Computational screening of FDA-approved drugs to identify potential TgDHFR, TgPRS, and TgCDPK1 proteins inhibitors against Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is one of the most successful parasites in the world, because about a third of the world's population is seropositive for toxoplasmosis. Treatment regimens for toxoplasmosis have remained unchanged for the past 20 years, and no new drugs have been introduced to the market recently. This study, performed molecular docking to identify interactions of FDA-approved drugs with essential residues in the active site of proteins of T. gondii Dihydrofolate Reductase (TgDHFR), Prolyl-tRNA Synthetase (TgPRS), and Calcium-Dependent Protein Kinase 1 (TgCDPK1). Each protein was docked with 2100 FDA-approved drugs using AutoDock Vina. Also, the Pharmit software was used to generate pharmacophore models based on the TgDHFR complexed with TRC-2533, TgPRS in complex with halofuginone, and TgCDPK1 in complex with a bumped kinase inhibitor, RM-1-132. Molecular dynamics (MD) simulation was also performed for 100 ns to verify the stability of interaction in drug-protein complexes. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis evaluated the binding energy of selected complexes. Ezetimibe, Raloxifene, Sulfasalazine, Triamterene, and Zafirlukast drugs against the TgDHFR protein, Cromolyn, Cefexim, and Lactulose drugs against the TgPRS protein, and Pentaprazole, Betamethasone, and Bromocriptine drugs against TgCDPK1 protein showed the best results. These drugs had the lowest energy-based docking scores and also stable interactions based on MD analyses with TgDHFR, TgPRS, and TgCDPK1 drug targets that can be introduced as possible drugs for laboratory investigations to treat T. gondii parasite infection.

Study of frequency and inheritance model of ACE1 I/D and ACE2 rs2285666 polymorphisms in COVID-19 patients with varying severity of lung involvement and its effect on serum cytokines levels.

The angiotensin-converting enzyme (ACE) has been shown to play a role as a receptor for the COVID-19 virus. This virus usually gets into cells and infects them by attaching to their glycoprotein receptors, which are found on the ACE2 receptor. The aim of this study was to evaluate the frequency and inheritance of ACE1 I/D and ACE2 rs2285666 polymorphisms in COVID-19 patients with varying severity of lung involvement and its effect on serum cytokines levels of interleukin (IL)-1 and IL-6 and laboratory parameters. One hundred eighty-five COVID-19 patients were grouped according to the severity of lung involvement. (I/D) polymorphism of the ACE1 gene and rs2285666 polymorphism of the ACE2 gene were determined by single specific primer-polymerase chain reaction and restriction fragment length reaction-polymerase chain reaction methods, respectively. Serum levels of IL-1 and IL-6 were also measured by the enzyme linked immunosorbent assay technique. No statistically significant association of ACE2 rs2285666 polymorphism genotypes and ACE1 I/D with the severity of lung involvement was noted. However, there was a statistically significant association between I/D ACE1 polymorphism genotypes and IL-6, white blood cells (WBC), and neutrophil-to-lymphocyte ratio (NLR) levels. Also, there was no statistically significant association between rs2285666 polymorphism genotypes and patients' blood oxygen saturation level, IL-6, IL-1ß, lactate dehydrogenase activity, WBC count, and NLR. In patients with COVID-19, the rs2285666 polymorphism of the ACE2 gene and the I/D polymorphism of the ACE1 gene were not significantly associated with the severity of COVID-19 disease and serum IL-6 and IL-1 cytokine levels.

Characterization of SARS-CoV-2 omicron variants from Iran and evaluation of the effect of mutations on the spike, nucleocapsid, ORF8, and ORF9b proteins function.

