Quasispecies Heterogeneity Research Articles

Hepatobiliary Quiz-7 (2013)

1.Regarding the molecular virology of hepatitis E virus (HEV) the following are TRUE:1.HEV genome consists of three open reading frames (ORF)2.ORF1 codes for structural proteins3.The 5′ untranslated region is important for viral encapsidation4.The 3′ untranslated region is important for initiation of viral replication5.Unlike other positive strand ribonucleic acid (RNA) viruses, replication of HEV does not require a negative sense RNA intermediary2.Regarding the immunology of HEV the following are TRUE:1.First sign of infection is the detection of IgM antibodies2.Shedding of virus in feces commonly persists after onset of clinical disease3.IgG antibodies persist for years after infection and may protect from future infection4.Liver damage is immune mediated rather than due to direct cytotoxic effect of HEV5.Antibody mediated liver injury is more important than cellular mechanisms3.Regarding chronic HEV infection the following are TRUE:1.Chronic HEV infection is exclusively seen with genotype 3 in immunocompromised individuals2.HEV RNA positivity in solid organ transplant recipients is about 30%3.HEV infection in immunocompromised patients is severe with higher bilirubin and aminotransferase levels4.Chronic HEV infection can progress to cirrhosis5.Risk factors for chronic HEV infection include use of tacrolimus, higher degree of immunosuppression and large heterogeneity of quasispecies of HEV4.Regarding immunosuppressive agents used after liver transplantation the following are TRUE EXCEPT:1.Tacrolimus is more likely to cause diabetes than cyclosporine2.Gingival hyperplasia and hypertrichosis are common with tacrolimus3.Main advantage of using mycophenolate is absence of haematological side effects4.Sirolimus is has additional anti-tumour effects making it useful in cases of transplantation for hepatocellular carcinoma5.Basiliximab and daclizumab are IL-2receptor blocking antibodies5.Regarding involvement of the liver in sarcoidosis the following are TRUE EXCEPT:1.Majority of patients of sarcoidosis will have liver involvement on investigation2.Symptomatic liver disease is common in sarcoidosis3.Pulmonary involvement is absent in about 25% of hepatic sarcoidosis4.Portal hypertension is a common finding in hepatic sarcoidosis5.Sarcoidosis is not a cause for cirrhosis of the liver6.Regarding non-alcoholic fatty liver disease (NAFLD) in Asia the following are TRUE:1.South Asians like Indians are at a higher risk of insulin resistance than their Western counterparts2.India has a low prevalence of NAFLD3.Prevalence of NAFLD is increasing in Asian countries4.PNPLA3 gene polymorphisms are not associated with NAFLD in Asians5.APO C3 gene polymorphisms are associated with NAFLD predominantly in Caucasians7.Regarding liver transplantation for cirrhosis due to non-alcoholic steatohepatitis (NASH) the following are TRUE EXCEPT:1.Recurrence of NAFLD is very common2.Overall survival after transplantation is poorer compared to other etiologies of cirrhosis3.Renal dysfunction after transplantation is less common compared to other etiologies of cirrhosis4.Cardiovascular events are a more common cause of death after transplantation in NASH patients5.Pre-transplant diagnosis of NASH related cirrhosis is an independent predictor of development of metabolic syndrome after transplantation8.Regarding nutritional management of patients with cirrhosis the following are TRUE:1.Resting energy expenditure is lower in patients with cirrhosis2.Late evening snack of complex carbohydrates may improve quality of life and survival3.Protein restriction is not advisable even in patients with hepatic encephalopathy4.Animal protein is preferred to vegetable protein5.Branched chain amino acids improve event free survival9.Regarding management of ascites in cirrhosis the following are TRUE:1.Mean arterial pressure is a predictor of survival in patients of cirrhosis with ascites2.Angiotensin converting enzyme inhibitors are good anti-hypertensive drugs in patients with ascites3.Standard medical therapy consisting of sodium restriction and diuretics is successful in controlling ascites in 90% of cases4.Midodrine is a treatment option for patients with refractory ascites5.Peritoneovenous shunts are a favoured treatment option for refractory ascites10.Regarding spontaneous bacterial peritonitis (SBP) the following are TRUE:1.Culture-negative neutrocytic ascites has better outcome than culture positive SBP2.Monomicrobial non-neutrocytic bacterascites frequently resolves spontaneously3.Albumin infusions decrease mortality in patients with SBP4.Primary prophylaxis for SBP is indicated if Child score is ≥9 and serum bilirubin is ≥3 mg/dl5.Short-term prophylaxis for SBP in patients with gastrointestinal bleeding reduces infection rate but does not affect mortality

