manochehr makvandi

Oncolytic Herpes simplex virus type 1 (HSV-1) has emerged as a promising strategy for cancer therapy. However, development of novel oncolytic mutants has remained a major challenge owing to low efficiency of conventional genome editing methods. Recently, CRISPR-Cas9 has revolutionized genome editing. In this study, we aimed to evaluate the capability of CRISPR-Cas9 to manipulate the UL39 gene to create oncolytic HSV-1. Herein, three sgRNAs were designed against the UL39 gene and transfected into HEK-293 cell line followed by infection with HSV-1 KOS. After three rounds of plaque purification, several HSV-1 mutants were identified by PCR analysis and sequencing. One of these mutations in which 55 nucleotides were deleted resulted in a frameshift mutation that in turn produced a truncated protein with only 167 amino acids from 1137 amino acids. Functional analysis in Vero and primary fibroblast cells revealed that viral replication was significantly lower and plaque size was smaller in the HSV-1 mutant compared with HSV-1 KOS. Moreover, the relative amount of viral genome present in the supernatants of infected cells (Vero and primary fibroblast cells) with HSV-1 mutant was significantly decreased compared with those of HSV-1 KOS. Our data revealed that targeting UL39 with CRISPR-Cas9 could develop oncolytic HSV-1.

Hepatitis G virus (HGV) is a member of Flaviviridae. Prevalence of HGV in healthy people is very low, but this virus is more prevalent in patients with hepatitis. Besides, relative frequency of HGV in patients undergoing hemodialysis, and kidney recipients is very high. The role of HGV in pathogenesis is not clear. Since this virus cannot be cultivated, molecular techniques such as Revers Transcription Polymerase Chain Reaction (RT-PCR) is applied to detect HGV.. The current study aimed to investigate the prevalence of HGV using determination of E2, viral envelope antigen, antibodies and the RNA by Enzyme Linked Immunosorbent Assay (ELISA) and RT-PCR techniques. The rational of the study was to determine the prevalence of HGV in patients undergoing hemodialysis and kidney transplantation in Khuzestan province, Iran..Patients and Five hundred and sixteen serum samples of the patients undergoing hemodialysis and kidney transplantation from various cities of Khuzestan province were collected. Anti-hepatitis G E2 antibodies were investigated by ELISA method. RNAs were extracted from serums and Hepatitis G RNA was detected by RT-PCR.. Of the 516 samples, 38 (7.36%) specimens were positive for anti-HGV by ELISA. All of these ELISA positive samples were negative for HGV genome by RT-PCR. Of the remaining 478 ELISA negative samples, 16 (3.14%) samples were positive by RT-PCR.. Hepatitis G Virus was not prevalent in the patients undergoing hemodialysis and kidney transplantation in Khuzestan province. Although reports indicated high frequency of co-infection of HGV with hepatitis B and C viruses, in the current research, co-infection of HGV with B and C was not considerable. Since different groups and subtypes of HGV are reported, periodic epidemiologic evaluation of HGV and its co-infection with other hepatitis viruses is suggested in other populations such as the patients with thalassemia; however, periodic epidemiologic monitoring of HGV may be helpful to control future potential variations of the virus..

