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Background & Aims HBV genotype G (HBV/G) is mainly found in co-infections with other HBV genotypes and was identified as an independent risk factor for liver fibrosis. This study aimed to analyse the prevalence of HBV/G co-infections in healthy European HBV carriers and to characterize the crosstalk of HBV/G with other genotypes. Methods A total of 560 European HBV carriers were tested via HBV/G-specific PCR for HBV/G co-infections. Quasispecies distribution was analysed via deep sequencing, and the clinical phenotype was characterized regarding qHBsAg-/HBV-DNA levels and frequent mutations. Replicative capacity and expression of HBsAg/core was studied in hepatoma cells co-expressing HBV/G with either HBV/A, HBV/D or HBV/E using bicistronic vectors. Results Although no HBV/G co-infection was found by routine genotyping PCR, HBV/G was detected by specific PCR in 4%-8% of patients infected with either HBV/A or HBV/E but only infrequently in other genotypes. In contrast to HBV/E, HBV/G was found as the quasispecies major variant in co-infections with HBV/A. No differences in the clinical phenotype were observed for HBV/G co-infections. In vitro RNA and DNA levels were comparable among all genotypes, but expression and release of HBsAg was reduced in co-expression of HBV/G with HBV/E. In co-expression with HBV/A and HBV/E expression of HBV/G-specific core was enhanced while core expression from the corresponding genotype was markedly diminished. Conclusions HBV/G co-infections are common in European inactive carriers with HBV/A and HBV/E infection, but sufficient detection depends strongly on the assay. HBV/G regulated core expression might play a critical role for survival of HBV/G in co-infections.

Chronic HBV infection results in various clinical manifestations due to different levels of immune response. In recent years, hepatitis B treatment has improved by long-term administration of nucleos(t)ide analogues (NUCs) and peg-interferon. Nucleic acid polymers (NAPs; REP 2139-Ca and REP 2139-Mg) are new antiviral drugs that block the assembly of subviral particles, thus preventing the release of HBsAg and allowing its clearance and restoration of functional control of infection when combined with various immunotherapies. In the REP 102 study (NCT02646189), 9 of 12 patients showed substantial reduction of HBsAg and seroconversion to anti-HBs in response to REP 2139-Ca, whereas 3 of 12 patients did not show responses (>1 log reduction of HBsAg and HBV DNA from baseline). We characterized the dynamic changes of HBV quasispecies (QS) within the major hydrophilic region (MHR) of the 'pre-S/S' open reading frame including the 'a' determinant in responders and nonresponders of the REP 102 study and four untreated matched controls. HBV QS complexity at baseline varied slightly between responders and nonresponders (P = .28). However, these responders showed significant decline in viral complexity (P = .001) as REP 2139-Ca therapy progressed but no significant change in complexity was observed among the nonresponders (P = .99). The MHR mutations were more frequently observed in responders than in nonresponders and matched controls. No mutations were observed in 'a' determinant of major QS population which may interfere with the detection of HBsAg by diagnostic assays. No specific mutations were found within the MHR which could explain patients' poor HBsAg response during REP 2139-Ca therapy.

OBJECTIVES This study aimed to describe the differences between next-generation sequencing (NGS) and cloning-based sequencing (CBS) in HBX quasispecies research and primitively investigate the relationship between the dominant HBX quasispecies and hepatocellular carcinoma (HCC). METHODS A total of 12 serum samples were collected. Serum hepatitis B virus (HBV) DNA was extracted, and the HBV X-region (HBX) was amplified by nested polymerase chain reaction (PCR). The PCR products were simultaneously tested with NGS and CBS to detect quasispecies of the HBX. RESULTS A total of 9348 eligible quasispecies sequences were obtained by NGS, which were much larger than the 98 of that by CBS. By the phylogenetic tree, the dominant quasispecies sequence of each sample could be found, although they had several nucleotides differences between the dominant quasispecies sequences found by CBS and NGS. By comparing the quasispecies heterogeneity, it was found that the quasispecies complexity value of HBV X-region obtained by NGS was higher than CBS (P

Nucleoside and nucleotide analogues (NUCs) targeting hepatitis B virus are capable of selecting resistant viruses upon long-term administration as monotherapies. The prevalence of resistance-associated substitutions (RASs) and fitness-associated substitutions at baseline of NUC therapy and their impact on treatment responses remain unknown. A total of 232 treatment-naïve patients chronically infected with hepatitis B virus (HBV) consecutively referred for the first time to one of French reference centres were included. The nearly full-length HBV reverse transcriptase was sequenced by means of deep sequencing, and the sequences were analysed. RASs were detected in 25% of treatment-naïve patients, generally representing low proportions of the viral quasispecies. All amino acid positions known to be associated with HBV resistance to currently approved NUCs or with increased fitness of resistant variants were affected, except position 80. RASs at positions involved in lamivudine, telbivudine and adefovir resistance were the most frequently detected. All patients with RASs detectable by next-generation sequencing at baseline who were treatment-eligible and treated with currently recommended drugs achieved a virological response. The presence of pre-existing HBV RASs has no impact on the outcome of therapy if potent drugs with a high barrier to resistance are used.

The emergence of resistance-associated substitutions (RASs) can compromise the high efficacy of direct-acting antivirals (DAAs). Little is known about RASs selection at very early time points during DAA treatment. Therefore, we analyzed the potential emergence of RASs immediately after therapy initiation. Samples of 71 patients treated with different DAAs were collected at baseline, during therapy (hours 4 and 8; days 1-7; weeks 2-4) or until target not detected. HCV-RNA levels were determined by qPCR, and RASs were detected by deep sequencing. Sixty-three (89%) patients achieved a sustained virological response (SVR), 7 (10%) relapsed, and 1 (1%) experienced a breakthrough. Almost all non-SVR (7/8, 88%) showed RASs either at baseline or relapse. High-frequency RASs detected at baseline (Y93H and L159F+C316N) remained detectable at early time points during therapy and reappeared as most prevalent substitutions at relapse. Conversely, emergent RASs at relapse (Q80R, D168E/V, R155K and L31V) were not observed during the first hours-days, before HCV-RNA became undetectable. HCV-RNA decay and genetic evolution of the quasispecies followed a similar pattern during the first hours of therapy in SVR and non-SVR patients. In conclusion, the absence of early RASs selection and the similar dynamics of HCV kinetics and quasispecies in SVR and non-SVR patients after therapy initiation suggest that RASs selection may occur at later stages in the remaining reservoir, where viral populations persist hidden at very low replication levels. Nevertheless, we cannot completely exclude very early selection, when RASs are present below the sensitivity limit of deep sequencing.

