Genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity

Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ～75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection.     

Assessing the effects of human mixing patterns on human immunodeficiency virus-1 interhost phylogenetics through social network simulation

Geneticists seeking to understand HIV-1 evolution among human hosts generally assume that hosts represent a panmictic population. Social science research demonstrates that the network patterns over which HIV-1 spreads are highly nonrandom, but the effect of these patterns on the genetic diversity of HIV-1 and other sexually transmitted pathogens has yet to be thoroughly examined. In addition, interhost phylogenetic models rarely account explicitly for genetic diversity arising from intrahost dynamics. This study outlines a graph-theoretic framework (exponential random graph modeling, ERGM) for the estimation, inference, and simulation of dynamic partnership networks. This approach is used to simulate HIV-1 transmission and evolution under eight mixing patterns resembling those observed in empirical human populations, while simultaneously incorporating intrahost viral diversity. Models of parametric growth fit panmictic populations well, yielding estimates of total viral effective population on the order of the product of infected host size and intrahost effective viral population size. Populations exhibiting patterns of nonrandom mixing differ more widely in estimates of effective population size they yield, however, and reconstructions of population dynamics can exhibit severe errors if panmixis is assumed. I discuss implications for HIV-1 phylogenetics and the potential for ERGM to provide a general framework for addressing these issues.     

Intercompartmental recombination of HIV-1 contributes to env intrahost diversity and modulates viral tropism and sensitivity to entry inhibitors

HIV-1 circulates within an infected host as a genetically heterogeneous viral population. Viral intrahost diversity is shaped by substitutional evolution and recombination. Although many studies have speculated that recombination could have a significant impact on viral phenotype, this has never been definitively demonstrated. We report here phylogenetic and subsequent phenotypic analyses of envelope genes obtained from HIV-1 populations present in different anatomical compartments. Assessment of env compartmentalization from immunologically discrete tissues was assessed utilizing a single genome amplification approach, minimizing in vitro-generated artifacts. Genetic compartmentalization of variants was frequently observed. In addition, multiple incidences of intercompartment recombination, presumably facilitated by low-level migration of virus or infected cells between different anatomic sites and coinfection of susceptible cells by genetically divergent strains, were identified. These analyses demonstrate that intercompartment recombination is a fundamental evolutionary mechanism that helps to shape HIV-1 env intrahost diversity in natural infection. Analysis of the phenotypic consequences of these recombination events showed that genetic compartmentalization often correlates with phenotypic compartmentalization and that intercompartment recombination results in phenotype modulation. This represents definitive proof that recombination can generate novel combinations of phenotypic traits which differ subtly from those of parental strains, an important phenomenon that may have an impact on antiviral therapy and contribute to HIV-1 persistence in vivo.     

High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections

Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in sequence data in domain III (DIII) of the envelope (E) gene in serial plasma samples (n = 49) taken from 17 patients infected with DENV type 1 (DENV-1), totaling some 8,458 clones. Statistically rigorous approaches were employed to account for artifactual variants resulting from amplification and sequencing, which we suggest have played a major role in previous studies of intrahost genetic variation. Accordingly, nucleotide sequence diversities of viral populations were very low, with conservative estimates of the average levels of genetic diversity ranging from 0 to 0.0013. Despite such sequence conservation, we observed clear evidence for mixed infection, with the presence of multiple phylogenetically distinct lineages present within the same host, while the presence of stop codon mutations in some samples suggests the action of complementation. In contrast to some previous studies we observed no relationship between the extent and pattern of DENV-1 genetic diversity and disease severity, immune status, or level of viremia.     

