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Background and Objectives: Human parechoviruses (HPeV) and Human enteroviruses (EV) frequently cause a sepsis-like illness in young infants (younger than three months). Therefore, this study was conducted to determine the frequency of HPeV and EV among the young infants with clinical signs and symptoms of sepsis in Ahvaz city, Iran. Materials and Methods: The blood specimens were collected from 100 (younger than 90 days hospitalized infants) including 54 (56.25%) males and 46 (43.75%) females with clinical signs and symptoms of sepsis-like disease. The RNA was extracted and tested for detection of VP1 region of HPeV and 5 UTR (Untranslated Region) of EV by RT-PCR. The sequences of positive of HPeV were further analyzed to determine HPeV genotyping. Results: 5/100 (5%) of patients including 2/46 (2%) females and 3/54 (3%) males tested positive for HPeV (P=0.85). The analysis of 5 positive VP1 region of HPeV revealed the genotype 1. The analysis of sequencing and phylogenetic tree revealed that the isolated HPeVs were genotype 1. While 38/100 (38%) specimens including 16 (16%) females and 22 (22%) males were tested positive for EV (P=0.68). Conclusion: The frequency of HPeV genotype 1 was 5% among the young infants with sepsis. While frequency of EV was 38% among the young infants with sepsis. This study showed HPeV genotype 1 and EV are dominant in this region.

Rotavirus is known to be responsible for remarkable numbers of severe diarrheal episodes and even death in infants and young children. In this study, we aimed to survey genetic diversity and variation analysis of viroporin, which is encoded by the rotavirus NSP4 segment. Thirty-five rotavirus-positive specimens were obtained, and RNA extraction and polymerase chain reaction amplification were performed. After the sequencing process, four specimens were excluded, and the final 31 samples remained for genetic diversity and variation analysis. The predominant single G/P combination was G1P[8] (~78%), followed by G2P[8] (~13%), and equal percentages (3%) of G2P[4], G3P[8], and G-non-typeable-P[8]. Further analyses revealed that variations could be found in the three regions of NSP4, including VP4 binding site (aa 112-146), double-layered particle binding site (aa 161-175), and finally, in the predicted amphipathic alpha-helix. Phylogenic tree analysis demonstrated that the mentioned samples clustered with genotype E1 and E2 reference sequences. As previously reported in the literature, in this study, it was revealed that no apparent correlation exists in the deduced amino acid sequences corresponding to this region between the rotaviruses collected from patients with and without diarrhea.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has been the world’s driving fatal bacterial contagious disease globally. It continues a public health emergency, and around one-third of the global community has been affected by latent TB infection (LTBI). This is mostly due to the difficulty in diagnosing and treating patients with TB and LTBI. Exosomes are nanovesicles (40–100 nm) released from different cell types, containing proteins, lipids, mRNA, and miRNA, and they allow the transfer of one’s cargo to other cells. The functional and diagnostic potential of exosomal miRNAs has been demonstrated in bacterial infections, including TB. Besides, it has been recognized that cells infected by intracellular pathogens such as Mtb can be secreting an exosome, which is implicated in the infection’s fate. Exosomes, therefore, open a unique viewpoint on the investigative process of TB pathogenicity. This study explores the possible function of exosomal miRNAs as a diagnostic biomarker. Moreover, we include the latest data on the pathogenic and therapeutic role of exosomal miRNAs in TB.

Introduction. Herpes simplex virus type 1 (HSV-1) is a virus that causes serious human disease and establishes a long-term latent infection. The latent form of this virus has shown to be resistant to antiviral drugs. Clustered Regularly Interspace Short Palindromic Repeats (CRISPR), is an important tool in genome engineering and composed of guide RNA (gRNA) and Cas9 nuclease that makes an RNA-protein complex to digest exclusive target sequences implementation of gRNA. Moreover, CRISPR-Cas9 system effectively suppresses HSV-1 infection by knockout of some viral genes. Materials and methods. To survey the efficacy of Cas9 system on HSV-1 genome destruction, we designed several guide RNAs (gRNAs) that all packaged in one vector. Additionally, we performed a one-step restriction using BamHI and Esp3I enzymes. Results. CRISPR/Cas9 system targeted against the gD gene of HSV-1 was transfected into HEK-AD cells that showed a significant reduction of HSV-1 infection by plaque assay and real-time PCR. Conclusion. The pCas-Guide-EF1a-GFP CRISPR vector can create a fast and efficient method for gRNA cloning by restriction enzymes (Esp3I (BsmBI) and BamHI). Therefore, the CRISPR/Cas9 system may be utilized for the screening of genes critical for the HSV-1 infection and developing new strategies for targeted therapy of viral infections caused by HSV-1.

Changes in the concentrations of trace metals such as zinc (Zn) and selenium (Se) can pathologically lead to neurodegenerative conditions such as the Alzheimer's disease (AD). Previous studies have shown that mitochondrial dysfunction plays an important role in the pathogenesis of AD. Several male Wistar rats were randomly divided into five groups: sham group, AD group that received 3 mg/kg of streptozotocin (STZ) intracerebroventricularly, AD + Zn group that received 10 mg/kg of Zn intraperitoneally (i.p.) for 1 week, AD + Se group that received 0.1 mg/kg of Se i.p. for 1 week, and AD + Zn + Se group that received 10 mg/kg of Zn i.p. plus 0.1 mg/kg of Se i.p. for 1 week. At end of the study, behavioral tests and mitochondrial oxidative stress and GPR39 gene expression evaluations were carried out. Co-administration of Zn and Se significantly decreased the potential collapse of mitochondrial membrane, reactive oxygen species levels, and lipid peroxidation levels while significantly increased cognitive performance, superoxide dismutase (SOD), glutathione peroxidase, and catalase activity in the brain mitochondria compared with the STZ group. In addition, no significant changes were observed in GPR39 expression in the co-treated group. Findings of the current study showed that ZnR/GPR39 receptor, mitochondrial dysfunction, and oxidative stress play important roles in the pathogenesis of AD. Co-treatment of Zn and Se improved the cognitive performance, mitochondrial dysfunction, and oxidative stress caused by STZ-induced AD. Therefore, therapeutic approaches to improve mitochondrial function could be effective in preventing the initiation and progression of AD.

