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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.

Introduction

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] .
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] .
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] .
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] .
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] .
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] .
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] .
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] .
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] .
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] .
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.
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.
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.
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.
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] .

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] .
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] .

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] .
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] .

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] .

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.

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] .

Chikungunya Virus (CHIKV)

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] .

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.
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] .
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] .
43 section matches

Abstract

Abstract SARS-CoV-2 has caused tens of thousands of infections and more than one thousand deaths. There are currently no registered therapies for treating coronavirus infections. Because of time consuming process of new drug development, drug repositioning may be the only solution to the epidemic of sudden infectious diseases. We systematically analyzed all the proteins encoded by SARS-CoV-2 genes, compared them with proteins from other coronaviruses, predicted their structures, and built 19 structures that could be done by homology modeling. By performing target-based virtual ligand screening, a total of 21 targets (including two human targets) were screened against compound libraries including ZINC drug database and our own database of natural products. Structure and screening results of important targets such as 3-chymotrypsin-like protease (3CLpro), Spike, RNA-dependent RNA polymerase (RdRp), and papain like protease (PLpro) were discussed in detail. In addition, a database of 78 commonly used anti-viral drugs including those currently on the market and undergoing clinical trials for SARS-CoV-2 was constructed. Possible targets of these compounds and potential drugs acting on a certain target were predicted. This study will provide new lead compounds and targets for further in vitro and in vivo studies of SARS-CoV-2, new insights for those drugs currently ongoing clinical studies, and also possible new strategies for drug repositioning to treat SARS-CoV-2 infections.
Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods, Acta Pharmaceutica Sinica B, https://doi.
Twenty structures including 19 SARS-CoV-2 targets and 1 human target were built by homology modeling. Library of ZINC drug database, natural products, 78 anti-viral drugs were screened against these targets plus human ACE2. This study provides drug repositioning candidates and targets for further in vitro and in vivo studies of SARS-CoV-2. (Mengzhu Zheng), xingzhouli@aliyun.com (Xingzhou Li). † These authors made equal contributions to this work.

. The Middle East Respiratory Syndrome Coronavirus

(MERS-CoV) broke out in the Arabian Peninsula in 2012 with a fatality rate of 35% 3, 4 . Both SARS-CoV and MERS-CoV are zoonotic viruses, and their hosts are bat/civet and dromedary, respectively 5, 6 . To date, no specific therapeutic drug or vaccine has been approved for the treatment of human coronavirus. Therefore, CoVs are considered to be a kind of viruses, of which the outbreak poses a huge threat to humans. Because Wuhan Viral Pneumonia cases were discovered at the end of 2019, the coronavirus was named as 2019 novel coronavirus or "2019-nCoV" by the World Health Organization (WHO) on January 12, 2020 7, 8 . Since 2019-nCoV is highly homologous with SARS-CoV, it is considered a close relative of SARS-CoV. The
The first strategy is to test existing broad-spectrum anti-virals 18 . Interferons, ribavirin, and cyclophilin inhibitors used to treat coronavirus pneumonia fall into this category. The advantages of these therapies are that their metabolic characteristics, dosages used, potential efficacy and side effects are clear as they have been approved for treating viral infections. But the disadvantage is that these therapies are too "broad-spectrum" and cannot kill coronaviruses in a targeted manner, and their side effects should not be underestimated. The second strategy is to use existing molecular databases to screen for molecules that may have therapeutic effect on coronavirus 19, 20 . High-throughput screening makes this strategy possible, and new functions of many drug molecules can be found through this strategy, for example, the discovery of anti-HIV infection drug lopinavir/ritonavir. The third strategy is directly based on the genomic information and pathological characteristics of different coronaviruses to develop new targeted drugs from scratch.
For the development of medicines treating SARS-CoV-2, the fastest way is to find potential molecules from the marketed drugs. Once the efficacy is determined, it can be approved by the Green Channel or approved by the hospital ethics committee for rapid clinical treatment of patients. Herein, bioinformatics analysis on the proteins encoded by the novel coronavirus genes was systematically conducted, and the proteins of SARS-CoV-2 were compared with other coronaviruses, such as SARS-CoV and MERS-CoV. We conducted homology modeling to build all possible protein structures, including viral papain like protease (PLpro), main protease (3CLpro, also named 3-chymotrypsin-like protease), RNA-dependent RNA polymerase (RdRp), helicase, Spike, etc. Further, we used these proteins and human relative proteins [human ACE2 and type-II transmembrane serine protease (TMPRSS2) enzymes] as targets to screen ZINC U. S Food and Drug Administration (FDA)-approved drug database (ZINC drug database, ZDD), our own database of traditional Chinese medicine and natural products (including reported common anti-viral components from traditional Chinese medicine), and the database of commonly used anti-viral drugs (78 compounds) by virtual ligand screening method. This study predicts a variety of compounds that may inhibit novel coronaviruses and provides scientists with information on compounds that may be effective. Subsequent validation of anti-viral effects in vitro and in vivo will provide useful information for clinical treatment of novel coronavirus pneumonia.

Papain-like proteinase (PLpro)

PLpro was also confirmed to be significant to antagonize the host's innate immunity [28] [29] [30] . As an indispensable enzyme in the process of coronavirus replication and infection of the host, PLpro has been a popular target for coronavirus inhibitors. It is very valuable for targeting PLpro to treat coronavirus infections, but no inhibitor has been approved by the FDA for marketing.

Coronaviruses (CoVs) have caused a major outbreak of human fatal pneumonia since the beginning of the 21st century. Severe acute respiratory syndrome coronavirus (SARS-CoV) broke out and spread to five continents in 2003 with a lethal rate of 10% 1, 2

. The Middle East Respiratory Syndrome Coronavirus

International Virus Classification Commission (ICTV) classified 2019-nCoV as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on February 11, 2020. At the same time, WHO named the disease caused by 2019-nCoV as COVID-19. Common symptoms of a person infected with coronavirus include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, infection can cause pneumonia, severe acute respiratory syndrome, kidney failure, and even death. There is currently no specific medicine or treatment for diseases caused by SARS-CoV-2 9 .
CoVs are enveloped viruses with a positive RNA genome, belonging to the Coronaviridae family of the order Nidovirales, which are divided into four genera (α, β, γ, and δ). The SARS-CoV-2 belongs to the β genus.
Potential anti-coronavirus therapies can be divided into two categories depending on the target, one is acting on the human immune system or human cells, and the other is on coronavirus itself. In terms of the human immune system, the innate immune system response plays an important role in controlling the replication and infection of coronavirus, and interferon is expected to enhance the immune response 11 .
Blocking the signal pathways of human cells required for virus replication may show a certain anti-viral effect.
In addition, viruses often bind to receptor proteins on the surface of cells in order to entering human cells, for example, the SARS virus binds to the angiotensin-converting enzyme 2 (ACE2) receptor [12] [13] [14] and the MERS binds to the DPP4 receptor 15, 16 . The therapies acting on the coronavirus itself include preventing the synthesis of viral RNA through acting on the genetic material of the virus, inhibiting virus replication through acting on critical enzymes of virus, and blocking the virus binding to human cell receptors or inhibiting the virus's self-assembly process through acting on some structural proteins.
In the fight against coronavirus, scientists have come up with three strategies for developing new drugs 17 .
Theoretically, the drugs found through these therapies would exhibit better anti-coronavirus effects, but the research procedure of new drug might cost several years, or even more than 10 years 11 .

Analysis, structure prediction and homology modeling of SARS-CoV-2 encoded proteins

We obtained the SARS-CoV-2 genome from Gene Bank. The genome sequence of Wuhan-Hu-1 was aligned with whole database using BLASTn to search for homology viral genomes. After phylogenetic analysis and sequence alignment of 23 coronaviruses from various species. We found three coronaviruses from bat (96%, 88% and 88% for Bat-Cov RaTG13, bat-SL-CoVZXC12 and bat-SL-CoVZC45, respectively) have the highest genome sequence identity to SARS-CoV-2 (Fig. 1A) . Moreover, as shown in Fig is helicase. As a new coronavirus, structure biology study about these proteins still at early stage. Until now, only one crystal structure of 3CLpro has been deposited in PDB (pdb code: 6LU7).
In order to acquire more three-dimensional structure information of proteins about these new coronaviruses for subsequent drug screening, we aligned all protein sequences from SARS-CoV-2 with all sequences in PDB1018 database in Fold & Function Assignment System (Supporting Information Figs. S19-S34). Fortunately, most of these proteins have found their high homology proteins that have three-dimensional structure, with homology between 72%-99% (Supporting Information Table S1 ). Those PDB codes were labeled below the corresponding sequences in Fig. 2B . Unsurprisingly, all these proteins with the highest homology are from SARS. Nevertheless, there are some proteins still have not high homologous in the database. After the prediction of transmembrane helices in these proteins carried out in TMHMM Server, as expected, we found that all these proteins are transmembrane proteins except for Nsp2 (Supporting Information Figs. S35-S41). So far, we have found as much structural information of this viral proteins as possible, which provides the basis for subsequent homology modeling and drug screening. Before homology modeling, all these proteins sequences were aligned with model sequences derived from SARS-CoV and predicted secondary structure as the same time ( Fig. 3A and Table S1 ). Interestingly, as shown in Fig. 3A , important anti-virus drug target protein like 3CLpro, PLpro, and RdRp are highly conserved between those two human coronaviruses, especially in functional region. As shown in Table S1 , all these potential drug target proteins have been homologously modeled, and all generated protein models were provided as the PDB files in Supporting Information. All other coordinates of target-screening hit complexes can be provided upon request.

Virtual ligand screening of potential drug targets of SARS-CoV-2

The therapies that act on the coronavirus can be divided into several categories based on the specific pathways:
(1) some acting on enzymes or functional proteins that are critical to virus, preventing the virus RNA synthesis and replication; (2) some acting on structural proteins of virus, blocking virus from binding to human cell receptors, or inhibiting the virus's self-assembly process; (3) some producing virulence factor to restore host's innate immunity; (4) some acting on host's specific receptors or enzymes, preventing virus from entering into host's cells. The related target proteins include Nsp1, Nsp3 (Nsp3b, Nsp3c, PLpro, and Nsp3e), Based on homology models of the above 18 viral proteins (19 models) and 2 human targets, we resorted to structure-based virtual ligand screening method using ICM 3.7.3 modeling software (MolSoft LLC) to screen potential small-molecule compounds from a ZINC Drug Database (2924 compounds) and a small in-house database of traditional Chinese medicine and natural products (including reported common anti-viral components from traditional Chinese medicine) and derivatives (1066 compounds). Compounds with lower calculated binding energies (being expressed with scores and mfScores) are considered to have higher binding affinities with the target protein.

. The Middle East Respiratory Syndrome Coronavirus

CoVs contain at least four structural proteins: Spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein 10 . Among them, Spike promotes host attachment and virus-cell membrane fusion during virus infection. Therefore, Spike determines to some extent the host range.

Papain-like proteinase (PLpro)

PLpro is responsible for the cleavages of N-terminus of the replicase poly-protein to release Nsp1, Nsp2 and Nsp3, which is essential for correcting virus replication 27 .

