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1.  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
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 emergence and evolution of the novel epidemic norovirus GII.4 variant Sydney 2012 
Virology  2013;0:106-113.
Norovirus is the leading cause of acute gastroenteritis with most infections caused by GII.4 variants. To understand the evolutionary processes that contribute to the emergence of GII.4 variants, we examined the molecular epidemiology of norovirus-associated acute gastroenteritis in Australia and New Zealand from 893 outbreaks between 2009 and 2012. Throughout the study GII.4 New Orleans 2009 was predominant; however, during 2012 it was replaced by an emergent GII.4 variant, Sydney 2012. An evolutionary analysis of capsid gene sequences was performed to determine the origins and selective pressures driving the emergence of these recently circulating GII.4 variants. This revealed that both New Orleans 2009 and Sydney 2012 share a common ancestor with GII.4 Apeldoorn 2007. Furthermore, pre-epidemic ancestral variants of each virus were identified up to two years before their pandemic emergence. Adaptive changes at known blockade epitopes in the viral capsid were also identified that likely contributed to their emergence.
doi:10.1016/j.virol.2013.12.005
PMCID: PMC3984403  PMID: 24503072
Norovirus; Gastroenteritis; Emergence; Evolution; Sydney 2012
4.  Recombination Is Associated with an Outbreak of Novel Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Viruses in China 
Journal of Virology  2013;87(19):10904-10907.
In 2009 to 2010, there was a marked increase in the number of infections with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in China. Through phylogenetic analysis, we show that viruses from this outbreak originated from a single recombination event, illustrating the potential importance of this process for disease emergence.
doi:10.1128/JVI.01270-13
PMCID: PMC3807407  PMID: 23885071
5.  Contrasting the epidemiological and evolutionary dynamics of influenza spatial transmission 
In the past decade, rapid increases in the availability of high-resolution molecular and epidemiological data, combined with developments in statistical and computational methods to simulate and infer migration patterns, have provided key insights into the spatial dynamics of influenza A viruses in humans. In this review, we contrast findings from epidemiological and molecular studies of influenza virus transmission at different spatial scales. We show that findings are broadly consistent in large-scale studies of inter-regional or inter-hemispheric spread in temperate regions, revealing intense epidemics associated with multiple viral introductions, followed by deep troughs driven by seasonal bottlenecks. However, aspects of the global transmission dynamics of influenza viruses are still debated, especially with respect to the existence of tropical source populations experiencing high levels of genetic diversity and the extent of prolonged viral persistence between epidemics. At the scale of a country or community, epidemiological studies have revealed spatially structured diffusion patterns in seasonal and pandemic outbreaks, which were not identified in molecular studies. We discuss the role of sampling issues in generating these conflicting results, and suggest strategies for future research that may help to fully integrate the epidemiological and evolutionary dynamics of influenza virus over space and time.
doi:10.1098/rstb.2012.0199
PMCID: PMC3678324  PMID: 23382422
influenza; spatial diffusion; human mobility; phylogeography; source population; viral persistence
6.  Inferring the inter-host transmission of influenza A virus using patterns of intra-host genetic variation 
Influenza A viruses (IAVs) cause acute, highly transmissible infections in a wide range of animal species. Understanding how these viruses are transmitted within and between susceptible host populations is critical to the development of effective control strategies. While viral gene sequences have been used to make inferences about IAV transmission dynamics at the epidemiological scale, their utility in accurately determining patterns of inter-host transmission in the short-term—i.e. who infected whom—has not been strongly established. Herein, we use intra-host sequence data from the viral HA1 (hemagglutinin) gene domain from two transmission studies employing different IAV subtypes in their natural hosts—H3N8 in horses and H1N1 in pigs—to determine how well these data recapitulate the known pattern of inter-host transmission. Although no mutations were fixed over the course of either experimental transmission chain, we show that some minor, transient alleles can provide evidence of host-to-host transmission and, importantly, can be distinguished from those that cannot.
doi:10.1098/rspb.2012.2173
PMCID: PMC3574438  PMID: 23135678
influenza A virus; transmission; evolution; phylogeny; phylodynamics
7.  Epidemiological Dynamics and Phylogeography of Influenza Virus in Southern China 
The Journal of Infectious Diseases  2012;207(1):106-114.
