Evolution Of Viral Quasispecies Research Articles

Intrapatient Evolutionary Dynamics in an Individual Infected with HIV-1 CRF01_AE Who Experienced Periods of Treatment Failure.

Although previous studies have analyzed cross-level CRF01_AE viral genomic data in populations, less is known about intrapatient viral evolutionary dynamics during antiretroviral therapy (ART) failure. We longitudinally sampled plasma and peripheral blood mononuclear cells (PBMC) at different time points from one human immunodeficiency virus type 1 infected patient. The evolution of viral quasispecies was inferred from viral phylogenies. Before treatment, no drug-resistant mutations were found in this patient's plasma, and all viruses had C-C chemokine receptor type 5 (CCR5) tropism. Two months after treatment, the majority of the virus population in plasma and PBMC were drug resistant and X4-tropic. By 5 months after treatment, the viral load increased significantly, and viruses reversed tropism from X4 to R5 in plasma and PBMC. During treatment failure, the effective population of the pol DNA reservoir in PBMC remained stable, whereas the env DNA reservoir increased. The effective population of the R5 tropism virus increased more rapidly than that of the X4 tropism virus. The ratio of non-synonymous to synonymous substitutions in the env gene of R5 tropism virus (0.43) was lower than X4 tropism (0.52). However, four env positive selection sites were identified in R5 tropism viruses (HXB2: 364, 398, 399, and 400) but none were identified in X4 tropism viruses. Our data demonstrated the different intrapatient evolutionary dynamics patterns of env and pol genes in an individual who experienced periods of ART failure. Our findings also suggest the importance of the R5 tropism virus in the DNA reservoir during ART failure.

Evolution of viral quasispecies during SARS-CoV-2 infection

ObjectivesStudies are needed to better understand the genomic evolution of the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to describe genomic diversity of SARS-CoV-2 by next-generation sequencing (NGS) in a patient with longitudinal follow-up for SARS-CoV-2 infection. MethodsSequential samples collected between January 29th and February 4th, 2020, from a patient infected by SARS-CoV-2 were used to perform amplification of two genome fragments—including genes encoding spike, envelope, membrane and nucleocapsid proteins—and NGS was carried out with Illumina® technology. Phylogenetic analysis was performed with PhyML and viral variant identification with VarScan. ResultsMajority consensus sequences were identical in most of the samples (5/7) and differed in one synonymous mutation from the Wuhan reference sequence. We identified 233 variants; each sample harboured in median 38 different minority variants, and only four were shared by different samples. The frequency of mutation was similar between genes and correlated with the length of the gene (r = 0.93, p = 0.0002). Most of mutations were substitution variations (n = 217, 93.1%) and about 50% had moderate or high impact on gene expression. Viral variants also differed between lower and upper respiratory tract samples collected on the same day, suggesting independent sites of replication of SARS-CoV-2. ConclusionsWe report for the first time minority viral populations representing up to 1% during the course of SARS-CoV-2 infection. Quasispecies were different from one day to the next, as well as between anatomical sites, suggesting that in vivo this new coronavirus appears as a complex and dynamic distributions of variants.

Attenuation of Bluetongue Virus (BTV) in an in ovo Model Is Related to the Changes of Viral Genetic Diversity of Cell-Culture Passaged BTV.

The embryonated chicken egg (ECE) is routinely used for the laboratory isolation and adaptation of Bluetongue virus (BTV) in vitro. However, its utility as an alternate animal model has not been fully explored. In this paper, we evaluated the pathogenesis of BTV in ovo using a pathogenic isolate of South African BTV serotype 3 (BTV-3) derived from the blood of an infected sheep. Endothelio- and neurotropism of BTV-3 were observed by immunohistochemistry of non-structural protein 1 (NS1), NS3, NS3/3a, and viral protein 7 (VP7) antigens. In comparing the pathogenicity of BTV from infectious sheep blood with cell-culture-passaged BTV, including virus propagated through a Culicoides-derived cell line (KC) or ECE, we found virus attenuation in ECE following cell-culture passage. Genomic analysis of the consensus sequences of segments (Seg)-2, -5, -6, -7, -8, -9, and -10 identified several nucleotide and amino-acid mutations among the cell-culture-propagated BTV-3. Deep sequencing analysis revealed changes in BTV-3 genetic diversity in various genome segments, notably a reduction of Seg-7 diversity following passage in cell culture. Using this novel approach to investigate BTV pathogenicity in ovo, our findings support the notion that pathogenic BTV becomes attenuated in cell culture and that this change is associated with virus quasispecies evolution.