The SARS-CoV-2 'Omicron' strain, with 15 mutations in the receptor binding domain (RBD), was detected in South Africa and rapidly spread worldwide. SARS-CoV-2 ORF9b protein by binding to the TOM70 receptor and ORF8 protein by binding to MHC-I, IF3 receptors inhibit the host's immune response. In this study, genomics variations were evaluated for 96 samples isolated from Iran from March to July 2022 using the Nextclade web server and informatics tools. We identified the mutations occurring in the SARS-CoV-2 proteins. We also evaluated the effect of mutations on spike protein interaction with the ACE2 receptor, ORF9b protein interaction with the TOM70 receptor, and structural stability of ORF8 and nucleocapsid proteins using docking and molecular dynamics. Results indicated that during March and April 2022, the BA.2 strain was dominant in the south of Iran, while during June 2022, the BA.5 strain was dominant. BF.5 strain had the most divergence among SARS-CoV-2 strains reported from south of Iran. The binding affinity of BA.5 and BF.5 strains spike protein to ACE2 receptor is similar, and compared to BA.2 strain, was stronger. The BF.5 ORF9b K40R mutation causes a better binding affinity of the protein to the TOM70 receptor. Also, mutations that occurred in the ORF8 protein led to instability in the dimer formation of this protein and improved immune response for mutations that occurred in BA.2 strain, while this mutation did not occur in BF.5 strain. The mutations that were detected in nucleocapsid protein CTD and NTD domains caused the stability of these domains.Communicated by Ramaswamy H. Sarma.

Evaluation of the dual effects of antiviral drugs on SARS-CoV-2 receptors and the ACE2 receptor using structure-based virtual screening and molecular dynamics simulation.

The use of US FDA-approved drugs is preferred due to the need for lower costs and less time. In in silico medicine, repurposing is a quick and accurate way to screen US FDA-approved medications to find a therapeutic option for COVID-19 infection. Dual inhibitors possess dual inhibitory activity, which may be due to the inhibition of two different enzymes, and are considered better than combination therapy from the developmental and clinical perspectives. In this study, a molecular docking simulation was performed to identify the interactions of antiviral drugs with the critical residues in the binding site of the main SARS-CoV-2 protease, spike glycoprotein, and papain-like protease receptors compared to the angiotensin-converting enzyme-related carboxypeptidase (ACE2) receptor of host cells. Each of the receptors was docked with 70 US FDA-approved antiviral drugs using AutoDock Vina. A molecular dynamics (MD) simulation study was also used for 100 ns to confirm the stability behaviour of the ligand receptor complexes. Among the drugs that had the strongest interaction with the SARS-CoV-2 main protease, spike glycoprotein and papain-like protease receptors, and host cell ACE2 receptors, Simeprevir, Maraviroc and Saquinavir had dual inhibitory effects. The MD simulation study confirmed the stability of the strongest interactions between the antiviral drugs and the main protease, ACE2, spike glycoprotein, and papain-like protease receptors to 100 ns. However the results of MMPBSA analysis showed that the bond between Saquinavir and the ACE2 receptor was weak. Simeprevir and Maraviroc drugs had acceptable binding energies with dual receptors, especially the Simeprevir.Communicated by Ramaswamy H. Sarma.

Characterization of SARS-CoV-2 isolated from a patient in Iran compared to SARS-CoV-2 different variants.

Since the onset of the global epidemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), whole genome sequencing of virus in all countries has been considered to track and predict virus transmission and variation patterns. In the current study we reported a novel complete genome sequence of SARS-CoV-2 isolated from Iran. Genomics variations and protein sequences were evaluated for the isolated sequence and seven Iranian complete genome sequences of SARS-CoV-2 from NCBI using the reference genome of the SARS-CoV-2 Wuhan-Hu-1. The results showed six nucleotide substitutions. The multiple sequence alignment of the spike protein of the Wuhan-Hu-1 strain and the emerging variants indicated similar its residue pattern in the current sequence to the Wuhan-Hu-1 strain. There were relatively similar binding affinity and residues involved in the interactions of the spike receptor-binding domain (RBD) of the Wuhan-Hu-1 strain, the variants and Hormozgan With angiotensin-converting enzyme 2 (ACE2). Tracing the phylogeny of virus indicated distinct clustering of Iranian variants in branches close to the Asian countries. The mutation effect study on the function of proteins predicted neutral impact of all six nucleotide substitutions. However, the free energy calculations indicated a decreasing the protein stability related to the mutations. This data, consistent with similar studies, showed that despite the high similarity in the nucleotide sequence of the SARS-CoV-2, the mutation pattern varies from country to country. Therefore, any country can benefit from these studies to track and find appropriate strategies for treating and controlling the epidemic.Communicated by Ramaswamy H. Sarma.

