Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population.

Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals. IMPORTANCE: Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance.

Transgenic Virus Resistance in Crop-Wild Cucurbita pepo Does Not Prevent Vertical Transmission of Zucchini yellow mosaic virus.

Zucchini yellow mosaic virus (ZYMV) is an economically important pathogen of cucurbits that is transmitted both horizontally and vertically. Although ZYMV is seed-transmitted in Cucurbita pepo, the potential for seed transmission in virus-resistant transgenic cultivars is not known. We crossed and backcrossed a transgenic squash cultivar with wild C. pepo, and determined whether seed-to-seedling transmission of ZYMV was possible in seeds harvested from transgenic backcrossed C. pepo. We then compared these transmission rates to those of non-transgenic (backcrossed and wild) C. pepo. The overall seed-to-seedling transmission rate in ZYMV was similar to those found in previous studies (1.37%), with no significant difference between transgenic backcrossed (2.48%) and non-transgenic (1.03%) backcrossed and wild squash. Fewer transgenic backcrossed plants had symptom development (7%) in comparison with all non-transgenic plants (26%) and may be instrumental in preventing yield reduction due to ZYMV. Our study shows that ZYMV is seed transmitted in transgenic backcrossed squash, which may affect the spread of ZYMV via the movement of ZYMV-infected seeds. Deep genome sequencing of the seed-transmitted viral populations revealed that 23% of the variants found in this study were present in other vertically transmitted ZYMV populations, suggesting that these variants may be necessary for seed transmission or are distributed geographically via seeds.

Analysis of viral (zucchini yellow mosaic virus) genetic diversity during systemic movement through a Cucurbita pepo vine.

Determining the extent and structure of intra-host genetic diversity and the magnitude and impact of population bottlenecks is central to understanding the mechanisms of viral evolution. To determine the nature of viral evolution following systemic movement through a plant, we performed deep sequencing of 23 leaves that grew sequentially along a single Cucurbita pepo vine that was infected with zucchini yellow mosaic virus (ZYMV), and on a leaf that grew in on a side branch. Strikingly, of 112 genetic (i.e. sub-consensus) variants observed in the data set as a whole, only 22 were found in multiple leaves. Similarly, only three of the 13 variants present in the inoculating population were found in the subsequent leaves on the vine. Hence, it appears that systemic movement is characterized by sequential population bottlenecks, although not sufficient to reduce the population to a single virion as multiple variants were consistently transmitted between leaves. In addition, the number of variants within a leaf increases as a function of distance from the inoculated (source) leaf, suggesting that the circulating sap may serve as a continual source of virus. Notably, multiple mutational variants were observed in the cylindrical inclusion (CI) protein (known to be involved in both cell-to-cell and systemic movement of the virus) that were present in multiple (19/24) leaf samples. These mutations resulted in a conformational change, suggesting that they might confer a selective advantage in systemic movement within the vine. Overall, these data reveal that bottlenecks occur during systemic movement, that variants circulate in the phloem sap throughout the infection process, and that important conformational changes in CI protein may arise during individual infections.

Zucchini yellow mosaic virus (ZYMV, Potyvirus): vertical transmission, seed infection and cryptic infections.

The role played by seed transmission in the evolution and epidemiology of viral crop pathogens remains unclear. We determined the seed infection and vertical transmission rates of zucchini yellow mosaic virus (ZYMV), in addition to undertaking Illumina sequencing of nine vertically transmitted ZYMV populations. We previously determined the seed-to-seedling transmission rate of ZYMV in Cucurbita pepo ssp. texana (a wild gourd) to be 1.6%, and herein observed a similar rate (1.8%) in the subsequent generation. We also observed that the seed infection rate is substantially higher (21.9%) than the seed-to-seedling transmission rate, suggesting that a major population bottleneck occurs during seed germination and seedling growth. In contrast, that two thirds of the variants present in the horizontally transmitted inoculant population were also present in the vertically transmitted populations implies that the bottleneck at vertical transmission may not be particularly severe. Strikingly, all of the vertically infected plants were symptomless in contrast to those infected horizontally, suggesting that vertical infection may be cryptic. Although no known virulence determining mutations were observed in the vertically infected samples, the 5' untranslated region was highly variable, with at least 26 different major haplotypes in this region compared to the two major haplotypes observed in the horizontally transmitted population. That the regions necessary for vector transmission are retained in the vertically infected populations, combined with the cryptic nature of vertical infection, suggests that seed transmission may be a significant contributor to the spread of ZYMV.

