Background and Aim
Children chronically infected with hepatitis B virus (HBV) are at high risk of progressive liver disease. However, no treatment is available that is consistently effective in curing chronic hepatitis B (CHB) in children. Improved understanding of the natural course of disease is warranted. Identification of specific microRNA (miRNA) profiles in children chronically infected with HBV may provide insight into the pathogenesis of CHB and lead to advances in the management of children with CHB.
Patients and Methods
MiRNA PCR panels were employed to screen plasma levels of 739 miRNAs in pooled samples from HBeAg positive, HBeAg negative, and healthy children. The three groups’ plasma miRNA profiles were compared, and aberrantly expressed miRNAs were identified. The identified miRNAs were then validated. Individual RT-qPCRs were performed on plasma from 34 HBeAg positive, 26 HBeAg negative, and 60 healthy children.
A panel of 16 plasma miRNAs were identified as aberrantly expressed in HBeAg positive and HBeAg negative children (p<0.001). Levels of all of the miRNAs were upregulated in HBeAg positive children compared with in HBeAg negative children. A positive correlation was furthermore found between plasma levels of the identified miRNAs and HBV DNA (p<0.001).
We are the first to investigate the plasma miRNA profile of children chronically infected with HBV. Our data indicates the existence of a relationship between abundance of circulating miRNAs and immunological stages in the natural course of disease. Certain miRNAs may contribute to the establishment and maintenance of CHB in children. Further studies are warranted to advance understanding of miRNAs in the pathogenesis of CHB, hopefully leading to the identification of future therapeutic targets.
GB virus C (GBV-C) is a member of the Flaviviridae family and the most closely related human virus to HCV. However, GBV-C does not replicate in hepatocytes, but rather in lymphocytes. GBV-C has a worldwide distribution and is transmitted sexually, parenterally and through mother-to-child transmission. Thus, co-infection with HCV and HIV is common. Until now, no human disease has been associated with GBV-C infection. However, there are several reports of a beneficial effect of GBV-C on HIV disease progression in vivo. Different mechanisms to explain these observations have been proposed, including modification of antiviral cytokine production, HIV co-receptor expression, direct inhibition of HIV-1 entry, T-cell activation and Fas-mediated apoptosis. Further understanding of these mechanisms may open new strategies for the treatment of HIV/AIDS.
co-infection; GBV-C; HCV; HIV
DNA methylation is being increasingly recognized to play a role in regulation of hepatitis B virus (HBV) gene expression. The aim of this study was to compare the CpG island distribution among different HBV genotypes. We analyzed 176 full-length HBV genomic sequences obtained from the GenBank database, belonging to genotypes A through J, to identify the CpG islands in the HBV genomes. Our results showed that while 79 out of 176 sequences contained three conventional CpG islands (I–III) as previously described, 83 HBV sequences harbored only two of the three known islands. Novel CpG islands were identified in the remaining 14 HBV isolates and named as CpG island IV, V, and VI. Among the eight known HBV genotypes and two putative genotypes, while HBV genomes containing three CpG islands were predominant in genotypes A, B, D, E, and I; genotypes C, F, G, and H tended to contain only two CpG islands (II and III). In conclusion, the CpG islands, which are potential targets for DNA methylation mediated by the host functions, differ among HBV genotypes, and these genotype-specific differences in CpG island distribution could provide new insights into the understanding of epigenetic regulation of HBV gene expression and hepatitis B disease outcome.
Viral diversity is a hallmark of hepatitis C virus (HCV) infection; however, only limited data are available regarding HCV variability in extrahepatic sites, and none have systematically compared diversity in non-structural and structural genomic regions. Therefore, HCV diversity in the NS5B and envelope 1 (E1) hypervariable region 1 (HVR1) genes was evaluated in matched sera and peripheral blood mononuclear cells (PBMCs) obtained from 13 HCV-infected women. Multiple clonal sequences were compared to evaluate quasispecies diversity and viral compartmentalization in PBMCs.
Genetic distances were higher for E1/HVR1 compared to NS5B in both the sera and PBMCs (p = 0.0511 and p = 0.0284). Genetic distances were higher in serum NS5B compared to PBMC NS5B (p = 0.0003); however, they were not different when comparing E1/HVR1 in sera to PBMCs. By phylogenetic analysis of NS5B, evidence of possible PBMC compartmentalization was observed for 1 woman, while statistical methods were consistent with PBMC compartmentalization for 6 women. Evidence of compartmentalization within a non-structural genomic region may suggest that viral adaptation to a unique extracellular microenvironment(s) may be required for efficient replication and could contribute to HCV persistence.
