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Background: The human immunodeficiency virus type 1 [HIV-1] is an infectious viral agent that gradually extinguishes the immune system, resulting in acquired immune deficiency syndrome [AIDS]. The aim of this study was to construct an RNA-positive control based on armored [AR] RNA technology, using HIV-1 RNA as a model
Methods: The MS2 maturase, a coat protein gene [at positions 1765 to 1787] and HIV-1 pol gene were cloned into pET-32a plasmid. The prepared plasmid was transformed into Escherichia coli strain BL2 [DE3], and the expression of the construct was induced by 1 mM of isopropyl-L-thio-D-galactopyranoside [IPTG] at 37 °C for 16 h to obtain the fabricated AR RNA. The AR RNA was precipitated and purified using polyethylene glycol and Sephacryl S-200 chromatography
Results: The stability of AR RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy and gel agarose electrophoresis. Tenfold serial dilution of AR RNA from 101 to 105 was prepared. Real-time PCR assays had a range of detection between 101 and 105. In addition, R2 value was 0.998, and the slope of the standard curve was -3.33
Conclusions: Prepared AR RNA, as a positive control, could be used as a basis for launching an in-house HIV-1 virus assay and other infectious agents. It can be readily available to laboratories and HIV research centers. The AR RNA is non-infectious and highly resistant to ribonuclease enzyme and can reduce the risk of infection in the clinical laboratory
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Aim: The aim of this study was to determine the effect of inhibition of TGF-beta/smad signaling on the expression profiles of miR-335, miR-150, miR-194, miR-27a, miR-199a of hepatic stellate cells [HSCs]
Background: Liver fibrosis is excessive deposition of extracellular matrix proteins due to ongoing inflammation and HSC activation that occurs in most types of chronic liver diseases. Recent studies have shown the importance of microRNAs in the pathogenesis of chronic liver diseases
Methods: In this study, for inhibition of TGF-beta smad-signaling pathway, expressing Smad4 shRNA plasmids were transfected into HSCs. Subsequently, using Real Time-PCR, we measured the expression levels of miR-335, miR-150, miR-194, miR-27a and miR- 199a
Results: Gene expression analysis showed that downregulation of Smad4 by vector Smad4shRNA significantly increased the expression levels of miR-335 [P<0.01] and miR-150 [P<0.001] and decreased the expression level of miR-27a [P<0.05]
Conclusion: The results of this study suggest that blocking TGF-beta smad-signaling can also differentially modulate microRNA expression in support of activation and fibrogenesis of HSCs
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Background: Detection and quantification of human Papillomavirus [HPV] genome in oral carcinoma play an important role in diagnosis, as well as implications for progression of disease
Methods: We evaluated tissues from 50 esopharyngeal cancers collected from different regions of Iran for HPV E6 using the two type-specific primers sets. E6 gene of HPV genotypes was amplified by specific primers. The sensitivity of PCR assay was analyzed and determined using HPV-DNA-containing plasmids. Real-time PCR was utilized to determine the prevalence and HPV viral load in patients with oral cavity squamous cell carcinoma
Results: Eighteen [36%] specimens were positive for HPV. Among the 18 positive specimens, 10 showed HPV-18 [55.55%], and 8 specimens were positive for HPV-11 [44.44%]. Of the 18 infected specimens, 6 [33.32%] and 12 [66.65%] were identified as high-titer and low-titer viral load, respectively
Conclusions: The PCR-based assay, developed in the current study, could be used for HPV detection, quantification, and genotyping in epidemiological and clinical studies
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Objective: To survey the level and patterns of reverse transcriptase-based drug resistance and subtype distribution among antiretroviral-treated HIV-infected patients receiving only reverse transcriptase inhibitors in Iran. Methods: A total of 25 samples of antiretroviral therapy experienced patients with no history of using protease inhibitors were collected. After RNA extraction, reverse transcriptase-nested PCR was performed. The final products were sequenced and then analysed for drug-resistant mutations and subtypes. Results: No drug resistant mutations were observed among the 25 subjects. The results showed the following subtypes among patients: CRF 35_AD (88%), CRF 28_BF (8%), and CRF 29_BF (4%). Conclusions: A significant increase in drug resistance has been noted in recently-infected patients worldwide. Subtype distributions are needed to perform properly-designed surveillance studies to continuously monitor rates and patterns of transmitted drug resistance and subtypes to help guide therapeutic approaches and limit transmission of these variants.