The evolutionary dynamics of influenza A virus adaptation to mammalian hosts.

Few questions on infectious disease are more important than understanding how and why avian influenza A viruses successfully emerge in mammalian populations, yet little is known about the rate and nature of the virus' genetic adaptation in new hosts. Here, we measure, for the first time, the genomic rate of adaptive evolution of swine influenza viruses (SwIV) that originated in birds. By using a curated dataset of more than 24 000 human and swine influenza gene sequences, including 41 newly characterized genomes, we reconstructed the adaptive dynamics of three major SwIV lineages (Eurasian, EA; classical swine, CS; triple reassortant, TR). We found that, following the transfer of the EA lineage from birds to swine in the late 1970s, EA virus genes have undergone substantially faster adaptive evolution than those of the CS lineage, which had circulated among swine for decades. Further, the adaptation rates of the EA lineage antigenic haemagglutinin and neuraminidase genes were unexpectedly high and similar to those observed in human influenza A. We show that the successful establishment of avian influenza viruses in swine is associated with raised adaptive evolution across the entire genome for many years after zoonosis, reflecting the contribution of multiple mutations to the coordinated optimization of viral fitness in a new environment. This dynamics is replicated independently in the polymerase genes of the TR lineage, which established in swine following separate transmission from non-swine hosts.

Deep sequencing reveals persistence of intra- and inter-host genetic diversity in natural and greenhouse populations of zucchini yellow mosaic virus.

The genetic diversity present in populations of RNA viruses is likely to be strongly modulated by aspects of their life history, including mode of transmission. However, how transmission mode shapes patterns of intra- and inter-host genetic diversity, particularly when acting in combination with de novo mutation, population bottlenecks and the selection of advantageous mutations, is poorly understood. To address these issues, this study performed ultradeep sequencing of zucchini yellow mosaic virus in a wild gourd, Cucurbita pepo ssp. texana, under two infection conditions: aphid vectored and mechanically inoculated, achieving a mean coverage of approximately 10 ,000×. It was shown that mutations persisted during inter-host transmission events in both the aphid vectored and mechanically inoculated populations, suggesting that the vector-imposed transmission bottleneck is not as extreme as previously supposed. Similarly, mutations were found to persist within individual hosts, arguing against strong systemic bottlenecks. Strikingly, mutations were seen to go to fixation in the aphid-vectored plants, suggestive of a major fitness advantage, but remained at low frequency in the mechanically inoculated plants. Overall, this study highlights the utility of ultradeep sequencing in providing high-resolution data capable of revealing the nature of virus evolution, particularly as the full spectrum of genetic diversity within a population may not be uncovered without sequence coverage of at least 2500-fold.

Segmental configuration and putative origin of the reassortant orbivirus, epizootic hemorrhagic disease virus serotype 6, strain Indiana.

In 2006, an exotic reassortant orbivirus, epizootic hemorrhagic disease virus serotype 6 (EHDV-6) [strain (Indiana)], was first detected in the United States. To characterize the reassortment configuration of this virus and to conclusively determine the parental virus of each RNA segment, the complete genome of EHDV-6 (Indiana) was sequenced, in addition to the genomes of representative EHDV-6 and EHDV-2 isolates. Based on genomic comparisons to all other EHDV serotypes, we determined that EHDV-6 (Indiana) originated from a reassortment event between the Australian prototype strain of EHDV-6 (CSIRO 753) and the North American topotype of EHDV-2 (Alberta). Additionally, phylogenetic analysis of all EHDV-6 (Indiana) isolates detected in the United States from 2006 to 2010 suggests that the virus may be undergoing continual reassortment with EHDV-2 (Alberta). In 2010, EHDV-6 (CSIRO 753) was detected in Guadeloupe, demonstrating that the parental virus of the reassortment event is circulating in the Caribbean.

Experimental Verification of Seed Transmission of Zucchini yellow mosaic virus.