NS5B; HVR1; diversity; quasispecies; extrahepatic replication
Wuwei City has the highest prevalence of hepatitis B virus (HBV) in China. From 2007 to 2011, the average reported incidence rate of hepatitis B was 634.56/100,000 people. However, studies assessing the epidemic features and risk factors of HCV in the general population of Wuwei City are limited.
A total of 7189 people were interviewed and screened for HCV antibodies. HCV RNA and HCV genotypes were analyzed by PCR. Relevant information was obtained from the general population using a standardized questionnaire, and association and logistic regression analyses were conducted.
The anti-HCV prevalence was 1.64% (118/7189), and HCV-RNA was detected in 37.29% (44/118) of the anti-HCV positive samples. The current HCV infection rate was 0.61% (44/7189) in the Wuwei general population. Hepatitis C infection rate was generally higher in the plains regions (χ2 = 27.54,P<0.05), and the most predominant HCV genotypes were 2a (59.1%) and 1b (34.1%). The concurrent HCV and HBV infection rate was 1.37%, and a history of blood transfusion (OR = 17.9, 95% CI: 6.1 to 52.6, p<0.001) was an independent risk factor for HCV positivity.
Although Wuwei is a highly endemic area for HBV, the anti-HCV positive rate in the general population is low. More than one-third of HCV-infected people were unaware of their infection; this may become an important risk factor for hepatitis C prevalence in the general population. Maintaining blood safety is important in order to help reduce the burden of HCV infection in developing regions of China.
Molecular mechanisms related to occult hepatitis B virus (HBV) infection, particularly those based on genotype C infection, have rarely been determined thus far in the ongoing efforts to determine infection mechanisms. Therefore, we aim to elucidate the mutation patterns in the surface open reading frame (S ORF) underlying occult infections of HBV genotype C in the present study. Nested PCRs were applied to 624 HBV surface antigen (HBsAg) negative Korean subjects. Cloning and sequencing of the S ORF gene was applied to 41 occult cases and 40 control chronic carriers. Forty-one (6.6%) of the 624 Korean adults with HBsAg-negative serostatus were found to be positive for DNA according to nested PCR tests. Mutation frequencies in the three regions labeled here as preS1, preS2, and S were significantly higher in the occult subjects compared to the carriers in all cases. A total of two types of deletions, preS1 deletions in the start codon and preS2 deletions as well as nine types of point mutations were significantly implicated in the occult infection cases. Mutations within the “a” determinant region in HBsAg were found more frequently in the occult subjects than in the carriers. Mutations leading to premature termination of S ORF were found in 16 occult subjects (39.0%) but only in one subject from among the carriers (2.5%). In conclusion, our data suggest that preS deletions, the premature termination of S ORF, and “a” determinant mutations are associated with occult infections of HBV genotype C among a HBsAg-negative population. The novel mutation patterns related to occult infection introduced in the present study can help to broaden our understanding of HBV occult infections.
Due to shared transmission routes, hepatitis C virus (HCV) infection is highly prevalent among people infected with human immunodeficiency virus (HIV). Highly active antiretroviral therapy (HAART) is associated with hepatotoxicity, leading to the negative effects on patients with HIV/HCV co-infection. In order to provide valuable information for HCV management in this particular population, we investigated the HCV genotypes in HIV-infected individuals from Henan and Guangxi, the two provinces with the most HIV-infected cases in China.
Individuals, who acquired HIV infection through various risk routes, were recruited from Henan and Guangxi. Test of antibodies against HCV (anti-HCV) was conducted, and detection of HCV RNA was performed by PCR amplification. HCV subtypes were determined by direct sequencing of amplicons, followed by phylogenetic analysis.