Influence of Polyproline Region and Macro Domain Genetic Heterogeneity on HEV Persistence in Immunocompromised Patients

Hepatitis E virus (HEV) can chronically infect immunocompromised patients. The polyproline region (PPR) and the macro domain of ORF1 protein may modulate virus production and/or the host immune response. We investigated the association between the genetic heterogeneity of HEV quasispecies in ORF1 and the outcome of infection in solid-organ transplant patients. Both sequence entropy and genetic distances during the hepatitis E acute phase were higher in patients whose infection became chronic than in those who cleared the virus. Hence, great quasispecies heterogeneity in the regions encoding the PPR and the macro domain may facilitate HEV persistence.

Dynamics of hepatitis B virus quasispecies heterogeneity and virologic response in patients receiving low‐to‐moderate genetic barrier nucleoside analogs

We characterized the early dynamics of hepatitis B virus (HBV) quasispecies evolution during the first weeks of antiviral therapy with low-to-moderate genetic barrier antiviral drugs and associated these data with antiviral response patterns. Fifteen chronic hepatitis B patients (men, 10; mean age, 34; HBeAg positive, 6) who received lamivudine or telbivudine for at least 52 weeks were included. HBV DNA was extracted from serum, and a 910-bp fragment covering domains A-F of the reverse transcriptase region was amplified, cloned and sequenced. Parameters of quasispecies heterogeneity, genetic diversity and complexity were calculated and were correlated with complete virologic response, defined as undetectable HBV DNA at week 52. Nine patients achieved complete virologic response during the observational period. While baseline HBV DNA levels and HBeAg status were associated with virologic response, baseline quasispecies complexity and diversity of responders showed no significant difference to those of nonresponders (P > 0.05). However, at week 4, quasispecies complexity of nonresponders was significantly higher compared with that of responders on the nucleotide level (P = 0.01) and the aa level (P = 0.04). The number of synonymous substitutions per synonymous site dropped significantly in responders at week 4 (P = 0.04), while there was no difference in nonresponders. The HBV quasispecies complexity at the early stage of antiviral therapy (week 4) with the low-to-moderate genetic barrier nucleoside analogs lamivudine or telbivudine was associated with subsequent virologic response. Further studies are needed to confirm HBV quasispecies evolution as additional predictive marker for beneficial treatment outcome.

Mutations in different regions of the genome of hepatitis C virus genotype 1b and association with response to interferon therapy

The aim of this study was to investigate the association of mutations in the E2/NS1 [hypervariable regions 1 and 2 (HVR1 and HVR2)] and NS5A regions of the hepatitis C virus (HCV) genome and the effectiveness of interferon (IFN) therapy, and assess whether the degree of heterogeneity of HCV quasispecies predicts response to IFN treatment. Fourteen patients infected with HCV genotype 1b (HCV-1b) who were treated with pegylated IFN-α-2a and ribavirin for 24 weeks, were studied. E2/NS1 and NS5A gene segments were amplified by reverse-transcription polymerase chain reaction. HCV quasispecies heterogeneity in the E2/NS1 region was determined by cloning and sequencing. Mutations in the NS5A region were detected by direct sequencing. The heterogeneity of HCV quasispecies in the HVR1 was significantly greater in the non-responder group than in the responder group, but was not significant for HVR2 or NS5A. The correlation between mutations in IFN sensitivity-determining region (ISDR, NS5A2209-2248) and IFN sensitivity could not be supported. The degree of quasispecies heterogeneity in HVR1, but not in HVR2 and NS5A, may be predictive of response to IFN therapy. An ISDR may not apply to patients infected with HCV-1b.