3C protease is one of the most important proteases in enteroviruses, which is responsible for several cleavages of the viral polyprotein, leading to virus maturation. It has several harmful effects on host cells, such as the induction of caspase-mediated apoptosis and the cleavage of various proteins like P53, TATA-binding protein (TBP), histone H3, octamer-binding protein 1 (Oct-I) and DNA polymerase III. In this study, we aimed to construct a structure to express enterovirus 71 3C protease, consisting of a 3C protease sequence and two kinds of linkers including rigid and cleavable. As a result of cloning the structure into a pEGFP-N1 vector, two forms of constructions were expressed and verified by western blotting analysis including a 3C-Linker-Cleavage site (3C-L-CL) and a 3C-Linker (3C-L) without a cleavage site. In 3C-L-CL construction, 3C protease is released from fused GFP by cleave cleavage site. In 3C-L construction, 3C protease was expressed in fusion with GFP. In fact, one of the constructions produced two forms of 3C proteases, a free 3C and a 3C fused with GFP. This study shows that free 3C is preferred over the second form (3C with linker) because it is not connected to other peptides, and its protease activity is not affected. The designed structures could be used as useful tools for 3C protease expression and investigation of its interactions with cellular signaling pathways such as interferon pathway.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in several patients who traveled to Wuhan or went to a seafood wholesale market in Wuhan. The phylogenetic tree showed that SARS-CoV-2 was 96.2% identical to bat β-coronaviruses from lineage B. Also, several studies reported that SARS-CoV-2 uses the SARS-CoV receptor, angiotensin-converting enzyme 2, for entry to target cells. Lung alveolar and small intestine are potential targets for SARS-CoV-2 due to the high expression of the angiotensin-converting enzyme 2 receptor. In this review, we focused on the zoonotic β-coronaviruses and given there is no specific drug or vaccine for coronavirus disease 2019, we reviewed the literature on the therapy options for SARS and Middle East respiratory syndrome coronavirus infection, in order to discover their possible use in the treatment of SARS-CoV-2 infections.

Background Functional constipation is a common and annoying gastrointestinal disorder among children worldwide in which the intestinal microbiota composition plays a fundamental role. This study aimed to compare the quantity of main intestinal Lactobacillus species in constipated children and healthy controls. Materials and Methods Fecal samples were collected from 40 children fulfilling Rome IV criteria for functional constipation and 40 healthy volunteers. Quantitative real-time polymerase chain reaction (qPCR) method with species-specific primers was used to investigate seven main Lactobacillus species in fecal samples. Results Lactobacillus strains of the patient group were different from the healthy controls, main differences being noticeable decrease in the population quantity of Lactobacillus reuteri (mean 102.61 CFU/gram feces) and lower abundance of Lactobacillus fermentum (p

Background 5-Fluorouracil (5-FU) is regarded as the first line treatment for colorectal cancer; however, its effectiveness is limited by drug resistance. The ultimate goal of cancer therapy is induction of cancer cell death to achieve an effective outcome with minimal side effects. The present work aimed to assess the anti-cancer activities of mitocans which can be considered as an effective anticancer drug due to high specificity in targeting cancer cells. Methods MTT (3–4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay was performed to determine the effects of our mitocans on cell viability and cell death. Apoptosis and necrosis, caspase 3 activity, mitochondrial membrane potential and ROS production in HT29 cell lines were analyzed by ApopNexin™ FITC/PI Kit, Caspase- 3 Assay Kit, MitoTracker Green and DCFH-DA, respectively. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression level of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) genes in HT29 cell lines. Results Treatment with mitocans (3Br-P + DCA) inhibited the growth of HT29. Moreover, 3Br-P + DCA significantly induced apoptosis and necrosis, activation of caspase 3 activity, depolarize the mitochondrial membrane potential, and ROS production. At a molecular level, 3Br-P + DCA treatment remarkably down-regulated the expression of Bcl-2, while up-regulated the expression of Bax. Conclusion Mitocans, in particular the combined drug, 3Br-P + DCA, could be regarded and more evaluated as a safe and effective compound for CRC treatment. Targeting hexokinase and pyruvate dehydrogenase kinase enzymes may be an option to overcome 5-FU -mediated chemo-resistant in colorectal cancer.

During the past decade, accumulating evidence from the research highlights the suggested effects of bacterial communities of the human gut microbiota and their metabolites on health and disease. In this regard, microbiota-derived metabolites and their receptors, beyond the immune system, maintain metabolism homeostasis, which is essential to maintain the host's health by balancing the utilization and intake of nutrients. It has been shown that gut bacterial dysbiosis can cause pathology and altered bacterial metabolites' formation, resulting in dysregulation of the immune system and metabolism. The short-chain fatty acids (SCFAs), such as butyrate, acetate, and succinate, are produced due to the fermentation process of bacteria in the gut. It has been noted remodeling in the gut microbiota metabolites associated with the pathophysiology of several neurological disorders, such as Alzheimer's disease, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, stress, anxiety, depression, autism, vascular dementia, schizophrenia, stroke, and neuromyelitis optica spectrum disorders, among others. This review will discuss the current evidence from the most significant studies dealing with some SCFAs from gut microbial metabolism with selected neurological disorders.

Despite the fact that high-dose radiotherapy is a main therapeutic modality in cancer treatment, recent evidence suggests that it might confer radioresistance. Hyper-radiosensitivity (HRS) is one of the important biological effects of low-dose ionizing radiation (LDIR) in mammalian cell lines. LDIR is considered as a promising assistant method of clinical cancer therapy. The purpose of this study was to evaluate the efficiency of intermittent LDIR followed by a high-dose radiation therapeutic approach compared with the conventional high-dose radiotherapy in the breast cancer MDA-MB-231 cell line. MDA-MB-231 cells were divided into four experimental groups—intermittent LDIR group: cells were irradiated for 10 fractions with a dose of 30 mGy at each time (interval 24 h) followed by 2 Gy, single LDIR group: cells have accepted a dose of 300 mGy LDIR and after 24 h a high dose of 2 Gy, high-dose ionizing radiation (HDIR) group: cells were exposed to a single high dose of 2 Gy, and control group. MTT and flow cytometry assay were used for cell proliferation and apoptosis after 24 h of the last irradiation dose (2 Gy). Also, we examined p21 and cespase3 gene expression by RT-qPCR. We observed that intermittent LDIR significantly increased the killing effect of radiotherapy (viability, 71.95 + 1.25%) (P

Introduction: Colorectal cancer is the most repetitious malignancies with high mortality worldwide. JC virus (JCV) is ubiquitous Polyomavirus, with seroprevalence rates ranging from 70% to 90% in adult population. Recently the role of JCV have been reported in many malignant tumors worldwide. The association of JCV was reported in patients with colon and rectum cancers. Thus this study was conducted to evaluate the association of JCV DNA in patients with colon cancer type Adenocarcinoma. Material and Methods: A total of 120 formalin-fixed paraffin-embedded tissue blocks samples were collected including 20/40(50%) males, 20/40(50%) females patients with Colorectal Cancer(CRC), and 80 (50% males, 50% females) patients with benign tumor as a control. DNA was extracted for all the samples. Nested PCR was carried out for detection of Vp1/T-Ag junction genome in JCV genome by Nested-PCR assay. Randomly, PCR products of 6 samples were sequenced to analysis the partial JCV DNA. The phylogeny tree was constructed to determine homology identity with other JCV. Results: 4/40(10%) samples of test group and 10/80 (12.5%) of control samples were positive for JCV DNA (P= 0.69). Out of 4 samples positive for JC DNA, 3(7.5%) were males and 1(2.4%) female (P=0.29). The frequency of JCV DNA in age group> 50 years was 4/32(10%), while in age group 50 years and 0% of age group