Recent studies suggest that withdrawal of hepatitis B immune globulin (HBIG) and nucleos(t)ide analogues (NA) prophylaxis may be considered in HBV surface antigen (HBsAg)-negative liver transplant (LT) recipients with a low risk of disease recurrence. However, the frequency of occult HBV infection (OBI) and HBV variants after LT in the current era of potent NA therapy is unknown. Twelve LT recipients on prophylaxis were tested in matched plasma and peripheral blood mononuclear cells (PBMCs) for HBV quasispecies by in-house nested PCR and next-generation sequencing of amplicons. HBV covalently closed circular DNA (cccDNA) was detected in Hirt DNA isolated from PBMCs with cccDNA-specific primers and confirmed by nucleic acid hybridization and Sanger sequencing. HBV mRNA in PBMC was detected with reverse-transcriptase nested PCR. In LT recipients on immunosuppressive therapy (10/12 male; median age 57.5 [IQR: 39.8-66.5]; median follow-up post-LT 60 months; 6 pre-LT hepatocellular carcinoma [HCC]), 9 were HBsAg-. HBV DNA was detected in all plasma and PBMC tested; cccDNA and/or mRNA was detected in the PBMC of 10/12 patients. Significant HBV quasispecies diversity (ie 143-2212 nonredundant HBV species) was noted in both sites, and single nucleotide polymorphisms associated with cirrhosis and HCC were detected at varying frequencies. In conclusion, OBI and HBV variants associated with severe liver disease persist in LT recipients on prophylaxis. Although HBV control and cccDNA transcriptional silencing may occur despite immunosuppression, complete virological eradication does not occur in LT recipients with a history of HBV-related end-stage liver disease.

Background The association between baseline human immunodeficiency virus (HIV) sequence diversity and HIV DNA decay after the initiation of antiretroviral therapy (ART) remains uncharacterized during the early stages of HIV infection. Method Samples were obtained from a cohort of 17 patients with early HIV infection ( 0.99), but the Homogenous group experienced more prominent decay than the Heterogeneous group after six months (P = 0.037). The Heterogeneous group had higher CD4 cell counts after ART initiation; however, both groups had comparable recovery in terms of CD4/CD8 ratios and CD8 T cell activation levels. Conclusion Viral population homogeneity upon the initiation of ART is associated with a decrease in HIV DNA levels during ART. This article is protected by copyright. All rights reserved

Background and Aim Hepatitis B virus (HBV) full-length genomic mutations and quasispecies characteristics in hepatocellular carcinoma (HCC) were investigated. Methods Hepatitis B virus DNA was extracted from the tumor and non-tumor tissues of 16 HCC patients. Overlapping DNA fragments covering the entire HBV genome were amplified and sequenced. To study HBV sequence at the quasispecies level, the preS region was amplified and clonally sequenced. HBV mutation profiles, quasispecies complexity and diversity, and phylogenetic characteristics were assessed. Results Fourteen patients had full-length HBV amplification. Hot-spot mutations at HBx aa130–131 and pre-S deletions were detected in 13 (93%) and 6 (43%) patients, respectively. Deletions in the X/preC/C regions were more frequently detected in the tumor than in the non-tumor tissues (P = 0.031). Compared with the non-tumor tissues, the tumor tissues had a lower quasispecies complexity (P = 0.014 and 0.043, at the nucleotide and amino acid levels, respectively) and diversity (P = 0.048 and 0.022, at the nucleotide and amino acid levels, respectively). Phylogenetic analysis showed that HBV sequences derived from tumor and non-tumor tissues were separately clustered, suggesting the occurrence of compartmentalization, which was confirmed by the correlation coefficient testing on both the number and length of branches of viral populations (all P

The spike glycoprotein of the Middle East respiratory coronavirus (MERS-CoV) facilitates receptor binding and cell entry. During investigation of a multi-facility outbreak of MERS-CoV in Taif, Saudi Arabia, we identified a mixed population of wild-type and variant sequences with a large 530 nucleotide deletion in the spike gene from the serum of one patient. The out of frame deletion predicted loss of most of the S2 subunit of the spike protein leaving the S1 subunit with an intact receptor binding domain. This finding documents human infection with a novel genetic variant of MERS-CoV present as a quasispecies. J. Med. Virol. 89:542-545, 2017. © 2016 Wiley Periodicals, Inc.

Chronic HBV infection affects more than 240 million people worldwide and is associated with a broad range of clinical manifestations including liver cirrhosis, liver failure and hepatocellular carcinoma. Because of the lack of an efficient cure for chronic hepatitis B, the main goal of antiviral therapy is the prevention of liver disease progression coupled with prolonged survival of patients. Because HBV viral load has been shown to be a crucial determinant of the progression of liver damage, these goals can be achieved as long as HBV replication can be suppressed. Unfortunately, long-term therapy with the low-to-moderate genetic barrier drugs, which are still recommended in a majority of developing countries, are strongly associated with HBV resistance development and treatment failure. In such cases, the precise and accurate determination of drug-resistant variants in an individual patient before treatment is important for a proper choice of first-line potent therapy. Nowadays, a number of techniques are available to study HBV quasispecies evolution. This review describes the advantages and limitations of various assays detecting drug-resistant HBV variants. Copyright © 2016 John Wiley & Sons, Ltd.

Sustained virological responses (SVR) by daclatasvir (DCV) and asunaprevir (ASV) therapy for genotype 1b hepatitis C virus (HCV) infected patients has been significantly affected by pre-existence of Y93 H resistance-associated variants (RAVs) in the non-structural protein 5A (NS5A) region. The aim of this study was to elucidate the dominancy of naturally occurring RAVs in viral quasispecies on treatment outcomes in patients with HCV. In total, 138 patients were prospectively selected from 152 patients treated with DCV and ASV, where evaluation of treatment outcomes at 12 weeks post-treatment was possible. Pre-treatment RAVs in the non-structural protein 3 and NS5A regions were detected by polymerase chain reaction (PCR)-Invader assays, and the ratio of Y93H RAVs in viral quasispecies was measured by quantitative PCR-Invader assay. Among 25 patients detected the Y93H RAV, the Y93H ratio was 1-25% in 5 patients, 26-75% in 7 patients, and ≥76% in 13 patients. Overall, SVR at 12 weeks after the completion of treatment (SVR12) was 91% (125/138), and those with Y93H ratios of

Aim The virologic characteristics of resistance-associated variants (RAVs) developing in patients receiving dual oral therapy with daclatasvir/asunaprevir, including those with previous triple therapy with simeprevir, were evaluated. Methods A total of 206 patients with genotype-1b HCV infection, including 5 patients with previous simeprevir therapy, were treated with daclatasvir/asunaprevir for 24 weeks. Resistance-associated variants in the NS5A regions at baseline and during/after therapy were evaluated using cycling-probe real-time polymerase chain reaction combined with direct sequencing. The dynamics of rare RAVs were also assessed using ultra-deep sequencing. Results A sustained virologic response (SVR12) was achieved in 180 patients (87%); the rates were 95% in patients without baseline NS5A-RAVs and 83%, 59%, and 77% in those with hepatitis C virus (HCV) strains carrying NS5A-L31M, NS5A-Y93H/C, and NS5A-R30Q/H/L mutations, respectively. A multivariate analysis revealed baseline NS5A-R30Q/H/L mutation and NS5A-Y93H mutations as significant factors associated with SVR12. Virologic failure developed in all 5 patients with previous simeprevir treatment, and rare RAVs (HCV strains with NS5A-R30H, NS5A-A92K, NS5A-P29del, and NS5A-P32del) developed at virologic failure. Ultra-deep sequencing revealed that HCV strains with NS5A-P29del or NS5A-P32del were absent at baseline and emerged within 4 weeks of dual oral therapy among the strains appearing after simeprevir administration. Conclusion NS5A-R30Q/H/L and NS5A-Y93H mutations at baseline determined the therapeutic efficacy of dual oral therapy with daclatasvir/asunaprevir, but rare NS5A-RAVs developed frequently in patients with previous simeprevir treatment. Such RAVs may develop in a two-hit manner, with simeprevir altering the quasispecies of HCV strains in the NS5A regions, leading to the emergence of HCV strains with NS5A-P29del and NS5A-P32del during exposure to daclatasvir/asunaprevir.