Characterization of EIAV env Quasispecies during Long-Term Passage In Vitro: Gradual Loss of Pathogenicity.

As the only widely used live lentiviral vaccine, the equine infectious anima virus (EIAV) attenuated vaccine was developed by in vitro passaging of a virulent strain for 121 generations. In our previous study, we observed that the attenuated vaccine was gradually selected under increased environmental pressure at the population level (termed a quasispecies). To further elucidate the potential correlation between viral quasispecies evolution and pathogenesis, a systematic study was performed by sequencing env using several methods. Some key mutations were identified within Env, and we observed that increased percentages of these mutations were accompanied by an increased passage number and attenuated virulence. Phylogenetic analysis revealed that env mutations related to the loss of virulence might have occurred evolutionarily. Among these mutations, deletion of amino acid 236 in the V4 region of Env resulted in the loss of one N-glycosylation site that was crucial for virulence. Notably, the 236-deleted sequence represented a “vaccine-specific” mutation that was also found in wild EIAVLN40 strains based on single genome amplification (SGA) analysis. Therefore, our results suggest that the EIAV attenuated vaccine may originate from a branch of quasispecies of EIAVLN40. Generally, the presented results may increase our understanding of the attenuation mechanism of the EIAV vaccine and provide more information about the evolution of other lentiviruses.

Correction: Characterization of intra- and inter-host norovirus P2 genetic variability in linked individuals by amplicon sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0201850.].

Applying Unique Molecular Identifiers in Next Generation Sequencing Reveals a Constrained Viral Quasispecies Evolution under Cross-Reactive Antibody Pressure Targeting Long Alpha Helix of Hemagglutinin.

To overcome yearly efforts and costs for the production of seasonal influenza vaccines, new approaches for the induction of broadly protective and long-lasting immune responses have been developed in the past decade. To warrant safety and efficacy of the emerging crossreactive vaccine candidates, it is critical to understand the evolution of influenza viruses in response to these new immune pressures. Here we applied unique molecular identifiers in next generation sequencing to analyze the evolution of influenza quasispecies under in vivo antibody pressure targeting the hemagglutinin (HA) long alpha helix (LAH). Our vaccine targeting LAH of hemagglutinin elicited significant seroconversion and protection against homologous and heterologous influenza virus strains in mice. The vaccine not only significantly reduced lung viral titers, but also induced a well-known bottleneck effect by decreasing virus diversity. In contrast to the classical bottleneck effect, here we showed a significant increase in the frequency of viruses with amino acid sequences identical to that of vaccine targeting LAH domain. No escape mutant emerged after vaccination. These results not only support the potential of a universal influenza vaccine targeting the conserved LAH domains, but also clearly demonstrate that the well-established bottleneck effect on viral quasispecies evolution does not necessarily generate escape mutants.

RNA Virus Evolution via a Quasispecies-Based Model Reveals a Drug Target with a High Barrier to Resistance.

The rapid occurrence of therapy-resistant mutant strains provides a challenge for anti-viral therapy. An ideal drug target would be a highly conserved molecular feature in the viral life cycle, such as the packaging signals in the genomes of RNA viruses that encode an instruction manual for their efficient assembly. The ubiquity of this assembly code in RNA viruses, including major human pathogens, suggests that it confers selective advantages. However, their impact on viral evolution cannot be assessed in current models of viral infection that lack molecular details of virus assembly. We introduce here a quasispecies-based model of a viral infection that incorporates structural and mechanistic knowledge of packaging signal function in assembly to construct a phenotype-fitness map, capturing the impact of this RNA code on assembly yield and efficiency. Details of viral replication and assembly inside an infected host cell are coupled with a population model of a viral infection, allowing the occurrence of therapy resistance to be assessed in response to drugs inhibiting packaging signal recognition. Stochastic simulations of viral quasispecies evolution in chronic HCV infection under drug action and/or immune clearance reveal that drugs targeting all RNA signals in the assembly code collectively have a high barrier to drug resistance, even though each packaging signal in isolation has a lower barrier than conventional drugs. This suggests that drugs targeting the RNA signals in the assembly code could be promising routes for exploitation in anti-viral drug design.