We recruited a total of 1,112 HIV-infected people in this present study. Anti-HCV was detected from 218 (50.1%) patients from Henan and 81 (12.0%) patients from Guangxi, respectively. The highest prevalence of HIV/HCV co-infection was observed from FBDs (former blood donors) (87.2%) in Henan and IDUs (intravenous drug users) (81.8%) in Guangxi, respectively. The seroprevalence rate of HCV among people with sexual contact was significantly higher in Henan than in Guangxi (18.7% vs. 3.5%, P<0.05). The positive rate of HCV RNA in Henan and Guangxi was 30.6% (133/435) and 11.2% (76/677), respectively. Moreover, we found that 20 anti-HCV negative samples were HCV positive by PCR amplification. HCV subtype 1b (52.7%) was predominant in Henan, followed by subtype 2a (41.9%). The most frequently detected subtypes in Guangxi were 6a (35.6%) and 3b (32.9%).
The HCV genotype distributions were different in HIV-infected people from Henan and Guangxi. HIV/HCV co-infection was not only linked to the transmission routes, but also associated with the geographic position.
Areas endemic for malaria and Hepatitis B virus (HBV) infection largely overlap geographically. A recent study has suggested the existence of an interaction between the two pathogens in symptomatic co-infected individuals on the South-American continent. We examined this issue in a hyperendemic area for both pathogens in sub-Saharan Africa.
Methodology and Findings
Pre-transfusion samples from a retrospective cohort of 154 blood transfusion recipients were screened for both serological and molecular markers of HBV and Plasmodium genomes using species-specific nested PCR and quantitative real-time PCR. Thirty-seven individuals met exclusion criteria and were subsequently eliminated from further analysis. Of 117 participants, 90% of recipients exhibited evidence of exposure to HBV, 42% with HBsAg and/or HBV DNA and 48% anti-HBc reactive without detectable HBV DNA. Plasmodium genome prevalence by NAT was 50%. Parasitemic individuals were significantly younger than non-parasitemic individuals (P = 0.04). Parasitemia level was not significantly lower in individuals with HBV DNA positive infections compared to those with HBV DNA negative exposures. HBV DNA load was not significantly different in parasitemic and non-parasitemic individuals.
The data presented suggests that, in sub-Saharan Africa, asymptomatic co-infections with these two ubiquitous pathogens do not appear to significantly affect each other and evolve independently.
A novel multiplex real-time PCR assay for concurrent detection of hepatitis viruses was evaluated for its clinical performance in screening patients with acute hepatitis. A total of 648 serum samples were collected from patients with acute symptoms of hepatitis. Concurrent detection of nucleic acids of HAV, HBV and HCV was performed using the Magicplex™ HepaTrio Real-time Detection test. Serum nucleic acid levels of HBV and HCV were also quantified by the Cobas® AmpliPrep/Cobas® TaqMan® (CAP/CTM) HBV and HCV tests. Patients’ medical records were also reviewed. Concordance rates between the results from the HepaTrio and the CAP/CTM tests for the detection of HBV and HCV were 94.9% (k = 0.88) and 99.2% (k = 0.98), respectively. The cycle threshold values with the HepaTrio test were also correlated well with the levels of HBV DNA (r = −0.9230) and HCV RNA (r = −0.8458). The sensitivity and specificity of the HepaTrio test were 93.8% and 98.2%, respectively, for detecting HBV infection, and 99.1% and 100.0%, respectively, for HCV infection. For the HepaTrio test, 21 (3.2%) cases were positive for both HBV and HCV. Among the positive cases, 6 (0.9%) were true coinfections. This test also detected 18 (2.8%) HAV positives. The HepaTrio test demonstrated good clinical performance and produced results that agreed well with those of the CAP/CTM assays, especially for the detection of HCV. This assay was also able to detect HAV RNA from anti-HAV IgM-positive individuals. Therefore, this new multiplex PCR assay could be useful for the concurrent detection of the three hepatitis viruses.
