae

Of the Flaviviridae family (genera Flavivirus, Pestivirus, Pegivirus and Hepacivirus), there are 89 animal viruses with a small, positive-sense, single stranded RNA genome [31] . The virions are 40-60 nm in diameter, spherical in shape and contain a lipid envelope (Figure 2A ). The majority of these viruses are arthropod-borne and transmitted via infected mosquitoes and ticks. They are considered as emerging and re-emerging pathogens such as dengue virus (DENV), West Nile Virus (WNV), Zika Virus (ZIKV) and these viruses pose a global threat to public health by causing significant mortality [32] . The flaviviral genome is approximately 11 kb and has a single open reading frame (ORF), which is flanked by untranslated regions (5 and 3 NTR). The ORF encodes three structural proteins (C, M and E) and 7 non-structural proteins (NS). The non-structural proteins include large, highly conserved proteins NS1, NS3 and NS5 and four small hydrophobic proteins NS2A, NS2B and NS4A and NS4B ( Figure 2B ) [33] . quasispecies utilized the dynamic proportion of varying haplotype populations to co-exist, sustained the ability of the population to adapt and enabled the propagation in different tissues. Lastly, the study concluded that the selection of haplotype(s) might be a driving factor in viral dissemination and severity of infections in humans as well as the virulence in EV-A71 infected patients [30] .
Of the Flaviviridae family (genera Flavivirus, Pestivirus, Pegivirus and Hepacivirus), there are 89 animal viruses with a small, positive-sense, single stranded RNA genome [31] . The virions are 40-60 nm in diameter, spherical in shape and contain a lipid envelope (Figure 2A ). The majority of these viruses are arthropod-borne and transmitted via infected mosquitoes and ticks. They are considered as emerging and re-emerging pathogens such as dengue virus (DENV), West Nile Virus (WNV), Zika Virus (ZIKV) and these viruses pose a global threat to public health by causing significant mortality [32] . The flaviviral genome is approximately 11kb and has a single open reading frame (ORF), which is flanked by untranslated regions (5′ and 3′ NTR). The ORF encodes three structural proteins (C, M and E) and 7 non-structural proteins (NS). The non-structural proteins include large, highly conserved proteins NS1, NS3 and NS5 and four small hydrophobic proteins NS2A, NS2B and NS4A and NS4B ( Figure 2B ) [33] .

Zika Virus (ZIKV)

Zika Virus was first discovered in 1947 when it was isolated from Aedes Africanus mosquitoes [42] . It belongs to the Flavivirus genus within the Flaviviridae family. Zika infections have been reported in Egypt [43] , East Africa [44] , India [45] , Thailand, Vietnam [46] , Philippines and Malaysia [47] .
An Asian/American lineage ZIKA virus (ZIKV) formed 2 types of plaques-large and small. The large plaque variant was observed to have faster growth kinetics compared to the small plaque variant. Sequencing of the plaque variants showed that the large plaque variant had a guanine nucleotide at position 796 (230 Gln ) while the small plaque clone had an adenine at the same position. A recombinant clone carrying the G796A mutation was produced using an infectious molecular clone of the ZIKV MR766 strain. The plaque size produced by the recombinant clone was smaller when compared to the parental strain and its growth rate was significantly reduced in Vero cells. In vivo studies demonstrated that the virulence of the MR766 strain in IFNAR1 mice had decreased, showing that the mutation at position 230 in the -M protein is a molecular determinant of plaque morphology, growth property and virulence in mice [48] .

West Nile Virus (WNV)

West Nile Virus was first characterized in 1937 in the West Nile district of Uganda and was taxonomically placed in the genus Flavivirus within the Flaviviridae family. The virus later appeared in New York in 1999, where it caused 59 hospitalized infections and 7 deaths before its spread to other parts of the USA between 1999-2001 [50] . WNV survives naturally in a mosquito-bird-mosquito transmission cycle involving the Culex sp. mosquitoes [51] .

Togaviridae

Viruses from the Togaviridae family can be further classified into the genus Alphavirus and Rubivirus. Alphaviruses are anthropod-borne viruses [54] and they formed icosahedral particles of about 70 nm with a lipid envelope ( Figure 3A ) [55] . The spikes of the virion are made up of E1 and E2 glycoproteins organized in a T4 icosahedral lattice of 80 trimers. The alphavirus virion carries a positive single stranded RNA of approximately 11-12kb as the genetic material [54] . The RNA has a 5′-methylated nucleotide cap and a polyadenylated 3′ end. The viral genome is translated into three structural proteins (CP, E2 and E1) and four non-structural proteins (NSP1, NSP2, NSP3 and NSP4) ( Figure 3B ).

Chikungunya Virus (CHIKV)

Chikungunya virus (CHIKV) is an arthropod-borne virus transmitted to humans by mosquitoes and has caused significant human morbidity in many parts of the world [56] . Chikungunya virus causes an acute febrile illness with high fever, severe joint pain, polyarthralgia, myalgia, maculopapular rash and edema. While the fever and rash are self-limiting and are able to resolve within a few days, arthralgia can be prolonged from months to years [57, 58] . Some cases of CHIKV
Chikungunya virus (CHIKV) is an arthropod-borne virus transmitted to humans by mosquitoes and has caused significant human morbidity in many parts of the world [56] . Chikungunya virus causes an acute febrile illness with high fever, severe joint pain, polyarthralgia, myalgia, maculopapular rash and edema. While the fever and rash are self-limiting and are able to resolve within a few days, arthralgia can be prolonged from months to years [57, 58] . Some cases of CHIKV disease were associated with neurological complications [59] . The virus has been associated with frequent outbreaks in tropical countries of Africa and Southeast Asia and also in temperate zones around the world. A major outbreak in 2013 affected several countries of the Americas, involving approximately 2 million people [60] .
The original geographical distributions of the CHIKV indicated that there are 3 distinct groups and phylogenetic analysis confirmed the West African, the Asian and the East/Central/South African (ECSA) genotypes. The ECSA virus with an A226V substitution in the E2 envelope gene had caused multiple massive outbreaks in various regions starting in the La Reunion Islands in 2005. The virus then spread to Asia and caused over a million cases in the following years [61] [62] [63] . The Asian genotype started invading the Americas in 2013, causing massive outbreaks in various countries in Central, South America and the Caribbean. The ECSA virus is now the dominant virus all over Africa and Asia and the Asian genotype is the dominant virus in the Americas [62, [64] [65] [66] [67] . Even though a number of CHIKV vaccine candidates are being developed, no effective vaccine is currently available for clinical use [68] .
The P5 CHIKV-NoLS clone remained genetically stable after five passages in Vero cells or insect cells when compared to the CHIKV-WT. Sequence analysis of the P5 CHIKV-NoLS plaques showed that the two plaque variants had no mutations in the capsid protein. A single non-synonymous change in the nucleotide of the capsid caused an alanine to serine substitution at position 101 in the third plaque variant. However, the substitution did not cause any change in the small plaque phenotype or replication kinetics of the CHIKV-NoLS clone after ten passages in vitro [74] . The in vivo study showed that the CHIKV-NoLS-immunized mice were able to produce long-term immunity against CHIKV infection following immunization with a single dose of the CHIKV-NoLS small plaque variant. Attenuation of CHIKV-NoLS through the NoLS mutation is most likely due to the disruption of the replication of viruses after viral RNA synthesis, however, the precise mechanism of reduced viral titer remained unsolved [76] . The NoLS mutation caused a considerable change in the very basic capsid region involving two nucleotides which could affect the structure of RNA binding, assembly of nucleocapsid and interaction with the envelope proteins [77] . Since the CHIKV-NoLS small plaque variant was attenuated in immunized mice and produced sera which could effectively neutralize CHIKV infection in vitro, it could serve as a promising vaccine candidate needed to control the explosive large-scale outbreaks of CHIKV [76] .

Filoviridae

Viruses found within the Filoviridae family can be further classified into five genera-Marburgvirus, Ebolavirus, Cuevavirus, Striavirus and Thamnovirus. The virions are 80 nm in diameter and appear as branched, circular or filamentous ( Figure 4A ). Filoviruses contain a linear negative sense single stranded RNA of approximately 19 kb. The genome of the filoviruses encodes for four structural proteins, namely nucleoprotein (NP), RNA-dependent RNA polymerase co-factor (VP35), transcriptional activator (VP30) and a RNA-dependent RNA polymerase (L). There are also three non-structural membrane-associated proteins, namely a spike glycoprotein (GP1,2), a primary matrix protein (VP40) and a secondary matrix protein (VP24) present within the virion membrane [78] ( Figure 4B ).

Ebola Virus (EboV)

The Ebolavirus genus belongs to the Filoviridae family within the order Mononegavirales. Five species have been identified within the genus of Ebolavirus-Zaire (EBOV), Bundibugyo (BDBV), Sudan (SUDV), Tai Forest (TAFV) and Reston (RESTV) [81] . Among them, only the Reston virus (RESTV) is assumed to be non-pathogenic for humans. The other four classified as Ebolaviruses are well-known to cause the Ebola virus disease (EVD). The virus causes a severe fever along with systemic inflammation and damage to the endothelial cell barrier, leading to shock and multiple organ failure with high mortality rates in humans and animals [82] . It is transmitted to people from wild animals and spreads in the human population through human-to-human transmission [83] . However, the natural host reservoirs of Ebola viruses are unknown. The average Ebola virus disease (EVD) case fatality rate is around 50%. So far, the largest recorded EVD with 28,652 infections had killed 11,325 people [84] . The Zaire, Bundibugyo and Sudan Ebola viruses are involved in large outbreaks in Africa.
The glycoprotein (GP) is responsible for cell attachment, fusion and cell entry. The broad cellular tropism of the GP resulted in multisystem involvement that led to high mortality [85] . The Ebola virus has a high frequency of mutation within a host during the spread of infection and in the reservoir in the human population [86] . Alignment of the Glycoprotein (GP) sequences of 66 Ebola virus isolates from the previous outbreaks (old Ebola outbreak of 1976 to 2005) with the new Ebola outbreak isolates (2014) showed some differences in the positions and frequency of the amino acid replacements. Comparative analysis between the isolates from the old epidemic with the new epidemic isolates showed that 19 out of the 22 amino acid mutations were consistently present in the latter [87] .
In addition, nucleotide mutations at positions A82V and P382T were present only in the Ebola virus glycoprotein from the new Ebola epidemic 2014 isolates. Mutation at position W291R was also found only in the 2014 isolate but at a very low frequency of occurrence [87] . Having a large number of mutations from previous outbreaks present with more than 90% frequency in the sequence of the new Ebola epidemic isolates as well as the emergence of new mutations (A82V, P382T and W291R) indicated the presence of viral quasispecies in the population. The high mutation rates found in a RNA quasispecies increased the probability of escape mutations and this could explain the escape of the 2014 viral isolates from neutralizing antibodies elicited by the old Ebola epidemic isolates. The structural analysis of the Ebola virus revealed the strong contribution of these residues in the three-
The Ebolavirus genus belongs to the Filoviridae family within the order Mononegavirales. Five species have been identified within the genus of Ebolavirus-Zaire (EBOV), Bundibugyo (BDBV), Sudan (SUDV), Tai Forest (TAFV) and Reston (RESTV) [81] . Among them, only the Reston virus (RESTV) is assumed to be non-pathogenic for humans. The other four classified as Ebolaviruses are well-known to cause the Ebola virus disease (EVD). The virus causes a severe fever along with systemic inflammation and damage to the endothelial cell barrier, leading to shock and multiple organ failure with high mortality rates in humans and animals [82] . It is transmitted to people from wild animals and spreads in the human population through human-to-human transmission [83] . However, the natural host reservoirs of Ebola viruses are unknown. The average Ebola virus disease (EVD) case fatality rate is around 50%. So far, the largest recorded EVD with 28,652 infections had killed 11,325 people [84] . The Zaire, Bundibugyo and Sudan Ebola viruses are involved in large outbreaks in Africa.
The glycoprotein (GP) is responsible for cell attachment, fusion and cell entry. The broad cellular tropism of the GP resulted in multisystem involvement that led to high mortality [85] . The Ebola virus has a high frequency of mutation within a host during the spread of infection and in the reservoir in the human population [86] . Alignment of the Glycoprotein (GP) sequences of 66 Ebola virus isolates from the previous outbreaks (old Ebola outbreak of 1976 to 2005) with the new Ebola outbreak isolates (2014) showed some differences in the positions and frequency of the amino acid replacements. Comparative analysis between the isolates from the old epidemic with the new epidemic isolates showed that 19 out of the 22 amino acid mutations were consistently present in the latter [87] .
In addition, nucleotide mutations at positions A82V and P382T were present only in the Ebola virus glycoprotein from the new Ebola epidemic 2014 isolates. Mutation at position W291R was also found only in the 2014 isolate but at a very low frequency of occurrence [87] . Having a large number of mutations from previous outbreaks present with more than 90% frequency in the sequence of the new Ebola epidemic isolates as well as the emergence of new mutations (A82V, P382T and W291R) indicated the presence of viral quasispecies in the population. The high mutation rates found in a RNA quasispecies increased the probability of escape mutations and this could explain the escape of the 2014 viral isolates from neutralizing antibodies elicited by the old Ebola epidemic isolates. The structural analysis of the Ebola virus revealed the strong contribution of these residues in the three-dimensional rearrangement of the glycoprotein and they played an important role in the re-emergence of the new epidemic Ebola isolates in 2014.
Dietzel et al. studied the functional significance of three non-synonymous mutations in the Ebola virus (EBOV) isolates from the outbreak in West Africa. Among 1000 sequenced Ebola virus genomes, approximately 90% carried the signature three mutations at positions 82, 111 and 759 of the Ebola virus genome. The impact of specific mutations on the role of each viral proteins and on the growth of recombinant EBOVs was analyzed by recently engineered virus-like particles and reverse genetics. A D759G substitution in proximity to a highly conserved region of the GDN motif in the enzymatically active center (amino acid 741 to 743) of the L polymerase was able to increase viral transcription and replication. On the other hand, a R111C substitution in the multifunctional region of the nucleoprotein which is essential for homo-oligomerization and nucleocapsid formation was found to reduce viral transcription and replication. Furthermore, the A82V replacement in the glycoprotein region was able to enhance the efficacy of GP-mediated viral entry into target cells. The combination of the three mutations in the recombinant Ebola virus affected the functional activity of viral proteins and enhanced the growth of the recombinant virus in the cell culture when compared to the prototype isolate [93] . A pilot epidemiological NGS study with a substantial sample size suggested that high mortality in the host was not changed by these three mutations since the rate of mortality in the overall study was not considerably altered throughout the outbreak [95] .
Furthermore, Fedewa et al. showed that genomic adaptation was not crucial for efficient infection of the Ebola virus. The genomes were characterized after serial-passages of EBOV in Boa constrictor kidney JK cells. Deep sequencing coverage (>×10,000) confirmed the presence of only one single nonsynonymous variant (T544I) of unknown significance within the viral population that demonstrated a shift in frequency of at least 10% over six serial passages. However, passaging the EBOV in other cell lines, such as HeLa and DpHt cheek cells, showed different mutations in the genomes of the viral population [96] . This brings forth the question as to whether the viral strains of the Ebola virus should be directly isolated from patients in order to determine the quasispecies of the Ebola virus.

Coronaviridae

Viruses within the Coronaviridae family are positive sense, single-stranded RNA viruses capable of infecting three vertebrate classes comprising mammals (Coronavirus and Torovirus), birds (Coronavirus) and fish (Bafinivirus). Coronaviruses are the largest RNA viruses identified so far with the enveloped spherical virions of about 120-160 nm and the viral genome is about 31 kb in length ( Figure 5A ) [97] . The genome consists of many ORFs. Two thirds of the 5 end is occupied by a replicase gene comprising two overlapping ORFs namely-ORF1a and ORF1b. The four structural proteins are spike glycoprotein (S), small envelope protein (E), membrane glycoprotein (M) and nucleocapsid (N). Accessory regions that are group specific ORFs are designated as ORF3, ORF4a, ORF4b and ORF5 [97] (Figure 5B ).

Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

Middle East respiratory syndrome (MERS) coronavirus is an enveloped, positive-sense, singlestranded RNA virus that was identified for the first time in 2012 in Saudi Arabia. The viral respiratory disease was caused by a novel coronavirus. The causative coronaviruses (CoV) belong to the lineage C of the Betacoronavirus within the family Coronaviridae. MERS-CoV can infect a broad range of mammals, including humans and is transmitted by the infected dromedary camels [98, 99] . Typical MERS symptoms are similar to the common flu but in some patients, pneumonia and gastrointestinal symptoms including diarrhea and organ failure were reported [100] . Since September 2012 to August 2018, 2253 MERS-CoV cases including 840 deaths were reported in 27 countries worldwide [101] . Approximately 35% of patients with MERS-CoV infection have died.
Alterations in the coronavirus spike glycoprotein by means of natural and experimentally induced mutations changed cell and organ tropism and virus pathogenicity. The wild-type MERS-CoV spike glycoprotein precursor contains 1353 amino acids arranged into two subunits-an aminoterminal subunit (S1) carrying the receptor binding domain (RBD) and a carboxy-terminal subunit (S2) containing the putative fusion peptide (FP/IFP), two heptad repeat domains (HR1/HR2) and the transmembrane (TM) and intracellular domains ( Figure 6 ).
Middle East respiratory syndrome (MERS) coronavirus is an enveloped, positive-sense, single-stranded RNA virus that was identified for the first time in 2012 in Saudi Arabia. The viral respiratory disease was caused by a novel coronavirus. The causative coronaviruses (CoV) belong to the lineage C of the Betacoronavirus within the family Coronaviridae. MERS-CoV can infect a broad range of mammals, including humans and is transmitted by the infected dromedary camels [98, 99] . Typical MERS symptoms are similar to the common flu but in some patients, pneumonia and gastrointestinal symptoms including diarrhea and organ failure were reported [100] . Since September 2012 to August 2018, 2253 MERS-CoV cases including 840 deaths were reported in 27 countries worldwide [101] . Approximately 35% of patients with MERS-CoV infection have died.
Alterations in the coronavirus spike glycoprotein by means of natural and experimentally induced mutations changed cell and organ tropism and virus pathogenicity. The wild-type MERS-CoV spike glycoprotein precursor contains 1353 amino acids arranged into two subunits-an amino-terminal subunit (S1) carrying the receptor binding domain (RBD) and a carboxy-terminal subunit (S2) containing the putative fusion peptide (FP/IFP), two heptad repeat domains (HR1/HR2) and the transmembrane (TM) and intracellular domains ( Figure 6 ).
Middle East respiratory syndrome (MERS) coronavirus is an enveloped, positive-sense, singlestranded RNA virus that was identified for the first time in 2012 in Saudi Arabia. The viral respiratory disease was caused by a novel coronavirus. The causative coronaviruses (CoV) belong to the lineage C of the Betacoronavirus within the family Coronaviridae. MERS-CoV can infect a broad range of mammals, including humans and is transmitted by the infected dromedary camels [98, 99] . Typical MERS symptoms are similar to the common flu but in some patients, pneumonia and gastrointestinal symptoms including diarrhea and organ failure were reported [100] . Since September 2012 to August 2018, 2253 MERS-CoV cases including 840 deaths were reported in 27 countries worldwide [101] . Approximately 35% of patients with MERS-CoV infection have died.