Background. Understanding the epidemiological dynamics of influenza virus is central to surveillance and vaccine strain selection. It has been suggested that tropical and subtropical regions represent the global source of influenza epidemics. However, our understanding of the epidemiological dynamics of influenza virus in these regions is limited by a relative lack of long-term data.
Methods. We analyzed epidemiological and virological data on influenza recorded over a period of 15 years from the metropolitan city of Shenzhen in subtropical southern China. We used wavelet analysis to determine the periodicity of influenza epidemics and molecular phylogeographic analysis to investigate the role of Shenzhen and southern China in the global evolution of influenza virus.
Results. We show that southern China is unlikely to represent an epicenter of global influenza activity, because activity in Shenzhen is characterized by significant annual cycles, multiple viral introductions every year, limited persistence across epidemic seasons, and viruses that generally are not positioned on the trunk of the global influenza virus phylogeny.
Conclusions. We propose that novel influenza viruses emerge and evolve in multiple geographic localities and that the global evolution of influenza virus is complex and does not simply originate from a southern Chinese epicenter.
doi:10.1093/infdis/jis526
PMCID: PMC3523792  PMID: 22930808
influenza; epidemiology; southern China; seasonality; phylogeography
8.  Presence of Oseltamivir-Resistant Pandemic A/H1N1 Minor Variants Before Drug Therapy With Subsequent Selection and Transmission 
The Journal of Infectious Diseases  2012;206(10):1504-1511.
A small proportion (1%–1.5%) of 2009 pandemic influenza A/H1N1 virus strains (A[H1N1]pdm09) are oseltamivir resistant, almost exclusively because of a H275Y mutation in the neuraminidase protein. However, many individuals infected with resistant strains had not received antivirals. Whether drug-resistant viruses are initially present as minor variants in untreated individuals before they emerge as the dominant strain in a virus population is of great importance for predicting the speed at which resistance will arise. To address this issue, we used ultra-deep sequencing of viral populations from serial nasopharyngeal specimens from an immunocompromised child and from 2 individuals in a household outbreak. We observed that the Y275 mutation was present as a minor variant in infected hosts before the onset of therapy. We also found evidence for the transmission of this drug-resistant variant with drug-susceptible viruses. These observations provide important information on the relative fitness of the Y275 mutation in the absence of oseltamivir treatment.
doi:10.1093/infdis/jis571
PMCID: PMC3475640  PMID: 22966122
9.  Endogenous Hepadnaviruses in the Genome of the Budgerigar (Melopsittacus undulatus) and the Evolution of Avian Hepadnaviruses 
Journal of Virology  2012;86(14):7688-7691.
Endogenous hepadnaviruses (hepatitis B viruses [HBVs]) were recently discovered in the genomes of passerine birds. We mined six additional avian genomes and discovered multiple copies of endogenous HBVs in the budgerigar (order Psittaciformes), designated eBHBV. A phylogenetic analysis reveals that the endogenous hepadnaviruses are more diverse than their exogenous counterparts and that the endogenous and exogenous hepadnaviruses form distinct lineages even when sampled from the same avian order, indicative of multiple genomic integration events.
doi:10.1128/JVI.00769-12
PMCID: PMC3416299  PMID: 22553337
10.  Adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of Middle East respiratory syndrome coronavirus 
Virology Journal  2013;10:304.
Background
The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) that first appeared in Saudi Arabia during the summer of 2012 has to date (20th September 2013) caused 58 human deaths. MERS-CoV utilizes the dipeptidyl peptidase 4 (DPP4) host cell receptor, and analysis of the long-term interaction between virus and receptor provides key information on the evolutionary events that lead to the viral emergence.
Findings
We show that bat DPP4 genes have been subject to significant adaptive evolution, suggestive of a long-term arms-race between bats and MERS related CoVs. In particular, we identify three positively selected residues in DPP4 that directly interact with the viral surface glycoprotein.
