Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles.

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

Hepatitis B virus genotypes in acute and fulminant hepatitis patients from north India using two different molecular genotyping approaches.

Data from India on hepatitis B virus (HBV) genotype related differences in clinical progression and outcome of acute and fulminant hepatitis B are limited. Sera from patients with acute hepatitis B (AHB) (n=80), fulminant hepatitis B (FHB) (n=40) and asymptomatic HBsAg carriers (ASC) (n=40) were tested for HBV genotype using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and type-specific primers-based PCR (TSP-PCR). The genotype distribution for 160 patients with HBV related hepatitis/carriers were as follows: A, 3/80 (3.7%) in AHB, 2/40 (5%) in FHB and 7/40 (17.5%) in ASC; D, 77/80 (96.2%) in AHB, 38/40 (95%) in FHB and 33/40 (82.5%) in ASC. C, 0; B, 0; E, 0; F, 0 (p<0.01, genotype D versus A). Compared with genotype D, genotype A patients had no significant clinical or biochemical differences (p>0.05). HBV genotypes A and D were found to be prevalent in patients with HBV related acute and fulminant hepatitis from New Delhi, India. Genotype D was the dominant genotype prevalent in all patient categories while genotype A was solely responsible for AHB leading to chronic hepatitis B in 3.7% of the cases from this region.

Hepatitis B virus genotypes in chronic liver disease patients from New Delhi, India.

AIM: To study the Hepatitis B virus (HBV) genotypes and their effect on the progression and outcome in patients with chronic liver diseases from New Delhi, India. METHODS: Sera from 100 HBV-related chronic liver disease (CLDB) cases were tested for HBV genotype using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Type-specific primers-based PCR (TSP-PCR) targeting to the surface (S) gene encoding hepatitis B surface antigen. RESULTS: Only genotypes A and D were present and genotype D was dominant. Genotype D was present in all CLDB patient categories. The genotype distribution for the 100 patients with CLDB was as follows: genotype A, 16/100 (16%) (7/40- 17% chronic hepatitis B (CHB); 8/47, 17%, HBV-related cirrhosis (CRB); 1/13, 7.6%, HBV-related hepatocellular carcinoma (HCCB); genotype D- 84/100 (84%) (32/40- 80% CHB; 38/47- 81%, CRB; 11/13, 85%, HCCB); genotype A+D, 3/100 (3%) (1/40- 3% CHB; 1/47- 2%, CRB; 1/13, 7.6%, HCCB); C, 0; B, 0; E, 0; F, 0; G 0, H 0; (P<0.01, genotype D vs A). CONCLUSION: Only HBV genotypes A and D were present in patients with CLDB from New Delhi, India. Compared with genotype D, genotype A patients had no significant clinical or biochemical differences (P>0.05). Mixed infection with genotype A and D were seen in 3% of the cases. Genotype D was the dominant genotype prevalent in all patient categories.

Hepatitis A viral genotypes and clinical relevance: Clinical and molecular characterization of hepatitis A virus isolates from northern India.

Viral hepatitis caused by hepatitis A virus (HAV) infection is a worldwide disease; in most cases, it causes an acute self-limited illness. The nucleotide sequence analysis of HAV has classified the virus in seven different genotypes, which include human (I-III and VII) and simian (IV-VI) groups. Most human strains belong to the genotype I, which has been divided into sub-genotypes IA and IB. The present study has been carried out to determine the prevalence of HAV genotypes from northern India and to correlate with their clinical characteristics. Peripheral venous blood collected from 546 cases of acute viral hepatitis was employed for enzyme-linked immunosorbent assays (ELISA) for the serological detection of hepatitis A-C and E viruses. A nested reverse transcription RT-PCR was performed to detect HAV genome, and the positive samples were sequenced to determine the HAV genotypes. Of 73 (13.4%) cases positive for IgM anti-HAV, 29 (39.7%) were positive for HAV RNA. Genotyping was done for 27 (93%) positive cases by direct nucleotide sequencing. Phylogenetic analysis revealed that 15 (55.6%) isolates belonged to genotype 1A, while 12 (44.4%) isolates to IIIA genotype. The results suggest that both genotypes IA and IIIA are almost equally prevalent in northern India. A significant difference was observed with respect to the mean liver-function profile between the IgM anti-HAV-positive and the IgM anti-HAV-negative (includes hepatitis B (153), hepatitis C (57), hepatitis E (153) and unclassified (136)) cases. There is a need for further research on HAV transmission and genotype distribution in Indian sub-continent.

Presence of TT virus infection in chronic hepatitis patients from a hospital in New Delhi, India.

