Ultradeep HIV-1 Proviral Envelope Sequencing Reveals Complex Population Structure within and between Brain and Splenic Tissues.

Understanding tissue-based HIV-1 proviral population structure is important for improving treatment strategies for individuals with HIV-associated neurological disorders (HAND). Previous analyses have revealed HIV-1 envelope (env) population structure between brain and peripheral tissues as well as Env functional differences, especially in individuals with HAND. Furthermore, population structure has been detected among different anatomical locations in the brain itself, although such patterns are inconsistent across individuals and less strongly associated with the presence/absence of HAND. Here, we utilized the Pacific Biosciences single-molecule real-time (SMRT) high-throughput technology to generate thousands of sequences for each tissue, along with phylogenetic and distance-based analyses, to investigate env sequences from paired brain and spleen samples from eight individuals with/without HAND. To account for the high error rate associated with SMRT sequencing, we used a clustering approach to identify high-quality consensus sequences representative of ≥10 reads ("HQCS10"). In parallel, we characterized variable regions from nonclustered sequences to identify potential functional differences. We found evidence for significant population structure between brain and spleen tissues, as well as among brain tissues and within the same brain tissue, in individuals both with and without HAND. Variable region analysis showed differences in length and charge among brain and nonbrain tissues as well as within the brain, suggesting possible functional differences. Our results demonstrate the complexity of HIV-1 env structure/gene flow among tissues and support the concept that selective pressures in different tissue microenvironments drive viral evolution and adaptation. IMPORTANCE Understanding the evolution of HIV-1 in the brain compared to other tissues is important for improving treatment strategies for individuals with HIV-associated neurological disorders (HAND). We utilized high-throughput sequencing technology to generate thousands of full-length env sequences from paired brain and spleen samples from eight individuals with/without HAND. We found significant viral population structure for participants both with and without HAND, providing robust evidence for the brain as a compartmentalized tissue and potentially a viral reservoir. We also found striking genetic differences between virus populations, even from the same tissue, suggesting the potential for functional differences and the possibility for multiple evolutionary pathways that result in similar tropisms and/or other tissue-adapted characteristics. Our results demonstrate the complexity of viral population structure within the brain and suggest that analysis of peripheral blood samples alone may not be fully informative with respect to improving strategies to treat or eradicate HIV-1.

Molecular characterization of Equine Infectious Anemia Viruses using targeted sequence enrichment and next generation sequencing.

Equine infectious anemia virus (EIAV) is responsible of acute disease episodes characterized by fever, anemia, thrombocytopenia and anorexia in equids. The high mutation rate in EIAV genome limited the number of full genome sequences availability. In the present study, we used the SureSelect target enrichment system with Illumina Next Generation Sequencing to characterize the proviral DNA of Equine Infectious Anemia Virus (EIAV) from asymptomatic horses. This approach allows a direct sequencing of the EIAV whole genome without cloning or amplification steps and we could obtain for the first time the complete genomic DNA sequences of French EIAV strains. We analyzed their phylogenetic relationship and genetic variability by comparison with 17 whole EIAV genome sequences from different parts of the world. The results obtained provide new insights into the molecular detection of EIAV and genetic diversity of European viral strains.

Origin and recent expansion of an endogenous gammaretroviral lineage in domestic and wild canids.

BACKGROUND: Vertebrate genomes contain a record of retroviruses that invaded the germlines of ancestral hosts and are passed to offspring as endogenous retroviruses (ERVs). ERVs can impact host function since they contain the necessary sequences for expression within the host. Dogs are an important system for the study of disease and evolution, yet no substantiated reports of infectious retroviruses in dogs exist. Here, we utilized Illumina whole genome sequence data to assess the origin and evolution of a recently active gammaretroviral lineage in domestic and wild canids. RESULTS: We identified numerous recently integrated loci of a canid-specific ERV-Fc sublineage within Canis, including 58 insertions that were absent from the reference assembly. Insertions were found throughout the dog genome including within and near gene models. By comparison of orthologous occupied sites, we characterized element prevalence across 332 genomes including all nine extant canid species, revealing evolutionary patterns of ERV-Fc segregation among species as well as subpopulations. CONCLUSIONS: Sequence analysis revealed common disruptive mutations, suggesting a predominant form of ERV-Fc spread by trans complementation of defective proviruses. ERV-Fc activity included multiple circulating variants that infected canid ancestors from the last 20 million to within 1.6 million years, with recent bursts of germline invasion in the sublineage leading to wolves and dogs.