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Background: Enteric viruses, particularly human rotavirus and norovirus, have been shown to replace bacteria and parasites, as the most common pathogens responsible for acute diarrhea. However, there are still few epidemiological data on the simultaneous occurrence of these viruses in Iran. In this regard, the aim of this study was to assess the useful epidemiological data on the gastroenteritis associated with rotavirus-norovirus mixed infection and to examine the prevalence of norovirus genogrouping among children aged less than five years old in Iran
Methods: A total of 170 stool samples were collected from children under five years of age with the clinical signs and symptoms of acute gastroenteritis, from May 2013 to May 2014. For the detection of both rotavirus and norovirus, total RNA was extracted from all samples, followed by reverse transcription polymerase chain reaction [RT-PCR]. For both detected rotaviruses and noroviruses, genogrouping was performed
Results: Of 170 samples, 49 [28.8%] and 15 [8.8%] samples were found to be positive for rotavirus and norovirus infections by RT-PCR. Interestingly, 6 [3.5%] patients were positive for both infections. Among the 15 norovirus-positive patients, 13 [86.6%] and 2 [13.3%] belonged to genogroups GII and GI
Conclusions: The norovirus genogroup GII and rotavirus lead to the serious infections in children with acute gastroenteritis. However, more well-designed studies are needed to further elucidate the role of other enteric viruses in acute gastroenteritis
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Objective: Hepatitis C virus [HCV] is considered to be a worldwide health problem. In most cases, HCV infection becomes chronic and may proceed to fibrosis, cirrhosis, and hepatocellular carcinoma. Many pathological effects in cells may occur by viral proteins. The purpose of this study is to evaluate the effect of the HCV core protein on cells to induction of the fibrogenesis process
Methods: We use the LX-2 cell line that originated from hepatic stellate cells. Plasmid which expressed HCV core protein was transfected to the cells. After 72 h, RNA was extracted and treated with DNase, followed by synthesis of cDNA. Positive control cells were treated with the leptin fibrotic hormone. We used real-time PCR to measure and statistically analyze alpha-SMA gene expression
Results: The HCV core protein significantly increased alpha-SMA gene expression [p<0.05]. There was more alpha-SMA gene expression in cells treated with leptin compared to cells treated with the HCV core protein
Conclusion: HCV infection is an impressive factor in the development of chronic hepatitis to hepatic fibrogenesis. The HCV core protein can induce a fibrogenesis process in HCV infection
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In this study, to clarify the SMAD4 blocking impact on fibrosis process, we investigated its down regulation by shRNA on activated human LX-2 cell, in vitro. Liver fibrosis is a critical consequence of chronic damage to the liver that can progress toward advanced diseases, liver cirrhosis and hepatocellular carcinoma [HCC]. Different SMAD proteins play as major mediators in the fibrogenesis activity of hepatic stellate cells through TGF-beta pathways, but the extent of SMAD4 as a co-SMAD protein remained less clear. vector expressing verified shRNA targeting human SMAD4 gene was transfected into LX-2 cells. The GFP expressing plasmid was transfected in the same manner as a control group while leptin treated cells were employed as positive controls. Subsequently, total RNA was extracted and real-time PCR was performed to measure the mRNA levels of SMAD4, COL-1A1, alpha-SMA, TGF-beta and TIMP-1. Furthermore, trypan blue exclusion was performed to test the effect of plasmid transfection and SMAD4 shutting-down on cellular viability. The results indicated that the expression of SMAD4was down-regulated following shRNA transfection into LX-2 cells [P<0.001]. The gene expression analysis of fibrotic genes in LX-2 cells showed that SMAD4 blocking by shRNA significantly reduced the expression level of fibrotic genes when compared to control plasmids [P<0.001]. Vector expressing SMAD4-shRNA induced no significant cytotoxic or proliferative effects on LX-2 cells as determined by viability assay [P<0.05]. The results of this study suggested that knockdown of SMAD4 expression in stellate cell can control the progression of fibrogenesis through TGF-beta pathway blocking