Hepatitis E Virus Quasispecies and the Outcome of Acute Hepatitis E in Solid-Organ Transplant Patients

Hepatitis E virus (HEV) infections are responsible for chronic hepatitis in immunocompromised patients, and this can evolve to cirrhosis. Like all RNA viruses, HEV exists as a mixture of heterogeneous viruses defining quasispecies. The relationship between the genetic heterogeneity described as a quasispecies, cytokine secretion, and the outcome of acute hepatitis in immunocompromised patients remains to be elucidated. We cloned and sequenced the region encoding the M and P capsid domains of HEV from eight solid-organ transplant (SOT) patients with acute HEV infection who subsequently cleared the virus and from eight SOT patients whose infection became chronic. We analyzed the cytokines and chemokines in the sera of these SOT patients by multianalyte profiling. The nucleotide sequence entropy and genetic distances were greater in patients whose infections became chronic. A lower K(a)/K(s) ratio was associated with the persistence of HEV. The patients who developed chronic infection had lower serum concentrations of interleukin-1 (IL-1) receptor antagonist and soluble IL-2 receptor. Increased concentrations of the chemokines implicated in leukocyte recruitment to the liver were associated with persistent infection. Those patients with chronic HEV infection and progressing liver fibrosis had less quasispecies diversification during the first year than patients without liver fibrosis progression. Great quasispecies heterogeneity, a weak inflammatory response, and high serum concentrations of the chemokines involved in leukocyte recruitment to the liver in the acute phase were associated with persistent HEV infection. Slow quasispecies diversification during the first year was associated with rapidly developing liver fibrosis.

Longitudinal analysis of the 5′UTR, E2-PePHD and NS5A-PKRBD genomic regions of hepatitis C virus genotype 1a in association with the response to peginterferon and ribavirin therapy in HIV-coinfected patients

BackgroundThe rate of non-response to pegylated interferon plus ribavirin (peg-IFN+RBV) in HCV/HIV coinfected patients is higher than in HCV-monoinfected patients. In this sense, the contribution of HCV genetic variability is unknown. The 5′ untranslated (5′UTR), the nonstructural 5A (NS5A) and the second envelope (E2) HCV genomic regions have been implicated to peg-IFN therapy response. The proteins appear to block interferon (IFN)-induced RNA-dependent protein kinase (PKR) and the 5′UTR may influence the viral lymphotropism. MethodsWe examined comparatively the pretreatment HCV variability between HIV coinfected and HCV monoinfected patients as well as assessed longitudinally the impact of peg-IFN+RBV on HCV variability when HIV is co-present. For this purpose, 15 HIV coinfected and 20 HCV monoinfected patients were compared. They were peg-IFN+RBV non-responders and infected with HCV 1a. ResultsIrrespectively of the HIV-coexistence, at baseline the amino acid variation in the NS5A-related domains was significantly higher than in the E2-PePHD (p<0.001). The number of amino acid variations (mean±SD) at the NS5A-ISDR domain was higher among HCV/HIV patients than HCV-monoinfected ones (1.80±0.77 vs. 0.95±1.05; p=0.009) but such difference was slightly lower when comparing NS5A-PKRBD sequences (2.47±1.13 vs. 1.60±1.57; p=0.06). No differences were found at the E2-PePHD (0±0 vs. 0.2±0.4). At intra-HIV coinfected patient level, only minor (HCV genetic analysis) or no (HCV substitution rate and quasispecies heterogeneity) changes were observed during therapy (basal, 24h, 4weeks, and 12weeks). ConclusionsAmong HCV-1a/HIV coinfected and HCV-monoinfected peg-IFN+RBV non-responder patients, the HCV variability at the 5′UTR, E2-PePHD and NS5A-PKRBD/ISDR domains was mostly comparable exhibiting a low number of variations. Four well-defined amino acid substitutions in NS5A-ISDR domain appeared most frequently when HIV coexists. The interferon-based therapy did not exert any effect in the variation, selection or diversity in the above mentioned HCV regions that could influence clinical responsiveness to IFN therapy.

Complexity and Catalytic Efficiency of Hepatitis C Virus (HCV) NS3 and NS4A Protease Quasispecies Influence Responsiveness to Treatment with Pegylated Interferon plus Ribavirin in HCV/HIV-Coinfected Patients

