Abstract: The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

Abstract: Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site associated with viral production, storage of viral particles in immune complexes, and viral persistence. Although combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. We present a spatial and dynamic model of persistent viral replication and spread that indicates why the development of drug resistance is not a foregone conclusion under conditions in which drug concentrations are insufficient to completely block virus replication. These data provide new insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and replenish the viral reservoir despite potent antiretroviral therapy.

Abstract: Segmented RNA viruses are widespread in nature and include important human, animal and plant pathogens, such as influenza viruses and rotaviruses Although the origin of RNA virus genome segmentation remains elusive, a major consequence of this genome structure is the capacity for reassortment to occur during co-infection, whereby segments are exchanged among different viral strains Therefore, reassortment can create viral progeny that contain genes that are derived from more than one parent, potentially conferring important fitness advantages or disadvantages to the progeny virus However, for segmented RNA viruses that package their multiple genome segments into a single virion particle, reassortment also requires genetic compatibility between parental strains, which occurs in the form of conserved packaging signals, and the maintenance of RNA and protein interactions In this Review, we discuss recent studies that examined the mechanisms and outcomes of reassortment for three well-studied viral families - Cystoviridae, Orthomyxoviridae and Reoviridae - and discuss how these findings provide new perspectives on the replication and evolution of segmented RNA viruses

Abstract: Viruses of the family Flaviviridae are important pathogens of humans and other animals, and currently classified into four genera. To better understand their diversity, evolutionary history and genomic flexibility, we used RNA-seq to search for the viruses related to the Flaviviridae in a range of potential invertebrate and vertebrate hosts. Accordingly, we recovered the full genomes of 5 segmented Jingmenviruses and 12 distant relatives of the known Flaviviridae (‘flavi-like9 viruses) from a range of arthropod species. Although these viruses are highly divergent, they share a similar genomic plan and common ancestry with the Flaviviridae in the NS3 and NS5 regions. Remarkably, while these viruses fill in major gaps in the phylogenetic diversity of the Flaviviridae , genomic comparisons reveal important changes in genome structure, genome size, and replication/gene regulation strategy during evolutionary history. In addition, the wide diversity of flavi-like viruses found in invertebrates, as well as their deep phylogenetic positions, suggests that they may represent the ancestral forms from which the vertebrate-infecting viruses evolved. For the vertebrate viruses, we expanded the previously mammal-only pegivirus-hepacivirus group to include a virus from the graceful catshark ( Proscyllium habereri ), which in turn implies that these viruses possess a larger host range than is currently known. In sum, our data show that the Flaviviridae infect a far wider range of hosts and exhibit greater diversity in genome structure than previously anticipated. IMPORTANCE The family Flaviviridae of RNA viruses contains several notorious human pathogens, including dengue virus, West Nile virus, and hepatitis C virus. To date, however, our understanding of the biodiversity and evolution of the Flaviviridae has largely been directed toward vertebrate hosts and their blood-feeding arthropod vectors. Therefore, we investigated an expanded group of potential arthropod and vertebrate host species that have generally been ignored by surveillance programs. Remarkably, these species contained diverse flaviviruses and related viruses that are characterized by major changes in genome size and genome structure, such that these traits are more flexible than previously thought. More generally, these data suggest that arthropods may be the ultimate reservoir of the Flaviviridae and related viruses, harbouring considerable genetic and phenotypic diversity. In sum, this study revises the traditional view on the evolutionary history, host range, and genomic structures of a major group of RNA viruses.

Abstract: Influenza genes evolve mostly via point mutations, and so knowing the effect of every amino-acid mutation provides information about evolutionary paths available to the virus. We and others have combined high-throughput mutagenesis with deep sequencing to estimate the effects of large numbers of mutations to influenza genes. However, these measurements have suffered from substantial experimental noise due to a variety of technical problems, the most prominent of which is bottlenecking during the generation of mutant viruses from plasmids. Here we describe advances that ameliorate these problems, enabling us to measure with greatly improved accuracy and reproducibility the effects of all amino-acid mutations to an H1 influenza hemagglutinin on viral replication in cell culture. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm at much higher resolution the results of previous studies suggesting that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. We also show that other regions of hemagglutinin—including the stalk epitopes targeted by broadly neutralizing antibodies—have a much lower inherent capacity to tolerate point mutations. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution.

