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1.  Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses 
eLife  null;4:e05378.
Although arthropods are important viral vectors, the biodiversity of arthropod viruses, as well as the role that arthropods have played in viral origins and evolution, is unclear. Through RNA sequencing of 70 arthropod species we discovered 112 novel viruses that appear to be ancestral to much of the documented genetic diversity of negative-sense RNA viruses, a number of which are also present as endogenous genomic copies. With this greatly enriched diversity we revealed that arthropods contain viruses that fall basal to major virus groups, including the vertebrate-specific arenaviruses, filoviruses, hantaviruses, influenza viruses, lyssaviruses, and paramyxoviruses. We similarly documented a remarkable diversity of genome structures in arthropod viruses, including a putative circular form, that sheds new light on the evolution of genome organization. Hence, arthropods are a major reservoir of viral genetic diversity and have likely been central to viral evolution.
DOI: http://dx.doi.org/10.7554/eLife.05378.001
eLife digest
Many illnesses, including influenza, hemorrhagic fever, and rabies, are caused by a group of viruses called negative-sense RNA viruses. The genetic information—or genome—of these viruses is encoded in strands of RNA that must be copied before they can be translated into the proteins needed to build new viruses. It is currently known that there are at least eight different families of these viruses, which have a wide range of shapes and sizes and arrange their RNA in different ways.
Insects, spiders, and other arthropods carry many different RNA viruses. Many of these viruses have not previously been studied, and those that have been studied so far are mainly those that cause diseases in humans and other vertebrates. Researchers therefore only know a limited amount about the diversity of the negative-sense RNA viruses that arthropods harbor and how these viruses evolved. Studying how viruses evolve helps scientists to understand what makes some viruses deadly and others harmless and can also help develop treatments or vaccines for the diseases caused by the viruses.
Li, Shi, Tian, Lin, Kang et al. collected 70 species of insects, spiders, centipedes, and other arthropods in China and sequenced all the negative-sense RNA viruses in the creatures. This revealed an enormous number of negative-sense RNA viruses, including 112 new viruses. Many of the newly discovered arthropod viruses appear to be the ancestors of disease-causing viruses, including influenza viruses and the filoviruses—the group that includes the Ebola virus. Indeed, it appears that arthropods host many—if not all—of the negative-sense RNA viruses that cause disease in vertebrates and plants.
While documenting the new RNA viruses and how they are related to each other, Li et al. found many different genome structures. Some genomes were segmented, which may play an important role in evolution as segments can be easily swapped to create new genetic combinations. Non-segmented and circular genomes were also found. This genetic diversity suggests that arthropods are likely to have played a key role in the evolution of new viruses by acting as a site where many different viruses can interact and exchange genetic information.
DOI: http://dx.doi.org/10.7554/eLife.05378.002
doi:10.7554/eLife.05378
PMCID: PMC4384744  PMID: 25633976
RNA virus; evolution; arthropods; segmentation; negative-sense; phylogeny; viruses
2.  Pandemics–Keep Calm and Carry On 
PLoS Biology  2014;12(2):e1001780.
Evolutionary biologist Edward Holmes reviews Peter Doherty's book, Pandemics: What Everyone Needs to Know.
doi:10.1371/journal.pbio.1001780
PMCID: PMC3913555
3.  The contrasting phylodynamics of human influenza B viruses 
eLife  null;4:e05055.
A complex interplay of viral, host, and ecological factors shapes the spatio-temporal incidence and evolution of human influenza viruses. Although considerable attention has been paid to influenza A viruses, a lack of equivalent data means that an integrated evolutionary and epidemiological framework has until now not been available for influenza B viruses, despite their significant disease burden. Through the analysis of over 900 full genomes from an epidemiological collection of more than 26,000 strains from Australia and New Zealand, we reveal fundamental differences in the phylodynamics of the two co-circulating lineages of influenza B virus (Victoria and Yamagata), showing that their individual dynamics are determined by a complex relationship between virus transmission, age of infection, and receptor binding preference. In sum, this work identifies new factors that are important determinants of influenza B evolution and epidemiology.
DOI: http://dx.doi.org/10.7554/eLife.05055.001
eLife digest
To develop new therapies against infections caused by a virus, it is important to understand the virus's history—where, when, and why it has caused disease and how it has changed over time. For example, new human strains of the influenza type A virus originate from strains that infect animals and rapidly can become common in human populations. In contrast, influenza type B virus strains almost exclusively infect humans and are continuously present in human populations. Both types have a detrimental impact on global health, but the type B viruses are less well understood, partly because outbreaks have not been as extensively documented.