Preparation and pre-clinical evaluation of flagellin-adjuvanted NOM vaccine candidate formulated with Spike protein against SARS-CoV-2 in mouse model.

From December 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was started as a cluster of pneumonia cases in Wuhan, Hubei Province, China. The disturbing statistics of SARS-CoV-2 promoted scientists to develop an effective vaccine against this infection. NOM protein is a multi-epitope protein that designed based on Nucleocapsid, ORF3a, and Membrane proteins of SARS-CoV-2. Flagellin is a structural protein that binds to the Toll-like receptor 5 and can enhance the immune response to a particular antigen. In this study, NOM protein as vaccine candidate was linked to the carboxyl and amino terminals of flagellin adjuvant derived from Salmonella enterica subsp. enterica serovar Dublin. Then, informatics evaluations were performed for both NOM protein and NOM protein linked to flagellin (FNOM). The interaction between the NOM and FNOM proteins with the TLR5 were assessed using docking analysis. The FNOM protein, which compared to the NOM protein, had a more suitable 3D structure and a stronger interaction with TLR5, was selected for experimental study. The FNOM and Spike (S) proteins expressed and then purified by Ni-NTA column as vaccine candidates. For analysis of immune response, anti-FNOM and anti-S proteins total IgG and IFN-γ, TNF-α, IL-6, IL-10, IL-22 and IL-17 cytokines were evaluated after vaccination of mice with vaccine candidates. The results indicated that the specific antisera (Total IgG) raised in mice that received FNOM protein formulated with S protein were higher than mice that received FNOM and S proteins alone. Also, IFN-γ and TNF-α levels after the spleen cells stimulation were significantly increased in mice that received the FNOM protein formulated with S protein compared to other groups. Immunogenic evaluations showed that, the FNOM chimeric protein could simultaneously elicit humoral and cell-mediated immune responses. Finally, it could be concluded that the FNOM protein formulated with S protein could be considered as potential vaccine candidate for protection against SARS-CoV-2 in the near future.

Active site-based analysis of structural proteins for drug targets in different human Coronaviruses.

Seven types of Coronaviruses (CoVs) have been identified that can cause infection in humans, including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, HCoV-MERS, and SARS-CoV-2. In this study, we investigated the genetic structure, the homology of the structural protein sequences, as well as the investigation of the active site of structural proteins. The active site of structural proteins was determined based on the previous studies, and the homology of their amino acid sequences and structure was compared. Multiple sequence alignment of Spike protein of HCoVs showed that the receptor-binding domain of SARS-CoV-2, SARS-CoV, and MERS-CoV was located at a similar site to the S1 subunit. The binding motif of PDZ (postsynaptic density-95/disks large/zona occludens-1) of the envelope protein, was conserved in SARS-CoV and SARS-CoV-2 according to multiple sequence alignment but showed different changes in the other HCoVs. Overall, spike protein showed the most variation in its active sites, but the other structural proteins were highly conserved. In this study, for the first time, the active site of all structural proteins of HCoVs as a drug target was investigated. The binding site of these proteins can be suitable targets for drugs or vaccines among HCoVs.

A triple-blind, placebo-controlled, randomized clinical trial to evaluate the effect of curcumin-containing nanomicelles on cellular immune responses subtypes and clinical outcome in COVID-19 patients.