Abstract: The natural evolution of rabies virus (RABV) provides a potent example of multiple host shifts and an important opportunity to determine the mechanisms that underpin viral emergence. Using 321 genome sequences spanning an unprecedented diversity of RABV, we compared evolutionary rates and selection pressures in viruses sampled from multiple primary host shifts that occurred on various continents. Two major phylogenetic groups, bat-related RABV and dog-related RABV, experiencing markedly different evolutionary dynamics were identified. While no correlation between time and genetic divergence was found in bat-related RABV, the evolution of dog-related RABV followed a generally clock-like structure, although with a relatively low evolutionary rate. Subsequent molecular clock dating indicated that dog-related RABV likely underwent a rapid global spread following the intensification of intercontinental trade starting in the 15th century. Strikingly, although dog RABV has jumped to various wildlife species from the order Carnivora, we found no clear evidence that these host-jumping events involved adaptive evolution, with RABV instead characterized by strong purifying selection, suggesting that ecological processes also play an important role in shaping patterns of emergence. However, specific amino acid changes were associated with the parallel emergence of RABV in ferret-badgers in Asia, and some host shifts were associated with increases in evolutionary rate, particularly in the ferret-badger and mongoose, implying that changes in host species can have important impacts on evolutionary dynamics.

Abstract: The H1 subtype of influenza A viruses (IAVs) has been circulating in swine since the 1918 human influenza pandemic. Over time, and aided by further introductions from nonswine hosts, swine H1 viruses have diversified into three genetic lineages. Due to limited global data, these H1 lineages were named based on colloquial context, leading to a proliferation of inconsistent regional naming conventions. In this study, we propose rigorous phylogenetic criteria to establish a globally consistent nomenclature of swine H1 virus hemagglutinin (HA) evolution. These criteria applied to a data set of 7,070 H1 HA sequences led to 28 distinct clades as the basis for the nomenclature. We developed and implemented a web-accessible annotation tool that can assign these biologically informative categories to new sequence data. The annotation tool assigned the combined data set of 7,070 H1 sequences to the correct clade more than 99% of the time. Our analyses indicated that 87% of the swine H1 viruses from 2010 to the present had HAs that belonged to 7 contemporary cocirculating clades. Our nomenclature and web-accessible classification tool provide an accurate method for researchers, diagnosticians, and health officials to assign clade designations to HA sequences. The tool can be updated readily to track evolving nomenclature as new clades emerge, ensuring continued relevance. A common global nomenclature facilitates comparisons of IAVs infecting humans and pigs, within and between regions, and can provide insight into the diversity of swine H1 influenza virus and its impact on vaccine strain selection, diagnostic reagents, and test performance, thereby simplifying communication of such data. IMPORTANCE A fundamental goal in the biological sciences is the definition of groups of organisms based on evolutionary history and the naming of those groups. For influenza A viruses (IAVs) in swine, understanding the hemagglutinin (HA) genetic lineage of a circulating strain aids in vaccine antigen selection and allows for inferences about vaccine efficacy. Previous reporting of H1 virus HA in swine relied on colloquial names, frequently with incriminating and stigmatizing geographic toponyms, making comparisons between studies challenging. To overcome this, we developed an adaptable nomenclature using measurable criteria for historical and contemporary evolutionary patterns of H1 global swine IAVs. We also developed a web-accessible tool that classifies viruses according to this nomenclature. This classification system will aid agricultural production and pandemic preparedness through the identification of important changes in swine IAVs and provides terminology enabling discussion of swine IAVs in a common context among animal and human health initiatives.

Abstract: Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.