Vijaykrishna et al. have now investigated the history of the two strains of the influenza type B virus—called Victoria and Yamagata—that currently circulate in humans. To do this, they inspected the genetic sequences of 908 viruses taken from samples of confirmed type B infections collected across Australia and New Zealand over 13 years.
Individual virus particles of the same strain have genetic sequences that are very similar, but not completely identical. Vijaykrishna et al. showed that the diversity of the genetic sequences from the Victoria strain fluctuated between seasons, and particular genetic variants of Victoria only persisted in the population for 1–3 years. This indicates that Victoria viruses are under a lot of pressure to evolve, which results in so-called ‘bottlenecks’ whereby only the viruses carrying particular varieties of genetic sequence survive. This fluctuating pattern resembles that of the better-understood type A seasonal flu strain H3N2.
On the other hand, there was little change in the genetic diversity of the Yamagata strains sampled over the same 13-year period. The Yamagata viruses have diversified to a greater extent and several different ‘varieties’ of the virus tend to circulate together for long periods of time. For example, the three varieties of Yamagata virus circulating in 2013 evolved from a common parent virus that was circulating around 10 years ago.
Vijaykrishna et al. found that between disease outbreaks, there was greater variation in the ability of Victoria viruses to be transmitted in humans, but that they were generally more easily transmitted than the Yamagata viruses. Victoria viruses tend to infect younger patients than Yamagata viruses, which is thought to be due to differences in the molecules that help the viruses enter the cells of the respiratory tract.
These findings suggests that it might be possible to eradicate the more slowly evolving influenza B Yamagata virus through mass vaccination programs using existing vaccines. This would then allow researchers to focus on developing effective vaccines to target the other strains of influenza virus.
DOI: http://dx.doi.org/10.7554/eLife.05055.002
doi:10.7554/eLife.05055
PMCID: PMC4383373  PMID: 25594904
influenza virus; evolution; epidemiology; antigenic drift; human; viruses
4.  An Allometric Relationship between the Genome Length and Virion Volume of Viruses 
Journal of Virology  2014;88(11):6403-6410.
ABSTRACT
Virions vary in size by at least 4 orders of magnitude, yet the evolutionary forces responsible for this enormous diversity are unknown. We document a significant allometric relationship, with an exponent of approximately 1.5, between the genome length and virion volume of viruses and find that this relationship is not due to geometric constraints. Notably, this allometric relationship holds regardless of genomic nucleic acid, genome structure, or type of virion architecture and therefore represents a powerful scaling law. In contrast, no such relationship is observed at the scale of individual genes. Similarly, after adjusting for genome length, no association is observed between virion volume and the number of proteins, ruling out protein number as the explanation for the relationship between genome and virion sizes. Such a fundamental allometric relationship not only sheds light on the constraints to virus evolution, in that increases in virion size but not necessarily structure are associated with concomitant increases in genome size, but also implies that virion sizes in nature can be broadly predicted from genome sequence data alone.
IMPORTANCE Viruses vary dramatically in both genome and virion sizes, but the factors responsible for this diversity are uncertain. Through a comparative and quantitative investigation of these two fundamental biological parameters across diverse viral taxa, we show that genome length and virion volume conform to a simple allometric scaling law. Notably, this allometric relationship holds regardless of the type of virus, including those with both RNA and DNA genomes, and encompasses viruses that exhibit more than 3 logs of genome size variation. Accordingly, this study helps to reveal the basic rules of virus design.
doi:10.1128/JVI.00362-14
PMCID: PMC4093846  PMID: 24672040
5.  Phylogeography of Influenza A(H3N2) Virus in Peru, 2010–2012 
Emerging Infectious Diseases  2015;21(8):1330-1338.
Peru did not fit the source–sink model for the global spread of this virus.