Within-host genetic diversity of endemic and emerging parvoviruses of dogs and cats

Viral emergence can result from the adaptation of endemic pathogens to new or altered host environments, a process that is strongly influenced by the underlying sequence diversity. To determine the extent and structure of intrahost genetic diversity in a recently emerged single-stranded DNA virus, we analyzed viral population structures during natural infections of animals with canine parvovirus (CPV) or its ancestor, feline panleukopenia virus (FPV). We compared infections that occurred shortly after CPV emerged with more recent infections and examined the population structure of CPV after experimental cross-species transmission to cats. Infections with CPV and FPV showed limited genetic diversity regardless of the analyzed host tissue or year of isolation. Coinfections with genetically distinct viral strains were detected in some cases, and rearranged genomes were seen in both FPV and CPV. The sporadic presence of some sequences with multiple mutations suggested the occurrence of either particularly error-prone viral replication or coinfection by more distantly related strains. Finally, some potentially organ-specific host effects were seen during experimental cross-species transmission, with many of the mutations located in the nonstructural protein NS2. These included residues with evidence of positive selection at the population level, which is compatible with a role of this protein in host adaptation.     

Alternating Host Cell Tropism Shapes the Persistence,Evolution and Coexistence of Epstein–Barr Virus Infections in Human

Epstein–Barr virus (EBV) infects and can persist in a majority of people worldwide. Within an infected host, EBV targets two major cell types, B cells and epithelial cells, and viruses emerging from one cell type preferentially infect the other. We use mathematical models to understand why EBV infects epithelial cells when B cells serve as a stable refuge for the virus and how switching between infecting each cell type affects virus persistence and shedding. We propose a mathematical model to describe the regulation of EBV infection within a host. This model is used to study the effects of parameter values on optimal viral strategies for transmission, persistence, and intrahost competition. Most often, the optimal strategy to maximize transmission is for viruses to infect epithelial cells, but the optimal strategy for maximizing intrahost competition is for viruses to mainly infect B cells. Applying the results of the within-host model, we derive a model of EBV dynamics in a homogeneous population of hosts that includes superinfection. We use this model to study the conditions necessary for invasion and coexistence of various viral strategies at the population level. When the importance of intrahost competition is weak, we show that coexistence of different strategies is possible.     

Genotype and sex-based host variation in behaviour and susceptibility drives population disease dynamics

Host heterogeneity in pathogen transmission is widespread and presents a major hurdle to predicting and minimizing disease outbreaks. Using Drosophila melanogaster infected with Drosophila C virus as a model system, we integrated experimental measurements of social aggregation, virus shedding, and disease-induced mortality from different genetic lines and sexes into a disease modelling framework. The experimentally measured host heterogeneity produced substantial differences in simulated disease outbreaks, providing evidence for genetic and sex-specific effects on disease dynamics at a population level. While this was true for homogeneous populations of single sex/genetic line, the genetic background or sex of the index case did not alter outbreak dynamics in simulated, heterogeneous populations. Finally, to explore the relative effects of social aggregation, viral shedding and mortality, we compared simulations where we allowed these traits to vary, as measured experimentally, to simulations where we constrained variation in these traits to the population mean. In this context, variation in infectiousness, followed by social aggregation, was the most influential component of transmission. Overall, we show that host heterogeneity in three host traits dramatically affects population-level transmission, but the relative impact of this variation depends on both the susceptible population diversity and the distribution of population-level variation.     

Hierarchical spatial structure of genetically variable nucleopolyhedroviruses infecting cyclic populations of western tent caterpillars

The cyclic population dynamics of western tent caterpillars, Malacosoma californicum pluviale, are associated with epizootics of a nucleopolyhedrovirus, McplNPV. Given the dynamic fluctuations in host abundance and levels of viral infection, host resistance and virus virulence might be expected to change during different phases of the cycle. As a first step in determining if McplNPV virulence and population structure change with host density, we used restriction fragment length polymorphism (RFLP) analysis to examine the genetic diversity of McplNPV infecting western tent caterpillar populations at different spatial scales. Thirteen dominant genetic variants were identified in 39 virus isolates (individual larvae) collected from field populations during one year of low host density, and another distinct variant was discovered among nine additional isolates in two subsequent years of declining host density. The distribution of these genetic variants was not random and indicated that the McplNPV population was structured at several spatial levels. A high proportion of the variation could be explained by family grouping, which suggested that isolates collected within a family were more likely to be the same than isolates compared among populations. Additionally, virus variants from within populations (sites) were more likely to be the same than isolates collected from tent caterpillar populations on different islands. This may indicate that there is limited mixing of virus among tent caterpillar families and populations when host population density is low. Thus there is potential for the virus to become locally adapted to western tent caterpillar populations in different sites. However, no dominant genotype was observed at any site. Whether and how selection acts on the genetically diverse nucleopolyhedrovirus populations as host density changes will be investigated over the next cycle of tent caterpillar populations.     