Longitudinal HIV sequencing reveals reservoir expression leading to decay which is obscured by clonal expansion.

After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.

Relationship between intact HIV-1 proviruses in circulating CD4+ T cells and rebound viruses emerging during treatment interruption.

Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4+ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti-HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound.

Promoter expression of HERV-K (HML-2) provirus-derived sequences is related to LTR sequence variation and polymorphic transcription factor binding sites.

BACKGROUND: Increased transcription of the human endogenous retrovirus group HERV-K (HML-2) is often seen during disease. Although the mechanism of its tissue-specific activation is unclear, research shows that LTR CpG hypomethylation alone is not sufficient to induce its promoter activity and that the transcriptional milieu of a malignant cell contributes, at least partly, to differential HML-2 expression. RESULTS: We analyzed the relationship between LTR sequence variation and promoter expression patterns in human breast cancer cell lines, finding them to be positively correlated. In particular, two proviruses (3q12.3 and 11p15.4) displayed increased activity in almost all tumorigenic cell lines sampled. Using a transcription factor binding site prediction algorithm, we identified two unique binding sites in each 5' LTR that appeared to be associated with inducing promoter activity during neoplasia. Genomic analysis of the homologous proviruses in several non-human primates indicated post-integration genetic drift in two transcription factor binding sites, away from the ancestral sequence and towards the active form. Based on the sequences of 2504 individuals from the 1000 Genomes Project, the active form of the 11p15.4 site was found to be polymorphic within the human population, with an allele frequency of 51%, whereas the activating mutation in the 3q12.3 provirus was fixed in humans but not present in the orthologous provirus in chimpanzees or gorillas. CONCLUSIONS: These data suggest that stage-specific transcription factors at least partly contribute to LTR promoter activity during transformation and that, in some cases, transcription factor binding site polymorphisms may be responsible for the differential HML-2 expression often seen between individuals.

Application of the SureSelect target enrichment system for next-generation sequencing to obtain the complete genome sequence of bovine leukemia virus.

In this study, the SureSelect target enrichment system for Illumina Multiplexed Sequencing was applied to proviral DNA sequencing of bovine leukemia virus (BLV). The complete genomic DNA sequences of four Vietnamese BLV strains were successfully obtained with high read depth values and a genome coverage of 100% across all sequenced samples, in less than one week. This study provides the first complete Vietnamese BLV genome sequences. Their genetic variability and phylogenetic relationship were also analyzed and compared with those of 28 whole BLV genome sequences from different parts of the world. The results obtained provided new insights into the genetic diversity of the BLV tax gene, and further enabled us to identify nucleotide mutations in the gene that might not have been detected with the commercial detection kit that is currently available.

HIV-1 latent reservoir size and diversity are stable following brief treatment interruption.

BACKGROUND: The effect of a brief analytical treatment interruption (ATI) on the HIV-1 latent reservoir of individuals who initiate antiretroviral therapy (ART) during chronic infection is unknown. METHODS: We evaluated the impact of transient viremia on the latent reservoir in participants who underwent an ATI and at least 6 months of subsequent viral suppression in a clinical trial testing the effect of passive infusion of the broadly neutralizing Ab VRC01 during ATI. RESULTS: Measures of total HIV-1 DNA, cell-associated RNA, and infectious units per million cells (IUPM) (measured by quantitative viral outgrowth assay [QVOA]) were not statistically different before or after ATI. Phylogenetic analyses of HIV-1 env sequences from QVOA and proviral DNA demonstrated little change in the composition of the virus populations comprising the pre- and post-ATI reservoir. Expanded clones were common in both QVOA and proviral DNA sequences. The frequency of clonal populations differed significantly between QVOA viruses, proviral DNA sequences, and the viruses that reactivated in vivo. CONCLUSIONS: The results indicate that transient viremia from ATI does not substantially alter measures of the latent reservoir, that clonal expansion is prevalent within the latent reservoir, and that characterization of latent viruses that can reactivate in vivo remains challenging. TRIAL REGISTRATION: ClinicalTrials.gov NCT02463227FUNDING. Funding was provided by the NIH.

Viral Discovery in the Invasive Australian Cane Toad (Rhinella marina) Using Metatranscriptomic and Genomic Approaches.