It remains unclear whether lineages of influenza A(H3N2) virus can persist in the tropics and seed temperate areas. We used viral gene sequence data sampled from Peru to test this source–sink model for a Latin American country. Viruses were obtained during 2010–2012 from influenza surveillance cohorts in Cusco, Tumbes, Puerto Maldonado, and Lima. Specimens positive for influenza A(H3N2) virus were randomly selected and underwent hemagglutinin sequencing and phylogeographic analyses. Analysis of 389 hemagglutinin sequences from Peru and 2,192 global sequences demonstrated interseasonal extinction of Peruvian lineages. Extensive mixing occurred with global clades, but some spatial structure was observed at all sites; this structure was weakest in Lima and Puerto Maldonado, indicating that these locations may experience greater viral traffic. The broad diversity and co-circulation of many simultaneous lineages of H3N2 virus in Peru suggests that this country should not be overlooked as a potential source for novel pandemic strains.
doi:10.3201/eid2108.150084
PMCID: PMC4517729  PMID: 26196599
Peru; H3N2; influenza virus; evolution; viruses; phylogeography; phylogeny; viral traffic; subtropical; tropical; gene pool; source population; surveillance; source–sink dynamic; source–sink model; interseasonal extinction; influenza A(H3N2) virus; human influenza; influenza
6.  Cyclic Avian Mass Mortality in the Northeastern United States Is Associated with a Novel Orthomyxovirus 
Journal of Virology  2014;89(2):1389-1403.
ABSTRACT
Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, MA, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here, we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein that was demonstrated to have fusion activity; this also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined.
IMPORTANCE The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here, we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.
doi:10.1128/JVI.02019-14
PMCID: PMC4300652  PMID: 25392223
7.  Analyses of evolutionary dynamics in viruses are hindered by a time-dependent bias in rate estimates 
Time-scales of viral evolution and emergence have been studied widely, but are often poorly understood. Molecular analyses of viral evolutionary time-scales generally rely on estimates of rates of nucleotide substitution, which vary by several orders of magnitude depending on the timeframe of measurement. We analysed data from all major groups of viruses and found a strong negative relationship between estimates of nucleotide substitution rate and evolutionary timescale. Strikingly, this relationship was upheld both within and among diverse groups of viruses. A detailed case study of primate lentiviruses revealed that the combined effects of sequence saturation and purifying selection can explain this time-dependent pattern of rate variation. Therefore, our analyses show that studies of evolutionary time-scales in viruses require a reconsideration of substitution rates as a dynamic, rather than as a static, feature of molecular evolution. Improved modelling of viral evolutionary rates has the potential to change our understanding of virus origins.
doi:10.1098/rspb.2014.0732
PMCID: PMC4046420  PMID: 24850916
evolutionary rates; virus evolution; phylogenetics; molecular clock
8.  What can we predict about viral evolution and emergence? 
Current opinion in virology  2012;3(2):180-184.
Predicting the emergence of infectious diseases has been touted as one of the most important goals of biomedical science, with an array of funding schemes and research projects. However, evolutionary biology generally has a dim view of prediction, and there is a danger that erroneous predictions will mean a misuse of resources and undermine public confidence. Herein, I outline what can be realistically predicted about viral evolution and emergence, argue that any success in predicting what may emerge is likely to be limited, but that forecasting how viruses might evolve and spread following emergence is more tractable. I also emphasize that a properly grounded research program in disease prediction must involve a synthesis of ecological and genetic perspectives.
doi:10.1016/j.coviro.2012.12.003
PMCID: PMC3626763  PMID: 23273851
9.  Multiyear Persistence of 2 Pandemic A/H1N1 Influenza Virus Lineages in West Africa 
The Journal of Infectious Diseases  2014;210(1):121-125.
Our understanding of the global ecology of influenza viruses is impeded by historically low levels of viral surveillance in Africa. Increased genetic sequencing of African A/H1N1 pandemic influenza viruses during 2009–2013 revealed multiyear persistence of 2 viral lineages within West Africa, raising questions about the roles of reduced air traffic and the asynchrony of seasonal influenza epidemics among West African countries in the evolution of independent lineages. The potential for novel influenza virus lineages to evolve within Africa warrants intensified influenza surveillance in Africa and other understudied areas.
doi:10.1093/infdis/jiu047
PMCID: PMC4162001  PMID: 24446525
human influenza A virus; pandemic; phylogenetic analysis; Africa
10.  Avian influenza virus exhibits distinct evolutionary dynamics in wild birds and poultry 
Background
Wild birds are the major reservoir hosts for influenza A viruses, occasionally transmitting to other species such as domesticated poultry. Despite an abundance of genomic data from avian influenza virus (AIV), little is known about whether AIV evolves differently in wild birds and poultry, although this is critical to revealing the dynamics and time-scale of viral evolution. In particular, because environmental (water-borne) transmission is more common in wild birds, which may reduce the number of replications per unit time, it is possible that evolutionary rates are systematically lower in wild birds than in poultry.
