Different Regions Of Viral Genome Research Articles

Occult hepatitis B virus infection in patients with chronic liver disease.

e15199 Background: Occult hepatitis B virus (HBV) infection is characterized by persistence of HBV DNA into the tissue of hepatitis B surface antigen-negative individuals. The clinical relevance of this infection is still under debate. In particular, the impact of occult HBV infection in cases of hepatocellular carcinoma (HCC) is uncertain. Methods: We investigated the prevalence of occult HBV in patients with chronic liver disease, with or without HCC, that were submitted to liver transplantation or partial hepatectomy in Alfa Institute of Gastroenterology from Clinical Hospital - UFMG - Brazil. We tested the presence of VHB DNA in liver sample using nested PCR in four different regions of viral genome (pre-S/S, pre –core/core, polymerase and X). We also tested these patients’serum for HVB antigens (HBsAg and HBeAg)and antibodies (anti-HBs, anti-HBe and anti-HBC). Results: Our studied population included 71 patients, 50 (70.4%) were male and had median age of 51±12.5 years. Cirrhosis etiology was alcoholic (22 cases, 32.4%), viral hepatitis (17 cases, 25%), cryptogenic (16 cases, 23.5%), autoimmune (10 cases, 14.7%) and 3 cases of other etiologies. HCC was found in 22 patients (31.4%). Viral DNA was detected in 4 cases (5.6%), three of them with HCC. Among these three cases viral DNA was found in non-tumoral area in two of them and in HCC sample in the other one. Conclusions: The prevalence of occult hepatitis B infection was high in our population of cirrhotic patients submitted to liver transplant, especially with associated HCC.

2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.

Unique lentivirus infecting feral simians from forests of Rajasthan, India

Lentivirus infection of wild rhesus macaques (Macaca mulatta) and langurs (Semnopithecus entellus) from forests of Rajasthan was suspected based on testing of simian plasma samples by commercial HIV-1 WB in 1998. Though simians have a religious connotation in India, aggressive monkey bites are frequently reported from urban and rural areas due to habitat fragmentation of wild simians. Blood samples from 34 feral rhesus macaques and 9 langurs were collected recently from forests of Rajasthan. Plasma samples were tested using monoclonal antibody against capsid proteins of SIVmac (p27) and HIV-1 (p24). Mitochondrial gene analyses confirmed the species of origin. Integrated provirus was detected by Alu-LTR amplification. Lentiviral genes, gag, pol, env, nef, vif, vpr and LTR were amplified and sequenced. Viral load assay was performed independently. Virus purified from simian plasma by ultracentrifugation was used to infect human PBMC, H9 (CD4+, CXCR4+) and U87 (CD4+, CCR5+) cell lines. Virus replication in vitro was detected by p24 antigen capture ELISA and microscopic syncytia formation. Phylogenetic analyses of viral sequences demonstrated intriguing homology to subtype B HIV-1 rather than any known SIV. Significant sequence divergence from HXB2 existed in different regions of viral genome except for conserved pol (RT) region, ruling out inadvertent laboratory contamination. Serological activity was observed against HIV-1 p24 but not SIVmac p27. Reverse transmission of subtype B HIV-1, which is transmitted in north India from infected humans to feral simians due to bleeding monkey bite followed by horizontal spread of the infection in troops of wild simians is proposed.

Performance of three PCR methods targeting different regions of viral genome for the detection of TTV in Non A-E hepatitis, chronic B and C hepatitis and healthy blood donors

Abstract TT virus (TTV) was suggested to be the etiologic agent for non A-E hepatitis but this could not yet be proven due to high detection rates not only in hepatitis but also in healthy persons and sensitivity differences of PCR methods employed. The aim of this study was to evaluate TTV DNA positivity in non A-E hepatitis cases, chronic HBV and HCV hepatitis cases and healthy blood donors via PCR systems that target all regions of the viral genome used for viral detection. 23 non A-E hepatitis, 28 chronic HCV, 21 chronic HBV cases and 56 healthy blood donors were included in the study and evaluated by PCR protocols that target 5′-UTR, 3′-UTR and N22 (ORF1) regions. As a result, 3′-UTR and 5′-UTR PCR had comparable detection rates that were higher than N22 PCR. Differences in detection rates among study groups were not statistically significant for any PCR method. Hepatic enzyme levels of the patients were not correlated with the presence of TTV DNA. Detection rate was significantly higher for Non A-E hepatitis group when positivity rates from all methods were combined. These results suggest an alteration of viral genotypes in Non A-E hepatitis which might be associated with pathogenesis.