3C-like main protease (3CLpro)

The 3CLpro, also known as Nsp5, is first automatically cleaved from poly-proteins to produce mature enzymes, and then further cleaves downstream Nsps at 11 sites to release Nsp4-Nsp16 31 . 3CLpro directly mediates the maturation of Nsps, which is essential in the life cycle of the virus. The detailed investigation on the structure and catalytic mechanism of 3CLpro makes 3CLpro an attractive target for anti-coronavirus drug development. Inhibitors targeting at SARS-CoV 3CLpro mainly include peptide inhibitors and small-molecule inhibitors [32] .
domain III (residues 201-303), and a long loop (residues 185-200) connects domains II and III. The active site of 3CLpro is located in the gap between domains I and II, and has a CysHis catalytic dyad (Cys145 and His41) 33 . As shown in Table 3 and Supporting excel file 3CLpro.xlsx), anti-bacterial drugs (lymecycline, demeclocycline, doxycycline and oxytetracycline), anti-hypertensive drugs (nicardipine and telmisartan), and conivaptan treating hyponatremia show highest binding affinity to 3CLpro. Several natural compounds and derivatives with anti-virus and anti-inflammatory effects also exhibited high binding affinity to 3CLpro (Table 4 and Supporting excel file 3CLpro_NP.xlsx), including a series of andrographolide derivatives (chrysin-7-O-β-glucuronide from Scutellaria baicalensis, betulonal from Cassine xylocarpa, 2β-hydroxy-3,4-seco-friedelolactone-27-oic acid, isodecortinol and cerevisterol from Viola diffusa, hesperidin and neohesperidin from Citrus aurantium, kouitchenside I and deacetylcentapicrin from the plants of Swertia genus. The above results suggest that these small-molecule compounds might be the potential 3CLpro
It's worth mentioning, anti-asthmatic drug montelukast also showed low binding energy to 3CLpro. As shown in Fig. 5A , montelukast was well fitted into the active pocket of 3CLpro, in which lots of hydrophobic amino acids, just like Thr24, Leu27, His41, Phe140, Cys145, His163, Met165, Pro168 and His172 compose a relatively hydrophobic environment to contain the compound and stabilize its conformation. Hydrogen bonding was predicted between Asn142 and the carbonyl group of the compound (Fig. 5B ). DNA and RNA along the 5′-3′ direction in an NTP-dependent manner 37 . Importantly, it has been reported that the SARS-Nsp13 sequence is conserved and indispensable, and is a necessary component for the replication of coronavirus. Therefore, it has been identified as a target for anti-viral drug discovery, but there are few reports about Nsp13 inhibitors 38, 39 .
Based on structure modeling of helicase protein, anti-bacterial drugs (lymecycline, cefsulodine and rolitetracycline), anti-fungal drug itraconazole, anti-human immunodeficiency virus-1 (HIV-1) drug saquinavir, anti-coagulant drug dabigatran, and diuretic drug canrenoic acid were predicted to be helicase inhibitors with high mfScores through virtual ligand screening. The natural products, such as many flavanoids from different sources (α-glucosyl hesperidin, hesperidin, rutin, quercetagetin 6-O-β-D-glucopyranoside and homovitexin), showed high binding affinity to this target.
Besides the above targets, some non-structural proteins, including Nsp3b, Nsp3e, Nsp7_Nsp8 complex, Nsp9, Nsp10, Nsp14, Nsp15, and Nsp16, also play an important role in the virus RNA synthesis and replication, suggesting these proteins may be useful targets for the anti-viral drug discovery. The virtual screening results showed many anti-bacterial, anti-viral, or anti-inflammatory drugs from ZINC drug database and our in-house natural products/derivatives database displayed potential good affinity to these targets, and the detailed information of virtual screening results is shown in Supporting excel files (for ZDD screening results, file names as target.xlsx; for natural products screening results, file names as target_NP.xlsx).

Targets inhibiting virus structural proteins

Spike is the main structural protein of coronavirus and assembles into a special corolla structure on the surface of the virus as a trimer. Spike is a main protein that interacts with the host by binding to host cell receptors to mediate virus invasion and determine viral tissue or host tropism 40 . Spike is cleaved into S1 and S2 by the host cell protease like TMPRSS2, etc. The main function of S1 is to bind with host cell surface receptors, and the However, most of above compounds were not predicted to bind with the binding interface of the Spike-ACE2 complex. The only compound that could target the binding interface between Spike and ACE2 was hesperidin, as shown in Fig. 6A . Hesperidin was predicted to lie on the middle shallow pit of the surface of RBD of Spike, where the dihydroflavone part of the compound went parallel with the β-6 sheet of RBD.
Except for Spike protein, E protein (E-channel) possesses important biological functions for the structural integrity of coronavirus and host virulence. NRBD and CRBD of coronavirus N protein are needed for N proteins in host cells to bind with coronavirus RNA efficiently. Therefore, E protein or N protein (NRBD and CRBD domains) can be used as targets for the discovery of anti-viral drugs. Through virtual screening, many anti-bacterial, anti-viral, anti-tumor, anti-asthmatic, and anti-inflammatory drugs, etc. from ZINC database and our in-house natural products/derivatives database were found to display relatively good affinity to these targets. And the detailed results of virtual screening are given in Supplementary excel files.

Targets inhibiting virulence factor

There are three coronavirus virulence factors Nsp1, Nsp3c and ORF7a related to interfering host's innate immunity and assisting coronavirus immune escape. Nsp1 interacts with host 40S ribosomal subunit that induces specifically host mRNA degradation 42 and also inhibits type-I interferon production 43 . Nsp3c has ability to bind with host's ADP-ribose to help coronavirus resist host innate immunity 44 . Bone marrow matrix antigen 2 (BST-2) can inhibit the release of newly-assembled coronavirus from host cells. SARS-CoV ORF7a directly binds to BST-2 and inhibits its activity by blocking the glycosylation of BST-2 45 . These evidences suggest that Nsp1, Nsp3c and ORF7a may be potential targets for anti-viral drug discovery.

Targets blocking hose specific receptor or enzymes

Based on the current research progress, ACE2 is considered as a host target for the treatment of coronavirus infection to block SARS-CoV-2 from entering host cells.
In addition, TMPRSS2 was known to cut the Spike to trigger the infection of SARS-CoV and MERS-CoV. Studies have shown that inhibiting the enzyme activity of TMPRSS2 can prevent some coronaviruses from entering host cells 46 . As a possible target for anti-viral drug discovery, the virtual screen results (shown in Supporting excel files) predicted many anti-bacterial drugs (pivampicillin, hetacillin, cefoperazone and clindamycin) and anti-virus natural compounds (phyllaemblicin G7, neoandrographolide, kouitchenside I), etc. to be potential TMPRSS2 inhibitors.

Virtual screening and target identification of common anti-viral drugs

showing that these selected anti-viral drugs are unlikely to acting on the above targets of the new coronavirus, which provides a meaningful reference for our future research.

Discussion

The ongoing SARS-CoV-2 epidemic makes us painfully realize that our current For those targets which are difficult to find direct inhibitors, or non-druggable targets, just like Nsp1, Nsp3b, Nsp3c, and E-channel, etc., currently popular PROTAC technology may be a good strategy to degrade these proteins and then inhibit the virus. The potential binding compounds found in this study for these targets might be a good start point.
Whether the screened anti-viral drugs really work on these targets needs further verification. We also do not recommend the application of new coronavirus pneumonia to compounds for which no target has been predicted.
The triphosphate nucleotide product of remdesivir, remdesivir-TP, competes with RdRp for substrate ATP, so it can interfere with viral RNA synthesis. Our docking results show that remdesivir-TP binds to SARS-CoV-2 RdRp, with a score of -112.8, and the docking results are consistent with its original anti-viral mechanism, so we think remdesivir may be good in treating SARS-CoV-2 pneumonia. In addition, remdesivir also predicted to bind with the human TMPRSS2, a protein facilitating the virus infection, this is a new discovery and provides ideas for subsequent research.

Papain-like proteinase (PLpro)

The screening results (Table 1 and Anti-viral drug ribavirin was predicted to bind to PLpro with low binding energy (Scores=-38.58). From generated docking model, ribavirin was bound in the active site of the enzyme as reported SARS-PLpro inhibitors (PDB code 3e9s). Hydrogen bonds were predicted between Gly164, Gln270, Tyr274, Asp303 and the compound. Also, π-π stacking was found between Tyr265 and triazolering in the compound ( Fig. 4A and B). The strong hydrogen bonding and hydrophobic interaction between ribavirin with the enzyme imply it may be a potent PLpro inhibitor.

Targets inhibiting virulence factor

The detailed screening results of Nsp1, Nsp3c, and ORF7a showed that a series of clinical drugs and natural products with anti-bacterial and anti-inflammatory effects exhibited relatively high binding affinity to these three target proteins, such as piperacillin, cefpiramide, streptomycin, lymecycline, tetracycline, platycodin D from Platycodon grandifloras, wogonoside from Scutellaria baicalensis, vitexin from Vitex negundo, andrographolide derivatives, and xanthones from Swertia genus. The detailed results of virtual screening are shown in Supporting excel files.

Targets blocking hose specific receptor or enzymes

Based on the virtual screening results of ACE2 protein, anti-diabetes drug troglitazone, anti-hypertensive drug losartan, analgesia drug ergotamine, anti-bacterial drug cefmenoxime, and hepatoprotective drug silybin, etc., were predicted to bind with ACE2 with low energy. The natural products, such as phyllaemblicin G7

Virtual screening and target identification of common anti-viral drugs

In order to further verify the screening results of ZINC drug database and utilize the current resource of anti-viral drugs immediately, we constructed a database of 78 anti-viral drugs for deep calculation, including compounds already on the market and currently undergoing clinical trials to treat SARS-CoV-2 infections.
Interestingly, remdesivir was predicted to bind with the target TMPRSS2 with low binding energy for both score and mfScore. As shown in Fig. 8A , remdesivir was bound in a relatively positively-charged allosteric pocket which is far away from the enzyme active center. Asn84 and Arg405 formed two hydrogen bonds with the phosphate groups of the compound. Weak hydrophobic interaction between the pyrrolotriazine ring of remdesivir with Tyr131 and Try401, and side chains of some polar amino acids may further stabilize the compound conformation (Fig. 8B) . We also performed the longitudinal analysis on the drugs against 21 targets, and the results showed that only tenofovir, disoproxil, fumarate, and beclabuvir may bind to Nsp1. Fewer compounds were predicted to act on some targets, such as Nsp1, Nsp3e, Nsp9, Nsp10, Nsp16, NRBD, CRBD, ORF7a, and TMPRSS2,
helicase, E-channel, Spike and ACE2, more anti-viral drugs were predicted to bind with them, especially E-channel, RdRp, 3CLpro and PLpro, indicating that these targets are more likely to be useful for the discovery of SARS-CoV-2 therapeutic drugs from known anti-virals, and should be the focus of our subsequent research.

Discussion

Also, we dock existing anti-viral drugs with our targets, analyze the possible targets of each anti-viral drug horizontally, and analyze the drugs that may interact with 21 targets vertically. We analyzed 21 targets based on the docking results and found that Nsp3b, Nsp3c, Nsp7_Nsp8 complex, Nsp14, Nsp15, PLpro, 3CLpro, RdRp, helicase, E-channel, Spike and ACE2 are more likely to be therapeutic targets of anti-viral drugs. The three targets Nsp3b, Nsp3c, and E-channel are screened more anti-viral drugs. This may be due to the model problem because of flexible small protein (Nsp3b and Nsp3c) or partial model (E-channel).
Chloroquine phosphate has shown better anti-SARS-CoV-2 effects in recent studies, but this drug has no clear target of action. In our docking results, chloroquine phosphate is predicted to possibly combine with
38 section matches

Abstract

Pathogen resistance and development costs are major challenges in current approaches to antiviral therapy. The high error rate of RNA synthesis and reverse-transcription confers genome plasticity, enabling the remarkable adaptability of RNA viruses to antiviral intervention. However, this property is coupled to fundamental constraints including limits on the size of information available to manipulate complex hosts into supporting viral replication. Accordingly, RNA viruses employ various means to extract maximum utility from their informationally limited genomes that, correspondingly, may be leveraged for effective host-oriented therapies. Host-oriented approaches are becoming increasingly feasible because of increased availability of bioactive compounds and recent advances in immunotherapy and precision medicine, particularly genome editing, targeted delivery methods and RNAi. In turn, one driving force behind these innovations is the increasingly detailed understanding of evolutionarily diverse hostvirus interactions, which is the key concern of an emerging field, neo-virology. This review examines biotechnological solutions to disease and other sustainability issues of our time that leverage the properties of RNA and DNA viruses as developed through coevolution with their hosts.