Results
We estimated rates of nucleotide substitution in two AIV subtypes that are strongly associated with infections in wild birds – H4 and H6 – and compared these to rates in the H5N1 subtype that has circulated in poultry for almost two decades. Our analyses of three internal genes confirm that H4 and H6 viruses are evolving significantly more slowly than H5N1 viruses, suggesting that evolutionary rates of AIV are reduced in wild birds. This result was verified by the analysis of a poultry-associated H6 lineage that exhibited a markedly higher substitution rate than those H6 viruses circulating in wild birds. Interestingly, we also observed a significant difference in evolutionary rate between H4 and H6, despite frequent reassortment rate among them.
Conclusions
AIV experiences markedly different evolutionary dynamics between wild birds and poultry. These results suggest that rate heterogeneity among viral subtypes and ecological groupings should be taken into account when estimating evolutionary rates and divergence times.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-015-0410-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12862-015-0410-5
PMCID: PMC4481119  PMID: 26111936
11.  Exploring Host–Pathogen Interactions through Biological Control 
PLoS Pathogens  2015;11(6):e1004865.
doi:10.1371/journal.ppat.1004865
PMCID: PMC4482387  PMID: 26110272
12.  Inter-Seasonal Influenza is Characterized by Extended Virus Transmission and Persistence 
PLoS Pathogens  2015;11(6):e1004991.
The factors that determine the characteristic seasonality of influenza remain enigmatic. Current models predict that occurrences of influenza outside the normal surveillance season within a temperate region largely reflect the importation of viruses from the alternate hemisphere or from equatorial regions in Asia. To help reveal the drivers of seasonality we investigated the origins and evolution of influenza viruses sampled during inter-seasonal periods in Australia. To this end we conducted an expansive phylogenetic analysis of 9912, 3804, and 3941 hemagglutinnin (HA) sequences from influenza A/H1N1pdm, A/H3N2, and B, respectively, collected globally during the period 2009-2014. Of the 1475 viruses sampled from Australia, 396 (26.8% of Australian, or 2.2% of global set) were sampled outside the monitored temperate influenza surveillance season (1 May – 31 October). Notably, rather than simply reflecting short-lived importations of virus from global localities with higher influenza prevalence, we documented a variety of more complex inter-seasonal transmission patterns including “stragglers” from the preceding season and “heralds” of the forthcoming season, and which included viruses sampled from clearly temperate regions within Australia. We also provide evidence for the persistence of influenza B virus between epidemic seasons, in which transmission of a viral lineage begins in one season and continues throughout the inter-seasonal period into the following season. Strikingly, a disproportionately high number of inter-seasonal influenza transmission events occurred in tropical and subtropical regions of Australia, providing further evidence that climate plays an important role in shaping patterns of influenza seasonality.
Author Summary
Human influenza virus commonly causes disease in the winter months of temperate countries, but exhibits more complex patterns in tropical localities. Most studies of this complex seasonality have only considered viruses sampled within the “normal” influenza season. To help reveal the drivers of influenza seasonality we utilized viruses sampled outside of the normal influenza season, focusing on Australia which is characterized by a wide range of climates. Using a phylogenetic approach we revealed more complex patterns of influenza transmission than previously anticipated, particularly that the virus is able to transmit for extended periods and even persist locally within Australia throughout the virus “off-season”. In addition, we found that inter-seasonal influenza was more frequent in tropical and sub-tropical than temperate regions, adding weight to theories that climate likely plays an important role in influenza seasonality.
