Diversity Of Virus Populations Research Articles

Infectious Bronchitis Virus Population Structure Defines Immune Response and Protection.

A commercial Arkansas (Ark) Delmarva Poultry Industry (DPI)-type vaccine and a more homogeneous population of that vaccine obtained previously through adaptation to chicken embryo kidney (CEK) cells (CEK-ArkDPI) were used as a model to further understand the impact of population genetic structure on generation of immune responses and protection. In a first experiment, vaccinated chickens were challenged with an IBV Ark99-type virulent strain (AL/4614/98). Despite extensive sequence similarity between the vaccines, the more heterogeneous commercial ArkDPI was more efficient at reducing viral loads in challenged chickens, while respiratory signs and tracheal lesions were reduced similarly by either vaccine. A distinct subpopulation of the Ark challenge virus showing asparagine at S1 position 56 was consistently negatively selected by immune pressure originating from vaccination with either vaccine. Antibody levels and antibody avidity to Ark-type S1 protein were greater in CEK-ArkDPI-vaccinated chickens compared to chickens vaccinated with the more diverse commercial ArkDPI vaccine. Synchronous replication of a homogeneous virus population likely elicits clonal expansion and affinity maturation of a greater number of responding B cells compared to a diverse virus population continuously changing its proportion of phenotypes during replication. The results of a second experiment showed that during initial vaccine virus replication (24 and 48 hr postvaccination), the virus population showing increased diversity (commercial ArkDPI) achieved higher concentrations of IBV RNA in the trachea compared to the more homogenous virus. mRNA expression of genes associated with innate immune responses in the trachea 48 hr postvaccination generally showed greater upregulation in chickens vaccinated with the heterogeneous commercial ArkDPI vaccine compared to the CEK-adapted virus. The greater upregulation of these genes is likely associated with higher virus replication achieved by the heterogeneous commercial vaccine. Thus, while the adaptive antibody response was favored by the more homogenous structure of the CEK-ArkDPI vaccine population (higher antibody levels and antibody avidity), the innate immune response was favored by the more diverse viral population of the commercial ArkDPI. We confirmed previous results that distinct subpopulations in wild Ark challenge virus become selected by immune pressure originating from vaccination, and we concluded that the population structure of IBV vaccines impacts innate immune response, antibody avidity, and protection.

A Viral Metagenomic Analysis Reveals Rich Viral Abundance and Diversity in Mosquitoes from Pig Farms

Mosquitoes harbor a diversity of viruses and are responsible for several mosquito-borne viral diseases of humans and animals, thereby leading to major public health concerns and significant economic losses across the globe. The viral metagenomics offers a great opportunity for bulk analysis of viral genomes retrieved directly from environmental samples. In this study, we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes, Anopheles and Culex species, collected from different pig farms in the vicinity of Shanghai, China to explore the viral community carried by mosquitoes. The resulting metagenomic data revealed that viral community in the mosquitoes was highly diverse and varied in abundance among pig farms, which comprised of more than 48 viral taxonomic families, specific to vertebrates, invertebrates, plants, fungi, bacteria, and protozoa. The read sequences related to animal viruses included parvoviruses, anelloviruses, circoviruses, flavivirus, rhabdovirus, and seadornaviruses, which might be taken by mosquitoes from viremic animal hosts during blood feeding. Notably, sample G1 contained the most abundant sequence related to Banna virus, which is of public health interest because it causes encephalitis in humans. Furthermore, non-classified viruses also shared considerable virus sequences in all the samples, presumably belonging to unexplored virus category. Overall, the present study provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms, which is a potential source of diseases for mammals including humans and animals. These viral metagenomic data are valuable for assessment of emerging and re-emerging viral epidemics.

Electrostatic interactions at the five-fold axis alter heparin-binding phenotype and drive enterovirus A71 virulence in mice.

Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly understood. In mice, adaptation and virulence determinants have been mapped to mutations at VP2-149, VP1-145 and VP1-244. We investigate how these amino acids alter heparin-binding phenotype and shapes EV-A71 virulence in one-day old mice. We constructed six viruses with varying residues at VP1-98, VP1-145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 149K/98E/145Q, termed KEQ) to generate KKQ, KKE, KEE, IEE and IEQ variants. We demonstrated that the weak heparin-binder IEE was highly lethal in mice. The initially strong heparin-binding IEQ variant acquired an additional mutation VP1-K244E, which confers weak heparin-binding phenotype resulting in elevated viremia and increased virus antigens in mice brain, with subsequent high virulence. IEE and IEQ-244E variants inoculated into mice disseminated efficiently and displayed high viremia. Increasing polymerase fidelity and impairing recombination of IEQ attenuated the virulence, suggesting the importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico docking and deep sequencing approaches, we inferred that virus population diversity is shaped by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, which ultimately results in increased neurovirulence. The dynamic switching between heparin-binding and weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71.

Metagenomic analysis of virome cross-talk between cultivated Solanum lycopersicum and wild Solanum nigrum

Wild plants and weeds growing close to crops constitute a potential reservoir for future epidemies or for the emergence of novel viruses but the frequency and directionality of viral flow between cultivated and wild plants remains poorly documented in many cases. Here, we studied the diversity of viral populations between tomato (Solanum lycopersicum) and neighboring european black nightshade (Solanum nigrum) using high throughput sequencing (HTS) based metagenomics. A large variability in virome richness with only 17.9% shared Operational Taxonomy Units between tomato and nightshade, but this richness could not be linked to a particular host or to local conditions. A detailed population analysis based on assembled contigs for potato virus Y (PVY), broad wilt bean virus 1 and a new ilarvirus tentatively named Solanum nigrum ilarvirus 1 provides information on the circulation of these viruses between these two Solanum species and enriches our knowledge of the tomato virome.

A viral metagenomic analysis reveals rich viral abundance and diversity in mosquitoes from pig farms.

Mosquitoes harbour a diversity of viruses and are responsible for several mosquito-borne viral diseases of humans and animals, thereby leading to major public health concerns, and significant economic losses across the globe. Viral metagenomics offers a great opportunity for bulk analysis of viral genomes retrieved directly from environmental samples. In this study, we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes, Anopheles and Culex species, collected from different pig farms in the vicinity of Shanghai, China, to explore the viral community carried by mosquitoes. The resulting metagenomic data revealed that viral community in the mosquitoes was highly diverse and varied in abundance among pig farms, which comprised of more than 48 viral taxonomic families, specific to vertebrates, invertebrates, plants, fungi, bacteria and protozoa. In addition, a considerable number of viral reads were related to viruses that are not classified by host. The read sequences related to animal viruses included parvoviruses, anelloviruses, circoviruses, flavivirus, rhabdovirus and seadornaviruses, which might be taken up by mosquitoes from viremic animal hosts during blood feeding. Notably, sample G1 contained the most abundant sequence related to Banna virus, which is of public health interest because it causes encephalitis in humans. Furthermore, non-classified viruses also shared considerable virus sequences in all the samples, presumably belonging to unexplored virus category. Overall, the present study provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms, which is a potential source of diseases for mammals including humans and animals. These viral metagenomic data are valuable for assessment of emerging and re-emerging viral epidemics.

A proofreading-impaired herpesvirus generates populations with quasispecies-like structure.

RNA virus populations are composed of highly diverse individuals that form a cloud of related sequences commonly referred to as a 'quasispecies'1-3. This diversity arises as a consequence of low-fidelity genome replication4,5. By contrast, DNA virus populations contain more uniform individuals with similar fitness6. Genome diversity is often correlated with increased fitness in RNA viruses, while DNA viruses are thought to require more faithful genome replication. During DNA replication, erroneously incorporated bases are removed by a 3'-5' exonuclease, a highly conserved enzymatic function of replicative DNA but not RNA polymerases. This proofreading process enhances replication fidelity and ensures the genome integrity of DNA organisms, including large DNA viruses7. Here, we show that a herpesvirus can tolerate impaired exonucleolytic proofreading, resulting in DNA virus populations, which, as in RNA viruses8, are composed of highly diverse genotypes of variable individual fitness. This indicates that herpesvirus mutant diversity may compensate for individual fitness loss. Notably, in vivo infection with diverse virus populations results in a marked increase in virulence compared to genetically homogenous parental virus. While we cannot exclude that the increase in virulence is caused by selection of and/or interactions between individual genotypes, our findings are consistent with quasispecies dynamics. Our results contrast with traditional views of DNA virus replication and evolution, and indicate that a substantial increase in population diversity can lead to higher virulence.

