Pretreatment sequence diversity differences in the full-length hepatitis C virus open reading frame correlate with early response to therapy

Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.     

Evolution of human immunodeficiency virus type 1 env sequence variation in patients with diverse rates of disease progression and T-cell function.

We examined the relationship between env sequence variation and disease progression in 10 human immunodeficiency virus type 1 (HIV-1)-seropositive subjects selected from a longitudinal cohort receiving zidovudine therapy. Five subjects were chosen for stable clinical status and CD4 counts (slow progressors), and five were selected for rapid clinical deterioration and CD4 count decline (rapid progressors). The slow progressors had significantly lower plasma viral RNA loads and greater lymphoproliferative responses to mitogens than the rapid progressors. DNA sequences representing the C1 through C3 regions of env were amplified from two peripheral blood mononuclear cell DNA samples from each subject separated by an average of 2.5 years. Molecular clones of these amplicons were then sequenced, and DNA sequence and deduced amino acid sequence distances were compared. Inter-time point sequence comparison showed a higher rate of sequence evolution for the rapid progressors in three of five matched pairs of rapid progressors and slow progressors and for the slow progressors in the remaining two subject pairs. However, intra-time point sequence comparisons showed that four of five slow progressors developed a more diverse quasispecies over time and one showed no change. In contrast, four of five rapid progressors showed no change in quasispecies diversity over time and one showed a significant decrease in diversity. The overall C1 through C3 region quasispecies diversity in the slow progressors at baseline was lower than that for the rapid progressors, but this difference was not significant at the follow-up time points. These diversity relationships were obscured if sequence analyses were limited to the 300-bp C2 to V3 region. Thus, HIV-1 quasispecies diversity increased over time in subjects with more functional immune systems.     

Sequence analysis of NS3 protease gene in clinical strains of hepatitis C virus

The amino terminal region of the non structural gene 3 (NS3) of hepatitis C virus (HCV) is a chymotripsinlike serine-protease responsible for cleavage of the non structural proteins of Hepatitis C virus (HCV). In order to investigate the genetic variation of this region, we developed a nested PCR to obtain NS3 protease sequences from 54 patients chronically infected with HCV genotypes 1a, 1b and 3, respectively. Comparison of nucleotide and amino acids sequences of NS3 protease domain with consensus sequence obtained within the same genotype, showed 3.73% nucleotide divergence and 1.64% amino acid divergence in isolates of genotype 3a, whereas isolates 1a exhibited 4.45% nucleotide and 4% amino acid change, respectively. Finally, NS3 sequence from 1b isolates revealed 6.47% nucleotide and 3.5 % aa changes. Comparison of consensus amino acid sequences derived from isolates 1a, 1b and 3, with the HCV prototypes showed a low amino acid sequence diversity. However, the consensus sequence of HCV genotype 3 isolates showed an amino acid changed from the prototype, that was located within a region important for enzyme structure and activity. These results indicated that the NS3 protease gene is highly conserved within the same HCV genotype. The domains involved in enzyme function were highly conserved in 1a and 1b strains, whereas consensus sequence of isolates 3a showed that the majority of these strains were not perfectly conserved in one of such regions. These findings altogether suggested that the NS3 protease enzyme of HCV may constitute an important target for antiviral therapy, but the NS3 protease variability of isolates 3 within a region that is a potential target for antiviral therapy could pose a problem for structure based drug development.     