Paramyxoviridae

Paramyxoviridae is a family of viruses in the order Mononegavirales that uses vertebrates as their natural hosts. Currently, 72 species are placed in this family and they are divided amongst 14 genera [109] . Diseases associated with Paramyxoviridae included measles (MeV), mumps and Newcastle disease (NDV). Paramyxoviridae virions are enveloped and pleomorphic which are presented as spherical or filamentous particles with diameters of around 150 to 350 nm ( Figure 7A ). The genome is linear, negative-sense single-stranded RNA, about 15-19 kb in length and encode 9-12 proteins through the production of multiple proteins from the P gene ( Figure 7B ) [110] . On the external surface of the virion, glycoproteins possessing hemagglutinin, neuraminidase and cell fusion activities are present. The middle component of the envelope is a lipid bilayer acquired from the host cell as the virus buds off the cytoplasmic membrane. The innermost surface of the envelope is a non-glycosylated membrane protein layer that maintains the outer structure of the virus. The paramyxoviruses can be characterized by the gene order of the viral proteins and by the biochemical characteristics of the proteins associated with viral attachment. Figure 7A ). The genome is linear, negative-sense single-stranded RNA, about 15-19 kb in length and encode 9-12 proteins through the production of multiple proteins from the P gene ( Figure 7B ) [110] . On the external surface of the virion, glycoproteins possessing hemagglutinin, neuraminidase and cell fusion activities are present. The middle component of the envelope is a lipid bilayer acquired from the host cell as the virus buds off the cytoplasmic membrane. The innermost surface of the envelope is a nonglycosylated membrane protein layer that maintains the outer structure of the virus. The paramyxoviruses can be characterized by the gene order of the viral proteins and by the biochemical characteristics of the proteins associated with viral attachment. The L protein which is the catalytic subunit of RNA-dependent RNA polymerase (RDRP) is associated with the nucleocapsid protein (N) and phosphoprotein (P) to form part of the RNA polymerase complex. The RNA polymerase complex is covered by the viral envelope consisting of a matrix protein (M) and two glycosylated envelope spike proteins, a fusion protein (F) and cell attachment protein. Cell attachment protein is different based on the genera and it could be hemagglutinin (H in Measles), hemagglutinin-neuraminidase (HN in Mumps and NDV viruses) or glycoprotein G (Henipavirus). Some genera within the Paramyxoviridae family also contain various conserved proteins including the non-structural proteins (C, NS1, NS2), a cysteine-rich protein (V), a small integral membrane protein (SH) and transcription factors M2-1 and M2-2 [111] .
Fusion and cell attachment proteins are large glycoprotein spikes that are present on the surface of the virion. Both of these proteins play important roles in the pathogenesis of viruses from Paramyxoviridae family and are responsible for attachment to the cellular receptor(s), whereas the F protein mediates cell entry by inducing fusion between the viral envelope and the host cell membrane. The matrix protein organizes and sustains the virion structure. The nucleocapsid associates with genomic RNA and protects the RNA from nucleases. Extracistronic (noncoding) regions include a 3′ leader sequence with 50 nucleotides in length, which works as a transcriptional promoter and a 5′ trailer with 50-161 nucleotides [111] .
The genomes of viruses within the family Paramyxoviridae are non-segmented and thus cannot undergo genetic reassortment. Like many other RNA viruses, the RNA-dependent RNA polymerase does not have an error proofreading capability and hence many mutations can occur when the RNA is processed. These mutations can build up in the genome and eventually give rise to new variants. Since each protein has an important function, the mutant viruses will exhibit a loss in viral fitness and are eliminated, leaving only those exhibiting good viral fitness [111] . Within the Paramyxoviridae family, mutations leading to a spectrum of mutant distributions among Measles virus, Mumps virus and Newcastle disease virus are reviewed. The L protein which is the catalytic subunit of RNA-dependent RNA polymerase (RDRP) is associated with the nucleocapsid protein (N) and phosphoprotein (P) to form part of the RNA polymerase complex. The RNA polymerase complex is covered by the viral envelope consisting of a matrix protein (M) and two glycosylated envelope spike proteins, a fusion protein (F) and cell attachment protein. Cell attachment protein is different based on the genera and it could be hemagglutinin (H in Measles), hemagglutinin-neuraminidase (HN in Mumps and NDV viruses) or glycoprotein G (Henipavirus). Some genera within the Paramyxoviridae family also contain various conserved proteins including the non-structural proteins (C, NS1, NS2), a cysteine-rich protein (V), a small integral membrane protein (SH) and transcription factors M2-1 and M2-2 [111] .
Fusion and cell attachment proteins are large glycoprotein spikes that are present on the surface of the virion. Both of these proteins play important roles in the pathogenesis of viruses from Paramyxoviridae family and are responsible for attachment to the cellular receptor(s), whereas the F protein mediates cell entry by inducing fusion between the viral envelope and the host cell membrane. The matrix protein organizes and sustains the virion structure. The nucleocapsid associates with genomic RNA and protects the RNA from nucleases. Extracistronic (noncoding) regions include a 3 leader sequence with 50 nucleotides in length, which works as a transcriptional promoter and a 5 trailer with 50-161 nucleotides [111] .
The genomes of viruses within the family Paramyxoviridae are non-segmented and thus cannot undergo genetic reassortment. Like many other RNA viruses, the RNA-dependent RNA polymerase does not have an error proofreading capability and hence many mutations can occur when the RNA is processed. These mutations can build up in the genome and eventually give rise to new variants. Since each protein has an important function, the mutant viruses will exhibit a loss in viral fitness and are eliminated, leaving only those exhibiting good viral fitness [111] . Within the Paramyxoviridae family, mutations leading to a spectrum of mutant distributions among Measles virus, Mumps virus and Newcastle disease virus are reviewed.

Measles Virus (MeV)

Measles virus belongs to the genus Morbillivirus within the family Paramyxoviridae of the order Mononegavirales. Measles is transmitted by air or by direct contact with body fluids. The initial site of viral infection is the respiratory tract, followed by dispersions in the lymphoid tissue, liver, lungs, conjunctiva and skin. The measles virus (MeV) may persist in the brain, causing fatal neurodegenerative diseases. This virus can only infect humans and causes subacute sclerosing panencephalitis and encephalitis [112] [113] [114] . Measles often lead to fatality in young children (below 5 years) due to complications in respiratory tract infections like pneumonia, brain swelling or encephalitis, dehydration, diarrhea and ear infections [115] .
The MeV is a negative sense single stranded RNA virus and the genome is composed of six contiguous, non-overlapping transcription units separated by three untranscribed nucleotides. The genes which code for eight viral proteins are in the order of 5 -N-P/V/C-M-F-H-L-3 [116] . The second transcription unit (P) codes for two non-structural proteins, C and V, which interfere with the host immune response [117] [118] [119] [120] .

Mumps

Mumps virus belongs to the genus nus Rubulavirus within the Paramyxoviridae family of the order Mononegavirales. Mumps is an extremely contagious, acute, self-limited, systemic viral infection that primarily affects swelling of one or more of the salivary glands, typically the parotid glands. The infection could cause pain in the swollen salivary glands on one or both sides of patient face, fever, headache, muscle aches, weakness, fatigue and loss of appetite. Complications of mumps are rare but they can be potentially serious involving inflammation and body swelling in testicles, brain, spinal cord or pancreas. Infections can lead to hearing loss, heart problems and miscarriage. In the United States, mumps was one of common disease prior to vaccination became routine. Then a dramatic decrease was observed in the number of infections. However, mumps outbreaks still occur in the United States and there was an increase in the number of cases recently. Majority of those who are not vaccinated or are in close-contact with the viruses in schools or college campuses are at high risk. There is currently no specific treatment for mumps [122] .

Newcastle Disease Virus (NDV)

Newcastle disease virus (NDV) belongs to the genus Avulavirus in the family Paramyxoviridae of the order Mononegavirales. NDV is an avian pathogen that can be transmitted to humans and cause conjunctivitis and an influenza like disease [124] . Clinical diseases affecting the neurological, gastrointestinal, reproductive and respiratory systems are detected in naïve, unvaccinated or poorly vaccinated birds [125] . NDV is a continuous problem for poultry producers since it was identified ninety years ago. It has negatively impacted the economic livelihoods and human welfare through reducing food supplies and many countries were affected since 1926 with NDV outbreaks [126] .
The NDV genome codes for seven major viral proteins in the order of 5 -N-P(V)-M-F-HN-L-3 . In NDV, the hemagglutinin neuraminidase (HN) and fusion (F) glycoproteins are presented on the surface of the virion envelope and contribute to viral infection [127] . The fusion protein is expressed as an inactive precursor (F0) prior to activation by proteolytic cleavage. The cleavage of F0 is crucial for infectivity and works as a key virulence indicator for certain viruses such as virulent strains of avian paramyxovirus 1 (NDV). The F0 cleavage site contains several basic residues which cause the cleavage of the F protein by furin, an endopeptidase present in the trans-Golgi network [110] .

Pneumoviridae

The Pneumoviridae family contains large enveloped negative-sense RNA viruses. Previously, this taxon was known as a subfamily of the Paramyxoviridae but it was reclassified in 2016 as a family of its own with two genera, Orthopneumovirus and Metapneumovirus. Some viruses belonging to Pneumoviridae family are only pathogenic to humans, such as the human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). Human pneumoviruses do not have animal reservoirs and their primary site of infection is the superficial epithelial cells of the respiratory tract. There are no known vectors for pneumoviruses and transmission is thought to be primarily by aerosol droplets [132] .

Respiratory Syncytial Virus (RSV)

Respiratory syncytial virus (RSV) belongs to the genus Orthopneumovirus under the family Pneumoviridae of the order Mononegavirales. Human respiratory syncytial virus (HRSV) is the primary cause of infection of the upper and lower respiratory tracts with mild, cold-like symptoms in infants and young children. The virus spreads through tiny air droplets. Globally, there are 4-5 million children younger than 4 years with HRSV infections and more than 125,000 are hospitalized every year in the United States. Although the risk of hospital admission is higher in known risk groups such as prematurely born infants. RSV is also responsible for 14,000 deaths in the elderly > 65 years of age annually in the United State [133, 134] . On the other hand, bovine respiratory syncytial virus (BRSV) is a common source of pneumonia in calves. Clinical infections stem from yearly outbreaks of the disease during winter and primarily affect calves less than 6 months of age. The target infection site of the viruses are the epithelial layer of the upper and lower respiratory tracts that can damage the bronchioles, leading to severe onset of bronchiolitis in caws [135] .
Palivizumab (PZ) is the sole humanized monoclonal antibody against an infectious disease that recognizes the fusion protein of respiratory syncytial virus (RSV). Zhao et al. selected a PZ resistant virus by passaging of RSVA2 strain in the presence of PZ in HEp-2 cell culture [136] . Utilization of PZ provided the opportunities to gain new insights into the transmission dynamics and the quasispecies nature of RSV. Protein sequence analysis of a single plaque (MP4) isolated from the fifth passage revealed the substitution of lysine by methionine 272. The mutation caused the cell culturederived virus to be completely resistant to PZ prophylaxis in cotton rats. Dramatic reduction in replication of the parental strain A2 virus was observed at PZ concentrations ranging from 4 to 40
Respiratory syncytial virus (RSV) belongs to the genus Orthopneumovirus under the family Pneumoviridae of the order Mononegavirales. Human respiratory syncytial virus (HRSV) is the primary cause of infection of the upper and lower respiratory tracts with mild, cold-like symptoms in infants and young children. The virus spreads through tiny air droplets. Globally, there are 4-5 million children younger than 4 years with HRSV infections and more than 125,000 are hospitalized every year in the United States. Although the risk of hospital admission is higher in known risk groups such as prematurely born infants. RSV is also responsible for 14,000 deaths in the elderly > 65 years of age annually in the United State [133, 134] . On the other hand, bovine respiratory syncytial virus (BRSV) is a common source of pneumonia in calves. Clinical infections stem from yearly outbreaks of the disease during winter and primarily affect calves less than 6 months of age. The target infection site of the viruses are the epithelial layer of the upper and lower respiratory tracts that can damage the bronchioles, leading to severe onset of bronchiolitis in caws [135] .
Palivizumab (PZ) is the sole humanized monoclonal antibody against an infectious disease that recognizes the fusion protein of respiratory syncytial virus (RSV). Zhao et al. selected a PZ resistant virus by passaging of RSVA2 strain in the presence of PZ in HEp-2 cell culture [136] . Utilization of PZ provided the opportunities to gain new insights into the transmission dynamics and the quasispecies nature of RSV. Protein sequence analysis of a single plaque (MP4) isolated from the fifth passage revealed the substitution of lysine by methionine 272. The mutation caused the cell culture-derived virus to be completely resistant to PZ prophylaxis in cotton rats. Dramatic reduction in replication of the parental strain A2 virus was observed at PZ concentrations ranging from 4 to 40 µg/mL. The replication of the MP4 mutant was not affected by PZ. The growth kinetics of both the parental strain and the variant were almost similar with maximum titers above 10 7 PFU/mL during the third and fourth day post infection. Hence, it was proposed that the fusion protein supported the entry of the MP4 mutants in HEp-2 cells in an early phase of the replication cycle through a fusion step. The A2 parental strain exhibited limited growth in HEp-2 cells due to its reactivity with PZ. However, the lack of reactivity of the MP4 mutants with PZ suggested that the F1 protein of the MP4 mutant caused a loss of antigenic reactivity with the humanized monoclonal antibody. Preclinical studies in cotton rats predicted the efficacy of PZ in humans. However, the usage of PZ up to 40 µg/mL, especially in immunosuppressed patients, could provide opportunities for the emergence of resistant viruses. Therefore, the PZ resistant viruses in humans could cause the PZ prophylaxis to be ineffective.
Larsen et al. analyzed the nucleotides coding for the extracellular part of the G glycoprotein and the full SH protein of bovine respiratory syncytial virus (BRSV) from several outbreaks from the same herd in different years in Denmark. Identical viruses were isolated within a herd during outbreaks but viruses from recurrent infections were found to vary up to 11% in sequences even in closed herds. It is possible that a quasispecies variant of BRSV persisted in some of the calves in each herd and this persistent variant displayed high viral fitness and became dominant. However, based on the high level of diversity, the most likely explanation is that BRSV was reintroduced into the herd prior to each new outbreak. These findings are highly relevant to understand the transmission patterns of BRSV among calves [138] .

Orthomyxoviridae

The family Orthomyxoviridae belongs to the order of Articulavirales and contains seven genera-Influenza A-D, Isavirus, Thogotovirus and Quaranjovirus. The virions within the Orthomyxoviridae family are usually spherical but can be filamentous, 80-120 nm in diameter ( Figure 9A ). The influenza virus genome is 12-15 kb and contains 8 segments of negative-sense, single-stranded RNA which encodes for 11 proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1 and PB2) ( Figure 9B ). Influenza viruses are pathogenic and they can cause influenza in vertebrates, including birds, humans and other mammals [139] . The genome fragments contain both the 5 and 3 terminal repeats which are highly conserved throughout all eight fragments.

Influenza Virus (IV)

The epidemiology and molecular characterization of low and highly pathogenic avian influenza virus strains (LPAIV & HPAIV, respectively) isolated from Germany were investigated. The complete genome analysis of the two strains showed that both LPAIV and HPAIV had high nucleotide similarity with only ten mutations outside the hemagglutinin cleavage site (HACS) which were Orthomyxoviruses employ many different splicing techniques to synthesize their viral proteins while making full use of the coding capacity of the genome. The virion envelope originates from the cell membrane with the addition of one to three virus glycoproteins and one to two non-glycosylated proteins. The viral RNA polymerase (PB1, PB2 and PB3) is involved in the transcription of a single mRNA from every fragment of the genome. The transcription is triggered by cap snatching and the poly(A) tail is added by the viral polymerase stuttering on the poly U sequence. Alternative splicing of the MP and NS mRNA led to the mRNA coding for M2 and NEP proteins. PB1-F2 is translated by leaky scanning from the PB1 mRNA. The structural proteins common to all genera include three polypeptides, the hemagglutinin which is an integral type I membrane glycoprotein involved in virus attachment, the envelope fusion and the non-glycosylated matrix protein (M1 or M) [140] .
The epidemiology and molecular characterization of low and highly pathogenic avian influenza virus strains (LPAIV & HPAIV, respectively) isolated from Germany were investigated. The complete genome analysis of the two strains showed that both LPAIV and HPAIV had high nucleotide similarity with only ten mutations outside the hemagglutinin cleavage site (HACS) which were spread along the six genome segments of the HPAIV. Of the ten mutations, five were previously identified as minor variants in the quasispecies population of the progenitor virus, LPAIV, with 18-42% significant variable frequency [144] . However, studies focusing on the diversity of quasispecies of avian influenza in the human host are few. Watanabe et al. successfully demonstrated that infections caused by a single-virus in vitro produced an evident spectra of mutants in the H5N1 progeny viruses. Analysis of the genetic diversity of the hemagglutinin (HA) revealed that variants with mutated HA had lower thermostability leading to higher binding specificity. Both traits were deemed beneficial for viral infection. On the other hand, other variants with higher thermostability also emerged but were unable to thrive against mutants with lower thermostability [145] . The quasispecies population of influenza A virus was also reported to be in a state of continuous genetic drift in a given subtype population. A viral single nucleotide polymorphism (vSNP) was reported to be important and was shared by more than 15% of the variants within the quasispecies population of the subtype strain in a given season. However, between the season 2010-2011, various vSNPs in the PB2, PA, HA, NP, NA, M and NS segments were shared among variants with more than 58-80% of the sample population and less than 50% of the shared vSNPs were located within the PB1 segment [146] .

Hepadnaviridae

Hepadnaviruses can be found within the family Hepadnaviridae. They are further classified into two genera-the mammalian genus Orthohepadnavirus and the avian genus Avihepadnavirus [149] . These viruses are spherical with 42-50 nm diameter and replicate their genomes with the help of a reverse transcriptase (RT) ( Figure 10A ). The approximate size of the DNA genome is 3.3 kb with a relaxed circular DNA (rcDNA) supported by base pairing complementary overlaps [150] . The DNA genome is made up of four partly or completely overlapping ORFs that encode for the core protein (Core and preCore), surface antigen protein (PreS1, PreS2 and S), the reverse transcriptase (Pol protein) and the X transcriptional transactivator protein [151] (Figure 10B ). Replication occurs by reverse transcription of the progenitor RNA by the RNA polymerase II from the covalently close circular form of the HBV DNA [152] .

Hepatitis B Virus (HBV)

Hepatitis B virus (HBV) is the prototype of hepadnaviruses. It infects humans and can be classified into 8 genotypes. More than one billion people have contracted hepatitis B virus (HBV) and more than 200 million patients are chronically infected with hepatitis B (CHB) [153] . CHB infections result in the development of hepatocellular carcinoma and chronic liver failure [151] and every year CHB causes 880,000 deaths worldwide [153] . Analysis of the immunodominant motifs of the HBV core region from the amino acids 40 to 95 indicated that the positions exhibiting peak rates of variability were found in the main core epitopes, thereby confirming their role in stimulating the immune system. Moreover, the distribution of the variability was observed to occur in a genotypedependent manner. For instance, HBV isolated from genotype A had higher variability within the core epitope regions but no significant differences in genotype D were observed in the core epitopes and other positions. Further sequential analysis of the samples put forth the dynamic nature of the HBV quasispecies whereby a strong selection for a single baseline variant was linked to a lower variability within the core region pre-and post-treatment. Leucine (L) at position 76 was determined to be the most highly conserved residue and the role of this amino acid was assessed by substitutions of Valine (V) or Proline (P) at position 76. Proline at position 76 was shown to drastically lower the production of Hepatitis B core antigen protein (HBsAg), likely due to the chemical and physical properties of the amino acid. However, substitution with Valine (V) at a similar position brought about a four-fold increase in the Hepatitis B e antigen protein (HBeAg) production when compared to Leucine at position 76. The decrease in the variability observed was associated with a stable quasispecies population after positive selection of the variant exhibiting high fitness level [154] .
Although a significant number of RNA viruses demonstrated the existence of quasispecies in their populations due to their low-fidelity polymerases, the phenomenon of quasispecies has been reported to exist in DNA viruses such as Hepatitis B virus (HBV) that replicates via a RNA intermediate.
Hepatitis B virus (HBV) is the prototype of hepadnaviruses. It infects humans and can be classified into 8 genotypes. More than one billion people have contracted hepatitis B virus (HBV) and more than 200 million patients are chronically infected with hepatitis B (CHB) [153] . CHB infections result in the development of hepatocellular carcinoma and chronic liver failure [151] and every year CHB causes 880,000 deaths worldwide [153] . Analysis of the immunodominant motifs of the HBV core region from the amino acids 40 to 95 indicated that the positions exhibiting peak rates of variability were found in the main core epitopes, thereby confirming their role in stimulating the immune system. Moreover, the distribution of the variability was observed to occur in a genotype-dependent manner. For instance, HBV isolated from genotype A had higher variability within the core epitope regions but no significant differences in genotype D were observed in the core epitopes and other positions. Further sequential analysis of the samples put forth the dynamic nature of the HBV quasispecies whereby a strong selection for a single baseline variant was linked to a lower variability within the core region pre-and post-treatment. Leucine (L) at position 76 was determined to be the most highly conserved residue and the role of this amino acid was assessed by substitutions of Valine (V) or Proline (P) at position 76. Proline at position 76 was shown to drastically lower the production of Hepatitis B core antigen protein (HBsAg), likely due to the chemical and physical properties of the amino acid. However, substitution with Valine (V) at a similar position brought about a four-fold increase in the Hepatitis B e antigen protein (HBeAg) production when compared to Leucine at position 76. The decrease in the variability observed was associated with a stable quasispecies population after positive selection of the variant exhibiting high fitness level [154] .
Although a significant number of RNA viruses demonstrated the existence of quasispecies in their populations due to their low-fidelity polymerases, the phenomenon of quasispecies has been reported to exist in DNA viruses such as Hepatitis B virus (HBV) that replicates via a RNA intermediate.
Another challenge to overcome is to accurately determine the origin and spread of a founder population of the virus. Hence, the discrepancies in the evolution of HBV was investigated. Eight related patients with acquired chronic HBV through mother-to-infant transmission were selected and the viral genomes isolated were analyzed. Sequence analysis indicated that the samples originated from a single source of HBV genotype B2 (HBV-B2) which diverged from a tiny common ancestral pool regardless of the route of acquisition. Between individuals, viral strains obtained from a time point showed evidence that they originated from a small pool of the previous time point. This conferred the strain an advantage over other strains with regards to the recovery of the founder state shortly after transmission to the new host and the adaptation to the local environment within the host. Natural selection rather than genetic drift was hypothesized to be the root cause for the evolution of HBV, due to the observed varying patterns of divergence at synonymous and non-synonymous sites. This was in line with the higher rate of substitutions within the host rather than between hosts. Approximately 85/88 amino acid residues changed from common to rare residues. Since these changes were shown not to be a random process, it is concluded that the HBV was able to evolve and change but was limited to a defined range of phenotypes. It can be argued that the mechanism observed thus far suggest that the adaptive mutations accumulated in one individual would not be maintained in another individual and might revert after transmission. Hence, within the host, substitutions were higher than between hosts [156] .