Quantifying perinatal transmission of Hepatitis B viral quasispecies by tag linkage deep sequencing

Despite full immunoprophylaxis, mother-to-child transmission (MTCT) of Hepatitis B Virus still occurs in approximately 2–5% of HBsAg positive mothers. Little is known about the bottleneck of HBV transmission and the evolution of viral quasispecies in the context of MTCT. Here we adopted a newly developed tag linkage deep sequencing method and analyzed the quasispecies of four MTCT pairs that broke through immunoprophylaxis. By assigning unique tags to individual viral sequences, we accurately reconstructed HBV haplotypes in a region of 836 bp, which contains the major immune epitopes and drug resistance mutations. The detection limit of minor viral haplotypes reached 0.1% for individual patient sample. Dominance of “a determinant” polymorphisms were observed in two children, which pre-existed as minor quasispecies in maternal samples. In all four pairs of MTCT samples, we consistently observed a significant overlap of viral haplotypes shared between mother and child. We also demonstrate that the data can be potentially useful to estimate the bottleneck effect during HBV MTCT, which provides information to optimize treatment for reducing the frequency of MTCT.

Hepatitis C virus quasispecies and pseudotype analysis from acute infection to chronicity in HIV-1 co-infected individuals

HIV-1 infected patients who acquire HCV infection have higher rates of chronicity and liver disease progression than patients with HCV mono-infection. Understanding early events in this pathogenic process is important. We applied single genome sequencing of the E1 to NS3 regions and viral pseudotype neutralization assays to explore the consequences of viral quasispecies evolution from pre-seroconversion to chronicity in four co-infected individuals (mean follow up 566 days). We observed that one to three founder viruses were transmitted. Relatively low viral sequence diversity, possibly related to an impaired immune response, due to HIV infection was observed in three patients. However, the fourth patient, after an early purifying selection displayed increasing E2 sequence evolution, possibly related to being on suppressive antiretroviral therapy. Viral pseudotypes generated from HCV variants showed relative resistance to neutralization by autologous plasma but not to plasma collected from later time points, confirming ongoing virus escape from antibody neutralization.

A systematic analysis of a broadly neutralizing antibody AR3C epitopes on Hepatitis C virus E2 envelope glycoprotein and their cross-reactivity.

BackgroundHepatitis C virus (HCV) belongs to Flaviviridae family of viruses. HCV represents a major challenge to public health since its estimated global prevalence is 2.8% of the world's human population. The design and development of HCV vaccine has been hampered by rapid evolution of viral quasispecies resulting in antibody escape variants. HCV envelope glycoprotein E1 and E2 that mediate fusion and entry of the virus into host cells are primary targets of the host immune responses.ResultsStructural characterization of E2 core protein and a broadly neutralizing antibody AR3C together with E1E2 sequence information enabled the analysis of B-cell epitope variability. The E2 binding site by AR3C and its surrounding area were identified from the crystal structure of E2c-AR3C complex. We clustered HCV strains using the concept of "discontinuous motif/peptide" and classified B-cell epitopes based on their similarity.ConclusionsThe assessment of antibody neutralizing coverage provides insights into potential cross-reactivity of the AR3C neutralizing antibody across a large number of HCV variants.