Baseline CD4+ T Cell Counts Correlates with HIV-1 Synonymous Rate in HLA-B*5701 Subjects with Different Risk of Disease Progression

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although risk of progression may vary among patients carrying this allele. The interplay between HIV-1 evolutionary rate variation and risk of progression to AIDS in HLA-B*5701 subjects was studied using longitudinal viral sequences from high-risk progressors (HRPs) and low-risk progressors (LRPs). Posterior distributions of HIV-1 genealogies assuming a Bayesian relaxed molecular clock were used to estimate the absolute rates of nonsynonymous and synonymous substitutions for different set of branches. Rates of viral evolution, as well as in vitro viral replication capacity assessed using a novel phenotypic assay, were correlated with various clinical parameters. HIV-1 synonymous substitution rates were significantly lower in LRPs than HRPs, especially for sets of internal branches. The viral population infecting LRPs was also characterized by a slower increase in synonymous divergence over time. This pattern did not correlate to differences in viral fitness, as measured by in vitro replication capacity, nor could be explained by differences among subjects in T cell activation or selection pressure. Interestingly, a significant inverse correlation was found between baseline CD4⁺ T cell counts and mean HIV-1 synonymous rate (which is proportional to the viral replication rate) along branches representing viral lineages successfully propagating through time up to the last sampled time point. The observed lower replication rate in HLA-B*5701 subjects with higher baseline CD4⁺ T cell counts provides a potential model to explain differences in risk of disease progression among individuals carrying this allele.     

Deep-Sequencing Analysis of the Association between the Quasispecies Nature of the Hepatitis C Virus Core Region and Disease Progression

Variation of core amino acid (aa) 70 of hepatitis C virus (HCV) has been shown recently to be closely correlated with liver disease progression, suggesting that the core region might be present as a quasispecies during persistent infection and that this quasispecies nature might have an influence on the progression of disease. In our investigation, the subjects were 79 patients infected with HCV genotype 1b (25 with chronic hepatitis [CH], 29 with liver cirrhosis [LC], and 25 with hepatocellular carcinoma [HCC]). Deep sequencing of the HCV core region was carried out on their sera by using a Roche 454 GS Junior pyrosequencer. Based on a plasmid containing a cloned HCV sequence (pCV-J4L6S), the background error rate associated with pyrosequencing, including the PCR procedure, was calculated as 0.092 ± 0.005/base. Deep sequencing of the core region in the clinical samples showed a mixture of “mutant-type” Q/H and “wild-type” R at the core aa 70 position in most cases (71/79 [89.9%]), and the ratio of mutant residues to R in the mixture increased as liver disease advanced to LC and HCC. Meanwhile, phylogenetic analysis of the almost-complete core region revealed that the HCV isolates differed genetically depending on the mutation status at core aa 70. We conclude that the core aa 70 mixture ratio, determined by deep sequencing, reflected the status of liver disease, demonstrating a significant association between core aa 70 and disease progression in CH patients infected with HCV genotype 1b.     

Contribution of Genome-Wide HCV Genetic Differences to Outcome of Interferon-Based Therapy in Caucasian American and African American Patients

Background

Hepatitis C virus (HCV) has six major genotypes, and patients infected with genotype 1 respond less well to interferon-based therapy than other genotypes. African American patients respond to interferon α-based therapy at about half the rate of Caucasian Americans. The effect of HCV''s genetic variation on treatment outcome in both racial groups is poorly understood.

Methodology

We determined the near full-length pre-therapy consensus sequences from 94 patients infected with HCV genotype 1a or 1b undergoing treatment with peginterferon α-2a and ribavirin through the Virahep-C study. The sequences were stratified by genotype, race and treatment outcome to identify HCV genetic differences associated with treatment efficacy.

Principal Findings

HCV sequences from patients who achieved sustained viral response were more diverse than sequences from non-responders. These inter-patient diversity differences were found primarily in the NS5A gene in genotype 1a and in core and NS2 in genotype 1b. These differences could not be explained by host selection pressures. Genotype 1b but not 1a African American patients had viral genetic differences that correlated with treatment outcome.

Conclusions & Significance

Higher inter-patient viral genetic diversity correlated with successful treatment, implying that there are HCV genotype 1 strains with intrinsic differences in sensitivity to therapy. Core, NS3 and NS5A have interferon-suppressive activities detectable through in vitro assays, and hence these activities also appear to function in human patients. Both preferential infection with relatively resistant HCV variants and host-specific factors appear to contribute to the unusually poor response to therapy in African American patients.     