In COVID-19 patients, cytokine storm due to excessive immune responses can cause severe complications. In this study, we investigated the effect of curcumin nanomicelles on clinical outcome and cellular immune responses subtypes changes in COVID-19 patients. A randomized, triple-blinded, placebo-controlled study was done. Forty COVID-19 patients were included into two groups of nano-curcumin and placebo. The nano-curcumin group received 40 mg of nano-curcumin capsule, four times per day for 2 weeks. Clinical signs and gene expression of TBX21, GATA3, RORC and FOXP3 genes and IFN-γ, IL-4, IL-17 and TGF-ß cytokines serum levels were measured at time points of 0, 7 and 14 days. Serum levels of IFN-γ (p = .52) and IL-17 (p = .11) decreased, while IL-4 (p = .12) and TGF-ß (p = .14) increased in the nano-curcumin group compared with placebo on day 14. Moreover, gene expressions of TBX21 (p = .02) and FOXP3 (p = .005) genes were significantly decreased and increased between nano-curcumin and placebo groups on day 7, respectively. It can be concluded that administration of nano-curcumin in inflammatory phase of COVID-19 can accelerate recovering of the acute inflammatory phase by modulating inflammatory immune responses. Therefore, it is suggested that this supplement in inflammatory diseases, including COVID-19, can be effective in controlling the inflammatory responses.

Dysentery as the only presentation of COVID-19 in a child: a case report.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic has caused irreparable damage to society, and the damage continues. Pediatricians are confronted with COVID-19 in a variety of presentations, which may lead to delayed diagnosis and treatment. Early diagnosis of the disease plays an important role in preventing transmission of the virus in the community. CASE PRESENTATION: Here we report a 27-month-old previously healthy Iranian female child who presented with fever and bloody diarrhea, diagnosed with COVID-19 based on contact history, exclusion of enteric bacterial pathogens and parasites, and positive stool and nasopharyngeal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcriptase polymerase chain reaction (RT-PCR) tests. The patient had viral shedding for more than a month. CONCLUSIONS: The pediatric population usually does not present with typical clinical features of COVID-19, which are respiratory involvement. Dysentery may be the only presentation of this disease, and long-term isolation should be considered, as the viral shedding may last for more than a month.

Reverse vaccinology approach to design a novel multi-epitope vaccine candidate against COVID-19: an in silico study.

At present, novel Coronavirus (2019-nCoV, the causative agent of COVID-19) has caused worldwide social and economic disruption. The disturbing statistics of this infection promoted us to develop an effective vaccine candidate against the COVID-19. In this study, bioinformatics approaches were employed to design and introduce a novel multi-epitope vaccine against 2019-nCoV that can potentially trigger both CD4+ and CD8+ T-cell immune responses and investigated its biological activities by computational tools. Three known antigenic proteins (Nucleocapsid, ORF3a, and Membrane protein, hereafter called NOM) from the virus were selected and analyzed for prediction of the potential immunogenic B and T-cell epitopes and then validated using bioinformatics tools. Based on in silico analysis, we have constructed a multi-epitope vaccine candidate (NOM) with five rich-epitopes domain including highly scored T and B-cell epitopes. After predicting and evaluating of the third structure of the protein candidate, the best 3 D predicted model was applied for docking studies with Toll-like receptor 4 (TLR4) and HLA-A*11:01. In the next step, molecular dynamics (MD) simulation was used to evaluate the stability of the designed fusion protein with TLR4 and HLA-A*11:01 receptors. MD studies demonstrated that the NOM-TLR4 and NOM-HLA-A*11:01 docked models were stable during simulation time. In silico evaluation showed that the designed chimeric protein could simultaneously elicit humoral and cell-mediated immune responses. Communicated by Ramaswamy H. Sarma.

