What is the best laboratory method for diagnosis of Herpes Simplex Virus in genital infections?

  Genital infection caused by Herpes simplex virus (HSV) is one of the most common health problems, worldwide. Several methods such as cell culture, serological and molecular methods have been used to detect this virus. Currently, Real-Time Polymerase Chain Reaction (Real time-PCR) technique is widely used due to its high sensitivity and specificity. Besides, Real time-PCR can be employed in the follow-up of therapeutic effects in HSV-infected person who is being treated with antiretroviral drugs. We conducted a review on traditional and current diagnostic methods with a focus on their limitations in the diagnosis of HSV infection

Evaluation of autophagy induction and inhibition in the Huh7.5 cell line through flow cytometry

Background: Autophagy is a physiologic process in which double membrane vesicles engulf damaged proteins and organelles for delivering them to lysosomein order to degrade and recycle them via lysosomal digestion. Beclin1 is one of the basic proteins involved in the initial step of autophagosome formation. In the current study, the effect of exogenous Beclin1 to induce autophagy and the effect of 3MA to inhibit of autophagy was assessed in Huh7.5 cells as an in vitro models of hepatocellular carcinoma. Material and methods: The Recombinant pcDNA-Beclin1was transfected into Huh7.5 cells. Also, the cell treated with 3MA. Next, the autophagy induction and inhibition was conducted via LC3 staining as a main autophagy marker using flow cytometry. Results: The result of this study suggest that the over expression of exogenous Beclin1 in Huh7.5 cells elevated the autophagosome formation as shown by intracellular autophagosomal marker LC3-II staining for about 32.32 % and   3MA decreased  it up to2% in compared with control cells in which the stained LC3-II was12.08. Conclusion: Recombinant beclin1 may be used as a potential autophagy inducer agent and 3-methyl-Adenin inhibits autophagy formation in Huh7.5 cell. The staining autophagy formation marker LC3-II with specific antibody is a reliable method to measure autophagy activation via flow cytometry

Design, construction and evaluation of 1a/JFH1 HCV chimera by replacing the intergenotypic variable region

Peer reviewedPublisher PD

Epidemiology and Clinical Characteristics of Patients with Hepatocellular Carcinoma in North-East of Iran

Background: Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is a life-threatening disease worldwide. The aim of this study was to investigate the epidemiology and clinical features of HCC patients who referred to Omid hospital in Mashhad, northeast of Iran.Materials and Methods: In this cross sectional retrospective study, we reviewed the medical records of patients who referred to Omid hospital – a cancer research center– in Mashhad during 1991 to 2012. Medical records of 29 patients with primary liver cancer proven with biopsy, CT scan or MRI were analyzed in this study.Results: Of 25 eligible cases, 68% were men and the rest were women. The majority of HCC patients were in the 60-69 age group. Also, 44% of patients were found to be hepatitis B virus surface antigen (HBsAg) positive.Conclusion: The age distribution and male preponderance of HCC patients observed in the present study in line with other conducted studies in Iran and other countries. Since this is a retrospective study, a comprehensive study with a larger sample size in a case-control study is needed to establish other HCC-related factors in our province