Analysis of the complete open reading frame of genotype 2b hepatitis C virus in association with the response to peginterferon and ribavirin therapy

Background and Aims

Patients infected with genotype 2b hepatitis C virus (HCV) generally can achieve favorable responses to pegylated-interferon plus ribavirin therapy (PEG-IFN/RBV). However, a proportion of patients show poorer responses and the correlation between viral sequence variation and treatment outcome remains unclear.

Methods

The pretreatment complete open reading frame (ORF) sequences of genotype 2b HCV determined by direct sequencing were investigated for correlation with the final outcome in a total of 60 patients.

Results

In this study group, 87.5% (14/16) of non-sustained virological response (non-SVR) patients (n = 16) were relapsers. Compared to sustained virological response (SVR) patients (n = 44), non-SVR patients were older and could not achieve prompt viral clearance after the therapy induction. Comparing each viral protein between the two groups, viral sequences were more diverse in SVR patients and that diversity was found primarily in the E1, p7, and NS5A proteins. In searching for specific viral regions associated with the final outcome, several regions in E2, p7, NS2, NS5A, and NS5B were extracted. Among these regions, part of the interferon sensitivity determining region (ISDR) was included. In these regions, amino acid substitutions were associated with the final outcome in an incremental manner, depending upon the number of substitutions.

Conclusions

Viral sequences are more diverse in SVR patients than non-SVR patients receiving PEG-IFN/RBV therapy for genotype-2b HCV infection. Through systematic comparison of viral sequences, several specific regions, including part of the ISDR, were extracted as having significant correlation with the final outcome.     

In vivo sequence variability of human immunodeficiency virus type 1 envelope gp120: association of V2 extension with slow disease progression.

According to the rate of depletion of CD4 cell counts, we grouped 12 cases of human immunodeficiency virus type 1 (HIV-1) infection as 6 rapid (21.0 to 33.8 cells per microl per month) and 6 slow (0.9 to 7.9 cells per microl per month) progressors and determined the individual viral quasispecies patterns by sequencing the genome region encoding the V1, V2, and V3 loops of envelope protein. Although the quasispecies structures varied widely from one individual to another, a strong correlation was observed between a low rate of disease progression and a high degree of genetic diversity of HIV-1. Furthermore, the V2 loop extension was observed specifically in individuals with slow or no disease progression, whereas basic amino acid substitutions in V3 characteristic of a viral phenotype shift from non-syncytium inducing to syncytium inducing were observed in patients with advanced stages of disease regardless of their rate of disease progression. Studies with recombinant viruses suggested that elongation of V2 potentially restricts the capacity of HIV-1 to replicate in macrophages. Thus, our results suggest the association of distinct sequence features of both V3 and V2 with particular patterns of disease progression. Elongation of the V2 loop may be a good predictor of slow disease progression, while basic substitutions of V3 without elongation of V2 are characteristic of rapid progression.     

Acute Plasma Biomarkers of T Cell Activation Set-Point Levels and of Disease Progression in HIV-1 Infection

T cell activation levels, viral load and CD4⁺ T cell counts at early stages of HIV-1 infection are predictive of the rate of progression towards AIDS. We evaluated whether the inflammatory profile during primary HIV-1 infection is predictive of the virological and immunological set-points and of disease progression. We quantified 28 plasma proteins during acute and post-acute HIV-1 infection in individuals with known disease progression profiles. Forty-six untreated patients, enrolled during primary HIV-1 infection, were categorized into rapid progressors, progressors and slow progressors according to their spontaneous progression profile over 42 months of follow-up. Already during primary infection, rapid progressors showed a higher number of increased plasma proteins than progressors or slow progressors. The plasma levels of TGF-β1 and IL-18 in primary HIV-1 infection were both positively associated with T cell activation level at set-point (6 months after acute infection) and together able to predict 74% of the T cell activation variation at set-point. Plasma IP-10 was positively and negatively associated with, respectively, T cell activation and CD4⁺ T cell counts at set-point and capable to predict 30% of the CD4⁺ T cell count variation at set-point. Moreover, plasma IP-10 levels during primary infection were predictive of rapid progression. In primary infection, IP-10 was an even better predictor of rapid disease progression than viremia or CD4⁺ T cell levels at this time point. The superior predictive capacity of IP-10 was confirmed in an independent group of 88 HIV-1 infected individuals. Altogether, this study shows that the inflammatory profile in primary HIV-1 infection is associated with T cell activation levels and CD4⁺ T cell counts at set-point. Plasma IP-10 levels were of strong predictive value for rapid disease progression. The data suggest IP-10 being an earlier marker of disease progression than CD4⁺ T cell counts or viremia levels.     