doi:10.1371/journal.ppat.1004991
PMCID: PMC4479464  PMID: 26107631
13.  Ebola Virus Epidemiology, Transmission, and Evolution during Seven Months in Sierra Leone 
Park, Daniel J. | Dudas, Gytis | Wohl, Shirlee | Goba, Augustine | Whitmer, Shannon L.M. | Andersen, Kristian G. | Sealfon, Rachel S. | Ladner, Jason T. | Kugelman, Jeffrey R. | Matranga, Christian B. | Winnicki, Sarah M. | Qu, James | Gire, Stephen K. | Gladden-Young, Adrianne | Jalloh, Simbirie | Nosamiefan, Dolo | Yozwiak, Nathan L. | Moses, Lina M. | Jiang, Pan-Pan | Lin, Aaron E. | Schaffner, Stephen F. | Bird, Brian | Towner, Jonathan | Mamoh, Mambu | Gbakie, Michael | Kanneh, Lansana | Kargbo, David | Massally, James L.B. | Kamara, Fatima K. | Konuwa, Edwin | Sellu, Josephine | Jalloh, Abdul A. | Mustapha, Ibrahim | Foday, Momoh | Yillah, Mohamed | Erickson, Bobbie R. | Sealy, Tara | Blau, Dianna | Paddock, Christopher | Brault, Aaron | Amman, Brian | Basile, Jane | Bearden, Scott | Belser, Jessica | Bergeron, Eric | Campbell, Shelley | Chakrabarti, Ayan | Dodd, Kimberly | Flint, Mike | Gibbons, Aridth | Goodman, Christin | Klena, John | McMullan, Laura | Morgan, Laura | Russell, Brandy | Salzer, Johanna | Sanchez, Angela | Wang, David | Jungreis, Irwin | Tomkins-Tinch, Christopher | Kislyuk, Andrey | Lin, Michael F. | Chapman, Sinead | MacInnis, Bronwyn | Matthews, Ashley | Bochicchio, James | Hensley, Lisa E. | Kuhn, Jens H. | Nusbaum, Chad | Schieffelin, John S. | Birren, Bruce W. | Forget, Marc | Nichol, Stuart T. | Palacios, Gustavo F. | Ndiaye, Daouda | Happi, Christian | Gevao, Sahr M. | Vandi, Mohamed A. | Kargbo, Brima | Holmes, Edward C. | Bedford, Trevor | Gnirke, Andreas | Ströher, Ute | Rambaut, Andrew | Garry, Robert F. | Sabeti, Pardis C.
Cell  2015;161(7):1516-1526.
Summary
The 2013–2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic. We confirm sustained human-to-human transmission within Sierra Leone and find no evidence for import or export of EBOV across national borders after its initial introduction. Using high-depth replicate sequencing, we observe both host-to-host transmission and recurrent emergence of intrahost genetic variants. We trace the increasing impact of purifying selection in suppressing the accumulation of nonsynonymous mutations over time. Finally, we note changes in the mucin-like domain of EBOV glycoprotein that merit further investigation. These findings clarify the movement of EBOV within the region and describe viral evolution during prolonged human-to-human transmission.
Graphical Abstract
Highlights
•In Sierra Leone, transmission has primarily been within-country, not between-country•Infectious doses are large enough for intrahost variants to transmit between hosts•A prolonged epidemic removes deleterious mutations from the viral population•There is preliminary evidence for human RNA editing effects on the Ebola genome
Ebola virus genomes from 232 patients sampled over 7 months in Sierra Leone were sequenced. Transmission of intrahost genetic variants suggests a sufficiently high infectious dose during transmission. The human host may have caused direct alterations to the Ebola virus genome.
doi:10.1016/j.cell.2015.06.007
PMCID: PMC4503805  PMID: 26091036
14.  Molecular Surveillance for Lymphoproliferative Disease Virus in Wild Turkeys (Meleagris gallopavo) from the Eastern United States 
PLoS ONE  2015;10(4):e0122644.
Lymphoproliferative disease virus (LPDV) is a poorly understood, oncogenic avian retrovirus of domestic turkeys that has historically been restricted to Europe and Israel. However, a recent study reported LPDV in multiple wild turkey diagnostic cases from throughout the eastern United States of America (USA). To better understand the distribution of LPDV in the eastern USA, we surveyed 1,164 reportedly asymptomatic hunter-harvested wild turkeys from 17 states for the presence of LPDV proviral DNA by PCR. In total, 564/1,164 (47%) turkeys were positive for LPDV. Wild turkeys from each state had a relatively high prevalence of LPDV, although statewide prevalence varied from 26 to 83%. Phylogenetic analysis revealed two major clades of LPDV in the USA, although one was at a low frequency suggesting restricted transmission, as well as significant clustering by state of isolation. To determine the best tissue to target for diagnostic purposes, liver, spleen, and bone marrow were tested from a subset of 15 hunter-harvested wild turkeys and 20 wild turkey diagnostic cases. Overall, bone marrow provided the highest level of detection for both hunter-harvested turkeys and diagnostic cases. The sensitivity of LPDV detection between tissues was not significantly different for diagnostic cases, but was for hunter-harvested birds. These results indicate that LPDV infection is common and widespread in wild turkey populations throughout the eastern USA, even without overt signs of disease.
doi:10.1371/journal.pone.0122644
PMCID: PMC4405500  PMID: 25897755
15.  Intrahost Dynamics of Antiviral Resistance in Influenza A Virus Reflect Complex Patterns of Segment Linkage, Reassortment, and Natural Selection 
mBio  2015;6(2):e02464-14.
