Regulation of HSVtk gene by endogenous microRNA-122a in liver cell lines as suicide gene therapy.

AIM: Here, we use miR-122a that exhibits liver-specific expression for developing a post-transcriptional regulative system mediated by microRNAs. BACKGROUND: Gene therapy with adenovirus (Ad) vectors that express herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (GCV) have been suggested as a therapeutic strategy to cancer. However, Ad vectors injected into tumors are dispersed into the systemic circulation and transduce liver cells, resulting in severe hepatotoxicity. To be effective, the delivery and expression of suicide genes to cancer treatment ought to be specific to tumor cells, and avoid death of healthy cells. Researchers have demonstrated that expression of transgene could be suppressed in healthy cells with use of vectors that are reactive to microRNA regulation. METHODS: We constructed an Ad vector carrying four tandem copies of target sequences of miR-122a that were incorporated into 3'-UTR of HSVtk gene. The expression level of miR-122a was quantified in HepG2 and Huh7 cell lines. RESULTS: Quantitative RT- PCR analysis demonstrated that Huh7 cells express large amounts of miR-122a compared to HepG2 cells. The viability of Huh7 cells and HepG2 cells after infection by Ad-tk-122aT vector was 83% and 23.5%, respectively. The viability of Huh7 cells was not reduced in the presence of GCV after infection by Ad-tk-122a vector. In contrast, cytotoxicity of HSV-tk/GCV was similar in Huh7 cells and HepG2 cells by Ad-tk vector, with 35.3% and 27% viability, respectively. CONCLUSION: Inclusion of the miR-122a target sequences in the HSVtk expression cassette yielded a feasible strategy for reducing cytotoxicity of suicide gene in a liver cell line with high miR-122a expression.

The effect of bovine rotavirus and its nonstructural protein 4 on ER stress-mediated apoptosis in HeLa and HT-29 cells.

Endoplasmic reticulum (ER) plays important roles in multiple cellular processes as well as cell survival and apoptosis. Perturbation of ER functions leads to ER stress and unfolded protein response (UPR). The primary goal of this response is cell survival, but severe ER stress can trigger apoptosis signaling. In tumor cells, chronically activated UPR response provides tumor growth. So, apoptosis induced by the ER stress has been the target for anti-cancer therapy. In this in vitro study, we examined the apoptotic effect associated with ER stress of bovine rotavirus and its nonstructural protein 4 (NSP4) alone in two cancer cell lines. The plasmid pcDNA3.1 encoding NSP4 protein of bovine rotavirus transfected with lipofectamine 2000 into the HeLa and HT-29 cells for protein production. MTT, flow cytometry, and Western blot were used to evaluate the cell viability, apoptosis, and expression level of C/EBP-homologous protein (CHOP) and activated caspase-4. In parallel, the apoptotic effect of the bovine rotavirus associated with ER stress in the infected cells was examined too. The cytotoxic and apoptotic effect of NSP4 protein on the cells were statistically significant compared to the control groups. However, Western blot showed that the expression of the NSP4 protein by recombinant plasmid did not lead to high expression of CHOP and activation of caspase-4. Interestingly, rotavirus not only induced significant apoptosis but also caused an increase in CHOP expression and caspase-4 activation in the infected cells compared to control. As a result, NSP4 protein and bovine rotavirus can be considered a potential novel bio-therapeutic strategy for cancer treatment.

The pigeonhole of occult hepatitis B.

Occult hepatitis B (OHB), or persistent hepatitis B virus (HBV) viremia in surface-antigen-HBsAg-negative patients, has been recognized as a medical concern during the last decade. The exact magnitude, pathogenesis and clinical relevance of OHB are unclear. This review organizes the published data on OHB and presents an overview of the current hypotheses on OHB's pathogenesis and clinical relevance.Many explanations have been offered for the pathogenesis of OHB, ranging from the inability of standard immunoassays to diagnose OHB to the involvement of the versatile virus-host factors. Also, special care should be taken regarding the diagnosis of OBH. It seems that both shared viral-host factors are involved in the pathogenesis of OBH. Further molecular studies on cohort patients group need to explore such association.

Antiretroviral drug resistance among antiretroviral-naïve and treatment experienced patients infected with HIV in Iran.