Conclusions

RNA viruses are responsible for numerous outbreaks of viral infections with substantial levels of fatality. We discussed how genetic variants carrying spontaneous mutations could give rise to diverse plaque morphologies in different RNA viruses. How the specific mutations could affect viral replications and have an impact on the virulence of the plaque variants are reviewed. The existence of quasispecies in the viral RNA populations is also explored. Many of the RNA viruses displayed different plaque morphologies and these variants could have arisen from a genetically diverse quasispecies population. Such diverse quasispecies in a population could be a key contributing factor to elevated levels of virulence exhibited by the RNA viruses. Through an extensive analysis of different plaque variants and quasispecies within a population, this study could shed more light on the evolutionary pattern and virulence of RNA viruses. More intricate in vitro and in vivo examination of the phenomenon of quasispecies and the relationship between plaque size determinants and virulence should be undertaken to reveal if serious infections are caused by a single strain or through the combined action of diverse quasispecies carrying different mutations. This can be a valuable tool to characterize the mechanisms that led to viral evolution and adaptation in a host. Eventually, discovering an answer to these concerns might ultimately help to design effective vaccines against the ever-evolving RNA viruses.

Enterovirus 71 (EV-A71)

The course of evolution through which EV-A71 evolves to escape the central nervous system (CNS) was investigated by complete sequencing and haplotype analysis of the strains isolated from the digestive system and the CNS. A novel bottleneck selection was revealed in various environments such as the respiratory system and the central nervous system throughout the dissemination of EV-A71 in the host. Consequently, a dominant haplotype resulting from the bottleneck effect caused a change from viruses harboring VP1-3D to VP1-31G where the amino acid 31 was a favorable site of selection among the circulating EV-A71 sub-genotype C2. VP1-31G was present at elevated levels amongst the population of mutants of EV-A71 in the throat swabs of subjects with severe EV-A71 infections. Furthermore, in vitro studies showed that VP1-31D virus isolates had higher infectivity, fitness and virion stability, which sustained the virus infections in the digestive system. Speculations were that such factors benefitted the virus in gaining added viral adaptation and subsequently enabled viral spread to more tissues. These beneficial abilities could also justify the reduced number of VP1-31D viruses located in the brain following positive selection. The VP1-31G viruses presenting the major haplotype in the central nervous system displayed increased viral fitness and growth rates in neuronal cells. This implied that the VP1-31G mutations aided the spread of the mutant virus in the brain which resulted in serious neurological complications in patients. It was speculated that the fluctuating degree of tissue tropism of EV-A71 at diverse inoculation sites resulted in the bottleneck effect of the viral population having a mutant spectrum. Hence, the adaptive VP1-31G haplotype became dominant in neuronal tissues and once the infection was achieved, VP1-31G viruses expedited bottleneck selection and propagation into the skin and CNS. Among the three minor haplotypes (C to E) which co-existed in various tissues, the minor haplotype C was isolated in the intestinal mucosa and throat swab specimens. The minor haplotype D was isolated from specimens obtained from the respiratory and digestive systems. However, the minor haplotype E appeared in throat swabs and the basal ganglia but not the intestinal mucosa, hence, suggesting that the intestinal mucosa is the initial replication site of the EV-A71. Collectively, these data showed that the EV-A71 quasispecies utilized the dynamic proportion of varying haplotype populations to co-exist, sustained the ability of the population to adapt and enabled the propagation in different tissues. Lastly, the study concluded that the selection of haplotype(s) might be a driving factor in viral dissemination and severity of infections in humans as well as the virulence in EV-A71 infected patients [30] .

West Nile Virus (WNV)

A small-plaque (SP) variant was picked from a mutant population of WNV isolated from an American crow in New York in 2000. Characterization of this variant in mammalian, avian and mosquito cell lines led to the discovery that the SP variant contained four nucleotides in its genome that differed from the wild-type genome. Two nucleotide changes led to non-synonymous mutations where there was a P54S change in the prM protein and a V61A change in the NS2A protein. Further analysis of the mutations revealed that deletion at the cleavage site of the prM site did not affect virus replication and its release from mammalian BHK cells. However, the progeny of this virus was no longer able to infect BHK cells. A mutation in the prM region of the TBEV was also reported to cause decreased secretion of virus particles with no effect on protein folding. Lower neurovirulence and neuroinvasiveness were reported when mutation A30P occurred in the NS2A region of the isolate. Further sequencing of the isolate showed that most of the small plaque clones initially isolated reverted back to their wild-type sequence at position 625 in the prM region. Remaining isolates reverted at position 3707 in the NS2A region. These findings suggested that the mutation present in the prM region could be responsible for the phenotype of the small plaque. It is probable that the mutation in the NS2A region was responsible for the determination of the plaque size as the mutation in the prM region was sufficient to revert the isolate to the wild-type phenotype [52] .

Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

The nsp1 was reported to suppress protein synthesis by degrading the host mRNA but viral RNA could circumvent the nsp-1 mediated translational shutoff. Terada et al. showed that the double mutations (A9G/R13A) in the non-structural protein 1a (nsp1) affected viral propagation and the plaque morphology. The size of the plaque in the mutated MERS-CoV was smaller and the infectious titers and intracellular viral RNA were decreased in infected Huh7 or Vero cells when compared to the wild-type virus. The formation of the small plaque variant was due to impairment of viral replication via the disruption of the stem-loop (SL) structure of the RNA. In addition, analysis of the biological properties of the nsp1-A9G/R13A mutant showed that the mutant virus possessed low binding activity at the 5′-UTR and promoted translational shutoff against reporter plasmids with or without 5′-UTR [102] .
The nsp1 was reported to suppress protein synthesis by degrading the host mRNA but viral RNA could circumvent the nsp-1 mediated translational shutoff. Terada et al. showed that the double mutations (A9G/R13A) in the non-structural protein 1a (nsp1) affected viral propagation and the plaque morphology. The size of the plaque in the mutated MERS-CoV was smaller and the infectious titers and intracellular viral RNA were decreased in infected Huh7 or Vero cells when compared to the wild-type virus. The formation of the small plaque variant was due to impairment of viral replication via the disruption of the stem-loop (SL) structure of the RNA. In addition, analysis of the biological properties of the nsp1-A9G/R13A mutant showed that the mutant virus possessed low binding activity at the 5 -UTR and promoted translational shutoff against reporter plasmids with or without 5 -UTR [102] .
The nsp1 was reported to suppress protein synthesis by degrading the host mRNA but viral RNA could circumvent the nsp-1 mediated translational shutoff. Terada et al. showed that the double mutations (A9G/R13A) in the non-structural protein 1a (nsp1) affected viral propagation and the plaque morphology. The size of the plaque in the mutated MERS-CoV was smaller and the infectious titers and intracellular viral RNA were decreased in infected Huh7 or Vero cells when compared to the wild-type virus. The formation of the small plaque variant was due to impairment of viral replication via the disruption of the stem-loop (SL) structure of the RNA. In addition, analysis of the biological properties of the nsp1-A9G/R13A mutant showed that the mutant virus possessed low binding activity at the 5′-UTR and promoted translational shutoff against reporter plasmids with or without 5′-UTR [102] .

Newcastle Disease Virus (NDV)

NDV strains are categorized based on their pathogenicity in chickens as highly virulent (velogenic), intermediately virulent (mesogenic) or nonvirulent (lentogenic). These levels of pathogenicity can be differentiated by the amino acid sequence of the cleavage site in the fusion protein (F0). Lentogenic NDV strains have dibasic amino acids at the cleavage site whereas the velogenic strains contain polybasic residues. Meng et al. studied the changes in virulence of NDV strains, leading to a switch in lentogenic variant (JS10) to velogenic variant (JS10-A10) through 10 serial passages of the virus in chicken air sacs [128] . However, the lentogenic variants (JS10) remained lentogenic after 20 serial passages in chicken embryos (JS10-E20). The nearly identical genome sequences of JS10, JS10-A10 and JS10-E20 showed that after passaging, both variants were directly generated from the parental strain (JS10). Genome sequence analysis of the F0 cleavage site of the parental strain and the passaged variants revealed that the rise in virulence observed in the parental strain (JS10) stemmed from a build-up of velogenic quasispecies population together with a gradual disappearance of the lentogenic quasispecies. The decline of the lentogenic F0 genotypes of 112 -E(G)RQE(G)RL-117 from 99.30% to 0.28% and the rise of the velogenic F0 genotypes of 112 -R(K)RQR(K)RF-117 from 0.34% to 94.87% after 10 serial passages in air sacs was hypothesized to be due to the emergence of velogenic F0 genotypes. Subsequently, this led to the enhancement of virulence in JS10-A10. The data indicated that lentogenic NDV strains circulating among poultry could lead to evolution of the velogenic NDV strain. This velogenic NDV strain has the potential to cause outbreaks due to the difficulty in preventing contact between natural waterfowl reservoirs and sensitive poultry operations.
NDV quasispecies comprised lentogenic and velogenic genomes in various proportions. The change in virulence of the quasispecies composition of JS10 and its variants was investigated by analysis of viral population dynamics. The F0 cleavage site was reported to be the main region in which the majority of amino acid changes had occurred and resulting in an accumulation of variants exhibiting velogenic properties due to serial passages. Furthermore, passaging of the virus caused a transition in the degree of virulence of NDV strains from lentogenic to mesogenic and ultimately an increase of the velogenic type. Therefore, NDV pathogenesis could be controlled by the ratio of avirulent to virulent genomes and their interactions within the chicken air sacs and the embryo. The data clearly demonstrated that the status of the quasispecies population is dependent on the pathogenicity of the NDV [128] . Gould et al. reported the presence of the F0 cleavage sequences of 112 -RRQRRF- 117 and HN extensions of 45 amino acids in virulent Australian NDV strains [129] . Furthermore, the genome analysis of the avirulent field isolates of NDV puts forth the existence of viruses with virulent F0 sequences without causing obvious clinical signs of the disease [130] . Subsequently, Kattenbelt et al. studied the underlying causes that could affect the balance of virulent (pp-PR32 Vir) and avirulent (pp-PR32 Avir) variants throughout viral infections. The variability of the quasispecies population and the rate of accumulation of mutations in vivo and in vitro were analyzed. The in vivo analysis showed that both virulent and avirulent plaque-purified variants displayed a rise in the variability of quasispecies from 26% and 39%, respectively. The error rate in the viral sequences was observed to increase as well, such that one bird out of three displayed virulent viral characteristics ( 112 -RRQRRF-117 ) after passaging of the PR-32 Avir variant. Genome analysis following the in vivo study revealed that a single base mutation occurring in the F0 region led to the switch from RRQGRF to RRQRRF.
Quasispecies analysis of all the NDV field isolates in this study showed variable ratios (1:4-1:4000) of virulent to avirulent viral F0 sequences. However, these sequences remained constant in the quasispecies population during replication. It was concluded that the virulent strains present in the quasispecies population did not emerge from an avirulent viral population unless the quasispecies population was placed under direct selective pressure, either by previous infection of the host by other avian viruses or by transient immunosuppression [131] .

Influenza Virus (IV)

The annual influenza epidemics caused about 3 to 5 million cases of severe illness with 290,000 to 680,000 deaths worldwide [141] . Current influenza vaccines have sub-optimal efficacy, as there was a lack of antigenic proximity between the vaccine candidate and the circulating seasonal influenza virus strains. During the 2016-2017 influenza epidemic, the influenza A (H3N2) viruses from the clade 3c.2a were dominant and was associated with severe onset of the disease. The low vaccine efficacy of the 2016-2017 egg-adapted H3N2 (clade 3c.2a) vaccine strain A/Hong Kong/4801/2014 was reported to be due to altered antigenicity [142] . To understand the pathogenesis of A(H3N2) viruses from the 3a.2c clade, it would be of great interest to consider if each infection was being caused by an individual strain or by a swarm of genetically related viruses (quasispecies). This would help to provide an insight into the vaccine coverage and efficacy.
A study investigated the impact of antigenic proximity, genomic substitutions, quasispecies, diversity and reassortment in order to understand the molecular evolution of the influenza A (H3N2) isolated directly from clinical samples. Of the 155/176 whole genomes analyzed, several amino acid substitutions were found to substantially affect the severity of the infection caused by the clade specific viruses. Within the sample, 121 viruses belonged to the genetic clade 3c.2a.1 and eight belonged to 3c.2a2, twenty-four belonged to 3c.2a3, one belonged to 3c.2a4 and one belonged to a different clade 3c.3a. Many distinct substitutions spanning across the whole influenza proteome, HA, NA and non-structural protein 1 were found to be responsible for causing mild and severe disease. Interestingly, two substitutions, V261I and K196E, were found in the NA and the NS1, respectively. These two mutations were found to be particularly significant as they showed the distinction between the strains causing mild and severe infections. Analysis of the clinical isolates showed a difference in a single amino acid residue, 160K within the HA, whereby 14 cases of glycosylation loss was observed within the quasispecies population linked to severity of infection. Moreover, the degree of diversity within the quasispecies population was reported to be elevated in severe cases when compared to mild ones [143] .
The annual influenza epidemics caused about 3 to 5 million cases of severe illness with 290,000 to 680,000 deaths worldwide [141] . Current influenza vaccines have sub-optimal efficacy, as there was a lack of antigenic proximity between the vaccine candidate and the circulating seasonal influenza virus strains. During the 2016-2017 influenza epidemic, the influenza A (H3N2) viruses from the clade 3c.2a were dominant and was associated with severe onset of the disease. The low vaccine efficacy of the 2016-2017 egg-adapted H3N2 (clade 3c.2a) vaccine strain A/Hong Kong/4801/2014 was reported to be due to altered antigenicity [142] . To understand the pathogenesis of A(H3N2) viruses from the 3a.2c clade, it would be of great interest to consider if each infection was being caused by an individual strain or by a swarm of genetically related viruses (quasispecies). This would help to provide an insight into the vaccine coverage and efficacy.
A study investigated the impact of antigenic proximity, genomic substitutions, quasispecies, diversity and reassortment in order to understand the molecular evolution of the influenza A (H3N2) isolated directly from clinical samples. Of the 155/176 whole genomes analyzed, several amino acid substitutions were found to substantially affect the severity of the infection caused by the clade specific viruses. Within the sample, 121 viruses belonged to the genetic clade 3c.2a.1 and eight belonged to 3c.2a2, twenty-four belonged to 3c.2a3, one belonged to 3c.2a4 and one belonged to a different clade 3c.3a. Many distinct substitutions spanning across the whole influenza proteome, HA, NA and non-structural protein 1 were found to be responsible for causing mild and severe disease. Interestingly, two substitutions, V261I and K196E, were found in the NA and the NS1, respectively. These two mutations were found to be particularly significant as they showed the distinction between the strains causing mild and severe infections. Analysis of the clinical isolates showed a difference in a single amino acid residue, 160 K within the HA, whereby 14 cases of glycosylation loss was observed within the quasispecies population linked to severity of infection. Moreover, the degree of diversity within the quasispecies population was reported to be elevated in severe cases when compared to mild ones [143] .
A study aimed to identify the key mechanisms contributing towards co-pathogenesis of BALB/c mice infected with the A(H1N1) quasispecies. It was revealed that the co-evolution of the quasispecies brought about a complex response due to different expressions of the biphasic gene. A significant upregulation of the Ifng was associated with an increased majority of mutants expressing a differentially expressed gene (DEG) named HA-G222 gene. This correlated with the increased levels of pro-inflammatory response observed in the lungs of the mice infected with the quasispecies A(H1N1) [147] . Serial passages of the H1N1 virus was also carried out prior to the analysis of its sequential replication, virulence and rate of transmission. Sequence analysis of the quasispecies in the viral population revealed that from the ninth passage onwards, the presence of five amino acid mutations (A469T, 1129T, N329D, N205K and T48N) in the various gene segments (PB1, PA, NA, NS1 and NEP) was detected. Furthermore, mutations located within the HA region indicated that the genetic makeup of the viral quasispecies was distinctly different in the upper and lower respiratory tracts of the infected pigs [148] .
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Abstract

Oncolytic viruses (OVs) are engineered and/or evolved to propagate selectively in cancerous tissues. They have a dual mechanism of action; direct killing of infected cancer cells crossprimes anticancer immunity to boost the killing of uninfected cancer cells. The goal of the field is to develop OVs that are easily manufactured, efficiently delivered to disseminated sites of cancer growth, undergo rapid intratumoral spread, selectively kill tumor cells, cause no collateral damage and pose no risk of transmission in the population. Here we discuss the many virus engineering strategies that are being pursued to optimize delivery, intratumoral spread and safety of OVs derived from different virus families. With continued progress, OVs have the potential to transform the paradigm of cancer care.

viruses as anticancer drugs

It is well established in several rodent cancer models that a single dose of an effective OV can completely cure disease [8, 9] . This has been shown for DNA and RNA viruses in diverse tumor models. However, while the single shot cure is an exciting prospect for cancer therapy, to date clinical outcomes have typically fallen short of this, and repeat IT virus administration has proven to be a more reliable approach [6, 10] . But there are a number of anecdotal case reports that give credence to the idea that a single shot OV cure may be achievable in clinical practice [4, 11] suggesting that OVs have the potential to transform the practice of oncology.

engineering viruses: a diversity of platforms

In nature, viruses are continuously evolving and adapting to occupy almost every imaginable biological niche. Viruses infect bacterial, archaea, protist, fungal, plant and animal cells. Their genomes are composed of DNA or RNA, which may be single or double stranded, positive or negative sense, ranging in size from 2 to 300 kb, in complexity from 1 to 300 genes and in capacity for foreign genetic material from a few hundred bases to several kilobases. The particles of viruses from different families also vary enormously in size and structure, ranging from 20 to 1000 nm, from icosahedral to helical symmetry, with or without lipid envelope, integument or matrix and with variable susceptibility to physical disruption. Naturally occurring viruses also offer a vast diversity of virus life cycles, cell entry and replication mechanisms, cell and species tropisms, cycle times, burst sizes, innate immune evasion, apoptosis, antiviral state prevention and immune combat strategies, modes of transmission and pathogenic mechanisms. The diversity of viruses that have been investigated as oncolytic platforms and the different strategies used to improve their efficacy continue to be expanded (Table 1) .

Delivery of Ovs

OV therapy begins with the administration of the virus to the patient just as with any other drug. Several approaches can be taken, each with to optimize oncolytic virotherapy indicated that maximizing delivery, so more tumor cells are infected, has a profound impact on the degree to which the virus must propagate from each infected cell for curative therapy [60] . The higher the number and the more random the dispersion of infectious centers established in a tumor after delivery of virus, the more effective the virus will be, although these parameters must be optimized for each virus and model [61] . IV and IT administrations of OVs are common delivery methods that introduce large amounts of virus into one compartment in a very short time. This rapid delivery of a massive virus inoculum does not parallel what typically occurs in a natural infection and potentially allows the IT spread of the OV infection to outpace host defenses. Successful delivery in the context of an OV infection requires that a critical viral concentration is achieved within the tumor to allow for sufficient oncolysis and systemic spread of the virus to all sites of disease in a safe and reproducible pattern.
Mouse models with implanted flank tumors provide a convenient model for IT injection as the tumor is superficial, but in human patients with spontaneous malignancies tumors often develop internally in visceral organs. Melanoma, head and neck cancer, and lower gastrointestinal malignancies may be better suited for IT injections due to anatomical position, although with advanced interventional radiology using computed tomography or MRI image guidance to place the needle many more tumors may be accessible for IT virotherapy. One additional advantage of IT injection over the IV route is that the threshold concentration of input virus required to initiate a spreading infection in the tumor tissue can be more easily achieved.

