Differential, type I interferon-mediated autophagic trafficking of hepatitis C virus proteins in mouse liver.

BACKGROUND & AIMS: The hepatitis C virus (HCV) serine protease NS3/4A can cleave mitochondria-associated antiviral signaling protein (MAVS) and block retinoic acid-inducible gene I-mediated interferon (IFN) responses. Although this mechanism is thought to have an important role in HCV-mediated innate immunosuppression, its significance in viral persistence is not clear. METHODS: We generated transgenic mice that express the HCV NS3/4A proteins specifically in the liver and challenged the animals with a recombinant vesicular stomatitis virus, a synthetic HCV genome, IFN alfa, or IFN beta. We evaluated the effects of HCV serine protease on the innate immune responses and their interactions. RESULTS: Expression of HCV NS3/4A resulted in cleavage of intrahepatic MAVS; challenge of transgenic mice with vesicular stomatitis virus or a synthetic HCV genome induced strong, type I IFN-mediated responses that were not significantly lower than those of control mice. Different challenge agents induced production of different ratios of IFN alfa and beta, resulting in different autophagic responses and vesicular trafficking patterns of endoplasmic reticulum- and mitochondria-associated viral proteins. IFN beta promoted degradation of the viral proteins by the autolysosome. Variant isoforms of MAVS were associated with distinct, type I IFN-mediated autophagic responses; these responses have a role in trafficking of viral components to endosomal compartments that contain Toll-like receptor-3. CONCLUSIONS: IFN beta mediates a distinct autophagic mechanism of antiviral host defense. MAVS has an important role in type I IFN-induced autophagic trafficking of viral proteins.

Aberrant transcription and post-transcriptional processing of hepatitis C virus non-structural genes in transgenic mice.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease worldwide. Since several aspects of the infection remain unresolved, there is a pressing need for a convenient animal model that can mimic the clinical disease and aid the evaluation of treatment strategies. Although some success has been achieved in transgenic approaches for development of rodent models of HCV, transgenic expression of the complete HCV polyprotein or an entire set of the viral non-structural (NS) proteins continues to be a serious challenge. Using northern blot and 5' rapid amplification of cDNA ends (RACE), we unraveled two possible mechanisms that can impede HCV NS transgene expression in the mouse liver. Several truncated transcripts are produced from alternate transcription start sites along the HCV NS sequence within the murine environment, in vivo. Translation of these shorter transcripts is blocked either by the positioning of a contextual stop codon or through a shift in the reading frame. In addition, the complete NS transcript undergoes trans-splicing through 5' recombination with a non-transgene-derived, spliced leader sequence that appends a potential stop codon upstream of the translation start. These findings thus demonstrate that HCV NS-derived transgenes are subject to aberrant transcriptional initiation and post-transcriptional processing in the nucleus of a mouse host. Strategies to prevent such aberrant transcription start/RNA processing might be key to the development of a successful HCV transgenic mouse model.

GB virus C/hepatitis G virus infection in Indian blood donors and high-risk groups.

The hepatitis G virus (HGV) or GB virus C (GBV-C) was discovered in 1995 as a putative agent of post-transfusion, non-A-E hepatitis. The present study was carried out with the aim to find the prevalence of this virus among various subject groups at risk for parenteral transmission as well as in healthy control subjects both individually and along with other parenterally transmitted hepatitis viruses. Of the 402 subjects tested, 6.22% were positive for the HBsAg surface antigen, 7.21% were positive for HCV RNA while only 2.24% were seen to be carriers of the HGV/GBV-C RNA. All the HGV/GBV-C positive cases were either multi-transfused thalassaemic subjects or hemodialysis patients. None of the healthy control subjects showed presence of the virus. Seven of the HGV/GBV-C positive subjects showed co-infection with one or more additional virological markers. Also, of the 9 HGV/GBV-C positive subjects, 5 showed elevated ALT levels while 4 showed elevated alkaline phosphatase levels. Overall our findings seem to indicate that HGV infections generally are asymptomatic, transient and self-limiting and the virus does not seem to show a very high prevalence among the Indian population.