Evolution patterns of HBV QS between genotype B and C during vertical transmission are not well understood. In this study, we enrolled 10 HBV infected mother-infant pairs (four pairs with genotype B, four pairs with genotype C, and two with co-infection) without anti-viral therapy. Serum HBV DNA of mothers and infants were sequenced, HBV QS complexity and diversity were analyzed, polymorphisms and mutation sites were recorded, and phylogenetic trees were performed. Our result showed that the QS complexities in P (amino acid), C/PreC (amino acid), and PreS1 (nucleotide) gene were significantly higher in mothers than in infants in pairs with genotype C (P 0.05). Unlike genotype C, QS complexity of P gene (nucleotide) was significantly higher in infants than in mothers (P 0.05). QS diversities of full-length and most genes in genotype B were comparable between mothers and their infants (P > 0.05), in pairs with genotype C, dS of P, X, RT genes, genetic distance of Pre S1 gene (amino acid) and dN of Pre S1 gene were significant higher in mothers than in infants (P < 0.05). Several HBV mutations correlated with immune escape, e antigen loss and drug resistance were observed in infants. The results indicated that differences of HBV QS evolution patterns between genotype B and C during vertical transmission might contribute to distinct prognosis.

Minority variants (1.0-25.0%) were evaluated by deep sequencing (DS) at baseline and virological failure (VF) in a selection of antiretroviral treatment-naive, HIV-1-infected patients from the rilpivirine ECHO/THRIVE phase III studies. Linkage between frequently emerging resistance-associated mutations (RAMs) was determined. DS (llIumina®) and population sequencing (PS) results were available at baseline for 47 VFs and time of failure for 48 VFs; and at baseline for 49 responders matched for baseline characteristics. Minority mutations were accurately detected at frequencies down to 1.2% of the HIV-1 quasispecies. No baseline minority rilpivirine RAMs were detected in VFs; one responder carried 1.9% F227C. Baseline minority mutations associated with resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) were detected in 8/47 VFs (17.0%) and 7/49 responders (14.3%). Baseline minority nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs M184V and L210W were each detected in one VF (none in responders). At failure, two patients without NNRTI RAMs by PS carried minority rilpivirine RAMs K101E and/or E138K; and five additional patients carried other minority NNRTI RAMs V90I, V106I, V179I, V189I, and Y188H. Overall at failure, minority NNRTI RAMs and NRTI RAMs were found in 29/48 (60.4%) and 16/48 VFs (33.3%), respectively. Linkage analysis showed that E138K and K101E were usually not observed on the same viral genome. In conclusion, baseline minority rilpivirine RAMs and other NNRTI/NRTI RAMs were uncommon in the rilpivirine arm of the ECHO and THRIVE studies. DS at failure showed emerging NNRTI resistant minority variants in seven rilpivirine VFs who had no detectable NNRTI RAMs by PS.

© 2015 John Wiley & Sons Ltd. RNA viruses exist as complex mixtures of genotypes, known as quasispecies, where the evolution potential resides in the whole community of related genotypes. Quasispecies structure and dynamics have been studied in detail for virus infecting animals and plants but remain unexplored for those infecting micro-organisms in environmental samples. We report the first metagenomic study of RNA viruses in an Antarctic lake (Lake Limnopolar, Livingston Island). Similar to low-latitude aquatic environments, this lake harbours an RNA virome dominated by positive single-strand RNA viruses from the order Picornavirales probably infecting micro-organisms. Antarctic picorna-like virus 1 (APLV1), one of the most abundant viruses in the lake, does not incorporate any mutation in the consensus sequence from 2006 to 2010 and shows stable quasispecies with low-complexity indexes. By contrast, APLV2-APLV3 are detected in the lake water exclusively in summer samples and are major constituents of surrounding cyanobacterial mats. Their quasispecies exhibit low complexity in cyanobacterial mat, but their run-off-mediated transfer to the lake results in a remarkable increase of complexity that may reflect the convergence of different viral quasispecies from the catchment area or replication in a more diverse host community. This is the first example of viral quasispecies from natural aquatic ecosystems and points to ecological connectivity as a modulating factor of quasispecies complexity., Spanish Polar Programme and the Spanish Ministry of Economy and Competitiveness (CTM2008-05134-E/ANT and CTM2009-08644-E).

Little is known about the natural occurrence and genetic variability of nepovirus large satellite RNA (satRNA). This study screened 71 Grapevine fanleaf virus (GFLV) isolates mainly from Slovenia, but also from other countries in Europe and the USA, for the presence of satRNA, using a newly developed RT-PCR assay. GFLV satRNA (satGFLV) was detected in 72% of naturally GFLV-infected grapevines analysed, which is the highest frequency of occurrence of satGFLV reported to date. From 39 naturally GFLV-infected grapevines, 122 satGFLV clones were sequenced and compared to publicly available sequences of satGFLVs and the closely related satRNAs from Arabis mosaic virus (satArMVs). Phylogenetic analyses of these satRNAs revealed that their evolution was driven by substitutions, insertions, deletions, recombinations and reassortments between closely related helper viruses. Phylogenetic relationships of the satGFLVs and satArMVs show their separate and subsequent common evolution. Furthermore, the satGFLVs varied in size and showed higher variability at the amino acid level than at the nucleotide level, just as the 2AHP gene of their helper virus. This study shows that satGFLVs are also similar to their helper virus with respect to their quasispecies nature and their transmission route through anthropogenic exchange of propagation material.

Aims To investigate an optimal long-term prophylactic strategy for prevention of hepatitis B virus (HBV) recurrence after liver transplantation, we conducted a randomized study of 29 transplant recipients receiving a short course of hepatitis B immune globulin (HBIg) + lamivudine (LAM), followed by randomization to long-term prophylaxis with LAM with or without HBIg. Methods The efficacy and safety, and impact on survival and HBV recurrence of these 2 prophylactic regimens were compared over a mean period of 10 years. In patients with viral recurrence, the HBV quasispecies in the surface/polymerase region were studied by ultra-deep pyrosequencing (UDPS). Results The 10-year survival rate was 76% and was not affected by the type of prophylaxis. Four patients had hepatitis B surface antigen (HBsAg) recurrence within the first 48 months after orthotopic liver transplantation (OLT). HBsAg-positive and -negative patients showed similar mean survival times, with no differences between the 2 regimens. Low HBV DNA levels were transiently detected in 32% of HBsAg-negative patients. UDPS showed major changes after OLT in the HBV quasispecies of patients with viral recurrence, which may be explained by a “bottleneck” effect of OLT together with prophylactic therapy. Conclusion Long-term survival after OLT in end-stage chronic hepatitis B patients was good with both prophylactic strategies. However, low, transient HBV DNA levels were detected even in the absence of HBsAg, showing the importance of continuing HBV prophylaxis.