Abstract: The four human coronaviruses (HCoVs) are globally endemic respiratory pathogens. The Middle East respiratory syndrome (MERS) coronavirus (CoV) is an emerging CoV with a known zoonotic source in dromedary camels. Little is known about the origins of endemic HCoVs. Studying these viruses' evolutionary history could provide important insight into CoV emergence. In tests of MERS-CoV-infected dromedaries, we found viruses related to an HCoV, known as HCoV-229E, in 5.6% of 1,033 animals. Human- and dromedary-derived viruses are each monophyletic, suggesting ecological isolation. One gene of dromedary viruses exists in two versions in camels, full length and deleted, whereas only the deleted version exists in humans. The deletion increased in size over a succession starting from camelid viruses via old human viruses to contemporary human viruses. Live isolates of dromedary 229E viruses were obtained and studied to assess human infection risks. The viruses used the human entry receptor aminopeptidase N and replicated in human hepatoma cells, suggesting a principal ability to cause human infections. However, inefficient replication in several mucosa-derived cell lines and airway epithelial cultures suggested lack of adaptation to the human host. Dromedary viruses were as sensitive to the human type I interferon response as HCoV-229E. Antibodies in human sera neutralized dromedary-derived viruses, suggesting population immunity against dromedary viruses. Although no current epidemic risk seems to emanate from these viruses, evolutionary inference suggests that the endemic human virus HCoV-229E may constitute a descendant of camelid-associated viruses. HCoV-229E evolution provides a scenario for MERS-CoV emergence.

Abstract: Gene therapy is the straightforward approach for the application of recent advances in molecular biology into clinical practice. One of the major obstacles in the development of gene therapy is the delivery of the effector to and into the target cell. Unfortunately, most methods commonly used in laboratory practice are poorly suited for clinical use. Viral vectors are one of the most promising methods for gene therapy delivery. Millions of years of evolution of viruses have resulted in the development of various molecular mechanisms for entry into cells, long-term survival within cells, and activation, inhibition, or modification of the host defense mechanisms at all levels. The relatively simple organization of viruses, small genome size, and evolutionary plasticity allow modifying them to create effective instruments for gene therapy approaches. This review summarizes the latest trends in the development of gene therapy, in particular, various aspects and prospects of the development of clinical products based on viral delivery systems.

Abstract: HIV is notorious for its capacity to evade immunity and anti-viral drugs through rapid sequence evolution. Knowledge of the functional effects of mutations to HIV is critical for understanding this evolution. HIV’s most rapidly evolving protein is its envelope (Env). Here we use deep mutational scanning to experimentally estimate the effects of all amino-acid mutations to Env on viral replication in cell culture. Most mutations are under purifying selection in our experiments, although a few sites experience strong selection for mutations that enhance HIV’s replication in cell culture. We compare our experimental measurements of each site’s preference for each amino acid to the actual frequencies of these amino acids in naturally occurring HIV sequences. Our measured amino-acid preferences correlate with amino-acid frequencies in natural sequences for most sites. However, our measured preferences are less concordant with natural amino-acid frequencies at surface-exposed sites that are subject to pressures absent from our experiments such as antibody selection. Our data enable us to quantify the inherent mutational tolerance of each site in Env. We show that the epitopes of broadly neutralizing antibodies have a significantly reduced inherent capacity to tolerate mutations, rigorously validating a pervasive idea in the field. Overall, our results help disentangle the role of inherent functional constraints and external selection pressures in shaping Env’s evolution.

Abstract: Background Chikungunya virus (CHIKV), an alphavirus and member of the Togaviridae family, is capable of causing severe febrile disease in humans. In December of 2013 the Asian Lineage of CHIKV spread from the Old World to the Americas, spreading rapidly throughout the New World. Given this new emergence in naive populations we studied the viral genetic diversity present in infected individuals to understand how CHIKV may have evolved during this continuing outbreak. Methodology/Principle Findings We used deep-sequencing technologies coupled with well-established bioinformatics pipelines to characterize the minority variants and diversity present in CHIKV infected individuals from Guadeloupe and Martinique, two islands in the center of the epidemic. We observed changes in the consensus sequence as well as a diverse range of minority variants present at various levels in the population. Furthermore, we found that overall diversity was dramatically reduced after single passages in cell lines. Finally, we constructed an infectious clone from this outbreak and identified a novel 3’ untranslated region (UTR) structure, not previously found in nature, that led to increased replication in insect cells. Conclusions/Significance Here we preformed an intrahost quasispecies analysis of the new CHIKV outbreak in the Caribbean. We identified novel variants present in infected individuals, as well as a new 3’UTR structure, suggesting that CHIKV has rapidly evolved in a short period of time once it entered this naive population. These studies highlight the need to continue viral diversity surveillance over time as this epidemic evolves in order to understand the evolutionary potential of CHIKV.

