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1.  Modulation of HCV Replication After Combination Antiretroviral Therapy in HCV/HIV Coinfected Patients 
Science translational medicine  2014;6(246):246ra98.
The hepatitis C virus (HCV) is an important contributor to morbidity and mortality in patients coinfected with human immunodeficiency virus (HIV). Coinfection results in increased HCV replication and more rapid rates of liver disease progression. The effect of HIV combination antiretroviral therapy (cART) on HCV replication has not been studied in depth. To address this issue, we enrolled a small cohort of HCV/HIV coinfected patients into a cART initiation trial, and used dynamic modeling combined with evaluation of immune responses and microarray profiles to determine how effective treatment of HIV affects HCV. Treatment with cART resulted in HCV flare and alanine aminotransferase (ALT) increase (2× or more increase from baseline) in a subset of treated patients. Subjects with evidence of hepatic injury (increased ALT) were more likely to have HCV-specific immune responses directed against HCV epitopes. Over time, HCV viral loads declined. Reproducible and biologically important gene expression changes occurred in patients who underwent successful cART, particularly with respect to downregulation of genes with known antiviral roles. Our findings suggest that the effective suppression of HIV by cART initiates a cascade of early and late events in treated patients with HCV. Early events involving downregulation of interferon-stimulated genes may lead to transiently increased viral replication and hepatic injury. At later time points, HCV viral load declines to levels comparable to those seen in the setting of HCV monoinfection. These findings support early antiretroviral therapy in those with HCV/HIV coinfection.
doi:10.1126/scitranslmed.3008195
PMCID: PMC4326686  PMID: 25101888
2.  GB Virus C (GBV-C) Infection in Hepatitis C Virus (HCV) Seropositive Women with or at Risk for HIV Infection 
PLoS ONE  2014;9(12):e114467.
Background
GB virus C (GBV-C) may have a beneficial impact on HIV disease progression; however, the epidemiologic characteristics of this virus are not well characterized. Behavioral factors and gender may lead to differential rates of GBV-C infection; yet, studies have rarely addressed GBV-C infections in women or racial/ethnic minorities. Therefore, we evaluated GBV-C RNA prevalence and genotype distribution in a large prospective study of high-risk women in the US.
Results
438 hepatitis C virus (HCV) seropositive women, including 306 HIV-infected and 132 HIV-uninfected women, from the HIV Epidemiologic Research Study were evaluated for GBV-C RNA. 347 (79.2%) women were GBV-C RNA negative, while 91 (20.8%) were GBV-C RNA positive. GBV-C positive women were younger than GBV-C negative women. Among 306 HIV-infected women, 70 (22.9%) women were HIV/GBV-C co-infected. Among HIV-infected women, the only significant difference between GBV-negative and GBV-positive women was age (mean 38.4 vs. 35.1 years; p<0.001). Median baseline CD4 cell counts and plasma HIV RNA levels were similar. The GBV-C genotypes were 1 (n = 31; 44.3%), 2 (n = 36; 51.4%), and 3 (n = 3; 4.3%). The distribution of GBV-C genotypes in co-infected women differed significantly by race/ethnicity. However, median CD4 cell counts and log10 HIV RNA levels did not differ by GBV-C genotype. GBV-C incidence was 2.7% over a median follow-up of 2.9 (IQR: 1.5, 4.9) years, while GBV-C clearance was 35.7% over a median follow-up of 2.44 (1.4, 3.5) years. 4 women switched genotypes.
Conclusions
Age, injection drug use, a history of sex for money or drugs, and number of recent male sex partners were associated with GBV-C infection among all women in this analysis. However, CD4 cell count and HIV viral load of HIV/HCV/GBV-C co-infected women were not different although race was associated with GBV-C genotype.
doi:10.1371/journal.pone.0114467
PMCID: PMC4262414  PMID: 25493916
3.  Effects of HCV Treatment on Cytokine Expression During HCV/HIV Coinfection 
There is growing evidence that cytokine expression is linked to hepatitis C virus (HCV) pathogenesis and treatment response rates among HCV-monoinfected persons. However, because of the profound effects of human immunodeficiency virus (HIV) coinfection on HCV, it is not clear if these observations are also true for HCV/HIV-coinfected individuals. Serum expression of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) and the fibrogenic cytokine transforming growth factor-β1 (TGF-β1) were measured in HCV/HIV-coinfected persons at baseline and at week 24 of HCV therapy. Higher levels of IL-8 and TGF-β were demonstrated among nonwhite subjects at baseline. Increases in TNF-α and IL-8 expression were found at week 24 of HCV therapy, suggesting that enhanced proinflammatory cytokine production may occur during HCV treatment. However, cytokine levels were not predictive of HCV virologic, biochemical, or histologic response. Although previous studies conducted among HCV-monoinfected individuals have suggested that cytokine levels could predict the virologic response to therapy, no such associations were observed among HCV/HIV-coinfected persons, suggesting that they may respond differently to treatment than do their HCV-monoinfected counterparts.
doi:10.1089/jir.2006.26.834
PMCID: PMC4113398  PMID: 17115902
4.  Hepatitis C Virus Infection of a Thyroid Cell Line: Implications for Pathogenesis of Hepatitis C Virus and Thyroiditis 
Thyroid  2013;23(7):863-870.