It is hypothesized that social networks facilitate transmission of the hepatitis C virus (HCV). We tested for association between HCV phylogeny and reported injecting relationships using longitudinal data from a social network design study. People who inject drugs were recruited from street drug markets in Melbourne, Australia. Interviews and blood tests took place three monthly (during 2005–2008), with participants asked to nominate up to five injecting partners at each interview. The HCV core region of individual isolates was then sequenced and phylogenetic trees were constructed. Genetic clusters were identified using bootstrapping (cut-off: 70%). An adjusted Jaccard similarity coefficient was used to measure the association between the reported injecting relationships and relationships defined by clustering in the phylogenetic analysis (statistical significance assessed using the quadratic assignment procedure). 402 participants consented to participate; 244 HCV infections were observed in 238 individuals. 26 genetic clusters were identified, with 2–7 infections per cluster. Newly acquired infection (AOR = 2.03, 95% CI: 1.04–3.96, p = 0.037, and HCV genotype 3 (vs. genotype 1, AOR = 2.72, 95% CI: 1.48–4.99) were independent predictors of being in a cluster. 54% of participants whose infections were part of a cluster in the phylogenetic analysis reported injecting with at least one other participant in that cluster during the study. Overall, 16% of participants who were infected at study entry and 40% of participants with newly acquired infections had molecular evidence of related infections with at least one injecting partner. Likely transmission clusters identified in phylogenetic analysis correlated with reported injecting relationships (adjusted Jaccard coefficient: 0.300; p<0.001). This is the first study to show that HCV phylogeny is associated with the injecting network, highlighting the importance of the injecting network in HCV transmission.
Previous studies have proved the presence of several distinct types of mutations in hepatitis B virus (HBV) infections, which are related to the progression of liver disease. However, few reports have detailed the mutation frequencies and mutation patterns in the precore/core (preC/C) region, which are based on the clinical status and HBeAg serostatus. Our aim in this study is to investigate the relationships between the preC/C mutations and clinical severity or HBeAg serostatus from patients chronically infected with HBV genotype C. A total of 70 Korean chronic patients, including 35 with hepatocellular carcinoma (HCC), participated in this study. HBV genotyping and precore/core mutations were analyzed by direct sequencing. All patients were confirmed to have genotype C infections. Mutations in the C region were distributed in a non-random manner. In particular, mutations in the MHC class II restricted region were found to be significantly related to HCC. Six (preC-W28*, C-P5H/L/T, C-E83D, C-I97F/L, C-L100I and C-Q182K/*) and seven types (preC-W28*, preC-G29D, C-D32N/H, C-E43K, C-P50A/H/Y, C-A131G/N/P and C-S181H/P) of mutations in the preC/C region were found to be related to HCC and to affect the HBeAg serostatus, respectively. In conclusion, our data indicated that HBV variants in the C region, particularly in the MHC class II restricted region, may contribute to the progress of HCC in chronic patients infected with genotype C. In addition, we found several distinct preC/C mutations in the Korean chronic cohort, which affect the clinical status of HCC and HBeAg serostatus of patients infected with genotype C.
There is growing evidence to suggest that HIV may interact with several hepatic cell types; however, evaluation of HIV variability in liver tissue has not been addressed to date. Among 16 HIV-positive individuals examined, nine (56%) had detectable HIV RNA in the liver. The mean CD4 cell count for these nine individuals was 337 cells/mm3 (range: 0–601), while their mean plasma HIV RNA level was 106,974 copies/ml (range: 1200–320,740). Among individuals in this study with detectable HIV in both the plasma and the liver, the consensus gag nucleotide sequences for each tissue type were different for seven of seven (100%) individuals, while amino acid sequences were distinct for five of seven (71%). Consensus envelope (env) nucleotide and amino acid sequences were also distinct in the plasma and liver tissue for six of six (100%) individuals. Statistical evidence of compartmentalization between HIV in the plasma and in the liver was demonstrated, and multiple liver-specific amino acids were identified that may distinguish HIV variants replicating within the liver. These preliminary data demonstrate that HIV is frequently detectable in the liver of HIV-positive persons at various levels of immunosuppression. Possible compartmentalization may reflect tissue-specific selection pressures that drive viral adaptation to the liver microenvironment and may facilitate interactions with other hepatotropic viruses.
To estimate the cost, effectiveness, and cost effectiveness of HIV and HCV screening of injection drug users (IDUs) in opioid replacement therapy (ORT).
Dynamic compartmental model of HIV and HCV in a population of IDUs and non-IDUs for a representative U.S. urban center with 2.5 million adults (age 15–59).
We considered strategies of screening individuals in ORT for HIV, HCV, or both infections by antibody or antibody and viral RNA testing. We evaluated one-time and repeat screening at intervals from annually to once every 3 months. We calculated the number of HIV and HCV infections, quality-adjusted life years (QALYs), costs, and incremental cost-effectiveness ratios (ICERs).