基于微卫星标记的桃蚜种群寄主遗传分化

桃蚜Myzus persicae(Sulzer)是寄主范围最广、危害最大的蚜虫种类之一。为了探明桃蚜在不同寄主上的遗传分化特点,采用微卫星分子标记技术,对西兰花、桃树、辣椒上的桃蚜种群进行遗传多样性和遗传结构研究。结果表明,在所选用的5个微卫星位点上共检测到38个等位基因,平均每个位点的等位基因数达到7.6个,桃树种群遗传多样性最高,这可能是因为各种夏寄主上的桃蚜迁回桃树上越冬,从而使多种等位基因和基因型得以聚集的原因。等位基因频率差异分析显示西兰花种群、桃树种群和辣椒种群两两之间(除了桃树06种群和辣椒06种群之间没有遗传分化外)都出现了明显遗传分化,相比之下桃树种群和辣椒种群的分化程度要比桃树种群和西兰花种群的分化程度低,这可能预示着西兰花寄主上的桃蚜正在向远离桃树和辣椒种群的方向进化。     

Host species-dependent population structure of a pollen-borne plant virus, Cherry leaf roll virus

Cherry leaf roll virus (CLRV) belongs to the Nepovirus genus within the family Comoviridae. It has a host range which includes a number of wild tree and shrub species. The serological and molecular diversity of CLRV was assessed using a collection of isolates and samples recovered from woody and herbaceous host plants from different geographical origins. Molecular diversity was assessed by sequencing a short (375-bp) region of the 3' noncoding region (NCR) of the genomic RNAs while serological diversity was assessed using a panel of seven monoclonal antibodies raised initially against a walnut isolate of CLRV. The genomic region analyzed was shown to exhibit a significant degree of molecular variability with an average pairwise divergence of 8.5% (nucleotide identity). Similarly, serological variability proved to be high, with no single monoclonal antibody being able to recognize all isolates analyzed. Serological and molecular phylogenetic reconstructions showed a strong correlation. Remarkably, the diversity of CLRV populations is to a large extent defined by the host plant from which the viral samples are originally obtained. There are relatively few reports of plant viruses for which the genetic diversity is structured by the host plant. In the case of CLRV, we hypothesize that this situation may reflect the exclusive mode of transmission in natural plant populations by pollen and by seeds. These modes of transmission are likely to impose barriers to host change by the virus, leading to rapid biological and genetic separation of CLRV variants coevolving with different plant host species.     

Viral biogeography revealed by signatures in Sulfolobus islandicus genomes

Viruses are a driving force of microbial evolution. Despite their importance, the evolutionary dynamics that shape diversity in viral populations are not well understood. One of the primary factors that define viral population structure is coevolution with microbial hosts. Experimental models predict that the trajectory of coevolution will be determined by the relative migration rates of viruses and their hosts; however, there are no natural microbial systems in which both have been examined. The biogeographic distribution of viruses that infect Sulfolobus islandicus is investigated using genome comparisons among four newly identified, integrated, Sulfolobus spindle-shaped viruses and previously sequenced viral strains. Core gene sequences show a biogeographic distribution where viral genomes are specifically associated with each local population. In addition, signatures of host–virus interactions recorded in the sequence-specific CRISPR (clustered regularly interspaced short palindromic repeats) system show that hosts have interacted with viral communities that are more closely related to local viral strains than to foreign ones. Together, both proviral and CRISPR sequences show a clear biogeographic structure for Sulfolobus viral populations. Our findings demonstrate that virus–microbe coevolution must be examined in a spatially explicit framework. The combination of host and virus biogeography suggests a model for viral diversification driven by host immunity and local adaptation.     