Abstract: The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has generated enormous interest in the biodiversity, genomics and cross-species transmission potential of coronaviruses, especially those from bats, the second most speciose order of mammals. Herein, we identified a novel coronavirus, provisionally designated Rousettus bat coronavirus GCCDC1 (Ro-BatCoV GCCDC1), in the rectal swab samples of Rousettus leschenaulti bats by using pan-coronavirus RT-PCR and next-generation sequencing. Although the virus is similar to Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9) in genome characteristics, it is sufficiently distinct to be classified as a new species according to the criteria defined by the International Committee of Taxonomy of Viruses (ICTV). More striking was that Ro-BatCoV GCCDC1 contained a unique gene integrated into the 3'-end of the genome that has no homologs in any known coronavirus, but which sequence and phylogeny analyses indicated most likely originated from the p10 gene of a bat orthoreovirus. Subgenomic mRNA and cellular-level observations demonstrated that the p10 gene is functional and induces the formation of cell syncytia. Therefore, here we report a putative heterologous inter-family recombination event between a single-stranded, positive-sense RNA virus and a double-stranded segmented RNA virus, providing insights into the fundamental mechanisms of viral evolution.

Abstract: Since their commercialization, vaccines against Porcine circovirus type 2 (PCV2) have been the cornerstone control strategy. Nevertheless, the periodic emergence of new genotype waves and the recent reports of vaccine failure outbreaks have raised the question if widespread vaccination strategies could have driven viral evolution and affected different genotype fitness. To investigate this issue an in-deep analysis, based on a bioinformatics and biostatistics approach, has been implemented. ORF2 sequences from vaccinated and non-vaccinated populations (i.e. domestic pigs before and after vaccine introduction and wild boars) were considered. The action of selective forces on PCV2 strains has been analyzed and compared among groups. Remarkable differences were found in the selective forces acting on viral populations circulating in different "immune environments". Particularly for PCV2a, a directional selection promoting a change in the viral capsid away from the vaccine specific antigenic determinants has been detected after vaccine introduction. Involved amino acids were previously reported to be part of viral epitopes whose variability is responsible of immune escape. Our findings support a change in PCV2 evolutionary pattern after widespread vaccination introduction and stress once more the compulsoriness of a continuous monitoring of PCV2 epidemiology to promptly act in response to the emergence of possible vaccine-escaping mutants.

Abstract: Accumulating data suggest that tripartite-motif-containing (TRIM) proteins participate in host responses to viral infections, either by acting as direct antiviral restriction factors or through regulating innate immune signaling of the host. Of >70 TRIMs, TRIM56 is a restriction factor of several positive-strand RNA viruses, including three members of the family Flaviviridae (yellow fever virus, dengue virus, and bovine viral diarrhea virus) and a human coronavirus (OC43), and this ability invariably depends upon the E3 ligase activity of TRIM56. However, the impact of TRIM56 on negative-strand RNA viruses remains unclear. Here, we show that TRIM56 puts a check on replication of influenza A and B viruses in cell culture but does not inhibit Sendai virus or human metapneumovirus, two paramyxoviruses. Interestingly, the anti-influenza virus activity was independent of the E3 ligase activity, B-box, or coiled-coil domain. Rather, deletion of a 63-residue-long C-terminal-tail portion of TRIM56 abrogated the antiviral function. Moreover, expression of this short C-terminal segment curtailed the replication of influenza viruses as effectively as that of full-length TRIM56. Mechanistically, TRIM56 was found to specifically impede intracellular influenza virus RNA synthesis. Together, these data reveal a novel antiviral activity of TRIM56 against influenza A and B viruses and provide insights into the mechanism by which TRIM56 restricts these medically important orthomyxoviruses. IMPORTANCE Options to treat influenza are limited, and drug-resistant influenza virus strains can emerge through minor genetic changes. Understanding novel virus-host interactions that alter influenza virus fitness may reveal new targets/approaches for therapeutic interventions. We show here that TRIM56, a tripartite-motif protein, is an intrinsic host restriction factor of influenza A and B viruses. Unlike its antiviral actions against positive-strand RNA viruses, the anti-influenza virus activity of TRIM56 was independent of the E3 ligase activity. Rather, expression of a short segment within the very C-terminal tail of TRIM56 inhibited the replication of influenza viruses as effectively as that of full-length TRIM56 by specifically targeting viral RNA synthesis. These data reveal the remarkable multifaceted activity of TRIM56, which has developed multiple domains to inhibit multiple viral families. They also raise the possibility of developing a broad-spectrum, TRIM56-based antiviral approach for addition to influenza prophylaxis and/or control strategies.