ABSTRACT
Resistance following antiviral therapy is commonly observed in human influenza viruses. Although this evolutionary process is initiated within individual hosts, little is known about the pattern, dynamics, and drivers of antiviral resistance at this scale, including the role played by reassortment. In addition, the short duration of human influenza virus infections limits the available time window in which to examine intrahost evolution. Using single-molecule sequencing, we mapped, in detail, the mutational spectrum of an H3N2 influenza A virus population sampled from an immunocompromised patient who shed virus over a 21-month period. In this unique natural experiment, we were able to document the complex dynamics underlying the evolution of antiviral resistance. Individual resistance mutations appeared weeks before they became dominant, evolved independently on cocirculating lineages, led to a genome-wide reduction in genetic diversity through a selective sweep, and were placed into new combinations by reassortment. Notably, despite frequent reassortment, phylogenetic analysis also provided evidence for specific patterns of segment linkage, with a strong association between the hemagglutinin (HA)- and matrix (M)-encoding segments that matches that previously observed at the epidemiological scale. In sum, we were able to reveal, for the first time, the complex interaction between multiple evolutionary processes as they occur within an individual host.
IMPORTANCE
Understanding the evolutionary forces that shape the genetic diversity of influenza virus is crucial for predicting the emergence of drug-resistant strains but remains challenging because multiple processes occur concurrently. We characterized the evolution of antiviral resistance in a single persistent influenza virus infection, representing the first case in which reassortment and the complex patterns of drug resistance emergence and evolution have been determined within an individual host. Deep-sequence data from multiple time points revealed that the evolution of antiviral resistance reflects a combination of frequent mutation, natural selection, and a complex pattern of segment linkage and reassortment. In sum, these data show how immunocompromised hosts may help reveal the drivers of strain emergence.
doi:10.1128/mBio.02464-14
PMCID: PMC4453542  PMID: 25852163
16.  Correction: The Role of Selection in Shaping Diversity of Natural M. tuberculosis Populations 
PLoS Pathogens  2013;9(8):10.1371/annotation/cff22061-44d5-4301-b853-41702d160203.
doi:10.1371/annotation/cff22061-44d5-4301-b853-41702d160203
PMCID: PMC3779277
17.  Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru 
The Journal of General Virology  2014;95(Pt 4):787-792.
Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.
doi:10.1099/vir.0.058685-0
PMCID: PMC3973475  PMID: 24421116
18.  A generic assay for whole-genome amplification and deep sequencing of enterovirus A71 
Journal of Virological Methods  2015;215-216:30-36.
Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples.
doi:10.1016/j.jviromet.2015.02.011
PMCID: PMC4374682  PMID: 25704598
Enterovirus A71; Picornavirus; Hand foot and mouth disease; Deep sequencing; Phylogeny
19.  Frequent Cross-Species Transmission of Parvoviruses among Diverse Carnivore Hosts 
Journal of Virology  2013;87(4):2342-2347.
Although parvoviruses are commonly described in domestic carnivores, little is known about their biodiversity in nondomestic species. A phylogenetic analysis of VP2 gene sequences from puma, coyote, gray wolf, bobcat, raccoon, and striped skunk revealed two major groups related to either feline panleukopenia virus (“FPV-like”) or canine parvovirus (“CPV-like”). Cross-species transmission was commonplace, with multiple introductions into each host species but, with the exception of raccoons, relatively little evidence for onward transmission in nondomestic species.
doi:10.1128/JVI.02428-12
PMCID: PMC3571474  PMID: 23221559
20.  Declining transition/transversion ratios through time reveal limitations to the accuracy of nucleotide substitution models 
Background
Genetic analyses of DNA sequences make use of an increasingly complex set of nucleotide substitution models to estimate the divergence between gene sequences. However, there is currently no way to assess the validity of nucleotide substitution models over short time-scales and with limited mutational accumulation.