Host-oriented RNAi therapies

Nearly 20 years elapsed between the first patent filings and the realisation of an approved RNAi-based therapeutic. While challenges remain, the coming decade appears likely to mark the beginning of the growth curve for creative new approaches to RNA-based therapeutics for antiviral and immunotherapeutic applications.

INTRODUCTION

Most human-infective viruses are RNA viruses, 94% of which harbour a single-stranded RNA (ssRNA) genome. 1 These include established pathogens such as HIV and dengue virus (DenV), most high-profile emerging pathogens this decade [e.g. Zika virus (ZikV), SARS-coronavirus (SARS-CoV) and avian influenza], re-emerging pathogens including measles virus (MV) and every pathogen prioritised in the recent WHO R&D Blueprint. 2 Furthermore, climate change-related factors are likely to drive changes in future dispersion or transmission of viruses including mosquito-borne viruses such as DenV and ZikV. 3 The disease burden associated with many of the 214 human-infective RNA virus species is large and growing, yet only five have US Food and Drug Administration (FDA)-approved antivirals available and nearly all target virus proteins (Table 1) .

Other targets

While not themselves multifunctional, ubiquitin and ubiquitin-like modifiers undergo covalent and non-covalent association with other proteins and exert plethoric effects on their function, abundance or subcellular distribution. Thus, manipulating the ubiquitin and ubiquitin-like post-translational modification machinery, or the proteasome itself, also enables viruses to subvert multiple cellular processes. 8 Indeed, most multitasking host proteins examined here undergo extensive post-translational modification. 47 Numerous multitasking proteins of the ubiquitin-proteasome system, as well as ubiquitin and ubiquitin-like modifiers, are key targets of multiple viruses. These targets include the proteasome regulatory subunit PSMD2; the E3 ubiquitin-protein ligase CHIP, which modulates the activity of numerous protein chaperones including HSP90 48 ; and the small ubiquitin-like modifierconjugating enzyme Ubc9, which regulates numerous cellular functions including cell cycle by modifying p53 49 (Table 2 and Supplementary table 1) . Numerous inhibitors of the proteasome, ubiquitin E1, E2 or E3 enzymes and deubiquitinating enzymes are currently in clinical trials or approved for use as anticancer agents. 50 Therapeutically modulating the ubiquitin-proteasome system may present an indirect method of targeting multitasking proteins that are otherwise 'undruggable' at present ( Table 2 ).

Current and future therapeutic applications of RNAi

From the first RNAi patent filing in 1998 until the end of 2017,~8500 siRNA-based and 2000 miRNAbased therapeutic patents were filed in the United States. Most were for anticancer applications, followed by viral infections and inflammatory disorders. 91 At present, the US National Library of Medicine lists 87 'miRNA', 28 'siRNA' and 26 'RNAi' interventional clinical trials as underway or completed. Several trials involved patisiran, which, in 2018, became the first RNAi-based therapeutic approved by the US FDA. Patisiran is an LNPencapsulated siRNA (siRNA-LNP) delivered to hepatocytes, where it transiently induces RNAimediated silencing of wild-type and mutant transthyretin mRNA. Prior to infusion, patients receive a combination of oral acetaminophen, intravenous corticosteroid and histamine H1 and H2 receptor antagonists, yet infusion-related reactions remain one of the most frequent adverse events. 92 Antagonists to other immune receptors as outlined above may further suppress IFN stimulation by circulating siRNA-LNPs or their breakdown products.

Designer vaccines

To address production costs, RNAi and CRISPR/ Cas9 have recently been applied in attempts to engineer cells that produce greater viral yields. Using a genome-wide RNAi screen in HEp-2C cells and validation in the Vero cell line approved for vaccine development, van der Sanden et al. identified several gene knockdowns that drastically increased yields of multiple PV, enterovirus and rotavirus strains. However, these effects on viral replication were not recapitulated on follow-up. 105 As the reasons for these discrepant results remain unclear, challenges evidently remain in engineering cell lines that support greater viral yields for vaccine deployment.

INTRODUCTION

While virus-oriented approaches are efficacious, the genetic diversity of viruses often restricts such treatments to particular species or serotypes (Table 1) . Furthermore, these antivirals are often costly and are ultimately susceptible to escape mutant selection. Simple point substitutions are often responsible for treatment failure, 4,5 while fitness costs associated with harbouring these substitutions may be trivially absorbed by the escaped strain upon accumulating compensatory adaptations. 6 Tenofovir is an example of a highly effective single-regimen treatment for chronic hepatitis B infection, a retro-transcribing virus characterised by considerable genetic heterogeneity, by simultaneously imposing potent viral suppression, a high barrier for escape and reduced replicative fitness of escape strains. Despite these synergising effects, complex escape mutants harbouring multiple point substitutions in the viral reverse transcriptase have recently emerged. 7 One way of enhancing treatment efficacy while minimising viral escape is to deploy existing antivirals as combination therapies, a strategy used extensively in current HIV (e.g. tenofovir/emtricitabine) and hepatitis C virus (HCV) treatment regimens. 4, 5 While increasing the number of combinations increases the height of the escape barrier, proportional increases in treatment costs, adverse effects and counterindications make this strategy one of ever compounding challenges that ultimately remains exposed to the core problem of viral resistance. Treatment failure and the continuous need for the development of additional therapies are the realised costs of playing into such 'strengths' of virus evolution. As obligate intracellular parasites, all viruses must subvert key resources of permissive hosts in order to replicate. 8 Subverting multifunctional host proteins can confer significant fitness advantages by enabling RNA viruses to efficiently execute multiple steps in their replication strategy. Over time, these features are likely to be conserved within lineages and serve as foci of evolutionary convergence for viruses with a similar host range, while purifying selection eliminates steps rendered less efficient. Nevertheless, ideal targets of pathogenic viruses include those that are also vital to the host, thereby limiting its options for antiviral adaptation and driving more costly evolutionary innovation on its part. Similarly, the potential for adverse effects limits options for targeting such host proteins therapeutically.
Therapeutic drug availability, together with recent advances in areas including immunotherapy and precision medicine, is beginning to alleviate such constraints on host-oriented approaches. Significantly, many of these technologies arose through examining evolutionarily diverse host-virus and immune interactions, which are being increasingly uncovered with the advent of mass next-generation genome sequencing and machine learning-assisted metagenomic analysis technologies. Furthermore, such interactions are increasingly found to perform crucial roles throughout our biosphere. [9] [10] [11] As was once the case for the CRISPR/ Cas bacterial immune system proteins now used in genome editing, 12 these host-virus interactions often employ unique proteins of unknown function. 10, 13, 14 This review examines how hostvirus evolution may be leveraged towards solving disease and sustainability issues of our time. Multitasking or multifunctional host proteins as antiviral therapeutic targets, methods for targeting such proteins, vaccine design and neo-virology as an emerging source of biotechnological innovation, will be discussed.

EXPLOITING THE INFORMATION ECONOMY PARADOX IN RNA VIRUS EVOLUTION

RNA and retro-transcribing virus genomes are highly versatile, with errors occurring 2-4 orders of magnitude more frequently than in high-order eukaryotes. 15 This, combined with their rapid replication cycle, imbues such viruses with two key strengths: enormous genetic diversity and rapid escape mutant selection. Nevertheless, this same process that enables remarkable genome plasticity also appears to limit the incorporation of new information with which to achieve more favorable host manipulation. There exists an inverse relationship between viral genome size and mutation rate, with large coronaviruses the only known RNA virus family to possess 3 0 -exonuclease proofreading activity. 16 Thus, the probability of acquiring a lethal mutation increases as a function of both polymerase infidelity and genome size. This suggests lengthening the genome to accommodate a larger repertoire of gene products with which to better manipulate the host comes with considerable fitness trade-offs for RNA viruses. Indeed, excepting extremely small circular ssDNA viruses, RNA virus genomes are typically far shorter than DNA virus genomes in both average size (10.3 vs. 77.8 kb, respectively) and maximal size (51.3 vs. 2474 kb) and encode fewer proteins (1-28 vs. 1-1839; Figure 1 ). As a result, while various RNA viruses tolerate certain gene substitutions (e.g. recombinant reporter strains and segmented genome viruses), 17 most are poorly tolerant of additions of new genetic information. Correspondingly, mutagenic nucleoside analogues exhibit broad-spectrum antiviral activity by increasing the error rate in genome synthesis for nascent viral particles. 18 This effectively reduces the optimal genome length of the virus for productive infection to below the threshold of viability, thereby driving the population to extinction. Therefore, while the core 'strengths' in RNA virus evolution arise because of the nature of their genetic material and its error-prone mode of replication, these appear intractably coupled to limits on the size of information available to subvert their more complex hosts.
RNA virus evolution attempts to resolve this information economy paradox by extensively employing functional genomic secondary structures and noncoding regions, genome segmentation, compression (e.g. RNA editing, overprinting and frameshift reading) and gene product pleiotropy or multifunctionality (e.g. intrinsically disordered proteins). 17, [19] [20] [21] Yet another way is to manipulate host cell factors that are themselves multi-interacting or multifunctional 'hubs' of cellular activity, 22 whereby a single viral gene product subverts a single host factor to achieve net favorable control over numerous cellular processes. This can enable the virus to extract maximum utility from its informationally limited genome at minimal informational cost. Multiplying this effect across several viral and/or host gene products may enable the virus to extract a substantial 'return on investment' in terms of replicative fitness.

Measles virus (MV)

Measles virus is an extremely contagious and virulent pathogen undergoing a recent global resurgence. The non-structural V protein targets the single largest number of highly multitasking human proteins: HSP90a, PABP1, vimentin, hnRNPK and p53 ( Figure 2 ). In addition to V's established roles in suppressing multiple components of host interferon (IFN) signalling, 23 these interactions may allow MV to interface with the cytoskeleton (vimentin) and subvert numerous host cell processes including cell cycle (p53, hnRNPK), protein translation (PABP1), RNA metabolism (PABP1, hnRNPK), and transcription and protein expression (HSP90a). V is produced by editing of the P gene transcript, which also overlaps with the C gene. The largest number of amino acid substitutions between wild-type MV and attenuated vaccine strains occurs within the P/V/C gene region, 24 suggesting changes in this region serve an important basis for natural attenuation. Since attenuated MV strains possess very limited capacity for reversion, 24 MV strains engineered to harbour defects in V binding to these host proteins may be suitable designer vaccine candidates. As discussed in the following sections, re-purposing existing host-targeting bioactive compounds as antivirals may also yield therapeutic results.

Influenza A virus (IAV)

Influenza A virus (IAV) engages four gene products to manipulate two highly multifunctional host proteins. HSP90a is targeted by NS1 (virulence factor), PB2 (transcription and capping) and RNAdependent RNA polymerase (RdRP), while alphaenolase is targeted by NS1 as well as NS2/NEP (vRNP nuclear export; Figure 2 ). The NS1 proteins of avian influenza strain H5N1 as well as H3N2 reportedly bind HSP90 to modulate caspase-mediated apoptosis. 33 In addition to impairing HIV-1 replication, 30,31 HSP90 inhibitors reportedly inhibit IAV replication without apparent cytotoxicity in vitro. 34 It will be of interest to examine whether these effects translate in vivo using next-generation HSP90 inhibitors 27 (Table 2) .
Influenza A virus, together with SARS-CoV and multiple flaviviruses, also targets alpha-enolase, an enzyme with roles in glycolysis, cell growth and immunity (Figure 2) . A novel inhibitor, AP-III-a4 (ENOblock), was recently developed with the interesting property of blocking the non-glycolytic functions of alpha-enolase (Table 2 ). While this drug shows promise in treating obesity in animal models, 35 its antiviral effects remain unexplored. This warrants further study as a potential hostoriented antiviral approach to IAV and other viral infections.