Reverse Genetics of RNA Viruses: ISA-Based Approach to Control Viral Population Diversity without Modifying Virus Phenotype.

Reverse genetic systems are essential for the study of RNA viruses. Infectious clones remain the most widely used systems to manipulate viral genomes. Recently, a new PCR-based method called ISA (infectious subgenomic amplicons) has been developed. This approach has resulted in greater genetic diversity of the viral populations than that observed using infectious clone technology. However, for some studies, generation of clonal viral populations is necessary. In this study, we used the tick-borne encephalitis virus as model to demonstrate that utilization of a very high-fidelity, DNA-dependent DNA polymerase during the PCR step of the ISA procedure gives the possibility to reduce the genetic diversity of viral populations. We also concluded that the fidelity of the polymerase is not the only factor influencing this diversity. Studying the impact of genotype modification on virus phenotype is a crucial step for the development of reverse genetic methods. Here, we also demonstrated that the utilization of different PCR polymerases did not affect the phenotype (replicative fitness in cellulo and virulence in vivo) compared to the initial ISA procedure and the use of an infectious clone. In conclusion, we provide here an approach to control the genetic diversity of RNA viruses without modifying their phenotype.

Bypassing the bottleneck: intentional hepatitis C transmission with organ transplant.

Solid organ transplantation from hepatitis C virus-positive (HCV-positive) deceased donors into HCV-negative recipients is a recent approach aimed to expand the donor organ pool in the setting of severe shortage. Good short-term outcomes have been reported with this approach in combination with direct-acting antivirals. In this issue of the JCI, Zahid and colleagues have characterized early viral kinetics and the genetic landscape of donor-to-recipient HCV transmission using single-genome sequencing. In seven HCV-negative recipients of four HCV-positive donor organs, productive infection with a highly diverse viral population was seen by day three after transplantation. The degree of genetic diversity seen in recipients of HCV-positive organs was unlike the narrow genetic bottleneck typically observed with acute HCV acquisition from intravenous drug use or sexual activity. All recipients achieved HCV cure with treatment. The consequences of acute infection with a genetically diverse HCV population are unknown; however, early clinical experience with this transplantation strategy is promising.

The Utility of Data Transformation for Alignment, De Novo Assembly and Classification of Short Read Virus Sequences.

Advances in DNA sequencing technology are facilitating genomic analyses of unprecedented scope and scale, widening the gap between our abilities to generate and fully exploit biological sequence data. Comparable analytical challenges are encountered in other data-intensive fields involving sequential data, such as signal processing, in which dimensionality reduction (i.e., compression) methods are routinely used to lessen the computational burden of analyses. In this work, we explored the application of dimensionality reduction methods to numerically represent high-throughput sequence data for three important biological applications of virus sequence data: reference-based mapping, short sequence classification and de novo assembly. Leveraging highly compressed sequence transformations to accelerate sequence comparison, our approach yielded comparable accuracy to existing approaches, further demonstrating its suitability for sequences originating from diverse virus populations. We assessed the application of our methodology using both synthetic and real viral pathogen sequences. Our results show that the use of highly compressed sequence approximations can provide accurate results, with analytical performance retained and even enhanced through appropriate dimensionality reduction of sequence data.