Within two decades of its discovery, Zucchini yellow mosaic virus (ZYMV) achieved a global distribution. However, whether or not seed transmission occurs in this economically significant crop pathogen is controversial, and the relative impact of seed transmission on the epidemiology of ZYMV remains unclear. Using reverse transcription-polymerase chain reaction, we observed a seed transmission rate of 1.6% in Cucurbita pepo subsp. texana and show that seed-infected C. pepo plants are capable of initiating horizontal ZYMV infections, both mechanically and via an aphid vector (Myzus persicae). We also provide evidence that ZYMV-infected seeds may act as effective viral reservoirs, partially accounting for the current geographic distribution of ZYMV. Finally, the observation that ZYMV infection of C. pepo seeds results in virtually symptomless infection, coupled with our finding that an antibody test failed to detect vertically transmitted ZYMV in infected seed, highlights the urgent need to standardize current detection methods for seed infection.

Epidemic dynamics revealed in dengue evolution.

Dengue is an emerging tropical disease infecting tens of millions of people annually. A febrile illness with potentially severe hemorrhagic manifestations, dengue is caused by mosquito-borne viruses (DENV-1 to -4) that are maintained in endemic transmission in large urban centers of the tropics with periodic epidemic cycles at 3- to 5-year intervals. Puerto Rico (PR), a major population center in the Caribbean, has experienced increasingly severe epidemics since multiple dengue serotypes were introduced beginning in the late 1970s. We document the phylodynamics of DENV-4 between 1981 and 1998, a period of dramatic ecological expansion during which evolutionary change also occurs. The timescale of viral evolution is sufficiently short that viral transmission dynamics can be elucidated from genetic diversity data. Specifically, by combining virus sequence data with confirmed case counts in PR over these two decades, we show that the pattern of cyclic epidemics is strongly correlated with coalescent estimates of effective population size that have been estimated from sampled virus sequences using Bayesian Markov Chain Monte Carlo methods. Thus, we show that the observed epidemiologic dynamics are correlated with similar fluctuations in diversity, including severe interepidemic reductions in genetic diversity compatible with population bottlenecks that may greatly impact DENV evolutionary dynamics. Mean effective population sizes based on genetic data appear to increase prior to isolation counts, suggesting a potential bias in the latter and justifying more active surveillance of DENV activity. Our analysis explicitly integrates epidemiologic and sequence data in a joint model that could be used to further explore transmission models of infectious disease.

Genetic analysis of Israel acute paralysis virus: distinct clusters are circulating in the United States.

Israel acute paralysis virus (IAPV) is associated with colony collapse disorder of honey bees. Nonetheless, its role in the pathogenesis of the disorder and its geographic distribution are unclear. Here, we report phylogenetic analysis of IAPV obtained from bees in the United States, Canada, Australia, and Israel and the establishment of diagnostic real-time PCR assays for IAPV detection. Our data indicate the existence of at least three distinct IAPV lineages, two of them circulating in the United States. Analysis of representatives from each proposed lineage suggested the possibility of recombination events and revealed differences in coding sequences that may have implications for virulence.

The evolutionary genetics of viral emergence.

Despite the wealth of data describing the ecological factors that underpin viral emergence, little is known about the evolutionary processes that allow viruses to jump species barriers and establish productive infections in new hosts. Understanding the evolutionary basis to virus emergence is therefore a key research goal and many of the debates in this area can be considered within the rigorous theoretical framework established by evolutionary genetics. In particular, the respective roles played by natural selection and genetic drift in shaping genetic diversity are also of fundamental importance for understanding the nature of viral emergence. Herein, we discuss whether there are evolutionary rules to viral emergence, and especially whether certain types of virus, or those that infect a particular type of host species, are more likely to emerge than others. We stress the complex interplay between rates of viral evolution and the ability to recognize cell receptors from phylogenetically divergent host species. We also emphasize the current lack of convincing data as to whether viral emergence requires adaptation to the new host species during the early stages of infection, or whether it is largely a chance process involving the transmission of a viral strain with the necessary genetic characteristics.

The evolutionary dynamics of canid and mongoose rabies virus in Southern Africa.

Two variants of rabies virus (RABV) currently circulate in southern Africa: canid RABV, mainly associated with dogs, jackals, and bat-eared foxes, and mongoose RABV. To investigate the evolutionary dynamics of these variants, we performed coalescent-based analyses of the G-L inter-genic region, allowing for rate variation among viral lineages through the use of a relaxed molecular clock. This revealed that mongoose RABV is evolving more slowly than canid RABV, with mean evolutionary rates of 0.826 and 1.676 x 10(-3) nucleotide substitutions per site, per year, respectively. Additionally, mongoose RABV exhibits older genetic diversity than canid RABV, with common ancestors dating to 73 and 30 years, respectively, and while mongoose RABV has experienced exponential population growth over its evolutionary history in Africa, populations of canid RABV have maintained a constant size. Hence, despite circulating in the same geographic region, these two variants of RABV exhibit striking differences in evolutionary dynamics which are likely to reflect differences in their underlying ecology.