The role of the hepatitis C virus (HCV) NS3/4A protease in ablating the signaling pathway involved in the production of alpha/beta interferon (IFN-α/β) suggests a relationship between NS3/4A proteolytic activity and a patient's response to IFN-based therapy. To identify viral factors associated with the HCV treatment response, we analyzed the pretreatment NS3/4A protease gene quasispecies composition of 56 HCV genotype 1-HIV-1-coinfected patients treated in our clinic with pegylated IFN (pegIFN) plus ribavirin (RBV). The catalytic efficiency of the dominant (i.e., the most abundant) quasispecies was also assayed for Cardif cleavage and correlated with treatment outcome. A total of 1,745 clones were isolated and sequenced. Significantly less nucleotide quasispecies heterogeneity and lower Shannon entropy values were detected within the responder group (P < 0.05). A correlation was also found between the efficiency of NS3/4A protease Cardif cleavage and therapy outcome. Proteases from sustained responder patients were more efficient at processing Cardif (mean ± standard error of the mean [SEM], 0.8960 ± 0.05568; n = 19) than proteases from nonresponders (mean ± SEM, 0.7269 ± 0.05306; n = 37; P < 0.05). Finally, the amino acid p distance (the proportion [p] of nucleotide sites at which two sequences being compared are different) was significantly shorter in patients with an interleukin-28B (IL-28B) risk allele (P < 0.01), suggesting that IL-28B risk allele carriers exert a lower positive selection pressure on the NS3/4A protease. NS3/4A protease efficiency in cleaving Cardif may be associated with the pegIFN-RBV treatment response, as shown in our cohort of HIV-HCV-coinfected patients. Greater NS3/4A nucleotide heterogeneity and higher Shannon entropy values in nonresponders suggest that less HCV quasispecies complexity may favor a better response to pegIFN-RBV.

Dynamics of In Vitro Fitness Recovery of HIV-1

The study on the evolutionary consequences of an RNA viral population's fluctuations can be approached by in vitro experiments. This work describes the fitness recovery of HIV-1 after 20 large-population passages in 10 debilitated clones. The serial passages promoted an increase in viral fitness. In addition, we detected a significant number of mutations fixed in the complete genome consensus sequence of the final viral populations. Among the mutations, events of convergent evolution with important phenotypic characteristics occurred in several independent clones. One common change, V35I, in the nuclear localization signal of the p17 protein appeared in four viruses of three different lineages. Other common alterations mapped in position E196K of the reverse transcriptase or in position S316K of the V3 loop of the gp120 residue that is associated with the X4/R5 phenotype. Together with this mutational analysis, we studied the quasispecies heterogeneity of the initial and final viruses, revealing that fitness increase correlated with an augmentation in the genetic heterogeneity of viral quasispecies. However, while heterogeneity was mostly composed of synonymous (dS) mutations in the first 10 passages performed, at passage 21 it switched to nonsynonymous (dN) substitutions, with significant differences in dN - dS values between passages 11 and 21. In summary, the HIV-1 in vitro fitness recovery depicts a multiphase process occurring first by generation of mutations followed by fixation of the beneficial ones, depicting a classical Darwinian process.

Hepatitis A virus genetic diversity in Venezuela: Exclusive circulation of subgenotype IA and evidence of quasispecies distribution in the isolates

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.

Initial Fitness Recovery of HIV-1 Is Associated with Quasispecies Heterogeneity and Can Occur without Modifications in the Consensus Sequence

BackgroundFitness recovery of HIV-1 “in vitro” was studied using viral clones that had their fitness decreased as a result of plaque-to-plaque passages.Principal FindingsAfter ten large population passages, the viral populations showed an average increase of fitness, although with wide variations among clones. While 5 clones showed significant fitness increases, 3 clones showed increases that were only marginally significant (p<0.1), and 4 clones did not show any change. Fitness recovery was not accompanied by an increase in p24 production, but was associated with an increase in viral titer. Few mutations (an average of 2 mutations per genome) were detected in the consensus nucleotide sequence of the entire genome in all viral populations. Five of the populations did not fix any mutation, and three of them displayed marginally significant fitness increases, illustrating that fitness recovery can occur without detectable alterations of the consensus genomic sequence. The investigation of other possible viral factors associated with the initial steps of fitness recovery, showed that viral quasispecies heterogeneity increased between the initial clones and the passaged populations. A direct statistical correlation between viral heterogeneity and viral fitness was obtained.ConclusionsThus, the initial fitness recovery of debilitated HIV-1 clones was mediated by an increase in quasispecies heterogeneity. This observation, together with the invariance of the consensus sequence despite fitness increases demonstrates the relevance of quasispecies heterogeneity in the evolution of HIV-1 in cell culture.