• Limiting viral neutralization & clearance

Antibody neutralization has been shown to reduce the efficacy of systemically administered OVs such as measles, VSV and vaccinia in preclinical models [76] [77] [78] . When previous exposure has occurred, preformed antibody reduces the effectiveness of the initial treatment. However, the situation is typically far more problematic for second and subsequent doses, even for low seroprevalence viruses, because the first dose induces a powerful primary antiviral antibody response or boosts the pre-existing response [79, 80] . These troublesome responses can be constrained by coadministering immunosuppressive drugs such as cyclophosphamide. Cyclophosphamide administered concurrently with an OV has been shown to suppress or delay the development of humoral and cytotoxic antiviral T-cell responses [81] .
Mengovirus, VSV and Newcastle disease virus are examples of animal pathogens being developed as oncolytic agents. Their appeal as oncolytic platforms is due to the coupling of their ability to selectively propagate in human tumors with their low seroprevalence in the human population [77] . Unlike viruses such as measles which have almost exclusively human cell tropism, these zoonotic viruses are capable of infecting both rodent and human tumor cells allowing preclinical testing in more informative immunocompetent mouse cancer models.

Ov spread

Engineering tumor specificity can also be achieved by detargeting viruses to ablate unwanted tropisms that can cause off target pathology in normal tissues. MircroRNA targeting is the best example of this approach. For example, miRNA targeting was used to control the tropism of an oncolytic coxsackievirus A21 virus which caused rapid tumor regression followed by fatal myositis in murine models of myeloma and melanoma [119] . Insertion of muscle-specific miRNA targets into the viral genome eliminated muscle toxicity but left the antitumor potency of the virus intact. This was shown to be due to miRNA-mediated recognition and rapid destruction of the viral genome in muscle cells. miRNA targeting has since been applied to many OVs from diverse virus families and provides a convenient and economical strategy (using sequence insertions of only ∼100 bases) to control unwanted virus tropisms [25] .

• viral amplification & spread

After an appropriately targeted virus has infected a tumor cell, it is the extent of its subsequent propagation that becomes the key driver of potency. Data-driven mathematical models of systemic oncolytic virotherapy indicate that tumor eradication is dependent on two major parameters: the initial density and distribution of infectious foci in the tumor; and the ultimate size of the infectious centers arising from each individual infected cell (i.e., virus spread) [120] . Viral spread may occur by various mechanisms. Local spread may occur by intercellular fusion, by direct transfer of virus from infected to adjacent cells, or by release and local migration of progeny virions through the interstitial space. Systemic spread as free virus particles or as virusinfected migratory cells occurs via lymphatic channels or via the bloodstream.
Another factor that significantly impacts the kinetics of virus spread is the burst size, or the number of progeny viruses released by a productively infected cell, which varies widely between viral families. Picornaviruses, VSV and vaccinia virus can release up to 10,000 progeny from a single infected cell after a delay of only 6-18 h [131] [132] [133] . In addition to innate antiviral immunity, adaptive cell-mediated immune responses are typically required for the complete elimination of a viral infection and act by eliminating infected cells before progeny can be released. Oncolytic virotherapy can therefore be viewed as a race between the spreading virus and the responding immune system. For this reason, faster moving viral infections are often considered capable of inflicting greater damage to an infected tumor before they can be contained by the immune system [134] . However, in defense of the viruses with smaller burst sizes, or which release progeny by budding, they tend to be less review Maroun, Muñoz-Alía, Ammayappan, Schulze, Peng & Russell future science group rapidly controlled both by the innate and adaptive host immune responses [135] . Therefore, as with the classic race between the hare and the tortoise, it is very difficult to predict whether a fast or slow replicating virus will show superior efficacy in a given preclinical cancer model.

• • Arming Ovs to amplify antitumor immunity

Each step of the above process can be impacted by virus engineering. Not only can the kinetics and mode of cell killing be manipulated (see the previous section on virus spread), but also the OVs can be armed with a wide variety of immunoregulatory genes whose products will be secreted into the interstitial fluid space. Thus, OVs have been engineered to express the following: high levels of type I interferons to better drive the early innate/inflammatory response; GM-CSF to stimulate the phagocytic activity and lymph node trafficking of professional antigen presenting cells; chemokines to enhance the IT recruitment of immune and inflammatory effector cells, especially cytotoxic T cells; cytokines to drive the activation and proliferation of tumor-resident cytotoxic T cells; bispecific T-cell engagers to enable tumor cell killing by T cells not recognizing tumor antigens; checkpoint inhibitor antibodies to block the protective PD-L1/B7.1 shield that protects tumor cells from T-cell attack; cytokines to increase MHC-peptide neoantigen expression on uninfected tumor cells; or cloned tumor antigens to further amplify tumor-specific immunity [165] .
While each of these approaches has proven beneficial in selected preclinical animal models where immune destruction of the tumor is the dominant OV effect, they are equally likely to decrease potency by speeding virus elimination in situations where the spread of the virus is dominant.

• • Limiting pathogenicity

Viral cytotoxicity is the basis of tumor cell death necessary for oncolytic activity. Off-target infection and killing of normal cells by poorly targeted OVs or by OVs that have evolved new tropisms in a treated patient can cause unwanted normal tissue pathology. OVs are therefore delicately balanced between retaining enough virulence to substantially decrease tumor burden versus being sufficiently targeted (or attenuated) to not cause a new disease in the patient. Cancer-specific targeting is the most critical safety feature, but viruses evolve and viral populations are dynamic [172] . Evolution is a constant accompaniment of a spreading virus infection, whether or not the virus is oncolytic; in vivo progeny of the therapeutic OV differs on average by a single -point mutation per genome from the input virus [173] . Hence, as the input virus is amplified, it generates a swarm of quasispecies viruses, each one a slightly imperfect replica of the input virus. This swarm of progeny viruses is subjected to selective pressure as it encounters new biological niches in the treated cancer patient. Thus, if it so happens that a member of the swarm is capable of infecting a normal host tissue, the virus may have gained a new foothold from which to further evolve. Gaining new cell tropisms or losing restriction factors is therefore a significant theoretical concern in oncolytic infections, but has not yet been documented in human trials, nor in preclinical models. However, given the importance of this particular scenario, a great deal of attention is paid to the problem not just by investigators, but also by regulatory agencies.

• • Sodium iodide symporter (radioconcentrator)

Iodide is a critical component of thyroxine and is concentrated in thyroid follicular cells by the thyroidal sodium iodine symporter (NIS), a cell surface glycoprotein with 13 transmembrane domains [146] . Radioiodine is therefore used routinely in the clinic for thyroid imaging and for ablation of overactive thyroid tissue, including metastatic thyroid cancer. This is facilitated by the ready availability of several iodine radioisotopes, most notably 123 Ifor γ camera and single-photon emission computed tomography imaging, 124- for PET imaging and 131 I -, a β-emitting isotope, for thyroid ablation. Besides radioiodine, NIS can concentrate several related anions, of equal or greater value for single-photon emission computed tomography ( 99m TcO 4 -, pertechnetate), PET (B 18 F 4 -, tetrafluoroborate) and tissue ablation ( 211 At -, astatide, 188 ReO 4 -, perrhenate) [147] . The β emissions of 131 Ihave an average path length of approximately 1.8 mm in tissue and can therefore inflict significant damage on cells adjacent to an 131 I --loaded NIS expressing cell. For 188 ReO 4 the β emission path length is longer such that NIS negative tumor cells are even more likely to be damaged in the β particle crossfire when this radioisotope is used. Unsurprisingly, therefore, the NIS gene has been engineered into several OVs whose IT spread has been elegantly mapped and monitored in tumor-bearing mice by serial radiotracer imaging, and whose potency has been substantially boosted by appropriately timed administration of 131 I - [148] . At least two NIS-expressing OVs, a prostate-targeted oncolytic adenovirus and a CD46-targeted measles virus, have been advanced to human clinical trials with positive imaging data reported [11, 149] .

viruses as anticancer drugs

Oncolytic viruses (OVs), evolved and engineered for cancer specificity, are gaining momentum as a new drug class in the fight against cancer. Besides, causing the death of virus-infected cancer cells, the spreading intratumoral (IT) infection can also boost the anticancer immune response, leading to immune destruction of uninfected cancer cells. The paradigm of OVs has been reviewed extensively [1] [2] [3] [4] [5] [6] [7] .
Based on these clinical advances, development of OVs is rapidly accelerating. The purpose of this review is to discuss the virus engineering approaches and OV performance optimization strategies that are being pursued in the field, and to point out some of the challenges that remain. We believe this perspective will be particularly valuable to virologists entering the field. We apologize to the numerous investigators whose work has not been acknowledged; we are well aware of these omissions but due to space constraints our approach has been choosing illustrative examples that demonstrate the key principles. The review is divided into five main sections: (i) an introduction to viruses and virus engineering, (ii) delivery, (iii) spread, (iv) arming and (v) safety. All five topics are highly interdependent.

engineering viruses: a diversity of platforms

Aside from their differences, viruses share fundamental similarities including their dependence on the host cell to provide a suitable environment for genome amplification, gene expression and progeny virus production and their adaptation to a specific set of host cell conditions and factors such that propagation is precluded in cells that fail to provide the necessary environment. These characteristics allow viruses to be targeted to cancer cells as a self-replicating antineoplastic therapy.

Delivery of Ovs

• • iv delivery IV delivery of an OV seems advantageous in the setting of metastatic disease, allowing the virus to potentially access all sites of disease via the circulation soon after infusion. In a clinical setting, systemic delivery may be preferred since it is more broadly applicable than IT inoculation, regardless of tumor location and total tumor burden. Yet, attempts at systemic delivery have shown limited success; the administered virus is immediately diluted in the circulating blood volume (∼5 l), so extremely high doses are needed to achieve meaningful circulating titers. A clinical trial using an oncolytic pox virus, JX-594, demonstrated that virus could only be detected in tumors of patients that received at least 10 9 p.f.u. per IV dose without any virus being detected in tumors of the lower dose cohorts of patients [62] . Because of this important dilution effect, doses required for IV therapy may have to be up to 1000-fold higher than for IT administration, which results in significant manufacturing challenges as well as a unique set of toxicities [2] . Administration of high titers of virus intravenously can lead to hepatotoxicity, thrombocytopenia and lymphopenia [33, 63] .
Following the path of an OV from the IV line to the tumor will quickly illustrate some of the challenges for IV delivery. First, the virus is rapidly diluted in the circulation, where its infectivity is neutralized by serum proteins including antibodies and complement, and a large proportion is sequestered by reticuloendothelial phagocytes in the spleen and liver. Any virus particles that reach the tumor vasculature without first being neutralized must then extravasate through fenestrations, and pores between the vascular endothelial cells of the tumor capillaries, and once in the interstitial space of the tumor, must negotiate the extracellular matrix (ECM) to reach and infect the resident cancer cell [64] .
Tumor vasculature can both aid and limit viral access. Tumors typically have an unorganized growth pattern, producing vasculature that is tumultuous and insufficient resulting in heterogeneous perfusion throughout the parenchyma [65] . Decreased perfusion limits the formation of infectious centers evenly throughout a tumor. Manipulating physiological parameters such as blood pressure and systemic vascular resistance can preferentially increase tumor perfusion and promote better delivery of viral vectors [66] . Once in a tumor vessel, the virus must move from the intravascular compartment into the interstitium by crossing the vascular endothelium to continue its journey through the interstitial space until finally reaching a permissive tumor cell.
At least in theory, viruses can cross the endothelial lining of tumor neovessels by diffusion, active infection, or by trafficking inside of or on the surface of marrow-derived cells that are capable of diapedesis. Tumors generally have poor lymphatic drainage and can have leaky blood vessels with a wide range of pore sizes which helps to explain the phenomenon of enhanced permeability and retention of nanoparticles such as OVs in the tumor parenchyma [67] . In some tumors, the pores and fenestrations in tumor capillaries may be large enough to permit passive diffusion of viruses (depending on their size) but in many tumors review Maroun, Muñoz-Alía, Ammayappan, Schulze, Peng & Russell future science group the endothelium is sufficiently organized that this type of extravasation is simply not possible.
Another approach to help OVs' extravasate is by temporally creating pores throughout the tumor microvasculature to enhance transport of the virus into tumor interstitium. This can be done through application of focused ultrasound in the presence of a contrast agent or a polymer. The microbubbles or polymers are injected intravenously and can freely circulate without harming tissues, but when they are exposed to ultrasound waves they can oscillate and induce pore formation or cavitation in the surrounding tissue. The ultrasound can be focused on a tumor just after the infusion of the virus to increase viral extravasation. This has been shown to enhance vaccinia virus delivery to tumor xenografts and can increase viral transgene expression by more than a 1000-fold in the tumor [71, 72] . Many strategies are being developed to enhance OV IV delivery.
• • iT delivery IT administration directly delivers a high concentration of the OV into the parenchyma of the injected tumor but may not result in spread of the virus to distant sites of metastasis. T-Vec is administered by IT injection to accessible skin tumors of patients with metastatic malignant melanoma. The injection is repeated every 2 weeks until lesions have resolved or failed to respond [12, 73] . Interestingly, viremia was not documented in patients, suggesting that the regression of noninjected lesions was likely immune-mediated and not due to direct oncolysis [74] .
Immediately following IT delivery, small levels of virus can be detected in the blood from leakage through injured tumor vasculature [75] , but that small amount of virus is quickly cleared from the system. Also, virus that has been injected into a tumor is frequently extruded immediately from the injection track once the syringe is removed, especially in smaller higher pressure tumors. However, systemic viral spread can occur following IT administration if the initially exposed tumor cells are capable of amplifying the input virus and releasing progeny into the bloodstream. Such secondary viremia may not peak until several days after IT virus delivery and, with currently used OVs, is often not detected. Secondary viremia, when it occurs, more closely mimics the natural propagation of a viral infection from the site of inoculation to more distant target organs.

• Other routes of delivery

Intraperitoneal virus administration is often pursued in virotherapy studies aiming to impact ovarian cancer and other disseminated intraperitoneal malignancies, while intrapleural administration is pursued for mesothelioma therapy. Both of these approaches are similar to IT administration in that the virus comes into direct contact with tumor cells in the injected cavity, although there is a greater risk that input virus will be immediately neutralized by antibodies and other proteins in ascites or pleural fluid. Intravesical instillation of virus is the favored route of administration for treatment of early-stage bladder cancer, requiring only that future science group www.futuremedicine.com the input virus be stable in urine. Also, immediately after brain cancer surgery, in an attempt to control residual disease, virus is often instilled directly into the resection cavity.

• Limiting viral neutralization & clearance

Perhaps the most significant barrier to widespread OV delivery is rapid neutralization and clearance of circulating virus particles. Antibodies and complement proteins can coat the virus blocking its ability to interact with its cellular receptor and accelerate Fc receptor-mediated clearance by splenic macrophages and hepatic Kupffer cells.
For high seroprevalence viruses, one approach to circumvent viral neutralization by preformed antibodies is to engineer or switch the viral coat proteins. Some OVs (e.g., adenovirus) offer a menu of different serotypes, providing a basis for serotype switching between successive doses of the therapy to avoid antibody neutralization, although this does greatly complicate the product development pathway since each serotype is considered to be a distinct pharmaceutical product [82] . For monotypic viruses such as measles, serotype switching is not an option, but antimeasles antibodies can be circumvented by substituting the surface glycoproteins of measles with those of a related but noncross-reactive morbillivirus such as canine distemper virus [83] . Alternatively, the immunodominant epitopes of the measles surface glycoproteins can be modified by mutating key surface residues to eliminate them, or by introducing glycosylation signals so they are shielded by N-linked glycans [84] . It is worth noting that all of these virus engineering strategies have the potential to alter viral receptor usage and hence tumor cell tropism which may limit their utility [83, 85] .
Virus-infected cell carriers can also be used to transport viruses via the bloodstream to sites of tumor growth. In part, this is possible because there is an 'eclipse period' after the cell carriers have been infected during which they do not display viral proteins on their surface and are therefore not bound by virus neutralizing antibody, but are still able to extravasate from tumor neovessels and release infectious progeny into the tumor parenchyma. Several cell carrier/OV combinations have been used including mesenchymal stem cells, dendritic cells, T cells and endothelial progenitor cells [89] [90] [91] [92] . Some carrier cells are believed to home more efficiently to tumors responding to chemotactic signals arising from hypoxia or IT inflammation.

Ov spread

Replication and amplification in the tumor is the major feature that sets OVs apart from other anticancer drugs. No traditional chemotherapy, immunotherapy or small molecule inhibitor can target tumor cells and then amplify at the site of action and spread to other sites of tumor growth. Specificity as well as speed and extent of IT virus replication are the key determinants of therapeutic index (efficacy/toxicity ratio) for an OV therapeutic, and each of these parameters can be modified by virus engineering. Additionally, the kinetics of spread can be impacted by combining the virus with immunomodulatory drugs. The idea of a virus with selective tropism for cancerous tissue has obvious appeal. Even for naturally occurring viruses that have not been tropism modified, a tumor offers a favorable environment to support a productive infection, and this is borne out by case reports of temporary remission or regression of different cancers concurrent with viral infections [4, 93] . There are several reasons why tumors are generally more susceptible than normal tissues to virus attack; poorly developed lymphatics, high nonsuppressible metabolic activity, resistance to apoptosis, poor responsiveness to interferon and intrinsic suppression of immune effector cells. But as we move in the direction of intentionally using virus infections to mediate tumor destruction, it is apparent that a targeted virus with exquisite tumor specificity will be superior to its non targeted counterpart, allowing for the administration of higher tumor destructive doses without toxicity to normal tissues.
Virus tropism is determined by many factors, most of which, if understood sufficiently, can be manipulated to enhance tumor specificity. The receptor tropisms of naturally occurring viruses are rarely of interest for tumor targeting, but this is not true for tissue-culture-adapted vaccine lineage viruses, some of which have evolved in the laboratory to use receptors more abundantly expressed on cancer cells. Examples include the CD46 receptor tropism of vaccine lineage measles virus and the heparan sulfate tropism of laboratory-adapted Sindbis virus [94, 95] . Where greater specificity is desired, it may be possible to engineer new receptor tropisms by modifying the structure of a viral attachment protein, for example, by displaying polypeptide ligands at the extreme C-terminus of the measles H glycoprotein [96, 97] . However, for many viruses, displaying a polypeptide ligand on the surface does no more than redirect attachment and does not confer a new receptor tropism [98] [99] [100] . This remains an area of active investigation.
In addition to their dependence on specific entry receptors, the surface glycoproteins of many enveloped viruses (and sometimes other critical viral proteins) must be proteolytically activated before they are competent to mediate virus entry [101] . By engineering the protease target sequences in these viral glycoproteins it is possible to generate viruses whose propagation is now dependent on exposure to a specific protease with high IT abundance, such as urokinase, matrix metalloproteinase 1 or cathepsin D [102] [103] [104] .
Beyond the step of cell entry, viruses are exquisite sensors of intracellular processes and can therefore be adapted or engineered in several ways for intracellular targeting of cancer cells. This is best understood by considering some fundamental aspects of the interplay between a virus and an infected cell. The incoming virus aims to usurp the cellular synthetic machinery for generation of progeny viruses. The cell resists this takeover bid by rapidly detecting virus invasion, then triggering a signaling cascade that leads to establishment of an antiviral state and release of interferon which induces an anti viral state in adjacent cells [105, 106] . The antiviral state is very complex but suppression of protein translation is a key component. Apoptosis is also triggered by the virus detection machinery so that the infected cell dies before it is able to manufacture virus progeny [107] .
For a virus to be 'successful' it must combat these host cell responses, avoiding detection, as long as possible, for suppressing the establishment of an antiviral state and preventing apoptosis. Virtually all naturally occurring viruses therefore encode proteins that inhibit apop tosis and the antiviral state [108] . Removing these accessory functions from the viral genome leads to virus attenuation in normal tissues, but to a much lesser degree in cancerous tissues. This is because cancer cells are intrinsically resistant to apoptosis and to the establishment of an antiviral state, making them highly susceptible to attenuated viruses that are no longer able to control those processes. This has provided a mechanistic basis for physiologic targeting of several viruses; VSV by mutating the matrix gene whose encoded protein blocks the interferon response [109] ; HSV by mutating both copies of the γ-34.5 gene which interferes with interferonmediated shutoff of host protein synthesis and enhances neurovirulence [110, 111] ; adenovirus by mutating the E1B protein, one of whose actions is to inhibit the apoptotic activity of p53 [112] .
Viruses can be further engineered to exclusively replicate in tumor cells by combining a virus' needs with physiologic peculiarities intrinsic in tumorigenesis. For example, a virally encoded thymidine kinase (TK) is required for HSV and vaccinia virus infection to increase the supply of deoxynucleotide triphosphates required for synthesis of progeny virus genomes [113, 114] . Elimination of the TK future science group www.futuremedicine.com gene from the viral genome restricts viral replication to cancer cells where there is an upregulation of human TK [115, 116] . Another example of a virus that exploits the high replication rate of tumor cells is Toca-511, a replication competent C-type retrovirus encoding the drug activating enzyme cytosine deaminase (CD) [117] . Since the integration of C-type retroviruses is S-phase dependent [118] , this virus is selectively amplified in rapidly proliferating tumor tissue.

