Recombinant Coree1e2 Protein Expressed in Pichia pastoris Yeast a Candidate Vaccine for Hepatitis C Virus

Background: Hepatitis C virus (HCV) infection is a major health problem both in developed and developing countries and HCV infection is a global blood borne disease that affects almost 3% of the world’s population with a morbidity and mortality rates. The efficacy of hepatitis C treatment is less than satisfactory and development of an effective vaccine may be essential in the control of HCV infection. The E1 and E2 proteins, two heavily glycosylated enveloped proteins, which can elicit neutralizing antibodies against HCV infection in the host and Core, E1 and E2 proteins are the major vaccine candidates for vaccination and ELISA is one of routine testes which has been used in clinical laboratories and different studies to detect the rate of antibody in sera against HCV infection. Aim: Evolvement and gradual development of a useful vaccine can be the main point in the control and eradication of hepatitis C virus (HCV) infection. Recent studies have reported that HCV envelope glycoproteins can induce neutralizing antibodies against antigen domain of HCV. So HCV envelope proteins are considered as the main HCV vaccine candidate. Methods: In this study, we used Pichia pastoris yeast expression system to express recombinant HCV CoreE1E2 protein, which consists of Core (269 nt-841nt) E1 (842 nt-1417nt) and E2 (1418 nt-2506nt). The Pichia pastoris can produce high level of recombinant HCV CoreE1E2 protein. The protein has glycosylation and also by codon optimization based on pichia expression system we could increase the rate of recombinant proteins. Moreover, the purified protein can efficiently induce anti-CoreE1E2 antibodies in rabbits, and also by developing homemade ELISA kit we can detect antibody of HCV Iranian patients with 1a genotype. Results: Although little is known about the mechanism of hepatitis C virion assembly, in our study the virus like particle of rCoreE1E2 with 70 nm size, were shown by Electron microscopy and have proved the self-assembly in vitro in yeast expression system. Conclusion: These findings indicate that the recombinant CoreE1E2 glycoprotein is effective in inducing neutralizing antibodies, and is an influential HCV vaccine candidate

Prevalence and Type Distribution of Human Papillomavirus Infection Using the INNo-Lipa Assay, Kerman, Southeast Iran

The human papilloma virus (HPV) causes skin and mucous membrane infections. It crosses from one person to another by skin-to-skin contact, such as sexual contact. There are more than 100 types of HPV that can influence different parts of the body. Some types of HPV can cause cancer (such as cervical or anal cancer) and others can cause warts (such as genital or plantar warts). HPV infection is one of the most common sexually transmitted infections (STIs) in Iran and around the world. Considerable molecular evidence suggests a role for human papilloma virus (HPV) in the pathogenesis of carcinoma. Epidemiological studies on human papilloma viruses (HPVs) infections in general population are critical for the performing of health policy guidelines for developing the strategies to hinder the primary and secondary different cancer. In different parts of Iran, there is a lack of population-based studies to determine the prevalence of HPV in the general population. The aim of this population-based study was therefore to report the prevalence ratse of HPV types among Iranian patients. To study the risk of human papilloma virus (HPV) infection, we managed a retrospective study in Kerman province, southeast of Iran. For this purpose, 410 patients tested for the presence of HPV DNA using PCR and INNo- Lipa assays. HPV DNA was detected in 108 out of 410 patients (26.34%), while it was not detected in any of the control group samples. Patients included 23 (21.1%) males and 86 (78.8%) females. HPV type 6 was the most common (49%) followed by HPV type 16 (10.1%), and also HPV type11 (9.2%). The prevalence of HPV in Iran is comparable to those reported in other regions of the world. In a similar manner, it seems that HPV types 6, 16 and11 are the most common types in Kerman. Additional studies on larger group of patients, particularly in thos

Hepatitis C Virus - Proteins, Diagnosis, Treatment and New Approach for VaccineDevelopment

Background: Hepatitis C virus (HCV) causes acute and chronic human hepatitis infection and as such is an important global health problem. HCV was discovered in the USA in 1989 and it is now known that three to four million people are infected every year. The WHO estimates that 3 percent of the 180 million people worldwide are chronically infected. Humans are the natural hosts of HCV and this virus can eventually leads to permanent liver damage and carcinoma. HCV is a member of the Flaviviridae family and Hepacivirus genus. The diameter of the virus is about 50-60 nm and the virion containing a single-stranded positive RNA with approximately 10,000 nucleotides in length and consists of one ORF which is encapsulated by an external lipid envelope and icosahedral capsid. HCV is a heterogeneous virus, classified into 6 genotypes and more than 50 subtypes. Because of the genome variability, nucleotide sequences of genotypes differ by approximately 31-34%, and by 20-23% among subtypes. The quasispecies of mixed virus populations make survival advantage for virus to create multiple variant genomes and a high rate of generation of variants to allow rapid selection of mutants for new environmental conditions. Direct contact with infected blood and blood products, sexual relationships and availability of injectable drugs have had remarkable effects on HCV epidemiology. Hundreds of thousands of people die each year from hepatitis and liver cancer caused by HCV virus infection. Approximately 80% of patients with acute hepatitis C progress into a chronic disease state leading to serious hepatic disorders, 10-20% of which develop chronic liver cirrhosis and hepatocellular carcinoma. The incubation period of HCV is 6-8 weeks and the infection is often asymptomatic so it is very hard to detect at early stages, making early treatment very difficult. Therefore, hepatitis C is called a “silent disease”. Neutralizing antibodies are produced against several HCV proteins during infection but the virus mutates to escape from antibodies. Some patients with chronic hepatitis C may have some symptoms such as fatigue, muscle aches, nausea and pain. Autoimmune and immunecomplex-mediated diseases have also been reported with chronic HCV infection