Transmission of HIV-1 during Passage through a Bottleneck: Different Evolution of HIV-1 V3 and NEF Quasispecies

Early in the AIDS era, it was feared that HIV-1 might evolve into untreatable, pandemic killer strains, similar to the 1918 Influenza outbreak or to today’s MRSA bacterial strains. Epidemiologists wanted urgently to know the origins of HIV-1 and the likelihood of evolution of such strains. HIV-1 exists as quasispecies, and evolution of viral quasispecies is most clearly studied where an external pressure creates a bottleneck through which only some variants survive. We studied variants in hypervariable gene regions characteristic of HIV-1 strains: the V3 domain of env gp120 (the principal neutralizing determinant of HIV-1), and the duplication region of the nef gene (associated with pathogenesis in the host), in two mother-infant pairs after transmission of virus from the mother to her infant, and in nine AIDS patients before and after therapy with didanosine (ddI). Evolutionary drift (accumulation of point mutations) and shift (involving insertion/deletion mutations) to new genotypes were observed in both gene regions, but drift was favored in the V3 region, whereas shift was more prominent in nef. Co-selection of genotypes in env and nef appeared to be unrelated, suggesting the presence of mixed genotypes within natural HIV-1 quasispecies.

Deep sequencing and phylogenetic analysis of variants resistant to interferon-based protease inhibitor therapy in chronic hepatitis induced by genotype 1b hepatitis C virus.

Because of recent advances in deep sequencing technology, detailed analysis of hepatitis C virus (HCV) quasispecies and their dynamic changes in response to direct antiviral agents (DAAs) became possible, although the role of quasispecies is not fully understood. In this study, to clarify the evolution of viral quasispecies and the origin of drug-resistant mutations induced by interferon (IFN)-based protease inhibitor therapy, the nonstructural-3 (NS3) region of genotype 1b HCV in 34 chronic hepatitis patients treated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) was subjected to a deep sequencing study coupled with phylogenetic analysis. Twenty-six patients (76.5%) achieved a sustained viral response (SVR), while 8 patients did not (non-SVR; 23.5%). When the complexity of the quasispecies was expressed as the mutation frequency or Shannon entropy value, a significant decrease in the IFNL3 (rs8099917) TT group and a marginal decrease in the SVR group were found soon (12 h) after the introduction of treatment, whereas there was no decrease in the non-SVR group and no significant decrease in mutation frequency in the IFNL3 TG/GG group. In the analysis of viral quasispecies composition in non-SVR patients, major populations greatly changed, accompanied by the appearance of resistance, and the compositions were unlikely to return to the pretreatment composition even after the end of therapy. Clinically TVR-resistant variants were observed in 5 non-SVR patients (5/8, 62.5%), all of which were suspected to have acquired resistance by mutations through phylogenetic analysis. In conclusion, results of the study have important implications for treatment response and outcome in interferon-based protease inhibitor therapy. In the host, hepatitis C virus (HCV) consists of a variety of populations (quasispecies), and it is supposed that dynamic changes in quasispecies are closely related to pathogenesis, although this is poorly understood. In this study, recently developed deep sequencing technology was introduced, and changes in quasispecies associated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) triple therapy and their clinical significance were investigated extensively by phylogenetic tree analysis. Through this study, the associations among treatment response, changes in viral quasispecies complexity in the early stage of treatment, changes in the quasispecies composition, and origin of TVR-resistant variant HCV were elucidated.

Next-generation sequencing in veterinary medicine: how can the massive amount of information arising from high-throughput technologies improve diagnosis, control, and management of infectious diseases?

The development of high-throughput molecular technologies and associated bioinformatics has dramatically changed the capacities of scientists to produce, handle, and analyze large amounts of genomic, transcriptomic, and proteomic data. A clear example of this step-change is represented by the amount of DNA sequence data that can be now produced using next-generation sequencing (NGS) platforms. Similarly, recent improvements in protein and peptide separation efficiencies and highly accurate mass spectrometry have promoted the identification and quantification of proteins in a given sample. These advancements in biotechnology have increasingly been applied to the study of animal infectious diseases and are beginning to revolutionize the way that biological and evolutionary processes can be studied at the molecular level. Studies have demonstrated the value of NGS technologies for molecular characterization, ranging from metagenomic characterization of unknown pathogens or microbial communities to molecular epidemiology and evolution of viral quasispecies. Moreover, high-throughput technologies now allow detailed studies of host-pathogen interactions at the level of their genomes (genomics), transcriptomes (transcriptomics), or proteomes (proteomics). Ultimately, the interaction between pathogen and host biological networks can be questioned by analytically integrating these levels (integrative OMICS and systems biology). The application of high-throughput biotechnology platforms in these fields and their typical low-cost per information content has revolutionized the resolution with which these processes can now be studied.The aim of this chapter is to provide a current and prospective view on the opportunities and challenges associated with the application of massive parallel sequencing technologies to veterinary medicine, with particular focus on applications that have a potential impact on disease control and management.