The clinical effect of Nano micelles containing curcumin as a therapeutic supplement in patients with COVID-19 and the immune responses balance changes following treatment: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: To investigates the effectiveness of curcumin-containing Nanomicelles as a therapeutic supplement in the treatment of patients with COVID-19 and its effect on immune responses balance changes following treatment. TRIAL DESIGN: This study is conducted as a prospective, placebo-controlled with parallel group, single-center randomized clinical trial on COVID-19 patients. PARTICIPANTS: Patients are selected from the COVID-19 ward of Shahid Mohammadi Hospital in Bandar Abbas, Iran. INCLUSION CRITERIA: 1. Real time PCR-approved positive COVID-19 test. 2. Both gender 3. Age between 18 and 75 years 4. Signing a written consent 5. Lack of participation in other clinical trials Exclusion criteria: 1. Pregnancy or lactation 2. Allergy to turmeric or curcumin 3. Smoking 4. Patient connected to the ventilator 5. SaO2 less than 90% or PaO2 less than 8 kPa 6. Having comorbidities (such as severe renal failure, Glomerular filtration rate less than 30 ml/min, liver failure, Congestive heart failure, or Chronic obstructive pulmonary disease) 7. History of gallstones 8. History of gastritis or active gastrointestinal ulcer INTERVENTION AND COMPARATOR: In addition to the routine standard treatments for COVID-19, in the intervention group, 40mg nanomicelles containing curcumin (SinaCurcumin Capsule, Exir Nano Sina Company, Iran), four times per day (after breakfast, lunch, dinner and before bedtime) and in the placebo group as the control group, capsules with the same appearance and characteristics (Placebo capsules, Exir Nano Sina Company, Iran) are prescribed for two weeks. MAIN OUTCOMES: The effectiveness of Nano micelles containing curcumin treatment will be evaluated as daily clinical examinations of patients in both groups and, on days 0, 7 and 14, complete clinical symptoms and laboratory findings including peripheral blood and serum parameters such as inflammatory markers will be measured and recorded. Moreover, in order to evaluate the balance of immune responses changes following treatments, serum level of IFN-γ, IL-17, Il-4 and TGF-ß serum cytokines will be measured in both groups at time points of 0, 7 and 14 days post treatment. Gene expression of t-bet, GATA-3, FoxP3 and ROR- γT will also be measured at mentioned time points to assess the shift of T helper1, T helper2, T regulatory and T helper 17 immune responses following treatment. RANDOMISATION: Randomized trials will be performed on 40 COVID-19 patients which will be randomized using encoded sealed boxes with computer generated random digits with 1:1 allocation ratio. In order to randomization, placebo and SinaCurcumin Capsules will be numbered first by computer generated random digits. SinaCurcumin and placebo will then be stored and numbered in sealed packages based on generated random numbers. Finally, according to the order in which patients enter the study, packages are given to patients based on their number. BLINDING (MASKING): The present study will be blind for all patients, physicians and nurses, laboratory technicians and statisticians. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total of 40 patients will be included in the study, 20 of them will be randomly assigned to the intervention group and 20 to the placebo group. TRIAL STATUS: This is Version 1.0 of protocol dated 21 May 2020. The recruitment was started June 24, 2020 and is expected to be completed by October 31, 2020. TRIAL REGISTRATION: This present clinical trial has been registered in the Iranian Registry of Clinical Trials (IRCT) with the registration code of "IRCT20200611047735N1", https://www.irct.ir/trial/48843 . Dated: 19 June 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

HIV-1 Drug Resistance Mutations among Antiretroviral Drug-Experienced Patients in the South of Iran.

BACKGROUND: The therapeutic effect of antiretroviral therapy (ART) is adversely influenced by antiretroviral drug resistance, mainly due to mutations (DRMs) in the human immunodeficiency virus (HIV) genome. These mutations are commonly associated with HIV protease and reverse-transcriptase genes. We sought to determine the frequency of DRMs in a population of ART-experienced patients in the South of Iran. METHOD: A total of 44 HIV-1-positive participants under ART were selected from April 2016 to March 2017. Their DRMs, antiretroviral resistance status, and viral subtypes were determined. RESULTS: At least one DRM was detected in 61.4% of the participants. The highest frequency was related to nucleotide reverse-transcriptase inhibitor (NRTI) mutations (45.45%). In contrast, major protease inhibitor (PI) mutations had the lowest frequency (6.81%). M184V (40.9%) and K103N (25%), respectively related to NRTI and nonnucleoside reverse-transcriptase inhibitor (NNRTI), were the mutations with the highest frequencies. Susceptibility to PI drugs was higher compared to NRTIs and NNRTIs, which was consistent with the results of genotypic DRMs. CONCLUSION: The highest frequency of antiretroviral DRMs was related to NRTIs and NNRTIs. In contrast, PI resistance mutations had the lowest frequency. Laboratory-guided ART to avoid the expansion of mutants as well as investigating DRMs in other viral regions, such as integrase, are recommended.

Cell cytotoxicity, immunostimulatory and antitumor effects of lipid content of liposomal delivery platforms in cancer immunotherapies. A comprehensive in-vivo and in-vitro study.