IFN-α/ß and autophagy: tug-of-war between HCV and the host.

Hepatitis C virus (HCV) infects approximately 130 million people worldwide. The clinical sequelae of this chronic disease include cirrhosis, functional failure and carcinoma of the liver. HCV induces autophagy, a fundamental cellular process for maintaining homeostasis and mediating innate immune response, and also inhibits autophagic protein degradation and suppresses antiviral immunity. In addition to this ploy, the HCV serine protease composed of the viral non-structural proteins 3/4A (NS3/4A) can enzymatically digest two cellular proteins, mitochondria-associated anti-viral signaling protein (MAVS) and Toll/interleukin-1 receptor domain containing adaptor inducing IFN-ß (TRIF). Since these two proteins are the adaptor molecules in the retinoic acid-inducible gene I (RIG-I) and TLR3 pathways, respectively, their cleavage has been suggested as a pivotal mechanism by which HCV blunts the IFN-α/ß signaling and antiviral responses. Thus far, how HCV perturbs autophagy and copes with IFN-α/ß in the liver remains unclear.

Polymerase chain reaction for the rapid diagnosis of tuberculous meningitis.

Polymerase chain reaction (PCR) based on the amplification of a 169 bp DNA fragment specific for the Mycobacterium tuberculosis complex was evaluated for the rapid diagnosis of tuberculous meningitis (TBM). A total of 105 CSF specimens from clinically suspected cases of TBM were studied. Clinical details of the cases and cytochemical parameters of the CSF specimens were recorded. For PCR 10 CSF specimens from cases other than TBM, 4 non-mycobacterial culture isolates (one strain of E coli, one strain of proteus species and 2 strains of salmonella species) and one sample of sterile distilled water were processed as negative controls. For positive control standard culture of Mycobacterium tuberculosis H37Rv was processed with every batch of specimens. Besides PCR, smear for AFB by the Ziehl-Neelsen (ZN) and the fluorochrome method and culture on Lowenstein-Jensen medium was also carried out. By PCR, 31.42% specimens were found positive, whereas by conventional culture on Lowenstein-Jensen medium only 3.8% specimens were positive.

Molecular epidemiology and clinical implications of TT virus (TTV) infection in Indian subjects.

GOALS: This study was aimed at obtaining data on the epidemiology and clinical course of TT virus (TTV) infections among Indian subjects. BACKGROUND: The TTV is a nonenveloped DNA virus, first identified in the peripheral blood of individuals with posttransfusion hepatitis of unknown etiology. There has been much conjecture regarding the disease association of this virus. STUDY: A total of 494 serum specimens from various groups of high-risk and control subjects were screened for TTV DNA by a semi-nested PCR, using the ORF1-derived N22 primers. The sera were also screened for the HBsAg surface antigen by an ELISA, HCV RNA by a 5' NCR-based RT-PCR and GBV-C/HGV RNA by a 5' UTR-based RT-PCR. The clinical and hepatic profiles of the various subjects were also studied. Seventy-one randomly picked TTV isolates were directly sequenced and their phylogeny was studied. RESULTS: TTV showed an overall positivity rate of 45.34% with a significant higher prevalence of 52.9% among the high-risk subjects as against a prevalence of 28% among healthy control subjects (P < 0.001). Abnormal liver function profiles were frequent among TTV viremic individuals and among the acute hepatitis cases studied a higher mortality rate correlated with a superimposed TTV infection. The 71 TTV isolates sequenced were found to belong to genotype 1a being closely homologous to TTV prototype TA278. CONCLUSION: The TT virus shows a significant prevalence in the Indian population, particularly among subjects at risk for acquiring parenterally transmitted infections. Our study corroborates a putative role of the virus in the etiology of liver disease, particularly in coinfection with other agents.