Results
We show that quantifying the decline in the ratio of transitions to transversions (ti/tv) over time provides an in-built measure of mutational saturation and hence of substitution model accuracy. We tested this through detailed phylogenetic analyses of 10 representative virus data sets comprising recently sampled and closely related sequences. In the majority of cases our estimates of ti/tv decrease with time, even under sophisticated time-reversible models of nucleotide substitution. This indicates that high levels of saturation are attained extremely rapidly in viruses, sometimes within decades. In contrast, we did not find any temporal patterns in selection pressures or CG-content over these short time-frames. To validate the temporal trend of ti/tv across a broader taxonomic range, we analyzed a set of 76 different viruses. Again, the estimate of ti/tv scaled negatively with evolutionary time, a trend that was more pronounced for rapidly-evolving RNA viruses than slowly-evolving DNA viruses.
Conclusions
Our study shows that commonly used substitution models can underestimate the number of substitutions among closely related sequences, such that the time-scale of viral evolution and emergence may be systematically underestimated. In turn, estimates of ti/tv provide an effective internal control of substitution model performance in viruses because of their high sensitivity to mutational saturation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-015-0312-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12862-015-0312-6
PMCID: PMC4358783  PMID: 25886870
Transition/transversion ratio; Substitution model; Virus; Substitution rate; Saturation
21.  Migratory Flyway and Geographical Distance are Barriers to the Gene Flow of Influenza Virus among North American Birds 
Ecology letters  2011;15(1):24-33.
Despite the importance of migratory birds in the ecology and evolution of avian influenza virus (AIV), there is a lack of information on the patterns of AIV spread at the intra-continental scale. We applied a variety of statistical phylogeographic techniques to a plethora of viral genome sequence data to determine the strength, pattern, and determinants of gene flow in AIV sampled from wild birds in North America. These analyses revealed a clear isolation-by-distance of AIV among sampling localities. In addition, we show that phylogeographic models incorporating information on the avian flyway of sampling proved a better fit to the observed sequence data than those specifying homogeneous or random rates of gene flow among localities. In sum, these data strongly suggest that the intra-continental spread of AIV by migratory birds is subject to major ecological barriers, including spatial distance and avian flyway.
doi:10.1111/j.1461-0248.2011.01703.x
PMCID: PMC3228906  PMID: 22008513
avian influenza; phylogeography; evolution; gene flow; ecological barriers; flyways; spatial distance
22.  Introductions and Evolution of Human-Origin Seasonal Influenza A Viruses in Multinational Swine Populations 
Journal of Virology  2014;88(17):10110-10119.
ABSTRACT
The capacity of influenza A viruses to cross species barriers presents a continual threat to human and animal health. Knowledge of the human-swine interface is particularly important for understanding how viruses with pandemic potential evolve in swine hosts. We sequenced the genomes of 141 influenza viruses collected from North American swine during 2002 to 2011 and identified a swine virus that possessed all eight genome segments of human seasonal A/H3N2 virus origin. A molecular clock analysis indicates that this virus—A/sw/Saskatchewan/02903/2009(H3N2)—has likely circulated undetected in swine for at least 7 years. For historical context, we performed a comprehensive phylogenetic analysis of an additional 1,404 whole-genome sequences from swine influenza A viruses collected globally during 1931 to 2013. Human-to-swine transmission occurred frequently over this time period, with 20 discrete introductions of human seasonal influenza A viruses showing sustained onward transmission in swine for at least 1 year since 1965. Notably, human-origin hemagglutinin (H1 and H3) and neuraminidase (particularly N2) segments were detected in swine at a much higher rate than the six internal gene segments, suggesting an association between the acquisition of swine-origin internal genes via reassortment and the adaptation of human influenza viruses to new swine hosts. Further understanding of the fitness constraints on the adaptation of human viruses to swine, and vice versa, at a genomic level is central to understanding the complex multihost ecology of influenza and the disease threats that swine and humans pose to each other.