BACKGROUND & OBJECTIVE: The recent discovery of a novel parenterally transmitted, unenveloped, single-stranded DNA virus called TT virus (TTV) in chronic hepatitis patients with unclear pathogenesis throughout the world led us to investigate, its presence in chronic hepatitis patients attending a hospital in New Delhi, India, and to evaluate its role in liver disease. METHODS: TT virus DNA was investigated in serum samples of 70 patients with various types of chronic hepatitis, and 100 healthy subjects from New Delhi, India by nested PCR using the primers that belonged to UTR (A) region of the genome. RESULTS: TTV DNA was detected in 6 of 23 patients (26%) with type B chronic hepatitis, 3 of 20 patients (15%) with type C chronic hepatitis, and 12 of 100 subjects (12%) from healthy control group with normal liver function profile tests. None of the 27 non-B, non-C chronic hepatitis patients had TTV DNA positivity. The prevalence of TTV was significantly higher in type-B chronic hepatitis patients as compared to normal subjects (P< 0.05) but comparable to type C chronic hepatitis patients. The clinical course and biochemical profiles of type B, or type C chronic hepatitis patients co-infected with TTV did not differ significantly from those without TTV infection. INTERPRETATION & CONCLUSION: Interestingly, in chronic hepatitis patients, TTV was always associated with either hepatitis B or C virus indicating a likely parenteral route of transmission. All TTV-positive subjects in healthy control group showed normal clinical and biochemical profiles. Thus, the presence of TTV infection is unlikely to influence the course of chronic hepatitis related to hepatitis B virus (HBV) or hepatitis C virus (HCV) or cause liver diseases in healthy subjects.

Prevalence of transfusion-transmitted virus infection in patients on maintenance hemodialysis from New Delhi, India.

Transfusion-transmitted virus (TTV) has been reported from a number of hemodialysis (HD) units from various countries throughout the world. TTV has been associated with liver diseases, viral hepatitis B, and C. Clinical details and information regarding TTV prevalence from India are insufficient. The prevalence and clinical significance of TTV infection were studied in New Delhi, India in HD patients. Serum samples were derived from 75 patients on maintenance HD, and 75 age- and sex-matched voluntary blood donors were examined for TTV viremia by nested polymerase chain reaction (PCR) using primers derived from UTR (A) region of the TTV genome. The prevalence of TTV DNA in patients on HD (83%) was significantly (p<0.05) higher than in blood donors (43%). Clinical background including the mean age, sex, mean duration of HD, and mean alanine aminotransferase (ALT) levels did not differ significantly between TTV DNA-positive and -negative HD patients. Fifty-four (72%) TTV-positive HD patients and 7 (56%) TTV-negative HD patients had blood transfusion histories (p>0.05). Among TTV-positive patients, Hepatitis B virus (HBV) co-infection was present in 14.2% cases while hepatitis C virus (HCV) co-infection was absent. Persistent elevation of ALT levels was observed in 7(9.3%) HD patients; 3 (43%) of them were TTV positive and 4 (57%) were TTV negative (p>0.05). All 3 TTV-positive patients with elevated ALT levels were co-infected with HBV. Patients with TTV infection alone showed normal ALT levels. Prevalence of TTV infection is high in North Indian patients on maintenance HD. Also, none of the exclusively TTV DNA-positive patients had clinical or biochemical signs of liver disease. TTV seems to spread through parenteral routes. More often, TTV seems to be associated with parenterally transmitted virus HBV, indicating a parenteral mode of TTV transmission. The pathogenicity of TTV remains unclear from the present study.

Prevalence of hepatitis B virus genotype D in precore mutants among chronic liver disease patients from New Delhi, India.

Hepatitis B is one of the most important causes of chronic viral hepatitis world wide. Mutations in the precore region of the hepatitis B virus (HBV) genome are frequently found in hepatitis B envelope antigen-negative cases. Data from India on the HBV genotype-associated distribution of precore mutations are limited. Our objective in this study was to genotype and detect the precore mutant with a point mutation from G to A at nucleotide 1896 using ligase chain reaction (LCR) and direct sequencing. A total of 115 cases of chronic liver disease were screened. The cases were evaluated on the basis of history, clinical examination, liver function profile, and serological test for HBV infection, which includes HBsAg, anti HBcIgG, HBeAg using commercially available ELISA kits. The cases, which were HBeAg+, HBeAg-, and HBV DNA+, were subjected to LCR and confirmed by direct sequencing. Of 115 chronic liver disease cases, 50 (43.5%) cases were HBV DNA positive. All cases were subjected to LCR; 11 (22%) cases confirmed the presence of precore mutants, while the remaining 39 (78%) were classified as the wild form of the virus. HBV genotyping by direct sequencing revealed that genotype D was predominant in both wild and mutant forms of the virus. We conclude that the HBV genotype distribution was not significantly different between precore mutants and the wild form of the virus (P>0.05). North Indian patients with genotype D were more likely to have persistent HBV infection with precore mutants. HBV genotypes correlate with the clinical outcome of chronic HBV infection.