Human immunodeficiency virus type 1 env evolves toward ancestral states upon transmission to a new host

Selecting human immunodeficiency virus (HIV) sequences for inclusion within vaccines has been a difficult problem, as circulating HIV strains evolve relentlessly and become increasingly divergent over time. We report an assessment of this divergence from three perspectives: (i) across different hosts as a function of time of infection, (ii) between donors and recipients in known transmission pairs, and (iii) within individual hosts over time in relation to the initially replicating virus and to the deduced ancestral sequence of the intrahost viral population. Surprisingly, we consistently found less divergence between viruses from different individuals sampled in primary infection than in individuals sampled at more advanced stages of illness. Furthermore, longitudinal analysis of intrahost divergence revealed a 2- to 3-year period of evolution toward a common ancestral sequence at the start of infection, indicating that HIV recovers certain ancestral features when infecting a new host. These results have important implications for the study of HIV population genetics and rational vaccine design, including favoring the inclusion of viral gene sequences taken early in infection.     

Subclonal components of consensus fitness in an RNA virus clone.

Most RNA virus populations exhibit extremely high mutation frequencies which generate complex, genetically heterogeneous populations referred to as quasi-species. Previous work has shown that when a large spectrum of the quasi-species is transferred, natural selection operates, leading to elimination of noncompetitive (inferior) genomes and rapid gains in fitness. However, whenever the population is repeatedly reduced to a single virion, variable declines in fitness occur as predicted by the Muller's ratchet hypothesis. Here, we quantitated the fitness of 98 subclones isolated from an RNA virus clonal population. We found a normal distribution around a lower fitness, with the average subclone being less fit than the parental clonal population. This finding demonstrates the phenotypic diversity in RNA virus populations and shows that, as expected, a large fraction of mutations generated during virus replication is deleterious. This clarifies the operation of Muller's ratchet and illustrates why a large number of virions must be transferred for rapid fitness gains to occur. We also found that repeated genetic bottleneck passages can cause irregular stochastic declines in fitness, emphasizing again the phenotypic heterogeneity present in RNA virus populations. Finally, we found that following only 60 h of selection (15 passages in which virus yields were harvested after 4 h), RNA virus populations can undergo a 250% average increase in fitness, even on a host cell type to which they were already well adapted. This is a remarkable ability; in population biology, even a much lower fitness gain (e.g., 1 to 2%) can represent a highly significant reproductive advantage. We discuss the biological implications of these findings for the natural transmission and pathogenesis of RNA viruses.     

Sex and the evolution of intrahost competition in RNA virus phi6.

Sex allows beneficial mutations that occur in separate lineages to be fixed in the same genome. For this reason, the Fisher-Muller model predicts that adaptation to the environment is more rapid in a large sexual population than in an equally large asexual population. Sexual reproduction occurs in populations of the RNA virus phi6 when multiple bacteriophages coinfect the same host cell. Here, we tested the model''s predictions by determining whether sex favors more rapid adaptation of phi6 to a bacterial host, Pseudomonas phaseolicola. Replicate populations of phi6 were allowed to evolve in either the presence or absence of sex for 250 generations. All experimental populations showed a significant increase in fitness relative to the ancestor, but sex did not increase the rate of adaptation. Rather, we found that the sexual and asexual treatments also differ because intense intrahost competition between viruses occurs during coinfection. Results showed that the derived sexual viruses were selectively favored only when coinfection is common, indicating that within-host competition detracts from the ability of viruses to exploit the host. Thus, sex was not advantageous because the cost created by intrahost competition was too strong. Our findings indicate that high levels of coinfection exceed an optimum where sex may be beneficial to populations of phi6, and suggest that genetic conflicts can evolve in RNA viruses.     