Background
Autoimmune and non-autoimmune thyroiditis frequently occur in persons with hepatitis C virus (HCV) infection. Treatment with interferon alpha (IFNα) is also associated with significant risk for the development of thyroiditis. To explore HCV–thyroid interactions at a cellular level, we evaluated whether a human thyroid cell line (ML1) could be infected productively with HCV in vitro.
Methods and Results
ML1 cells showed robust surface expression of the major HCV receptor CD81. Using a highly sensitive, strand-specific reverse transcription polymerase chain reaction assay, positive-sense and negative-sense HCV RNA were detected in ML1 cell lysates at days 3, 7, and 14 postinfection with HCV. HCV core protein was expressed at high levels in ML1 supernatants at days 1, 3, 5, 7, and 14 postinfection. The nonstructural protein NS5A was also detected in ML1 cell lysates by Western blotting. HCV entry into ML1 cells was shown to be dependent on the HCV entry factors CD81 and SR-B1/CLA1, while IFNα inhibited HCV replication in ML1 cells in a dose-dependent manner. Supernatants from HCV-infected ML1 cells were able to infect fresh ML1 cells productively, suggesting that infectious virions could be transferred from infected to naïve thyroid cells in vivo. Additionally, HCV infection of ML1 cells led to increased expression of the pro-inflammatory cytokine IL-8.
Conclusions
For the first time, we have demonstrated that HCV can infect human thyroid cells in vitro. These findings strongly suggest that HCV infection of thyrocytes may play a role in the association between chronic HCV infection and thyroid autoimmunity. Furthermore, the thyroid may serve as an extrahepatic reservoir for HCV viral replication, thus contributing to the persistence of viral infection and to the development of thyroid autoimmunity.
doi:10.1089/thy.2012.0507
PMCID: PMC3704108  PMID: 23259732
5.  Viral Factors Associated with Cytokine Expression During HCV/HIV Co-Infection 
Co-infection with human immunodeficiency virus (HIV) is associated with reduced hepatitis C virus (HCV) treatment response and accelerated HCV disease. Cytokines, as mediators of immune responses, inflammation, and fibrogenesis, may underlie important differences in HCV pathogenesis during HIV co-infection. We previously found that serum interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) increased after HCV therapy with interferon (IFN) in HCV/HIV co-infected patients; however, cytokine levels were not predictive of HCV therapeutic response. Here, we examined viral factors associated with expression of IL-8, TNF-α, and transforming growth factor-β1 (TGF-β1) in uninfected, HCV mono-infected, HIV mono-infected, and HCV/HIV co-infected persons. HIV co-infection was associated with decreased IL-8 detection but not TNF-α detection. A significant interaction effect demonstrated that HIV infection was associated with elevated TGF-β1 in HCV-positive individuals but not in HCV-negative individuals. The induction of a sustained profibrotic signal, such as TGF-β1, by HIV may cause accelerated liver fibrosis during HCV/HIV co-infection and may hinder the host’s ability to mount an effective HCV-specific immune response. Further studies are warranted to identify noninvasive markers of liver disease for the clinical management of HCV disease, particularly when liver biopsies have not been performed or are contraindicated.
doi:10.1089/jir.2006.0147
PMCID: PMC4066618  PMID: 17477814
7.  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.
doi:10.1111/j.1872-034X.2012.01072.x
PMCID: PMC3505246  PMID: 22909008
HIV co-infection; Injection drug use; Occult HBV; Surface antigen negative
8.  HIV Infection of Hepatocytes Results in a Modest Increase in Hepatitis C Virus Expression In Vitro 
PLoS ONE  2014;9(2):e83728.