Abstract: Equine influenza viruses (EIVs) of H3N8 subtype are culprits of severe acute respiratory infections in horses, and are still responsible for significant outbreaks worldwide. Adaptability of influenza viruses to a particular host is significantly influenced by their codon usage preference, due to an absolute dependence on the host cellular machinery for their replication. In the present study, we analyzed genome-wide codon usage patterns in 92 EIV strains, including both H3N8 and H7N7 subtypes by computing several codon usage indices and applying multivariate statistical methods. Relative synonymous codon usage (RSCU) analysis disclosed bias of preferred synonymous codons towards A/U-ended codons. The overall codon usage bias in EIVs was slightly lower, and mainly affected by the nucleotide compositional constraints as inferred from the RSCU and effective number of codon (ENc) analysis. Our data suggested that codon usage pattern in EIVs is governed by the interplay of mutation pressure, natural selection from its hosts and undefined factors. The H7N7 subtype was found less fit to its host (horse) in comparison to H3N8, by possessing higher codon bias, lower mutation pressure and much less adaptation to tRNA pool of equine cells. To the best of our knowledge, this is the first report describing the codon usage analysis of the complete genomes of EIVs. The outcome of our study is likely to enhance our understanding of factors involved in viral adaptation, evolution, and fitness towards their hosts.

Abstract: Human cytomegalovirus (HCMV) infects most of the population worldwide, persisting throughout the host's life in a latent state with periodic episodes of reactivation. While typically asymptomatic, HCMV can cause fatal disease among congenitally infected infants and immunocompromised patients. These clinical issues are compounded by the emergence of antiviral resistance and the absence of an effective vaccine, the development of which is likely complicated by the numerous immune evasins encoded by HCMV to counter the host's adaptive immune responses, a feature that facilitates frequent super-infections. Understanding the evolutionary dynamics of HCMV is essential for the development of effective new drugs and vaccines. By comparing viral genomes from uncultivated or low-passaged clinical samples of diverse origins, we observe evidence of frequent homologous recombination events, both recent and ancient, and no structure of HCMV genetic diversity at the whole-genome scale. Analysis of individual gene-scale loci reveals a striking dichotomy: while most of the genome is highly conserved, recombines essentially freely and has evolved under purifying selection, 21 genes display extreme diversity, structured into distinct genotypes that do not recombine with each other. Most of these hyper-variable genes encode glycoproteins involved in cell entry or escape of host immunity. Evidence that half of them have diverged through episodes of intense positive selection suggests that rapid evolution of hyper-variable loci is likely driven by interactions with host immunity. It appears that this process is enabled by recombination unlinking hyper-variable loci from strongly constrained neighboring sites. It is conceivable that viral mechanisms facilitating super-infection have evolved to promote recombination between diverged genotypes, allowing the virus to continuously diversify at key loci to escape immune detection, while maintaining a genome optimally adapted to its asymptomatic infectious lifecycle.

Abstract: RNA viruses rapidly diversify into quasispecies of related genotypes. This genetic diversity has long been known to facilitate adaptation, but recent studies have suggested that cooperation between variants might also increase population fitness. Here, we demonstrate strong cooperation between two H3N2 influenza variants that differ by a single mutation at residue 151 in neuraminidase, which normally mediates viral exit from host cells. Residue 151 is often annotated as an ambiguous amino acid in sequenced isolates, indicating mixed viral populations. We show that mixed populations grow better than either variant alone in cell culture. Pure populations of either variant generate the other through mutation and then stably maintain a mix of the two genotypes. We suggest that cooperation arises because mixed populations combine one variant's proficiency at cell entry with the other's proficiency at cell exit. Our work demonstrates a specific cooperative interaction between defined variants in a viral quasispecies.