Reactivation of a former hepatitis B virus (HBV) infection can be triggered by immunosuppressive therapy, diseases associated with an immunocompromised state, organ transplantation or the withdrawal of antiviral drugs. Despite the absence of such risk factors, a spontaneous reactivation of HBV replication occurred in two elderly patients with resolved or occult HBV infection. A 73-year-old male underwent coronary artery bypass grafting in October 2008, and was negative for HBsAg but positive for anti-HBs. In July 2009, his serum became positive for HBsAg, HBeAg and HBV DNA (6.4 log copies/ml; genotype C), but negative for anti-HBc IgM, with abrupt elevation of the liver enzymes. The entire genomic sequence of HBV recovered from this patient revealed no mutations in the core promoter and precore regions that interfere with HBeAg production. A 76-year-old male with a history of endoscopic mucosal resection for esophageal cancer in 2002 and an initial diagnosis of diabetes mellitus in 2009, at which time he was negative for HBsAg. He was found to be positive for HBsAg in September 2012 during a laboratory examination performed prior to the resection of recurrent esophageal cancer, despite a low HBV load (2.1 log copies/ml). Three months later, without the administration of any anticancer drugs, the HBV DNA (genotype B) level increased to 5.1 log copies/ml. A precore G1896A variant with high quasispecies diversity was recovered from the patient. Aging, surgical stress and complication of disease(s) associated with compromised immunity, such as cancer, arteriosclerosis and diabetes mellitus may trigger spontaneous HBV reactivation. J. Med. Virol. 87:589–600, 2015. © 2015 Wiley Periodicals, Inc.

Lytic viruses were thought to kill the most numerous host (i.e., kill the winner). But persisting viruses/defectives can also protect against viruses, especially in a ubiquitous virosphere. In 1991, Yarmolinsky et al. discovered the addiction modules of P1 phage, in which opposing toxic and protective functions stabilize persistence. Subsequently, I proposed that lytic and persisting cryptic virus also provide addiction modules that promote group identity. In eukaryotes (and the RNA world), a distinct RNA virus-host relationship exists. Retrovirurses/retroposons are major contributors to eukaryotic genomes. Eukaryotic complexity appears to be mostly mediated by regulatory complexity involving noncoding retroposon-derived RNA. RNA viruses evolve via quasispecies, which contain cooperating, minority, and even opposing RNA types. Quasispecies can also demonstrate group preclusion (e.g., hepatitis C). Stem-loop RNA domains are found in long terminal repeats (and viral RNA) and mediate viral regulation/identity. Thus, stem-loop RNAs may be ancestral regulators. I consider the RNA (ribozyme) world scenario from the perspective of addiction modules and cooperating quasispecies (i.e., subfunctional agents that establish group identity). Such an RNA collective resembles a "gang" but requires the simultaneous emergence of endonuclease, ligase, cooperative catalysis, group identity, and history markers (RNA). I call such a collective a gangen (pathway to gang) needed for life to emerge.

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-naive 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. J. Med. Virol. 87:315–322, 2015. © 2014 Wiley Periodicals, Inc.

Viral diversity is an important predictor of hepatitis C virus (HCV) treatment response and may influence viral pathogenesis. HIV influences HCV variability in the plasma; however, limited data on viral variability are available from distinct tissue/cell compartments in patients co-infected with HIV and HCV. Thus, this exploratory study evaluated diversity of the hypervariable region 1 (HVR1) of HCV in the plasma and liver for 14 patients co-infected with HIV and HCV. Median intra-patient genetic distances and entropy values were similar in the plasma and liver compartments. Positive immune selection pressure was observed in the plasma for five individuals and in the liver for three individuals. Statistical evidence supporting viral compartmentalization was found in five individuals. Linear regression identified ALT (P = 0.0104) and AST (P = 0.0130) as predictors of viral compartmentalization. A total of 12 signature amino acids that distinguish liver from plasma E1/HVR1 were identified. One signature amino acid was shared by at least two individuals. These findings suggest that HCV compartmentalization is relatively common among patients co-infected with HIV and HCV. These data also imply that evaluating viral diversity, including drug resistance patterns, in the serum/plasma only may not adequately represent viruses replicating with in the liver and, thus, deserves careful consideration in future studies. J. Med. Virol. 86:1332–1341, 2014. © 2014 Wiley Periodicals, Inc.

Summary The growing number of cases of acute hepatitis C (AHC) infections among human immunodeficiency virus type 1 (HIV-1)-positive men who have sex with men (MSM) in the last 10 years has promoted the search for predictors of AHC clearance as well as for epidemiological networks of viral transmission. We characterized the diversity and catalytic efficiency of HCV NS3/4A protease quasispecies in AHC patients coinfected with HIV-1. Plasma samples obtained at HCV diagnosis from 18 MSM HIV-coinfected patients with AHC were studied. Five HCV monoinfected patient samples with AHC were also investigated. An average of 39 clones from each sample was analysed. The catalytic efficiency of the dominant quasispecies (i.e. the most abundant) from each quasispecies was also assayed for mitochondrial antiviral signalling protein (MAVS) cleavage. Phylogenetic analysis identified two clusters of patients with highly related viruses, suggesting a common source of HCV infection. None of the 18 MSM HIV-coinfected patients spontaneously cleared HCV, although 78% of the treated patients achieved a sustained virological response after early treatment with pegylated interferon (pegIFN) plus ribavirin (RBV). The synonymous-nonsynonymous (ds/dn) mutation ratio, a marker of selective pressure, was higher in AHC compared to 26 HIV-1-infected men with genotype 1a chronic hepatitis C (CHC) (P

This study sought to assess the antiviral efficacy of lamivudine (LMV) administered during third trimester to reduce maternal viraemia and to identify the emergence of LMV resistance. A prospective observational analysis was performed on 26 mothers with high viral load (>10⁷ IU/mL). Twenty-one women received LMV (treated group) for an average of 53 days (range 22-88 days), and the remaining five formed the untreated control group. Serum samples from two time points were used to measure HBV DNA levels and antiviral drug resistance. The LMV-treated women achieved a median HBV DNA reduction of 2.6-log10 IU/mL. Although end-of-treatment (EOT) HBV DNA in four (18%) LMV-treated women remained at >10(7) IU/mL (± 0.5 log IU/mL), no mother-to-baby transmission was observed. In contrast, a baby from the untreated mother was HBsAg positive at 9 months postpartum. Four technologies were used for drug resistance testing. Only ultra-deep pyrosequencing (UDPS) was sufficiently sensitive to detect minor viral variants down to

Antarctic ecosystems are dominated by micro-organisms, and viruses play particularly important roles in the food webs. Since the first report in 2009 (Lopez-Bueno et al. 2009), ‘omic’-based studies have greatly enlightened our understanding of Antarctic aquatic microbial diversity and ecosystem function (Wilkins et al. 2013; Cavicchioli 2015). This has included the discovery of many new eukaryotic viruses (Lopez-Bueno et al. 2009), virophage predators of algal viruses (Yau et al. 2011), bacteria with resistance to phage (Lauro et al. 2011) and mechanisms of haloarchaeal evasion, defence and adaptation to viruses (Tschitschko et al. 2015). In this issue of Molecular Ecology, Lopez-Bueno et al. (2015) report the first discovery of RNA viruses from an Antarctic aquatic environment. High sequence coverage enabled genome variation to be assessed for four positive-sense single-stranded RNA viruses from the order Picornavirales. By examining the populations present in the water column and in the lake's catchment area, populations of ‘quasispecies’ were able to be linked to local environmental factors. In view of the importance of viruses in Antarctic ecosystems but lack of data describing them, this study represents a significant advance in the field.