Adding HIV and HCV viral RNA testing to antibody testing averts 14.8–30.3 HIV and 3.7–7.7 HCV infections in a screened population of 26,100 IDUs entering ORT over 20 years, depending on screening frequency. Screening for HIV antibodies every 6 months costs $30,700/QALY gained. Screening for HIV antibodies and viral RNA every 6 months has an ICER of $65,900/QALY gained. Strategies including HCV testing have ICERs exceeding $100,000/QALY gained unless awareness of HCV-infection status results in a substantial reduction in needle-sharing behavior.
Although annual screening for antibodies to HIV and HCV is modestly cost effective compared to no screening, more frequent screening for HIV provides additional benefit at less cost. Screening individuals in ORT every 3–6 months for HIV infection using both antibody and viral RNA technologies and initiating ART for acute HIV infection appears cost effective.
Hepatitis C virus (HCV) is a genetically diverse pathogen infecting approximately 2–3% of the world's population. Herein, we describe results of a large, multicentre serological and molecular epidemiological study cataloguing the prevalence and genetic diversity of HCV in five regions of Vietnam; Ha Noi, Hai Phong, Da Nang, Khanh Hoa and Can Tho. Individuals (n = 8654) with varying risk factors for infection were analysed for the presence of HCV Ab/Ag and, in a subset of positive specimens, for HCV RNA levels (n = 475) and genotype (n = 282). In lower risk individuals, including voluntary blood donors, military recruits and pregnant women, the prevalence of infection was 0.5% (n = 26/5250). Prevalence rates were significantly higher (p<0.001) in intravenous drug users (IDUs; 55.6%, n = 556/1000), dialysis patients (26.6%, n = 153/575) commercial sex workers (CSWs; 8.7%, n = 87/1000), and recipients of multiple blood transfusions (6.0%, n = 32/529). The prevalence of HCV in dialysis patients varied but remained high in all regions (11–43%) and was associated with the receipt of blood transfusions [OR: 2.08 (1.85–2.34), p = 0.001], time from first transfusion [OR: 1.07 (1.01–1.13), p = 0.023], duration of dialysis [OR: 1.31 (1.19–1.43), p<0.001] and male gender [OR: 1.60 (1.06–2.41), p = 0.026]. Phylogenetic analysis revealed high genetic diversity, particularly amongst dialysis and multi-transfused patients, identifying subtypes 1a (33%), 1b (27%), 2a (0.4%), 3a (0.7%), 3b (1.1%), 6a (18.8%), 6e (6.0%), 6h (4.6%), 6l (6.4%) and 2 clusters of novel genotype 6 variants (2.1%). HCV genotype 1 predominated in Vietnam (60%, n = 169/282) but the proportion of infections attributable to genotype 1 varied between regions and risk groups and, in the Southern part of Vietnam, genotype 6 viruses dominated in dialysis and multi-transfused patients (73.9%). This study confirms a high prevalence of HCV infection in Vietnamese IDUs and, notably, reveals high levels of HCV infection associated with dialysis and blood transfusion.
Over 350 million people worldwide are infected with hepatitis B virus (HBV), a major cause of liver failure and hepatocellular carcinoma. Current therapeutic agents are highly effective, but are also associated with development of viral resistance. Therefore, strategies for identifying other anti-HBV agents with specific, but distinctive mechanisms of action are needed. The human La (hLa) protein, which forms a stabilizing complex with HBV RNA ribonucleoprotein to promote HBV replication, is a promising target of molecular therapy.
This study aimed to discover novel inhibitors of hLa that could inhibit HBV replication and expression.
A multistage molecular docking approach was used to screen a Specs database and an in-house library against hLa binding sites. Sequential in vitro evaluations were performed to detect potential compounds with high scores in HepG2.2.15 cells.
Of the 26 potential compounds with high scores chosen for experimental verification, 12 had HBV DNA inhibition ratios of less than 50% with P<0.05. Six had significant inhibition of HBV e antigen (HBeAg) levels, and 13 had significant inhibition of HBV surface antigen (HBsAg) levels by in vitro assays. Compounds HBSC-11, HBSC-15 and HBSC-34 (HBSC is system prefix for active compounds screened by the library) were selected for evaluation. HBSC-11 was found to have an obvious inhibitory effect on hLa transcription and expression.
Our findings suggest that anti-HBV activity of HBSC-11 may be mediated by a reduction in hLa levels. In addition, our data suggest the potential clinical use of hLa inhibitors, such as HBSC-11, for treating HBV infection.