Abstract: Dengue virus (DENV) is currently the most prevalent mosquito-borne viral pathogen. DENVs naturally exist as highly heterogeneous populations. Even though the descriptions on DENV diversity are plentiful, only a few studies have narrated the dynamics of intra-epidemic virus diversity at a fine scale. Such accounts are important to decipher the reciprocal relationship between viral evolutionary dynamics and disease transmission that shape dengue epidemiology. In the current study, we present a micro-scale genetic analysis of a monophyletic lineage of DENV-1 genotype III (epidemic lineage) detected from November 2012 to May 2014. The lineage was involved in an unprecedented dengue epidemic in Singapore during 2013-2014. Our findings showed that the epidemic lineage was an ensemble of mutants (variants) originated from an initial mixed viral population. The composition of mutant spectrum was dynamic and positively correlated with case load. The close interaction between viral evolution and transmission intensity indicated that tracking genetic diversity through time is potentially a useful tool to infer DENV transmission dynamics and thereby, to assess the epidemic risk in a disease control perspective. Moreover, such information is salient to understand the viral basis of clinical outcome and immune response variations that is imperative to effective vaccine design.

Abstract: Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997–2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens.

Abstract: Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014-2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.

Abstract: Identifying viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. Here we quantify how every amino-acid mutation to influenza hemagglutinin affects neutralization by monoclonal antibodies targeting several antigenic regions. Our approach involves creating all replication-competent protein variants of the virus, selecting these variants with antibody, and using deep sequencing to identify enriched mutations. These high-throughput measurements are predictive of the effects of individual mutations in traditional neutralization assays. At many residues, only some of the possible mutations escape from an antibody. For instance, at a single residue targeted by two different antibodies, we identify some mutations that escape both antibodies and other mutations that escape only one or the other. Therefore, our approach maps how viruses can escape antibodies with mutation-level sensitivity, and shows that only some mutations at antigenic residues actually alter antigenicity.

Abstract: The Flavivirus genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barre syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian/American ZIKV lineage.

Abstract: Highly pathogenic influenza A(H5N8) viruses from clade 2.3.4.4 were introduced to North America by migratory birds in the fall of 2014. Reassortment of A(H5N8) viruses with avian viruses of North American lineage resulted in the generation of novel A(H5N2) viruses with novel genotypes. Through sequencing of recent avian influenza viruses, we identified PB1 and NP gene segments very similar to those in the viruses isolated from North American waterfowl prior to the introduction of A(H5N8) to North America, highlighting these bird species in the origin of reassortant A(H5N2) viruses. While they were highly virulent and transmissible in poultry, we found A(H5N2) viruses to be low pathogenic in mice and ferrets, and replication was limited in both hosts compared with those of recent highly pathogenic avian influenza (HPAI) H5N1 viruses. Molecular characterization of the hemagglutinin protein from A(H5N2) viruses showed that the receptor binding preference, cleavage, and pH of activation were highly adapted for replication in avian species and similar to those of other 2.3.4.4 viruses. In addition, North American and Eurasian clade 2.3.4.4 H5NX viruses replicated to significantly lower titers in differentiated normal human bronchial epithelial cells than did seasonal human A(H1N1) and highly pathogenic A(H5N1) viruses isolated from a human case. Thus, despite their having a high impact on poultry, our findings suggest that the recently emerging North American A(H5N2) viruses are not expected to pose a substantial threat to humans and other mammals without further reassortment and/or adaptation and that reassortment with North American viruses has not had a major impact on viral phenotype. IMPORTANCE Highly pathogenic H5 influenza viruses have been introduced into North America from Asia, causing extensive morbidity and mortality in domestic poultry. The introduced viruses have reassorted with North American avian influenza viruses, generating viral genotypes not seen on other continents. The experiments and analyses presented here were designed to assess the impact of this genetic diversification on viral phenotypes, particularly as regards mammalian hosts, by comparing the North American viruses with their Eurasian precursor viruses.

Abstract: We are interested in the influence of nucleotide composition on the fundamental characteristics of the virus RNA genome. Most RNA viruses have genomes with a distinct nucleotide composition, e.g. ranging from minimally 12.9 % to maximally 40.3 % (C- and U-count, respectively, in coronavirus HKU). We present a global analysis of diverse virus types, including plus-strand, minus-strand and double-strand RNA viruses, for the impact of this nucleotide preference on the predicted structure of the RNA genome that is packaged in virion particles and on the codon usage in the viral open reading frames. Several virus-specific features will be described, but also some general conclusions were drawn. Without exception, the virus-specific nucleotide bias was enriched in the unpaired, single-stranded regions of the RNA genome, thus creating an even more striking virus-specific signature. We present a simple mechanism that is based on elementary aspects of RNA structure folding to explain this general trend. In general, the nucleotide bias was the major determinant of the virus-specific codon usages, thus limiting a role for codon selection and translational control. We will discuss molecular and evolutionary scenarios that may be responsible for the diverse nucleotide biases of RNA viruses.