Cane toads are a notorious invasive species, inhabiting over 1.2 million km2 of Australia and threatening native biodiversity. The release of pathogenic cane toad viruses is one possible biocontrol strategy yet is currently hindered by the poorly described cane toad virome. Metatranscriptomic analysis of 16 cane toad livers revealed the presence of a novel and full-length picornavirus, Rhimavirus A (RhiV-A), a member of a reptile- and amphibian-specific cluster of the Picornaviridae basal to the Kobuvirus-like group. In the combined liver transcriptome, we also identified a complete genome sequence of a distinct epsilonretrovirus, Rhinella marina endogenous retrovirus (RMERV). The recently sequenced cane toad genome contains 8 complete RMERV proviruses as well as 21 additional truncated insertions. The oldest full-length RMERV provirus was estimated to have inserted 1.9 million years ago (MYA). To screen for these viral sequences in additional toads, we analyzed publicly available transcriptomes from six diverse Australian locations. RhiV-A transcripts were identified in toads sampled from three locations across 1,000 km of Australia, stretching to the current Western Australia (WA) invasion front, while RMERV transcripts were observed at all six sites. Finally, we scanned the cane toad genome for nonretroviral endogenous viral elements, finding three sequences related to small DNA viruses in the family Circoviridae This shows ancestral circoviral infection with subsequent genomic integration. The identification of these current and past viral infections enriches our knowledge of the cane toad virome, an understanding of which will facilitate future work on infection and disease in this important invasive species.IMPORTANCE Cane toads are poisonous amphibians that were introduced to Australia in 1935 for insect control. Since then, their population has increased dramatically, and they now threaten many native Australian species. One potential method to control the population is to release a cane toad virus with high mortality rates, yet few cane toad viruses have been characterized. This study samples cane toads from different Australian locations and uses an RNA sequencing and computational approach to find new viruses. We report novel complete picornavirus and retrovirus sequences that were genetically similar to viruses infecting frogs, reptiles, and fish. Using data generated in other studies, we show that these viral sequences are present in cane toads from distinct Australian locations. Three sequences related to circoviruses were also found in the toad genome. The identification of new viral sequences will aid future studies that investigate their prevalence and potential as agents for biocontrol.

Characterization of HIV-1 Near Full-Length Proviral Genome Quasispecies from Patients with Undetectable Viral Load Undergoing First-Line HAART Therapy.

Increased access to highly active antiretroviral therapy (HAART) by human immunodeficiency virus postive (HIV⁺) individuals has become a reality worldwide. In Brazil, HAART currently reaches over half of HIV-infected subjects. In the context of a remarkable HIV-1 genetic variability, highly related variants, called quasispecies, are generated. HIV quasispecies generated during infection can influence virus persistence and pathogenicity, representing a challenge to treatment. However, the clinical relevance of minority quasispecies is still uncertain. In this study, we have determined the archived proviral sequences, viral subtype and drug resistance mutations from a cohort of HIV⁺ patients with undetectable viral load undergoing HAART as first-line therapy using next-generation sequencing for near full-length virus genome (NFLG) assembly. HIV-1 consensus sequences representing NFLG were obtained for eleven patients, while for another twelve varying genome coverage rates were obtained. Phylogenetic analysis showed the predominance of subtype B (83%; 19/23). Considering the minority variants, 18 patients carried archived virus harboring at least one mutation conferring antiretroviral resistance; for six patients, the mutations correlated with the current ARVs used. These data highlight the importance of monitoring HIV minority drug resistant variants and their clinical impact, to guide future regimen switches and improve HIV treatment success.

HIV Type 1 Integrase Natural Polymorphisms in Viral Variants Circulating in FSU Countries.