Liposome is one of the promising technologies for antigen delivery in cancer immunotherapies. It seems that the phospholipid content of liposomes can act as immunostimulatory molecules in cancer immunotherapy. In the present study, the immunological properties of different phospholipid content of liposomal antigen delivery platforms were investigated. To this aim, F1 to F4 naïve liposomes (without tumor-specific loaded antigens) of positively charged DOTAP/Cholesterol/DOPE (4/4/4 mol ratio), negatively charged DMPC/DMPG/Cholesterol/DOPE (15/2/3/5), negatively charged DSPC/DSPG/Cholesterol/DOPE (15/2/3/5) and PEGylated HSPC/mPEG2000-DSPE/Cholesterol (13/110) liposomal compositions were administered in mice bearing C26 colon carcinoma to assess tumor therapy. Moreover, In-vitro studies were conducted, including cytotoxicity assay, serum cytokines measurements, IFN-γ and IL-4 ELISpot assay, T cells subpopulation frequencies assay. The liposomes containing DOTAP and DOPE (F1 liposomes) were able to stimulate cytotoxic T lymphocytes signals such as IFN-γ secretions. In parallel, the aforementioned phospholipids stimulated secretion of IL-4 and IL-17 cytokines from T helper cells. However, these liposomes did not improve survival indices in mice. As conclusion, DOTAP and DOPE contained liposomes (F1 liposomes) stimulate a mixture of Th1 and Th2 immune responses in a tumor-specific antigens-free manner in mice bearing C26 colon carcinoma. Therefore, phospholipid composition of liposomes merits consideration in designing antigen-containing liposomes for cancer immunotherapy.

An overview of diarrheagenic Escherichia coli in Iran: A systematic review and meta-analysis.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) is a common enteric pathogen that causes a wide spectrum of gastrointestinal infections, particularly in developing countries. This is a systematic review and meta-analysis to determine the prevalence of DEC in various geographical regions in Iran. MATERIALS AND METHODS: English (PubMed, Web of Science, Scopus, Embase, Cochrane Library, and Google Scholar) and Persian (IranMedex, SID, Magiran, and Iran Doc) databases were comprehensively searched from January 1990 to April 2017. Study selection and data extraction were performed by two independent reviewers. After assessing heterogeneity among studies, a random effects model was applied to estimate pooled prevalence. Data analyses were done with the Stata software (version 12.0). This meta-analysis was registered with PROSPERO, number CRD42017070411. RESULTS: A total of 73 studies with 18068 isolates were eligible for inclusion within the meta-analysis. The results of random effects model showed that the most prevalent DEC pathotypes were enterotoxigenic E. coli (ETEC) (16%; 95% confidence interval [CI]: 11%-23%), enteroaggregative E. coli (11%; 95% CI: 8%-15%), atypical enteropathogenic E. coli (EPEC) (11%; 95% CI: 8%-14%), Shiga toxin-producing E. coli (9%; 95% CI: 6%-13%), diffuse adherent E. coli (6%; 95% CI: 6%-12%), enteroinvasive E. coli (4%; 95% CI: 2%-6%), and typical EPEC (3%; 95% CI: 1%-5%). CONCLUSION: This study showed that DEC infections in the Iranian population have low frequency. Our data suggest that the ETEC pathotype can be regarded as one of the most important etiological agents of diarrhea in this country. However, the prevalence of DEC pathotypes is diverse in different regions of Iran.

Development of Preventive Vaccines for Hepatitis C Virus E1/E2 Protein.

Hepatitis C virus (HCV) is responsible for a vast majority of liver failure cases. HCV is a kind of blood disease estimated to chronically infect 3% of the worlds population, causing significant morbidity and mortality. Therefore, a complete knowledge of humoral responses against HCV, resulting antibodies, and virus-receptor and virus-antibody interactions, are essential to design a vaccine. HCV epitopes or full sequence of HCV proteins can induce HCV specific immune responses. In fact, structural proteins are usually the main target of humoral responses and non-structural proteins are usually the main target of cellular responses. Hence, various vaccines based on distinct antigenic combinations are developed to prevent HCV infection and the current study tried to summarize them.