• viral amplification & spread

Direct cell-to-cell transfer of viruses has the advantage of stealth as the virus cannot be neutralized by antiviral antibodies in the interstitial fluid [121] . Nonfusogenic viruses can be armed with fusogenic membrane glycoproteins (FMGs) to enable stealthy spread through intercellular fusion leading to the formation of large, nonviable multinucleated syncytia which may also serve as excellent antigen presenting cells for amplification of the antitumor immune response [122] . By way of example, VSV encoding measles fusogenic glycoproteins and HSVs encoding the fusogenic gibbon ape leukemia virus glycoproteins have shown superior efficacy when compared with their corresponding parental viruses [123, 124] .
In the case of free virus particles, which are susceptible to antibody neutralization, the stroma of the tumor also has the potential to limit IT diffusion and block systemic release. Fibroblasts, endothelial cells, immune cells and the ECM make up the tumor stroma, and the exact composition varies widely depending on tumor type [125] . The ECM is a collagenous matrix of protein fibrils, adhesive proteins and proteoglycans that create a web with pore sizes similar or slightly smaller than virions [126] . Destruction of ECM components can facilitate viral spread throughout the tumor and can be achieved using conventional chemoradiotherapy or by delivering matrix-degrading enzymes such as collagenase and hyaluronidase [127] . An alternative approach is to encode a matrix-degrading enzyme or inducer of matrixdegrading enzymes in the viral genome. For example, hyaluronidase and relaxin encoding oncolytic adenoviruses have each been shown to spread more efficiently in experimental tumors [128] [129] [130] .

• • Secreted toxins

As a general rule, if a virus is to be armed with a gene encoding a secreted toxin, that toxin should be targeted so that it can kill only cancer cells. In the absence of such targeting, there would be little prospect of avoiding off-target toxicities. Immunotoxins are bifunctional proteins in which plant or bacterial toxins (typically ribosomal inhibitors) are fused to a single chain antibody or other polypeptide domain to target endocytosis in cancer cells [136] . In theory, such molecules that have been extensively investigated and advanced to human clinical trials particularly for the treatment of B-cell and T-cell malignancies [137] , could be expressed from an engineered OV genome.

• • Fusogenic membrane glycoproteins

Fusion of the lipid envelope of an incoming virus with the limiting membrane of its target cell is a necessary step in the life cycle of an enveloped virus, to deliver the encapsidated viral genome into the target cell cytoplasm. This virus-to-cell fusion reaction is mediated by FMGs embedded in the envelope of the virus, and may occur at the cell surface at neutral pH, or in the endo-somal compartment at acidic pH [150] . Neutral pH fusion is triggered by receptor attachment. Cells expressing certain virally encoded, neutral pH-active FMGs on their surface may fuse with neighboring receptor positive cells (cell-to-cell fusion), giving rise to multinucleated syncytia, the hallmark cytopathic signature of a fusogenic virus (e.g., measles) [151] . But more often the FMG is activated only after its incorporation into a budding virus particle, so cannot cause cell-tocell fusion, and must be modified, for example, by cytoplasmic tail truncation, to render it constitutively fusogenic. Either way, FMG-driven fusion of OV-infected cells with uninfected neighboring cells leads to increased bystander killing because multinucleated syncytia are nonviable as well as being highly immunogenic [152] . Based on these observations, nonfusogenic OVs have been armed with FMGs thereby conferring superior oncolytic potency [123, 153] . In one example, an oncolytic herpes virus was rendered highly fusogenic when engineered to encode a cytoplasmically truncated gibbon ape leukemia virus envelope glycoprotein [154] [155] [156] while in another study, an oncolytic VSV was rendered highly fusogenic by replacing its surface glycoprotein (G) with the hemagglutinin and fusion glycoproteins of measles virus.

Safety

Engineering viruses for increased stealth, greater specificity, faster spread and enhanced potency is not without risk. As OVs gain new properties, checks and balances must be applied to assure that they do not readily evolve into pathogens, let alone transmissible pathogens that could pose a risk not only to immediate contacts of the treated patient, but also to the greater population.

• • Limiting pathogenicity

Polymerase infidelity is not the sole driver of virus genome diversification and evolution. For many viruses, recombination between homologous viral genomes can occur when two related viruses infect the same cell [178] . This type of recombination is not a feature of negative strand RNA virus evolution because the nascent progeny viral genomes are cotranscriptionally incorporated into a helical ribonucleocapsid structure which prevents recombination. However, for positive sense RNA picornaviruses, and DNA viruses such as adenovirus, HSV and vaccinia, it is important to consider the possibility that the input virus may encounter and recombine with a homologous 'wild-type' virus in a treated patient. While such recombination has never been documented in OV trials, there are well described examples in the field of vaccinology, such as the re-emergence of pathogenic polioviruses through the recombination of vaccine genomes and naturally circulating picornavirus genomes [179] . In view of these risks, it is generally considered inadvisable to arm viruses with therapeutic transgenes that have theoretical potential to increase pathogenicity if transferred to a related naturally circulating virus. Genes encoding immunosuppressive, antiapoptotic or directly cytotoxic proteins are on this list.
Certain viruses with segmented genomes, most notably the orthomyxoviruses (e.g., influenza), are capable of rapid evolution by genome fragment reassortment in multiple infected cells. Reassortment of genome fragments in virus-infected birds and pigs is considered to be a key driver of the antigenic shifts that occur during evolution of new pathogenic influenza virus strains [180] . This important safety concern may explain why oncolytic influenza viruses have not yet been advanced to human clinical trials [181] . Because of the toxicity risks associated with the proliferative and evolutionary capacity of OVs, there is considerable interest in contingency plans to terminate the spread and/or transmission of an OV infection. Reliable antiviral medications are available for TK-expressing herpes viruses, but this is not the case for the majority of viruses being developed as OV platforms [182, 183] . Attention has therefore been directed to the development of safety switches that can be engineered into the OV genome and triggered on demand to terminate in vivo replication.

• Limiting transmissibility

For obvious reasons OV transmission from a treated patient to a caregiver, family member, coworker, pet or other species is highly undesir-able. Infectious virus particles may be present in the blood of a treated patient, and may be shed into the environment in urine, feces, saliva and other bodily secretions. Contact with the body fluids of an OV-treated patient therefore may have the potential to spread an infection to new hosts [190] .
Because the risks of OV transmission are typically unknown when first-in-human Phase I trials are initiated, it is usual to implement standard infection containment measures throughout these studies and to carefully monitor body fluids for the appearance and disappearance of viral genomes and infectious virus progeny [191] . For certain viruses, containment may be considered unnecessary, particularly when there is already widespread population immunity to the OV in question (e.g., measles) or when there has been extensive human experience of exposure to a related virus in the form of a live viral vaccine (e.g., vaccinia). Contingency plans may also be required calling for quarantine of OV-treated patients if treatment toxicity is associated with a longer period of shedding. In reality, to date there has been no instance in which transmission of an OV from a patient to a caregiver or other contact has been demonstrated, and there are no examples of long-term virus persistence or shedding in a treated patient.
In light of the inconvenience and undesirability of OV shedding, there is interest in engineering strategies that may selectively interfere with the process. As one example, wild-type measles virus is a highly transmissible airborne virus that uses the nectin-4 receptor to enter into airway epithelial cells from whence its progeny are shed into respiratory secretions [192, 193] . Eliminating the nectin-4 tropism by strategically mutating key surface residues in the hemagglutinin attachment protein results in a virus that still causes measles in nonhuman primates, but which is no longer shed into respiratory secretions or urine [194] . Measles virus RNA (but not infectious virus) was detected in the blood, urine and saliva of myeloma patients up to 3 weeks after IV administration of an oncolytic measles virus, especially at higher dose levels, and it is possible that this shedding might be eliminated by using a nectin-4 blind version of the virus [195] . Virus shedding has also been detected in human clinical trials of HSV, reovirus, vaccinia, reovirus and adenovirus oncolytics, but was less readily detected following IT therapy with oncolytic VSV and polioviruses [167] . However, future science group www.futuremedicine.com it should be noted that while genome sequences have been detected in shed material, infectious virus particles have not been recovered.
In an OV-treated patient pre-existing antiviral immunity can be a formidable barrier to efficacy, particularly if the virus is administered intravenously. However, pre-existing antiviral immunity in caregivers and patient contacts provides reassuring protection against virus transmission. Conversely, when this particular efficacy barrier is circumvented by using OVs engineered for antibody evasion or selected for low seroprevalence, the risk of epidemic spread in the human population, unchecked by pre-existing herd immunity, looms larger.
An alternative strategy often used to side step antibody neutralization, at least of the first dose of virus administered, is to use OVs derived from zoonotic animal viruses such as New Castle disease virus (chicken), VSV (cattle), myxomavirus (rabbit), Seneca Valley virus (pig) or mengovirus (e.g., mouse and monkey). Additional regulatory executive summary viruses as anticancer drugs • Viruses naturally possess many properties that favor infection of cancer cells. Enhancing these natural properties and adding new properties through directed evolution and genetic engineering are used to create oncolytic viruses (OVs), which are emerging as a new anticancer drug class.
• The diversity of virus families and engineering techniques allows for the creation of OVs with a wide range of properties that can be tailored for each type of cancer.

Delivery of Ovs

• Intravenous delivery allows a virus to reach distant sites of metastasis via the circulation, but extravasation into the tumor parenchyma is inefficient.
• Intratumoral injection can concentrate virus at a site of tumor growth, but regression of distant tumors requires that the virus spread systemically or induce a systemic antitumor immune response.
• Neutralizing antibodies, hepatosplenic sequestration of the virus by macrophages and dilution of the virus in blood or tissue may limit the effectiveness of treatment.

viral spread

• Targeting viral spread to tumor cells can be accomplished by transductional targeting (modifying receptor tropism), transcriptional targeting (controlling virus gene expression with tumor-specific promoters), physiologic targeting (disrupting viral immune combat proteins), apoptosis targeting (disrupting viral antiapoptotic proteins) or miRNA targeting.

Safety

• Ideally, OVs should be nontransmissible. scrutiny is generally required for these viruses (e.g., from the US Department of Agriculture) to address the additional risks of environmental release and epidemic spread in domestic animals, particularly as they relate to agricultural livestock.

Conclusion & future perspective

Viruses are at last being harnessed for the benefit of cancer patients. The OV field has moved well beyond proof of principle in human studies, and virus engineering will be the key to its continued advancement in the coming years. Virtually every component of every naturally occurring or laboratory-adapted virus can be engineered and/or evolved to enhance its suitability for cancer therapy and we are currently witnessing unstoppable creative activity in this area. Safety is obviously of paramount importance in this relatively new field, and is therefore closely regulated from the design stage to clinical implementation. Considering the current trajectory of OV research, there can be little doubt that viruses are on their way to becoming one of the foundational modalities of future cancer treatment regimens.

viruses as anticancer drugs

In light of recent clinical progress, interest in the approach is burgeoning. One critical milestone was the 2015 marketing approval granted in Europe and the USA for talimogene laherparepvec (T-Vec, Imlygic™), an engineered HSV encoding GM-CSF. This virus, administered intratumorally every 2 weeks for malignant melanoma, led to complete resolution in 47% of injected tumors and boosted systemic antitumor immunity leading to resolution of 9% of distant un infected visceral tumors [12] . Subsequent clinical studies have shown that responses are more frequent and more durable when T-Vec (and other OVs such as coxsackievirus A21) is combined with immune checkpoint For reprint orders, please contact: reprints@futuremedicine.com review Maroun, Muñoz-Alía, Ammayappan, Schulze, Peng & Russell future science group inhibitor antibody therapy [13] . Another significant milestone was the demonstration that a systemically administered oncolytic measles virus can target and destroy disseminated cancer in a human subject [11] .

engineering viruses: a diversity of platforms

As might be expected, proponents of a given OV are typically able to advance strong arguments to support their choice of platform, emphasizing unique features such as replication kinetics, genome plasticity, targetability, seroprevalence and stability that may lead to superior oncolysis. However, it is too early to determine which unique viral characteristics will be the critical drivers of clinical success for a given cancer type. Reverse genetics systems are available for virtually all virus families, and the rules of engagement for new virus creation are well established. In general, the most effective strategy is to combine rational design with evolution, allowing each engineered virus to mutate and fully adapt to its intended target cells after it has been rescued. Biosafety oversight is in place at all academic centers responsibly engaging in virus engineering activities, and it is now a relatively straightforward matter to generate and test new virus configurations using what now amounts to the world's best lego set. Viral gene and noncoding sequences can be modified in a variety of ways to add or eliminate functions and nonviral genes or noncoding regulatory elements, whether synthetic or naturally occurring, can be added into viral genomes to confer additional desirable properties. The overarching engineering goal for the oncolytic virotherapy field is to generate viruses that can be efficiently delivered to disseminated tumors in the body where they will spread and selectively kill both infected and uninfected tumor cells, without causing collateral damage and posing no risk of transmission to the population.

Delivery of Ovs

Viral infections are well understood but for OV applications must be viewed as drugs obeying pharmacological principles. A drug is typically administered to a patient in an extremely controlled way to produce a reliable, consistent and predictable pharmacokinetic profile (absorption, biodistribution, metabolism and excretion) and bioavailability. Natural viral infections do not obey these rules since the inoculum is of variable size; host resistance varies from person to person; and the kinetics of the adaptive immune response differ greatly between individuals. Thus, the outcomes of natural infections with a given pathogenic virus range from asymptomatic seroconversion to full blown disease. The primary virus inoculum must contain sufficient virus particles to overcome initial host defenses at the site of entry. Some viruses enter through mucous membranes in the GI or respiratory tracts, while others enter via direct inoculation into the blood stream following a needle stick or arthropod blood meal [58] . OVs are delivered in the same way as traditional drugs, by introducing a highly concentrated virus inoculum into the body via oral, intravenous (IV), intranasal, transdermal, subcutaneous or intramuscular routes whereupon the dispersion of the inoculated virus, or its progeny, takes it to the targeted cancerous tissues. A major factor distinguishing OVs from traditional drugs is that they self-amplify and spread after delivery so their peak concentration may not be reached until sometime after the treatment is administered. Biological amplification by viral replication is the most important difference between viral therapies and traditional drugs. The concentration of a drug diminishes over time at a very well described rate depending on the clearance and elimination from the body. Through the course of a viral infection the viral load is initially small and then increases and finally decreases rather than just following a specific rate law of elimination. Limited amounts of virus particles replicate at the site of inoculation [59] . Input or progeny viruses then can either drain with the lymphatic fluid to the nearest lymph node, go directly into circulation or spread locally before spreading systemically to eventually arrive at a specific target organ. Pathology is induced by high levels of viral replication in the target organ directly killing infected cells or recruiting the immune system to kill them, at the same time provoking a local inflammatory response.
Clearly, the ability to infect the activated endothelial cells of tumor neovessels would be an attractive targeting property for OVs to enhance entry into the tumor parenchyma by releasing viral progeny on the ablumenal side of the blood vessel. Intravascular coagulation in the capillary could also be provoked by virusinfected endothelial cells reacting with clotting and inflammatory factors. Virus engineering strategies have been pursued to achieve this goal, for example, by displaying polypeptide ligands echistatin and urokinase plasminogen activator on the surface of measles virus to target integrin αVβ3 and UPaR endothelial cell surface receptors, respectively [68, 69] . Also, at least one OV (vesicular stomatitis virus VSV) has been shown naturally capable of infecting neovessel endothelium in implanted mouse tumors, but the study did highlight the potential toxicity of the approach, namely intravascular coagulation requiring heparin therapy for its prevention [70] .

• Limiting viral neutralization & clearance

Another strategy for avoiding neutralization is to block the reticuloendothelial system with polyinosinic acid or with clodronate-loaded liposomes which poison or deplete splenic macrophages and Kupffer cells. This approach has been shown to slow the clearance of circulating virus particles that have been coated with antibodies or complement [86] [87] [88] .

Arming Ovs: extending the range

No matter how extensively an OV infection spreads through a tumor, a sizable percentage of the cancer cells will escape infection [61] . Killing of these uninfected (bystander) cancer cells is therefore critical if oncolytic virotherapy is to become a curative, as opposed to just a tumor debulking strategy. Bystander killing can be achieved, both locally at the site of a spreading infection and systemically at uninfected tumor sites, by genetically arming the virus using one of several possible approaches. For example, an OV can be engineered to encode a secreted protein that selectively mediates the destruction of neighboring and/or distant cancer cells. Alternatively, it can be armed with a 'suicide gene' or prodrug convertase whose encoded protein converts a harmless prodrug to a diffusible anticancer drug. In a third approach, the OV can be armed with a 'radioconcentrator gene' so that infected tumor cells are able to concentrate a β-emitting radioisotope whose emitted electrons damage adjacent uninfected tumor cells. Fourth, it can be engineered to fuse infected tumor cells with uninfected neighboring cells. And last, but by no means least, it can be engineered to express one or more genes capable of amplifying immune-mediated killing of uninfected tumor cells. Each of these approaches is discussed below using specific examples to illustrate the concepts.

• • Prodrug convertases

CD converts 5-flurocytosine (5-FC), an inert small molecule that is administered intravenously, to 5-flurouracil (5-FU), an antimetabolite that irreversibly inhibits thymidylate synthase and is an approved chemotherapeutic agent for a variety of cancers (anal, breast, colorectal, esophageal, stomach, pancreatic and skin) [29] . The argument supporting the CD/5-FC system is that local production of 5-FU in the OV-infected tumor will create a 5-FU concentration gradient that will expose tumor cells to a higher concentration of the drug compared with distant tissues, thereby ameliorating toxicity and enhancing the therapeutic index of the drug. However, 5-FU is freely diffusible so local production of the drug in a CD-positive tumor exposed to high concentrations of 5-FC can lead to systemic toxicity [140] . The dose of 5-FC must therefore be adjusted accordingly. CD has been incorporated into several OVs including adenoviruses, paramyxoviruses and poxviruses, but the one that has advanced ahead of all others in clinical testing is a C-type retrovirus, Toca-511, which is currently being evaluated in a Phase III clinical trial for the treatment of patients with malignant glioma [141] [142] [143] . Besides the TK and CD prodrug convertase systems, there are reports of OVs engineered to express the cyclophosphamide-activating protein CYP2B1, future science group www.futuremedicine.com the CPT11-activating secreted human intestinal carboxylesterase (shiCE) and the fludarabine phosphate activating purine nucleotide phosphorylase [144, 145] .

Safety

So far OVs have an exceptional safety record in the clinic with few serious adverse events and minimal mortality reported from human trials [166, 167] . However, since efficacy has also been limited, it remains possible that toxicity profiles may appear less favorable as doses are increased and as newer OVs or virus-drug combinations are advanced to clinical testing. Currently, there are over 40 active clinical trials ongoing using OVs alone or in combination with other therapies [168] . As the number of patients participating in trials grows so will the insight into rare adverse effects. The most common adverse effects reported in recent OV trials are fever and flu-like symptoms [169] . However, the potential for severe adverse reactions was much more clearly demonstrated in the earliest virotherapy trials undertaken in the 1950s and 1960s when deaths and severe adverse reactions were not infrequently recorded due to presumed OV replication in normal tissues, notably the brain, especially in immunocompromised cancer patients [4, 170, 171] .