Evolution of the human immunodeficiency virus envelope gene is dominated by purifying selection.

The evolution of the human immunodeficiency virus (HIV-1) during chronic infection involves the rapid, continuous turnover of genetic diversity. However, the role of natural selection, relative to random genetic drift, in governing this process is unclear. We tested a stochastic model of genetic drift using partial envelope sequences sampled longitudinally in 28 infected children. In each case the Bayesian posterior (empirical) distribution of coalescent genealogies was estimated using Markov chain Monte Carlo methods. Posterior predictive simulation was then used to generate a null distribution of genealogies assuming neutrality, with the null and empirical distributions compared using four genealogy-based summary statistics sensitive to nonneutral evolution. Because both null and empirical distributions were generated within a coalescent framework, we were able to explicitly account for the confounding influence of demography. From the distribution of corrected P-values across patients, we conclude that empirical genealogies are more asymmetric than expected if evolution is driven by mutation and genetic drift only, with an excess of low-frequency polymorphisms in the population. This indicates that although drift may still play an important role, natural selection has a strong influence on the evolution of HIV-1 envelope. A negative relationship between effective population size and substitution rate indicates that as the efficacy of selection increases, a smaller proportion of mutations approach fixation in the population. This suggests the presence of deleterious mutations. We therefore conclude that intrahost HIV-1 evolution in envelope is dominated by purifying selection against low-frequency deleterious mutations that do not reach fixation.

Cross-protective immunity can account for the alternating epidemic pattern of dengue virus serotypes circulating in Bangkok.

Dengue virus, the causative agent of dengue fever and its more serious manifestation dengue hemorrhagic fever, is widespread throughout tropical and subtropical regions. The virus exists as four distinct serotypes, all of which have cocirculated in Bangkok for several decades with epidemic outbreaks occurring every 8-10 years. We analyze time-series data of monthly infection incidence, revealing a distinctive pattern with epidemics of serotypes 1, 2, and 3 occurring at approximately the same time and an isolated epidemic of serotype 4 occurring in the intervening years. Phylogenetic analysis of virus samples collected over the same period shows that clade replacement events are linked to the epidemic cycle and indicates that there is an interserotypic immune reaction. Using an epidemic model with stochastic seasonal forcing showing 8- to 10-year epidemic oscillations, we demonstrate that moderate cross-protective immunity gives rise to persistent out-of-phase oscillations similar to those observed in the data, but that strong or weak cross-protection or cross-enhancement only produces in-phase patterns. This behavior suggests that the epidemic pattern observed in Bangkok is the result of cross-protective immunity and may be significantly altered by changes in the interserotypic immune reaction.

Passive sexual transmission of human immunodeficiency virus type 1 variants and adaptation in new hosts.

Human immunodeficiency virus type 1 (HIV-1) genetic diversity is a major obstacle for the design of a successful vaccine. Certain viral polymorphisms encode human leukocyte antigen (HLA)-associated immune escape, potentially overcoming limited vaccine protection. Although transmission of immune escape variants has been reported, the overall extent to which this phenomenon occurs in populations and the degree to which it contributes to HIV-1 viral evolution are unknown. Selection on the HIV-1 env gene at transmission favors neutralization-sensitive variants, but it is not known to what degree selection acts on the internal HIV-1 proteins to restrict or enhance the transmission of immune escape variants. Studies have suggested that HLA class I may determine susceptibility to HIV-1 infection, but a definitive role for HLA at transmission remains unproven. Comparing populations of acute seroconverters and chronically infected patients, we found no evidence of selection acting to restrict transmission of HIV-1 variants. We found that statistical associations previously reported in chronic infection between viral polymorphisms and HLA class I alleles are not present in acute infection, suggesting that the majority of viral polymorphisms in these patients are the result of transmission rather than de novo adaptation. Using four episodes of HIV-1 transmission in which the donors and recipients were both sampled very close to the time of infection we found that, despite a transmission bottleneck, genetic variants of HIV-1 infection are transmitted in a frequency-dependent manner. As HIV-1 infections are seeded by unique donor-adapted viral variants, each episode is a highly individual antigenic challenge. Host-specific, idiosyncratic HIV-1 antigenic diversity will seriously tax the efficacy of immunization based on consensus sequences.