Previous studies demonstrate that soluble HIV proteins impact both hepatocyte function and HCV replication in vitro. It has also been reported that HIV can productively infect hepatocytes. We therefore investigated the impact of HIV infection of hepatocytes on HCV expression. The Huh7.5JFH1 cell line that constitutively expresses infectious HCV was infected with the lab-adapted strains HIVNL4-3 or HIVYK-JRCSF. HCV expression was quantified via HCV core antigen ELISA, Western blot, and strand-specific real-time PCR for positive-sense and negative-sense HCV RNA. After HIVNL4-3 infection of Huh7.5JFH1 cells, positive-sense and negative-sense HCV RNA levels were elevated compared to HIV uninfected cells. Increased HCV RNA synthesis was also observed after infection of Huh7.5JFH1 cells with HIVYK-JRCSF. HIV-induced HCV core production was decreased in the presence of the anti-HIV drugs AZT, T20, and raltegravir, although these medications had a minimal effect on HCV expression in the absence of HIV. HCV core, NS3, and NS5A protein expression were increased after HIV infection of Huh7.5JFH1 cells. Chemically inactivated HIV had a minimal effect on HCV expression in Huh7.5JFH1 cells suggesting that ongoing viral replication was critical. These data demonstrate that HIV induces HCV RNA synthesis and protein production in vitro and complement previous in vivo reports that HCV RNA levels are elevated in individuals with HIV/HCV co-infection compared to those with HCV mono-infection. These findings suggest that HIV suppression may be a critical factor in controlling liver disease, particularly if the underlying liver disease is not treated.
doi:10.1371/journal.pone.0083728
PMCID: PMC3938396  PMID: 24586227
9.  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.
doi:10.1111/j.1365-2893.2012.01595.x
PMCID: PMC3442934  PMID: 22967103
G145A; HBV/HIV co-infection; HBsAg mutants; hepatitis B surface antigen (HBsAg); occult hepatitis B virus
10.  Effects of HCV on Basal and Tat-Induced HIV LTR Activation 
PLoS ONE  2013;8(6):e64956.
Hepatitis C virus (HCV) co-infection occurs in ∼30–40% of the HIV-infected population in the US. While a significant body of research suggests an adverse effect of HIV on HCV replication and disease progression, the impact of HCV on HIV infection has not been well studied. Increasing data suggest that hepatocytes and other liver cell populations can serve as reservoirs for HIV replication. Therefore, to gain insight into the impact of HCV on HIV, the effects of the HCV Core protein and infectious hepatitis C virions were evaluated on basal and Tat-induced activation of the HIV long terminal repeat (LTR) in hepatocytes. The HIV LTR was highly induced by the HIV transactivator protein Tat in hepatocytes. Activation varied according to the number of NF-kB binding sites present in the LTRs from different HIV subtypes. Involvement of the NF-kB binding pathway in LTR activation was demonstrated using an NF-kB inhibitor and deletion of the NF-kB binding sites. TNFα, a pro-inflammatory cytokine that plays an important role in HIV pathogenesis, also induced LTR activity in hepatocytes. However, HIV LTR activity was suppressed in hepatocytes in the presence of HCV Core protein, and the suppressive effect persisted in the presence of TNFα. In contrast, infectious hepatitis C virions upregulated HIV LTR activation and gene transcription. Core-mediated suppression remained unaltered in the presence of HCV NS3/4A protein, suggesting the involvement of other viral/cellular factors. These findings have significant clinical implications as they imply that HCV could accelerate HIV disease progression in HIV/HCV co-infected patients. Such analyses are important to elucidate the mechanisms by which these viruses interact and could facilitate the development of more effective therapies to treat HIV/HCV co-infection.
doi:10.1371/journal.pone.0064956
PMCID: PMC3677892  PMID: 23762271
11.  Role of GB virus C in modulating HIV disease 
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.
doi:10.1586/eri.12.37
PMCID: PMC3499065  PMID: 22702320
co-infection; GBV-C; HCV; HIV
12.  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.
doi:10.1002/jmv.22269
PMCID: PMC3243959  PMID: 22170544
NS5B; HVR1; diversity; quasispecies; extrahepatic replication
13.  HIV Variability in the Liver and Evidence of Possible Compartmentalization 
AIDS Research and Human Retroviruses  2011;27(10):1117-1126.
Abstract
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.
doi:10.1089/aid.2010.0329
PMCID: PMC3186706  PMID: 21417757
14.  Low-level HIV infection of hepatocytes 
Virology Journal  2012;9:157.
Background
There are only limited data on whether HIV infection occurs within the liver; therefore, we explored early and late stages of the HIV life cycle in two hepatocyte cell lines – Huh7.5 and Huh7.5JFH1 – as well as in primary human hepatocytes.
Results
Integrated HIV DNA was detected in Huh7.5 and Huh7.5JFH1 cells, as well as in primary hepatocytes, and was inhibited by the integrase inhibitor raltegravir in a dose-dependent manner. HIV p24 protein was also detected in cell culture supernatants at days 1, 3, 5, and 7 post-infection and was inhibited by AZT, although levels were modest compared to those in a lymphocyte cell line. Culture supernatants from HIV-infected hepatocytes were capable of infecting a non-hepatic HIV indicator cell line.
Conclusions
These results indicating low-level HIV replication in hepatoctyes in vitro complement evidence suggesting that HIV has deleterious effects on the liver in vivo.
doi:10.1186/1743-422X-9-157
PMCID: PMC3607931  PMID: 22877244
HIV; Hepatocyte; Liver; Integration; Huh7.5
15.  Evidence for extensive genotypic diversity and recombination of GB virus C (GBV-C) in Germany 
Journal of medical virology  2011;83(4):685-694.
