Characterization of liver specific promoters in a foamy viral vector pMD09.

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.

A comparative study of hepatitis B virus X protein mutants K130M, V131I and KV130/131MI to investigate their roles in fibrosis, cirrhosis and hepatocellular carcinoma.

Hepatitis B virus (HBV) genomic mutations A1762T, G1764A and AG1762/1764TA cause production of HBV X protein (HBx) mutants, namely K130M, V131I and KV130/131MI. These mutations are important biomarkers for the development of cirrhosis and hepatocellular carcinoma (HCC) in chronic HBV patients. This study comparatively analyses the impact of intracellular expression of HBx mutants on HCC cell line Huh7. It was found that expression of KV130/131MI induced: cell proliferation, altered expression of cell cycle regulatory genes in favour of cell proliferation, intracellular reactive oxygen species (ROS) production and mitochondrial depolarization. KV130/131MI may be directly involved in host cell proliferation and hepatocarcinogenesis via altering expression of cell cycle regulatory genes. KV130/131MI may also play pivotal roles in fibrosis and cirrhosis via inducing ROS production and mitochondrial depolarization. Furthermore, these might be the possible reasons for higher occurrence of AG1762/1764TA as compared to A1762T and G1764A in cirrhosis and HCC patients.

Occurrence of co-infection with dengue viruses during 2014 in New Delhi, India.

Dengue fever is an arthropod-borne viral infection that has become endemic in several parts of India including Delhi. We studied occurrence of co-infection with dengue viruses during an outbreak in New Delhi, India in 2014. For the present study, blood samples collected from symptomatic patients were analysed by RT-PCR. Eighty percent of the samples were positive for dengue virus. The result showed that DENV-1 (77%) was the predominant serotype followed by DENV-2 (60%). Concurrent infection with more than one serotype was identified in 43% of the positive samples. Phylogenetic analysis clustered DENV-1 strains with the American African and DENV-2 strains in Cosmopolitan genotypes. Four common amino-acid mutations were identified in the envelope gene of DENV-1 sequences (F337I, A369T, V380I and L402F) and one common mutation (N390S) in the DENV-2 sequences. Further analysis revealed purifying selection in both the serotypes. A significant number of patients were co-infected with DENV-1 and DENV-2 serotypes. Although we do not have direct evidence to demonstrate co-evolution of these two stereotypes, nonetheless their simultaneous occurrence does indicate that they are favoured by evolutionary forces. An ongoing surveillance and careful analysis of future outbreaks will strengthen the concept of co-evolution or otherwise. Whether the concurrent dengue viral infection is correlated with disease severity in a given population is another aspect to be pursued. This study is envisaged to be useful for future reference in the context of overall epidemiology.

Role of HBV genotype in predicting response to lamivudine therapy in patients with chronic hepatitis B.

BACKGROUND: Predictors of response of chronic hepatitis B (CHB) to lamivudine therapy need better definition. Whether hepatitis B virus (HBV) genotypes could serve as such a predictor has not been well studied. AIM: To study the association of HBV genotypes with the outcome of lamivudine treatment in patients with CHB. METHODS: Seventy-six patients with CHB (45 HBeAg +ve) received lamivudine 100 mg/day, orally for 12 mo. Infecting HBV genotypes were determined in pre-treatment specimens using restriction fragment length polymorphism. End-of-treatment response (ETR) and sustained viral response (SVR) were defined as undetectable HBV DNA (< 0.5 pg/mL) at 12 and 18 months, respectively. RESULTS: ETR was observed in 26 (34%) and SVR in 11 (14%) patients receiving lamivudine. The pre-treatment characteristics of the responders and non-responders were comparable. Genotypes A and D were observed in 28 (37%) and 48 (63%) patients, respectively. The frequency of genotypes A and D was comparable between responders (28.6% vs. 37.5%) and non-responders (71.4% vs. 62.5%), respectively (p=ns). Of the 26 responders, SVR could be evaluated in 20 subjects; 9 (45%) relapsed and 11 achieved SVR. Patients with genotype D achieved higher SVR rate than genotype A (10 of 48, 28.8% vs. 1 of 28, 3.5% p =0.0359). CONCLUSIONS: Forty-five percent of Indian patients with CHB who achieve ETR relapse, and SVR to lamivudine therapy is achieved in 14%. Patients with genotype D achieve higher SVR rate than with genotype A.

Base-pair alterations in the epsilon-lower stem due to a novel double substitution in the precore gene of HBV-e negative variant were recovered by secondary mutations.

The HBe negative phenotype, a natural precore mutant (G1896A/G1897A) of HBV with aborted HBeAg expression is known to cause chronic hepatitis. The destabilized C : G base-pairing in the lower stem of epsilon-hairpin due to G1896A substitution is reportedly compensated by a second C1858T mutation and suggested to play an important role in enhanced selection of the HBe negative variant. We undertook to investigate presence of such compensatory mutations at other positions by analyzing epsilon-sequences (nts. 1847-1907) as well as to look for their effect(s), if any, on the consensus sequence of the overlapping core-initiator of HBe negative HBV variants in CLD patients. Three of the 5 HBe negative patients had classical G1896A mutation having a second compensatory mutation at nt. 1858. One patient showed an additional G1897A substitution, presenting as a novel precore stop codon mutation (UGG-->UAA), followed by a compensatory mutation at position 1857. In the third patient, a G1899A substitution was seen which compensated the impaired U at position 1855. Other substitution and deletion mutations were also observed in the remaining epsilon-hairpin, which however, did not produce any compensatory mutation. Further, all the precore variants showed a conserved G at position 1904, important for the optimal context of their core-initiator which however, remained impaired with A (nt. 1850). Our results suggest that the nts. 1851-1859 and nts. 1895-1904 in the lower stem, and restoration of authentic base-pairings therein, maintain the structural integrity and stability of the epsilon-hairpin. This may have a role in the enhanced selection of the HBe negative variants and persistence of HBV infection in chronic liver disease patients.

Beneficial effects of lamivudine in hepatitis B virus-related decompensated cirrhosis.

BACKGROUND/AIMS: HBV-related chronic liver disease patients often present with hepatic decompensation and are not eligible for interferon therapy. Whether long-term lamivudine is effective in these patients was prospectively evaluated. METHODS: Eighteen patients with HBV-related decompensated cirrhosis, all with quantitative DNA +ve and 10 HBeAg +ve, were given lamivudine 150 mg/d. RESULTS: Each patient received at least 9 months (mean 17.9) of lamivudine. Three HBeAg+ve patients (30%) seroconverted to anti-HBe and one lost HBsAg during the follow-up. An improvement from baseline in the aspartate aminotransferase (130 vs. 72 IU/l, p<0.04); alanine aminotransferase (111 vs. 58 IU/l, p<0.01) and Child-Pugh score (8.3 vs 6.7, p<0.013) was seen. Lamivudine had no significant side-effects. HBV DNA became undetectable in all patients by 8 weeks of therapy. In three (17%) patients, HBV DNA again became positive at 9, 9 and 27 months. YMDD mutant was, however, detected in only one (6%). A significant reduction was noted in the morbidity and hospitalizations for complications of liver disease before and after starting lamivudine (1.5+/-0.7 vs. 0.6+/-0.7, p<0.002). CONCLUSIONS: In decompensated HBV-related cirrhosis, lamivudine: i) is effective in suppressing HBV DNA and seroconversion to anti-HBe (30%), ii) can achieve significant improvement in clinical and biochemical status of liver functions.