Human Bocavirus in Iranian children with acute gastroenteritis

Background: Human Bocavirus (HBoV) infection is of worldwide distribution. There is increasing evidence that HBoV is pathogenic for the human gastroenteric tract. However, less data are available on the role of HBoV in gastroenteritis. The present study was aimed to determine the prevalence of HBoV in children with gastroenteritis. Methods: Real-time PCR TaqMan was used to screen 200 stool specimens that had been referred to the virology laboratory for HBoV evaluation. All of samples were collected on viral transport media. Results: Of the 200 stool samples analyzed, 16 (8%) were positive for HBoV. Human Bocavirus positive samples from patients aged between 1 to 5 years with acute gastroenteritis infection suggest a minor role of HBoV in gastroenteritis (p=0.0001). Conclusion: The study showed a high prevalence of human Bocavirus in young children with acute gastroenteritis diseases in Iran, suggesting that HBoV play a role in the pathogenesis of gastroenteritis. Keywords: Gastroenteritis, Child, Human Bocavirus, Real-time PCR

Antiviral Activity of Sirna UL42 against Herpes Simplex Virus Type 1 in HeLa Cell Culture

RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. In this study, we applied siRNAs targeting the UL42 gene of human herpes simplex virus type 1 (HSV-1), which encodes a multifunctional polypeptide that is vital for virus DNA replication, binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. HeLa cell line was used for HSV 1 infection and SiRNA transfection was done to suppress UL-42 gene in cell culture. The decrease in titer of HSV 1 was observed by rReal Time PCR to detect the drop in HSV 1 DNA synthesis and translation. The inhibition rates of siRNA1 and siRNA2 on HSV-1 plaque formation were reported and Comparing with virus control, siRNA1 and siRNA2 reduced DNA replication HSV-1. The decision whether the decrease in the number of HSV-1 plaques was due to siRNA silencing expression of the UL42 gene, a real-time PCR indicating that UL42-specific siRNAs blocked the expression of the UL42 gene significantly. Comparing with virus control, siRNA1 and siRNA2 reduced the expression of UL42 gene. In this study the synthetic siRNA silenced UL42 mRNA expression effectively and specifically and inhibited HSV-1 replication and also our data offer new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication

Evaluation of the Frequency of the IL-28 Polymorphism (rs8099917) in Patients with Chronic Hepatitis C Using Zip Nucleic Acid Probes, Kerman, Southeast of Iran

Polymorphisms in the region of the interleukin IL-28 gene on chromosome 19 have been related with clearance of hepatitis C virus (HCV), a major human pathogen responsible for chronic hepatitis, cirrhosis and hepatocellular carcinoma. About 3% of the world’s population is infected with HCV. The long-term response to therapy is influenced by many host and viral factors, and recent evidence has indicated that some host genetic polymorphisms related to IL-28 are the most powerful predictors of virological response in patients with HCV. This study assessed frequency of the IL-28 polymorphism (rs8099917) in 50 patients (39 men and 11 women ) with chronic hepatitis C using ZNA probe real time PCR new method . All patients were tested for genotype of HCV and the HCV viral load. In parallel, the levels of SGOT, SGPT and ALK enzymes were assessed. Treatment using Peg-interferon alpha with ribavirin was conducted for patients and subsequently samples were collected to detect any change in viral load or liver enzyme rates. The overall frequency of the TT allele is 74%, TG allele 20% and GG allele 6% and the percent of patients who had T allele was 84%. Clear reduction in viral load and liver enzymes was reported in patients with the T allele. Especially for genotype 1 which is relatively resistant to treatment, these alleles may have a role in this decline. In conclusion, we showed that IL-28 polymorphism rs8099917 strongly predicts virological response in HCV infection and that real-time PCR with Zip nucleic acid probes is a sensitive, specific and rapid detection method for detection of SNPs which will be essential for monitoring patients undergoing antiviral therapy

Human Herpesvirus-6 and Human Herpesvirus-7 Infection in Iranian Patients with Neurological Illness