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.
doi:10.1002/jmv.22029
PMCID: PMC3101640  PMID: 21328384
GB virus C (GBV-C) HIV; diversity; genotype; recombination
16.  HIV Mono-infection Is Associated With FIB-4 – A Noninvasive Index of Liver Fibrosis – in Women 
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.
doi:10.1093/cid/ciq199
PMCID: PMC3106241  PMID: 21248367
17.  Compartmentalization of Hepatitis C Virus (HCV) during HCV/HIV Coinfection 
The Journal of infectious diseases  2007;195(12):1765-1773.
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.
doi:10.1086/518251
PMCID: PMC3210030  PMID: 17492592
18.  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.
doi:10.1128/JCM.01613-10
PMCID: PMC3020841  PMID: 20810773
19.  HCV QUASISPECIES COMPLEXITY AND SELECTION IN HCV/HIV COINFECTED SUBJECTS TREATED WITH INTERFERON-BASED REGIMENS 
The Journal of infectious diseases  2010;201(5):712-719.
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.
RESULTS
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.
CONCLUSION
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.
doi:10.1086/650490
PMCID: PMC2827649  PMID: 20105080
HCV; HIV; RNA; Quasispecies; Complexity; Pegylated-interferon; Coinfection
20.  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.
doi:10.1016/j.cyto.2009.06.005
PMCID: PMC3031085  PMID: 19625194
Cytokine; HBV/HIV co-infection; Occult hepatitis B virus; Soluble Fas (sFas)
21.  Screening for hepatitis C virus non-nucleotide resistance mutations in treatment-naive women 
Objectives
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.
Methods
Partial NS5B sequences from HCV-positive women were amplified by RT–PCR. Additionally, subcloning was performed to evaluate intrapatient variability in selected samples.
Results
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.
Conclusions
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.
doi:10.1093/jac/dkp328
PMCID: PMC2766823  PMID: 19767319
HCV; NS5B; C316Y
22.  Genotypic characterization of symptomatic hepatitis E virus (HEV) infections in Egypt 
Background
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.
Objectives
The objectives were to determine the HEV genotype(s) currently circulating in Egypt.
Study Design
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.
Results
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.
Conclusions
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.
doi:10.1016/j.jcv.2009.07.007
PMCID: PMC2753377  PMID: 19651539
Hepatitis E virus (HEV); Egypt; symptomatic; genotype; diversity
23.  Human Regulatory T Cells Are Targets for Human Immunodeficiency Virus (HIV) Infection, and Their Susceptibility Differs Depending on the HIV Type 1 Strain ▿  
Journal of Virology  2009;83(24):12925-12933.
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.
doi:10.1128/JVI.01352-09
PMCID: PMC2786841  PMID: 19828616
24.  Cell-based models of sustained, interferon-sensitive hepatitis C virus genotype 1 replication 
Journal of virological methods  2005;132(1-2):195-203.
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.
doi:10.1016/j.jviromet.2005.10.014
PMCID: PMC2865175  PMID: 16313977
Hepatitis C virus; HCV; Replication; Genotype 1; Adenovirus vector; Huh-T7
25.  HCV E2 PROTEIN BINDS DIRECTLY TO THYROID CELLS AND INDUCES IL-8 PRODUCTION: A NEW MECHANISM FOR HCV INDUCED THYROID AUTOIMMUNITY 
Journal of autoimmunity  2008;31(4):339-344.
HCV infection is well-known to be associated with autoimmune thyroiditis. However, the mechanisms by which HCV triggers thyroiditis are unknown. We hypothesized that HCV envelope proteins could induce thyroidal inflammation directly, thereby triggering thyroiditis by bystander activation mechanism. To test this hypothesis we examined whether the HCV receptor CD81 was expressed and functional on thyroid cells. We found significant levels of CD81 mRNA by QPCR analysis, as well as CD81 protein by flow cytometric (FACS) analysis. Incubation of thyroid cells with HCV envelope glycoprotein E2 resulted in E2 binding to thyroid cells and activation of IL-8, an important pro-inflammatory cytokine. Intriguingly, thyroid cells incubated with E2 continued to proliferate normally and did not undergo apoptosis, as was reported in hepatocytes. We conclude that: (1) HCV envelope glycoprotein E2 can bind to CD81 receptors which are expressed on thyroid cells and induce a cascade of signaling pathway leading to IL-8 release; (2) HCV may trigger thyroiditis in genetically susceptible individuals by bystander activation mechanisms.
doi:10.1016/j.jaut.2008.08.001
PMCID: PMC2641008  PMID: 18799285
Hepatitis C virus; thyroiditis; autoimmunity; infection thyroid

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