Conclusions
Our study suggests that the evolutionary lineage leading to MERS-CoV may have circulated in bats for a substantial time period.
doi:10.1186/1743-422X-10-304
PMCID: PMC3852826  PMID: 24107353
MERS-CoV; Bats; Arms-race; Adaptive evolution; Emergence
11.  Global transmission of influenza viruses from humans to swine 
The Journal of General Virology  2012;93(Pt 10):2195-2203.
To determine the extent to which influenza viruses jump between human and swine hosts, we undertook a large-scale phylogenetic analysis of pandemic A/H1N1/09 (H1N1pdm09) influenza virus genome sequence data. From this, we identified at least 49 human-to-swine transmission events that occurred globally during 2009–2011, thereby highlighting the ability of the H1N1pdm09 virus to transmit repeatedly from humans to swine, even following adaptive evolution in humans. Similarly, we identified at least 23 separate introductions of human seasonal (non-pandemic) H1 and H3 influenza viruses into swine globally since 1990. Overall, these results reveal the frequency with which swine are exposed to human influenza viruses, indicate that humans make a substantial contribution to the genetic diversity of influenza viruses in swine, and emphasize the need to improve biosecurity measures at the human–swine interface, including influenza vaccination of swine workers.
doi:10.1099/vir.0.044974-0
PMCID: PMC3541789  PMID: 22791604
12.  Evolution of Equine Influenza Virus in Vaccinated Horses 
Journal of Virology  2013;87(8):4768-4771.
Influenza A viruses are characterized by their ability to evade host immunity, even in vaccinated individuals. To determine how prior immunity shapes viral diversity in vivo, we studied the intra- and interhost evolution of equine influenza virus in vaccinated horses. Although the level and structure of genetic diversity were similar to those in naïve horses, intrahost bottlenecks may be more stringent in vaccinated animals, and mutations shared among horses often fall close to putative antigenic sites.
doi:10.1128/JVI.03379-12
PMCID: PMC3624384  PMID: 23388708
13.  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
14.  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
15.  Endogenous RNA viruses of plants in insect genomes 
Virology  2012;427(2):77-79.
Endogenous viral elements (EVEs) derived from RNA viruses with no DNA stage are rare, especially those where the parental viruses possess single-strand positive-sense (ssRNA+) genomes. Here we provide evidence that EVEs that share a sequence similarity to ssRNA+viruses of plants are integrated into the genomes of a number of insects, including mosquito, fruit flies, bees, ant, silkworm, pea aphid, Monarch butterfly, and wasps. A preliminary phylogenetic analysis places these EVEs as divergent relatives of the Virgaviridae and three currently unclassified plant viral species.
doi:10.1016/j.virol.2012.02.014
PMCID: PMC3725393  PMID: 22410578
Endogenous viral element; RNA virus; Plant viruses; Insects; Phylogeny; Evolution
16.  Trends in patterns of dengue transmission over four years of a pediatric cohort study in Nicaragua 
Background
Dengue is the most prevalent mosquito-borne viral disease in humans and a major urban public health problem worldwide.
Methods
A prospective cohort study of ~3,800 children initially aged 2-9 years old was established in Managua, Nicaragua, in 2004 to study the natural history of dengue transmission in an urban pediatric population. Blood samples from healthy subjects were collected annually prior to the dengue season, and identification of dengue cases occurred via enhanced passive surveillance at the study health center.
Results
Over the first four years of the study, seroprevalence of anti-dengue virus (DENV) antibodies increased from 22-40% in the 2-year-old cohort and 90-95% in the 9-year-old cohort. The incidence of symptomatic dengue cases and the ratio of inapparent to symptomatic DENV infection varied substantially year-to-year. The switch in dominant transmission from DENV-1 to DENV-2 was accompanied by an increase in disease severity but, paradoxically, a decrease in transmission. Phylogeographic analysis of full-length DENV-2 sequences revealed strong geographic clustering of dengue cases.