Hepatitis B virus (HBV) infection is a worldwide public health problem. Nine HBV genotypes (A-I) have been already discovered. HBV genotypes are important both in the clinical manifestation of disease and treatment response. Moreover, HBV DNA without HBs (Hepatitis B surface)-antigenemia was detected in some patients with chronic hepatitis (occult hepatitis). There is little information about HBV genotypes and its relation to occult infection despite the importance of this infection in Khuzestan Province. This study aimed to determine both occult hepatitis B infection and HBV genotypes among cirrhotic patients.Patients and Thirty-eight patients with liver cirrhosis, including 11 (28.9%) HBsAg-positive patients and 27 (71.1%) patients with cryptogenic cirrhosis participated in this study. The mean age of the patients at the time of cirrhosis diagnosis was 54.85 years (range 26-75 years). All patients were anti-HCV and anti-HIV negative. For all the samples, the serological Enzyme-Linked Immunosorbent Assay (ELISA) was performed for HBV markers including HBsAg, HBcAb, HBeAg, HBeAb tests. The common primer of S region of HBV was used for Nested PCR. The PCR products of the positive individuals were sequenced for genotyping and subtyping of HBV. Eleven (40.7%) out of 27 HBV cryptogenic cirrhosis and all 11 HBsAg-positive patients were positive for HBV DNA. The seroprevalences of Hepatitis B virus HBe antigen, anti-HBe and anti-HBc antibodies among the cryptogenic cirrhosis patients were 5 (18.5%), 1 (3.7%), and 5 (20.83), and among HBsAg-positive patients were 6 (54.5%), 5 (45.5%), and 7 (63.6%), respectively. In our study, only HBV genotype D was found among all the positive HBsAg and occult HBV infection. Moreover, high prevalence (40.7%) of occult HBV infection was determined among patients suffered from cryptogenic cirrhosis.

Introduction and Hepatitis B is a disease of public health importance in Iran. The aim of this study was to evaluate some possible risk factors for the spread of hepatitis B infection from the hepatitis B virus (HBV) carriers. A cross-sectional study was conducted among chronic HBV individuals who referred to gastrointestinal department, Imam Khomeini hospital, Ahvaz Jundishapur University of medical sciences from October 2009 to June 2010. All subjects were evaluated using a face-to-face questionnaire about demographic aspects. The analysis included data on past medical history, physical examination and periodic evaluation clinically and serologically. The control group consisted of the patients referred to the gastrointestinal clinics with negative HBV serologic markers of HBV infection. The risk factors among infected subjects (HBV-positive) were compared to those of subjects never exposed (HBV-negative) to HBV. A total of 560 subjects were studied for HBV, of which 272 were HBV-positive and 288 HBV negative cases comprised the control group. Mean age of the patients was 35±9 years. HBV-positive subjects were more likely to be of female gender (61.39% versus 41.31%, P 0.0001). Endoscopy 54.77%, major surgery 44.48%, and tattooing history 8.45% were found to be independent risk factors of being chronically infected with hepatitis B virus. Our data indicate that a history of endoscopy, major surgery and tattooing are important risk factors for spreading of HBV infection. Significance and impact of the study: Improvements in certain lifestyle patterns and customs in this area may be essential to prevent transmission of the infection.

Introduction and Hepatitis B virus (HBV) is a member of Hepadnaviridae and a major causative agent of chronic and acute hepatitis, liver cirrhosis and hepatocellular carcinoma. Genotype determination of HBV is based on PCR-RFLP and sequencing of genome of the virus. The genotype formation is mainly due to mutations of HBV precore, S, and YMDD (tyrosine-methionine-aspartate-aspartate motif in the C domain of the HBV DNA polymerase gene) genome area. Moreover, some of the mutant HBV remains undetectable by serological tests (occult hepatitis). Since the genotypes of HBV and occult hepatitis B has not been studied in our area, this study was conducted to determine both occult hepatitis B infection and genotypes among hemodialysis patients. Two hundred and fifty hemodialysis patients were selected in this study. The sera of the patients were collected and the extracted DNA was used as template of PCR to amplify a 479bp fragment of the viral genome. The PCR products were digested by Ava2 and Mbo1 restriction enzymes. Based on RFLP patterns, the genotypes were determined. The HBV markers including; HBV surface antigen (HBsAg), hepatitis Bc antibody (HBcIgG), hepatitis B virus e antigen (HBeAg) and hepatitis B virus e antibody (HBeAb) were carried out for all the patients by ELISA test. Fifty (20%) out of 250 sera showed positive HBV by PCR. Out of the 50 positive cases for HBV, 46(92%) belonged to genotype D2 and 2(4%) cases of them were B6 genotype. Ten cases were positive for HBV by PCR test but negative by ELISA test (4% occult hepatitis). Prevalence of HBV infection was high among the dialysis patients (20%), and occult hepatitis B was 4% in these patients. The dominant genotype of HBV was D2 (92%) followed by genotypes B6 (4%) in hemodialysis patients.