Prospective characterization of full-length hepatitis C virus NS5A quasispecies during induction and combination antiviral therapy

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR(237-276)) sequence associated with IFN resistance was not found, although the presence of Ala(245) within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P<0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P<0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P<0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.     

Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals

Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4-18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance.     

Conservation of the conformation and positive charges of hepatitis C virus E2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.     

HIV-1 Replication Fitness of HLA-B*57/58:01 CTL Escape Variants Is Restored by the Accumulation of Compensatory Mutations in Gag

Expression of HLA-B*57 and the closely related HLA-B*58:01 are associated with prolonged survival after HIV-1 infection. However, large differences in disease course are observed among HLA-B*57/58:01 patients. Escape mutations in CTL epitopes restricted by these HLA alleles come at a fitness cost and particularly the T242N mutation in the TW10 CTL epitope in Gag has been demonstrated to decrease the viral replication capacity. Additional mutations within or flanking this CTL epitope can partially restore replication fitness of CTL escape variants. Five HLA-B*57/58:01 progressors and 5 HLA-B*57/58:01 long-term nonprogressors (LTNPs) were followed longitudinally and we studied which compensatory mutations were involved in the restoration of the viral fitness of variants that escaped from HLA-B*57/58:01-restricted CTL pressure. The Sequence Harmony algorithm was used to detect homology in amino acid composition by comparing longitudinal Gag sequences obtained from HIV-1 patients positive and negative for HLA-B*57/58:01 and from HLA-B*57/58:01 progressors and LTNPs. Although virus isolates from HLA-B*57/58:01 individuals contained multiple CTL escape mutations, these escape mutations were not associated with disease progression. In sequences from HLA-B*57/58:01 progressors, 5 additional mutations in Gag were observed: S126N, L215T, H219Q, M228I and N252H. The combination of these mutations restored the replication fitness of CTL escape HIV-1 variants. Furthermore, we observed a positive correlation between the number of escape and compensatory mutations in Gag and the replication fitness of biological HIV-1 variants isolated from HLA-B*57/58:01 patients, suggesting that the replication fitness of HLA-B*57/58:01 escape variants is restored by accumulation of compensatory mutations.     

Human immunodeficiency virus (HIV)-specific gamma interferon secretion directed against all expressed HIV genes: relationship to rate of CD4 decline

Immune responses to human immunodeficiency virus (HIV) are detected at all stages of infection and are believed to be responsible for controlling viremia. This study seeks to determine whether gamma interferon (IFN-gamma)-secreting HIV-specific T-cell responses influence disease progression as defined by the rate of CD4 decline. The study population consisted of 31 subjects naive to antiretroviral therapy. All were monitored clinically for a median of 24 months after the time they were tested for HIV-specific responses. The rate of CD4+-T-cell loss was calculated for all participants from monthly CD4 counts. Within this population, 17 subjects were classified as typical progressors, 6 subjects were classified as fast progressors, and 8 subjects were classified as slow progressors. Peripheral blood mononuclear cells were screened for HIV-specific IFN-gamma responses to all expressed HIV genes. Among the detected immune responses, 48% of the recognized peptides were encoded by Gag and 19% were encoded by Nef gene products. Neither the breadth nor the magnitude of HIV-specific responses correlated with the viral load or rate of CD4 decline. The breadth and magnitude of HIV-specific responses did not differ significantly among typical, fast, and slow progressors. These results support the conclusion that although diverse HIV-specific IFN-gamma-secreting responses are mounted during the asymptomatic phase, these responses do not seem to modulate disease progression rates.     