Togaviruses

Similarly, by committing four of its proteins to manipulating hnRNPK (Figure 2) , Sindbis virus (SinV) reveals this multifunctional host protein to be crucial in its replication strategy. Co-immunoprecipitation experiments suggest hnRNPK associates with the SinV polyprotein processing products nsp1 (methyl/ guanylyltransferase), nsp2 (helicase/protease) and nsp3 (regulatory component) and may be required for viral transcription. 36 Other Togaviridae members including Semliki Forest virus (SFV) and chikungunya virus (ChikV) also manipulate hnRNPK (Figure 2 ), suggesting this host protein serves multiple, evolutionarily conserved roles in togavirus replication. While this hints at a potential therapeutic route against togaviruses, there are currently no selective hnRNPK-targeting drugs available (Table 2) . hnRNPK is also tumor suppressor, with mutated or constitutively downregulated hnRNPK being associated with tumorigenesis. 37 Nevertheless, short-term therapeutic targeting of hnRNPK as an antiviral strategy has yet to be explored.

Flaviviruses

Numerous flaviviruses including DenV, HCV, TBEV, AlkV and KunV/WNV manipulate alpha-enolase (Figure 2 ), an enzyme with many functions including catalysing the penultimate step in ATP synthesis via glycolysis. While viruses often reprogram cellular metabolic pathways, DenV drastically increases the rate of glycolysis to support its own replication. Metabolic acidosis is often associated with severe disease and may correlate with the subcellular redistribution of viral proteins to further compromise host stress responses. 42 Thus, DenV-infected patients who are simultaneously hyperglycaemic (e.g. diabetics) are at greater risk of severe disease. 43 Accordingly, inhibiting glycolysis impairs replication of DenV and other flaviviruses in vitro. 44, 45 In this context, it would be of interest to examine whether alphaenolase inhibitors such as AP-III-a4/ENOblock (Table 2) , which blocks the non-glycolytic functions of alpha-enolase as mentioned previously, may block subversion of this key enzyme by flaviviruses in vivo.

Other targets

One unexpected multiple viral target is VKORC1, an enzyme highly expressed in liver and crucial in activating blood clotting factors. 46 While relatively fewer RNA viruses target VKORC1 (Figure 2 ), it is targeted by several DNA viruses including the hepatotropic Epstein-Barr virus. It would be of interest to examine what effects, if any, infection by such viruses have in the context of treatment with vitamin K or VKORC1-targeting drugs such as warfarin ( Table 2) .

CHALLENGES AND STRATEGIES IN TARGETING MULTIFUNCTIONAL HOST PROTEINS

Therapeutically targeting host proteins that converge various cellular processes can elicit unwanted effects. This challenge is familiar to the very viruses that exploit such proteins in the first instance, yet their success also implies its soundness as an antiviral strategy. Illustrating that multifunctional host proteins can be 'druggable', 48% of the 694 human multitasking proteins annotated by Franco-Serrano et al. 51 are already targets of known compounds, compared with 9.8% of all 26 199 human proteins listed in UniProt. This also suggests potential for repurposing existing drugs for host-oriented antiviral therapy (Table 2) .
Since viruses typically infect and replicate best in only a limited set of host tissues, delivering therapies to specific tissues may mitigate adverse effects. Synthetic lipid nanoparticles (LNPs) protect and deliver small RNAs for RNAi-based therapy as well as synthetic vaccines and bioactive compounds, 52-56 while modified viruses or viruslike particles deliver RNA interference (RNAi)based therapies as well as nucleases and DNA for gene therapy. 57, 58 Despite additional challenges as outlined below, these technologies are currently applied to deliver specific treatments for viral infection, cardiovascular disease, inherited genetic disorders and cancer immunotherapy in animal models and humans. [52] [53] [54] [55] [56] [57] RNA-based therapies miRNA and siRNA biogenesis
Exosomes and the exoribonuclease XRN1 are both required for full degradation of RISC cleavage products in Drosophila. 71 Intriguingly, XRN1, which serves as a crucial viral restriction factor in totivirusharbouring (i.e. RNAi-deficient) yeasts, 72 and exosome core components RRP41, which associates with DGCR8, 65 and RRP42 are identified here as among the most highly multitasking proteins most frequently manipulated by human-infective viruses. Other such proteins include LSm3, a core component of U6 snRNA-protein complexes in spliceosomes, 73, 74 and UPF2 (Supplementary table 1) , a key mediator of the nonsense-mediated mRNA quality control pathways that recruits endonucleases and other factors to regulate aberrant mRNA decay. 75 Such interactions may enable evolutionarily diverse viruses to manipulate host/virus mRNA or ncRNA biogenesis and stability. The immune mechanisms and potential therapeutic applications of RNA post-transcriptional control are discussed further by Yoshinaga and Takeuchi 119 in another article in this Special Feature.

Current and future therapeutic applications of RNAi

RNAi-based approaches to antiviral therapy show both promise and new and familiar challenges. Most known primate-infective viruses manipulate IFN signalling 85 ; however, despite nearly two decades of study, the role of RNAi as a specific immune defence mechanism in somatic, IFNresponsive tissues remains controversial. 86, 87 If IFNand RNAi-mediated immunity are incompatible, human-infective viruses would likely be subjected to only weak, if any, specific RNAi-mediated immune selective pressure. This suggests the 'dormant' immune functions of the mammalian RNAi system could be re-engineered as a future antiviral or immunotherapeutic strategy. Alternatively, the gut microbiome, which is a crucial regulator of immune homeostasis, T-cell activation and predicts treatment outcomes in anti-PD-1 cancer immunotherapy, might be genetically modified to secrete therapeutic small RNAs. [88] [89] [90] RNAi-based strategies could be used to modulate host immune programme or selectively and reversibly block expression of key multifunctional host proteins in or near virus-infected tissues, thereby multiply regulating viral replication as discussed earlier.

Virus-oriented RNAi therapies

Remarkably, siRNA-LNP 'cocktails' of perfect complementarity to Ebola virus (EboV) RNA have been reported to confer 100% protection in nonhuman primates when administered as late as 3 days post-lethal challenge. 54 Nevertheless, nucleotide escape mutants and genetic variation between EboV strains in different geographic locations necessitate accurate, up-to-date sequencing data on circulating strains in order to continuously generate effective siRNA cocktails. Recently, a protocol employing the MinION portable sequencer was developed that enabled the direct sequencing of an intact RNA virus genome (IAV) for the first time. 95 Direct sequencing of field EboV strains would drastically reduce the current development time of new siRNA-LNPs. Nevertheless, further improvements in this sequencing technology will be required for accurate, cost-effective, routine sequencing of substantially lower-yielding and genetically diverse field strains.
As with other virus-oriented treatments, the problem of viral resistance to RNA-based therapeutics is perhaps best illustrated by HIV. Liu et al. produced a double long hairpin RNA (dlhRNA) that was processed endogenously to raise four anti-HIV shRNAs directed against gag, tat, vpu and env transcripts. Despite the cells stably expressing the dlhRNA, together with the combinatorial targeting approach and using virus produced from a single molecular clone, nucleotide escape mutants emerged, integrated and proliferated in as few as 8 days post-infection. 96 Notably, however, viral transcript knockdown was variable and incomplete, thereby creating an environment suitable for escape mutant propagation. Selecting RNAi targets in the virus that are even more highly conserved, as well as incorporating a larger number of these in an shRNA 'cocktail', may better resist escape mutants and yield longer-lasting efficacy in future.

Host-oriented RNAi therapies

Virus-oriented RNA therapies engage the virus on its own terms and in full view of its evolutionary strengths. One alternative approach is to sequester host miRNAs crucial for viral replication. miR-122 is highly expressed in liver and is necessary for HCV replication. This is targeted by two host-oriented therapeutics, miravirsen and RG-101. 97 Miravirsen showed some efficacy with few adverse effects in clinical trials, while RG-101 showed promising efficacy but remains subject to clinical hold because of adverse effects.
Besides safety, a clear limitation is that not all medically relevant viruses use host miRNAs as key elements in their replication strategy. One solution is to engineer such viruses to harbour endogenous miRNA-targeting sequences. This yields recombinant viruses resembling wild-type but with greatly reduced pathogenicity, restricted tissue tropism and impaired replicative fitness, with potential use as vaccines. Using poliovirus (PV), which replicates primarily in the pharynx and gastrointestinal tract but causes severe neurological disease, Barnes et al. first demonstrated that recombinant PV harbouring a complementary sequence of murine brain-specific miR-124a was severely compromised in its ability to replicate within this tissue. When this sequence was substituted for a sequence complementary to the ubiquitously expressed miRNA let-7a, PV replication was further reduced, indicating miRNAs may be used to control tissue tropism. Notably, similar effects were obtained in mice rendered genetically unresponsive to IFN, which nevertheless generated protective antibodies against reinfection by between 10 and 10 000 times the LD 50 of wild-type PV. 98 This approach was recently used by Yee et al. 99 towards developing a live attenuated vaccine for enterovirus 71. Similar results were also obtained by Kelly et al. 100 using Coxsackievirus, where a majority of mice inoculated with recombinant virus harbouring tissue-specific miRNA-targeting sequences showing greatly reduced morbidity and mortality up to 70 days post-infection. Benitez et al. showed that mice inoculated with as much as 2500 times the LD 50 of IAV, also modified to harbour murine miRNAtargeting sequences, remained asymptomatic up to 10 days post-infection. These mice were also IFNunresponsive, confirming that mammals can, in principle, elicit a highly effective RNAi-mediated antiviral response and immunological memory against evolutionary diverse viruses in the absence of IFN-I. 101 Nevertheless, other viruses harbouring similar modifications have shown mixed results. In contrast to their earlier work on Coxsackievirus, Kelly et al. found vesicular stomatitis virus engineered to contain various host miRNA-targeting sequences largely resisted miRNA-mediated restriction. Nevertheless, a decrease in neurotoxicity was observed with miR-125 that also preserved the virus' oncolytic activity, 102 properties that are crucial in cancer immunotherapy applications. DenV was successfully restricted from hematopoietic cells by introducing four miR-142 targeting sites, although the virus quickly reverted and continued proliferating at low levels after excising all four sites. 103 Since there appears to be no clear pattern that might explain these disparate effects between virus species, additional work remains in order to use recombinant miR-targeting viruses for routine therapeutic use.

Designer vaccines

To elicit humoral as well as long-lasting cellular immunity, live attenuated vaccines are the most effective therapy currently available. These are typically produced by passaging viral isolates in permissive immune-deficient hosts such as embryonated chicken eggs or non-human continuous cell lines (e.g. Vero), thereby forcing viral re-adaptation and loss of virulence in the original host. However, the basis for attenuation is usually ill-defined. Some species or clinical isolates are not readily amenable to current in vitro culturing methods (e.g. norovirus), 104 which often fail to recapitulate essential elements of the viral replication cycle and pathogenesis. Furthermore, attenuated strains are often so compromised that immune adjuvants are required to stimulate antigenicity upon inoculation. Collectively, these challenges increase production costs of many vaccines while limiting detailed studies and the number of viruses for which safe and effective live vaccines can be produced.
An alternative approach is to genetically reprogram cells derived from the host species that serve as the natural reservoir of the virus. One advantage of this approach is the likely greater amenability of previously uncultivable or pooryielding viruses for cultivation ex vivo. Additionally, the resulting strain will likely preserve some replicative competence upon inoculation, thereby eliciting stronger immunity in the absence of adjuvants. The challenge remains, however, to identify which parts of the cell or culture methods should be modified to generate broad permissiveness, high titres and greatly reduced virulence simultaneously. Obvious candidates include genes that restrict viral replication but are dispensable for cell survival, such as IFN genes and their signalling components, and potentially certain multifunctional proteins. Taken to its logical conclusion, it should be possible to genetically engineer an immune-null human cell substrate within which to passage virus free of virulence factor targets and immune selective pressure. In this way, the strain that emerges will likely exhibit strong antigenicity but severe degradation in mechanisms of host immune antagonism. Such an approach may also prove useful in the context of viruses that cause severe disease primarily through cytokine hyperactivation.
Additional modifications to this host-oriented approach may further improve vaccine yield or safety. These may include eliminating pro-apoptotic genes to limit virus-induced programmed cell death and increase viral titres. The culture system itself may be improved to better represent the threedimensional microarchitecture of the host tissue and other features necessary for efficient viral replication. Organoids and other stem cell-derived tissues represent one approach under recent and intensifying examination. Human lung organoids have been demonstrated to recapitulate key properties of RSV pathogenesis, 106 and human intestinal epithelium has been developed for previously uncultivable norovirus. 104 Additionally, incorporating multiple tissue-specific miRNAtargeting sequences within the attenuated viral genome may improve vaccine safety by impairing its ability to replicate within inflammation-sensitive or irreplaceable tissues. Inversely, this same strategy may be used to help guide infection of certain tissues such as oncolytic viruses in the case of cancer immunotherapy. 100 Another safety feature could involve a drug-selective 'kill switch', whereby key viral proteins are fused to the FK506-binding protein 12 destabilisation domain. Viral fusion proteins are 'rescued' from proteasomal degradation in the presence of the drug Shield-1 but efficiently degraded upon its removal, 107 thereby yielding a conditionally replicationincompetent strain. The coming decade appears likely to see two key transitions: from empirical to designer vaccines, and from viruses as pathogens to important tools in biotechnology.