This report explains the employing of a combination test of traditional cell culture with a quantitative real-time PCR for assessment of the antiviral effect of zinc sulfate (ZnSO4) on herpes simplex virus (HSV)-infected Vero cells. Our evidence showed that the treatment with 0.3 mM ZnSO4 strongly inhibited the replication of virus progeny (MOI 0.001) at least 68-fold less. On the other hand, the IC50 demonstrated that the highest activity of ZnSO4 was at the 0.23 mM concentration.

COVID-19 caused by SARS-CoV-2, is an international concern. This infection requires urgent efforts to develop new antiviral compounds. To date, no specific drug in controlling this disease has been identified. Developing the new treatment is usually time consuming, therefore using the repurposing broad-spectrum antiviral drugs could be an effective strategy to respond immediately. In this review, a number of broad-spectrum antivirals with potential efficacy to inhibit the virus replication via targeting the virus spike protein (S protein), RNA-dependent RNA polymerase (RdRp), 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) that are critical in the pathogenesis and life cycle of coronavirus, have been evaluated as possible treatment options against SARS-CoV-2 in COVID-19 patients.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichia coli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta-d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.

The pandemic coronavirus disease 2019 (COVID‐19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2), has affected millions of people worldwide. To date, there are no proven effective therapies for this virus. Efforts made to develop antiviral strategies for the treatment of COVID‐19 are underway. Respiratory viral infections, such as influenza, predispose patients to co‐infections and these lead to increased disease severity and mortality. Numerous types of antibiotics such as azithromycin have been employed for the prevention and treatment of bacterial co‐infection and secondary bacterial infections in patients with a viral respiratory infection (e.g., SARS‐CoV‐2). Although antibiotics do not directly affect SARS‐CoV‐2, viral respiratory infections often result in bacterial pneumonia. It is possible that some patients die from bacterial co‐infection rather than virus itself. To date, a considerable number of bacterial strains have been resistant to various antibiotics such as azithromycin, and the overuse could render those or other antibiotics even less effective. Therefore, bacterial co‐infection and secondary bacterial infection are considered critical risk factors for the severity and mortality rates of COVID‐19. Also, the antibiotic‐resistant as a result of overusing must be considered. In this review, we will summarize the bacterial co‐infection and secondary bacterial infection in some featured respiratory viral infections, especially COVID‐19.

Background and Objectives: Hepatitis C virus and Human Immunodeficiency Virus (HIV) share the same rate of transmis- sion. HIV/HCV co-infected individuals may result in faster progression of liver fibrosis and highly increase the risk of cir- rhosis, hepatocellular carcinoma development. Thus this study was conducted to determine co-infection of HCV genotypes in positive HIV patients in Ahvaz city, Iran. Materials and Methods: The sera samples were collected from confirmed 78 infected HIV, 67 (85.89%) males and 11 (14.1%) females. All sera samples were tested for HCV Ab using ELISA test. The HCV Ab positive samples were tested for detection of 5' untranslated (UTR) and core regions of HCV genome using nested RT-PCR. The PCR products of 5UTR and core regions were sequenced to determine HCV genotypes. Results: Among the 78 infected HIV, 25 (32.05%) cases including 20 (25.64%) males and 5 (6.41%) females were positive for HCV Ab (p=0.316). 53 (67.94%) of HIV patients were negative for HCV Ab. Among 25 positive HCV Ab, 19 (24.35%) cases including 15 (19.23%) males and 4 (5.12%) females were positive for HCV RNA (p=0.447). The PCR products of 5 positive samples were randomly sequenced. The results of sequences and alignments showed that the detected HCV geno- types were three 3a and two 1a. The occurrence of genotype HCV 1a was found in one male injecting drug user Injecting Drug User (IDU) and one female. The HCV 3a genotype was detected in the three males IDU. Conclusion: The results of this survey indicated that 32.05% of HIV patients were positive for HCV Ab, among them 24.35% were positive HCV RNA. HCV genotype 3a was dominant and detected in the three males IDU. Regarding the consequences of HIV/HCV co-infection, it is suggested that HCV RNA detection should be regularly checked in individuals infected with HIV.

Human adenovirus 36 (Ad-36) causes obesity with increased adiposity, in contrast, Ad-36 infection reduces glucose and lipid metabolism; the results, however, are not consistent. In the current study, the effects of Ad-36 infection on glucose and lipid profile and inflammatory markers in Wistar rats were investigated. Sixty male Wistar rats were randomly divided into infected and control groups. Ad-36 virus suspension was injected in the experimental group rats. Blood samples were collected in the beginning and after 12 weeks in both groups. After 12 weeks, a significant improvement was observed in fasting blood glucose, fasting serum insulin, insulin sensitivity, serum triglycerides and total cholesterol in the infected group compared with the non-infected groups. There were no significant differences in inflammatory biomarkers including tumor necrosis factor-α, interleukin 6 and monocyte chemoattractant protein-1 levels between infected and control groups. This study showed that Ad-36 had favorable effects on glycemic and lipid control in infected rats, but inflammatory biomarker levels were similar for 2 groups. Ad-36 infections could potentially be a new way to develop novel antidiabetic and antihyperlipidemic therapeutic agents.

Despite the fact that radiotherapy is a main therapeutic modality in cancer treatment, recent evidence suggests that fractionated radiotherapy (FR) might confer radioresistance through epithelial-mesenchymal transition (EMT). Nevertheless, the effects of FR on EMT phenotype and the potential link between EMT induction and radioresistance development yet to be clarified. The aim of this study was to assess whether FR could promote EMT, and to elucidate if induction of EMT contributes to the acquisition of radioresistance. To this end, two human cancer cell lines (A549 and HT-29) were irradiated (2 Gy/day) and analyzed using wound healing, transwell migration and invasion assays, real-time polymerase chain reaction (for E-cadherin, N-cadherin, Vimentin, CD44, CD133, Snail, and Twist), clonogenic assay, Annexin V/PI, and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Irradiation of A549 (for 5 or 10 consecutive days) resulted in morphological changes including elongation of cytoplasm and nuclei and pleomorphic nuclei. Also, irradiation-enhanced migratory and invasive potential of A549. These phenotypic changes were in agreement with decreased expression of the epithelial marker (E-cadherin), enhanced expression of mesenchymal markers (N-cadherin, Vimentin, Snail, and Twist) and increased stemness factors (CD44 and CD133). Moreover, induction of EMT phenotype was accompanied with enhanced radioresistance and proliferation of irradiated A549. However, FR (for 5 consecutive days) did not increase HT-29 motility. Furthermore, molecular alterations did not resemble EMT phenotype (downregulation of E-cadherin, Vimentin, ALDH, CD44, CD133, and Snail). Eventually, FR led to enhanced radiosensitivity and decreased proliferation of HT-29. Altogether, our findings suggest that FR might induce EMT and confer radioresistance in a cell context-dependent manner.