The evolution of a super-swarm of foot-and-mouth disease virus in cattle.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that severely impacts global food security and is one of the greatest constraints on international trade of animal products. Extensive viral population diversity and rapid, continuous mutation of circulating FMD viruses (FMDVs) pose significant obstacles to the control and ultimate eradication of this important transboundary pathogen. The current study investigated mechanisms contributing to within-host evolution of FMDV in a natural host species (cattle). Specifically, vaccinated and non-vaccinated cattle were infected with FMDV under controlled, experimental conditions and subsequently sampled for up to 35 days to monitor viral genomic changes as related to phases of disease and experimental cohorts. Consensus-level genomic changes across the entire FMDV coding region were characterized through three previously defined stages of infection: early, transitional, and persistent. The overall conclusion was that viral evolution occurred via a combination of two mechanisms: emergence of full-genomic minority haplotypes from within the inoculum super-swarm, and concurrent continuous point mutations. Phylogenetic analysis indicated that individuals were infected with multiple distinct haplogroups that were pre-existent within the ancestral inoculum used to infect all animals. Multiple shifts of dominant viral haplotype took place during the early and transitional phases of infection, whereas few shifts occurred during persistent infection. Overall, this work suggests that the establishment of the carrier state is not associated with specific viral genomic characteristics. These insights into FMDV population dynamics have important implications for virus sampling methodology and molecular epidemiology.

Estimating HIV-1 Genetic Diversity in Brazil Through Next-Generation Sequencing.

Approximately 36.7 million people were living with the human immunodeficiency virus (HIV) at the end of 2016 according to UNAIDS, representing a global prevalence rate of 0.8%. In Brazil, an HIV prevalence of 0.24% has been estimated, which represents approximately 830,000 individuals living with the virus. As a touristic and commercial hub in Latin America, Brazil harbors an elevated HIV genetic variability, further contributed by the selective pressure exerted by the host immune system and by antiretroviral treatment. Through the progress of the next-generation sequencing (NGS) techniques, it has been possible to expand the study of HIV genetic diversity, evolutionary, and epidemic processes, allowing the generation of HIV complete or near full-length genomes (NFLG) and improving the characterization of intra- and interhost diversity of viral populations. Greater sensitivity in the detection of viral recombinant forms represents one of the major improvements associated with this development. It is possible to identify unique or circulating recombinant forms using the near full-length viral genomes with increasing accuracy. It also permits the characterization of multiple viral infections within individual hosts. Previous Brazilian studies using NGS to analyze HIV diversity were able to identify several distinct unique and circulating recombinant forms and evidenced dual infections. These data unveiled unprecedented high rates of viral recombination and highlighted that novel recombinants are continually arising in the Brazilian epidemic. In the pooled analysis depicted in this report, HIV subtypes have been determined from HIV-positive patients in five states of Brazil with some of the highest HIV prevalence, three in the Southeast (Rio de Janeiro, São Paulo, and Minas Gerais), one in the Northeast (Pernambuco) and one in the South (Rio Grande do Sul). Combined data analysis showed a significant prevalence of recombinant forms (29%; 101/350), and a similar 26% when only NFLGs were considered. Moreover, the analysis was able to evidence the occurrence of multiple infections in some individuals. Our data highlight the great HIV genetic diversity found in Brazil and unveils a more accurate scenario of the HIV evolutionary dynamics in the region.

Chikungunya Virus Fidelity Variants Exhibit Differential Attenuation and Population Diversity in Cell Culture and Adult Mice.