BACKGROUND: Natural variability of integrase (IN) across HIV-1 variants may influence the emergence of resistant viruses. The most apparent explanation of this fact is the IN polymorphism and the associated differences in codon usage, which in turn, influence the probability and the terms of DRMs acquisition. Possible mechanisms by which polymorphisms affect DRMs emergence remain disputed and should still be clarified because these substitutions may be associated with a reduced activity of some INSTIs and may impact on ART regimen choice depending of HIV-1 subtype. OBJECTIVE: The aim of this work was to assess the prevalence of naturally occurring polymorphisms within the HIV-1 integrase gene, which might influence the susceptibility to INSTIs, among the patients from Russia and former USSR countries, according to HIV-1 subtypes. METHOD: A study involved 506 HIV-1 IN sequences of INSTI-naive patients from Russia, Ukraine, Armenia, Kyrgyzstan, Kazakhstan, Uzbekistan, Belarus, and Georgia. Among them, 194 sequences were newly obtained in this study and 312 were downloaded from Los-Alamos database. The proviral DNA was sequenced using an in-house PCR protocol designed on the basis of a well-conserved integrase region in order to detect all HIV-1 variants. RESULTS: The phylogenetic analyses based on IN population sequencing found subtype A6 being the most prevalent (259) (51.2%) in the collection studied, followed by subtype G (36) (7.1%), AGrecombinants (148) (29.3%), subtype B (50) (9.9%), and CRF03_AB (5) (1.0%). The major INSTI resistance-associated mutations (DRMs) were found only in two A6 samples. The prevalence of minor/ accessory substitutions depended on HIV-1 variants, while the most notable findings were L74I in subtype A6 (93.1%) and E157Q in subtype B (44.0%). Most of minor DRMs and polymorphic substitutions were concentrated in the central catalytic domain of the IN molecule. Both the DDE triad and HHCC zinc binding motifs were fully conserved. CONCLUSION: The results of the study suggest a very low risk of initiating INSTI-based therapy in patients with pre-existing polymorphic mutations in Russia and FSU countries. The therapy response in dominating HIV-1 genetic variants might be further studied in the future for a better understanding of their effect on INSTI susceptibility. The INSTI TDR is absent for the moment, but the risk may increase with expanded use of INSTIs, indicating the need for ongoing surveillance.

Molecular Characteristics of the Envelope of Vertically Transmitted HIV-1 Strains from Infants with HIV Infection.

Mother-to-child transmission (MTCT) of HIV offers a good opportunity to study the dynamics of early viral evolution in the host environment to which the virus has partially adapted. Such studies would throw light on the unique features of the infecting viruses, which will subsequently help to design preventive or therapeutic measures against the newly infecting and evolving strains of HIV. Therefore, we undertook a study to determine the genetic divergence of proviral envelope sequences from the HIV-infected infants (<2 years). Detailed analysis revealed unique features of potential N-linked glycosylation sites (PNGS) and their frequency of occurrence that built on the difference in length of the V1V2 region of the envelope sequences. Surprisingly, frequency of PNGS in the V5 region was found to revert rapidly, in about 75% of the sequences, which could surmise a fitness disadvantage in the variant forms. Further, a stable net charge was observed in the V2 and V3 regions prompting us to speculate on the established interaction of the transmitted variant with the integrin α4ß7 receptor and R5 co-receptor, respectively. In brief, our observations suggest that differences in the length of the variable regions and variation in the frequency of PNGS in the envelope of the viruses obtained from very recently infected individuals in our population could be important characteristics of the unique quasispecies that is responsible for the spread of HIV in the early stages of infection in MTCT.

Defective HIV-1 Proviruses Are Expressed and Can Be Recognized by Cytotoxic T Lymphocytes, which Shape the Proviral Landscape.

Despite antiretroviral therapy, HIV-1 persists in memory CD4+ T cells, creating a barrier to cure. The majority of HIV-1 proviruses are defective and considered clinically irrelevant. Using cells from HIV-1-infected individuals and reconstructed patient-derived defective proviruses, we show that defective proviruses can be transcribed into RNAs that are spliced and translated. Proviruses with defective major splice donors (MSDs) can activate novel splice sites to produce HIV-1 transcripts, and cells with these proviruses can be recognized by HIV-1-specific cytotoxic T lymphocytes (CTLs). Further, cells with proviruses containing lethal mutations upstream of CTL epitopes can also be recognized by CTLs, potentially through aberrant translation. Thus, CTLs may change the landscape of HIV-1 proviruses by preferentially targeting cells with specific types of defective proviruses. Additionally, the expression of defective proviruses will need to be considered in the measurement of HIV-1 latency reversal.

Romidepsin-induced HIV-1 viremia during effective antiretroviral therapy contains identical viral sequences with few deleterious mutations.