Use of Massive Parallel Pyrosequencing for Near Full-Length Characterization of a Unique HIV Type 1 BF Recombinant Associated with a Fatal Primary Infection

Near full length genome characterization of a BF recombinant from a patient who died from multiorgan failure during HIV-1 seroconversion is reported. Massive parallel pyrosequencing was used with the shotgun approach. Intrahost genetic variability along the whole genome was calculated and coreceptor usage of viral quasispecies was predicted. A consensus sequence was established to perform subtype assignment, phylogenetic analysis, and recombination tests. The sequence clustered with two recently described BF unique recombinant forms from Brazil, consistent with the recombination pattern, yielding breakpoints located at the same positions, with the exception of the second env breakpoint. The actual prevalence of recombinant forms is probably underestimated if partial genomic regions are considered. Here the first full length BF recombinant from Italy is described, together with an evaluation of quasispecies heterogeneity. Our data provide evidence that next generation sequencing may provide a major contribution to HIV-1 molecular epidemiology and to the comprehension of intrapatient heterogeneity.

Dynamic Hepatitis C Virus Genotypic and Phenotypic Changes in Patients Treated With the Protease Inhibitor Telaprevir

Telaprevir (VX-950), a hepatitis C virus (HCV) NS3.4A protease inhibitor, has shown strong antiviral activity in phase 1 clinical studies. Because of high levels of HCV replication and the low fidelity of HCV polymerase, selection of resistant isolates during therapy may occur. A highly sensitive sequencing method was developed in which approximately 80 clones/sample were analyzed to identify mutations in the NS3 protease catalytic domain in HCV genotype-1-infected patients dosed with 450 mg every 8 hours, 750 mg every 8 hours, or 1250 mg every 12 hours of telaprevir for 14 days. Mutations that confer low-level resistance (V36A/M, T54A, R155K/T, and A156S) and high-level resistance (A156V/T, 36+155, 36+156) to telaprevir were detected and correlated with telaprevir exposure and virologic response. Changes in the frequency of mutations after the end of dosing showed an inverse relationship between in vivo viral fitness and resistance. In the absence of telaprevir selective pressure the majority of resistant variants were replaced by wild-type virus within 3-7 months. Resistant HCV isolates are selected rapidly during therapy with the highly active protease inhibitor telaprevir. Combination therapy with pegylated interferon-alfa or other direct antiviral drugs seem mandatory to avoid developing resistance.

Is hepatitis C virus NS3 protease quasispecies heterogeneity predictive of progression from cirrhosis to hepatocellular carcinoma?

We investigated whether an HCV NS3 protease quasispecies heterogeneity was associated with progression from viral cirrhosis to hepatocellular carcinoma (HCC). The NS3 protease quasispecies structure of 10 HCV-1b cirrhotic patients (controls) was compared with that of 10 paired HCV-1b cirrhotic patients who displayed progression to HCC (cases). NS3 protease genetic complexity and diversity did not differ significantly between cases and controls. Amino acid substitutions were detected at 20 (11%) and 25 (14%) sites in at least two variants of the NS3 protease in cases and controls, respectively. Significant differences in the percentage of substituted clones were observed for 10 NS3 sites. Mutations Y56F, I71V, T72I, Q86P, P89S, S101G/D, R117H, S122G/T/N, V132I and V170I were more frequently observed in the NS3 protease sequences of controls than in those of cases. Residue V107 was substituted in NS3 cases but not in controls. However, these differences did not allow the definition of a specific NS3 profile related to HCC occurrence. The NS3 secondary structure B1-1 previously identified as potentially predictive of HCC was identified with a higher frequency in cases quasispecies (84.2%) than in controls (55.9%; P < 0.05). Our results suggest that there may be a relationship to fibrosis progression when diversity parameters are considered together with secondary structure profiles. Further investigations are required to determine the cellular interactions of HCV NS3 protease in the context of carcinogenesis.