Neo-virology and future biotechnologies

Of the 8.7 million known species on earth, viruses are likely the most ancient and prolific with at least 10 31 virions estimated to exist today. 10 Sampling only a fraction of these diverse host-virus interactions has already resulted in ground-breaking biotechnologies including biomolecule and bioactive compound delivery systems, RNAi-mediated antiviral therapies and genetic engineering using zinc finger nucleases, TALENs and CRISPR/Cas9. These have wide-ranging applications in antiviral therapy and vaccine development, immunotherapy, regenerative medicine, environmental science and numerous other fields.
Neo-virology is an emerging field aiming to further this trajectory of innovation by systematically characterising the roles of viruses and viral-mediated processes in the entire living biosphere. 10 As the unexplored genetic diversity of viruses is unlocked through improvements in sequencing technologies and big data analysis, the molecular basis of host-virus interactions and the evolutionary relationships between highly divergent species are becoming clearer.
One area of interest is the increasing number of nucleocytoplasmic large DNA viruses (NCLDVs) being discovered in prokaryote, protist and invertebrate hosts and in soil and water. These include two amoebal pathogens: pandoravirus, which harbours the largest known viral genome at 2.5 Mb, 108 and mimivirus, an emerging human pathogen harbouring a 1.2 Mb genome. 109 In stark contrast to RNA viruses, these giant DNA viruses appear capable of acquiring additional information without clear bound, presenting an alternative solution to the information economy paradox. These viruses are also suggested to readily generate genes de novo. 13 While few complete genome sequences of such viruses are currently available, most of the numerous proteins encoded by these vast viral genomes are entirely novel. 108, 110, 111 If the process for de novo generation of viral genes can be harnessed, this could support efforts at directed evolution of new and useful biological functions.
Nucleocytoplasmic large DNA viruses proteins with inferred functions reveal interesting patterns. A recently discovered NCLDV encodes a full set of eukaryote-like histones and a DNA polymerase, potentially placing it at the root of the eukaryotic clades. 110 An ancient NCLDV-like virus may have been responsible for the origin of the eukaryote nucleus itself. 112 Subsequently, eukaryote multicellularity, coupled with programmed cell death, may have emerged as an ancient antiviral defence mechanism, 113 possibly enabling the rise of complex life. Retroviral elements, in addition to driving formation of the mammalian placenta, control hormones involved in gestation and birth timing in some mammals. 114 Such elements comprisẽ 8% of the human genome, which also contains genetic material derived from viruses with no retrotranscription or integration functionality. 115 Thus, despite being strictly non-living, viruses have radically shaped the living biosphere. Understanding these processes could enable new approaches to control the basic functions of life.
Areas where improved understanding of such host-virus interactions could have immediate implications include human disease, bioremediation of harmful algal blooms and climate change. Contemporary viruses overwhelmingly infect marine microorganisms, turning over an estimated 20% of the ocean microbiome daily. 9 These infections have significant effects on carbon absorption by oceans as well as global nutrient and energy cycles. 11, 14 Nevertheless, the interactions between the ocean virome and microbiome and their effects climate remain poorly characterised. By examining evolutionarily diverse host-virus interactions in detail, immunology and virology may provide effective solutions to not only human disease but also cost, environmental and other sustainability issues of our time.

CONCLUDING REMARKS

As outlined in this review, current antivirals almost exclusively target virus proteins and have significant development costs, limited therapeutic range and are ultimately susceptible to escape mutant selection. Despite being intractably limited in informational size, RNA viruses are thorough problem solvers, often subverting multitasking host proteins to achieve favorable host subversion at minimal informational cost. Such solutions to the viral information economy paradox are often conserved, creating opportunities to leverage imbricated multidependency on key host proteins for host-oriented antiviral therapies that are more effective, broad-acting and ultimately more costeffective. Although such proteins can present challenging therapeutic targets, host-oriented therapies will synergise with increased therapeutic drug availability and developments in RNAi, precision medicine and immunotherapy. Additionally, methods of increasing viral antigenicity yet controlling replication and tissue tropism will increase the number of viruses for which safe and highly effective vaccines can be produced. Viruses such as NCLDVs appear to readily acquire new information with which to subvert their hosts. The fruits of problem-solving by such viruses include large numbers of proteins with unique and unknown biological functions. The influence and genetic hallmarks of viruses at both extremes are found in humans and throughout the living biosphere. By expanding the examination of evolutionarily diverse host-virus interactions, disease, cost, environmental and other sustainability issues of our time may be remedied by leveraging, rather than yielding to, the properties of RNA and DNA viruses as developed through co-evolution with their hosts.

EXPLOITING THE INFORMATION ECONOMY PARADOX IN RNA VIRUS EVOLUTION

Despite facilitating viral infectivity, these solutions to the information economy paradox cut both ways. Imbricated dependency on multifunctional host proteins for various key replication steps creates vulnerabilities that may be exploited for highly efficacious antiviral therapies. For example, denying such host proteins to the virus may disrupt multiple key elements in its replication strategy. Where these proteins represent foci of evolutionary convergence, such therapies may yield robust, broad-spectrum antiviral activity. The degree of innovation required to circumvent such a therapy and, especially in the case of RNA viruses, the informational barrier to realising this innovation may be high. Escape mutants that emerge may be forced to overcome multiple deficiencies simultaneously and suffer compounding fitness penalties in the process. Furthermore, host-oriented approaches present a much larger list of potential therapeutic targets than the dozen or so gene products produced by most human-infective RNA viruses, many of which are challenging targets in the first instance because of genetic diversity and intrinsic protein disorder. 19 Given the higher fidelity of DNA versus RNA replication, host therapeutic targets may prove resilient to certain mechanisms of viral resistance. Altogether, these advantages may reduce antiviral development costs over the long term, allowing for greater treatment accessibility and faster development of future therapies. But which host proteins are sufficiently multifunctional? And how might these present viable therapeutic substrates?

MULTIFUNCTIONAL HOST PROTEINS AS POTENTIAL ANTIVIRAL TARGETS

To canvass potential therapeutic targets, the 282 most multifunctional human proteins identified to date were used to interrogate available protein interaction data ( Figure 2 ). Strikingly, 77% (216/ 282) of these have been experimentally determined to interact with at least one viral protein (Supplementary table 1) . Of these, 74% interact with at least one ssRNA viral protein, highlighting how multifunctional host proteins represent key ssRNA viral manipulation targets. The three highly multifunctional host proteins targeted by the greatest diversity of ssRNA virus families are as follows: heat-shock protein 90a (HSP90a), HSP7C and polyadenylate-binding protein 1 (PABP1; Figure 2 ). These proteins appear to serve as key drivers of convergence between diverse human-infective viruses, suggesting these are potential targets of broad-spectrum antivirals. Additional targets of significant interest include alpha-enolase, heat-shock protein beta-1 (HSPB1, also termed HSP27), heterogeneous nuclear ribonucleoprotein K (hnRNPK), histone acetyltransferase (HAT) p300, vimentin, vitamin K epoxide reductase complex subunit 1 (VKORC1) and tumor antigen p53 ( Figure 2 and Table 2 ). Diverse RNA viruses targeting these proteins, as well as possible therapeutic avenues, are discussed below.

Human immunodeficiency virus (HIV)

The HIV-1 accessory protein viral infectivity factor (Vif), also crucial in suppressing host immunity, 25 targets all three highly multitasking heat-shock proteins HSP90a, HSPB1/HSP27 and HSP7C ( Figure 2 ). This suggests subversion of the cellular protein quality control pathways or HSP-mediated gene expression is vital in the HIV-1 replication cycle. Accordingly, HSP90 inhibitors 17-AAG/ tanespimycin and AUY922 (Table 2) were recently shown to inhibit HIV-1 transcription and suppress viral rebound in a humanised mouse model. 26 Although these and other HSP90 inhibitors have encountered efficacy and toxicity issues during clinical trials as anticancer therapies, aminoxyrone is novel, first-in-class HSP90 inhibitor that appears to alleviate both issues. 27 Its efficacy as an antiviral or antiretroviral therapeutic has yet to be studied. Inhibitors of HSPB1 and HSP7C (Table 2) represent additional avenues for effective hostoriented antiretroviral therapies that have also yet to be explored. HIV-1 tat (transactivating regulatory protein), which is required for efficient viral gene transcription, 28 targets PABP1, p53 and HAT p300 ( Figure 2 ). The latter protein is further manipulated by two additional HIV-1 proteins, viral protein R (Vpr) and Pol (DNA polymerase), as well as the transactivating regulatory protein (Tax) of human T-lymphotropic virus (HTLV; Figure 2 ), plausibly representing a conserved mechanism of host subversion. HATs and histone deacetylases (HDACs) are crucial effectors in epigenetic modulation of gene expression, connecting a large number of cell signalling inputs with transcriptional outputs through histone post-translational modifications. 29 HAT and HDAC inhibitors have been shown to suppress viral transcription ('kill') and re-activate latent virus ('shock-and-kill'), respectively, 30, 31 suggesting epigenetic remodulation of host gene activity is exceptionally important in the replication strategy of HIV-1 and other retroviruses. Alternatively, retroviral proteins may block or usurp the enzymatic activity of HAT p300 to direct acetylation of viral or other host proteins, which has been suggested for Tat. 32 The interactions between HAT p300 and HIV-1 proteins Tat, Vpr and Pol (Figure 2) , together with the large number of HAT p300 inhibitors currently available (Table 2) , expand the possibilities for targeting HAT p300 in host-oriented antiretroviral therapies.

Togaviruses

Viral infection often induces cytoskeletal remodelling, resulting in cytopathic morphologies including syncytia and tumor-like aggregates. Cells treated with actin depolymerising agents such as cytochalasin D show drastic reductions in production of numerous viruses, 38 Figure 2 ). Vimentin is reported to play a key role in replication complex assembly and modulating viral protein expression levels in DenV and HCV infection, respectively. 39, 40 Withaferin D targets the soluble form of vimentin 41 (Table 2) and has anticancer properties, although the effects of vimentin-targeting drugs in the context of infection have yet to be extensively studied.

CHALLENGES AND STRATEGIES IN TARGETING MULTIFUNCTIONAL HOST PROTEINS

The last eukaryote common ancestor likely possessed an RNAi system utilising endogenous or exogenous noncoding RNAs (ncRNAs) and an RdRP. 59 However, host-pathogen interactions have shaped RNAi utilisation throughout eukaryote diversification. Budding yeasts, including the prototypical Saccharomyces cerevisiae, harbouring endemic dsRNA viruses of the Totiviridae family lost RNAi while other yeasts lacking such viruses retained RNAi. 60 With the rise of jawed vertebrates, RdRP was lost while IFN was gained, enabling large, complex life to coordinate multifurcated, system-wide responses to infection and eventually supplanted RNAi as the primary antiviral defence mechanism in animals. 61, 62 Nevertheless, ncRNAs continue to perform crucial roles in fundamental mammalian cellular processes including pre-mRNA processing via spliceosomes (e.g. small nuclear RNA; snRNA). Small interfering RNA (siRNA) and microRNA (miRNA) are those most commonly applied in current RNAi biotechnology.
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Abstract

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21 st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drugresistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine-and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P,0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine-and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza.

