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1.  Evidence of Distinct Populations of Hepatitis C Virus in the Liver and Plasma of Patients Co-Infected With HIV and HCV 
Journal of medical virology  2014;86(8):1332-1341.
Viral diversity is an important predictor of hepatitis C virus (HCV) treatment response and may influence viral pathogenesis. HIV influences HCV variability in the plasma; however, limited data on viral variability are available from distinct tissue/cell compartments in patients co-infected with HIV and HCV. Thus, this exploratory study evaluated diversity of the hypervariable region 1 (HVR1) of HCV in the plasma and liver for 14 patients co-infected with HIV and HCV. Median intra-patient genetic distances and entropy values were similar in the plasma and liver compartments. Positive immune selection pressure was observed in the plasma for five individuals and in the liver for three individuals. Statistical evidence supporting viral compartmentalization was found in five individuals. Linear regression identified ALT (P = 0.0104) and AST (P = 0.0130) as predictors of viral compartmentalization. A total of 12 signature amino acids that distinguish liver from plasma E1/HVR1 were identified. One signature amino acid was shared by at least two individuals. These findings suggest that HCV compartmentalization is relatively common among patients co-infected with HIV and HCV. These data also imply that evaluating viral diversity, including drug resistance patterns, in the serum/plasma only may not adequately represent viruses replicating with in the liver and, thus, deserves careful consideration in future studies.
PMCID: PMC4562016  PMID: 24788693
HIV; HCV; co-infection; diversity; quasispecies; compartmentalization
2.  Hepatitis B Virus (HBV) X Gene Diversity and Evidence of Recombination in HBV/HIV Co-Infected Persons 
Journal of medical virology  2011;83(7):1142-1150.
The high frequency of mutation during hepatitis B virus (HBV) infection has resulted in 8 genotypes (A–H) with varying effects on disease severity and treatment efficacy. However, analysis of intrapatient HBV diversity is limited, especially during HIV co-infection. Therefore, a preliminary study was performed to analyze HBV X gene diversity in 17 HBV/HIV co-infected individuals. Phylogenetic analysis revealed HBV genotype A in 13 individuals (76.5%) or genotype E in 1 individual (5.9%). Additionally, 3 individuals were dually infected with HBV genotypes A and G (17.6%). Overall, higher genetic distance and entropy were observed in the X region and overlapping polymerase (Pol(X)) regions when compared to the PreS, S, and overlapping polymerase (Pol(PS) and Pol(S)) regions analyzed in the same patients as part of a previous study. In addition, multiple viral variants from 2 individuals with dual HBV infection did not group with either genotype A or G by phylogenetic analysis, indicating possible recombination. SimPlot bootscan analysis confirmed recombination breakpoints within the X gene in both individuals. Recombination between HBV genotypes may represent an important evolutionary strategy that enhances overall pathogenic potential and/or alters the downstream effects of the HBV X protein.
PMCID: PMC4557641  PMID: 21520141
intrapatient diversity; dual HBV infection; HBV/HIV co-infection; hepatitis B virusXprotein (HBx); quasispecies; recombination
3.  Limited infection with occult hepatitis B virus in drug users in the United States 
Occult HBV infection (O-HBV) is defined as low level HBV replication in the absence of detectable circulating HBV surface antigen. O-HBV has been implicated in HBV reactivation, advanced liver fibrosis and cirrhosis, reduced interferon response rates, elevated liver enzyme levels, and the development of hepatocellular carcinoma. However, prevalence of O-HBV has not been clearly established in certain at-risk populations, such as injection drug users. Therefore, the current pilot study examined the prevalence of O-HBV in a prospective cohort designed to assess the role of injection and non-injection drug use (IDU) on HIV-associated co-morbidities. Utilizing two distinct real-time PCR assays, HBV DNA was not detected in 99 participants examined. This finding is in contrast to other data from US IDU cohorts and suggests that the prevalence of O-HBV infection is very specific to the cohort studied, is sensitive to other confounding variables such as HCV and/or HIV serostatus, and should not be generalized across risk groups or distinct cohorts.
PMCID: PMC3505246  PMID: 22909008
HIV co-infection; Injection drug use; Occult HBV; Surface antigen negative
4.  Mutations Associated with Occult Hepatitis B Virus Infection Result in Decreased Surface Antigen Expression In Vitro 
Journal of viral hepatitis  2012;19(10):716-723.
Occult hepatitis B virus infection is characterized by the absence of detectable hepatitis B surface antigen (HBsAg) in the serum, despite detectable HBV DNA. Investigations of the mechanisms underlying the development of occult HBV infection are lacking in the current literature, although viral mutations in the surface region, resulting in decreased HBsAg expression or secretion, represent one potential mechanism. Wild-type HBsAg expression vectors were constructed from genotype-matched chronic HBV sequences. Site-directed mutagenesis was then utilized to introduce three genotype A mutations – M103I, K122R, and G145A – associated with occult HBV infection in vivo, alone and in combination, into the wild-type HBsAg vectors. Transfection of Huh7 and HepG2 cell lines was performed, and cell culture supernatants and cell lysates were collected over 7 days to assess the effects of these mutations on extracellular and intracellular HBsAg levels. The G145A mutation resulted in significantly decreased extracellular and intracellular HBsAg expression in vitro. The most pronounced reduction in HBsAg expression was observed when all 3 mutations were present. The mutations evaluated in vitro in the current study resulted in decreased HBsAg expression and potentially increased hepatic retention and/or decreased hepatic secretion of synthesized HBsAg, which could explain the lack of HBsAg detection that is characteristic of occult HBV infection in vivo.
PMCID: PMC3442934  PMID: 22967103
G145A; HBV/HIV co-infection; HBsAg mutants; hepatitis B surface antigen (HBsAg); occult hepatitis B virus
5.  Analysis of a non-structural gene reveals evidence of possible hepatitis C virus (HCV) compartmentalization 
Journal of medical virology  2012;84(2):242-252.
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.
PMCID: PMC3243959  PMID: 22170544
NS5B; HVR1; diversity; quasispecies; extrahepatic replication
6.  HIV Variability in the Liver and Evidence of Possible Compartmentalization 
AIDS Research and Human Retroviruses  2011;27(10):1117-1126.
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.
PMCID: PMC3186706  PMID: 21417757
7.  Variability of the Polymerase Gene (NS5B) in Hepatitis C Virus-Infected Women▿  
Journal of Clinical Microbiology  2010;48(11):4256-4259.
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.
PMCID: PMC3020841  PMID: 20810773
8.  Cytokine expression during chronic versus occult hepatitis B virus infection in HIV co-infected individuals 
Cytokine  2009;47(3):194-198.
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.
PMCID: PMC3031085  PMID: 19625194
Cytokine; HBV/HIV co-infection; Occult hepatitis B virus; Soluble Fas (sFas)
9.  Screening for hepatitis C virus non-nucleotide resistance mutations in treatment-naive women 
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.
PMCID: PMC2766823  PMID: 19767319
HCV; NS5B; C316Y

Results 1-9 (9)