• • Limiting pathogenicity

Understanding the selective pressures that operate within the tumor and the host, as well as the role of viral quasispecies in treatment outcomes is an active area of research. RNA and DNA viruses exist as a population of quasispecies or collections of related viral genomes undergoing variation and selective pressure [174] . Generation of quasispecies occurs when viral genomes are copied during the replication cycle. Viral polymerases typically have an error rate that introduces an average of one or more base mutations per progeny genome [175] . In general, the larger the virus genome, the lower the polymerase error rate. Picornaviruses are among the smallest viruses being developed for oncolytic therapy with positive sense RNA genomes ranging from 7 to 9 kb in length, and their RNA polymerases have a correspondingly high intrinsic error rate. Production of a virus for clinical application is a highly regulated process and involves multiple rounds of replication to achieve enough virus for patients. The viral product is therefore already a swarm of quasispecies at the time it is administered to the patient and mutates further as it undergoes additional rounds of replication in vivo. Studies of the mutation rates of viral polymerases, the generation of quasi species, the evolution of viral populations and the evolution between dominant subspecies within a virus population are therefore of great interest and relevance to the OV field.
Two so-called suicide genes, TK and inducible caspase 9, are of interest in this regard. Both of these genes have been used as a safety switches to eliminate genetically modified T cells that were causing graft versus host disease in human clinical trials [184] [185] [186] . Viruses engineered to encode TK have also been controlled with ganciclovir therapy [187] , but this cannot be considered a reliable safety switch because of the ever-present risk of the emergence of viral quasispecies with TK inactivating mutations. The development of a universal and highly reliable safety switch remains one of the significant research challenges facing the field of oncolytic virotherapy.

• • Prodrug convertases

Unlike secreted toxins, prodrug convertases do not pose a risk of increasing OV virulence because their toxic potential is manifest only in the presence of an exogenously added prodrug. For this reason, the approach has been extensively studied. Perhaps the most well-known convertase-prodrug combination is HSV TK, used with ganciclovir. TK converts ganciclovir to ganciclovir monophosphate which is further processed intracellularly to ganciclovir triphosphate, a DNA synthesis chain terminator that kills dividing cells as they enter S phase [138] . A major weakness of the TK-ganciclovir system, aside from its inability to kill nondividing tumor cells, is that it has very limited bystander killing potential. This is because ganciclovir monophosphate is not released from the cell in which it is generated, so does not impact uninfected tumor cells unless they are connected to the infected cell via gap junctions through which it can pass [139] . Encoding connexin, a gap junction protein, in the OV genome can enhance the bystander killing effect of TK, but attention is shifting to other convertases, most notably CD.

Arming viruses with transgenes

• Secreted toxins, prodrug convertases and immunostimulatory proteins have been incorporated into OVs to increase treatment efficacy.
62 section matches

Abstract

Nipah (Nee-pa) viral disease is a zoonotic infection caused by Nipah virus (NiV), a paramyxovirus belonging to the genus Henipavirus of the family Paramyxoviridae. It is a biosafety level-4 pathogen, which is transmitted by specific types of fruit bats, mainly Pteropus spp. which are natural reservoir host. The disease was reported for the first time from the Kampung Sungai Nipah village of Malaysia in 1998. Human-to-human transmission also occurs. Outbreaks have been reported also from other countries in South and Southeast Asia. Phylogenetic analysis affirmed the circulation of two major clades of NiV as based on currently available complete N and G gene sequences. NiV isolates from Malaysia and Cambodia clustered together in NiV-MY clade, whereas isolates from Bangladesh and India clusterered within NiV-BD clade. NiV isolates from Thailand harboured mixed population of sequences. In humans, the virus is responsible for causing rapidly progressing severe illness which might be characterized by severe respiratory illness and/or deadly encephalitis. In pigs below six months of age, respiratory illness along with nervous symptoms may develop. Different types of enzyme-linked immunosorbent assays along with molecular methods based on polymerase chain reaction have been developed for diagnostic purposes. Due to the expensive nature of the antibody drugs, identification of broad-spectrum antivirals is essential along with focusing on small interfering RNAs (siRNAs). High pathogenicity of NiV in humans, and lack of vaccines or therapeutics to counter this disease have attracted attention of researchers worldwide for developing effective NiV vaccine and treatment regimens.

Introduction

The NiV belongs to the Henipavirus genus under the family Paramyxoviridae. This genus alsocontains Cedar virus (CedPV) and Hendra virus (HeV). Molecular studies have significantly improved our understanding of the genetic diversity of Henipaviruses (Wang et al. 2001; Rockx et al. 2012) .The almost annual occurrence of Henipaviruses in South-Eastern Asia and Australia since the mid 1990s is noteworthy. In Australia alone 48 cases of Hendra viruses and in south eastern parts of Asia 12 outbreaks of Nipah viruses have been reported which not only hit the health sector but also the economic stability of these nations (Aljofan, 2013) . There have been a total 639 human cases of NiV infection reported from Bangladesh (261 cases), India (85 cases), Singapore (11 cases), Philippines (17 cases) and Malaysia (265 cases), with a mortality rate of about 59% (Ang et al. 2018) . This points to the survival efficiency of NiV in nature and the history on its species jumping/host adaptation pattern adds to the public health concerns posed by this virus. Detailed studies and clinical therapeutic trials on various animal models such as guinea pigs, hamsters, ferrets, cats, pigs and African green monkeys are being investigated for Henipaviruses . The Nipah disease outbreak in 2001 in Siliguri and latest in Kerala have emphazised the need for an efficacious vaccine against it. Moreover the virus imposes threat to health of public (Sharma et al. 2018) . Enhanced monitoring and surveillance for Nipah infection and the development of an efficacious vaccine are the needs of the hour.

Transmission of the Nipah virus

NiV transmission occurs via consumption of viruscontaminated foods and contact with infected animals or human body fluids. Risk factors include close proximity viz., touching, feeding or attending virus infected person, thus facilitating contact to droplet NiV infection. Recently, experimental studies with aerosolized NiV in Syrian hamsters revealed that NiV droplets (aerosol exposure) might play a role in transmitting NiV during close contact (Escaffre et al. 2018) . Three transmission pathways of the Nipah virus have been identified after investigation carried out in Bangladesh. Consumption of freshdate palm sap is the most frequent route, with the consumption of tari (fermented date palm juice) being a potential pathway of viral transmission. NiV infection associated with tari can be prevented by prevention of the access of bat to date palm sap (Islam et al. 2016) . Studies using infrared camera revealed that the date palm trees are often visited bats like Pteropus giganteus and during the process of collection of the sap, bats lick them. The virus can survive for days in sugar-rich solutions, viz., fruit pulp (Fogarty et al. 2008; Khan et al. 2008) . The Nipah viral outbreak reported from Tangail district, Bangladesh was found to be associated with drinking of raw date palm sap. Notably, symptoms have been recognized in patients in Bangladesh during the season of collection of date palm sap, i.e. during December to March . Data also revealed high seroprevalence of anti-Nipah viral antibodies among Pteropusspp. This is suggestive of the fact that the virus has undergone adaptation well enough to get transmitted among Pteropus bats. The modes of transmission of the Nipah virus are depicted in Figure 2 .
Changing resource landscapes, rapid change in fruit bat habitat, related shifts in their ecology and behavior, altered diet, roosting environment, movement and behaviors altogether constitute the ecological drivers causing increasing spillover risk of bat-borne viruses like Henipavirus to domestic animals and humans (Kessler et al. 2018) . Understanding virus-bat interactions is an exciting new area of research that could through new light on the different modes regulating NiV infection and to designing effective and novel therapeutics (Ench ery and Horvat 2017).

Phylogenetic analysis of NiV

A pair-wise-similarity analysis among the nucleotide sequences retrieved from the Nipah Virus (NiV) N and G genes was carried out after aligning the sequences by the Clustral V program in MegAlign software of the DNASTAR software package. For the genetic relatedness study, representative 1599-and 1809 bp-length for N gene (27 strains) and G gene (15 strains), respectively, were investigated. NiV strains from different countries including Malaysia, Cambodia, Bangladesh, India and Thailand, submitted during 2001-2018 were retrieved from the NCBI database. Phylogenetic analysis was performed using the maximum likelihood method (1000 bootstrap replicates) in MEGA 6 software (v 6.06) (Tamura et al. 2013) . The suitable dendrogram analysis model was identified by using the find best DNA/protein model tool available in MEGA 6 (v 6.06), confirmed with the FindModel online tool (Posada and Crandall 1998) . For N and G genes, the respective models were KHY þ G and T92.

Molecular epidemiology

For comparison of the open reading frame sequence of the NiV with those from other members of the Paramyxovirinae subfamily, phylogenetic analysis had been used widely and by such approach the closest relation between NiV and Henipavirus has been proven . It has been revealed by nucleotide sequencing technique that there exist very little difference in the nucleotide sequences of NiV isolated from throat secretion and cerebrospinal fluid (difference by just 4 out of 18,246 nucleotides) (Arankalle et al. 2011) . Nucleotide sequence homology has also been observed between the virus isolated from Bangladesh and Malaysia but it is interesting to note that nucleotide heterogeneity (inter-strain) had been found to be more obvious. It is interesting to note that differences in genetic variability certainly have relation with the mode of transmission. It is evident by molecular epidemiological studies that NiV had been introduced in pigs in Malaysia during 1998-1999 causing great loss to pig farming (Looi and Chua 2007) . However, the human and pig isolates in Malaysia during the later phase of outbreak showed nearly identical sequences. This is suggestive of the fact that there was rapid spread of only one variant in pig and such variant was responsible for most of the cases in man. In contrast, the introduction of NiV from fruit bats to humans for multiple times in Bangladesh might be responsible for the sequence heterogeneity of the NiV isolates (Chan et al. 2001; Chakraborty 2012) . Detailed phylogenetic analyses have been performed on thecomplete gene sequences of NiV strains from the year 2008 as well as 2010 outbreaks in Bangladesh. On the basis of a nucleotide sequence window (comprising of 729 nucleotides), a genotyping scheme has been introduced. An accurate and simple way for classification of current as well as future sequences of NiV has been provided by this genotyping scheme. A phylogenetic tree (with very high bootstrap values) has been constructed by such genotyping method. Phylogenetic analysis showed close similarity of sequences obtained from pigs and humans during the Malaysian outbreak. Analysis also revealed that the virus isolated from Bangladesh possesses an additional 6 nucleotides than the prototype Malysian strain . For classification of sequences of NiV such methodology and phylogenetic tree is very helpful . For investigating the viral genetic diversity, a phylogenetic study of the infection caused by NiV has helped in estimating the infection spread and its date of origin .

Immunobiology (immune response/immunity)

Immune response studies regarding Nipah virus have been conducted by various researchers, especially after each reported outbreak. Since the virus exhibits two dictinct types of association among its hosts (maintaining its persistence in the nature through reservoir hosts like bats and inflicting fatal clinical condition in humans as well as domestic animals like pig), the immune responses might be host-specific Negrete et al. 2005; Kulkarni et al. 2013) . Several proteins of Henipaviruses block host innate immune responses viz., P/phosphoprotein; V protein; the C and the W proteins. In response to several stimuli the IFNa/b production can be inhibited by V as well as W proteins whereas the ability of IFNs for signaling are blocked by P, V as well as W proteins, leading to induction of a state of cellular antiviral response (Basler 2012) . The innate immune system of pteropid bats is remarkable for its constitutive action of Type 1 interferon system (which can restrict the early viral replication within their body) (Zhou et al. 2016 ). This mode of action has been associated also with several interferon stimulated genes (ISG) particularly of those involved in noninflammatory pathways so that elevation in interferon response in bats is not allied with chronic inflammation unlike in case of rodents or humans (Halpin et al. 2011 ). Due to these differences, bat cells are primed to react to viral attack immediately but only upto a level of restricting replication (Zhou et al. 2011a,b) . Bats possess comparatively higher repertoire of naive immunoglobulins with more specifities, thereby favouring direct clonal selction of B lymphocytes for antibody production. In such condition there may be poor or no hypermutation and affinity maturation stages in B cells, leading to poorer responses and restricted production of high-titered antibodies than other species. These features contribute for the delay in viral clearance and persistence of virus for a pretty long period (Wellehan et al. 2009; Schountz et al. 2017) . Nipah virus comparative studies conducted in pteropid bats and hamster reinforce these points as virus showed lesser multiplication and shedding from bat endothelial cells as well as with poorer antibody responses upon challenge studies (Wong et al. 2003; Lo et al. 2010; de Wit et al. 2011) . Recently, tetherin (an IFN-induced protein from bats) has been reported to inhibit NiV replication in fruit bat cellsand to act as an innate immune antiviral protein that can facilitate the host to combat virus induced pathological changes (Hoffmann et al. 2018) .

Pathogenesis

In the initial stage of illness in man, detection of NiV can be done in epithelial cells of the bronchiole ). Viral antigens can be detected in bronchi and alveoli in experimental animal models; the primary targets being epithelium of bronchi and type II pneumocytes (Rockx et al. 2011) . Inflammatory cytokines are induced due to infection of the epithelium of the respiratory tract; thereby recruiting cells of the immune system and ultimately leading to development of acute respiratory distress syndrome (ARDS)-like disease (Rockx et al. 2011 ). Significant inflammatory mediators, viz., interleukin (IL)-1a, IL-6, IL-8; granulocyte-colony stimulating factor (G-CSF), C-X-C motif chemokine 10 (CXCL10), etc. are induced when the airway epithelium (smaller ones) get infected (Escaffre et al. 2013) .
Two pathways are distinctly involved in the process of viral entry into the central nervous system (CNS), viz., via hematogenous route (through choroid plexus or blood vessels of the cerebrum) and/or anterogradely via olfactory nerves (Weingartl et al. 2005) . The blood brain barrier (BBB) is disrupted andIL-1b along with tumor necrosis factor (TNF)-a are expressed due to infection of the CNS by the virus which ultimately leads to development of neurological signs (Rockx et al. 2011 ). There may be presence of inclusion bodies in case of infected CNS in man. In both the gray as well as white matter plaques may be evident along with necrosis (Escaffre et al. 2013) . It is quite noteworthy that the virus can directly enter the CNS in several experimental animal models via the olfactory nerve. The olfactory epithelium of the nasal turbinate is infected by NiV in such animal models. The viral infection subsequently extends through the cribiform plate into the olfactory bulb. Ultimately, the virus is disseminated throughout the ventral cortex along with olfactory tubercle (Weingartl et al. 2005; Munster et al. 2012; Escaffre et al. 2013) . A diagrammatic representation of pathogenesis of NiV has been depicted in Figure 4 .

Postmortem findings

There may be consolidation of varying degree along with hemorrhages (either petechiae or ecchymosis) in the lungs of affected pigs at necropsy. Froth-filled bronchi along with trachea are commonly observed. In certain instances, there may be presence of blood stained fluids in the trachea and bronchi. Congestion along with generalized edema is present in kidneys and brain. Both the cortex as well as suface of kidneys may become congested (Nor et al. 2000) . There may be pneumonia (moderate to high) along with formation of syncytial cells in the endothelial cell lining of the blood vasculatures as revealed histologically Nor et al. 2000) . In the CNS and other major organs like lungs and kidneys, there may be development of small vessel vasculopathy (disseminated) in case of acute infection ). Generalized vasculitis along with fibrinoid necrosis and mononuclear cell infiltration may be noticed in the brain, kidneys and lungs. Viral antigens at greater concentration may be present in the blood vascular endothelial cells (especially in the lungs) as is revealed immunohistologically. In the upper respiratory tract of pigs in the lumen viral antigens are evident amidst the cellular debris which is suggestive of the possible transmission of NiV through exhalation (Nor et al. 2000; Kulkarni et al. 2013) . In dogs, kidneys may show congestion with severe hemorrhage. Exudates may be present in the bronchi and trachea (Nor 1999; Kulkarni et al. 2013 ).

Public health significance and zoonotic aspects

NiV is the most recently emerging zoonotic and highly deadly virus having pandemic threat. As an emerging and recognized zoonotic pathogen discovered in modern times, NiV causes severe febrile illness and high fatality rates in affected persons and is posing an ongoing high risk to the health of humans worldwide (Clayton 2017; Mukherjee 2017; Thibault et al. 2017) . NiV is an uncommon but has become a deadly virus responsible for causing high fatality rates of 40-75%. Fruit bats (Pteropus) serve as natural hosts (wildlife reservoir) and pigs are the intermediate hosts for NiV zoonotic cycle (Paul 2018) . During a large outbreak of acute encephalitis in Malaysia in 1998, the virus was discovered in affected patients having contact with sick pigs. The pigs got infection from bats, and then NiV spread proficiently among pig-to-pig, and thereafter from pig-to-man. Moreover, it has been revealed that Pteropus vampyrus and Pteropus hypomelanus (flying foxes in the Malysian Islands) bear the virus in saliva as well as urine, indicating their potential to act as natural reservoir of the virus (Looi and Chua 2007) . It is interesting to note that there is always risk of spill over associated with NiV infection. Interaction of the molecular as well as ecological factors collectively that govern the susceptible nature of populations of animals (domestic) as well as humans are not understood yet well (Thibault et al. 2017) .
Besides Malaysia, the fruit bats of Pteropus genus serve as the main reservoir of NiV in Thailand and Cambodia. Apart from drinking raw date palm sap contaminated by bats as a cause of initial outbreak, man-to-man and animal-to-man transmission is also a major mode of spread of the infection during an ongoing outbreak. Further, it has been found that direct contact of the susceptible population with the respiratory and body secretions of the infected patients increases the risk of acquiring the infection. During the NiV outbreak in Thakurgaon district, northwest Bangladesh, anti-NiV antibodies were detected in half of the Pteropus bats tested (Chadha et al. 2006; Gurley et al. 2007; Homaira et al. 2010a,b; Clayton 2017; Thibault et al. 2017) . Other major public health threats appear to be acquiring NiV infection from the susceptible food and domestic animals. Many domesticated mammals seem to be susceptible to Nipah virus. This virus can be maintained in pig populations, but other domesticated animals such as sheep, goats, dogs, cats and horses appear to be incidental hosts acquiring the infection during outbreaks. Fruits punctured by the bat and contaminated with their saliva forma common source of transmission of NiV infection from bats to domestic animals. Consumption of fruits eaten partially by fruit bats may cause infection in pigs which may then transmit it to humans. Contact with sick cow was reported to have caused a case of human infection in Bangladesh (Chua 2003; Luby et al. 2012; Siddique et al. 2016 ; http://www.cfsph. iastate.edu/Factsheets/pdfs/nipah.pdf).
Consumption of fruits, vegetables or water contaminated with saliva, urine or fecal matter of infected bats could also be a possible mode of transmission to man and animals ). Date palm sap can be used to prepare alcoholic beverages and such beverages when consumed can lead to human infection (Harit et al. 2006; Simons et al. 2014) . Evidence from several NiV outbreaks indicate that consumption of undercooked meat from infected animals or handling of infected animals in the home, farm or slaughter houses may also pose risk of animal-to-man transmission (Chanchal et al. 2018) .
Close contact with symptomatic patients or their infectious secretions has been implicated for human transmission of NiV in Bangladesh. Specific exposures can pose a high risk of person-to-person transmission, though sustained transmission do not occur in humans. Studies conducted in animal models further support this fact (Clayton 2017) . The recent NiV outbreak in Kerala, India, which caused encephalitis in humans, raised global health concerns (Paul 2018) .

Vaccines

A recombinant measles virus (rMV) vaccine that expresses envelope glycoprotein of NiV has been found to be promisingfor use in man (Yoneda 2014) . A replication-competent, recombinant VSV-vectored vaccine encoding NiV glycoprotein was reported to show high efficiency in a hamster model. A single intramuscular dose of the vaccine conferred protective immunity in African green monkeys one month after vaccination (Prescott et al. 2015) . Healthcare workers and family contacts attending Nipah cases should be considered for Nipah vaccination, in order to limit human-to-human transmission and curb outbreaks (DeBuysscher et al. 2016) . A very strong virusspecific immune response is generated through vaccination which inhibits the virus replication and shedding. Such vaccine could provide protection from NiV in disease outbreaks. Attenuated live vaccines as well as subunit G (recombinant platforms) have also been tested (Satterfield et al. 2016a) .
Immunoinformatic advances have been utilized for developing peptide-based NiV vaccine by prediction and modeling of T-cell epitopes of NiV antigenic proteins. Specific epitopes, viz., VPATNSPEL, NPTAVPFTL and LLFVFGPNL of N, V and F proteins, respectively, showed substantial binding energy as well as score with HLA-B7, HLA-B Ã 2705 and HLA-A2 MHC class-I alleles, respectively (Kamthania and Sharma 2015) . Such predicted peptides can potentially stimulate T-cell-mediated immunity and could have utility in developing epitope-based vaccines to counter NiV. In silico epitope prediction tools which evaluated G and F protein of NiV indicated that either GPKVSLIDTSSTITI or EWISIVPNFILVRNT peptides could formulate an effective universal vaccine component, inducing both humoral and cell-mediated immunity (Sakib et al. 2014) . A more recent in silico analysis using bioinformatics tools indicated that the epitopes from G (VDPLRVQWRNNSVIS) and M (GKLEFRRNNAIAFKG) proteins can be helpful for designing common B-and T-cell epitope-based peptide vaccinesagainst HeV and NiV, and this approach needs to be evaluated (Saha et al. 2017) . From another epitope-based immunoinformatics and prediction study on the NiV associated RNA-dependent RNA polymerase protein complex, best-predicted Tcell epitopes identified are 'ELRSELIGY' (peptide of phosphoprotein) and 'YPLLWSFAM' (nucleocapsid protein). Such approach identified B-cell epitope sequences in phosphoprotein (421 to 471), polymerase enzyme gene (606 to 640) and nucleocapsid protein (496 to 517). These studies are oriented for the validation of potential vaccine candidate protein portions from Nipah virus which could then spearhead towards the development of fruitful subunit vaccines (Ravichandran et al. 2018) .
The development of animal models of NiV disease is another priority, in order to evaluate the preventive and therapeutic approaches. This will help in employing successful immunization strategies (both active as well as passive) by targeting the envelope glycoprotein of the virus ).