Introduction

Globally, influenza viruses cause substantial morbidity and mortality in humans and economic dislocation in afflicted nations. Each year in the United States, seasonal influenza virus infection result in an estimated 36,000 deaths and 200,000 hospitalizations [1] . Antiviral drugs are an important means to mitigate the impact of the yearly influenza epidemics and potential pandemics. Currently, two classes of antiviral drugs have been approved for the prevention and treatment of influenza infection -the M2 channel inhibitors (aminoadamantanes; amantadine and rimantadine) and the neuraminidase inhibitors (NAIs; oseltamivir and zanamivir). However, the prevalence of drug-resistant strains, which has been reported for both classes of antiviral drugs for seasonal influenza [2, 3] , could undermine their clinical benefit when utilized as monotherapy. Indeed, in 2009, the Centers for Disease Control and Prevention (CDC) reported that 100% of the seasonal H3N2 virus isolate tested were resistant to the adamantanes, and 99.6% of the seasonal H1N1 viruses tested were resistant to oseltamivir [4] .

Discussion

Against the 2009 H1N1 strains, the interactions of oseltamivir carboxylate, peramivir, and zanamivir in double combinations ranged from additive to moderately antagonistic, indicative that the activity of these drugs was not enhanced in combination compared to their activity as single agents. These results suggest that double combinations of NAIs may not provide any added benefit over the drugs as single agents. Given that all NAIs bind in the same substrate binding pocket in NA, the use of these drugs in combination in the absence of enhanced activity raises the risk of selecting for a single mutation that could confer resistance to both neuraminidase inhibitors simultaneously. Indeed, cross-resistance to oseltamivir and zanamivir resulting from a single amino acid change has been documented for seasonal influenza A and B viruses [30] . If this were to occur in the 2009 H1N1 background, the resulting virus would be resistant to all approved anti-influenza drugs.

Introduction

In April, 2009, a novel H1N1 virus of swine-origin capable of human-to-human transmission likely emerged in Mexico and was first isolated from patients enrolled in two separate surveillance activities in Southern California [5] . The emergence and spread of 2009 H1N1 to over 168 countries has led U.S. officials [6] and the World Health Organization (WHO) [7] to declare a public health emergency; on June 11, 2009 the WHO raised the influenza pandemic alert from phase 5 to phase 6, the official declaration of a pandemic [8] . Early published results from the CDC showed that 2009 H1N1 bears the amantadine-resistance associated S31N mutation in the M2 ion channel, but remains susceptible to oseltamivir and zanamivir [9] . More recently, however, the CDC has reported that ten 2009 H1N1 isolates tested in the United States have been found to be resistant to oseltamivir, raising the concern that dually resistant viruses may become prevalent [4] .
In this study, we sought to evaluate the activity and synergy of the TCAD regimen against influenza viruses which were resistant to amantadine or oseltamivir. Our data showed that against amantadine-resistant viruses -both seasonal and 2009 H1N1and oseltamivir-resistant seasonal viruses, the TCAD regimen was strongly synergistic, and the synergy of the TCAD regimen was greater than the synergy of any double combination. Surprisingly, we found that amantadine and oseltamivir contributed to the synergy of the TCAD regimen at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our findings highlight the utility of the TCAD regimen as a potential approach to address the current limitations of antiviral potency and drug resistance, and as a viable broad-spectrum therapeutic option for serious influenza virus infection.

Cell-Based Assays

EC 50 , TC 50 , and Synergy Calculations EC 50 , TC 50 , and synergy calculations were done as described previously [10] . Briefly, EC 50 and TC 50 calculations for single agents were made by normalizing the NR data for each well against the cell and virus controls, which was assumed to represent 100% cell viability and 0% cell viability, respectively. Normalized data were plotted as percent cell viability versus compound concentration. The data points were then fitted using fourparameter curve fitting in Graphpad Prism (Graphpad Software, La Jolla, CA) to derive the EC 50 and TC 50 . Statistical comparisons between best-fit EC 50 values for any two curves were performed in Prism using the extra sum-of-squares F test; differences in EC 50 values between two curves with a P-value of ,0.05 were considered significant.

Statistical Analysis of Synergy Volumes

Between three and nine independent experiments were conducted using identical dosing ranges for amantadine, ribavirin, and oseltamivir carboxylate against each virus. The experiments used a 3-replicate plate format, for a total of 9 to 27 observations for each condition. The data from the independent experiments were merged and subjected to statistical analysis using the random effects model. The raw data were imported into the program R and were normalized to the virus and cell controls as described above, and synergy (percent inhibition above calculated) is calculated as above. For synergy volume estimates, random effects models were applied to the data to account for variation between replicate measures within experiments. Therefore, the models took the form as given by:

Activity of Amantadine in the Context of the TCAD Regimen against Seasonal and Avian Amantadine-Resistant Viruses

As an extension of these studies we assessed the contribution of amantadine to the synergy of the TCAD regimen against . As a single agent, amantadine had no activity against these viruses at concentrations up to the highest concentration tested (32 mg/mL; data not shown). As shown in Figure 2 , against NC V27A and WI S31N, amantadine contributed to the synergy of the TCAD regimen in a concentration-dependent manner. The synergy of the TCAD regimen in the presence of amantadine was greater than the synergy of the ribavirin/oseltamivir carboxylate double combination without amantadine, and the increase in the synergy of the TCAD regimen was observed at 0.32 mg/mL amantadine for NC V27A and 1 mg/mL amantadine for WI S31N, when significance was evaluated at the level of ,0.05. Against DK A30T, however, amantadine did not make a contribution to the synergy of the TCAD regimen within the concentration range tested (0.1-3.2 mg/mL), and the synergy of the TCAD regimen was not greater than the synergy of the ribavirin/oseltamivir carboxylate double combination. Whether the lack of contribution from amantadine against the DK A30T virus was due to the specific subtype (H5N1), the M2 mutation (A30T), or the combination of both remain to be determined. However, it should be noted that while amantadine made no contribution to the activity of the TCAD regimen against DK A30T, both ribavirin and oseltamivir remain active and their interactions were additive, indicative that two out of three drugs contribute to the activity of the TCAD regimen against this virus (data not shown).

Activity of Oseltamivir in the Context of the TCAD Regimen against Oseltamivir-Resistant Viruses

Given that a large percentage of seasonal influenza circulating viruses are resistant to oseltamivir, we next assessed the activity and synergy of the TCAD regimen against oseltamivir-resistant viruses to evaluate the spectrum of activity of the TCAD regimen, and to determine whether oseltamivir contributed to the activity of the TCAD regimen against oseltamivir-resistant viruses. Two H1N1 oseltamivir-resistant viruses were used, both of which bear the H274Y substitution in NA which has been demonstrated to confer resistance to oseltamivir [20] : A/Mississippi/3/01 (MS H274Y) and A/Hawaii/21/07 (HI H274Y). As a single agent, oseltamivir carboxylate had no antiviral activity against either virus below 1 mg/mL (data not shown). The synergy volumes of double and triple combinations of amantadine, ribavirin, and oseltamivir were determined against both viruses, and the data are presented in Figure 3 . As double combinations, amantadine/ oseltamivir carboxylate, amantadine/ribavirin, and ribavirin/ oseltamivir carboxylate were all additive ( Figure 3A) . As was seen with the 2009 H1N1 viruses, the TCAD regimen was synergistic, and the synergy volume was greater than the synergy volume of any double combination, with each drug making a contribution to the synergy of the TCAD regimen against the oseltamivir-resistant viruses ( Figure 3B-D) . Importantly, oseltamivir carboxylate contributed to the synergy of the TCAD regimen starting at 0.1 mg/mL against MS H274Y (P,0.05) and at 0.32 mg/mL against HI H274Y (P,0.01). At these concentrations, which are achievable clinically, oseltamivir carboxylate is not active as a single agent against these resistant strains. Synergy plots for the TCAD regimen against MS H274Y and HI H274Y are provided in Figure S3 , and reveal increasing synergy with increasing concentrations of oseltamivir carboxylate. At the highest concentration of oseltamivir carboxylate tested (3.2 mg/mL), synergy occurred over wide concentrations of ribavirin and amantadine. At lower concentrations of oseltamivir carboxylate, synergy occurred at higher concentrations of amantadine (0.1 mg/mL or higher), and at wide concentrations of ribavirin. No significant antagonism was detected at any concentrations of the three drugs. We also determined the EC 50 of oseltamivir carboxylate as a single agent and in double and triple combinations against both oseltamivir-resistant viruses. As summarized in Table 3 , the EC 50 of oseltamivir carboxylate as a single agent was 74 mg/mL against MS H274Y and 15 mg/mL against HI H274Y. These represent 1480-and 300-fold reductions in susceptibility compared to the published values for a wild-type virus [11] . The EC 50 of oseltamivir carboxylate was not reduced in double combination with 1mg/mL ribavirin or 0.032 mg/mL amantadine against MS H274Y, and was reduced by only 1.7-and 1.4-fold, respectively, against HI H274Y. However, in triple combination with ribavirin and amantadine at the same concentrations used in double combination, the EC 50 of oseltamivir carboxylate was reduced by 21-fold against MS H274Y and by 5.8-fold against HI H274Y.

Inhibitory Quotients of Antiviral Drugs against Susceptible and Resistant Viruses

As representatives of the current circulating strains, we used A/ New Caledonia/20/99 (H1N1) (NC20) as the susceptible virus (susceptible to amantadine, ribavirin, and oseltamivir carboxylate), CA10 as the amantadine-resistant virus, and MS H274Y as the oseltamivir-resistant virus. Concentration-response curves for amantadine, ribavirin, oseltamivir carboxylate, and TCAD were generated against all three viruses, and the EC 50 s and IQs are summarized in Table 4 . Amantadine was effective at inhibiting NC20 and MS H274Y in vitro, resulting in IQs of 1.95 and 7.17, respectively. However, the IQ for amantadine was reduced to 0.02 when tested against CA10, indicative that an in vitro concentration representing the achievable plasma concentration at the recommended dose was not adequate to inhibit the amantadineresistant virus in vitro. An IQ of 0.02 represents a 100-fold reduction compared to NC20 and a 350-fold reduction compared to MS H274Y. Similarly, oseltamivir carboxylate was effective at inhibiting NC20 and CA10 in vitro resulting in IQs of 1.5 and 5.0, respectively, but not MS H274Y (IQ = 0.004). Similar to amantadine against the amantadine-resistant virus, the IQ for oseltamivir was reduced 350-to 2300-fold against the oseltamivirresistant virus compared to susceptible viruses. The IQs for ribavirin were uniformly low and below 1 for all three viruses (0.23 to 0.42). On the other hand, the IQs for the TCAD regimen as a fixed dose combination were consistently high against all three viruses (8.33 to 17.24), varying by no more than 2-fold between susceptible and resistant viruses. These data suggest that the TCAD regimen may have broad utility against all circulating influenza strains, including strains that are resistant to either amantadine or oseltamivir.