Arsenic (As) is a toxic and hazardous metalloid. Unfortunately, its presence in drinking water together with wrong nutritional patterns is associated with an increase in the occurrence of metabolic disorders in young people. Degradation of mitochondria is presented by a specific form of autophagy called mitophagy which is an important landmark leading to apoptosis during lipotoxicity. Lipotoxicity and cellular toxicity due to arsenic intake can lead to changes in mitophagy and apoptosis. The protein derived from SQSTM1 gene, also called p62, plays an important role in energy homeostasis in the liver, and it can contribute to the regulation of autophagic responses given its effect on signaling of mTOR, MAPK, and NF-KB. Consequently, changes in Sqstm1, mitophagy (BNIP3), and apoptotic (caspase 3) genes in the livers of NMRI mice were examined with the use of real-time RT-PCR Array followed by exposure to an environmentally relevant and negligible cytotoxic concentration of arsenite (50 ppm) in drinking water while being fed with a high-fat diet (HFD) or low-fat diet (LFD) for 20 weeks (LFD-As and HFD-As groups). While LFD-As and HFD groups showed a decrease in BNIP3 expression, a significant increase was observed in the HFD-As group. P62 gene showed downregulation in LFD-As and HFD groups, and upregeneration was observed in the HFD-As group. Caspase 3 showed increased expression as the key factor associated with apoptotic liver cell death in the three groups, with the highest value in HFD-As group. Overall, the changes observed in the expression of Sqstm1, BNIP3, and caspase 3 in this study can be related to the level of liver damage caused by exposure to arsenic and HFD and probably, BNIP3 pro-apoptotic protein is associated with an increased cell death due to HFD and As.

The key features of type 2 diabetes mellitus (T2DM) caused by high fat diet (HFD) in combination with arsenic (As) exposure (pronounced glucose intolerance despite a significant decrease in insulin resistance) are different from those expected for T2DM. Autophagy has been considered as a possible link between insulin resistance and obesity. Therefore in this study, we utilized autophagy gene expression profiling via real-time RT-PCR array analysis in livers of NMRI mice exposed to an environmentally relevant and minimally cytotoxic concentration of arsenite (50 ppm) in drinking water while being fed with a HFD for 20 weeks. Out of 84 genes associated with autophagy under study, 21 genes were related to autophagy machinery components of which 13 genes were downregulated when HDF diet was applied. In this study, for the first time, it was shown that the exposure to arsenic in the livers of mice chronically fed with HFD along with increased oxidative stress resulted in the restoration of autophagy [upregulation of genes involved in the early phase of phagophore formation, phagophore expansion and autophagosome-lysosome linkage stages]. Considering the role of arsenic in the induction of autophagy; it can be argued that reduced insulin resistance in HFD − As induced diabetes may be mediated by autophagy upregulation.

Background Flagellin is the major structural protein monomer of bacterial flagella. Flagellin through binding to its receptor and activation of antigen presenting cells stimulates the innate and adaptive immune responses. Flagellin is used as an effective systemic or mucosal adjuvant to stimulate the immune system. Recently, the therapeutic and protective role of flagellin in some infectious diseases and cancers has been investigated. In this study, we cloned the fliC genes from Salmonella typhimurium and Escherichia coli into pET-28a vector and investigated their expression in the prokaryotic system. Methods The fliC genes of S. typhimurium and E. coli were amplified by PCR with a specific oligonucleotide primer set. thse were cloned into the pET-28a vector and the recombinant pET-28a-fliC plasmids were successfully transformed into the E. coli strain BL-21(DE3). The expression of flagellin proteins in the prokaryotic cells were evaluated. Finally, Transcription of TNF-α mRNA was confirmed using Real-time PCR. Results The expression of proteins in the prokaryotic cells were approved by SDS-PAGE and western blotting method. Further, the functional characterization of flagellin proteins were evaluated using their ability to induce increased m-RNA expression of pro-inflammatory cytokine. Conclusions The flagellin proteins were expressed in the prokaryotic system. These proteins can be used to link target antigens as an effective adjuvant for future DNA vaccine studies. Purified recombinant proteins in this study can also be used for therapeutic and prophylactic purposes.

Background: Echovirus 6 (E6), is one of the main enteroviral serotypes, was initially isolated from patients with aseptic meningitis (AM) and is a major cause of hospitalization among children and adults worldwide. Methods: A cerebrospinal fluid (CSF) sample was collected from patient with clinically suspected aseptic meningitis (AM) in August 2011. Following detection of a virus and subsequent virus serotyping, the whole genome sequence was determined. The sequence of the VP1 region of the isolated strain E6 RA/E6/Ahvaz/Iran/2011 showed 79% (>75%) nucleotide and 94% (>85%) amino acid homology with prototype strain D'Amori. The isolated strain was identified as an E6 serotype. A specimen was cultured in a human rhabdomyosarcoma (RD) cell line. Following propagation, the virus was further analyzed using the plaque assay technique, reverse transcription PCR (RT-PCR), rapid amplification of CDNA ends (RACE), TA cloning, sequencing, phylogenetic analysis, Simplot and boot scanning analyses (ver. 3.5) were applied to find evidence of recombination in the isolated strain. Results: The isolated Echo6 strain RA/E6/Ahvaz/Iran/2011 has been recorded in GenBank with a partial and complete genome accession numbers (KX619440) (KX198605), respectively. The complete genomic sequence was 7435 nt, with a 742 bp 5′ UTR, 117 bp 3′ UTR, and an open reading frame (ORF) encoding a polypeptide of 2191 amino acids. The nucleotide analysis of the VP1 and structural genomic regions of the isolated strain showed high similarity with strain E6-10887-99 isolated from patient with facial nerve paresis in Russia in 1999. The recombinations evidence were observed in the isolated strain E6 RA/E6/Ahvaz/Iran/2011 and found to have a high levels of inter-serotypic exchanges in 2C and 3A-3C genomic regions with Echovirus13 and Echovirus14, respectively. Conclusion: Full genome sequence analysis of enteroviral is required to understand the epidemiological pattern and to evaluate the new enterovirus circulating in community. Keywords: Enterovirus B, Iran, Molecular sequence data, RD cells, Recombination

