Hepatic Transcriptome Analysis of Hepatitis C Virus Infection in Chimpanzees Defines Unique Gene Expression Patterns Associated with Viral Clearance

Hepatitis C virus infection leads to a high rate of chronicity. Mechanisms of viral clearance and persistence are still poorly understood. In this study, hepatic gene expression analysis was performed to identify any molecular signature associated with the outcome of hepatitis C virus (HCV) infection in chimpanzees. Acutely HCV-infected chimpanzees with self-limited infection or progression to chronicity were studied. Interferon stimulated genes were induced irrespective of the outcome of infection. Early induction of a set of genes associated with cell proliferation and immune activation was associated with subsequent viral clearance. Specifically, two of the genes: interleukin binding factor 3 (ILF3) and cytotoxic granule-associated RNA binding protein (TIA1), associated with robust T-cell response, were highly induced early in chimpanzees with self-limited infection. Up-regulation of genes associated with CD8+ T cell response was evident only during the clearance phase of the acute self-limited infection. The induction of these genes may represent an initial response of cellular injury and proliferation that successfully translates to a “danger signal” leading to induction of adaptive immunity to control viral infection. This primary difference in hepatic gene expression between self-limited and chronic infections supports the concept that successful activation of HCV-specific T-cell response is critical in clearance of acute HCV infection

Analysis of Duck Hepatitis B Virus Reverse Transcription Indicates a Common Mechanism for the Two Template Switches during Plus-Strand DNA Synthesis

The synthesis of the hepadnavirus relaxed circular DNA genome requires two template switches, primer translocation and circularization, during plus-strand DNA synthesis. Repeated sequences serve as donor and acceptor templates for these template switches, with direct repeat 1 (DR1) and DR2 for primer translocation and 5′r and 3′r for circularization. These donor and acceptor sequences are at, or near, the ends of the minus-strand DNA. Analysis of plus-strand DNA synthesis of duck hepatitis B virus (DHBV) has indicated that there are at least three other cis-acting sequences that make contributions during the synthesis of relaxed circular DNA. These sequences, 5E, M, and 3E, are located near the 5′ end, the middle, and the 3′ end of minus-strand DNA, respectively. The mechanism by which these sequences contribute to the synthesis of plus-strand DNA was unclear. Our aim was to better understand the mechanism by which 5E and M act. We localized the DHBV 5E element to a short sequence of approximately 30 nucleotides that is 100 nucleotides 3′ of DR2 on minus-strand DNA. We found that the new 5E mutants were partially defective for primer translocation/utilization at DR2. They were also invariably defective for circularization. In addition, examination of several new DHBV M variants indicated that they too were defective for primer translocation/utilization and circularization. Thus, this analysis indicated that 5E and M play roles in both primer translocation/utilization and circularization. In conjunction with earlier findings that 3E functions in both template switches, our findings indicate that the processes of primer translocation and circularization share a common underlying mechanism

The Clearance of Hepatitis C Virus Infection in Chimpanzees May Not Necessarily Correlate with the Appearance of Acquired Immunity

Clearance of hepatitis C virus (HCV) infection in humans and chimpanzees is thought to be associated with the induction of strong T-cell responses. We studied four chimpanzees infected with HCV derived from an infectious full-length HCV genotype 1b cDNA. Two of the chimpanzees cleared the infection to undetectable levels for more than 12 months of follow-up; the other two became persistently infected. Detailed analyses of HCV-specific immune responses were performed during the courses of infection in these chimpanzees. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. A comparison of the frequency of gamma interferon (IFN-γ)-producing CD4(+) and CD8(+) T cells in peripheral blood by ELISpot assay did not reveal any correlation between viral clearance and T-cell responses. In addition, analyses of IFN-γ, IFN-α, and interleukin-4 mRNA levels in liver biopsies, presumably indicative of intrahepatic T-cell responses, revealed no distinct pattern in these chimpanzees with respect to infection outcome. The present study suggests that the outcome of HCV infection in chimpanzees is not necessarily attributable to HCV sequence variation and that chimpanzees may recover from HCV infection by mechanisms other than the induction of readily detectable HCV-specific T-cell responses

Genes induced during the clearance phase of self-limited infection.

a<p>Expression levels of genes induced above the confidence interval at week 13 but below the confidence interval at weeks 4, 6 & 40 of X0190.</p><p>The bold and italicized values represent data above the 99% confidence interval as described in the text.</p

Genes induced during the early phase of self-limited infection as defined by average expression values.

a<p>Average expression values of biopsies taken during the 1^st 8 weeks of infection.</p><p>Bold-face genes are also in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003442#pone-0003442-t001" target="_blank">Table 1</a>.</p

Genes induced during the early phase of self-limited and persistent infections.

a<p>Expression levels of genes induced above the confidence interval at weeks 4 & 6 but below the confidence interval at weeks 13 & 40 in X0190.</p><p>The bold and italicized values represent data above the 99% confidence interval as described in the text.</p

Real-time PCR quantification of candidate genes involved in viral clearance and persistence.

<p>TaqMan real-time PCR was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003442#s4" target="_blank">Materials and Methods</a>. The y-axis shows the relative unit of a given gene normalized to GAPDH and 18s rRNA. Data are expressed as means±SEM. In all cases, average values obtained during the first eight weeks of infection were compared between recovered (X0190) and chronically infected chimpanzees (X 0234, X0142 and X6412). * <i>P<0.05</i>, ** <i>P<0.01</i>, *** <i>P<0.01</i>, **** <i>P<0.005</i>.</p