Mutagenic Effect of Ribavirin on Hepatitis C Nonstructural 5B Quasispecies In Vitro and During Antiviral Therapy

Addition of ribavirin to interferon alfa treatment has substantially increased sustained virologic response rates in patients with chronic hepatitis C (CHC). Ribavirin acts as an RNA virus mutagen in vitro, thereby leading to error catastrophe. However, data in CHC are controversial. The nonstructural (NS) 5B quasi-species heterogeneity was analyzed in Huh7 cells harboring a subgenomic hepatitis C virus (HCV) replicon system treated with ribavirin or levovirin. Accordingly, NS5B quasi-species were studied in 14 patients with CHC who received ribavirin alone or combined with pegylated interferon alfa both at baseline and during the first weeks of therapy. Analysis of NS3 quasi-species served as control. Cultivation of HCV replicon cells with ribavirin led to higher NS5B mutational frequencies compared with levovirin-treated or untreated cells (P < .05). Patients receiving ribavirin monotherapy showed higher overall mutational frequencies within NS3 and NS5B during therapy as compared with baseline (P < .01). Proportions of ribavirin-specific G-to-A and C-to-T transitions increased (P < .01). Paired analysis confirmed significant mean increases of mutational frequencies of approximately 5%. Ribavirin serum concentrations were positively correlated with mutational frequency changes (P < .05). In patients receiving combination therapy, a decrease of NS5B mutational frequencies ( approximately 10%) and lower proportions of G-to-A and T-to-C mutations (P < .01) were detectable. Ribavirin, but not its L-enantiomer levovirin, is a mutagen in HCV replicon cells. In patients with CHC, ribavirin monotherapy exhibits a moderate mutagenic effect early during therapy that is not detectable in combination with pegylated interferon alfa.

AIDS vaccine development and challenge viruses: getting real

AIDS vaccine development and challenge viruses: getting real

Evolution of Hepatitis C Virus Quasispecies in Children with Chronic Hepatitis C

Hepatitis C virus (HCV) circulates as a mixture of different but closely related genomes: this quasispecies nature could be essential for virus persistence and could induce resistance to interferon therapy. Since little is known on the behavior of HCV quasispecies in children and adolescents with chronic hepatitis C, we analyzed the virus population in six untreated children during a 5-year follow-up. Six children aged 1-8 years, infected early in life with HCV, were included in the study. From each of them, 2 or 3 sequential serum samples obtained over a 5-year follow-up period were examined. The HCV quasispecies heterogeneity and diversity in the E2 hypervariable region-1 (HVR-1) were analyzed among samples by the heteroduplex mobility assay, and the distance between variants was estimated by the heteroduplex mobility ratio (HMR). The HCV population was initially highly homogeneous in all six children. During follow-up, diversification of HVR-1 leading to a more complex viral population occurred in all cases, and was particularly evident in the three older children (HMR: 0.82-0.54). Changes in the HVR-1 sequence occurred without relation to the profile of ALT and HCV-RNA levels. HCV quasispecies diversification is a common event during chronic hepatitis C in childhood. Host and environmental pressure could be major determinants. The increasing viral heterogeneity could impair the response to antiviral therapy, thus indicating a rationale for early antiviral treatment in children with chronic hepatitis C.

HIV Infection and Antiretroviral Therapy: Effect on Hepatitis C Virus Quasispecies Variability

Hepatitis C virus (HCV) quasispecies variability has been associated with liver disease progression. The effects of human immunodeficiency virus (HIV) coinfection and highly active antiretroviral therapy (HAART) on HCV quasispecies variability have not been firmly established. We determined HCV quasispecies complexity and diversity in 69 subjects, 28 of whom were HIV infected, using clonal frequency analysis via heteroduplex mobility analysis of the second envelope gene hypervariable region. Nucleotide sequencing was performed for a small subset of subjects. HIV-positive, HAART-naive subjects had significantly lower HCV quasispecies complexity and diversity than did both HIV-negative and HIV-positive HAART-treated subjects. In multivariate analysis, HIV infection predicted decreased complexity (P < .0001) and diversity (P = .001) of HCV quasispecies, whereas HAART predicted increased complexity (P = .013) and diversity (P = .026). For 4 of 6 patients, sequence analysis yielded data supporting the model that positive host pressure drives HCV quasispecies heterogeneity, although data favoring the hypothesis of selective outgrowth of the most fit variants were also observed. HIV coinfection is associated with decreased HCV quasispecies variability, which appears to be reversed by effective HAART. Although HIV- and HAART-related effects on host immune pressure are likely to play a role in the observed differences in HCV genetic heterogeneity, other mechanisms may be operative.