HIV-1 coreceptor usage in paired plasma RNA and proviral DNA from patients with acute and chronic infection never treated with antiretroviral therapy.

Although an independent evolution of viral quasispecies in different body sites might determine a differential compartmentalization of viral variants, the aim of this paper was to establish whether sequences from peripheral blood mononuclear cells (PBMCs) and plasma provide different or complementary information on HIV tropism in patients with acute or chronic infection. Tropism was predicted using genotypic testing combined with geno2pheno (coreceptor) analysis at a 10% false positive rate in paired RNA and DNA samples from 75 antiretroviral-naïve patients (divided on the basis of avidity index into patients with a recent or long-lasting infection). A high prevalence of R5 HIV strains (97%) was observed in both compartments (plasma and PBMCs) in patients infected recently. By contrast, patients with a long-lasting infection showed a quite different situation in the two compartments, revealing more (46%) X4/DM in PBMCs than patients infected recently (3%) (P = 0.008). As- a knowledge of viral strains in different biological compartments might be crucial to establish a therapeutic protocol, it could be extremely useful to detect not only viral strains in plasma, but also viruses hidden or archived in different cell compartments to better understand disease evolution and treatment efficacy in patients infected with HIV.

HIV-1 tropism dynamics and phylogenetic analysis from longitudinal ultra-deep sequencing data of CCR5- and CXCR4-using variants.

ObjectiveCoreceptor switch from CCR5 to CXCR4 is associated with HIV disease progression. The molecular and evolutionary mechanisms underlying the CCR5 to CXCR4 switch are the focus of intense recent research. We studied the HIV-1 tropism dynamics in relation to coreceptor usage, the nature of quasispecies from ultra deep sequencing (UDPS) data and their phylogenetic relationships.MethodsHere, we characterized C2-V3-C3 sequences of HIV obtained from 19 patients followed up for 54 to 114 months using UDPS, with further genotyping and phylogenetic analysis for coreceptor usage. HIV quasispecies diversity and variability as well as HIV plasma viral load were measured longitudinally and their relationship with the HIV coreceptor usage was analyzed. The longitudinal UDPS data were submitted to phylogenetic analysis and sampling times and coreceptor usage were mapped onto the trees obtained.ResultsAlthough a temporal viral genetic structuring was evident, the persistence of several viral lineages evolving independently along the infection was statistically supported, indicating a complex scenario for the evolution of viral quasispecies. HIV X4-using variants were present in most of our patients, exhibiting a dissimilar inter- and intra-patient predominance as the component of quasispecies even on antiretroviral therapy. The viral populations from some of the patients studied displayed evidences of the evolution of X4 variants through fitness valleys, whereas for other patients the data favored a gradual mode of emergence.ConclusionsCXCR4 usage can emerge independently, in multiple lineages, along the course of HIV infection. The mode of emergence, i.e. gradual or through fitness valleys seems to depend on both virus and patient factors. Furthermore, our analyses suggest that, besides becoming dominant after population-level switches, minor proportions of X4 viruses might exist along the infection, perhaps even at early stages of it. The fate of these minor variants might depend on both viral and host factors.

Phylogenetic inferences on HIV-1 transmission

Phylogenetic inferences on HIV-1 transmission

Impact of immune escape mutations on HIV-1 fitness in the context of the cognate transmitted/founder genome.

BackgroundA modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method.ResultsThe T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region.ConclusionsThese findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.