Human cytomegalovirus (HCMV) may cause severe or fatal disease among immunocompromised patients. The first line prophylaxis and systemic HCMV disease therapy is ganciclovir (GCV). The presence of GCV-resistant virus has been linked to fatal HCMV disease. The implementation of rapid and sensitive techniques for the early detection and monitoring of GCV-resistance may be helpful to support antiviral therapy management. A pyrosequencing assay for the detection and quantitation of the most frequent mutations conferring moderate- and high-grade GCV resistance was implemented. The pyrosequencing achieved an analytical sensitivity for adequate interpretation of ≥103 copies/ml. The assay was validated with 18 whole blood samples taken over a 6-month period from an umbilical cord blood recipient infected persistently with HCMV and allowed the detection and monitoring of the M460I and A594V GCV-resistant mutations. The percentage of resistant quasispecies ranged from 7.9% to 55.2% for the M460I mutation and from 19.8% to 43% for the A594V mutation. Clearance of the M460I mutation occurred in parallel with a decrease in the HCMV viremia, while the A594V mutation persisted. The pyrosequencing method for detection of GCV is sensitive enough to be used directly on clinical samples for the early identification of resistance mutations and allows the quantitation of resistant and wild type virus quasispecies within hours. The quantitation of minor resistant variants is an important issue to understand their relationship with viral load modification, and potentially anticipate treatment adjustment. J. Med. Virol. 85:1250–1257, 2013. © 2013 Wiley Periodicals, Inc.

The evolution of hepatitis-B virus (HBV) drug resistance is characterized by the emergence of resistance conferring mutations and compensatory mutations. Therefore HBV drug-resistant isolates often harbor multiple mutations in the reverse transcriptase (RT) and corresponding mutations in the hepatitis B surface antigen (HBsAg). In this study mutational patterns of 60 HBV isolates harboring drug resistance mutations rtM204V or rtM204I were retrospectively analyzed. Both mutations, especially mutation rtM204V, were most often accompanied by compensatory mutations rtV173L and rtL180M but also by mutations conferring entecavir (n = 5) or adefovir resistance (n = 4). In addition, 22 (36.7%) drug resistant HBV isolates carried mutations related to immune escape in the HBsAg. In seven cases premature stop codons in HBsAg were detected resulting in the expression of truncated HBsAg. Clonal analysis of these seven quasispecies even disclosed the presence of HBV isolates carrying further stop codons and in one case the occurrence of resistance mutation rtA181T without the stop codon mutation sW172*. Interestingly, only one HBV clone carried the resistance mutations rtM204V and rtA181T. HBV drug resistant isolates frequently harbored HBsAg mutations associated with immune escape or disease progression pointing to a complex interaction of both proteins. HBV genotypic resistance tests based on population sequencing methods seemed to be inappropriate for determining the clinical relevance of stop codons in the HBsAg.

Objectives The aim of the study was to gain more insight into the relationship between transmitted singletons found at HIV diagnosis by population sequencing and the possible presence of clinically relevant viral minorities containing additional resistance mutations. Methods We studied the viral quasispecies and therapy response in 10 individuals with transmitted single nucleoside reverse transcriptase inhibitor (NRTI)-related resistance mutations as detected by population sequencing. Results Ultra-deep pyrosequencing did not reveal additional drug-resistance mutations in nine of 10 patients. In these nine patients, no breakthrough with resistant viruses was observed despite the use of low genetic nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens in the majority of patients. Conclusions These data suggest that viral minority variants containing additional resistance mutations may be rare in patients with transmitted NRTI singletons in the Netherlands. Larger studies are required to confirm these findings and to determine the therapeutic consequences.

Hepatitis B virus (HBV) infection is highly prevalent in circumpolar indigenous peoples. However, the clinical outcome is extremely variable, such that while hepatocellular carcinoma (HCC) is uncommon in Canadian Inuit, the incidence of HCC is slightly higher in Greenlanders than in Danes, and it is especially high in Alaskan Native people infected with HBV genotypes F (HBV/F) and C (HBV/C). These differences may be associated with the genomic variability of the predominant HBV genotype in each group. The purpose of this study was to determine the rate, nature and regional susceptibility of HBV genomic mutations among circumpolar indigenous individuals. Paired serum samples, separated by 5-6 years, were analysed from Canadian and Greenlandic Inuit infected with HBV genotype B6 (HBV/B6) and HBV/D, respectively, and from Alaskan Native people infected with HBV/F, each having subsequently developed HCC. Phylogenetic and mutational analyses were performed on full-genome sequences, and the dynamic evolution within the quasispecies population of each patient group was determined by clonal analysis of the non-overlapping core coding region. Mutations associated with severe outcomes predominated in HBV/F, mostly within the precore/core and PreS1 region. HBV/B6 genomes exhibited higher diversity compared to HBV/D and HBV/F, particularly within the core coding region. Thus, differing mutational profiles and genetic variability were observed among different HBV genotypes predominating in circumpolar indigenous patients. The unusual observation of persistently high genetic variability with HBV/B6 despite clinical inactivity could be due to the evolution of a host-pathogen balance, but other possible factors also need to be explored.

George Williams defined an evolutionary unit as hereditary information for which the selection bias between competing units dominates the informational decay caused by imperfect transmission. In this article, I extend Williams' approach to show that the ratio of selection bias to transmission bias provides a unifying framework for diverse biological problems. Specific examples include Haldane and Lande's mutation-selection balance, Eigen's error threshold and quasispecies, Van Valen's clade selection, Price's multilevel formulation of group selection, Szathmary and Demeter's evolutionary origin of primitive cells, Levin and Bull's short-sighted evolution of HIV virulence, Frank's timescale analysis of microbial metabolism, and Maynard Smith and Szathmary's major transitions in evolution. The insights from these diverse applications lead to a deeper understanding of kin selection, group selection, multilevel evolutionary analysis, and the philosophical problems of evolutionary units and individuality.

Polyomavirus BK is a recognized cause of nephropathy and hemorrhagic cystitis in kidney or allogeneic hematopoietic stem cell transplant recipients. This study explored a role of genetic variations in capsid protein VP-1 gene as a factor in viral pathogenesis. VP-1 was amplified from 7 healthy subjects with viruria, 7 transplant patients with viruria, and 11 patients with viremia or nephropathy. PCR products were cloned and a total of 558 clonal sequences were subjected to phylogenetic analysis using standard methods. VP-1 quasispecies were found in 25/25 and coinfection with different genotypes in 12/25 subjects. Genotype II was found as an unexpected minority species in 5/25 individuals. Recombinant strains of uncertain biologic significance, which frequently contained genotype II and IV sequences were identified in 9/25 subjects. Viremia/nephropathy group was characterized by (a) greater sequence complexity in whole VP-1 versus BC loop and BC loop compared to the HI loop, (b) greater intra-strain genetic diversity in the BC loop compared to whole VP-1 protein and HI loop, (c) more non-synonymous substitutions (dN) in the BC loop compared to whole VP-1 and HI loop, (e) fewer synonymous substitutions (dS) compared to healthy-viruria group, and (f) selection pressure (dN/dS

RNA viruses display the highest replication error rate in our biosphere, leading to highly diverse viral populations termed quasispecies. The gold standard method for detection and quantification of variants in a quasispecies is cloning and sequencing, but it is expensive, laborious and time consuming. Therefore, other mutation detection approaches, including SSCP, are often used. In this study, we demonstrate development and the usage of a CE-SSCP method for quantification of two nearly identical viral variants in heterogenic population of a mumps virus strain and its comparison to RFLP-CE-fragment length analysis (RFLP-CE-FLA). Analyzed PCR fragments were of the same size (245 bp) with one difference in their nucleotide sequence. The limit of detection of both methods was at 5% of the minor variant. When PCR amplicons of the two variants were pooled, methods' results were very similar. On the contrary, the quantification results of samples in which variants were mixed prior to PCR showed substantial difference between the two methods. Our results indicate that although both methods can be used for detection and monitoring of a specific mutation within a viral population, caution should be taken when quantitative analysis of complex samples is based solely on results of one method.