Discussion

In the present study, we examined the efficacy and synergy of the TCAD regimen against viruses which were resistant to oseltamivir or amantadine, including 2009 H1N1. Consistent with the previous findings [9] , we found that the 2009 H1N1 strains were susceptible to NAIs (oseltamivir carboxylate, zanamivir, and peramivir) and ribavirin, but were resistant to adamantanes (amantadine and rimantadine). Surprisingly, we found that amantadine as a single agent retained partial activity against these viruses (Table S2) , albeit the activity was reduced by 100-fold compared to a susceptible virus, whereas rimantadine had no activity below the 50% cytotoxic concentration. This observation suggests that phenotypic testing, in addition to determination of the genotype, may be necessary in order to fully understand the susceptibility profile of a novel virus and may have important implications in guiding the choice of antivirals for use in combinations.
In total, we tested the activity and synergy of the TCAD regimen against six amantadine-resistant viruses, including three strains of 2009 H1N1, and two oseltamivir-resistant viruses. The viruses tested in this study come from the three subtypes that cause significant morbidity and mortality in humans (H1N1, H3N2, and H5N1), and include seasonal, avian, and pandemic strains. The double combinations of amantadine/oseltamivir carboxylate, amantadine/ribavirin, and ribavirin/oseltamivir carboxylate were additive against 2009 H1N1, and ranged from additive to moderately synergistic against the other viruses (data not shown). In contrast, with the exception of the duck H5N1 virus, we found that the TCAD regimen was synergistic at clinically achievable concentrations of all three drugs, and that the synergy of the TCAD regimen was greater than that of any double antiviral drug combination. These data suggest that the TCAD regimen may have broad-spectrum antiviral activity against circulating influenza A viruses, including strains that are resistant to either classes of antivirals. To date, most influenza A strains in circulation (,99%) are resistant to either the adamantanes or oseltamivir, and not to both [31] , and thus are expected to be susceptible to the TCAD regimen. Currently, rapid diagnostic tests are not available to determine the susceptibility profile of influenza viruses in real time, and thus clinicians do not often have the necessary information with which to guide appropriate antiviral use. The availability of a broad-spectrum antiviral therapy that would be effective against the majority of circulating strains regardless of the susceptibility would be of high clinical utility.
Importantly, we found that amantadine and oseltamivir contributed to the synergy of the TCAD regimen against amantadine-resistant and oseltamivir-resistant viruses. The contributions from both drugs to the synergy of the TCAD regimen were significant at clinically achievable concentrations where they had little or no antiviral activity as a single agent. For instance, a comparison of the synergy volume of the TCAD regimen at 0.32 mg/mL amantadine to the synergy volume of the ribavirin/ oseltamivir carboxylate double combination (no amantadine) revealed that amantadine contributed 39%, 24%, and 44% to the total synergy of the TCAD regimen against CA04, CA05, and CA10, respectively. Similarly, against the oseltamivir-resistant viruses, oseltamivir carboxylate at 0.32 mg/mL contributed 76% and 83% to the total synergy of the TCAD regimen against MS H274Y and HI H274Y, respectively. Thus, all three drugs contributed to the synergy and activity of the TCAD regimen against amantadine-and oseltamivir-resistant viruses, and the activities of amantadine and oseltamivir were restored in the context of the TCAD regimen against influenza strains that were resistant to these drugs, thereby maximizing the clinical utility of these drugs. The mechanism(s) by which amantadine and oseltamivir carboxylate contribute to the synergy of the TCAD regimen against resistant strains is unclear. The interactions between M2, HA, and NA on the surface of the influenza particle are complex and not well understood, and a number of studies have demonstrated that HA-M2 and HA-NA interactions can affect the susceptibility to amantadine and oseltamivir, respectively [32, 33] . Furthermore, amantadine has been demonstrated to exert antiviral activity via interactions with HA at higher concentrations [34, 35] . It is conceivable that, as the result of protein-protein interactions between M2, HA, and NA, the binding of a drug at one site may affect the conformation and therefore affinity for another drug at another site. The mechanism by which ribavirin contributes to the synergy of the TCAD regimen is also unclear. Ribavirin has been documented to act through multiple mechanisms affecting both virus replication and host immune response [36] [37] [38] , and it remains to be elucidated which of these mechanisms are responsible for the synergy with amantadine and oseltamivir.
Finally, we evaluated the activity and inhibitory quotient (IQ) of TCAD against susceptible and resistant viruses representing the currently circulating strains. While the correlation between IQ and clinical efficacy has not been demonstrated for influenza, it is valuable to construct a relative ranking of the IQ of different antiviral regimens against susceptible and resistant viruses in order to assess the spectrum of their activity. When tested against a seasonal susceptible H1N1 virus, an amantadine-resistant 2009 H1N1, and a seasonal oseltamivir-resistant H1N1 virus, TCAD was uniformly active against all three viruses with significantly high IQs (8.33 to 17.24; Table 4 ). This suggests that TCAD may have broad antiviral activity against all currently circulating influenza strains and may have good efficacy in the clinical setting against these strains.

Supporting Information

Table S1 Concentration ranges (mg/mL) of each drug tested in double and triple combinations against different influenza A viruses. Drugs were titrated at half log 10 Figure S1 Synergy plot of double and triple combinations of amantadine, ribavirin, and oseltamivir carboxylate against 2009 H1N1 A/California/05/09 (CA05) replication as determined by Neutral Red assay in MDCK cells. Calculated additive interactions were subtracted from the experimentally determined inhibition to reveal regions of synergy (inhibition above expected) or antagonism (inhibition below expected). Values were derived from mean triplicate data and presented at 95% confidence. This experiment was repeated a total of six times with similar results. Blue areas indicate concentrations of each drug that are synergistic, gray areas indicate concentrations that are additive, and red areas indicate concentrations that are antagonistic. The intensity of the color (blue or red) corresponds to percent inhibition above or below expected. Figure S4 Viability of MDCK cells treated with the TCAD regimen. MDCK cells were incubated with the TCAD regimen at the highest concentrations of all three drugs used in the synergy experiments (3.2 mg/mL amantadine, 10 mg/mL ribavirin, and 3.2 mg/mL oseltamivir carboxylate), and cell viability was determined by Neutral Red assay after 72 hours. Values are the mean of nine replicates from three experiments, with standard deviations. The difference in viability between the TCAD treated cells and the cell controls was not statistically significant (P = 0.47, Student's t-test). Found at: doi:10.1371/journal.pone.0009332.s006 (1.00 MB TIF)

Introduction

In an earlier study, we explored the in vitro antiviral activity and synergy of single, double, and triple combinations of amantadine, ribavirin and oseltamivir against a panel of influenza A viruses that were susceptible to these drugs [10] . Our hypothesis was that a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action, and which act at different stages in the viral life cycle, could result in synergistic antiviral activity. Our results showed that these drugs did indeed act synergistically, with the triple combination showing significantly greater synergy than any of the double combinations evaluated. Furthermore, we found that the synergy of the TCAD regimen was maintained across multiple seasonal and avian influenza A strains, including the three major subtypes -A/H1N1, A/H3N2, and the avian A/H5N1 -that currently cause significant morbidity and mortality in humans.

Cell-Based Assays

To obtain single agent concentration-response curves, individual drugs were added to MDCK cells in 96-well microplates (8610 4 cells/well) using three wells for each concentration used. The compounds were added at the following concentrations: oseltamivir carboxylate, zanamivir, and peramivir at 0, 0.000032, 0.0001, 0.00032, 0.001, 0.0032, 0.01, 0.032, 0.1, 1.0, 10.0 and 100 mg/mL; amantadine, rimantadine, and ribavirin at 0, 0.001, 0.0032, 0.01, 0.032, 0.1, 0.32, 1, 3.2, 10, 32 and 100 mg/mL. Untreated wells of infected cells (virus controls), uninfected cells (cell controls), and drug cytotoxicity controls (cells and drugs only, using the same dilution range for each drug as the test wells) were included on each test plate. At three days after infection, the virus control wells exhibited 100% cytopathology. The 50% effective concentration (EC 50 ) and 50% cytotoxic concentration (TC 50 ) was determined for each drug as outlined below.
For double and triple combination studies, each drug was tested in triplicate at five or six concentrations (including no drug), in which the high concentration for each drug was set to approximate the EC 50 of the drug as a single agent. The concentrations of each drug used in double and triple combinations against each virus are provided in Table S1 . The cytotoxicity of the double and triple drug combinations were determined using the same experimental format in three separate experiments, and using the same concentration ranges as outlined in Table S1 .

Activity of Antiviral Drugs as Single Agents against 2009 H1N1

The susceptibility of three 2009 H1N1 influenza strains -A/ California/04/09 (CA04), A/California/05/09 (CA05) and A/ California/10/09 (CA10) -to each of six antiviral drugs (amantadine, rimantadine, oseltamivir carboxylate, zanamivir, peramivir, and ribavirin) as single agents was determined by measuring the inhibition of virus-induced CPE in MDCK cells. Against the three strains, oseltamivir carboxylate, zanamivir, peramivir, and ribavirin produced concentration-dependent inhibition of cytopathic effect (data not shown). Amantadine was active only at higher concentrations (EC 50 of 16-20 mg/mL; 85-106 mM) (Table S2) , which represents a .250-fold reduction in susceptibility compared to the published values for a wild-type virus [17] . Rimantadine did not produce inhibition up to the 50% cytotoxic concentration (EC 50 .12 mg/mL; .55 mM). The EC 50 values for the six drugs as single agents against all three strains are summarized in Table S2 , and confirm that the three virus strains remain susceptible to oseltamivir carboxylate, zanamivir, peramivir, and ribavirin. These results are consistent with the results previously published that demonstrated that 2009 H1N1 contained a mutation (S31N) in the M2 channel that has been associated with resistance to adamantanes, but remained susceptible to NAIs [18] .

Synergy of Double and Triple Combinations of Amantadine, Ribavirin, and Oseltamivir Carboxylate against 2009 H1N1

We next assessed the synergistic activity of double and triple combinations of amantadine, ribavirin, and oseltamivir carboxylate over a range of concentrations of each drug against each 2009 H1N1 isolate. A quantitative measure of the total synergy (or antagonism) of a drug combination can be expressed in terms of synergy volumes, which represents the cumulative synergy and antagonism across all concentrations for all the drugs in a combination. Based on the empirically determined criterion of synergy volume .100 mg/mL 2 % using the lower confidence interval, all three double combinations were additive against the 2009 H1N1viruses (Table 1) . By contrast, the TCAD regimen was synergistic against all three viruses over multiple concentrations of all three drugs, with the synergy occurring at 0.1 mg/mL and above for amantadine, 0.32 mg/mL and above for ribavirin, and 0.0032 mg/mL and above for oseltamivir carboxylate (Table 1) . These data show that the synergy of the TCAD regimen occurred at clinically achievable concentrations for all three drugs, given that the expected average plasma concentrations based on the recommended doses are 0.43 mg/mL for amantadine, 1.3 mg/mL for ribavirin, and 0.3 mg/mL for oseltamivir carboxylate. Furthermore, the synergy of the TCAD regimen was greater than the synergy of any double combination tested for all three 2009 H1N1 strains. Figure 1B -1D shows the synergy of the TCAD regiment as a function of increasing concentrations of amantadine, ribavirin, or oseltamivir carboxylate as the third drug, representing the contribution of each drug to the synergy of the double combination without the third drug. These data reveal that the addition of each drug as the third drug to the double combinations resulted in a concentration-dependent increase in synergy, indicative that each drug contributed to the synergy of the TCAD regimen.
Importantly, despite the fact that amantadine had no significant antiviral activity as a single agent below 3.2 mg/mL (data not shown), we find that the amantadine contributed to the activity of the TCAD regimen at clinically achievable concentrations (0.43 mg/mL). Statistical analysis of the variability across all replicates from the six experiments for each virus revealed that amantadine made a significant contribution to the synergy of the TCAD regimen at concentrations 0.1 mg/mL and 0.32 mg/mL and above against CA05 and CA10, respectively, compared to the double combination of ribavirin/oseltamivir carboxylate without amantadine ( Figure 1B ). For CA04, while only the 3.2 mg/mL amantadine concentration had statistically significant greater synergy volume than the double combination without amantadine, there was a trend toward increasing synergy volume starting at 0.32 mg/mL. Thus, amantadine contributed to the activity of the TCAD regimen at concentrations where it was inactive as a single agent, and at concentrations that were clinically achievable.