Conclusions
This large-scale cohort study of dengue in the Americas demonstrates year-to-year variation of dengue within a pediatric population, revealing expected patterns in transmission while highlighting the impact of interventions, climate, and viral evolution.
doi:10.1086/648592
PMCID: PMC3724236  PMID: 19929380
Dengue; transmission; cohort; Nicaragua; pediatric; phylogeography; seroprevalence; seroincidence; infection incidence; serotypes
17.  Rapid turnover of intra-host genetic diversity in Zucchini yellow mosaic virus 
Virus research  2010;155(2):389-396.
Genetic diversity in RNA viruses is shaped by a variety of evolutionary processes, including the bottlenecks that may occur at inter-host transmission. However, how these processes structure genetic variation at the scale of individual hosts is only partly understood. We obtained intra-host sequence data for the coat protein (CP) gene of Zucchini yellow mosaic virus (ZYMV) from two horizontally transmitted populations – one via aphid, the other without – and with multiple samples from individual plants. We show that although mutations are generated relatively frequently within infected plants, attaining similar levels of genetic diversity to that seen in some animal RNA viruses (mean intra-sample diversity of 0.02%), most mutations are likely to be transient, deleterious, and purged rapidly. We also observed more population structure in the aphid transmitted viral population, including the same mutations in multiple clones, the presence of a sub-lineage, and evidence for the short-term complementation of defective genomes.
doi:10.1016/j.virusres.2010.11.007
PMCID: PMC3026904  PMID: 21138748
evolution; intra-host diversity; Zucchini yellow mosaic virus; plant RNA virus; potyvirus; complementation
18.  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
19.  Evolutionary Conservation of the PA-X Open Reading Frame in Segment 3 of Influenza A Virus 
Journal of Virology  2012;86(22):12411-12413.
PA-X is a fusion protein of influenza A virus encoded in part from a +1 frameshifted X open reading frame (X-ORF) in segment 3. We show that the X-ORFs of diverse influenza A viruses can be divided into two groups that differ in selection pressure and likely function, reflected in the presence of an internal stop codon and a change in synonymous diversity. Notably, truncated forms of PA-X evolved convergently in swine and dogs, suggesting a strong species-specific effect.
doi:10.1128/JVI.01677-12
PMCID: PMC3486490  PMID: 22951836
20.  Identification of diverse full-length endogenous betaretroviruses in megabats and microbats 
Retrovirology  2013;10:35.
Background
Betaretroviruses infect a wide range of species including primates, rodents, ruminants, and marsupials. They exist in both endogenous and exogenous forms and are implicated in animal diseases such as lung cancer in sheep, and in human disease, with members of the human endogenous retrovirus-K (HERV-K) group of endogenous betaretroviruses (βERVs) associated with human cancers and autoimmune diseases. To improve our understanding of betaretroviruses in an evolutionarily distinct host species, we characterized βERVs present in the genomes and transcriptomes of mega- and microbats, which are an important reservoir of emerging viruses.
Results
A diverse range of full-length βERVs were discovered in mega- and microbat genomes and transcriptomes including the first identified intact endogenous retrovirus in a bat. Our analysis revealed that the genus Betaretrovirus can be divided into eight distinct sub-groups with evidence of cross-species transmission. Betaretroviruses are revealed to be a complex retrovirus group, within which one sub-group has evolved from complex to simple genomic organization through the acquisition of an env gene from the genus Gammaretrovirus. Molecular dating suggests that bats have contended with betaretroviral infections for over 30 million years.
Conclusions
Our study reveals that a diverse range of betaretroviruses have circulated in bats for most of their evolutionary history, and cluster with extant betaretroviruses of divergent mammalian lineages suggesting that their distribution may be largely unrestricted by host species barriers. The presence of βERVs with the ability to transcribe active viral elements in a major animal reservoir for viral pathogens has potential implications for public health.
doi:10.1186/1742-4690-10-35
PMCID: PMC3621094  PMID: 23537098
Retrovirus; Betaretrovirus; Endogenous; Evolution; Bats; Pteropus; Myotis; Rhinolophus
21.  Malpais spring virus is a new species in the genus vesiculovirus 
Virology Journal  2013;10:69.