The hepatitis B virus (HBV) replicates via an error-prone reverse transcriptase generating potential drug-resistant quasispecies. The degree of HBV variability in liver vs peripheral blood mononuclear cells (PBMC) in patients on long-term suppressive antivirals is unclear. We characterized HBV replication, drug resistance and molecular diversity in patients with plasma HBV DNA undetectable by clinical assays. Explant liver (n=9), PBMC (n=6) and plasma (n=7) from nine such patients undergoing liver transplantation were evaluated for HBV genomes by sensitive PCR/nucleic acid hybridization assay. Cases with HBV DNA in liver and PBMC were tested for covalently closed circular DNA (HBV cccDNA). HBV polymerase (P) amplicons were cloned, sequenced and both P and overlapping surface (S) gene sequences were analysed. HBV DNA was detected in 43% (3/7) of plasma, 100% (9/9) of liver and 83% (5/6) of PBMC samples. HBV cccDNA was detected in all liver and one PBMC sample. Four patients had a clinical diagnosis of resistance. HBV P gene sequencing revealed 100% wild type (wt) in plasma (2/2), 83% wt in PBMC (5/6) but livers of 3/9 (33%) contained wt and 6/9 (66%) carried resistance to lamivudine and/or adefovir. The translated S gene revealed no changes affecting HBV antigenicity. Sequences from livers with antiviral resistant mutants revealed greater interpatient quasispecies diversity. Despite apparent HBV suppression, the liver continues to support HBV replication and extrahepatic HBV can be detected. PBMC may be a sanctuary for wt virus during antiviral therapy, while the liver harbours more drug-resistant viruses. Drug resistance correlates with intrahepatic viral diversity.

As an RNA virus, hepatitis C virus (HCV) shows a characteristically high level of nucleotide diversity. Accumulation of nucleotide substitutions in the virus has resulted in diversification into quasispecies, subtypes and distinct genotypes. Pathobiological studies linking nucleotide and amino acid sequences with clinical findings have identified relationships between certain genotypes and characteristic biological properties. Genotype 3 HCV infection was found to be associated with a high level of liver steatosis. Genotypes 1 and 4 were found to be more resistant to interferon (IFN) based therapies than genotypes 2 and 3. Studies of genotype 1 sequences obtained from patients treated with IFN have identified a relationship between favorable response to interferon therapy and amino acid substitutions in the NS5A region (interferon response determining region; ISDR). Further studies have identified a relationship between the effect of IFN therapy and other regions of the NS5A protein. More recently, a relationship has been found between poor response to peg-IFN plus ribavirin combination therapy and substitutions at amino acid 70 and 91 in the core protein. Furthermore, a correlation between human genetic variation in the IL28B (IFN-lamda 3) locus and core amino acid substitutions has been characterized. In this review we briefly summarize the discovery, classification and nomenclature of HCV genotypes and subtypes. We also discuss amino acid substitutions within specific regions that have been reported to be associated with outcome of IFN and peg-IFN plus ribavirin combination therapy.

Hepatitis A virus (HAV) infection is highly prevalent in Latin America, including Venezuela. Subgenotype IA seems to circulate in an almost exclusive fashion, except in Brazil. The aim of this study was the molecular characterization of the HAV infection in Venezuela, in order to characterize the circulating strains and to analyze the presence of quasispecies in sporadic cases and an epidemic outbreak. A total of 125 (113 sera and 12 feces) samples positive for anti-HAV IgM from sporadic cases and epidemic outbreak, were submitted to hemi-nested RT-PCR for amplification of the VP1 N terminus or complete region of the HAV genome. Sequences obtained from 96 Venezuelan isolates were used for phylogenetic analysis. The quasispecies distribution was evaluated by cloning of HAV amplicons. Phylogenetic analysis of HAV sequences from Venezuela showed the exclusive circulation of subgenotype IA, but with co-circulation of two lineages, not found in other countries. The genetic variability found among Venezuelan strains was also analyzed by single-strand conformation polymorphism (SSCP). This technique allowed the detection of intra-strain variability, which was indeed related to the presence of quasispecies populations in the isolates. The quasispecies heterogeneity was higher in some isolates derived from sporadic cases compared to the one observed in the outbreak. The molecular characterization of HAV isolates from Venezuela showed the circulation of a unique subgenotype IA, but with the presence of diverse strains and quasispecies inside the viral populations.

Summary. The pathogenesis of hepatitis B virus (HBV) is complex and it appears that molecular variants play a role in this process. HBV undergoes numerous rounds of error prone production within an infected host. The resulting quasispecies are heterogeneous and in the absence of archaeological records of past infection, the evolution of HBV can only be inferred indirectly from its epidemiology and by genetic analysis. This review gathered the controversies about the HBV origin and factors influencing its quasispecies. Also, it provided some evidence on how HBV genotypes correlated with human history and patterns of migration. It is our belief that this topic deserves further attention and thus it is likely that more critical research work will be performed to elucidate the unknown mechanisms and processes in this area.

The mechanisms of synergy in antiviral activity of interferon-alpha and ribavirin in treating chronic hepatitis C virus (HCV) infection are still unknown. Interferon-alpha indirectly induces cleavage of viral RNA by RNase L at UU/UA dinucleotides. There is evidence that HCV genomes with a higher number of UU/UA dinucleotides are more sensitive to interferon-alpha. As a guanosine analogue, ribavirin exerts a mutagenic effect promoting G-to-A and C-to-U transitions. This study investigates whether ribavirin-induced mutagenesis causes a higher frequency of UU/UA dinucleotides in the viral progeny sequences. Increased mutational frequencies in favour of G-to-A and C-to-U transitions during ribavirin treatment was reported by Hofmann et al. (Gastroenterology 2007;132:921-930). Overall, 937 nucleotide sequences from that publication were reanalysed for RNase L cleavage sites. These included HCV NS3 quasispecies from three patients with ribavirin monotherapy and NS5B quasispecies from patients who received ribavirin alone (n = 7) or in combination with interferon-alpha (n = 7) at baseline and during treatment; NS5B quasispecies from a subgenomic HCV replicon system after 24, 48 and 72 h of cultivation with or without ribavirin or with levovirin. For NS3 quasispecies during ribavirin monotherapy and NS5B quasispecies from patients who received ribavirin alone or in combination with interferon-alpha, analysis of RNase L cleavage sites did not reveal changes during treatment or differences between treatment regimes. Similarly, RNaseL cleavage sites from NS5B quasispecies of the HCV replicon did not differ significantly between time points or treatments. In conclusion, Ribavirin-induced mutagenesis did not increase RNase L cleavage sites (UU/UA dinucleotides) within the HCV NS3 or NS5B encoding regions.