Background Herpes simplex virus type 2 (HSV-2) infectious disease is one of the most common viral sexually transmitted diseases. As regards, vaginal lactobacilli play an important role in protecting host against the urogenital pathogens; here we assessed the potential antiviral activity of Lactobacillus crispatus against HSV-2 infection in vitro. Methods Both Vero and HeLa cell lines were treated by L. crispatus before, during and after HSV-2 infection. The pre-incubation assay was also performed for the evaluating of virus adsorption by L. crispatus . Virus titer reduction in each stage was determined by a plaque reduction assay. Results L. crispatus significantly decreased the infectivity of the HSV-2 in initial steps on both cell lines; however, no significant inhibition was ascertained during adsorption and multiplication process. The lactobacilli adhere on Vero cells two-fold stronger than HeLa and subsequently protect the Vero cells nearly 2.5 fold higher than HeLa cell against the virion. Co-incubation of HSV-2 with bacterial cells prior to virus inoculation significantly decreased the virus titer. Conclusion L. crispatus appears to inhibit the entry of the virus into cells by trapping HSV-2 particles. In addition, formation of L . crispatus microcolonies in the cell surface could block HSV-2 receptors and prevent viral entry to cells in initial infection steps.

Context: Host factors including single-nucleotide polymorphisms (SNPs) in or near interferon lambda (IFNL) gene are the important factors in predicting response to treatment of chronic hepatitis C (CHC). Aims: The aim of this study was to determine the frequency and association of IFNL4 rs368234815 with IFNL3 SNPs rs12979860, rs8099917 and other factors including cholesterol, alanine aminotransferase, fibrosis, viral load, age and body mass index in genotype 1a treated CHC patients, to achieve rapid virologic response (RVR) and sustained virologic response (SVR). Subjects and Methods: A total of 71 hepatitis C virus genotype 1a patients were enrolled from 2013 to 2015. The genotypes of rs12979860, rs8099917 were identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism while the genotype rs368234815 detected by amplification-refractory mutation system-PCR. Results: The rate of RVR and SVR were 43/71 (60.6%) and 46/71 (64.8%), respectively. To achieve an SVR in patients with rs368234815, TT/TT genotype 20/24 (83.3%) was found to be higher than other SNPs. The correlation coefficient of rs368234815 was strongly associated with rs12979860 (r = 0.788, P < 0.001). Multivariate logistic regression showed that the cholesterol (odds ratio [OR]: 0.205, confidence interval [CI] 95%: 0.047–0.891, P = 0.035), age (OR: 0.160, CI 95%: 0.035–0.730, P = 0.018), baseline viral load (OR: 0.167, CI 95%: 0.032–879, P < 0.035) and IFNL4 (OR: 5.453, CI 95%: 1.015–29.293, P < 0.048) could be independent predictors of SVR. Conclusions: The results of these findings emphasise that factors such as age, cholesterol, baseline viral load and IFNL4 rs368234815 are better predictive factors and should be evaluated before CHC treatment.

Acute gastroenteritis caused by Rotavirus remains the leading cause of child mortality worldwide. Rotavirus genotype G9 circulates in humans throughout the world. Antibodies against the outer glycoproteins VP7 and VP4 Rotavirus capsid are the main neutralization antibodies in the vaccine assessment. This study aimed to select an epitope to evoke T and B cells' response, as a favorable candidate for vaccine development using in Silico evaluation. In the present study, Rotavirus genotypes were determined in 100 stools specimens collected from children with acute diarrhea. The results showed predominant G genotype, G9 (38.5%) followed by G2 (22.9%), G1 (16.5%), G12 (11.4%), G4 (6.4%), and G3 (4.3%). The G9 was dominant in this study and other regions of Iran; thus, this study was conducted to select an epitope from Rotavirus genotype G9 as a promising epitope candidate for future vaccine development. For this reason, several works including a complete sequence of VP7 G9, phylogenetic analysis, Prediction of Protein Structure, Physicochemical Properties of Protein and Epitope prediction were carried out. The outcomes of this study revealed that the complete sequence of VP7 (G9) was comprised of 1062 nt with 326 amino acids (accession number MH824633). The selected epitope contained amino acid sequence of STLCLYYPTEASTQIGDTEWKN with the best score for T and B cells response. Based on data of computational biology, the selected epitope can optimistically have considered as an epitope candidate for Rotavirus vaccine development.

Autoimmune hepatitis (AIH) is recognized as a serious disease in which the body's immune system attacks liver cells so untreated patients may consequently suffer from liver cirrhosis, hepatocellular carcinoma (HCC) and liver failure. The role of viral infection may be involved in AIH. Presence of anti-HBc alone is a predictive signal of potential OBI. Thus, this study was conducted to evaluate the rate OBI among the patients with AIH. Methods The sera of 20 consecutive patients with AIH were collected and tested for LFT (ALT, AST, ALP elevation), Immunoglobulin (IgG) level, bilirubin, anti -LKM-1, ASMA, ANA in titer, HBsAg, HBcIgG. The patients' sera were also tested for HBV DNA by nested PCR and Real-time PCR. Results Out of 20 patients, 10 (50%) were males and 10 (50%) females. The patients' ages ranged from 25 to 71 years with the mean age of 44.5±13.4. All patients' had elevated abnormal ALT and AST but their level of alkaline phosphatase was normal among the patients. All patients had IgG level>1.5 times upper than the normal limit. The patients' sera were negative for HBsAg and HBV DNA (by nested PCR and real- time PCR). Only 2 (10%) females with AHI type 1 (positive ANA, ASMA in titers >1:100 were positive for HBcIgG while no OBI detection was found among the males (p=0.005)). All diagnosis of the AHI was confirmed by pathologist. The level of ALT, AST among the cases with positive and negative OBI were (p=0.000) and (p=0.003), respectively. Conclusion In the present study, two OBI female patients with type 1 AIH were positive for anti-HBc but negative for HBsAg and HBV DNA. With regard to the consequences of OBI, prior to prophylactic treatment, it is recommended to screen HBV markers including anti-HBc in all diagnosed patients with AIH. .