A multigene analysis of the phylogenetic relationships among the flaviviruses (Family: Flaviviridae) and the evolution of vector transmission.

The genus Flavivirus (family Flaviviridae) presently comprises around 70 single-strand positive-sense RNA viruses. These replicate in a range of vertebrate and invertebrate cells and may be mosquito-borne, tick-borne or have no-known-vector. Since transmission mode correlates strongly with phylogeny, the flaviviruses constitute a valuable model for the evolution of vector-borne disease. Attempts to resolve the higher-level taxonomic relationships of the flaviviruses through molecular phylogenetics have thus far proved inconclusive because of conflicting positions for the three main transmission groups. We conducted the most comprehensive phylogenetic study to date, involving maximum likelihood analyses of the NS3 and NS5 genes and the entire genome sequences available at present. For the first time, we use and test a variety of more robust methods of sequence alignment and appropriate models of amino acid replacement to study these highly divergent sequences, and explicitly test specific hypotheses of tree topology. We show that (i) the NS5 gene contains insufficient phylogenetic signal to choose between competing topological hypotheses, (ii) the NS3 gene and whole genome data indicate that the mosquito-borne flaviviruses represent an outgroup to the remaining flaviviruses, and (iii) that tick-borne transmission is probably a derived trait within the genus.

A phylogenetic survey of recombination frequency in plant RNA viruses.

The severe economic consequences of emerging plant viruses highlights the importance of studies of plant virus evolution. One question of particular relevance is the extent to which the genomes of plant viruses are shaped by recombination. To this end we conducted a phylogenetic survey of recombination frequency in a wide range of positive-sense RNA plant viruses, utilizing 975 capsid gene sequences and 157 complete genome sequences. In total, 12 of the 36 RNA virus species analyzed showed evidence for recombination, comprising 17% of the capsid gene sequence alignments and 44% of the genome sequence alignments. Given the conservative nature of our analysis, we propose that recombination is a relatively common process in some plant RNA viruses, most notably the potyviruses.

Phylogenetic relationships among rhabdoviruses inferred using the L polymerase gene.

RNA viruses of the family Rhabdoviridae include arthropod-borne agents that infect plants, fish and mammals, and also include a variety of non-vector-borne mammalian viruses. Herein is presented a molecular phylogenetic analysis, the largest undertaken to date, of 56 rhabdoviruses, including 20 viruses which are currently unassigned or assigned as tentative species within the Rhabdoviridae. Degenerate primers targeting a region of block III of the L polymerase gene were defined and used for RT-PCR amplification and sequencing. A maximum-likelihood phylogenetic analysis of a 158-residue L polymerase amino acid sequence produced an evolutionary tree containing the six recognized genera of the Rhabdoviridae and also enabled us to identify four more monophyletic groups of currently unclassified rhabdoviruses that we refer to as the 'Hart Park', 'Almpiwar', 'Le Dantec' and 'Tibrogargan' groups. The broad phylogenetic relationships among these groups and genera also indicate that the evolutionary history of rhabdoviruses was strongly influenced by mode of transmission, host species (plant, fish or mammal) and vector (orthopteran, homopteran or dipteran).

HIV evolution: CTL escape mutation and reversion after transmission.

Within-patient HIV evolution reflects the strong selection pressure driving viral escape from cytotoxic T-lymphocyte (CTL) recognition. Whether this intrapatient accumulation of escape mutations translates into HIV evolution at the population level has not been evaluated. We studied over 300 patients drawn from the B- and C-clade epidemics, focusing on human leukocyte antigen (HLA) alleles HLA-B57 and HLA-B5801, which are associated with long-term HIV control and are therefore likely to exert strong selection pressure on the virus. The CTL response dominating acute infection in HLA-B57/5801-positive subjects drove positive selection of an escape mutation that reverted to wild-type after transmission to HLA-B57/5801-negative individuals. A second escape mutation within the epitope, by contrast, was maintained after transmission. These data show that the process of accumulation of escape mutations within HIV is not inevitable. Complex epitope- and residue-specific selection forces, including CTL-mediated positive selection pressure and virus-mediated purifying selection, operate in tandem to shape HIV evolution at the population level.