Prevention and control measures

When the genomes of Squirrel monkey, Cynomolgus macaques and African green monkey are analyzed it sheds light on protection against the infection caused by NiV with the aid of immune factors of man. Cynomolgus macaques do not develop signs of NiV infection while Squirrel monkeys and African green monkeys develop symptomatic infection. NiV is endemic in Southeast Asia where co-evolution of the virus has taken place likely with other Henipaviruses apart from NiV. Squirrel monkeys are found in central as well as south America while African green monkeys inhabit Africa. NiV is not reported in any of these regions hence theSquirrel monkeys and African green monkeysarenaïve to NiV. Full genome sequencing along with annotation of such species of primates is possible due to the availability of improved DNA sequencing technology. This can yield insights on the host genetics conferring susceptibility of certain primate species to NiV infection and might inform on therapeutic and preventive targets in humans (Satterfield 2017) . The National Centre for Disease Control (NCDC) reported that implementation of infection control and precaution at both household and hospital levels helps to limit the NiV disease outbreak. Active surveillance and contact tracing are important along with quarantine of health professionals and peopleat high risk (http://www.ncdc.gov.in/showfile. php?lid=241). Sporadic nature of NiV outbreaks, lack of information of exact correlates of protective immunity, lack of interest among private pharmaceutical companies, and inadequate availability of BSL4 laboratory facilities to test the vaccines or therapeutics against NiV in preclinical models pose challenges to NiV vaccine development. Development of diagnostics suitable for field conditions, immunotherapeutic approaches, vaccines and antiviral drugs are urgent priorities for long-term measures aimed at prevention and control of NiV disease. Utmost care should be taken in order to avoid direct contact with the persons infected by NiV. Personal protection devices, viz., masks, glasses and gloves, should be used properly. Hydration of the patient is also important (https://www.ndtv.com/health/nipah-virussome-preventive-measures-for-nipah-virus-1855891). In nations having no past history of Nipah viral outbreak, anticipatory preparedness for rural as well as urban outbreaks of the disease will ultimately help in prevention and control of potential outbreaks (Donaldson and Lucey 2018) . Control measures adopted during the NiV outbreaks in Malaysia have shown the involvement of multidisciplinary, multiministerial teams in a close collaborative and cooperative manner with various agencies at international level (Chua 2010) . Such approach needs to be adopted in case of the unpredictable disease outbreaks and deadly epidemics for controlling the spread of the virus (Kumar and Anoop Kumar 2018) .
A detailed understanding of the biogeography of the disease is required to comprehend the potential distribution of the NiV disease. Deka and Morshed (2018) carried out a study implementing certain means of modelling the risk of regional disease transmission viz., ENMeval and BIOMOD2. Such approaches help in measuring niche similarity between the ecological features and the Pteropus bats (as reservoirs of NiV). A recent bibliometric study identified a sudden increase in the number of publications referring to the eight pathogens of global concern identified by WHO, viz., Lassa, Rift Valley, Marburg, Ebola, Middle Eastern Respiratory Syndrome, Severe Acute Respiratory Syndrome, and Crimean-Congo Hemorrhagic Fever viruses (Sweileh 2017) . Almost two decades after the first report of NiV, a fruitful development in the therapeutical and preventive aspect of this deadly disease is still lacking which adds to the public health threat out of it. According to the reports from the Centers for Disease Control and Prevention (CDC), several developing as well as economically deprived countries are at high risk of Nipah outbreak (Ramphul et al. 2018 ).

Therapeutics and treatment modalities

The essence of treatment modalities along with effective therapeutics is understood, once there is an outbreak of an infectious disease. There is a need for administering therapeutics to manage the patients during NiV outbreaks and to prevent the mortality. No specific drug has been yet approved for the treatment of this important disease. Limited work has been done to develop therapeutics against NiV infection. In preclinical studies, monoclonal antibodies have been used for treatment purposes. Due to the expensive nature of the drugs based on antibodies, identification of broad spectrum antivirals is essential along with focusing on small interfering RNAs (siRNAs) (Satterfield 2017) . In animal models, the NiV pathogenesis has been understood by shedding light on the crucial nature of phospho-matrix as well as accessory proteins. For the development of novel anti-NiV drugs, such viral proteins, fusion protein and glycoprotein of the virion surface are attractive targets (Mathieu et al. 2012; Satterfield et al. 2015 Satterfield et al. , 2016b Watkinson and Lee 2016; Satterfield 2017) . A monoclonal antibody targeting the viral G glycoprotein has been shown beneficial in a ferret model of the NiV disease (Bossart et al. 2009 ). A successful outcome of an in vivo study using an investigational therapeutic, i.e. fully humanized monoclonal antibody m102.4 against NiV, in a nonhuman primate model highlights the availability of potential drug for NiV treatment in future (Geisbert et al. 2014 ). All the 12 African green monkeys that received m102.4 survived the NiV infection, whereas the untreated control subjects succumbed to disease between days 8 and 10 after infection. It has been noticed in the recent outbreak in Kerala in South India that the antiviral drug ribavirincould be explored as anti-NiV agent (https://indianexpress.com/ article/india/nipah-virus-outbreak-in-kerala-everythingyou-need-to-know-5194341/). Supportive therapies such as hydration and ventilator support constitute important aspects of clinical management of NiV cases.
Properties like virulence, cell tropism, viral entry into the host cell (that includes virus attachment and receptor identification and the process of fusion of membranes of the virus and host cell), etc. have been studied extensively to develop therapeutics effective against infection caused by Henipavirus including NiV Bossart and Broder 2006) . The G as well as F proteins of NiV (as well as HeV) can be targeted for inhibition of the viral entry into the host cell (Steffen et al. 2012) . For treating infections caused by Henipaviruses, there were no efficacious therapeutic or prophylactic measures as is evident from the report of Vigant and Lee (2011) . Even though in case of NiV outbreaks the empirical use of ribavirin has been proven to be beneficial, but its use has got dispute due to its inefficacy in case of infection caused by other Henipaviruses in various animal models (Vigant and Lee 2011) .
In animal models, the recent therapeutic approaches against NiV have been validated targeting the early steps in the infection caused by the virus. These include: use of the virus neutralizing antibodies and blocking the fusion of membrane with peptides binding the fusion protein of the virus (Mathieu and Horvat 2015) . Full protection has been provided by the drug favipiravir (T-705) when used for 2 weeks (either orally two times a day or through subcutaneous route once a day) in Syrian hamsters challenged with Nipah viral lethal dose (Dawes et al. 2018) . The use of monoclonal antibodies, immunomodulators, convalescent plasma along with intensive supportive care are in vogue for treating severe complications associated with respiratory and nervous system (Chattu et al. 2018) . Experiments have been conducted to develop concept of prophylactic use of antifusion lipopeptides against the lethal NiV. As far as developing effective lipopeptide inhibitors (with convincing biodistribution as well as pharmacokinetic features) and efficacious delivery method are concerned results of such experiments are very much crucial (Mathieu et al. 2018) . Nevertheless, the pathogenesis of the Henipavirus infection including NiV should be understood in a better way for advancing the field of therapy against such kind of viral infection further (Mathieu and Horvat 2015) .
Monoclonal antibodies of mouse origin or polyclonal antiserum which is glycoproteins G or F specific are used for passive immunotherapy in hamster modelwhich is found to be protective in nature (Guillaume et al. 2004b (Guillaume et al. , 2006 (Guillaume et al. , 2009 .

Transmission of the Nipah virus

Bats serve as reservoir hosts for several high risk pathogens, including Nipah, rabies and Marbug viruses. Such viruses are not associated with any significant pathological changes in the bat population (O'Shea et al. 2014; Schountz 2014) . Detailed studies are needed to understand the mechanisms of NiV transmission from bats-to-pigs, pigs-to-man, and from date palm sap to human and viral circulation between fruit bats, pigs and human beings. Fruit bats act as natural reservoir of Nipah viruses and among various outbreaks documented from different geographical parts of the globe these bats have been associated in one or other way for transmission of the virus and associated infection (Clayton et al. 2016; Yadav et al. 2018) . From bats, the virus has crossed its species-barrier frequently to several other species including man through spilled over transmission, but with limited transmission from person to person thereafter (Gurley et al. 2017) . Transmission of NiV to man occurs mainly in places where man, pigs and bats come in close proximity. People rear pigs for economic benefits and fruit bearing trees are also cultivated in and around the farm for shade. Bats of Pteropus spp. which are NiV reservoirs, are attracted by the fruits, hence NiV gets spilled over to pigs/animals and also to man. Infected pig meat travels across continents which led to transmission of virus from animals in one part of the world to people in another part of globe. This combination of close surroundings of fruiting trees, fruits-like date palm, fruit bats, pigs and man altogether form the basis of emergence and spread of new deadly zoonotic virus infections like Nipah (Pulliam et al. 2012) .

Introduction

Viral diseases like Avian/bird flu, Swine flu, Middle East respiratory syndrome coronavirus (MERS-CoV), Severe acute respiratory syndrome (SARS), Crimean-Congo haemorrhagic fever (CCHF), Lassa fever, Rift Valley fever (RVF), Marburg virus disease, Ebola, Zika, Nipah and Henipaviral diseases pose considerable risk of an international public health emergency, when these spread rapidly (Rizzardini et al. 2018) . After the recent emergency situations created by Ebola and Zika virus during past five years (Singh et al. 2016 , now Nipah virus disease outbreaks have created panic in the public. Ebola virus disease (EVD) outbreaks and epidemics (2014-2016) led a massive mobilization of researchers to seek new technologies in terms of developing efficient and rapid diagnostics, vaccines, therapies and drug targets to combat EVD and save lives of large human population across the globe. Like Zika, scientists are on the way to counter Nipah virus.
Nipah (Nee-pa) viral disease is a zoonotic infection and an emerging disease caused by Nipah virus (NiV), an RNA virus of the genus Henipavirus, family Paramyxoviridae, which is transmitted by specific types of fruit bats, mainly Pteropus spp. (Halpin et al. 2000; Vandali and Biradar, 2018) . NiV is a highly fatal virus posing potential threat to global health security. The Pteropus bats, viz., P. vampyrus, P. hypomelanus, P. lylei and P. giganteu, were associated with outbreaks of the Nipah viral disease in various countries of South and Southeast Asia, including Bangladesh, Cambodia, East Timor, Indonesia, India, Malaysia, Papua New Guinea, Vietnam and Thailand (Hayman et al. 2008; Sendow et al. 2010; Wacharapluesadee et al. 2010; Halpin et al. 2011; Hasebe et al. 2012; Yadav et al. 2012; Field et al. 2013; de Wit and Munster, 2015a; Majid and Majid Warsi 2018) . Fruit bats are the major reservoirs of the virus and it is the contact with such bats (infected) or intermediate hosts like pigs which are responsible for infection in man. It is to be remembered that various biologic as well as genetic features of various paramyxoviruses are retained by Nipah virus (Bellini et al. 2005) . Dependence on animal rearing as a source of additional income in many Asian countries is a predisposing factor for emergence of novel zoonoses like Nipah (Bhatia and Narain 2010) . Various studies reported that major factor responsible for emergence of NiV was thorough interaction between wildlife reservoir particularly fruit bats of the Pteropus spp. with animal population reared and managed under intensive conditions (Daszak et al. 2013) . The high fatality rate associated with Nipah disease and the lack of efficacious treatment and vaccines against it, classify it as a global threat (Epstein et al. 2006; Rahman and Chakraborty 2012) . The disease was recognized for the first time in 1998 in Kampung Sungai Nipah village, state of Perak, Malaysia. The causative agent was characterized and since then has been named as "Nipah virus (NiV)". The zoonotic potential of NiV was unknown before 1999 till Malaysia experienced Nipah viral outbreak. Such an outbreak had created alarming situation in the public health community globally as far as the potential of severe pathogenicity as well as viral distribution in widespread fashion are concerned (Chua 2012) . Considerable uncertainty exists about the patterns of Nipah virus circulation in bats and the epidemiological factors associated with its spill-over into pigs and horses (McCormack 2005) .
Encephalitis (acute) along with high mortality is the main manifestation of infection due to NiV. Apart from this there may be development of pulmonary illness and sometimes the infection may be asymptomatic in nature (Kitsutani and Ohta 2005) . Myoclonus (segmental) along with tachycardia may become evident. The involvement of brain stem, which locates the major vital centres, is probably responsible for death and mortality may vary between 32% and 92%. From a diagnostic point of view serology is quite helpful but discrete, high signal lesions can be visualized best by fluid-attenuated inversion recovery (FLAIR) where the effect of cerebrospinal fluid (CSF) is reduced, so that an enhanced MRI image can be obtained (Arif et al. 2012) . Nipah virus was first isolated in 1999 (Farrar 1999; . Gene sequencing of the isolates showed that the outbreak involved two different NiV strains, probably with different origins (AbuBakar et al. 2004 ). The clinical signs and symptoms of the NiV disease include fever along with laboured breathing, cough and headache. Encephalitis along with seizures are the complications involved (Broder et al. 2013) . Survivors of NiV infection develop symptoms of neurological malfunction such as encephalopathy, cerebral atrophy, change in behavior, ocular motor palsies, cervical dystonia, weakness and facial paralysis, which remain for several years (Sejvar et al. 2007) . Despite an increasing risk, rigorous studies that collate data from Nipah infections of pigs, bats and humans have been scarce (Hsu et al. 2004; Chadha et al. 2006; Pulliam et al. 2012) . Serosurveillance studies in multiple host species may yield important insights into NiV epidemiology (Weingartl et al. 2009; Li et al. 2010; Rockx et al. 2010; Pallister et al. 2011; Fischer et al. 2018) .

Nipah virus

Nipah virus (NiV) is a paramyxovirus (Henipavirus genus, Paramyxovirinae subfamily, Paramyxoviridae family, order Mononegavirales), an emerging virus that can cause severe respiratory illness and deadly encephalitis in humans. It is a negative sense, singlestranded, nonsegmented, enveloped RNA virus possessing helical symmetry. The RNA genome, from the 3-5, contains consecutive arrangement of six genes, viz., nucleocapsid (N), phosphoprotein (P), matrix (M), fusion glycoprotein (F), attachment glycoprotein (G) and long polymerase (L). The N, P and L attached to the viral RNA forming the virus ribonucleoprotein (vRNP). F and G proteins are responsible for cellular attachment of the virion and subsequent host cell entry (Ternhag and Penttinen 2005; Ciancanelli and Basler 2006; Bossart et al. 2007 ). The newly produced precursor F protein (F0) is cleaved into two subunits, viz., F1 and F2, by host protease. The fusion peptide of the virus contained in the F1 subunit drives the viral and host cellular membrane fusion for the virus entry (Eaton et al. 2006) . The virus M protein mediates morphogenesis and budding. Antibody to the G protein is essential for neutralization of the NiV infectivity (Bossart et al. 2005; White et al. 2005) . It is quite noteworthy that through the coordinated efforts of the fusion (F) (class I) and attachment (G) glycoproteins the target cell (i.e. host cell) is entered upon after binding by the enveloped Henipaviruses including NiV. Interactions between Class B ephrins (viral receptors) on host cells and the NiV glycoprotein (G) trigger conformational changes in the latter, leading to activation of F glycoprotein and membrane fusion (Steffen et al. 2012) . It is believed that the strategies of replication as well as fusion of the ephrin receptors are responsible for greater pathogenicity of these viruses. Multiple accessory proteins encoded by Henipaviruses aid in host immune evasion .
Nipah virus can survive for up to 3 days in some fruit juices or mango fruit, and for at least 7 days in artificial date palm sap (13% sucrose and 0.21% BSA in water, pH 7.0) kept at 22 C. The virus has a halflife of 18 h in the urine of fruit bats. NiV is relatively stable in the environment, and remains viable at 70 C for 1 h (only the viral concentration will be reduced). It can be completely inactivated by heating at 100 C for more than 15 min (de Wit et al. 2014) . However, the viability of the virus in its natural environment may vary depending on the different conditions. NiV can be readily inactivated by soaps, detergents and commercially available disinfectants such as sodium hypochlorite (Hassan et al. 2018) .

Transmission of the Nipah virus

Investigations during NiV outbreaks in Malaysia revealed that pigs are the intermediate as well as amplifying hosts for the virus (Nor et al. 2000; de Wit and Munster 2015b) . In Bangladesh, domestic animals represented another route of transmission of NiV. Foraging for fruits (contaminated with infectious saliva) was observed among domestic animals in Bangladesh. There has been a report of spread of the disease from sick cows during the year 2001 in a place called Meherpur in Bangladesh (Hsu et al. 2004 ). Illness acquired from pigs or saliva of goats and secretions of bats infected with Nipah virus has also been recorded in Naogaon (International Centre for Diarrhoeal Disease Research, Bangladesh; ICDDRB 2003; Montgomery et al. 2008; Hughes et al. 2009 ). In ferrets, systemic disease was induced when the animals are exposed to certain doses of NiV particles (Clayton et al. 2016) . NiV is more likely to be transmitted from patients suffering from infection of the respiratory tract (Escaffre et al. 2013) . A case-control study of risk factors for human infection with NiV during the outbreak in Malaysia showed that direct close contact with pigs was the primary source of human NiV infections, where only 8% of patients had no contact with pigs. The outbreak was stopped after pigs in the affected areas were slaughtered and proper disinfection measures were taken (Parashar et al. 2000; Chua 2010) .
Domesticated animals play key roles in major spill-over events of bat-borne viruses but their exact rolesas bridging or amplifying species remain unclear (Glennon et al. 2018) . Their susceptibility to zoonotic viruses and potential for disease transmission to humans needs to be studied in depth in order to diminish spill-over risks of viruses like NiV and others, especially in view of global intensification of agriculture.

Epidemiology and disease outbreaks

In Malaysia in 1999, human cases of Nipah viral encephalitis were initially confused with Japanese encephalitis or Hendra-like viral encephalitis. However, the Ministry of Health confirmed that NiV was the causative agent of the infection in pigs and man and morbidity was higher (231 cases out of 283 cases reported) in Negri Sembilan region of Malaysia. Genome of the NiV was sequenced at the CDC, Atlanta, Georgia, USA. The Ministry of Health declared total of 101 human deaths and approximately 900,000 pigs were culled (Uppal 2000) . Researchers confirmed that Nipah infections in pigs and man that occurred in peninsular Malaysia in 1998-1999 spilled over from Chiropteran bats (Yob et al. 2001 ). In peninsular Malaysia, an epidemiological study was conducted for three years to assess the seroprevalence of anti-NiV antibodies and the presence of virus among Pteropus vampyrus and P. hypomelanus bats of different age groups and physiological status [involving adults, especially pregnant lactating and juvenile bats (6-24 months)]. Various risk factors for NiV infection in pteropid bats were also explored. Among the two bat species, the risk of NiV and seroprevalence were higher for P. vampyrus (33%) than P. hypomelanus (11%). NiV seroprevalence and distribution showed variation (1-20%) in the P. hypomelanus batsand also in between the years 2004-2006 irrespective of seasons (Rahman et al. 2013 ). The surveillance study was performed to assess the distribution of Henipaviruses in Southeast Asia, Australasia, Papua New Guinea, East Timor, Indonesia and neighboring countries. NiV RNA was detected in P. vampyrus bats of Pteropodidae family and non-Pteropid Rousettus amplexicaudatus bats from East Timor (Breed et al. 2013) .
In Bangladesh, outbreaks of Nipah virus were initially confirmed only by the presence of anti-NiV antibodies in serum samples. However after 2004, researchers started genetic characterization of Nipah virus by detecting viral nucleic acid .Till the year 2010, overall 9 outbreaks have been recorded in Bangladesh. Raw date palm was the source of infection of the outbreak recorded during the year 2011 ). Such finding is further strengthened by the fact that raw date palm consumption was common in patients with fatal infection ($65% mortality rate) (Olson et al. 2002; Luby et al. 2006; ICDDRB 2010) . Another outbreak during 2011 in a remote town named Hatibandha in the Lalmonirhat district, northern Bangladesh, reported 15 deaths due to NiV infection (Wahed et al. 2011) . Studies performed in pigs in Ghana suggested that serum antibodies against Henipaviruses including Hendra and Nipah viruses and viral nucleic acid were also present in another species of fruit bat, i.e. Eidolon helvum, reflecting the exposure of pigs to these bats (Hayman et al. 2011) .
NiV disease outbreak investigation in Kerala, India, during May-June 2018, elucidated virus transmission dynamics and epidemiological analysis by employing real-time RT PCR testing to detect presence of virus in throat swabs, blood, urine and CSF. A total of 23 cases were identified including the index case, and 18 laboratory confirmed cases. The incubation period was recorded to be 9.5 days (6-14 days). Twenty cases (87%) showed respiratory symptoms and the case fatality rate was 91% with only two survivors. Sequencing and phylogenetic analysisrevealed NiV isolate to be closer to the Bangladesh lineage (Arunkumar et al. 2018 ). Nevertheless, there is a growing demand for increasing public awareness regarding the transmission pattern and risk of NiV infection which will ultimately aid in the potential reduction ofoccurrence and associated spread/ outbreaks of the disease (Yu et al. 2018) .
A firefly luciferase that expresses NiV has been generated for facilitating studies (spatiotemporal) on the pathogenesis of Henipaviruses. Herein bioluminescence imaging technique has been used for monitoring of the replication of the virus as well as spread in knockout mice. This reverse genetics system may be a useful tool to investigate Henipa-like viruses (Yun et al. 2015) .