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising anticancer cytokine with minimal toxicity towards normal cells. Nevertheless, most primary cancers are often intrinsically TRAIL-resistant or can acquire resistance after TRAIL therapy. This study aimed to investigate the inhibitory effect of co-treatment of 3-bromopyruvate (3-BP) as a potent anticancer agent with TRAIL on colon cancer cells (HT-29). The results of present study indicated that combined treatment with 3-BP and TRAIL inhibited the proliferation of HT-29 cells to a greater extent (88.4%) compared with 3-BP (54%) or TRAIL (11%) treatment alone. In contrast, the combination of 3-BP and TRAIL had no significant inhibitory effect on the proliferation of normal cells (HEK-293) (8.4%). At a cellular mechanistic level, the present study showed that 3-BP sensitized human colon cancer cells to TRAIL-induced apoptosis via reactive oxygen species generation, upregulation of Bax, downregulation of Bcl-2 and survivin, release of cytochrome c into the cytosol, and activation of caspase-3. In normal cells, 3-BP, TRAIL, or combination of both had no significant effect on the reactive oxygen species levels, release of cytochrome c, and caspase-3 activity. Therefore, the combination of 3-BP and TRAIL can be a promising therapeutic strategy for treatment of colon cancer.

Background: The 3C protease is one of the most important proteases in enteroviruses which is responsible for several cleavages on viral polyproteins and maturation processes. Some reports indicated that this protease disrupts the interferon (IFN) signaling pathway; however, another report shows that 3C protease does not have such inhibitory effects on IFN signaling. Objectives: In this study, a construction was designed consisting of 3C protease to test its effects on Interferon type I signaling pathway. Methods: The designed construction containing 3C protease was transfected to rhabdomyosarcoma (RD) cells for evaluation of its effects on interferon signaling pathway. Rhabdomyosarcoma cells were treated with Interferon β for 4 hours and then the expression of two ISGs including MxA and OAS were analyzed by real-time PCR. All the proteins in interferon type I signaling pathway were also evaluated by Western blot analysis. Results: The results showed that 3C protease does not affect ISG expression after IFN induction. Western blot analysis also indicated that there is no difference in IFN pathway protein level between 3C protease treated and control groups. Conclusions: This report suggests that 3C protease alone does not have a significant effect on IFN pathway, in contrast to any of the previous reports.

Objectives: The aim of this study was to evaluate the etiologic agents of a hand, foot, and mouth disease (HFMD) outbreak in Ahvaz, Southwest Iran and their evolutionary analysis by phylogenetic construction and Simplot analysis. Methods: We collected 16 serum samples in an outbreak of HFMD in Ahvaz in October 2013. RNA was extracted from the samples and subjected to reverse transcription polymerase chain reaction for detection of Enterovirus group A and B. Positive cases were sequenced and subjected to phylogenetic and Simplot analysis for detecting the signs of recombination. Results: Of the 16 specimens, nine (56.25) were PCR-positive with the universal primers for the Enterovirus 5‘UTR region. Coxsackievirus A6 was detected as a predominant agent of HFMD with two cases of Echovirus 6 and Echovirus 30. In the case of Echovirus 6, the signs of recombination in the 5‘UTR region were observed based on phylogenetic and Simplot analysis. Conclusions: Coxsackievirus A6 is the main agent of HFMD in Ahvaz. The evidence of recombination in this isolate of Echovirus 6 in this study emphasizes common hygiene practices and sanitation to prevent the circulation of this isolate in community and the advent of new strains. © 2019, Archives of Clinical Infectious Diseases.

Background The pattern and distribution of human rotavirus genotypes in young children in developing countries play an important role in epidemiological studies, as well as providing a strategy for the development of future rotavirus vaccine. Methods We evaluated stool samples from 349 children with acute gastroenteritis from Northern Iran (Gorgan city, Golestan province). Polyacrylamide Gel Electrophoresis (PAGE) and Latex Agglutination Test (LAT) were utilized to determine the prevalence of human rotavirus in fecal samples. Moreover semi-multiplex RT-PCR technique was carried out in order to determine the P and G genotypes of human rotavirus in rotavirus-positive samples. Results A total of 46 rotavirus-positive samples were G and P genotyped. Whereas 28 (60.8%) fecal specimens contained only one rotavirus strain, 14 (30.4%) were mixed rotavirus infections and 4 (8.8%) was non-typeable. Overall, during the study, 57.82% of strains identified as genotype G1, G2 (18.70%), G3 (4.69%), G4 (3.13%), G8 (3.13%), G9 (6.26%) and non-typeable G (6.26%). From all these mentioned rotavirus strains, 46 were characterized as P [8] (97.80%) and P [4] (2.20%).Our analysis of the G and P genotyping of strains from all 46 rotavirus-infected children has revealed that 4/46(6.26%) of G type strains were non-typeable. The predominant single G/P combination was G1P [8] (57.82%), followed by, G2P [8] (16.98%), G2P [4] (1.72%), G3P [8] (4.69%), G4P [8] (3.13%) G8P [8] (3.13%), G9P [8] (6.26%) and four cases of non-typeable G (6.26%). Rotavirus was detected in 39 specimens (11.17%) by PAGE and in 38 specimens (10.88%) by LAT. Both tests were 100% specific; however, the LAT was 82.61% sensitive compared to the PAGE, which was 84.78% sensitive. Conclusions The results suggest that to characterize rotavirus strains as well as design new effective vaccines for children with acute gastroenteritis, a large-scale study is needed in future.

Background To explore the prevalence, transmission routes and genotypes distribution of HCV in HIV-1/HCV co-infected individuals in Ahvaz, Iran. Methods The present cross-sectional study was conducted among HIV adult voluntary counseling and testing (VCT) clients, from September to November 2016. Reverse transcription (RT) nested PCR was performed to amplify the HCV core and 5′UTR regions from 90 HIV/HCV co-infected individuals. The PCR products were then sequenced for HCV subtyping. Genetic analysis was done by MEGA6 software. Results The prevalence of HCV in HIV-1-infected individuals was 58.7%. Injection drug use (IDU) was the most common route (99.1%) of transmission, and most of the patients (97.8%) had a history of imprisonment. The HCV subtypes were identified as 1a (55.2%), 3a (35.8%), 3 h (4.5%), 1b (3%) and 4a (1.5%) respectively, HCV 1a and 3a subtypes were predominant. Conclusions The diversity of HCV subtypes in HIV-1/HCV co-infected individuals in Ahvaz city was high, although two subtypes (1a and 3a) are predominant.

Background and Objectives: Adenoviral keratoconjunctivitis is an extremely frequent ophthalmological disease caused by various serological subtypes of human adenovirus (HAdV) worldwide. Adenoviruses serotypes 8, 11, 19, 37 frequently cause epidemic keratoconjunctivitis (EKC). This study was conducted to evaluate the frequency of adenovirus serotypes in patients with EKC in Ahvaz , Iran. Materials and Methods: Eighty- eight ocular swabs were collected from patients with EKC. The specimens were analyzed for detection of adenovirus by standard PCR. The PCR products were further sequenced and analyzed to determine the serotypes. Results: The study population consisted of 49/88 (55.7%) males and 39/88 (44.3%) females. Among them 25 (51.02%) males and 22 (56.41%) females were positive for HAdV serotype 8 (p= 0.488). Overall forty-seven (53.4%) samples were positive for AdV serotype 8 while forty- one patients (46.59%) were negative for the adenovirus serotypes. Conclusion: The results of this study revealed predominanance of HAdV 8 with high prevalence of 53.4% among patients with Keratoconjunctivitis. Forty- one patients (46.59%) were negative for adenovirus. Still, the role for other related viruses such as enteroviruses need to be investigated in patients with EKC.