Chikungunya virus (CHIKV) is a reemerging global health threat that produces debilitating arthritis in people. Like other RNA viruses with high mutation rates, CHIKV produces populations of genetically diverse genomes within a host. While several known CHIKV mutations influence disease severity in vertebrates and transmission by mosquitoes, the role of intrahost diversity in chikungunya arthritic disease has not been studied. In this study, high- and low-fidelity CHIKV variants, previously characterized by altered in vitro population mutation frequencies, were used to evaluate how intrahost diversity influences clinical disease, CHIKV replication, and antibody neutralization in immunocompetent adult mice inoculated in the rear footpads. Both high- and low-fidelity mutations were hypothesized to attenuate CHIKV arthritic disease, replication, and neutralizing antibody levels compared to wild-type (WT) CHIKV. Unexpectedly, high-fidelity mutants elicited more severe arthritic disease than the WT despite comparable CHIKV replication, whereas a low-fidelity mutant produced attenuated disease and replication. Serum antibody developed against both high- and low-fidelity CHIKV exhibited reduced neutralization of WT CHIKV. Using next-generation sequencing (NGS), the high-fidelity mutations were demonstrated to be genetically stable but produced more genetically diverse populations than WT CHIKV in mice. This enhanced diversification was subsequently reproduced after serial in vitro passage. The NGS results contrast with previously reported population diversities for fidelity variants, which focused mainly on part of the E1 gene, and highlight the need for direct measurements of mutation rates to clarify CHIKV fidelity phenotypes.IMPORTANCE CHIKV is a reemerging global health threat that elicits debilitating arthritis in humans. There are currently no commercially available CHIKV vaccines. Like other RNA viruses, CHIKV has a high mutation rate and is capable of rapid intrahost diversification during an infection. In other RNA viruses, virus population diversity associates with disease progression; however, potential impacts of intrahost viral diversity on CHIKV arthritic disease have not been studied. Using previously characterized CHIKV fidelity variants, we addressed whether CHIKV population diversity influences the severity of arthritis and host antibody response in an arthritic mouse model. Our findings show that CHIKV populations with greater genetic diversity can cause more severe disease and stimulate antibody responses with reduced neutralization of low-diversity virus populations in vitro The discordant high-fidelity phenotypes in this study highlight the complexity of inferring replication fidelity indirectly from population diversity.

Metagenomic Sequencing of HIV-1 in the Blood and Female Genital Tract Reveals Little Quasispecies Diversity during Acute Infection.

Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is associated with a significant bottleneck in the viral quasispecies population, yet the timing of that bottleneck is poorly understood. We characterized HIV-1 diversity in the blood and female genital tract (FGT) within 2 weeks after detection of infection in three women enrolled in a unique prospective cohort in South Africa. We assembled full-length HIV-1 genomes from matched cervicovaginal lavage (CVL) samples and plasma. Deep sequencing allowed us to identify intrahost single-nucleotide variants (iSNVs) and to characterize within-sample HIV-1 diversity. Our results demonstrated very little HIV-1 diversity in the FGT and plasma by the time viremia was detectable. Within each subject, the consensus HIV-1 sequences were identical in plasma and CVL fluid. No iSNV was present at >6% frequency. One subject had 77 low-frequency iSNVs across both CVL fluid and plasma, another subject had 14 iSNVs in only CVL fluid from the earliest time point, and the third subject had no iSNVs in CVL fluid or plasma. Overall, the small amount of diversity that we detected was greater in the FGT than in plasma and declined over the first 2 weeks after viremia was detectable, compatible with a very early HIV-1 transmission bottleneck. To our knowledge, our study represents the earliest genomic analysis of HIV-1 in the FGT after transmission. Further, the use of metagenomic sequencing allowed us to characterize other organisms in the FGT, including commensal bacteria and sexually transmitted infections, highlighting the utility of the method to sequence both HIV-1 and its metagenomic environment.IMPORTANCE Due to error-prone replication, HIV-1 generates a diverse population of viruses within a chronically infected individual. When HIV-1 is transmitted to a new individual, one or a few viruses establish the new infection, leading to a genetic bottleneck in the virus population. Understanding the timing and nature of this bottleneck may provide insight into HIV-1 vaccine design and other preventative strategies. We examined the HIV-1 population in three women enrolled in a unique prospective cohort in South Africa who were followed closely during the earliest stages of HIV-1 infection. We found very little HIV-1 diversity in the blood and female genital tract during the first 2 weeks after virus was detected in the bloodstream. These results are compatible with a very early HIV-1 population bottleneck, suggesting the need to study the HIV-1 population in the female genital tract before virus is detectable in the bloodstream.

Viral population analysis of the taiga tick, Ixodes persulcatus, by using Batch Learning Self-Organizing Maps and BLAST search.