Synergy of Double Combinations of Neuraminidase Inhibitors against 2009 H1N1

We also evaluated the interactions of double combinations of neuraminidase inhibitors (NAIs) against 2009 H1N1. As shown in Table 1 , the synergy volumes for the zanamivir/oseltamivir carboxylate were 2246116 mg/mL 2 %, 2155689 mg/mL 2 %, and 2197698 mg/mL 2 % against CA04, CA05, and CA10, respectively. These values suggest that the zanamivir/oseltamivir carboxylate combination was additive against these viruses. For the zanamivir/peramivir combination, synergy volumes were 2356112 mg/mL 2 %, 21976108 mg/mL 2 % and 2239693 mg/ mL 2 % against CA04, CA05, and CA10, respectively, indicative of additivity to moderate antagonism. Consistent with these values, the synergy plots for the NAI double combinations against CA05 revealed regions of antagonism (red areas), which occurred at higher concentrations of zanamivir (0.01-0.1 mg/mL) and at variable concentrations of the second NAI (oseltamivir or peramivir, Figure S2 ). Similar results were found with the other two 2009 H1N1 strains. Furthermore, evaluation of the EC 50 of each NAI in combination with a fixed concentration of a second NAI revealed that the antiviral activity of each drug was not enhanced in combination with a second drug (data not shown). The observation that double combinations of neuraminidase inhibitors were not synergistic is consistent with the fact that all three drugs are known to target the same enzyme, and all bind in the same substrate binding pocket in a similar manner [19] .

Discussion

Given that virtually all seasonal H3N2 and 2009 H1N1 strains are resistant to amantadine, and virtually all currently circulating seasonal H1N1 strains are resistant to oseltamivir, the pharmacologic rationale for the development of a triple combination antiviral drug (TCAD) composed of amantadine, ribavirin, oseltamivir is that at least two, and possibly three drugs, in the TCAD regimen will be active against all of these viruses. A number of studies have evaluated double combinations of antivirals [21] [22] [23] [24] [25] [26] [27] against influenza A infection in vitro, and Hayden et al. have tested a triple combination of two antivirals with human interferon a [28] . However, there have been few reports on the effects of drug combinations on resistant influenza viruses [26, 27, 29] . Recently, Smee et al. evaluated the effects of double combinations of amantadine, ribavirin, and oseltamivir against the same amantadine-resistant H5N1 virus used in this study (A/Duck/MN/1525/81) [26] . The authors found that the presence of amantadine in double combinations did not provide added benefit over the second drug alone, either in cell culture or in mouse models.
Our data suggests that a triple combination antiviral drug (TCAD) composed of amantadine, ribavirin, and oseltamivir may be an effective and viable therapeutic option for the treatment of pandemic and seasonal influenza infection. The body of data presented in this report validates the TCAD hypothesis, which states that for any given susceptible or resistant circulating influenza virus, at least two, and in some cases all three, drugs in TCAD will be active. Furthermore, the TCAD regimen appears to overcome baseline drug resistance and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza. The safety, pharmacokinetics, distribution, and metabolism of amantadine, ribavirin, and oseltamivir as single agents are well understood, and it is not expected that co-administration of the three drugs will result in substantially increased risk to patients compared to the administration of the individual drugs. In addition, all three double combinations have been tested in humans without adverse effects, including amantadine plus oseltamivir [39] , amantadine plus ribavirin [40] , and ribavirin plus oseltamivir [41] . Clinical trials to assess the efficacy and safety of TCAD for the treatment of influenza have been initiated, and will provide important data on the use of TCAD against both pandemic and seasonal influenza.

Cell-Based Assays

Synergy was calculated using the MacSynergy II software developed by Prichard and Shipman, which was modified to accommodate a three-drug combination [12] and is similar to that reported previously describing this approach [13] . The theoretical additive interactions were calculated from the concentrationresponse curves of each drug as a single agent. This calculated additive surface was then subtracted from the observed, experimental surface to reveal regions that deviate from the calculated additive effects. Purely additive interactions are represented graphically as areas in grey, indicating that they do not differ from the calculated additive effects. Synergistic interactions result in greater inhibition than the calculated inhibition, and are represented as areas in blue. Conversely, antagonism is represented as areas in red. Synergy plots are shown as the percent inhibition above or below expected (calculated additive inhibition), and are presented as the mean of three replicates at a level of 95% confidence, which eliminates insignificant deviations from the additive surface.
The synergy of the cytotoxic effects of double and triple drug combinations were calculated in a similar manner.

Determination of Inhibitory Quotient

The inhibitory quotient (IQ) is defined herein as the ratio of the expected average total (free and protein-bound) plasma concentration (C ave ) of each drug at recommended dosage to the EC 50 (C ave /EC 50 ). Thus, an IQ of 1 or greater means that the achievable total plasma concentration of the drug is equal to or greater than the in vitro 50% effective concentration, and the higher the IQ translates to a greater predicted efficacy. The C ave of each drug was determined by pharmacokinetic modeling using a non-compartmental model in the progam WinNonLin. The recommended doses, along with the pharmacokinetic parameters, used for modeling were obtained from the package inserts for amantadine [14] , oseltamivir [15] , and ribavirin [16] , and the references therein. For ribavirin, since the plasma concentration does not achieve steady-state until ,4 weeks after the start of dosing, the C ave was determined for the first 10-day window. Based on these parameters, the expected C ave for amantadine was determined to be 0.43 mg/mL based on the recommended dosage of 100 mg twice daily for the treatment of influenza infection; the expected C ave for oseltamivir was determined to be 0.3 mg/mL based on the recommended dosage of 75 mg twice daily for the treatment of influenza infection; and the expected C ave of for ribavirin was determined to be 1.3 mg/mL after 10 days of treatment based on the recommended dosage of 600 mg twice daily for the treatment of hepatitis C infection. To determine the IQ of the triple combination, amantadine, ribavirin, and oseltamivir carboxylate were tested as a fixed ratio combination, wherein the ratio of the three drugs was kept constant even as the total concentration of drugs varied. The ratio of drugs in the TCAD regimen was based on the expected C ave of each drug. A dilution curve of TCAD regimen was created by first preparing a solution of all three drugs at 100-fold the C ave of each drug (43 mg/ mL amantadine, 30 mg/mL oseltamivir carboxylate, and 130 mg/ mL ribavirin), and then serially diluting this solution in 0.5-log 10 increments. In this manner, the EC 50 of TCAD regimen as a fixed dose combination was determined as ratio of the C ave and expressed in units of fold-C ave .

Synergy of Double and Triple Combinations of Amantadine, Ribavirin, and Oseltamivir Carboxylate against 2009 H1N1

Synergy plots, which reveal the extent of synergy at each concentration of each drug in the combinations, are shown in Figures S1 for CA05. The data are presented as contour plots, in which regions where inhibition is greater (synergy) or less (antagonism) than expected are identified by subtracting the theoretical additive inhibition from the observed inhibition. Synergy plots for TCAD regimen showed a concentrationdependent increase in synergy with respect to amantadine ( Figure S1B , top plane). At the highest concentration tested (3.2 mg/mL), synergy was observed over a wide range of concentrations of ribavirin and oseltamivir carboxylate tested. At lower concentrations of amantadine, synergy occurred at 1 mg/mL ribavirin and higher, and at 0.0032 mg/mL oseltamivir carboxylate and higher. No significant antagonism was observed at any dose of any drug in the double combinations or the TCAD regimen. Similar patterns of synergy were observed with double and triple combinations of these antiviral drugs against CA04 and CA10 (data not shown).

Enhancement of Antiviral Drug Activity in Triple Combination against 2009 H1N1

One notable consequence of synergy is that the antiviral activities of each drug in the combination is enhanced compared to the activities of the drugs as single agents. To demonstrate this, we compared the antiviral activity of each of the three drugsamantadine, ribavirin, and oseltamivir carboxylate -as single agents and in the presence of fixed concentrations of the second and third drugs against CA10 replication. For each drug, the EC 50 was reduced in triple combination compared to the EC 50 as a single agent, indicative that each drug was active at lower concentrations (greater potency). For example, the EC 50 for amantadine as a single agent was reduced by 3.2-fold in combination with 1 mg/mL ribavirin and 0.0032 mg/mL oseltamivir carboxylate; the EC 50 for ribavirin as a single agent was reduced by 2.7-fold in combination with 0.0032 mg/mL oseltamivir carboxylate and 1 mg/mL amantadine; and the EC 50 for oseltamivir carboxylate as a single agent was reduced 16.2-fold in combination with 1 mg/mL ribavirin and 1 mg/mL amantadine ( Table 2) . For all three drugs, the reduction in EC 50 values observed in the triple combination compared to the single agent was statistically significant (P,0.05), indicative that each drug had greater antiviral potency and was effective at lower concentrations. In addition, the EC 50 of all three drugs in triple combination were reduced 1.5-to 6.5-fold compared to the EC 50 in double combinations, indicative of that the antiviral activity of the drugs in triple combination were enhanced compared to double combinations. Importantly, the data presented here do not represent the maximum reductions in EC 50 values for the three drugs. Due to the dynamic range of the assay, we were only able to obtain precise dose-response curves for each drug at fixed concentrations of the second and third drugs which were well below their EC 50 values and well below concentrations where maximum synergy occurred. At higher concentrations, the antiviral activity of the second and third drug contributed significantly to the inhibition, which decreased the linear range of the assay and reduced the accuracy of the curve-fitting (data not shown). A comprehensive assessment of the interaction of two or three drugs in combination requires the evaluation of multiple concentrations of each drug in order to quantify synergy over the entire dosing range, as was done in the section above.

Cell-Based Assays

Synergy volume for each double and triple combination was also calculated, which represents the sum of the synergy or antagonism across all concentrations of a combination. Synergy volumes are presented as a quantitative measure of the overall interaction of the drugs within a combination. As determined by cytopathic effect (Neutral Red assay), synergy volumes .100 mg/ mL 2 % for double combinations or .100 mg/mL 3 % for triple combinations are considered to be strongly synergistic. Conversely, combinations with synergy volumes ,2100 mg/mL 2 % or mg/ mL 3 % are considered to be strongly antagonistic.

Cytotoxicity of Antiviral Drugs as Single Agents and in Double and Triple Combinations

The TC 50 of amantadine as a single agent was 37-40 mg/mL, whereas the TC 50 of ribavirin and oseltamivir carboxylate as single agents were .100 mg/mL (Table S2) . These values are more than 10-fold higher than the highest concentration of each drug used in the combination experiments (Table S1 ). Furthermore, synergy analysis of the double and triple combinations revealed no synergistic cytotoxicity for any double combination or the TCAD regimen within the concentration ranges tested (data not shown). For example, cells treated with the TCAD regimen at the highest concentrations tested for all three drugs used in the combination experiments (3.2 mg/mL amantadine, 10 mg/mL ribavirin, and 3.2 mg/mL oseltamivir carboxylate) exhibited 97% viability, which was not statistically different than the cell control (P = 0.47 as determined by Student's t-test, Figure S4 ).

Inhibitory Quotients of Antiviral Drugs against Susceptible and Resistant Viruses

One indicator of the expected clinical antiviral activity is the inhibitory quotient (IQ), defined herein as the ratio between the average total plasma concentration (C ave ) and the 50% effective inhibitory concentration (EC 50 ). In order to predict the effectiveness of the TCAD regimen against the circulating influenza strains in the clinical setting, we calculated and compared the IQs for amantadine, ribavirin, and oseltamivir carboxylate as single agents and the TCAD regimen against susceptible and resistant influenza viral strains (including 2009 H1N1). To determine the IQ of the TCAD regimen, amantadine, ribavirin, and oseltamivir carboxylate was tested as a fixed ratio combination, wherein the ratio of the three drugs was kept constant even as the total concentration of drugs varied. A dilution curve of the TCAD regimen was created by first preparing a solution of all three drugs at 100-fold the C ave of each drug (43 mg/mL amantadine, 130 mg/mL ribavirin, 30 mg/mL oseltamivir carboxylate), and then serially diluting this solution in half-log 10 increments. In this manner, the EC 50 of the TCAD regimen was determined as a ratio of the C ave and expressed in units of fold change from C ave .
40 section matches

Abstract

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

Baculovirus Vectors

Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors.

Veterinary Pox Vectors

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

Introduction

Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] .
Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] .

Virus-Vectored Vaccines

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