Human herpes virus-6 (HHV-6) and HHV-7 have been implicated as causes of meningitis and encephalitis in children and adults. In this study the presence HHV-6 and HHV-7 DNA were tested in cerebrospinal fluid (CSF) sample taken from Iranian children, suffered from meningoencephalitis. From 2007 to 2009, 150 patients from Tehran with meningoencephalits who were referred to a pediatric ward in Rasoul Akram hospital, Tehran Iran, were enrolled in the present study. Conventional and BACTEC Ped Plus medium were used in conjunction with latex agglutination test and real time PCR for detection of HHV-6 and HHV-7 DNA in clinical specimen. All type of human herpes virus DNA was detected in 12 % (18/150) cases. HHV-6 DNA was detected in 4.7% and HHV-7 DNA was detected in 2 cases (1.4%). Human herpes virus-6 and HHV-7 DNA was detected in 6% of all studied cases. HHV-6 was slightly more frequent than HHV-7. Our findings were lower than the rate of other references but were higher than the findings of previous study in Iran. This variation might be due to differences in methods, age of study cases or epidemiologic and geographic variation

Congenital rubella infection in neonatal cord blood samples of newborns in hospitals affiliated to Tehran University of Medical Sciences

Rubella is a disease caused by the rubella virus and it is usually mild and self-limiting. Infection of a developing fetus is serious and important because the child may be born with congenital rubella syndrome. Its symptoms include mental retardation, heart defects, cataract, etc. In 2003, mass vaccination against measles and rubella in individuals 5-25 years old was done. One of the main objectives of this study was to survey congenital rubella infection status with the presence of IgM antibodies against rubella virus in cord blood samples and also the immunity assessment of maternal IgG antibodies against rubella virus in the above samples. Methods: The cross-sectional study was to determine the transfer of congenital rubella in 358 cord blood samples collected in hospitals affiliated to the Tehran University of Medical Sciences that was done in 2008-2009 The collected samples were analyzed by two ELISA methods for detection of IgG and IgM antibodies, RT-Nested PCR tests was applied on samples of IgG–negative and IgM-positive and also some of randomly IgG-positive samples for identifying the presence of the virus genome. In this study two groups of mothers were tested, one consisted above 29 years of age (at the time of vaccination) with the frequency of 73.4% and the other one below 29 years of age with the frequency of 26.6%.Results: Of the 358 samples, 91.1% IgG and 2.8% were found to be positive. None of the 31 samples were positive according to the presence of the virus genome via the method of RT-Nested PCR. Conclusion: According to high immunity of mothers, the probability of congenital rubella transmission was low, but because of low immunity of mothers of >29 years of age, it is much better to upgrade the age of vaccination to 28 years old

Detection of cytomegalovirus (CMV) antibodies or DNA sequences from ostensibly healthy Iranian mothers and their neonates

Cytomegalovirus (CMV) remains the most common cause of viral intrauterine infection. The objective of this research was to determine the prevalence of at-risk pregnancies for congenital cytomegalovirus transmission in a randomly selected pregnant women and their newborns. Enzyme Link Immunosorbent Assay (ELISA) and real-time polymerase chain reaction (PCR) were utilized to screen the sera of mothers (n = 100) and consecutive umbilical cord blood samples from their newborn (n = 100). Of the 100 mother's sera analyzed, 100 (100%) and 3 (3%) were positive for cytomegalovirus IgG and IgM antibodies, respectively. Of the 100 cord serum specimens analyzed, 99 (99%) and 2 (2%) were positive for cytomegalovirus IgG and IgM antibodies, respectively. Cytomegalovirus DNA was detected in 4 out of 100 (4%) cord blood samples of newborns. From four CMV DNA positive cases, Case 1 had no IgM in cord serum, but had IgM in mother's sera. Cases 2 and 4 were positive for IgM in both mother's sera and cord serum. Case 3 had no detectable CMV IgM in sera and cord serum. As many as 66 and 100% of CMV IgM-positive women in this study also had CMV IgM and CMV DNA in their delivery cord blood samples, respectively suggesting an increased risk of congenital CMV infection in those pregnancies. A paired women sera/cord blood CMV IgM-negative was found to be positive for CMV DNA. The data may also suggest the utility of PCR in place of CMV IgM as a diagnostic method for congenital CMV infection

Detection of human T-cell lymphotropic virus Type-1 among patients with malignant hematological diseases in Capital of Iran, Tehran

Human T-cell lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus linked causally to adult T-cell leukemia or lymphoma (ATL), and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The aim of this study was to detect HTLV-1 infection in patients with malignant hematological diseases and also determining the prevalence of HTLV-1 in these patient groups. Sixty patients with malignant hematological diseases were included in the study and tested by enzyme-linked immunosorbent assay (ELISA) for anti-HTLV-1, and Real time-PCR for the sequences from HTLV-1 tax gene. The mean age of patients was 33.9 ± 18.3 years. 18 of the subjects were found HTLV-1 seropositive using ELISA and the viral prevalence by Real time-PCR was 12%. HTLV-1 was found in 25% of patients with acute myelogenous leukemia (AML), 58.3% of patients with chronic myelogenous leukemia (CML), 16.7% of patients with acute lymphoblastic leukemia (ALL), and no detected in patients with lymphoma. The present study revealed that HTLV-1 is prevalent in patients with malignant hematological diseases and in our study. The major HTLV-1 associated syndromes were chronic myelogenous leukemia and acute lymphoblastic leukemia