Multiple genotypes of GB virus C (GBV-C) – a non-pathogenic flavivirus – have been identified to date, although they are not uniformly distributed worldwide. It has also been suggested that GBV-C genotype may play a role in modulating HIV disease; however, the prevalence and genotype distribution of GBV-C has not been adequately studied in most countries. Among 408 HIV positive subjects in Germany, 97 (23.8%) had detectable GBV-C RNA. Based on sequencing of the 5′ untranslated region (5′UTR), the GBV-C genotypes were 1 (n = 8; 8.2%), 2 (n = 81; 83.5%), and 3 (n = 2; 2.1%), as well as a unique genotype not previously reported (n = 6; 6.2%). Among 17 samples also sequenced in the envelope 2 (E2) region, 14 had concordant genotype results when comparing the 5′UTR and E2, while evidence of intergenotypic recombination was observed among E2 sequences from 3 individuals. These results suggest that genotypic diversity and viral recombination contribute to the overall genetic variability of GB virus C.
GB virus C (GBV-C) HIV; diversity; genotype; recombination
Predictors of liver fibrosis were evaluated in women using a noninvasive index (FIB-4). HIV RNA levels were associated with increased FIB-4 in the absence of viral hepatitis, alcohol use, or antiretroviral therapy. These data complement evidence suggesting a potential relationship between HIV infection and hepatic fibrosis.
Background. FIB-4 represents a noninvasive, composite index that is a validated measure of hepatic fibrosis, which is an important indicator of liver disease. To date, there are limited data regarding hepatic fibrosis in women.
Methods. FIB-4 was evaluated in a cohort of 1227 women, and associations were evaluated in univariate and multivariate regression models among 4 groups of subjects classified by their human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection status.
Results. The median FIB-4 scores were 0.60 in HIV-/HCV- women, 0.83 in HIV-/HCV+ women, 0.86 in HIV+/HCV- women, and 1.30 in HIV+/HCV+ women. In the HIV/HCV co-infected group, multivariate analysis showed that CD4+ cell count and albumin level were negatively associated with FIB-4 (P <.0001), whereas antiretroviral therapy (ART) was positively associated with FIB-4 score (P =.0008). For the HIV mono-infected group, multivariate analysis showed that CD4+ cell count (P <.0001) and albumin level (P =.0019) were negatively correlated with FIB-4 score, ART was positively associated with FIB-4 score (P =.0008), and plasma HIV RNA level was marginally associated with FIB-4 score (P =.080). In 72 HIV mono-infected women who were also hepatitis B surface antigen negative, ART naive, and reported no recent alcohol intake, plasma HIV RNA level was associated with increased FIB-4 score (P =.030).
Conclusions. HIV RNA level was associated with increased FIB-4 score in the absence of hepatitis B, hepatitis C, ART, or alcohol use, suggesting a potential relationship between HIV infection and hepatic fibrosis in vivo. A better understanding of the various demographic and virologic variables that contribute to hepatic fibrosis may lead to more effective treatment of HIV infection and its co-morbid conditions.
Extrahepatic replication has important implications for the transmission and treatment of hepatitis C virus (HCV). We analyzed longitudinal HCV diversity in peripheral-blood mononuclear cells (PBMCs) and serum during HCV monoinfection and HCV/HIV coinfection to determine whether distinct amino acid signatures characterized HCV replicating within PBMCs. Analysis of E1-HVR1 sequences demonstrated higher serum genetic distances among HCV/human immunodeficiency virus (HIV)–coinfected persons. Moreover, consensus PBMC sequences were rarely identical to those in the corresponding serum, suggesting divergence in these 2 compartments. Three of 5 HCV/HIV-coinfected participants showed evidence of HCV compartmentalization in PBMCs. Additionally, signature sequence analysis identified PBMC-specific amino acids in all HCV/HIV-coinfected persons. To our knowledge, this is the first study to identify specific amino acids that may distinguish HCV variants replicating in PBMCs. It is provocative to speculate that extrahepatic HCV diversity may be an important determinant of treatment response and thus warrants additional study, particularly during HCV/HIV coinfection.