Extensive genome-wide variability of human cytomegalovirus in congenitally infected infants

Research has shown that RNA virus populations are highly variable, most likely due to low fidelity replication of RNA genomes. It is generally assumed that populations of DNA viruses will be less complex and show reduced variability when compared to RNA viruses. Here, we describe the use of high throughput sequencing for a genome wide study of viral populations from urine samples of neonates with congenital human cytomegalovirus (HCMV) infections. We show that HCMV intrahost genomic variability, both at the nucleotide and amino acid level, is comparable to many RNA viruses, including HIV. Within intrahost populations, we find evidence of selective sweeps that may have resulted from immune-mediated mechanisms. Similarly, genome wide, population genetic analyses suggest that positive selection has contributed to the divergence of the HCMV species from its most recent ancestor. These data provide evidence that HCMV, a virus with a large dsDNA genome, exists as a complex mixture of genome types in humans and offer insights into the evolution of the virus.     

The recent spread of a vertically transmitted virus through populations of Drosophila melanogaster

The sigma virus is a vertically transmitted pathogen that commonly infects natural populations of Drosophila melanogaster. This virus is the only known host-specific pathogen of D. melanogaster, and so offers a unique opportunity to study the genetics of Drosophila-viral interactions in a natural system. To elucidate the population genetic processes that operate in sigma virus populations, we collected D. melanogaster from 10 populations across three continents. We found that the sigma virus had a prevalence of 0-15% in these populations. Compared to other RNA viruses, we found that levels of viral genetic diversity are very low across Europe and North America. Based on laboratory measurements of the viral substitution rate, we estimate that most European and North American viral isolates shared a common ancestor approximately 200 years ago. We suggest two explanations for this: the first is that D. melanogaster has recently acquired the sigma virus; the second is that a single viral type has recently swept through D. melanogaster populations. Furthermore, in contrast to Drosophila populations, we find that the sigma viral populations are highly structured. This is surprising for a vertically transmitted pathogen that has a similar migration rate to its host. We suggest that the low structure in the viral populations can be explained by the smaller effective population size of the virus.     

Adaptation in an insect host–plant pathogen interaction

Selection on parasites to adapt to local host populations may be direct or through other components of the system such as vectors or the food plant on which the parasite is ingested. To test for local adaptation of nucleopolyhedrovirus among island populations of western tent caterpillars, Malacosoma californicum pluviale, we compared virus isolates from three geographically distinct sites with different dominant host plants. Pathogenicity, speed of kill and virus production of each isolate were examined on the three food plants. Virus isolates from the two permanent host populations had the fastest speed of kill on the host plant from which they were isolated. This was not the case for a caterpillar population that goes extinct when populations are regionally low. Virus isolates on some plant species combined rapid speed of kill with high virus yield. Infection of hosts by mixed microparasite populations could facilitate local adaptation in response to differing food plant chemistry.     