Evolution of human immunodeficiency virus type 1 in perinatally infected infants with rapid and slow progression to disease.

We addressed the relationship between the origin and evolution of human immunodeficiency virus type 1 (HIV-1) variants and disease outcome in perinatally infected infants by studying the V3 regions of viral variants in samples obtained from five transmitting mothers at delivery and obtained sequentially over the first year of life from their infected infants, two of whom (rapid progressors) rapidly progressed to having AIDS. Phylogenetic analyses disclosed that the V3 sequences from each mother-infant pair clustered together and were clearly distinct from those of the other pairs. Within each pair, the child's sequences formed a monophyletic group, indicating that a single variant initiated the infection in both rapid and slow progressors. Plasma HIV-1 RNA levels increased in all five infants during their first months of life and then declined within the first semester of life only in the three slow progressors. V3 variability increased over time in all infants, but no differences in the pattern of V3 evolution in terms of potential viral phenotype were observed. The numbers of synonymous and nonsynonymous substitutions varied during the first semester of life regardless of viral load, CD4+-cell count, and disease progression. Conversely, during the second semester of life the rate of nonsynonymous substitutions was higher than that of synonymous substitutions in the slow progressors but not in the rapid progressors, thus suggesting a stronger host selective pressure in the former. In view of the proposal that V3 genetic evolution is driven mainly by host immune constraints, these findings suggest that while the immune response to V3 might contribute to regulating viral levels after the first semester of life, it is unlikely to play a determinant role in the initial viral decline soon after birth.     

Dynamic behavior of hepatitis C virus quasispecies in patients undergoing orthotopic liver transplantation.

We have studied the distribution of viral sequences from the 5' noncoding region and from a fragment of the E2/NS2 region of the hepatitis C virus (HCV) genome in samples obtained before and after liver transplantation in two patients with HCV cirrhosis. The population of viral sequences in both regions were established by sequencing cloned PCR products. In both cases, the complexity of the viral quasispecies decreased after transplantation, although the consensus nucleotide and amino acid sequences remained unchanged. It is suggested that both positive and negative selection and random sampling events contribute substantially in shaping the genetic composition of HCV quasispecies and that recurrence of HCV infection may occur under equilibrium conditions.     

A novel genotype of GB virus C: its identification and predominance among injecting drug users in Yunnan, China

GB virus C (GBV-C) is prevalent globally and particularly among individuals at risk of parental exposures. Based on genetic diversity, this virus is now classified into six genotypes and many subtypes with distinct geographical distribution. In this study, 120 Injecting Drug Users (IDUs) were recruited from Yunnan province, China. Among them, 43 (35.8%) were positive for GBV-C RNA, 70 (58.3%) and 103 (85.8%) sero-positive for HIV-1 and HCV respectively. This revealed 18.3% of IDUs having GBV-C/HIV/HCV triple infection, which is significantly higher than 7.5% of GBV-C/HIV-1 and 10% of GBV-C/HCV dual infection rates (P<0.05). Based on 5'UTR sequences, the identified 43 viral isolates can be classified into three phylogenetic groups: one (2.3%) and two (4.7%) belonged to genotype 3 and 4, respectively, and the remaining 40 (93%) formed a new group with 97% of bootstrap support. This new GBV-C group was further confirmed by characterizing the E2 region and full-length genome sequences. Analysis of 187 nt 5'UTR sequence showed three previous reported isolates from Southeast Asia were re-classified into this new group. It implies they have the same origin with strains from Yunnan. Although we provisionally assigned this new group as GBV-C genotype 7, a simpler five groups of GBV-C nomenclature is recommended. Genotype 4, 6 and the newly designated genotype 7 could be reclassified as one group, which may represent a single GBV-C genotype. The classification of the other four groups was corresponding to that of previous reported genotype 1, 2, 3 and 5. Furthermore, the diversity of amino acid sequence in the E2 region was analyzed. The inhibitory effect of GBV-C genotype 7 on HIV-1 cell entry could be deduced. Since GBV-C may have a beneficial effect on AIDS disease progression and interact with HCV during co-infection, this finding may raise interests in future studies on this virus that was previously thought to be a "non-pathogenic virus".     