OBJECTIVE: To investigate the origin of the HIV-1 viremia induced by the latency-reversing agent romidepsin. DESIGN: Six individuals on suppressive antiretroviral therapy received romidepsin administered intravenously once weekly for 3 consecutive weeks. CD4 T cells were obtained at baseline, following the second and third romidepsin infusion, and 10 weeks after the final romidepsin treatment. Plasma samples were collected 24 and 72 h after romidepsin infusions. METHODS: Single-genome sequencing of the env and p24-RT region was used to genetically characterize the virus from proviral DNA, the transcribed cell-associated RNA and the plasma RNA pool. RESULTS: In three of six participants with available plasma samples we identified plasma HIV-1 RNA sequences that were identical to DNA and/or cell-associated RNA sequences from peripheral blood CD4 T cells. In two participants, plasma RNA sequences contained expansions of identical sequences, corresponding to 62 and 100% of the total sequences, respectively. Plasma HIV-1 RNA had very low amounts of defective viruses compared to cell-associated RNA (odds ratio 20.85, P < 0.001) and to DNA (odds ratio 7.07, P = 0.011) during romidepsin therapy. CONCLUSIONS: Romidepsin induced transcription from proviruses in peripheral blood cells, which contributed to viremia in patients on suppressive therapy. The intermingling of these cell-associated HIV-1 RNA with DNA sequences indicates transcription from a diverse range of proviruses, but the expansions of identical viral plasma sequences with few defects indicate that the romidepsin-induced viremia arises from intact proviruses with highly similar or identical genetic backgrounds.

Origin of Rebound Plasma HIV Includes Cells with Identical Proviruses That Are Transcriptionally Active before Stopping of Antiretroviral Therapy.

UNLABELLED: Understanding the origin of HIV variants during viral rebound may provide insight into the composition of the HIV reservoir and has implications for the design of curative interventions. HIV single-genome sequences were obtained from 10 AIDS Clinical Trials Group participants who underwent analytic antiretroviral therapy (ART) interruption (ATI). Rebounding variants were compared with those in pre-ART plasma in all 10 participants and with on-ART peripheral blood mononuclear cell (PBMC)-associated DNA and RNA (CA-RNA) in 7/10 participants. The highest viral diversities were found in the DNA and CA-RNA populations. In 3 of 7 participants, we detected multiple, identical DNA and CA-RNA sequences during suppression on ART that exactly matched plasma HIV sequences. Hypermutated DNA and CA-RNA were detected in four participants, contributing to diversities in these compartments that were higher than in the pre-ART and post-ATI plasma. Shifts in the viral rebound populations could be detected in some participants over the 2- to 3-month observation period. These findings suggest that a source of initial rebound viremia could be populations of infected cells that clonally expanded prior to and/or during ART, some of which were already expressing HIV RNA before treatment was interrupted. These clonally expanding populations of HIV-infected cells may represent an important target for strategies aimed at achieving reservoir reduction and sustained virologic remission. IMPORTANCE: Antiretroviral therapy alone cannot eradicate the HIV reservoir, and viral rebound is generally rapid after treatment interruption. It has been suggested that clonal expansion of HIV-infected cells is an important mechanism of HIV reservoir persistence, but the contribution of these clonally proliferating cells to the rebounding virus is unknown. We report a study of AIDS Clinical Trials Group participants who underwent treatment interruption and compared rebounding plasma virus with that found within cells prior to treatment interruption. We found several incidences in which plasma HIV variants exactly matched that of multiple proviral DNA copies from infected blood cells sampled before treatment interruption. In addition, we found that these cells were not dormant but were generating unspliced RNA transcripts before treatment was interrupted. Identification of the HIV reservoir and determining its mechanisms for persistence may aid in the development of strategies toward a cure for HIV. (This study was presented in part at the Conference on Retroviruses and Opportunistic Infections, Seattle, WA, February 23 to 26 2015.).

HTLV-1 subgroups associated with the risk of HAM/TSP are related to viral and host gene expression in peripheral blood mononuclear cells, independent of the transactivation functions of the viral factors.

Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, the risk of developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) across lifetime differs between ethnic groups. There is an association between HTLV-1 tax gene subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. In this study, we investigated the full-length proviral genome sequences of various HTLV-1-infected cell lines and patient samples. The functional differences in the viral transcriptional regulators Tax and HTLV-1 bZIP factor (HBZ) between each subgroup and the relationships between subgroups and the clinical and laboratory characteristics of HAM/TSP patients were evaluated. The results of these analyses indicated the following: (1) distinct nucleotide substitutions corresponding to each subgroup were associated with nucleotide substitutions in viral structural, regulatory, and accessory genes; (2) the HBZ messenger RNA (mRNA) expression in HTLV-1-infected cells was significantly higher in HAM/TSP patients with subgroup-B than in those with subgroup-A; (3) a positive correlation was observed between the expression of HBZ mRNA and its target Foxp3 mRNA in HAM/TSP patients with subgroup-B, but not in patients with subgroup-A; (4) no clear differences were noted in clinical and laboratory characteristics between HAM/TSP patients with subgroup-A and subgroup-B; and (5) no functional differences were observed in Tax and HBZ between each subgroup based on reporter gene assays. Our results indicate that although different HTLV-1 subgroups are characterized by different patterns of viral and host gene expression in HAM/TSP patients via independent mechanisms of direct transcriptional regulation, these differences do not significantly affect the clinical and laboratory characteristics of HAM/TSP patients.