There are limited data on diversity within the hepatitis C virus polymerase (NS5B). In concordance with its key functional role during the life cycle, NS5B intrapatient variability was low. Moreover, differences between NS5B nonsynonymous (dN) and synonymous (dS) mutation rates (dN − dS) were positively correlated with CD4 cell count, while nonsynonymous mutations were strongly correlated with reduced replication in vivo.
HCV/HIV coinfection has emerged as a major cause of morbidity and mortality due to liver disease. Interferon-based therapy response rates have been disappointingly low. Baseline HCV complexity and the relationship between complexity and viral kinetic parameters has not been well described in HCV/HIV subjects. A subset of patients enrolled in ACTG 5071 underwent sampling to evaluate viral kinetics and HCV complexity changes. Early kinetic parameters, baseline complexity, and treatment outcomes, including rapid (RVR), early (EVR), and sustained (SVR) viral response were evaluated. HCV monoinfected subjects were matched to HCV/HIV coinfected subjects.
Baseline complexity was determined in 108 HCV/HIV coinfected subjects and 13 HCV controls. Quasispecies complexity was 2.24 in HCV/HIV and 1.90 in monoinfected subjects (p=0.14). Lower baseline complexity was associated with EVR (p=0.04) and approached significance for SVR. In patients who underwent viral kinetic modeling, complexity decrease was associated with RVR (p= 0.03), and was independent of the correlation between first phase viral decline efficiency and RVR.
Baseline HCV complexity is an independent predictor of early viral response in HCV/HIV subjects. Complexity decrease occurs by 4 weeks of interferon-based therapy and is associated with RVR. These findings may enhance predictive modeling of treatment outcomes in HCV/HIV patients.
HCV; HIV; RNA; Quasispecies; Complexity; Pegylated-interferon; Coinfection
Chronic hepatitis B virus infection is characterized by persistent detectable levels of hepatitis B surface antigen (HBsAg) and HBV DNA in the serum. In contrast, HBsAg is not detectable during occult HBV infection, despite the presence of HBV DNA. An altered host immune response could play a role in the development of occult HBV infection; however, potential differences in immune responses among chronic and occult HBV-infected patients have not been evaluated in vivo. In the current study, we evaluated serum levels of regulatory, apoptotic, and fibrotic/anti-fibrotic cytokines/markers as indicators of immune responses in 25 chronic and 12 occult HBV-infected patients. More than half of the patients in both chronic and occult HBV infection groups had IL-2, IL-4, IL-13, and IFN-γ levels below detectable limits. In contrast, most patients had detectable levels of IL-8, IL-10, IP-10, sFas, sFasL, and TGF-β1. Of these, only sFas was significantly different between the two groups, with lower levels observed during occult compared to chronic HBV infection (p = 0.01). As a surrogate marker of apoptotic inhibition, decreased sFas during occult HBV infection suggests that apoptosis occurs at different rates in occult compared to chronic HBV infection and therefore, may contribute to persistence of occult HBV infection.
Cytokine; HBV/HIV co-infection; Occult hepatitis B virus; Soluble Fas (sFas)
Hepatitis C virus (HCV) non-nucleoside inhibitors (NNIs) target the viral RNA-dependent RNA polymerase encoded by the NS5B gene. Several NNIs share a similar allosteric binding site, and their antiviral efficacy is attenuated by a cysteine-to-tyrosine mutation at amino acid 316 (C316Y). In the current study, we assessed NS5B resistance mutations in treatment-naive individuals from a prospective natural history study of viral infections in women.
Partial NS5B sequences from HCV-positive women were amplified by RT–PCR. Additionally, subcloning was performed to evaluate intrapatient variability in selected samples.
HCV NS5B genotypes were 45 genotype 1a (57.0%), 11 genotype 1b (13.9%), 5 genotype 2a (6.3%), 3 genotype 2b (3.8%), 9 genotype 3a (11.4%) and 6 genotype 4a (7.6%). One HCV genotype 1a-infected patient was found to have the C316Y mutation (1.3%). Clonal analysis further revealed that all NS5B sequences from this individual—representing three serum samples collected 4 years apart—contained the C316Y mutation. In contrast, the S282T resistance mutation was not found in any samples.
The C316Y polymerase resistance mutation was found in 1.3% of samples from HCV-infected women. The presence of this mutation over time suggests significant replicative fitness of this variant and has implications for development of new specifically targeted antiviral therapies against HCV (STAT-C) targeting this region.