Background
Malpais Spring virus (MSPV) is a mosquito-borne rhabdovirus that infects a variety of wild and feral ungulates in New Mexico, including horses and deer. Although, initial serologic tests and electron microscopy at the time of isolation nearly 25 years ago provided evidence that MSPV is a novel virus, possibly related to vesiculoviruses, the virus still has not been approved as a new species.
Findings
Use of the illumina platform allowed us to obtain the complete genome of MSPV. Analysis of the complete 11019 nt genome sequence of the prototype 85-488NM strain of MSPV indicates that it encodes the five common rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (> 180 nt) in the N, M and G genes, including a 249 nt ORF in the G gene predicted to encode a 9.26 kDa highly basic transmembrane protein. Although antigenically very distant, phylogenetic analysis of the L gene indicates that MSPV is most closely related to Jurona virus, also isolated from mosquitoes in Brazil, as well as a number of other vesiculoviruses.
Conclusions
In sum, our analysis indicates MSPV should be classified as a member of the genus Vesiculovirus, family Rhabdoviridae. The complete genome sequence of MSPV will be helpful in the development of a reverse genetics system to study the unique aspects of this vesiculovirus in vivo and in vitro, and will assist development of specific diagnostic tests to study the epidemiology of MSPV infection.
doi:10.1186/1743-422X-10-69
PMCID: PMC3599451  PMID: 23497016
Malpais spring virus (MSPV); Family rhabdoviridae; Genus vesiculovirus; Genome sequence; Phylogeny
22.  Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe 
Nature genetics  2012;44(9):1056-1059.
Shigella are human-adapted Escherichia coli that have gained the ability to invade the human gut mucosa and cause dysentery1,2, spreading efficiently via low-dose fecal-oral transmission3,4. Historically, S. sonnei has been predominantly responsible for dysentery in developed countries, but is now emerging as a problem in the developing world, apparently replacing the more diverse S. flexneri in areas undergoing economic development and improvements in water quality4-6. Classical approaches have shown S. sonnei is genetically conserved and clonal7. We report here whole-genome sequencing of 132 globally-distributed isolates. Our phylogenetic analysis shows that the current S. sonnei population descends from a common ancestor that existed less than 500 years ago and has diversified into several distinct lineages with unique characteristics. Our analysis suggests the majority of this diversification occurred in Europe, followed by more recent establishment of local pathogen populations in other continents predominantly due to the pandemic spread of a single, rapidly-evolving, multidrug resistant lineage.
doi:10.1038/ng.2369
PMCID: PMC3442231  PMID: 22863732
23.  Phylodynamic Analysis of the Emergence and Epidemiological Impact of Transmissible Defective Dengue Viruses 
PLoS Pathogens  2013;9(2):e1003193.
Intra-host sequence data from RNA viruses have revealed the ubiquity of defective viruses in natural viral populations, sometimes at surprisingly high frequency. Although defective viruses have long been known to laboratory virologists, their relevance in clinical and epidemiological settings has not been established. The discovery of long-term transmission of a defective lineage of dengue virus type 1 (DENV-1) in Myanmar, first seen in 2001, raised important questions about the emergence of transmissible defective viruses and their role in viral epidemiology. By combining phylogenetic analyses and dynamical modeling, we investigate how evolutionary and ecological processes at the intra-host and inter-host scales shaped the emergence and spread of the defective DENV-1 lineage. We show that this lineage of defective viruses emerged between June 1998 and February 2001, and that the defective virus was transmitted primarily through co-transmission with the functional virus to uninfected individuals. We provide evidence that, surprisingly, this co-transmission route has a higher transmission potential than transmission of functional dengue viruses alone. Consequently, we predict that the defective lineage should increase overall incidence of dengue infection, which could account for the historically high dengue incidence reported in Myanmar in 2001–2002. Our results show the unappreciated potential for defective viruses to impact the epidemiology of human pathogens, possibly by modifying the virulence-transmissibility trade-off, or to emerge as circulating infections in their own right. They also demonstrate that interactions between viral variants, such as complementation, can open new pathways to viral emergence.