Hepatitis C virus (HCV) infection is a frequent problem in hemodialysis units. The prevalence and incidence of HCV infection over a decade were studied in a nephrology unit affected by previous nosocomial HCV transmission. The HCV non-structural 5B protein gene was sequenced to achieve phylogenetic analysis of a new (incident) case of infection. Proportions of patients who were and were not infected with HCV remained similar over the period, as did the inflow and outflow of patients infected previously. In 1997, 12/157 (8%) of patients at the unit (treatment: hemodialysis, peritoneal dialysis, and renal transplant recipients) were positive in HCV RNA, whereas in 2007 the overall number was 9/239 (4%). One patient acquired an HCV infection, and the NS5B sequence in that case clustered with genotype 2b sequences found in patients from an earlier outbreak. Comparing the HCV from the incident patient with several stored longitudinal samples and cloned PCR products from the most likely source patient revealed close phylogenetic relationship with an HCV quasispecies member from the possible source. The source patient and the incident newly infected patient were not scheduled on the same dialysis shift, although the records showed that simultaneous treatment occurred on two occasions during the months preceding transmission. In conclusion, over the 10-year period, the proportion of HCV-infected patients at the unit was unchanged. Only one new infection occurred, which originated from a fellow patient's quasispecies. This establishes phylogenetic analysis as a valuable tool for tracing patient sources of HCV transmission.

Occult hepatitis B virus (O-HBV) infection is characterized by the presence of HBV DNA without detectable hepatitis B surface antigen (HBV DNA+/HBsAg-) in the serum. Although O-HBV is more prevalent during HBV/HIV co-infection, analysis of HBV mutations in co-infected patients is limited. In this preliminary study, HBV PreSurface (PreS) and surface (S) regions were amplified from 33 HIV-positive patient serum samples - 27 chronic HBV (C-HBV) and six O-HBV infections. HBV genotype was determined by phylogenetic analysis, while quasispecies diversity was quantified for the PreS, S and overlapping polymerase regions. C-HBV infections harboured genotypes A, D and G, compared to A, E, G and one mixed A/G infection for O-HBV. Interestingly, nonsynonymous-synonymous mutation values indicated positive immune selection in three regions for O-HBV vs one for C-HBV. Sequence analysis further identified new O-HBV mutations, in addition to several previously reported mutations within the HBsAg antigenic determinant. Several of these O-HBV mutations likely contribute to the lack of detectable HBsAg in O-HBV infection by interfering with detection in serologic assays, altering antigen secretion and/or decreasing replicative fitness.

Acute exacerbations in HBeAg negative patients with chronic hepatitis B virus (HBV) infection are invariably associated with concurrent increases in the index of IgM class antibodies against the core protein (anti-HBc) of the virus. This study aimed to investigate whether this was related to the clearance of variants from the quasispecies pool and the appearance of new ones, with aminoacid substitutions in well recognized B-cell epitopes. In this study, 5 HBeAg negative patients (A to E) with 13 sequential serum samples (A1-A2, B1-B2-B3, C1-C2, D1-D2-D3, E1-E2-E3) were investigated after amplification of the entire core encoding region followed by cloning/sequencing studies. The sequences at different time points were compared with those from a single HBeAg positive patient with no apparent acute exacerbations. The results from sequence comparison showed that virus variants emerged in all (A2, B3, C2, D3, E2, and E3) but two (B2 and D2) subsequent sera with amino-acid substitutions affecting B-cell epitopes. It is concluded that the rise in the values of IgM anti-HBc may be attributed to the alteration of the antigenic epitopes leading to new antibody production in the majority of the cases. However, it appears that increases in IgM anti-HBc indexes in a few cases may relate to other possible mechanisms which are discussed.

The cellular source(s) and the clinical significance of persistent low-level viremia, below 50 HIV RNA copies per ml of plasma, achieved in many patients with high adherence to highly active antiretroviral therapy (HAART) remain unclear. Also, it is not clear if residual plasma HIVs during HAART can become predominant populations in the rebounding plasma viral loads after therapy interruption. Since, different HIV quasispecies tend to compartmentalize in various cell types and tissue locations in patients during chronic infection, the phylogenetic relationships between HIV sequences amplified from residual plasma viruses and CD4 T cells of five patients on long-term suppressive therapy were examined. Three of these patients stopped therapy voluntarily for 3 weeks, but only one of them demonstrated viral load rebound in plasma. In phylogenetic analyses, the residual plasma viruses were found to be distinct genetically from the majority of CD4 T cell-associated virus populations in four of five patients. The compartmental analyses revealed that in all patients, plasma- and CD4 T cell-derived viral sequences were compartmentalized separately. Interestingly, the plasma sequences obtained before and after HAART-off in two patients were produced apparently from the same compartment, which was different from the circulating CD4 T cell-compartment. These results suggest the possibility that residual plasma viruses in patients on long-term suppressive HAART may be produced persistently from a cellular source yet to be identified, and are capable of spreading quickly in vivo, accounting for the rapid rebound of viral loads in plasma after therapy interruption.

A recent empirical study by Turner and Chao on the evolution of competitive interactions among phage virus strains revealed that a strain grown at high rates of co-infection evolved towards lowered fitness relative to an ancestral strain. The authors went on to show that the fitness pay-off matrix between the evolved and ancestral strain conforms to the prisoners’ dilemma. In this paper, I use Turner and Chao’s data to parameterize a simple model of parasite collective action. The prisoners’ dilemma is based on pairwise interactions of a discrete cooperate/defect nature. In contrast, the collective action model explicitly deals with individual–group interactions where the extent of cooperation is a continuous variable. I argue here that the ‘collective action’ modelling approach is more appropriate than the prisoners’ dilemma for the biology of virus evolution, and hence better able to form a predictive framework for further work on related strains of virus, linking mixing ecology, cooperative phenotype and fitness. Furthermore, the collective action model is used to motivate discussion on the evolutionary ecology of viruses, with a focus on the ‘levels of selection’ debate and the evolution of virulence.

Aim: A series of recent studies have indicated the presence of natural intergenotypic recombinant hepatitis C virus (HCV) strains in distinct parts of the world. The majority of the current genotyping methods are based on analysis of either 5′UTR, structural (Core/E1/E2) or non-structural (NS5B) genomic regions of the virus. Methods: In the present study, based on both structural and non-structural regions, we determined the genotype of 55 anti-HCV-positive intravenous drug users (IDUs) in Uzbekistan. Results: HCV-3a (67.3%) was the most prevalent genotype in this cohort, followed by HCV-1b (27.3%). A discrepancy in results was observed between structural and non-structural regions in one case (1.8%). Phylogenetically this strain was related to the previously reported RF1_2k/1b variant. Based on accumulated sequences, specific primers were designed for polymerase chain reaction (PCR) spanning the tentativeintergenotypic crossover point of RF1_2k/1b. The sensitivity and specificity of the method were assessed using generated template clones of HCV-1b, 2a, 2 k and RF1_2k/1b. The method was applied to 55 cases in the present study and only one case showed a positive result, indicating that in these individuals, the variant is not present as a minor quasispecies clone. Conclusion: In conclusion, the finding of RF1_2k/1b in Central Asia indicates that the variant has wide geographic distribution. The PCR-based screening method developed in this study should be useful in further epidemiological and clinical studies on the recombination phenomenon in HCV.

