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

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

RNAi and miRNA in Viral Infections and Cancers

Since the first report of RNA interference (RNAi) less than a decade ago, this type of molecular intervention has been introduced to repress gene expression in vitro and also for in vivo studies in mammals. Understanding the mechanisms of action of synthetic small interfering RNAs (siRNAs) underlies use as therapeutic agents in the areas of cancer and viral infection. Recent studies have also promoted different theories about cell-specific targeting of siRNAs. Design and delivery strategies for successful treatment of human diseases are becomingmore established and relationships between miRNA and RNAi pathways have been revealed as virus-host cell interactions. Although both are well conserved in plants, invertebrates and mammals, there is also variabilityand a more complete understanding of differences will be needed for optimal application. RNA interference (RNAi) is rapid, cheap and selective in complex biological systems and has created new insight sin fields of cancer research, genetic disorders, virology and drug design. Our knowledge about the role of miRNAs and siRNAs pathways in virus-host cell interactions in virus infected cells is incomplete. There are different viral diseases but few antiviral drugs are available. For example, acyclovir for herpes viruses, alpha-interferon for hepatitis C and B viruses and anti-retroviral for HIV are accessible. Also cancer is obviously an important target for siRNA-based therapies, but the main problem in cancer therapy is targeting metastatic cells which spread from the original tumor. There are also other possible reservations and problems that might delay or even hinder siRNA-based therapies for the treatment of certain conditions; however, this remains the most promising approach for a wide range of diseases. Clearly, more studies must be done to allow efficient delivery and better understanding of unwanted side effects of siRNA-based therapies. In this review miRNA and RNAi biology, experimental design, anti-viral and anti-cancer effects are discussed

High Resolution Melting Curve Assay for Detecting rs12979860 IL28B Polymorphisms Involved in Response of Iranian Patients to Chronic Hepatitis C Treatment

Background: A recent genome-wide association study (GWAS) on patients with chronic hepatitis C (CHC) treated with peginterferon and ribavirin (pegIFN-α/RBV) identified a single nucleotide polymorphism (SNP) on chromosome 19 (rs12979860) which was strongly associated with a sustained virological response (SVR). The aim of this study was twofold: to study the relationship between IL28B rs12979860 and sustained virological response (SVR) to pegIFN-α/RVB therapy among CHC patients and to detect the rs12979860 polymorphism by high resolution melting curve (HRM) assay as a simple, fast, sensitive, and inexpensive method. Materials and Methods: The study examined outcomes in 100 patients with chronic hepatitis C in 2 provinces of Iran from December 2011 to June 2013. Two methods were applied to detect IL28B polymorphisms: PCR-sequencing as a gold standard method and HRM as a simple, fast, sensitive, and inexpensive method. Results: The frequencies of IL28B rs12979860 CC, CT, and TT alleles in chronic hepatitis C genotype 1a patients were 10% (10/100), 35% (35/100), and 6% (6/100) and in genotype 3a were 13% (13/100), 31% (31/100), and 5% (5/100), respectively. In genotype 3a infected patients, rs12979860 (CC and CT alleles) and in genotype 1a infected patients (CC allele) were significantly associated with a sustained virological response (SVR). The SVR rates for CC, CT and TT (IL28B rs12979860) were 18%, 34% and 4%, respectively. Multiple logistic regression analysis identified two independent factors that were significantly associated with SVR: IL-28B genotype (rs 12979860 CC vs TT and CT; odds ratio [ORs], 7.86 and 4.084, respectively), and HCV subtype 1a (OR, 7.46). In the present study, an association between SVR rates and IL28B polymorphisms was observed. Conclusions: The HRM assay described herein is rapid, inexpensive, sensitive and accurate for detecting rs12979860 alleles in CHC patients. This method can be readily adopted by any molecular diagnostic laboratory with HRM capability and will be clinically beneficial in predicting treatment response in HCV genotype 1 and 3 infected patients. In addition, it was demonstrated that CC and CT alleles in HCV-3a and the CC allele in HCV-1a were significantly associated with response to pegIFN-α/RBV treatment. The present results may help identify subjects for whom the therapy might be successful

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 Human Papilloma Virus Infection in Patients with Esophageal Squamous Cell Carcinoma from the Caspian Sea Area, North of Iran