Comparison of Therapeutic Response and Clinical Outcome between HCV Patients with Normal and Abnormal Alanine Transaminase Levels

Background and Aims

Patients with chronic hepatitic C (HCV) infection and normal serum alanine transaminase (ALT) levels were considered to have mild disease. In Taiwan, these patients were not suggested for interferon (IFN) based therapies. The aim of study is to compare therapeutic outcomes between HCV patients with normal and elevated ALT levels.

Methods

We conducted a retrospective study on 3241 HCV patients treated by IFN based therapies. Patients with normal ALT levels were classified as group A (n = 186) while those with elevated ALT levels were group B (n = 3055).

Results

At baseline, incidence of diabetes mellitus, low platelet counts and cirrhosis were significantly higher in group B patients. The sustained virologic response (SVR) rate was comparable between the 2 groups (65.3% vs. 65.3%, P = .993). But significantly higher incidence of HCC development after HCV treatment was observed in group B (7.4% vs. 3.2%, P = .032). No significant differences with respect to the outcome of liver decompensation, spontaneous bacterial peritonitis, and mortality were noted between 2 groups. Multivariate analysis showed younger age, female gender, non-HCV genotype 1, lower viral load, higher platelet counts and non-cirrhosis were favorable factors for achieving SVR, rather than ALT levels. Further analysis revealed older age, cirrhosis, lower platelet levels and non- peg-interferon treatment are risk factors of HCC development.

Conclusions

HCV patients with normal ALT levels had similar response to antiviral therapy and low rate of HCC development after therapy. Antiviral therapies begun at early course of HCV infection may be beneficial to prevent disease progression.     

Hepatitis C virus nonstructural 5A protein and interferon resistance: a new model for testing the reliability of mutational analyses

Presumably due to the capability of the hepatitis C virus (HCV) to evade the antiviral effects of alpha interferon, treatment is ineffective in more than half of chronically genotype HCV type 1 (HCV-1)-infected patients. Previous approaches to correlate the number of amino acid mutations within regions of HCV nonstructural (NS)-5A protein with virologic treatment response provided conflicting results. In the present study, we developed a new mathematical model to investigate NS5A sequences of HCV-1-infected patients. The mean number of all mutations within the complete NS5A protein was significantly higher in virologic responders compared to nonresponders (P = 0.008 and P = 0.0001 for amino acid residues predicted on the surface of the NS5A protein). Differences did not achieve statistical significance for NS5A regions that are currently assumed to be functionally relevant (e.g., the interferon sensitivity-determining region, the RNA-activated protein kinase-binding domain, etc.). Analyses of smoothed mutational frequencies showed that the number of mutations in other NS5A regions correlated with virologic response. Such a correlation was observed for both genuine and randomly generated NS5A sequences. The existence of local accumulations of mutations within genuine NS5A isolates that truly correlated with treatment response was defined by a refined test procedure. Upon considering the predicted residue accessibility, we identified the main focus of mutations correlating with treatment response to be the sequence from amino acids 2350 to 2370. Thus, evaluation of NS5A mutations in correlation with treatment response is improved by consideration of functional and predicted conformational amino acid properties. As shown by simulations with randomly generated sequences, multiple analyses of simple counts of local NS5A amino acid mutations and correlation with treatment response are insufficient. For improvement of mutational analysis, a refined specific functional data test procedure is proposed.