Prevalence of HIV-1 pre-treatment drug resistance in a southern province of Iran, 2016-2017.

HIV-1 transmitted drug resistance (TDR) occurs when primary viruses bear drug resistance mutations (DRMs). TDR causes first-line antiretroviral (ARV) therapy (ART) failure and is becoming more pronounced due to the widespread use of ART. With the absence of routine individual-level drug resistance testing, the World Health Organization (WHO) recommends the tracking of TDR mutations and optimizing the first-line ART following pre-treatment drug resistance (PDR) surveys. Here, we report the PDR frequency for the first time in Hormozgan, a southern province of Iran. In this study, 41 blood samples from HIV-1-positive ART-candidate volunteers were collected across the province between April 2016 and March 2017. Phylogenetic analysis of the sequenced protease (PR) and reverse transcriptase (RT) regions showed that 39 out of 41 samples (95%) were CRF35_AD and the two remaining cases were subtype B (2.5%) and C (2.5%). D67G (2.4%), a mutation that reduces susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs) was the only detectable TDR mutation in this population. Two other DRMs, including E138A (9.7%) and V179T (4.9%), which confer resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), were also identified. Although no major protease inhibitor (PI) resistance mutations were detected, the minor mutations L10F and L33F (2.5% each) as well as several highly frequent polymorphic mutations were identified. Our results show a PDR frequency of 17% in infected individuals from Hormozgan, classified further as 2.4% NRTIs and 14.6% NNRTIs. These results suggest that first-line ART should be practiced carefully in Hormozgan province, and alternative regimens may become necessary for all starters.

Prevalence and Characteristics of Precore Mutation in Iran and Its Correlation with Genotypes of Hepatitis B.

INTRODUCTION: Mutation of the HBV precore gene prevents the production of HBeAg, which is an important target for immune responses. Distribution of this mutation varies along with frequency of HBV genotypes in accordance with geographic and ethnic variations. The general objective of this study was to evaluate the prevalence and characteristics of precore mutation in Iran and its correlation with genotypes of hepatitis B. METHODS: In this cross-sectional study, viral DNA of 182 Iranian hepatitis B surface antigen positive patients who were admitted to Bandar Abbas Blood Transfusion Organization in 2012 and 2013 was retrieved from their serum samples. HBeAg, anti-HBe, and anti-HBc IgM diagnostic tests were performed using ELISA kits. Precore and Pre-S regions were amplified using specific primers and PCR thereafter to determine the genotypes; precore mutation, PCR, and restriction fragment length polymorphism (RFLP) methods also were applied. SPSS version 12 was used for data analysis by Mann-Whitney U test, Fisher's exact probability test, and t-test. RESULTS: A total of 62 patients (34.1%) had precore mutation (A1896G), and genotype D was the predominant genotype in these patients, which was followed by an unknown genotype that was suspected for genotype B. Interestingly, the relationships between precore mutation and HBeAg (p=0.037) and genotype D (p=0.005) were significant; however, no correlation was observed between this mutation and acute or chronic hepatitis and sex of patients. CONCLUSION: This study found high prevalence of precore mutations in southern Iran, which was significantly associated with HBeAg and genotype D.

Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region.

BACKGROUND: The E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions. OBJECTIVES: The aim of the present study was to construct an HCV 1a/JFH1 chimeric virus by replacing the intergenotypic variable region (igVR) fragment of the highly variable region of the E2 gene of the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCV genotype 1a. This chimera was produced as a model virus with the ability to be cultured. We analyzed the adapted virus and the variations of nucleic acids within it. METHODS: Specific primers were designed for the igVR of HCV genotype 1a followed by the overlap-PCR method for the synthesis of the desired DNA fragment. The amplified igVR-1a chimera gene and pFL-J6/JFH were digested by KpnI and BsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed into Escherichia coli JM109 strain competent cells. All clones were confirmed by colony PCR using specific primers, and the confirmed recombinant vector was sequenced. The recombinant vector was targeted for RNA synthesis by T7 RNA polymerase enzyme. RNA transfection was performed in the Huh7.5 cell line. Virus production in several passages and the evaluated viral load were studied using quantitative real-time PCR and ELISA methods. After 30 passages, the RNA virus was extracted and cloned in PCDNA3.1 vector, and was then sequenced. RESULTS: Quantitative real-time PCR results showed 11,292,514 copies/mL of chimeric virus production in cell culture. The virus production was confirmed using ELISA, which showed a virus core production of 808.2 pg/mL. The results of cloning and sequencing showed that some of the nucleic acids in the chimera virus were changed, affecting the viral behavior in the cell culture. CONCLUSIONS: Real-time PCR and ELISA showed high levels of production of 1a/JFH1 chimeric HCV in the Huh7.5 cell culture. The constructed virus can be used for future studies, including the development of new HCV drugs and vaccines.