Introduction: HPV has been found repeatedly in esophageal squamous cell carcinoma (ESCC) tissues. However, reported detection rates of HPV DNA in these tumors have varied markedly. Differences in detection methods, sample types, and geographic regions of sample origin have been suggested as potential causes of variation. We have reported that infection of HPV DNA in ESCC tumors depends on anatomical sites of esophagus of the patients from Mazandaran, north of Iran. Materials and Methods: HPV DNA was examined in 46 upper, 69 middle and 62 lower third anatomical sites of esophageal squamous cell carcinoma specimens collected from Mazandaran province in north Iran, near the Caspian Littoral as a region with high incidence of ESCC. HPV L1 DNA was detected using Qualitative Real time PCR and MY09/MY11 primers. Results: 28.3% of upper, 29% of middle and 25.8% of lower third of ESCC samples were positive for HPV DNA. 13.6% for males and 14.1% for females were HPV positive in all samples. Conclusions: HPV infection is about one third of ESCC in this area. Findings in this study increase the possibility that HPV is involved in esophageal carcinogenesis. Further investigation with a larger sample size is necessary

Frequency of Influenza-A-H1N1 in Patients with Community-Acquired Pneumonia Admitted to Loghman Hakim Hospital

Background: Here we assessed the incidence of Influenza-A-H1N1-related pneumonia in community-acquired pneumonia (CAP) at Loghman Hakim Hospital, Tehran, Iran.Materials and Methods: In this prospective study from November 22, 2016, to June 21, 2017, patients with CAP and suspected to seasonal influenza were included. Rapid Antigen test and quantitative real-time PCR assay were performed on samples. P-value < 0.05 was considered significant. In addition, radiologic patterns of them were evaluated.Results: a total of 29 admitted CAP patients were suspected of seasonal influenza. Two cases out of them were positive for influenza by real-time PCR, similar to result of influenza rapid test. The most common finding in their chest X ray was consolidation in one lobe. None of them vaccinated against influenza. Only nine patients received empiric Oseltamivir treatment. The amount of irrational antibiotic administration was significant.Conclusion: Despite low statistical numbers, admitted influenza CAP patients did not have unusual symptoms and radiologic patterns. Other results in this study showed need for antibiotic stewardship program and better training about necessity of vaccination

High-Risk and Low-Risk Human Papillomavirus in Esophageal Squamous Cell Carcinoma at Mazandaran, Northern Iran

Cancers are the second most common cause of nonaccidental deaths in Iran, following cardiovascular deaths. Mazandaran, near the Caspian Littoral at north of Iran have identified as a several-high incidence area for Esophageal Squamous Cell Carcinoma (ESCC) in the world. Several associated risk factors, such as dietary and cultural habits, infectious agents, nutritional deficiencies, too much use of tobacco and alcohol and infection to certain DNA tumor viruses (HPVs), including environmental and genetic factors are attributed to this disease. To explore this issue, we analyzed HPV DNA prevalence and HPV types together in relation to tumor sites a high-incidence population. Archived tissue blocks from 46, 69 and 62 upper, middle and lower third of esophagus, respectively from ESCC patients were evaluated for the presence of HPV DNA by PCR using the degenerate HPV L1 consensus primer pairs MY09/MY11. The positive specimens were evaluated by Real-time PCR to determine HPV genotypes. From the 49 HPV positive cases, of ESCC patients, 5 (23.1%), 11 (55 %) and 9 (56.3 %) of upper, middle and lower third of ESCC specimens, respectively were positive by at least one high and one low-risk HPV genotypes. In general, HPV45 and HPV11 were the most common high- risk and low-risk HPV genotypes in HPV L1 positive cases, respectively, followed by HPV6, HPV52 and HPV39. Therefore, the high prevalence of HPV DNA in different anatomical sites of ESCC patients from the Mazandaran region in North of Iran provides more evidence for a role of HPV in this cancer

Human Papillomavirus Type 16 Integration Analysis by Real-time PCR Assay in Associated Cancers

Human papillomavirus (HPV) is a common viral infection worldwide associated with a variety of cancers. The integration of the HPV genome in these patients causes chromosomal instability and triggers carcinogenesis. The aim of this study was to investigate the HPV-16 genome physical status in four major cancers related to HPV infection. Formalin-fixed paraffin-embedded blocks from our previous projects on head and neck, colorectal, penile, and cervical cancers were collected, and HPV-16–positive specimens were used for further analysis. The DNA extraction copy number of E2 and E7 genes was calculated by qualitative real-time PCR method. Serially diluted standards that were cloned in PUC57 plasmid were used. Standard curve and melting curve analysis was used for quantification. Of the 672 specimens studied, 76 (11.3%) were HPV-16 positive. We found that 35.6% (16/45) were integrated. Statistical analysis showed that there were significant correlations between integration of HPV-16 and cervical cancer end-stage carcinogenesis (P < .0001), episomal form, and ASCUS lesions (P = .045). Significant correlation in penile cancer patients was seen between the episomal form and high-grade cancer stage (P = .037). Integration is a major factor in the carcinogenesis mechanism of HPV and has different prevalence in various cancers with a higher rate in progression except in penile cancer