Background: Oral candidiasis is a common disease in cancer patients subject to chemotherapy. The aim of this study was to evaluate the risk factors of rising oral candidiasis incidence and to identify the Candida species isolated from oral lesions of cancer patients and their antifungal sensitivity. Materials and Methods: A total of 645 patients with cancer were examined. Several Candida species were isolated from specimens and identified by morphological and molecular methods. The susceptibility of isolates to amphotericin B, fluconazole, and nystatin was also investigated. Results: A total of 74 isolates of Candida were recovered from oral cavity of 61 cancer patients with oral candidiasis. The isolates included Candida albicans (n = 56; 75.5%), Candida glabrata (n = 4; 5.4%), Candida krusei (n = 5; 7%), Candida tropical (n = 7; 9.4%), and Candida kefyr (n = 2; 2.7%). A total (n = 72; 98.65%) of isolates were susceptible to nystatin, (n = 58; 78.4%) of them were susceptible to fluconazole, and (n = 8; 10.8%) of susceptible dose-dependent isolates were specified, (n = 46; 62.16%) of isolates were susceptible to amphotericin B. Conclusion: Finally, in addition to emphasis on topical nystatin application in the first stage of oral candidiasis in these patients, using alternative systemic drugs such as fluconazole and amphotericin B can be considered for the resistant candida isolates to nystatin.

Giardia duodenalis, one of the most common intestinal protozoa, infects a wide range of vertebrates, including humans and animals. In this study, 84 Giardia duodenalis positive stool samples were collected from 3580 patients attending the Imam Ali Hospital and two public health centers in Andimeshk County, southwestern Iran. Stool samples were examined initially by microscopy, and then G. duodenalis was confirmed by SSU rRNA gene and genotypes were determined by amplification of the gdh and β-giardin genes. The SSU rRNA, gdh, and β-giardin genes were successfully amplified in 89.3%, 58.3%, and 51.2% samples, respectively. Of the positive samples for gdh and β-giardin, 40 isolates were successfully sequenced. Twenty-three isolates belonged to assemblage A, sub-assemblage AII, and 17 belonged to assemblage B. Of the 24 successfully amplified asymptomatic cases, 12 belonged to assemblage A and 12 belonged to assemblage B. The current study found that 64.3% of the patients were asymptomatic. From an epidemiological point of view, the high percentage of asymptomatic patients is important because of their role in the transmission of Giardia. The predominant assemblage was assemblage A, sub-assemblage AII. In general, therefore, it seems that most infections are probably transmitted by anthroponotic pathways in the region.

Rotaviruses (RV) are the leading cause of acute infantile gastroenteritis, associated with elevated mortality in low-income countries. Morbidity and mortality, length and rates of hospitalization due to RV gastroenteritis are dropping. Improving the quality of newborns life is an ongoing challenge for health-care providers. In this study, homemade reassortant human-bovine rotavirus was developed and biological activity and molecular characterization of candidate vaccine were evaluated for the vaccine stability. Virus titration and purification of reassortant rotavirus strains were evaluated by plaque assays, electropherotyping. The genetic stability after first, third and sixth passage was by sequencing. Due to WHO recommendation, developingment of national capacity for vaccine production in appropriate quantities and at affordable prices is the cornerstone of developing global vaccination policies. Such studies are critical to producing national vaccines and modeling herd protection.

The present study was designed to elucidate the mechanism(s) of the gastro-protective effect of crocin against indomethacin-induced gastric lesions. Crocin or pantoprazole was administered to rats 30 min before indomethacin. Five hours later, the animals were killed and their stomachs were removed and examined macroscopically. Samples of gastric mucosa were collected for microscopic evaluation, mRNA expression of caspase-3, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 was quantified by RT-PCR, and protein levels of COX-1, COX-2, iNOS and caspase-3 were assessed by Western blotting. The pH, volume of gastric effluent and antioxidant activity were measured in 5 separate groups of rats following pylorus ligation. Indomethacin induced significant increases in mRNA and protein expression of iNOS and caspase-3 and increased MDA levels, and reduced the pH of the gastric effluent and protein and mRNA expression of COX-2 and protein expression of COX-1 and mucus content associated with gastric ulceration. Crocin and pantoprazole significantly inhibited mRNA and protein expression of iNOS, caspase-3 and MDA, and reduced mucus content induced by indomethacin. However, unlike pantoprazole, crocin failed to increase COX-1 and pH, but had variable increasing effects on mRNA and protein expression of COX-2. Macroscopic and microscopic observations showed that mucosal erosions induced by indomethacin were significantly inhibited by pantoprazole and crocin. These findings suggest that crocin exerts its gastro-protective effects mainly by inhibition of MDA, reduction in iNOS and caspase-3, and inhibition of the reduction in mucus content induced by indomethacin. Crocin is a novel agent that has potential in the prevention of ulceration induced by NSAIDs.

Background: Cutaneous leishmaniasis is one of the most important tropical diseases in many parts of the world caused by two main species of parasites including Leishmania major and L. tropica in the old world. Several attempts have been made to reach an effective and reliable vaccine against the disease with a spectrum of success. Selection of an effective target for vaccination needs to recognize a protein antigen with minor diversity. Leishmania activated C kinase (LACK) antigen is one of the good targets for this purpose that needs to be evaluated for its diversity in local study areas. Objectives: This study was conducted to evaluate the diversity in LACK protein among L. major species. Methods: We collected 30 L. major isolates from different parts of Khuzestan province, southwest of Iran. Leishmania activated C kinase gene was selected for amplifying by PCR and sequencing. Results: Phylogenetic tree showed that there is no diversity in LACK gene in this area. Conclusions: Leishmania activated C kinase gene is a very conservative gene, suitable for vaccination.