Abstract: UNLABELLED Influenza A virus (IAV) of the H3 subtype is an important respiratory pathogen that affects both humans and swine. Vaccination to induce neutralizing antibodies against the surface glycoprotein hemagglutinin (HA) is the primary method used to control disease. However, due to antigenic drift, vaccine strains must be periodically updated. Six of the 7 positions previously identified in human seasonal H3 (positions 145, 155, 156, 158, 159, 189, and 193) were also indicated in swine H3 antigenic evolution. To experimentally test the effect on virus antigenicity of these 7 positions, substitutions were introduced into the HA of an isogenic swine lineage virus. We tested the antigenic effect of these introduced substitutions by using hemagglutination inhibition (HI) data with monovalent swine antisera and antigenic cartography to evaluate the antigenic phenotype of the mutant viruses. Combinations of substitutions within the antigenic motif caused significant changes in antigenicity. One virus mutant that varied at only two positions relative to the wild type had a >4-fold reduction in HI titers compared to homologous antisera. Potential changes in pathogenesis and transmission of the double mutant were evaluated in pigs. Although the double mutant had virus shedding titers and transmissibility comparable to those of the wild type, it caused a significantly lower percentage of lung lesions. Elucidating the antigenic effects of specific amino acid substitutions at these sites in swine H3 IAV has important implications for understanding IAV evolution within pigs as well as for improved vaccine development and control strategies in swine. IMPORTANCE A key component of influenza virus evolution is antigenic drift mediated by the accumulation of amino acid substitutions in the hemagglutinin (HA) protein, resulting in escape from prior immunity generated by natural infection or vaccination. Understanding which amino acid positions of the HA contribute to the ability of the virus to avoid prior immunity is important for understanding antigenic evolution and informs vaccine efficacy predictions based on the genetic sequence data from currently circulating strains. Following our previous work characterizing antigenic phenotypes of contemporary wild-type swine H3 influenza viruses, we experimentally validated that substitutions at 6 amino acid positions in the HA protein have major effects on antigenicity. An improved understanding of the antigenic diversity of swine influenza will facilitate a rational approach for selecting more effective vaccine components to control the circulation of influenza in pigs and reduce the potential for zoonotic viruses to emerge.

Abstract: A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation.

Abstract: Aleutian mink disease virus (AMDV) causes plasmacytosis, an immune complex-associated syndrome that affects wild and farmed mink. The virus can also infect other small mammals (e.g., ferrets, skunks, ermines, and raccoons), but the disease in these hosts has been studied less. In 2007, a mink plasmacytosis outbreak began on the Island of Newfoundland, and the virus has been endemic in farms since then. In this study, we evaluated the molecular epidemiology of AMDV in farmed and wild animals of Newfoundland since before the beginning of the outbreak and investigated the epidemic in a global context by studying AMDV worldwide, thereby examining its diffusion and phylogeography. Furthermore, AMDV evolution was examined in the context of intensive farming, where host population dynamics strongly influence viral evolution. Partial NS1 sequences and several complete genomes were obtained from Newfoundland viruses and analyzed along with numerous sequences from other locations worldwide that were either obtained as part of this study or from public databases. We observed very high viral diversity within Newfoundland and within single farms, where high rates of co-infection, recombinant viruses and polymorphisms were observed within single infected individuals. Worldwide, we documented a partial geographic distribution of strains, where viruses from different countries co-exist within clades but form country-specific subclades. Finally, we observed the occurrence of recombination and the predominance of negative selection pressure on AMDV proteins. A surprisingly low number of immunoepitopic sites were under diversifying pressure, possibly because AMDV gains no benefit by escaping the immune response as viral entry into target cells is mediated through interactions with antibodies, which therefore contribute to cell infection. In conclusion, the high prevalence of AMDV in farms facilitates the establishment of co-infections that can favor the occurrence of recombination and enhance viral diversity. Viruses are then exchanged between different farms and countries and can be introduced into the wild, with the rapidly evolving viruses producing many parallel lineages.