Host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures

The mechanisms by which RNA arboviruses, including chikungunya virus (CHIKV), evolve and maintain the ability to infect vertebrate and invertebrate hosts are poorly understood. To understand how host specificity shapes arbovirus populations, we studied CHIKV populations passaged alternately between invertebrate and vertebrate cells (invertebrate ↔ vertebrate) to simulate natural alternation and contrasted the results with those for populations that were artificially released from cycling by passage in single cell types. These CHIKV populations were characterized by measuring genetic diversity, changes in fitness, and adaptability to novel selective pressures. The greatest fitness increases were observed in alternately passaged CHIKV, without drastic changes in population diversity. The greatest increases in genetic diversity were observed after serial passage and correlated with greater adaptability. These results suggest an evolutionary trade-off between maintaining fitness for invertebrate ↔ vertebrate cell cycling, where maximum adaptability is possible only via enhanced population diversity and extensive exploration of sequence space.Emergence of pathogenic RNA viruses is associated with their genomic variability and environmental changes that lead to novel host contacts. Many new and reemerging RNA viruses have been introduced into humans (10). Arthropod-borne viruses (arboviruses), including dengue virus (DENV), have caused recent epidemics by changing their host ranges to increase infection of humans (54). Adaptation to the urban mosquito Aedes albopictus may have expanded a 2005-2006 outbreak of Chikungunya virus (CHIKV) in Reunion Island, France (46), that subsequently circulated among humans in the absence of other amplifying hosts.Despite these emergence events, the evolutionary processes that mediate arbovirus host range changes are poorly understood, partly since arbovirus evolution is understudied. Arboviruses are transmitted horizontally between arthropod vectors and vertebrate reservoir hosts. They replicate rapidly and achieve large population sizes. Polymerases of RNA viruses lack proofreading to repair errors, leading to one substitution per ∼10⁻⁴ nucleotides (nt) copied (11, 36), corresponding to one error per 10-kb genome. This polymerase infidelity leads to diversification that produces closely related but nonidentical RNAs that together form a spectrum of mutants. Although arbovirus mutant spectra have been observed in nature (1, 8, 20, 55, 57), the diversity and divergence within the spectrum are not well described, and the phenotypic roles of minority RNAs are unknown. Understanding the mutation distribution in a heterogeneous arbovirus population is important, given that any variant can be favored by selection and ultimately affect fitness (12, 13). However, the relationships between fitness and RNA virus population diversity are poorly understood.Studies with other RNA viruses, including human immunodeficiency virus (HIV) (3, 58, 60), hepatitis C virus (14, 15, 25), and poliovirus (30, 52), indicate that intrahost population diversity is important for virus evolution, fitness, and pathogenesis. Unlike these vertebrate-only RNA viruses, arboviruses obligately cycle between vertebrates and arthropods, a process that imposes additional selective constraints on evolution and adaptation. Sequence comparisons of RNA arbovirus isolates show that they are relatively stable (18, 19), and genetic studies reveal that evolution is dominated by purifying selection (20-22, 57). This constancy of consensus sequence may derive from the need to infect disparate hosts that present conflicting demands for adaptation where sequence changes that improve fitness in one host may not be maintained in the alternate organism.Experimental evolution studies have been performed to study fitness trade-offs and the unique ability of arboviruses to simultaneously evolve to vertebrate and invertebrate hosts. In vitro evolution studies reveal three general patterns of arbovirus evolution: (i) fitness gains after serial passage in vertebrate or invertebrate cells (except in certain cases [7]) and losses in bypassed host cell types (DENV, Eastern equine encephalitis virus [EEEV], Sindbis virus [SINV], and vesicular stomatitis virus [VSV]) (17, 28, 51, 56); (ii) reduced fitness in new cells (VSV) (28), and (iii) fitness increases after alternating (invertebrate ↔ vertebrate) passage (DENV, EEEV, SINV, VSV) (17, 28, 51, 56). Together, these studies suggest that constraints on fitness differ in insect and vertebrate cells and can be virus specific but that arbovirus fitness is not limited by alternating between vertebrate and invertebrate hosts.An in vivo study revealed a similar pattern of arbovirus evolution: vertebrate-passaged Venezuelan equine encephalitis virus (VEEV) was five times more fit than its unpassaged parent, and mosquito-passaged VEEV was more infectious for vectors (6). Consensus sequences revealed that, despite becoming more fit, mutations in passaged populations were slight or absent. This suggests that fitness increases were mediated by minority genomes in the mutant spectrum. However, a major limitation of the in vivo experiments and other arbovirus evolution studies is that sequencing of individual RNAs from the mutant spectrum in passaged intrahost populations was not performed (although notable exceptions for flaviviruses exist [5, 22]). The identity of minority variants within intrahost arbovirus populations and their influences on phenotype have not been extensively examined.The goal of this study, therefore, is to understand how obligate host cycling shapes an intrahost arbovirus population. RNA viruses can tolerate increased niche breadth when they evolve in heterogeneous environments (48), a trait which may promote easier emergence and therefore help explain why arboviruses frequently emerge or reemerge to cause human and veterinary disease. One limitation of increasing breadth may be restricted genetic diversity, where only genomes accepted in both hosts can be maintained. To explore this possibility, we described arbovirus populations after invertebrate ↔ vertebrate cell cycling and compared them to populations that were artificially released from alternating passage via serial vertebrate or invertebrate cell transfer. Since previous studies indicate that intrahost RNA virus population diversity can determine phenotype (3, 37, 52) and given that increases in arbovirus fitness are not always associated with consensus genome changes (6, 27), we determined how arbovirus population diversity (mutation frequency) and distance (number of mutations by which each RNA differs from the consensus) relate to fitness. We predicted that genetic diversity and distance between RNAs in the mutant spectrum and the corresponding consensus are correlated with fitness and that diverse populations better escape varied selective pressures than genetically homogeneous populations since, by chance, they are more likely to contain an advantageous mutation(s) when the pressure is applied.Chikungunya virus (family Togaviridae, genus Alphavirus) was selected primarily because of its medical relevance. CHIKV has caused outbreaks of human disease characterized by arthralgia and myalgia since the 18th century and since 2004 in Africa, Indian Ocean islands, Southeast Asia, and Italy (32). Furthermore, no adaptation studies have been performed for CHIKV, and no intrahost population studies have been conducted for an alphavirus. An infectious clone was generated from the strain used for passages and was genetically marked to differentiate its RNAs from passaged CHIKV, so that after direct competition, the fitnesses of passaged populations could be compared to those for progenitors.     