Ticks transmit a wide range of viral, bacterial, and protozoal pathogens, which are often zoonotic. Several novel tick-borne viral pathogens have been reported during the past few years. The aim of this study was to investigate a diversity of tick viral populations, which may contain as-yet unidentified viruses, using a combination of high throughput pyrosequencing and Batch Learning Self-Organizing Map (BLSOM) program, which enables phylogenetic estimation based on the similarity of oligonucleotide frequencies. DNA/cDNA prepared from virus-enriched fractions obtained from Ixodes persulcatus ticks was pyrosequenced. After de novo assembly, contigs were cataloged by the BLSOM program. In total 41 different viral families and order including those previously associated with human and animal diseases such as Bunyavirales, Flaviviridae, and Reoviridae, were detected. Therefore, our strategy is applicable for viral population analysis of other arthropods of medical and veterinary importance, such as mosquitos and lice. The results lead to the contribution to the prediction of emerging tick-borne viral diseases. A sufficient understanding of tick viral populations will also empower to analyze and understand tick biology including vector competency and interactions with other pathogens.

Viruses control dominant bacteria colonizing the terrestrial deep biosphere after hydraulic fracturing.

The deep terrestrial biosphere harbours a substantial fraction of Earth's biomass and remains understudied compared with other ecosystems. Deep biosphere life primarily consists of bacteria and archaea, yet knowledge of their co-occurring viruses is poor. Here, we temporally catalogued viral diversity from five deep terrestrial subsurface locations (hydraulically fractured wells), examined virus-host interaction dynamics and experimentally assessed metabolites from cell lysis to better understand viral roles in this ecosystem. We uncovered high viral diversity, rivalling that of peatland soil ecosystems, despite low host diversity. Many viral operational taxonomic units were predicted to infect Halanaerobium, the dominant microorganism in these ecosystems. Examination of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) spacers elucidated lineage-specific virus-host dynamics suggesting active in situ viral predation of Halanaerobium. These dynamics indicate repeated viral encounters and changing viral host range across temporally and geographically distinct shale formations. Laboratory experiments showed that prophage-induced Halanaerobium lysis releases intracellular metabolites that can sustain key fermentative metabolisms, supporting the persistence of microorganisms in this ecosystem. Together, these findings suggest that diverse and active viral populations play critical roles in driving strain-level microbial community development and resource turnover within this deep terrestrial subsurface ecosystem.

Whole genome sequencing, variant analysis, phylogenetics, and deep sequencing of Zika virus strains

The recent emergence of Zika virus (ZIKV) has been concentrated in the Caribbean, Southeastern United States, and South- and Central America; resulting in travel-based cases being reported around the globe. As multi-disciplinary collaborations are combatting the ZIKV outbreak, the need to validate the sequence of existing strains has become apparent. Here, we report high-quality sequence data for multiple ZIKV strains made publicly available through the National Institutes of Health- (NIH) funded biorepository, BEI Resources (www.beiresources.org). Next-generation sequencing, 3′ rapid amplification of cDNA ends (RACE), and viral genome annotation pipelines generated GenBank sequence records for 16 BEI Resources strains. Minor variants, consensus mutations, and consensus insertions/deletions were identified within the viral stocks using next-generation sequencing (NGS) and consensus changes were confirmed with Sanger sequencing. Bioinformatics analyses of the sequencing results confirm that the virus stocks available to the scientific research community through BEI Resources adequately represent the viral population diversity of ZIKV.

Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies.

Many broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1) were shown effective in animal models, and are currently evaluated in clinical trials. However, use of these antibodies in humans is hampered by the rapid emergence of resistant viruses. Here we show that soft-randomization can be used to accelerate the parallel identification of viral escape pathways. As a proof of principle, we soft-randomized the epitope regions of VRC01-class bNAbs in replication-competent HIV-1 and selected for resistant variants. After only a few passages, a surprisingly diverse population of antibody-resistant viruses emerged, bearing both novel and previously described escape mutations. We observed that the escape variants resistant to some VRC01-class bNAbs are resistant to most other bNAbs in the same class, and that a subset of variants was completely resistant to every well characterized VRC01-class bNAB, including VRC01, NIH45-46, 3BNC117, VRC07, N6, VRC-CH31, and VRC-PG04. Thus, our data demonstrate that soft randomization is a suitable approach for accelerated detection of viral escape, and highlight the challenges inherent in administering or attempting to elicit VRC01-class antibodies.