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A recent field of research in epigenetics is DNA methylation which involves the CpG island in the genome that subsequently controls transcription and translation of targeted genes. In the hepatitis B virus [HBV] genome, there are three CpG islands which tend to be methylated. The aim of the current study is to determine the methylation pattern of the HBV X gene in chronically infected HBV patients. Study participants comprised 45 chronically infected HBV patients. According to the presence of the HBeAg, patients were divided into two groups, HBeAg positive [n=24] and HBeAg negative [n=21]. Initially, viral DNA was treated with natrium bisulfate. Then, analysis was performed with two sets of methylated and non-methylated primers by the MSP method. The overall methylation rate in serum samples of hepatitis B infected patients was 35.5%; the rate in the HBeAg positive patients was 20.8%, whereas it was 52.3% in HBeAg negative patients. There was a significantly higher rate of methylation in serum samples of HBeAg negative patients compared to HBeAg positive patients [student's t-test; P=0.02]. Methylation of HBV can be used as a new mechanism to control the progression of viral infection. This methodology can be useful for determining the characteristics of clinical stages of this infection
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Identification of drug resistant mutations is important in the management of HIV-1 infected patients. The aim of the current study was to evaluate drug resistance profile of RT gene and assess subtypes among the HIV-1 circulating strains and intensification of physician's options for the best therapy. HIV-1 RNA of 25 samples was extracted from plasma and RT Nested- PCR was performed and the final products were sequenced and phylogenetically analyzed. Stanford HIV drug resistance sequence database was used for interpretation of the data. The results of phylogenetic analysis showed subtypes A1 and B in 14 [58%] and 10 [42%] patients respectively. Of the 24 patients, 16 [66.6%] had resistance to NRTIs, 8 individuals [32%] to NNRTIs and one patient was susceptible to NRTIs as well as NNRTIs. The drug resistance interpretation in this study showed: 87.7% susceptible for AZT, 70.8% susceptible, and 25% high-level resistance for 3TC, 87.7% susceptible for TDF, 29.1% high-level resistance for NVP and 70.8% susceptible and 25% high-level resistance for EFV. Our data suggests that probably, the use of 2 NRTIs plus 1 protease inhibitor [PI] regimen is more effective than 2 NRTIs plus 1 NNRTI regimen in Iranian patients that use 2 NRTI plus NNRTI regimen and also continuous surveillance should be perform to evaluate resistance patterns for more effective therapeutic approaches
Subject(s)
Humans , Male , Female , Drug Resistance , Reverse Transcriptase Polymerase Chain Reaction , Phylogeny , Reverse Transcriptase Inhibitors , Nucleosides , DNA, Complementary , RNA , Sequence Analysis, DNAABSTRACT
GBV-C is a virus transmissible via blood transfusion and sexual routes. Because of the shared transmission routes in GBV-C and hepatitis B virus [HBV] we have attempted to detect GBV-C in HBsAg-positive patients using the more sensitive semi-nested PCR. We used restriction fragment length polymorphism [RFLP] to determine genotype. A total of 100 serum samples were collected from HBsAg-positive patients from 1389-1390. After designing specific primers and optimizing the semi-nested PCR, sequences of PCR products were analyzed by Neb Cutter software. Restriction enzyme sites were determined and suitable enzymes selected for RFLP. Semi-nested PCR shows acceptable sensitivity for the detection of GBV-C and its genotype determination. This technique is more affordable than other techniques. There appears to be a high rate of GBV-C infection among Iranian HBV patients. In this study, the majority of patients co-infected with GBV-C were of HBV genotype 2, which is similar to the pattern seen in the US and Europe
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In this project, our aim was to construct a novel expressing vector harboring a new sequence from overlapping region of NS3 gene of HCV from infected Iranian patient. The partial NS3 [pNS3] gene was amplified by Nested-RT-PCR method using sera of HCV infected patients harboring genotype 1a. After purification and cloning the pNS3 into TA-cloning vector, the best colony was selected based on Blue/White screening and colony-PCR following by confirmation with sequencing and restriction digestion with BglII. The sequenced gene was compared with other reference sequences using alignment softwares. The resultant pNS3 gene subcloned into the expression vector, IRES vector, followed by selection the suitable clones by 2 different colony-PCRs. The gene expression was evaluated using GFP detection, RT-PCR and western blotting techniques