Multiple invasions of an infectious retrovirus in cat genomes.

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of host germ-line cells. While most ERVs are defective, some are active and express viral proteins. The RD-114 virus is a replication-competent feline ERV, and several feline cell lines produce infectious RD-114 viral particles. All domestic cats are considered to have an ERV locus encoding a replication-competent RD-114 virus in their genomes; however, the locus has not been identified. In this study, we investigated RD-114 virus-related proviral loci in genomes of domestic cats, and found that none were capable of producing infectious viruses. We also found that all domestic cats have an RD-114 virus-related sequence on chromosome C2, termed RDRS C2a, but populations of the other RDRSs are different depending on the regions where cats live or breed. Our results indicate that RDRS C2a, the oldest RD-114-related provirus, entered the host genome before an ancestor of domestic cats started diverging and the other new RDRSs might have integrated into migrating cats in Europe. We also show that infectious RD-114 virus can be resurrected by the recombination between two non-infectious RDRSs. From these data, we conclude that cats do not harbor infectious RD-114 viral loci in their genomes and RD-114-related viruses invaded cat genomes multiple times.

Molecular characteristics of the complete genome of a J-subgroup avian leukosis virus strain isolated from Eurasian teal in China.

The J-subgroup avian leukosis virus (ALV-J) strain WB11098J was isolated from a wild Eurasian teal, and its proviral genomic sequences were determined. The complete proviral sequence of WB11098J was 7868 nt long. WB11098J was 95.3.9 % identical to the prototype strain HPRS-103, 94.2 % identical to the American strain ADOL-7501, 94.5-94.7 % identical to Chinese broiler isolates, 94.8-97.5 % identical to layer chicken isolates, and 94.4-95.0 % identical to Chinese local chicken isolates at the nucleotide level. Phylogenetic analysis showed that the WB11098J isolate shared the greatest homology with the layer strain SD09DP03 and was included in the same cluster. Interestingly, two 19-bp insertions in the U3 regions of the 5'LTR and 5'UTR that were most likely derived from other retroviruses were found in the WB11098J isolate. These insertions separately introduced one E2BP-binding site in the U3 region of the 5'LTR and a RNA polymerase II transcription factor IIB and core promoter motif of ten elements in the 5'UTR. A 5-bp deletion was identified in the U3 region of the 5'LTR. No nucleotides were deleted in the rTM or DR-1 regions in the 3'UTR. A 1-bp deletion was detected in the E element and introduced a specific and distinct binding site for c-Ets-1. Our study is the first to report the molecular characteristics of the complete genome of an ALV-J that was isolated from a wild bird and will provide necessary information for further understanding of the evolution of ALV-J.

Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group.

PURPOSE: We have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification. RESULTS: The overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/10(6) PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /10(6) PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification. CONCLUSIONS: Our results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.

Genome-wide characterization of endogenous retroviruses in the bat Myotis lucifugus reveals recent and diverse infections.

Bats are increasingly recognized as reservoir species for a variety of zoonotic viruses that pose severe threats to human health. While many RNA viruses have been identified in bats, little is known about bat retroviruses. Endogenous retroviruses (ERVs) represent genomic fossils of past retroviral infections and, thus, can inform us on the diversity and history of retroviruses that have infected a species lineage. Here, we took advantage of the availability of a high-quality genome assembly for the little brown bat, Myotis lucifugus, to systematically identify and analyze ERVs in this species. We mined an initial set of 362 potentially complete proviruses from the three main classes of ERVs, which were further resolved into 13 major families and 86 subfamilies by phylogenetic analysis. Consensus or representative sequences for each of the 86 subfamilies were then merged to the Repbase collection of known ERV/long terminal repeat (LTR) elements to annotate the retroviral complement of the bat genome. The results show that nearly 5% of the genome assembly is occupied by ERV-derived sequences, a quantity comparable to findings for other eutherian mammals. About one-fourth of these sequences belong to subfamilies newly identified in this study. Using two independent methods, intraelement LTR divergence and analysis of orthologous loci in two other bat species, we found that the vast majority of the potentially complete proviruses identified in M. lucifugus were integrated in the last ~25 million years. All three major ERV classes include recently integrated proviruses, suggesting that a wide diversity of retroviruses is still circulating in Myotis bats.