HCV; NS5B; C316Y
Hepatitis E virus (HEV) is a common cause of acute viral hepatitis (AVH) in many developing countries. In Egypt, HEV seroprevalence is among the highest in the world; however, only a very limited number of Egyptian HEV sequences are currently available.
The objectives were to determine the HEV genotype(s) currently circulating in Egypt.
AVH patients without serologic evidence of hepatitis A, B, and C viruses were evaluated for possible HEV infection using serologic assays for anti-HEV IgM and anti-HEV IgG and real-time PCR for HEV RNA. Stool suspensions from suspected cases were inoculated into rhesus macaques to confirm the presence of HEV. Sequence analysis was utilized to determine HEV genotype.
Of 287 subjects with AVH enrolled, 58 had serologic evidence of acute HEV infection. Stool samples for two of these patients were repeatedly positive for HEV RNA by real-time PCR. Macaques experimentally inoculated with these human stools also developed viremia. Sequence analysis of open reading frame (ORF) 1 demonstrated that these isolates belonged to HEV genotype 1 and were 3.9% – 9.5% divergent from other genotype 1 isolates. ORF2 was 5.3% – 8.7% divergent from previously reported Egyptian isolates.
This study strongly suggests that genotype 1 HEV related to other North African isolates is circulating in acute symptomatic patients in Egypt. Further evaluation of genotypic variability is underway in this highly endemic cohort and is considered an important component of our increased understanding of HEV pathogenesis.
Hepatitis E virus (HEV); Egypt; symptomatic; genotype; diversity
Regulatory T cells (Treg) are a subpopulation of CD4+ T cells characterized by the suppressive activity they exert on effector immune responses, including human immunodeficiency virus (HIV)-specific immune responses. Because Treg express CXCR4 and CCR5, they represent potential targets for HIV; however, Treg susceptibility to HIV infection is still unclear. We therefore performed an extensive study of Treg susceptibility to HIV, using lab strains and primary isolates with either CCR5 or CXCR4 tropism. Furthermore, we quantified HIV infection at early and late time points of the virus life cycle. We found that Treg were clearly susceptible to HIV infection. Circulating Treg were not preferentially infected with HIV compared to effector T cells (Teff) in vivo. Conversely, in vitro infection with either CCR5-using (R5) or CXCR4-using (X4) viruses occurred with different dynamics. For instance, HIV infection by R5 viruses (lab strains and primary isolates) resulted in lower levels of infection in Treg compared with Teff at both early and late time points. In contrast, X4 viruses induced higher levels of infection in Treg compared to Teff at early time points, but this difference disappeared at the late time points of the virus life cycle. Our results suggest that the relative susceptibility of Treg to HIV infection compared to Teff varies, depending on both viral and host factors. These variations may play an important role in HIV pathogenesis.
We have previously reported hepatitis C virus (HCV) replication using a novel binary expression system in which mammalian cells were transfected with a T7 polymerase-driven full-length genotype 1a HCV cDNA plasmid (pT7-flHCV-Rz) and infected with vaccinia-T7 polymerase. We hypothesized that the use of replication-defective adenoviral vectors expressing T7 (Ad-T7pol) or cell lines stably transfected with T7 (Huh-T7) would alleviate cell toxicity and allow for more sustained HCV replication.
CV-1, Huh7, and Huh-T7 cells were transfected with pT7-flHCV-Rz and treated with Ad-T7pol (CV-1 and Huh7 only). Protein and RNA were harvested from cells on days 1, 2, 3, 5, 7, and 9 post-infection. No cytotoxicity was observed at 9 days post-infection in any cell type. HCV positive- and negative-strand RNA expression were strongest during days 1–3 post-infection; however, HCV RNA remained detectable throughout the 9-day observation period. Furthermore, transfection with a replication-incompetent plasmid suggested that efficient HCV replication is dependent upon NS5B gene expression. Finally, after 1–2 days of IFN treatment, HCV positive-strand levels decreased significantly compared to HCV-infected but untreated samples (p < 0.05).
In conclusion, these refined binary systems offer more durable and authentic models for identification of host cellular processes critical to HCV replication and will permit longer-term analysis of virus–host interactions critical to HCV pathogenesis and the treatment of genotype 1 infections.
Hepatitis C virus; HCV; Replication; Genotype 1; Adenovirus vector; Huh-T7