Immunobiology (immune response/immunity)

Another immune mechanism within bats to prevent complete elimination of Nipah virus is the modulation of bat antiviral responses towards virus survival. In the reservoir host, the virusemploys immune evasion strategies especially against innate immune system so as to escape from the immune attack and maintain perpetuation within the host by retaining replication at a minimum level (Rodriguez and Horvath 2004; Rupprecht et al. 2011) . Such evasion strategies are mediated through accessory proteins encoded within the virus which may also have effect over other hosts through spillover adaptation (Schountz 2017) . The NiV P gene (coding for polymerase-associated phosphoprotein) playsa key role in evading interferon mediated immune response from the host (Shaw, 2009 ). This gene encodes accessory proteins such as P, V, W and C; all of these were reported to inhibit host antiviral responses through blockage of interferon mediated signaling pathways, especially STAT1 stimulated JAK-STAT signaling pathway (Shaw, 2009; Prescott et al. 2012) .
In case of hosts exhibiting clinical disease from Nipah virus, various virus associated immune antagonistic proteins subvert host immune responses, thus leading to pathogenesis and clinical condition. Wild type virus uses an unique RNA editing mechanism for the controlled transcription and translation of multiple antagonistic proteins which may be delayed in some hosts, so that the protein production may be slightly delayed. In such situations antiviral responses would be strong with associated inflammatory responses thus partially restricting viral replication and pathogenesis in some hosts (Seto et al. 2010 ). Although Nipah virus effectively suppresses antiviral cytokine production at early phase of infection, release of some amount of inflammatory cytokines has been suggested which can be attributed to the elevation in vascular permeability, ultimately favoring viral spread (Schountz 2014) .

Pathogenesis

From the respiratory epithelium, the virus is disseminated to the endothelial cells of the lungs in the later stage of the disease. Subsequently, the virus can gain entry into the blood stream followed by dissemination, either freely or in host leukocyte bound form. Apart from lungs, spleen and kidneys along with brain may act as target organs leading to multiple organ failure (Rockx et al. 2011; Escaffre et al. 2013 ). There is development of lethal infection in hamsters when leukocytes loaded with NiV are passively transferred (Mathieu et al. 2011 ). In pigs, there is productive infection of monocytes, natural killer (NK) cells along with CD6 þ CD8þ T lymphocytes (Stachowiak and Weingartl 2012) .

In humans

The virus is responsible for causing severe and rapidly progressing illness in humans with the respiratory system as well as the central nervous system (CNS) mainly getting affected ). The signs and symptoms of the disease appear 3-14 days post NiV exposure. Initially, there is a high rise of temperature along with drowsiness and headache. This is followed by mental confusion as well as disorientation, ultimately progressing towards coma within 1-2 days. A critical complication of the NiV infection is encephalitis. During initial phase, the respiratory problems may become evident. There is development of atypical pneumonia. Coughing along with acute respiratory distress may be evident in certain patients Williamson and Torres-Velez 2010) . There may be sore throat, vomiting, along with muscle aches (www.medicinenet.com). There may be development of septicemia along with impairment of the renal system and bleeding from the gastrointestinal tract. In severe cases within a period of 24-48 h, there may be development of encephalitis along with seizures that ultimately leads to coma (Giangaspero 2013) . It is crucial to note that transmission of the virus is more common from patients having labored breathing than those having no respiratory problems (www. cdc.gov; Luby et al. 2009 ). NiV antigen can be detected in bronchi and alveoli. 3. Inflammatory mediators are activated as a result of infection to the airway epithelium. 4. Virus is disseminated to the endothelial cells of the lungs in the later stage of the disease. 5, 6. Virus enter the blood stream followed by dissemination, either freely or in host leukocyte bound form, reach brain, spleen and kidneys. 7. Two pathways are involved in the process of viral entry into the central nervous system (CNS), via hematogenous route and anterogradely via olfactory nerve nerves. 8. The blood brain barrier (BBB) is disrupted and IL-1b along with tumor necrosis factor (TNF)-a are expressed due to infection of the CNS by the virus which ultimately leads to development of neurological signs. Red font shows the symptoms in human.

Laboratory diagnosis

Confirmation of the human as well as animal NiV infections can be done by isolation of the virus along with performing serological tests and tests to amplify viral nucleic acids. Biosafety level-4 (BSL-4) laboratory facilities are required for NiV isolation as well as propagation. However, BSL-3 may prove to be sufficient to primarily isolate the virus from suspected clinical materials. Following confirmation of the virus in infected cells (fixed by acetone) by immunofluorescent technique, there should be immediate transfer of the culture fluid in BSL-4 laboratory Ksiazek et al. 2011) . It is crucial to note in this aspect that International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDRB) along with Institute of Epidemiology Disease Control and Research (IECDR) are the institutes involved in handling NiV in Bangladesh. In India, BSL-4 laboratory has been established in Pune at National Institute of Virology (NIV) (Kulkarni et al. 2013) . In Japan, National Institute of Animal Health has developed immunohistochemical diagnostic technique based on monoclonal antibodies (Tanimura et al. 2004 ).
In order to screen the serum samples of pigs, a recombinant N protein based-ELISA has been developed at the High Security Animal Disease Laboratory (HSADL), Bhopal. By the use of pseudotyped particles, a serum neutralization test for NiV can be performed under BSL-2 conditions. This test uses a recombinant vesicular stomatitis virus that expresses secreted alkaline phosphatase (SEAP). Neutralization titer can be obtained by measurement of SEAP activity . Microsphere assay (luminex based) has been used for detection of antibodies against a glycoprotein of NiV, namely NiV sG, in the sera of pigs and ruminants like goats and cattle (Chowdhury et al. 2014) . Recently, ELISA has also been developed using recombinant full length N protein and truncated G protein for detecting virus specific antibodies in serum samples of porcines (Fischer et al. 2018) . NiV N ELISA was employed for initial screening of serum samples for henipavirus infection, while NiV G ELISA detected specifically the NiV infections. Such ELISAs are valuable diagnostic methods for seromonitoring of swine population and probably livestock and wildlife animals.

Vaccines

Nipah virus-like particles (NiV-VLPs) composed of three NiV proteins G, F and M derived from mammalian cells have been produced and validated as vaccine in BALB/c mice. The immunogenicity of the NiV-VLP vaccine was high because the VLPs possess the native characteristics of the virus including the size, morphology and surface composition (Jegerlehner et al. 2002; Jennings & Bachmann 2007; Walpita et al. 2011; Liu et al. 2013) . A recent work reported a novel strategy of adding a cholesterol group to the C-terminal heptad repeat (HRC) of the F protein that facilitated membrane targeting and fusion of the peptide. Enhanced penetration of the central nervous system and significant increase in antiviral effects were observed with these peptides (Porotto et al. 2010) . NiV-VLPs derived from mammalian cells transfected with plasmids containing NiV G, F and M genes have also been produced yielding VLPs with the three proteins. These VLPs are composed of G, M and F proteins of the virus. Golden Syrian hamsters immunized with these VLPs developed high titres of neutralizing antibody in serum, and showed complete protection upon viral challenge (Walpita et al. 2017) .
An overview on different vaccine strategies available for Nipah virus (NiV) is presented in Table 1 and few important vaccine platforms are depicted in Figure 5 .

Prevention and control measures

Bat-borne viruses are posing high risks to human and animal health, and the present scenario demands a 'One Health' approach to comprehend their frequently complex spill-over routes (Glennon et al. 2018 ). There is an urgent need to create multidisciplinary teams as far as 'One Health' approach is concerned. Such team should include: medical doctors, veterinarians and agriculturists; officers from public health sectors; vector biologists as well as ecologists and phylogeneticists who can altogether put combined effort for preventing any major outbreak (Zumla et al. 2016) .
The role of bats in transmission and spread of the pathogens need to be understood in depth so as to avoid cross-species spill over especially of the deadly virusesat wild and domestic animals as well as human interface. As an illustration, screening of fecal samples of bats in caves often visited by local residents to gather manure or for hunting in Zimbabwe revealed the significance of virus monitoring and surveillance in bats at sites with high zoonotic diseases transmission ability and to strengthen appropriate prevention and control measures to curtail and check the dissemination of virus to other places (Bourgarel et al. 2018) .

Conclusion and future directions

The 'One Health' approach is also the utmost importance. There is requirement of coordination between institutes as well as at the international level among virologists from both medical and veterinary fields as well as ecologists for understanding to the fullest the period and mechanism involved in excretion of the virus by the bats. At the same time, the common people should be educated about food hygiene as well as hygiene at personal level. Inspection of all the imported livestock at the time of arrival and also before travel at the point of origin is essential. Proper isolation, quarantine and disinfection protocol including infrastructure facilities and trained personnel with protective clothing should be in place to respond quickly upon identification of any new case. There should be maintenance of proper hygiene at maximum level for slaughtering such livestock. For preventing future NiV outbreaks, a continuous surveillance in the area of human health, animal health, and reservoir hosts should be carried out to determine the prevalence and to predict risk of virus transmission in human and swine populations. Successful accelerated development of preventive vaccines and therapeutic antibodies or antivirals are need of the hour to control the spread and treat the infected patients during an outbreak. Collaborative efforts such as CEPI and biotech companies will accelerate the vaccine or therapeutic development for NiV.

Nipah virus

NiV infects its host cells via two glycoproteins, i.e. G and F proteins. The G glycoprotein mediates attachment to host cell surface receptors and the fusion (F) protein makes fusion of virus-cell membranes for cellular entry. The G protein of NiV binds to host ephrin B2/3 receptors and induces conformational changes in G protein that trigger the F protein refolding (Liu et al. 2015) . Wong et al. (2017) have demonstrated that monomeric ephrinB2 binding leads to allosteric changes in NiV G protein that pave the way to its full activation and receptor-activated virus entry into the host cells. Recently, viral regulation of host cell machinery has been revealed to target nucleolar DNA-damage response (DDR) pathway by causing inhibition of nucleolar Treacle protein that increases Henipavirus (Hendra and Nipha virus) production (Rawlinson et al. 2018) . A diagrammatic structure of Nipah virus is depicted in Figure 1 .
NiV infection produces severe respiratory symptoms in pigs compared to humans. A rapid spread of NiV is seen in human airway epithelia which express high levels of the NiV entry receptor ephrin-B2, and the expression levels vary between cells of different donors (Sauerhering et al. 2016) . NiV infection upregulates IFN-k in human respiratory epithelial cells. IFN-k pretreatment can proficiently demonstrate antiviral activity by hindering NiV replication and thus variations in its receptor expression can participate in a useful role in NiV replication kinetics in different donors (Sauerhering et al. 2017) . The NiV V protein, which is one of the three accessory proteins encoded by the viral P gene, plays crucial role in pathogenesis of the virus in experimental infection in hamster. NiV V protein has been shown to increase the level of a host protein UBXN1 (UBX domain-containing protein 1, a negative controller of RIG-I-like receptor signaling) by restraining its proteolysis and thus regulating (suppressing) induction of innate interferons (Uchida et al. 2018) . Analyzing viral proteins, their structure and biological functions would help in designing possible strategies for designing appropriate drugs and vaccines (Sun et al. 2018) . A variety of cellular machinery is recruited by matrix protein of NiV in order to scaffold the viral structure as well as facilitate the assembly and coordinatevirion budding. The matrix protein also highjacks ubiquitination pathways to facilitate transient nuclear localization. It is crucial to note that amongst the matrix proteins there is conservation of the molecular details of the virus (Watkinson and Lee 2016) . Production of viral RNA as well as regulation of viral polymerase activity is governed by overexpression of the nucleocapsid protein of NiV. There is inhibition of transcription (of viral specific proteins) due to overexpression of such protein but definitely synthesis of genome of the virus is increased. Ultimately, the progeny of the virus is inhibited due to the bias of the activity of polymerase towards production of genome (Ranadheera et al. 2018) . Super-resolution microscopy revealedrandom distribution of F as well as G proteins on the NiV plasma membrane irrespective of the presence of matrix (M) protein. Virus like particles (VLPs) are formed due to the assembly of M molecules at the plasma membrane . G protein recruitment into VLPs is augmented by formation of viral particles that are driven by F, M as well as M/F. Such studies on viral proteins aid in improving the knowledge regarding the process of virus assembly which can ultimately spearhead researchers to design effective and specific therapeutics (Johnston et al. 2017) . Further for developing prophylactic as well as therapeutic agents it is necessary to know the interaction between host and NiV. The microRNA processing machinery along with the PRP19 complex are the host targets of the virus. The p53 control along with expression of genesisgets altered by W protein of the virus. Affinity purification coupled with mass spectrometry has helped to identify interaction between the human as well as NiV proteins (Martinez-Gil et al. 2017) . VLPs consisting of M, G and F proteins have been produced in humanderived cells, and have been characterized by liquid chromatography and mass spectrometry (Vera-Velasco et al. 2018 ).

Transmission of the Nipah virus

Spatial and temporal distribution studies of NiV spillover events in Bangladesh (2007-2013) revealed bat-to-man spillovers every winter with 36% annual variation and the distance to surveillance hospitals showed 45% of spatial heterogeneity (Cortes et al. 2018) . Therefore, strategies to prevent NiV infections in humans need to be strengthened all through colder winters. Dynamics of bat infections and spillover risk need to be understood in depth, for which purpose the evolutionary studies based on codonusage pattern can throw some lights.A recent study on the systematic evolutionary set up and codon usage pattern by both Hendra and Nipah viruses revealed that Henipaviruses are highly adapted within bats belonging to the genus Pteropus and this is strongly influenced by natural selection ).

Epidemiology and disease outbreaks

In 1998, NiV disease was recognized for the first time in Malaysia in persons who were in contact with swine population. In March 1999, one outbreak of acute Nipah virus infection was recorded in 11 male abattoir workers (average age of 44 years) in Singapore where pig meat was imported from Malaysia, with one dead. Patients showed higher level of IgM in serum and some unusual symptoms of atypical pneumonia and encephalitis with characteristic focal areas of increased signal intensity in the cortical white matter in MRI. Symptoms of hallucination along with abnormal laboratory results including low lymphocyte and platelet counts, high levels of CSF proteins and of aspartate aminostransferase were present. The patients were treated by intravenous acyclovir and eight were cured (Paton et al. 1999; Abdullah and Tan 2014) . FromSeptember 1998 to June 1999, 94 patients (both males and females), with anaverage age of 37 years, reporting close contact with swine population and diagnosed with severe viral encephalitis were investigated. Results showed a direct transmission of Nipah virus from pigs to human beings. The illness showed a very short incubation period and the symptoms includedheadache, dizziness, fever, vomiting, doll's-eye reflex, hypotonia, tachycardia, lowering of consciousness, areflexia (loss of all spinal reflexes), hypertension and high mortality (Goh et al. 2000) . Surveillance studies on Malaysian wild life species like island flying foxes (Pteropus hypomelanus) initially revealed the seropositivity of Nipah viral antibodies in them and laterconfirmed the existence of virus also by isolation studies .
In Singapore and Malaysia, febrile encephalitis due to NiV has been reported from 246 patients between1998 and 1999 and in farmed pigs during the same period, as an epidemic with neurological as well as respiratory signs (CDC 1999a,b; Nor et al. 2000; Pulliam et al. 2012) . Farmers associated with pig farming and abattoir workers were found to be in the high risk group (Pulliam et al. 2012) , and the human mortality was about 40% (Lo and Rota, 2008) . NiV infection has not been reported directly in man or pig in Indonesia, but exposure of Pteropus vampyrus bats to NiV has been reported. Thus in Indonesia, there is every possibility of disease spread from the carrier bats to pig or man (Woeryadi and Soeroso 1989; Mounts et al. 2001; Kari et al. 2006 ). Presence of anti-NiV antibodies in serum indicated an early exposure of bats to the virus. In India, a sero-surveillance study conducted over 41 pteropid fruit bats in North Indian region showed seropositivity in twenty bats (Epstein et al. 2008) .
NiV was detected for the first time in Siliguri, West Bengal, India in the year 2001 during an outbreak characterized by febrile illness in association with altered sensorium (poor thinking capability or poor concentrating capacity). A close resemblance had been found between the isolates of Siliguri outbreak and those obtained during the outbreak in Bangladesh. Such resemblance is justified, as Siliguri is located at the vicinity of Bangladesh (Harit et al. 2006; ICDDRB 2011) . Another outbreak was reported from Nadia district, West Bengal in the year 2007 (Chadha et al. 2006) . Most recently in the year 2018, Nipah viral disease outbreak has been reported in Kozhikode district, northern Kerala, India and the fruit bats have been identified as the source of the outbreak (Chatterjee 2018; Paul 2018) . During this outbreak, deaths occurred in the infected subjects as well as in healthcare personnel who were involved in treatment of patients. On May 19, 2018, 4 infected people died and on 23 May, 2018 13 more subjects deceased (3 from Malappuram and 10 from Kozhikode district). NiV was confirmed upon laboratory testing using RT-PCR. Genetic analysis at the early stage confirmed NiV etiology and that the epidemic strain showed close resemblance to the BD strain of NiV (http://gvn.org/update-on-the-nipahvirus-outbreak-in-kerala-india/). In both outbreaks, circumstantial evidences suggested the human-tohuman transmission, as most people who acquired the infection were either care-givers, or family members of infected persons.

Immunobiology (immune response/immunity)

Presence of antigen-positive inclusions in the brain tissues of patients with Nipah Viral encephalitis points to the inadequacy of both innate and adaptive responses for preventing viral spread. These findings suggest the inability of dendritic cells residing at primary entry point of virus; especially respiratory tract and lungs, rendering inefficient antigen capturing and tissue restriction (Chua et al., 1999; Chong and Tan 2003) .
Evidence also suggest the suppression of MHC-I expression in immune cells by the viral proteins, leading to a repression in both antigen presentation by antigen presenting cells and stimulation for mounting adaptive responses, ultimately resulting in viral spread and persistence in other target organs (Dasgupta et al. 2007; Seto et al. 2010) . Besides these, the virus induced immune evasion for long time also accounts for the persistence of virus in brain tissues and ensuing relapsed and late onset fatal encephalitis in man (Tan et al. 2002) . Apart from these findings, typical interaction pattern of the virus with other critical genes of the host such as TLR genes of host defence, Notch genes of neurogenesis, and other genes like TJP1, FHL1 and GRIA3 concerned with blood-brain barrier and encephalitis, etc. have been reported by computational prediction. Crucial role of miRNAs present in NiV genome in inhibiting these host genes, thereby aiding the viral spread and pathogenesis has been reported (Saini et al. 2018) . The pathogenecity of Nipah virus in pigs and man can be correlated with its ability and magnitude to evade immune responses in reservoir host. Though the virus has undergone frequent species jumping involving various hosts, higher fatality rates are being associated with human outbreaks so far, which warrants a comprehensive study to elucidate and explore the viral evolution and adaptation in different hosts.