Increased viral quasispecies evolution in HBeAg seroconverter patients treated with oral nucleoside therapy

Increased viral diversity and evolution appear to be a pre-HBeAg-seroconversion feature in spontaneous and interferon-treated seroconverters. The aim of this study was to examine the viral evolution pattern in nucleoside analogue related HBeAg-seroconversion. This was a case control study consisting of ten lamivudine-treated HBeAg-seroconverters and ten lamivudine-treated non-seroconverters as matching controls. All patients in this study were followed as long as 6 years after starting lamivudine, and cases had three serum time points before HBeAg-seroconversion while controls had three matching serum time points. Nested PCR, cloning and sequencing of HBV precore/core gene were performed. Sequences were aligned with Clustal X 2.0. Phylogenetic trees were constructed and viral diversity, evolutionary rates and patterns of positive selection were evaluated. After starting lamivudine treatment, HBV viral diversity increased in both seroconverters and non-seroconverters, but seroconverters showed a significantly higher level of viral diversity that persisted over time by 2.1-fold (p = 0.009). The increased viral diversity correlated with reduced HBV DNA levels (p <0.001). Lamivudine-treated seroconverters had significant reduced HBV DNA concurrent with increased viral diversity after starting treatment (p = 0.001, compared to non-seroconverters, and resembled those of interferon-seroconverters published previously). There was evidence of positive selection in seroconverters with significantly increased amino acid changes compared to non-seroconverters (p <0.001), occurring in recognized T-cell and B-cell epitopes. Lamivudine-treated HBeAg-seroconverters showed a higher viral diversity than non-seroconverters, and the pattern resembled that of interferon-treated seroconverters. The findings strengthen the evidence that increased viral diversity is strongly associated with HBeAg-seroconversion.

Evolution of hepatitis B genotype C viral quasi-species during hepatitis B e antigen seroconversion

Although the evolution of viral quasi-species may be related to the pathological status of disease, little is known about this phenomenon in hepatitis B, particularly with respect to hepatitis B e antigen (HBeAg) seroconversion. Nucleotide sequences of the hepatitis B virus (HBV) X/precore/core region was analyzed at five time-points in four groups of chronic hepatitis B patients, interferon-induced seroconverters (IS, N = 9), interferon non-responders (IN, N = 9), spontaneous seroconverters (SS, N = 9), and non-seroconverters (SN, N = 9) followed during 60 months on an average. Only patients with genotype C were studied. Analysis of 1800 nucleotide sequences showed that there was no statistical difference between the nucleotide genetic distances of seroconverters (IS and SS; 6.9 × 10⁻³ substitutions (st)/site and 6.7 × 10⁻³ st/site, respectively) and those of non-seroconverters (IN and SN; 5.3 × 10⁻³ st/site and 3.8 × 10⁻³ st/site, respectively) before seroconversion. Compared to non-seroconverters (IN and SN; 5.1 × 10⁻³ st/site and 5.9 × 10⁻³ st/site, respectively), the sequence diversity of seroconverters (IS and SS; 10.9 × 10⁻³ st/site and 9.9 × 10⁻³ st/site, respectively) was significantly higher after seroconversion (p < 0.05), and was higher in seroconverters after seroconversion than before seroconversion (p < 0.05), while this changed very little in non-seroconverters during the observation period. Phylogenetic trees showed greater complexity in secoconverters than non-seroconverters. Parsimony-based estimation of the direction of sequence change between descendants and ancestors before HBeAg seroconversion, revealed higher frequencies of transversional A to T substitution in seroconverters (0.06 vs. 0.02, p = 0.0036) that coincided with the dynamics of quasi-species possessing A1762T mutation. The distinctly greater viral diversity in HBeAg seroconverters after seroconversion could be related to escape mutants resulting from stronger selection pressure.

Accelerated evolution of SIV env within the cerebral compartment in the setting of morphine-dependent rapid disease progression

Human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) have been shown to compartmentalize within various tissues, including the brain. However, the evolution of viral quasispecies in the setting of drug abuse has not been characterized. The goal of this study was to examine viral evolution in the cerebral compartment of morphine-dependent and control macaques to determine its role in rapid disease progression. To address this issue, we analyzed the envelope (env) gene from proviral DNA in our SIV/SHIV macaque model of morphine dependence and AIDS. Analyses of proviral DNA revealed a direct correlation between total genetic changes and survival time. However, the rate of evolution during disease progression was higher in morphine-dependent and rapid-progressor macaques than was the rate of evolution in the control animals. This study provides additional insight into SIV envelope variation in the CNS of morphine-dependent macaques and genotypes that may have evolved in the brain and contributed to disease progression.