Functional interactions of structural and NS proteins of hepatitis c virus

Hepatitis C virus (HCV) is a small enveloped virus with a positive-sense single stranded RNA genome. Based on its molecular genetic characteristics, the virus has been classified into the Hepacivirus genus of the family Flaviviridae. HCV is one of the major causes of chronic hepatitis which can lead to liver cirrhosis and hepatocellular carcinoma. According to the recent WHO published data, 123 million individuals are infected with HCV (approximately 3% of the world’s population) throughout the world. Due to its highly variable nature, HCV is classified into six major genotypes. The HCV genome encodes a single polyprotein that is cleaved to yield at least 10 mature proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The recently developed HCV cell culture system, based on the JFH1 strain of HCV, has provided an opportunity to study the role of the viral proteins in the complete HCV replication cycle in human hepatoma cells. How the viral proteins functionally interact during replication of HCV in cell culture is not completely understood. Passage of cell cultures transfected with HCV genomic RNA containing attenuating mutations allows for the selection of genomes with second site compensatory mutations that restore replication to wild type levels. Using this approach, the functional interactions of p7 and E2 with other viral proteins during HCV replication was investigated. A small protein of 63 amino acids, p7 is encoded at the junction of the structural and non-strucutural region. p7 is a highly hydrophobic, integral membrane protein and is classified in the viroporin family. In this thesis, it is shown that p7 is critical for production of viral particles and is implicated in a late step of particle assembly. Since the protein plays a critical role in the virus life cycle, chemical compounds that block p7 function are potential candidates for anti-viral therapy. In this thesis, a chimeric JFH1 genome that encodes the p7 protein of genotype (GT) 1b strain J4 was generated. The intergenotypic chimeric genome was nonviable in human hepatoma cells and infectious chimeric virions were only produced after cells harboring the chimeric genomes were passaged several times. To investigate the emergence of compensatory mutations in the viral proteins during cell passaging, the consensus sequences of the entire polyprotein coding regions of the wild type JFH1 and three chimeric viruses were determined. Sequence analysis revealed mutations in core, NS2, NS5A and NS5B. Reverse genetic analysis demonstrated that any one of the single mutations restored the infectivity of the defective chimeric genomes. These data suggest that there are critical genetic interactions between p7 with core, NS2, NS5A and NS5B. In addition, a stable physical interaction between p7 and NS2 is shown in a transient expression system. The HCV glycoproteins E1 and E2 are present on the surface of virions as a heterodimer that attach virions to host cell receptors and facilitate virus fusion and entry. HCV entry proceeds via attachment to glycosaminoglycans followed by binding to scavenger receptor type B class I, and the tetraspanin CD81. Recently, claudin-1 and occludin have emerged as additional receptors required for entry. E2 has a receptor binding domain (E2661RBD) that conatins three variable regions, hypervariable regions 1 (HVR1), HVR2 and the intergenotypic variable region (igVR). In this thesis, HVR1 of E2 was deleted in the context of full-length replication comptetent HCV. Deletion of HVR1 increases CD81-binding ability of the mutant and also increases its susceptibility to neutralizing antibody MAb 24. The infectivity of the HVR1 deleted virions was attenuated approximately 10-fold prior to accumulation of compensatory mutations. Sequencing of cDNA obtained from reverted virions revealed mutations in E1 (I262L) and E2 (N415D). Reverse genetic studies revealed that I262L improved the infectivity of HVR1 deleted virions 2.5 fold while N415D restored infectivity to wild type levels. These data suggest that mutations within E1 or E2 can compensate for the reduction in infectivity observed for HVR1 deleted viruses. In summary, this thesis demonstrates the importance of functional interactions between HCV proteins during virus morphogenesis and infectivity

Prevalence and Characteristics of Precore Mutation in Iran and Its Correlation with Genotypes of Hepatitis B

Introduction: Mutation of the HBV precore gene prevents the production of HBeAg, which is an important target for immune responses. Distribution of this mutation varies along with frequency of HBV genotypes in accordance with geographic and ethnic variations. The general objective of this study was to evaluate the prevalence and characteristics of precore mutation in Iran and its correlation with genotypes of hepatitis B. Methods: In this cross-sectional study, viral DNA of 182 Iranian hepatitis B surface antigen positive patients who were admitted to Bandar Abbas Blood Transfusion Organization in 2012 and 2013 was retrieved from their serum samples. HBeAg, anti-HBe, and anti-HBc IgM diagnostic tests were performed using ELISA kits. Precore and Pre-S regions were amplified using specific primers and PCR thereafter to determine the genotypes; precore mutation, PCR, and restriction fragment length polymorphism (RFLP) methods also were applied. SPSS version 12 was used for data analysis by Mann–Whitney U test, Fisher’s exact probability test, and t-test. Results: A total of 62 patients (34.1%) had precore mutation (A1896G), and genotype D was the predominant genotype in these patients, which was followed by an unknown genotype that was suspected for genotype B. Interestingly, the relationships between precore mutation and HBeAg (p=0.037) and genotype D (p=0.005) were significant; however, no correlation was observed between this mutation and acute or chronic hepatitis and sex of patients. Conclusion: This study found high prevalence of precore mutations in southern Iran, which was significantly associated with HBeAg and genotype D