Background: Hepatitis E virus (HEV) is able to induce fulminant hepatitis E infection in immunosuppressed individuals. Previous studies showed that the Baculovirus expression system (BES) could be used for developing hepatitis E virus-like particles (VLPs). Objectives: In the present study, the formation of VLPs of the recombinant proteins of HEV truncated open reading frame 2 (ORF2; 112 - 607) and Rotavirus non-structural protein 4 (NSP4) (OSU-a) were investigated in BES and Escherichia. coli. Methods: To construct VLPs, the truncated ORF2-NSP4 protein was expressed in BES. The expression of protein was confirmed by western blot. This protein was expressed in E. coli. The truncated ORF2-NSP4 gene was subcloned in pET28a, and then transformed into E. coli DH5α. The confirmed colonies were transformed into E. coli BL21. The solubility of protein were checked by SDS-PAGE and western blot analysis. In the final step, to verify the VLP formation in BES and E. coli, the recombinant proteins were stained with 2% uranyl acetate and checked by transmission electron microscopy (TEM). Results: The 75 KDa truncated ORF2-NSP4 protein was successfully expressed in Sf9 cells, assembled, and formed VLP according to TEM results. In the prokaryotic expression system (E. coli), the ORF2-NSP4 gene was successfully subcloned and expressed in BL21 but VLP was not detected in TEM analysis. Conclusions: The truncated ORF2-NSP4 VLP were efficiently expressed in the SF9 cells, as a potential mucosal vaccine against HEV and Rotavirus. In the prokaryotic expression system (E. coli), the ORF2-NSP4 gene was successfully expressed.

Infections caused by non-tuberculous mycobacteria (NTM) is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM) analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates) were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%), were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates), followed by M. fortuitum (20 isolates), M. tuberculosis (15 isolates), M. kansassi (10 isolates), M. abscessus group (5 isolates), M. avium (5 isolates), and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus-M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens.

Background MDR1 is a highly polymorphic gene that encodes P-glycoprotein (P-gp). This protein anchor to the cell membrane and transports toxins, xenobiotic, chemicals, and drugs from the intracellular to extracellular and thus protect cells. Polymorphism of the MDR1 gene seems to be effective in gene expression and response to treatment. Since one of the main mechanisms of drug resistance is the removal of the drug from the cell by ATP-dependent efflux proteins, thus MDR1, single nucleotide polymorphism (SNP) C3435T can be used as a predictor for treatment outcomes. Methods The peripheral blood-EDTA samples were collected from 71 patients with chronic hepatitis C. The total genomic DNA extraction was carried out. The PCR was performed for detection of the MDR1 gene in HCV patients and MDR1 gene polymorphism was genotyped by the PCR-RFLP method. Results Out of 71 patients 52 (73.3%) were male, 19 (26.7%) female with mean age-min-max; 41.17 ± 8.3-(26–59). The distribution of MDR1 genotype in 48(67.6%) responders were CC 13 (27%), CT 34 (71%) and TT 1(2%), while MDR1 genotypes in 8 (11.3%) non responders were CC 2(25%), CT 1(12.5%) TT 5(62.5%) and in 15(21.1%) recurrence were 5 (33%) CC, 6 (40%) CT and 4 (27%) TT genotype. The patients with heterozygous CT (C3435T) genotype 34/48(71%) were found better response than non-responders with TT 5/8(62.5%) genotype (p Conclusion Our result reveals that 71% of the responders were CT genotypes (C3435T) and 62.5% of non-responders were TT genotype (T3435T). With aforementioned results, determination of different forms of SNPs in MDR1 gene should be considered as a predictor in the treatment of all chronic HCV patients. The homozygous TT genotype and high prevalence of T allele may be related to low antiviral response during combined therapy in treatment of chronic HCV patients.

Several viruses are responsible for aseptic meningitis; however, in the region of Southwest Iran, the role played by each virus is still not very well known. The aim of this study is to determine the relative frequencies of mumps virus, herpes viruses, and enteroviruses, as well as coinfections among them, in patients with aseptic meningitis. In this cross-sectional study, samples of cerebrospinal fluid were collected between December 2012 and December 2013 from patients under 14 years, who were hospitalized in Abuzar Children's Hospital in Ahvaz, Southwest Iran (the only children's hospital in Khuzestan province and Southwest Iran). All 66 cerebrospinal fluid samples and corresponding clinical data were collected from patients with aseptic meningitis by specialists, and with the patients’ consent. The DNA and RNA were extracted from these samples and subjected to polymerase chain reaction as well as reverse transcription polymerase chain reaction (RT-PCR) for detection of mumps virus, herpes viruses, and enteroviruses. Nine of the samples (3 mumps-positive and 6 enterovirus-positive) were sequenced. The mumps virus sequences were investigated for possible mutations in the SH and partial HN regions. Up to 39 patients (59.09%) were found to be positive for enteroviruses, 3 (4.5%) for mumps virus, and 1 (1.5%) for herpes viruses (specifically, the varicella-zoster virus). Two patients (3.03%) had a mumps virus and enterovirus coinfection. Among the 3 detected mumps virus samples, 1 belonged to genotype B, while the others belonged to genotype N. Six sequenced enteroviruses indicated the highest similarity with Echovirus 30. An amino acid substitution at position 51 (N→T) was detected in the HN region of genotype N mumps virus samples, in comparison to the reference strain.

Background: The hepatitis E virus (HEV) accounts for hepatitis E infection with relatively high mortality rate in pregnant women that can lead to fulminant hepatitis. The baculovirus expression system (BES) has the capability to produce high-level recombinant proteins and could be useful for vaccine designing. Objectives: The aim of this study was designing a recombinant hepatitis E virus ORF2 and Rotavirus NSP4 (ORF2-NSP4) and to evaluating construction these recombinant proteins in the BES. Methods: The truncated ORF2 gene (112-607) and truncated ORF2-NSP4 were subcloned in pFastBac1 plasmid, separately, followed by digestion and confirmed by digestion and sequencing. Then the products were transformed into Escherichia coli DH5 alpha and retransformed in DH10Bac competent cells. Finally the white colonies containing Bacmid DNA subjected to PCR for confirming transformation. Bacmid DNA containing HEV truncated ORF2 and HEV truncated ORF2-NSP4 genes were transfected into SF9 cells using BES. The expressed proteins in the cell lysate were evaluated by SDS-PAGE and determined by the western blot assay. Results: The lengths of the subcloned genes, truncated ORF2 and truncated ORF2-NSP4 were 1500 and 2000bp, respectively. After retransforming in DH10Bac, the size of PCR products were 300 bp in Bacmid DNA without recombination while it was 4300 and 3800 bp in Bacmid truncated ORF2-NSP4 and Bacmid truncated ORF2 PCR products. The analysis of protein expression by SDS-PAGE and immunoblotting revealed the presence of 56 KDa for truncated ORF2 and 74.5 KDa for truncated ORF2-NSP4 proteins. Conclusions: The results of the present study showed that the baculovirus expression system (SF9 cells) was able to express truncated ORF2 and truncated ORF2-NSP4 proteins as a potential candidate vaccine.