Hepatitis Viruses B and D and Human Immunodeficiency Virus Infections in Hemodialysis Patients in the South of Iran: Prevalence and Genotypes.

BACKGROUND: Hepatitis B virus (HBV), hepatitis D virus (HDV), and human immunodeficiency virus (HIV) are transmitted by blood transfusion. Thus, hemodialysis (HD) patients are more prone to become the carriers of these infections due to their treatment demands. OBJECTIVES: The aim of this study was to assess the prevalence of HBV and HIV infections among HD patients in Bandar Abbas, Iran, 2015. PATIENTS AND METHODS: A total of 153 patients with chronic renal failure undergoing HD at Shahid Mohammadi hospital in Bandar Abbas were examined for hepatitis B surface antigen (HBsAg), anti- hepatitis C virus, and anti-HIV over a period of 2 months. Thereafter, all of the specimens were evaluated for HBV-DNA, HDV-RNA and HIV-RNA using polymerase chain reaction (PCR) and further techniques. All statistical analyses were carried out using SPSS version 12 for Windows with the t-test and chi-square (χ(2)) test. RESULTS: Both kinds of assay determined that nine (5.88%) patients were HBV positive (HBsAg-positive), whereas no HIV- and HDV-positive patients were diagnosed. All of the diagnosed HBV samples belonged to genotype D; the prevalence of HBV is associated with age, duration of HD, history of blood transfusion, and using shared HD devices. CONCLUSIONS: In conclusion, the prevalence of HBV infection was low in the south of Iran, but genotype D represented the major HBV genotype in this population. Among the variables, age, duration of HD, history of blood transfusion, and using shared HD devices influenced the prevalence of HBV among HD patients.

Prevalence and Characteristics of Basal Core Promoter Mutations in Iran and its Correlation with Acute and Chronic Hepatitis B Infection.

INTRODUCTION: Manifestations of HBV infection differ in chronic and acute phases. Therefore, identifying the determinants such as mutations has a vital role in the treatment of the disease. A dual transversion in the basal core promoter (BCP) region is common among HBV patients. Thus, the present study was conducted with the objective of determining the prevalence of basal core promoter (BCP) mutations and its correlation with the outcome of HBV infection. METHOD: In this cross-sectional study, samples were obtained from 182 Iranian HBsAg positive patients who were admitted to the Bandar Abbas Blood Transfusion Organization in 2012 and 2013. They were screened by ELISA test using commercial kits to detect serological marker anti-HBc IgM for distinct chronic hepatitis from acute infection. Thereafter, the extracted DNA was used for determination of the BCP mutations by PCR-RFLP technique. Data analyses were performed with SPSS 12 by Mann-Whitney U test, Fisher's exact probability test, and t-test. RESULTS: BCP mutations were observed in 15 samples (8.24%) of the study population, and serological tests determined that, among the BCP mutants, one sample (6.67%) was HBeAg positive, 14 samples (93.33%) were HBeAg negative, and four samples (2.2%) were positive for anti-HBc IgM test. Data analysis indicated a statistically significant association between BCP mutations and acute hepatitis (p=0.002). However, no relationship was detected between the prevalences of the BCP mutations and gender of subjects (p>0.567). CONCLUSIONS: The prevalence of BCP variants was low in the south of Iran, and this mutation can lead to acute phase of viral hepatitis.