Author Summary
Defective viruses are viral particles with genetic mutations or deletions that eliminate essential functions, so that they cannot complete their life cycles independently. They can reproduce only by co-infecting host cells with functional viruses and ‘borrowing’ their functional elements. Defective viruses have been observed for many human pathogens, but they have not been thought to impact epidemiological outcomes. Recently it was reported that a lineage of defective dengue virus spread through humans and mosquitoes in Myanmar for at least 18 months in 2001–2002. In this study, we investigate the emergence and epidemiological impact of this defective lineage by combining genetic sequence analyses with mathematical models. We show that the defective lineage emerged from circulating dengue viruses between June 1998 and February 2001, and that it spreads because—surprisingly—its presence causes functional dengue viruses to transmit more efficiently. Our model shows that this would cause a substantial rise in total dengue infections, consistent with historically high levels of dengue cases reported in Myanmar during 2001 and 2002. Our study yields new insights into the biology of dengue virus, and demonstrates a previously unappreciated potential for defective viruses to impact the epidemiology of infectious diseases.
doi:10.1371/journal.ppat.1003193
PMCID: PMC3585136  PMID: 23468631
24.  Random Codon Re-encoding Induces Stable Reduction of Replicative Fitness of Chikungunya Virus in Primate and Mosquito Cells 
PLoS Pathogens  2013;9(2):e1003172.
Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.
Author Summary
Emerging arthropod-borne viruses (arboviruses) are a major cause of human and animal morbidity and mortality. Climatic and anthropological activities are responsible for the dispersal of arbovirus transmission vectors into new territories. Chikungunya virus (CHIKV) is an important example of a re-emerging pathogen for which no licensed vaccine exists. One of the vectors of CHIKV, the mosquito Aedes albopictus, has dispersed into new temperate regions resulting in outbreaks where they had not been previously observed. Here, we demonstrate that random codon re-encoding, a method that modifies the nucleic acid composition of large coding regions without modifying the encoded proteins, can significantly decrease the replicative fitness of CHIKV. This powerful method of attenuating viruses has several potential advantages for vaccine development, including the possibility to modulate precisely the degree of replicative fitness loss and to generate safe, live-attenuated vaccines that confer long-term protection, in a cost effective manner. Our studies also demonstrate that these re-encoded viruses exhibit a stable phenotype, and that the response to codon re-encoding was largely compensatory in nature, with little reversion of mutations. Finally, we provide further evidence that many synonymous sites in RNA viruses are not neutral and clearly impact viral fitness.
doi:10.1371/journal.ppat.1003172
PMCID: PMC3578757  PMID: 23436995
25.  Evolution of Novel Reassortant A/H3N2 Influenza Viruses in North American Swine and Humans, 2009–2011 
Journal of Virology  2012;86(16):8872-8878.
Novel H3N2 influenza viruses (H3N2v) containing seven genome segments from swine lineage triple-reassortant H3N2 viruses and a 2009 pandemic H1N1 (H1N1pdm09) matrix protein segment (pM) were isolated from 12 humans in the United States between August and December 2011. To understand the evolution of these novel H3N2 viruses in swine and humans, we undertook a phylogenetic analysis of 674 M sequences and 388 HA and NA sequences from influenza viruses isolated from North American swine during 2009–2011, as well as HA, NA, and M sequences from eight H3N2v viruses isolated from humans. We identified 34 swine influenza viruses (termed rH3N2p) with the same combination of H3, N2, and pM segments as the H3N2v viruses isolated from humans. Notably, these rH3N2p viruses were generated in swine via reassortment events between H3N2 viruses and the pM segment approximately 4 to 10 times since 2009. The pM segment has also reassorted with multiple distinct lineages of H1 virus, especially H1δ viruses. Importantly, the N2 segment of all H3N2v viruses isolated from humans is derived from a genetically distinct N2 lineage that has circulated in swine since being acquired by reassortment with seasonal human H3N2 viruses in 2001–2002, rather than from the N2 that is associated with the 1998 H3N2 swine lineage. The identification of this N2 variant may have implications for influenza vaccine design and the potential pandemic threat of H3N2v to human age groups with differing levels of prior exposure and immunity.
doi:10.1128/JVI.00259-12
PMCID: PMC3421719  PMID: 22696653

Results 1-25 (176)