Changes in mumps virus neurovirulence phenotype associated with quasispecies heterogeneity

Mumps virus is a highly neurotropic virus with evidence of central nervous system invasion (CNS) in approximately half of all cases of infection. In countries where live attenuated mumps virus vaccines were introduced, the number of mumps cases declined dramatically; however, recently, the safety of some vaccine strains has been questioned. For example, one of the most widely used vaccines, the Urabe AM9 strain, was causally associated with meningitis, leading to the withdrawal of this product from the market in several countries. This highlights the need for a better understanding of the attenuation process and the identification of markers of attenuation. To this end, we further attenuated the Urabe AM9 strain by serial passage in cell culture and compared the complete nucleotide sequences of the parental and passaged viruses. Interestingly, despite a dramatic decrease in virus virulence (as assayed in rats), the only genomic changes were in the form of changes in the level of genetic heterogeneity at specific genome sites, i.e., either selection of one nucleotide variant at positions where the starting material exhibited nucleotide heterogeneity or the evolution of an additional nucleotide to create a heterogenic site. This finding suggests that changes in the level of genetic heterogeneity at specific genome sites can have profound neurovirulence phenotypic consequences and, therefore, caution should be exercised when evaluating genetic markers of virulence or attenuation based only on a consensus sequence.

Evolution of HVR-1 Quasispecies after 1-Year Treatment in HIV/HCV-Coinfected Patients According to the Pattern of Response to Highly Active Antiretroviral Therapy

Hepatitis C virus (HCV) variability is mainly attributed to the ability of the virus to respond to host immune pressure, acting as a driving force for the evolution of quasispecies. This study was aimed at studying the changes in HVR-1 heterogeneity and the evolution of HCV quasispecies in HIV/HCV-coinfected patients according to the pattern of response to highly active antiretroviral therapy (HAART). Sixteen HIV/HCV-coinfected patients harbouring HCV genotype 1 and who had been on HAART for at least 1 year, 8 showing increasing CD4+ T-cell counts (immunological responders) and 8 showing a stable or decreasing CD4+ T-cell counts (immunological nonresponders), were selected from a prospective cohort study. After 1 year of HAART, 11 patients showed HIV viral load <2.6 log10 cp/ml (virological responders), and 5 showed HIV viral load above this value (virological non-responders). Plasma samples, collected before starting therapy and after 1 year of HAART, underwent clonal sequence analysis for HVR-1 region of HCV. Nonsynonymous/synonymous substitutions ratio (Ka/Ks), aminoacidic complexity (normalized Shannon entropy) and diversity (p-distance), were considered as parameters of quasispecies heterogeneity. After 1 year of HAART, heterogeneity of HVR-1 quasispecies significantly decreased in virological non-responders, whereas the heterogeneity tended to increase in virological responders. The differences in the evolution were less stringent, when considering immunological response. On the other hand, profound qualitative modifications of HVR-1 quasispecies were observed only in patients with both immunological and virological HAART response. On the whole, these findings suggest that, in patients undergoing HAART, the extent of HCV variability and the evolution of HVR-1 quasispecies is influenced by the pattern of response to antiretroviral therapy.

Clinical Significance of In Vitro Replication–Enhancing Mutations of the Hepatitis C Virus (HCV) Replicon in Patients with Chronic HCV Infection

Mutations in nonstructural (NS) hepatitis C virus (HCV) proteins enhance replication in HCV-1a/b replicons. The prevalence of such mutations and their clinical significance in vivo are unknown. Parts of HCV NS3 and NS4B-NS5B genes that included 31 in vitro replication-enhancing sites were sequenced for 26 patients with chronic HCV genotype 1 infection. Five patients showed specific mutations within NS3 at sites enhancing replication in the replicon. Those mutations were associated with a slower decrease in HCV RNA concentration during interferon (IFN)- alpha -based therapy (P = .007). Neither specific nor other mutations within NS3 and NS4B-NS5B were associated with baseline HCV RNA concentrations. Within NS5A, fewer mutations in the major HCV strain (P = .001) and increased quasi-species complexity (P = .02) and diversity (P = .02) correlated with increasing baseline HCV RNA concentrations. In silico analyses of NS3 protein structures suggested that the majority of observed mutations did not lead to major conformational changes. Specific mutations leading to enhanced replication in the replicon system were detected in 5 of 26 patients in vivo and were not associated with baseline HCV RNA concentrations but were associated with a slower decrease in HCV RNA concentration during IFN- alpha -based therapy. Quasi-species heterogeneity of NS5A correlated with baseline HCV RNA concentrations.