Evolutionarily related Sindbis-like plant viruses maintain different levels of population diversity in a common host

The levels of population diversity of three related Sindbis-like plant viruses, Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), and Cowpea chlorotic mottle virus (CCMV), in infections of a common host, Nicotiana benthamiana, established from genetically identical viral RNA were examined. Despite probably having a common evolutionary ancestor, the three viruses maintained different levels of population diversity. CMV had the highest levels of diversity, TMV had an intermediate level of diversity, and CCMV had no measurable level of diversity in N. benthamiana. Interestingly, the levels of diversity were correlated to the relative host range sizes of the three viruses. The levels of diversity also remained relatively constant over the course of serial passage. Closer examination of the CMV and TMV populations revealed biases for particular types of substitutions and regions of the genome that may tolerate fewer mutations.     

Tracking Dengue Virus Intra-host Genetic Diversity during Human-to-Mosquito Transmission

Dengue virus (DENV) infection of an individual human or mosquito host produces a dynamic population of closely-related sequences. This intra-host genetic diversity is thought to offer an advantage for arboviruses to adapt as they cycle between two very different host species, but it remains poorly characterized. To track changes in viral intra-host genetic diversity during horizontal transmission, we infected Aedes aegypti mosquitoes by allowing them to feed on DENV2-infected patients. We then performed whole-genome deep-sequencing of human- and matched mosquito-derived DENV samples on the Illumina platform and used a sensitive variant-caller to detect single nucleotide variants (SNVs) within each sample. >90% of SNVs were lost upon transition from human to mosquito, as well as from mosquito abdomen to salivary glands. Levels of viral diversity were maintained, however, by the regeneration of new SNVs at each stage of transmission. We further show that SNVs maintained across transmission stages were transmitted as a unit of two at maximum, suggesting the presence of numerous variant genomes carrying only one or two SNVs each. We also present evidence for differences in selection pressures between human and mosquito hosts, particularly on the structural and NS1 genes. This analysis provides insights into how population drops during transmission shape RNA virus genetic diversity, has direct implications for virus evolution, and illustrates the value of high-coverage, whole-genome next-generation sequencing for understanding viral intra-host genetic diversity.