Hepatitis B virus (HBV) circulates in blood as closely related, but genetically diverse molecules called quasispecies. During replication, HBV production may approach 10(11) molecules/day, although during peak activity this rate may increase 100-1000 times. Generally, DNA polymerases have excellent fidelity in reading DNA templates because they are associated with an exonuclease which removes incorrectly added nucleotides. However, the HBV-DNA polymerase lacks fidelity and proofreading function partly because exonuclease activity is either absent or deficient. Thus, the HBV genome and especially the envelope gene, is mutated with unusually high frequency. These mutations can affect more than one open reading frame because of overlapping genes. The S gene contains an exposed major hydrophilic region (residues 110-155), which encompasses the 'a' determinant that is important for inducing immunity. Nucleotide substitutions in this region are common and result in reduced binding or failure to detect hepatitis B surface antigen (HBsAg) in diagnostic assays. Adaptive immunity also depends on the recognition of HBsAg by specific antibody and variants pose a threat if they interfere with binding to antibody. Finally, genomic hypervariability allows HBV to escape selection pressures imposed by antiviral therapies, vaccines and the host immune system, and is responsible for creating genotypes, subgenotypes and subtypes.

Background: The interaction between the hepatitis C virus (HCV) non-structural 5A (NS5A) protein of HCV and the protein kinase R (PKR), which is an effector of the cellular antiviral response and has been defined as a tumour suppressor, may affect the control of protein synthesis and cell growth. Aim: We investigated the genetic evolution of the NS5A region in the NS5A PKR-binding domain (NS5A-PKRbd) of patients with HCV 1b-related cirrhosis who subsequently developed or not hepatocellular carcinoma (HCC). Patients and Methods: The quasispecies composition of NS5A-PKRbd was inferred by sequencing an average of 15 clones per sample in specimens obtained from 26 patients with cirrhosis who developed or not HCC during a follow-up of 5 years. Results: At baseline, 13/17 patients with final HCC and six out of nine patients with cirrhosis who subsequently did not develop HCC harboured a wild-type (wt) strain master sequence. Over time, the prevalence of wt strain was higher in patients who developed HCC with respect to those who maintained the cirrhosis status (15/17 vs 4/9, respectively; P=0.0166). Conclusion: The maintenance of or evolution to the wt strain of the NS5A domain in cirrhotic patients with final HCC highlights the central role of NS5A protein in the viral life cycle and in the progression of liver disease.

Background Hepatitis C virus (HCV) easily undergoes genomic changes, especially in the hypervariable region (HVR) in the N-terminus of the E2/NS1 region. The quasispecies nature of HCV may have important biological implications in relation to viral persistence; however, the relationship between disease activity of chronic HCV infection and development of the genomic complexity have yielded conflicting results. We explored the changes in the complexity of the HVR-1 in the natural course of chronic HCV infection with and without elevation of serum alanine transaminase (ALT) levels. Materials and methods Ten patients with chronic hepatitis C proven by liver biopsy, who showed persistent elevation of the serum ALT levels, and 15 patients with chronic HCV infection and persistently normal serum ALT levels (PNAL) were enrolled in this study. The number of the HCV quasispecies was determined twice for each patient at an interval of mean 2·5 years by fluorescence single-strand conformation polymorphism and sequence analysis. Results There was no significant difference in the changes in the number of quasispecies during the follow-up period between chronic hepatitis C and PNAL. There was also no significant difference in the change in the number of variable nucleotides sites between the two groups. In these patients, the number of quasispecies and the diversity of HVR-1 were correlated with platelet counts and serum hyaluronic acid levels previously shown to be associated with disease progression. Conclusion Our results suggested that the disease activity is not always related to the generation of the HVR-1 quasispecies complexity.

Although Korea is a hepatitis B virus (HBV) endemic area, relatively few full-length genome sequences are available. In particular, no comparative analysis has been performed on the full-genome sequences of different HBV quasispecies from a single Korean patient. This report describes the full-length sequences of five HBV clones (two clones with shorter PCR amplicons and three clones with longer amplicons). Large deletions, that is, 685-bp, 487-bp, and 144-bp, that might interfere with the production of normal proteins were observed in four of five clones. Double mutations in the basal core promoter (BCP) region (T1762/A1764) were detected in two clones but no precore mutations (A1896) were detected in any of the five clones. These data support previous results that genotype C, in particular Korean clones of this genotype, is prone to mutations. Two independent mechanisms, namely, the deletions of long lengths and amino acid substitutions followed by BCP double mutations might contribute to the diversity of HBV quasispecies. Considering the importance of HBV quasispecies as HBV variant sources, the distribution of HBV quasispecies in mutation prone genotype C prevalent areas like Korea, should be monitored to improve the management of chronic HBV infections and to control HBV variants that arise due to the administration of vaccine or antiviral therapy.

This study aimed to define clinical and virological parameters associated with spontaneous control of HIV replication in patients having initiated HAART during primary HIV infection, who underwent structured therapy interruption by two protocols with either fixed or HIV viremia-guided scheme. At the end of the protocol all patients were changed to viremia-guided scheme and observed for 12 months (follow-up). Patients maintaining HIV viremia below the indications for resumption of HAART during the follow-up, were defined controllers, those who had to resume HAART were defined non-controllers. The following parameters were examined: pre-interruption therapy duration, CD4+, HIV RNA, proviral DNA, evolution of viral quasispecies. No specific advantage was conferred by either interruption of structured therapy in the proportion of controllers and non-controllers. Pre-HAART and zenith CD4+, pre-therapy interruption, HAART duration, but not pre-HAART HIV RNA, were significantly higher in controllers as compared to non-controllers. HIV RNA levels after the first interruption cycle of therapy were significantly lower in controllers than in non-controllers. Proviral DNA levels were also lower in controllers at this time point. HIV RNA and proviral DNA levels associated with the last interruption of therapy cycle were not different from those associated with the first cycle, and, in spite of multiple waves of virus rebound, very few gag quasispecies variants emerged in each patient. The data suggest that pre-treatment clinical parameters and virological events associated with the first viral rebound are crucial factors in determining the ability to control viral replication after multiple cycles of interruption of treatment. J. Med. Virol. 79: 1047–1054, 2007. © 2007 Wiley-Liss, Inc.

The active replication phases of the hepatitis B virus (HBV) are marked by a high frequency of mutational events resulting from an enormous daily viral turnover rate and an error-prone polymerase. Because HBV is not directly cytopathic, it is the host's immune response that accounts for most of the liver disease associated with persistent infection. HBV escape mutants can be found at the transitional phases of chronic hepatitis B, such as hepatitis B e antigen (HBeAg)-positive versus HBeAg-negative infection, and these mutants are selected out from the diverse quasispecies pool comprising the HBV population at that time. Not surprisingly, the introduction of nucleoside and nucleotide analog therapy has also seen the emergence of drug resistance, which has become the major factor limiting the long-term application of antiviral agents for patients with chronic hepatitis B. Thus, the prevention of resistance requires the adoption of strategies that effectively control virus replication and exploit a detailed understanding of the mechanisms and processes that drive the emergence of drug resistance.