Evolution of HIV-1 within untreated individuals and at the population scale in Uganda.

HIV-1 undergoes multiple rounds of error-prone replication between transmission events, resulting in diverse viral populations within and among individuals. In addition, the virus experiences different selective pressures at multiple levels: during the course of infection, at transmission, and among individuals. Disentangling how these evolutionary forces shape the evolution of the virus at the population scale is important for understanding pathogenesis, how drug- and immune-escape variants are likely to spread in populations, and the development of preventive vaccines. To address this, we deep-sequenced two regions of the HIV-1 genome (p24 and gp41) from 34 longitudinally-sampled untreated individuals from Rakai District in Uganda, infected with subtypes A, D, and inter-subtype recombinants. This dataset substantially increases the availability of HIV-1 sequence data that spans multiple years of untreated infection, in particular for different geographical regions and viral subtypes. In line with previous studies, we estimated an approximately five-fold faster rate of evolution at the within-host compared to the population scale for both synonymous and nonsynonymous substitutions, and for all subtypes. We determined the extent to which this mismatch in evolutionary rates can be explained by the evolution of the virus towards population-level consensus, or the transmission of viruses similar to those that establish infection within individuals. Our findings indicate that both processes are likely to be important.

Comparison of chikungunya viruses generated using infectious clone or the Infectious Subgenomic Amplicons (ISA) method in Aedes mosquitoes.

Reverse genetics systems provide the opportunity to manipulate viral genomes and have been widely used to study RNA viruses and to develop new antiviral compounds and vaccine strategies. The recently described method called ISA (Infectious Subgenomic Amplicons) gives the possibility to rescue RNA viruses in days. We demonstrated in cell culture that the use of the ISA method led to a higher genetic diversity of viral populations than that observed using infectious clone technology. However, no replicative fitness difference was observed. In the present study, we used the chikungunya virus as a model to compare in Aedes aegypti and Aedes albopictus mosquitoes the genotypic and phenotypic characteristics of viruses produced either from an infectious clone or using the ISA method. We confirmed the results found in cellulo corroborating that the use of the ISA method was associated with higher genetic diversity of viral populations in mosquitoes but did not affect the vector competence validating its use for in vivo experiments.

Agricultural Freshwater Pond Supports Diverse and Dynamic Bacterial and Viral Populations.

Agricultural ponds have a great potential as a means of capture and storage of water for irrigation. However, pond topography (small size, shallow depth) leaves them susceptible to environmental, agricultural, and anthropogenic exposures that may influence microbial dynamics. Therefore, the aim of this project was to characterize the bacterial and viral communities of pond water in the Mid-Atlantic United States with a focus on the late season (October–December), where decreasing temperature and nutrient levels can affect the composition of microbial communities. Ten liters of freshwater from an agricultural pond were sampled monthly, and filtered sequentially through 1 and 0.2 μm filter membranes. Total DNA was then extracted from each filter, and the bacterial communities were characterized using 16S rRNA gene sequencing. The remaining filtrate was chemically concentrated for viruses, DNA-extracted, and shotgun sequenced. Bacterial community profiling showed significant fluctuations over the sampling period, corresponding to changes in the condition of the pond freshwater (e.g., pH, nutrient load). In addition, there were significant differences in the alpha-diversity and core bacterial operational taxonomic units (OTUs) between water fractions filtered through different pore sizes. The viral fraction was dominated by tailed bacteriophage of the order Caudovirales, largely those of the Siphoviridae family. Moreover, while present, genes involved in virulence/antimicrobial resistance were not enriched within the viral fraction during the study period. Instead, the viral functional profile was dominated by phage associated proteins, as well as those related to nucleotide production. Overall, these data suggest that agricultural pond water harbors a diverse core of bacterial and bacteriophage species whose abundance and composition are influenced by environmental variables characteristic of pond topology and the late season.