Resistance to antiretroviral therapy (ART) threatens the success of programs to reduce HIV morbidity and mortality, particularly in countries with few treatment options. In the present study, genotype and phenotype data from ART-naïve and experienced hospitalized patients infected with HIV in Tehran, Iran were used to assess the prevalence and types of transmitted (TDR) and acquired drug resistance (ADR) mutations. All 30 participants naïve to ART and 62 of 70 (88.6%) participants receiving ART had detectable viral loads. Among participants receiving ART with sequencing data available (n = 62), 36 (58.1%) had at least one drug resistance mutation; the most common mutations were K103N (21.0%), M184V (19.4%), and the thymidine analogue mutations. Seven (11.3%), 27 (43.5%), and two (3.2%) of these participants had resistance to one, two, and three drug classes, respectively. High-level resistance to efavirenz (EFV) was more common among participants on EFV-based regimens than high-level lopinavir/ritonivar (LPV/r) resistance among those on LPV/r-based regimens (55.3% vs. 6.7%, P < 0.0001). Two (6.7%) antiretroviral-naïve participants had K103N mutations. These findings document an alarmingly high frequency of multiple HIV drug class resistance in Iran, confirm the presence of TDR, and highlight the need for systematic viral load monitoring and drug resistance testing, including at diagnosis. Expanded access to new antiretroviral medications from additional drug classes is needed.

Drug-related mutational patterns in hepatitis B virus (HBV) reverse transcriptase proteins from Iranian treatment-naïve chronic HBV patients.

BACKGROUND: Immunomodulators and Nucleotide analogues have been used globally for the dealing of chronic hepatitis B virus (HBV) infection. However, the development of drug resistance is a major limitation to their long-term effectiveness. OBJECTIVES: The aim of this study was to characterize the hepatitis B virus reverse transcriptase (RT) protein variations among Iranian chronic HBV carriers who did not receive any antiviral treatments. MATERIALS AND METHODS: Hepatitis B virus partial RT genes from 325 chronic in active carrier patients were amplified and directly sequenced. Nucleotide/amino acid substitutions were identified compared to the sequences obtained from the database. RESULTS: All strains belonging to genotype D.365 amino-acid substitutions were found. Mutations related to lamivudine, adefovir, telbivudine, and entecavir occurred in (YMDD) 4% (n = 13), (SVQ) 17.23% (n = 56), (M204I/V + L180M) 2.45% (n = 8) and (M204I) 2.76% (n = 9) of patients, respectively. CONCLUSIONS: RT mutants do occur naturally and could be found in HBV carriers who have never received antiviral therapy. However, mutations related to drug resistance in Iranian treatment-naïve chronic HBV patients were found to be higher than other studies published formerly. Chronic HBV patients should be monitored closely prior the commencement of therapy to achieve the best regimen option.

The prevalence of hepatitis B virus surface antigen (HBsAg) variations and correlation with the clinical and serologic pictures in chronic carriers from Khorasan Province, North-East of Iran.

This study was designed to determine the correlation of hepatitis B virus surface Ag (HBsAg) variations with the clinical/serological pictures among chronic HBsAg positive patients. The surface gene (S-gene) was amplified and directly sequenced in twenty-five patients. Eight samples (group I) contained at least one mutation at the amino acid level. Five showed alanine aminotransferase (ALT) levels above the normal range of which only one sample was anti-HBe positive. Group II (17 samples) did not contain any mutation, 4 were anti-HBe positive and 9 had increased ALT levels. In both groups, from a total of 18 mutations, 5 (27.5%) and 13 (72.5%) occurred in anti-HBe and HBeAg positive groups respectively. The small number of amino acid mutations might belong to either the initial phase of chronicity in our patients; or that even in anti-HBe positive phase in Iranian genotype D-infected patients, a somehow tolerant pattern due to the host genetic factors may be responsible.

Multiplex reverse transcriptase-PCR assay for typing and subtyping of influenza A (H5 & H9) virus in Iran.

BACKGROUND: Avian influenza virus (AIV) infection is a major cause of bird or human mortality and morbidity, therefore the rapid identification of the virus is of important clinical and epidemiological implication. METHODS: A multiplex Reverse Transcriptase PCR (RT-PCR) was optimized for the detection of influenza A virus and the H5 and H9 subtypes. The influenza type A specific primers were directed to the region of the influenza A matrix gene that is conserved among most type A influenza viruses. The H5 and H9 primers were directed to H5 and H9 hemagglutinin (HA) gene regions that are conserved among H5 and H9 subtypes. The selected primer sets were used in the RT-PCR for simultaneous detection of matrix, H5 and H9 responding specific sequences in a multiplex format. RESULTS: Three reaction conditions were optimized which include: i) RT-PCR typing using matrix gene primers for five subtypes of flu A (H1, H3, H5, H7 and H9), ii) RT-PCR subtyping for H5 and H9 subtypes, and iii) multiplex subtyping of H5 and H9. In this study, the multiplex RT-PCR was applied to 147 cloacal and tracheal swabs of clinical poultry cases with similar influenza symptoms. CONCLUSIONS: These results suggest that multiplex RT-PCR assay can be a useful test for rapid detection and subtyping of AIV in clinical samples.