Expression, purification and immunogenic description of a hepatitis c virus recombinant coreE1E2 protein expressed by yeast pichia pastoris

Background: Gradual development of a useful vaccine can be the main point in the control and eradication of Hepatitis C virus (HCV) infection. Hepatitis C Virus envelope glycoproteins are considered as the main HCV vaccine candidate. Objectives: In this study, the Pichia pastoris expression system was used to express a recombinant HCV CoreE1E2 protein, which consists of Core (269 nt-841nt) E1 (842 nt-1417nt) and E2 (1418 nt-2506nt). Materials and Methods: By a codon optimization technique based on the P. pastoris 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 a homemade Enzyme-Linked ELISA kit we can detect antibody of HCV Iranian patients with genotype 1a. Results: In our study, the virus-like particle of rCoreE1E2 with 70 nm size, was shown by Electron microscopy and proved the self-assembly in vitro in a yeast expression system. Conclusions: These findings of the present study indicate that the recombinant CoreE1E2 glycoprotein is effective in inducing neutralizing antibodies, and is an influential HCV vaccine candidate. © 2015, Kowsar Medical Publishing Company. All rights reserved

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 those with pre-invasive forms of disease, are needed to explain the roles of different HPV types in this location of Iran

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

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

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

Stereochemical trajectories of a two-component regulatory system pmra/b in a colistin-resistant acinetobacter baumannii clinical isolate

Background: There is limited information on the 3D prediction and modeling of the colistin resistance-associated proteins PmrA/B TCS in Acinetobacter baumannii. We aimed to evaluate the stereochemical structure and domain characterization of PmrA/B in an A. baumannii isolate resistant to high-level colistin, using bioinformatics tools. Methods: The species of the isolate and its susceptibility to colistin were confirmed by PCR-sequencing and MIC assay, respectively. For 3D prediction of the PmrA/B, we used 16 template models with the highest quality (e-value <1 � 10�50). Results: Prediction of the PmrA structure revealed a monomeric non-redundant protein consisting of 28 α-helices and 22 β-sheets. The PmrA DNA-binding motif displayed three antiparallel α-helices, followed by three β-sheets, and was bond to the major groove of DNA by intermolecular van der Waals bonds through amino acids Lys, Asp, His, and Arg, respectively. Superimposition of the deduced PmrA 3D structure with the closely related PmrA protein model (GenBank no. WP071210493.1) revealed no distortion in conformation, due to Glu�Lys substitution at position 218. Similarly, the PmrB protein structure displayed 24 α-helices and 13 β-sheets. In our case, His251 acted as a phosphate receptor in the HisKA domain. The amino acid substitutions were mainly observed at the putative N-terminus region of the protein. Furthermore, two substitutions (Lys21�Ser and Ser28�Arg) in the transmembrane domain were detected. Conclusion: The DNA-binding motif of PmrA is highly conserved, though the N-terminal fragment of PmrB showed a high rate of base substitutions. This research provides valuable insights into the mechanism of Col-R in A. baumannii. © 2021, Pasteur Institute of Iran. All rights reserved

Molecular characterization of lpxACD and pmrA/B two-component regulatory system in the colistin resistance Acinetobacter baumannii clinical isolates

Colistin is drug of choice for treatment of carbapenem-resistant Acinetobacter baumannii infections. Unfortunately, global increase in clinical outbreaks of colistin and carbapenem resistant A. baumannii infections is on the rise and cause public health concern. In the present study, a total of 187 A. baumannii recovered from specimens of 240 patients admitted to intensive care units (ICUs) of two hospitals in Kerman, Iran during 2017�2018. Among the isolates, we found four extensive drug-resistant (XDR) with Minimum Inhibitory Concentration (MIC) �4 μg/mL against colistin. The colistin-resistant (Col-R) isolates harbored blaOXA�51 and blaOXA-23 carbapenemase genes, exhibited resistance to all antibiotic classes except tigecycline and ampicillin-sulbactam. They belonged to clonal complex 2, a new MLST type 1752 and displayed identical RAPD-PCR fingerprints. Phylogenetic tree analysis suggested that, the Col-R A. baumannii emerged by endogenous mutations rather than acquisition of preexisting clones. Expressions of pmrCAB by quantitative real-time PCR (qRT-PCR) revealed 8 and 7 folds increased in the transcription levels of pmrB/C genes in strain 1 grown in presence of 16 μg/mL colistin (p � 0.01). However, no change in the expression of the pmrA was observed. Furthermore, DNA sequencing of Col-R genes illustrated three nonsynonymous substitutions in the LpxA (N136 � K), LpxC (P293 � Q), and PmrB (V21 � F, S28 � R, I149 � F) in the strains 1 and 3, respectively showing MIC 32 μg/mL against colistin. Multiple amino acids alignments demonstrated several substitutions in N-terminal region of PmrB. In conclusion, the above results provide valuable insights into the mechanism of Col-R in A. baumannii and the expressions of relative genes. © 2020 Elsevier Inc