Application of bacterial shuttle vectors in designing new vaccines against infectious diseases: brief report

Background: Today, several vaccines have been developed to prevent infectious diseases. The older first-generation vaccines may have many problems. In this regard, genetic engineering plays an important role using tools such as shuttle vectors to develop recombinant DNA vaccines that usually include plasmid constructed so that can propagate in two different host species. The present study reviews a variety of shuttle vectors, their structures, productions, pathogenicity and more importantly their applications in the production of novel vaccines. Methods: A systematic review was performed based on search in international databases with no time limit including Scopus, PubMed and Google Scholar. All databases were searched using the standard (English and Persian) keywords. Relevant articles from 1996 to 2018 were collected from search of international databases including Science Direct, Google Scholar, and PubMed using keywords such as “shuttle vectors”, “recombinant plasmids” and “DNA vaccines”. Results: In this study, a total of 31 full texts were used. A shuttle vector typically contains similar components to replication origins and promoters and can propagate in various hosts. Nowadays, they are used in designing and constructing of new vaccines against infectious diseases including tuberculosis and viral hepatitis. Also, Multi-epitope peptide DNA vaccines are effective against some viruses and they are potentially effective against some bacteria such as Helicobacter pylori. Conclusion: Shuttle vectors as a powerful genetic engineering tool have a high ability to study the mechanisms of pathogenic microorganisms and make new vaccines such as DNA vaccines and multi-epitope vaccines. The hope is that such multi-epitope DNA vaccines might induce immunity against multiple antigenic targets, multiple strain variants, and/or even multiple pathogens. However, the ability of DNA vaccination to co-deliver a series of antibody and/or CD4 T cell epitopes remains largely unexplored

An overview of diarrheagenic Escherichia coli in Iran: A systematic review and meta-analysis

Background: Diarrheagenic Escherichia coli (DEC) is a common enteric pathogen that causes a wide spectrum of gastrointestinal infections, particularly in developing countries. This is a systematic review and meta-analysis to determine the prevalence of DEC in various geographical regions in Iran. Materials and Methods: English (PubMed, Web of Science, Scopus, Embase, Cochrane Library, and Google Scholar) and Persian (IranMedex, SID, Magiran, and Iran Doc) databases were comprehensively searched from January 1990 to April 2017. Study selection and data extraction were performed by two independent reviewers. After assessing heterogeneity among studies, a random effects model was applied to estimate pooled prevalence. Data analyses were done with the Stata software (version 12.0). This meta-analysis was registered with PROSPERO, number CRD42017070411. Results: A total of 73 studies with 18068 isolates were eligible for inclusion within the meta-analysis. The results of random effects model showed that the most prevalent DEC pathotypes were enterotoxigenic E. coli (ETEC) (16%; 95% confidence interval [CI]: 11%–23%), enteroaggregative E. coli (11%; 95% CI: 8%–15%), atypical enteropathogenic E. coli (EPEC) (11%; 95% CI: 8%–14%), Shiga toxin-producing E. coli (9%; 95% CI: 6%–13%), diffuse adherent E. coli (6%; 95% CI: 6%–12%), enteroinvasive E. coli (4%; 95% CI: 2%–6%), and typical EPEC (3%; 95% CI: 1%–5%). Conclusion: This study showed that DEC infections in the Iranian population have low frequency. Our data suggest that the ETEC pathotype can be regarded as one of the most important etiological agents of diarrhea in this country. However, the prevalence of DEC pathotypes is diverse in different regions of Iran