IMPORTANCE The swine origin of the 2009 A/H1N1 pandemic virus underscored the importance of understanding how influenza A virus evolves in these animals hosts. While the importance of reassortment in generating genetically diverse influenza viruses in swine is well documented, the role of human-to-swine transmission has not been as intensively studied. Through a large-scale sequencing effort, we identified a novel influenza virus of wholly human origin that has been circulating undetected in swine for at least 7 years. In addition, we demonstrate that human-to-swine transmission has occurred frequently on a global scale over the past decades but that there is little persistence of human virus internal gene segments in swine.
doi:10.1128/JVI.01080-14
PMCID: PMC4136342  PMID: 24965467
23.  Myxoma Virus and the Leporipoxviruses: An Evolutionary Paradigm 
Viruses  2015;7(3):1020-1061.
Myxoma virus (MYXV) is the type species of the Leporipoxviruses, a genus of Chordopoxvirinae, double stranded DNA viruses, whose members infect leporids and squirrels, inducing cutaneous fibromas from which virus is mechanically transmitted by biting arthropods. However, in the European rabbit (Oryctolagus cuniculus), MYXV causes the lethal disease myxomatosis. The release of MYXV as a biological control for the wild European rabbit population in Australia, initiated one of the great experiments in evolution. The subsequent coevolution of MYXV and rabbits is a classic example of natural selection acting on virulence as a pathogen adapts to a novel host species. Slightly attenuated mutants of the progenitor virus were more readily transmitted by the mosquito vector because the infected rabbit survived longer, while highly attenuated viruses could be controlled by the rabbit immune response. As a consequence, moderately attenuated viruses came to dominate. This evolution of the virus was accompanied by selection for genetic resistance in the wild rabbit population, which may have created an ongoing co-evolutionary dynamic between resistance and virulence for efficient transmission. This natural experiment was repeated on a continental scale with the release of a separate strain of MYXV in France and its subsequent spread throughout Europe. The selection of attenuated strains of virus and resistant rabbits mirrored the experience in Australia in a very different environment, albeit with somewhat different rates. Genome sequencing of the progenitor virus and the early radiation, as well as those from the 1990s in Australia and Europe, has shown that although MYXV evolved at high rates there was no conserved route to attenuation or back to virulence. In contrast, it seems that these relatively large viral genomes have the flexibility for multiple pathways that converge on a similar phenotype.
doi:10.3390/v7031020
PMCID: PMC4379559  PMID: 25757062
myxoma virus; leporipoxvirus; poxvirus; myxomatosis; rabbit; coevolution
24.  Evolution of Genome Size and Complexity in the Rhabdoviridae 
PLoS Pathogens  2015;11(2):e1004664.
RNA viruses exhibit substantial structural, ecological and genomic diversity. However, genome size in RNA viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. Here we conduct a large-scale analysis of the genome sequences of 99 animal rhabdoviruses, including 45 genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. All but seven of the rhabdoviruses clustered into 17 well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. We show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive ORFs within the major structural protein genes, and the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. Changes in the lengths of gene junctions accounted for as much as 48.5% of the variation in genome size from the smallest to the largest genome, and the frequency with which new ORFs were observed increased in the 3’ to 5’ direction along the genome. We also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving TURBS-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. We conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the Rhabdoviridae.
Author Summary
Understanding the patterns and mechanisms of genome evolution is one of the most important, yet least understood, aspects of RNA virus biology. The evolutionary challenge faced by RNA viruses is to maximize functional diversity within severe constraints on genome size. Here we show that rhabdoviruses, a family of RNA viruses that infect hosts as diverse as plants, insects and vertebrates, have an unusual capacity for genomic plasticity. By analysing the complete or near-complete genome sequences of 99 animal rhabdoviruses, we show that genome expansion and contraction has likely occurred frequently throughout the evolution of the family. Genomic plasticity has been associated with the evolution of alternative, overlapping and consecutive ORFs within the major structural protein genes, as well as the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. This has resulted in remarkable diversity in genome organisation and gene expression strategies that is reflective of the broad ecological diversity of rhabdoviruses. We conclude that genomic plasticity in rhabdoviruses may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome and propose a general model that accounts for both gains and losses in genome size and complexity.
doi:10.1371/journal.ppat.1004664
PMCID: PMC4334499  PMID: 25679389
25.  Avian oncogenesis induced by lymphoproliferative disease virus: a neglected or emerging retroviral pathogen? 
Virology  2013;0:2-12.
Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown.
doi:10.1016/j.virol.2013.11.037
PMCID: PMC3925403  PMID: 24503062
Lymphoproliferative disease virus (LPDV); alpharetrovirus; avian tumor viruses; exogenous retrovirus; oncogenesis; Order Galliformes

Results 1-25 (191)