after transfection of the IRES-pNS3 vector into the 293 cell line. After pNS3 sequence amplification by RT-PCR, sequencing results showed high homology among the sequences with other reference sequences. This result also showed that it belonged to genotype 1 of HCV. Colony-PCR showed the insertion of gene into expressing vector with the right orientation. GFP expression, RT-PCR and western blotting confirmed transfection of vector, expression of pNS3 gene and production of its protein in 293 cells respectively. This novel expressing vector harboring partial region of NS3 gene in compare to full NS3 gene maybe more useful in immune induction by antigen presenting cells due to absence of genes responsible for protease activity of the protein in the setting of HCV vaccine
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Hepatitis B virus [HBV] infection is an important health problem worldwide with critical outcomes. The nucleoside analog lamivudine [LMV] is a potent inhibitor of HBV polymerase and impedes HBV replication in patients with chronic hepatitis B. Treatment with LMV for long periods causes the appearance and reproduction of drug-resistant strains, rising to more than 40% after 2 years and to over 50% and 70% after 3 and 4 years, respectively. Artificial neural networks [ANNs] were used to make predictions with regard to resistance phenotypes using biochemical and biophysical features of the YMDD sequence. The study population comprised patients who were intended for surgery in various hospitals in Tehran-Iran. An ACRS-PCR method was performed to distinguish mutations in the YMDD motif of HBV polymerase. In the training and testing stages, these parameters were used to identify the most promising optimal network. The ideal values of RMSE and MAE are zero, and a value near zero indicates better performance. The selection was performed using statistical accuracy measures, such as root mean square error [RMSE], coefficient of determination [R2], and mean absolute error [MAE]. The main purpose of this paper was to develop a new method based on ANNs to simulate HBV drug resistance using the physiochemical properties of the YMDD motif and compare its results with multiple regression models. The results of the MLP in the training stage were 0.8834, 0.07, and 0.09 and 0.8465, 0.160.04 in the testing stage; for the total data, the values were 0.8549, 0.115, and 0.065, respectively. The MLP model predicts lamivudine resistance in HBV better than the MLR model. The ANN model can be used as an alternative method of predicting the outcome of HBV therapy. In a case study, the proposed model showed vigorous clusterization of predicted and observed drug responses. The current study was designed to develop an algorithm for predicting drug resistance using chemiophysical data with artificially created neural networks. To this end, an intelligent and multidisciplinary program should be developed on the basis of the information to be gained on the essentials of different applications by similar investigations. This program will help design expert neural network architectures for each application automatically
Subject(s)
Humans , Hepatitis B virus/drug effects , Drug Resistance , Drug Resistance, Viral , Neural Networks, Computer , Polymerase Chain ReactionABSTRACT
Human CMV is the most causative agent of a very common viral infection contacted by most adults that have no noticeable or with only mild uncharacteristic symptoms. However when a pregnant women is infected with CMV as a primary infection, there is a risk for transmission of virus to the fetus as well as reactivation of virus in rare case. HCMV antibodies were already described in spontaneous abortion and fetal abnormalities cases. Also antibodies against HCMV in fetal abnormalities as well as abortion had been reported by several studies in different part of the world. Due to lack of published data about CMV epidemiology in Ilam, the aim of current study was to determine the seroprevalence of specific viral IgM and IgG in spontaneous abortion cases as well as the age and socioeconomic status in the studied population in Ilam. Sera sample from 42 patients in abortion process as well as 30 healthy pregnant and 30 healthy women as negative control were collected and quantitative serological test to assess IgM/IgG against HCMV was performed using a commercial ELISA assay. SPSS software was used to analysis the results and demographic information. Among 42 patients in abortion process, IgG was found in 6 [14.28%] patients and IgM in 12 [28.58%] cases. Based on demographical information, it was showed that IgG seropositivity correlate with the increase of age, but there is no correlation between IgM and age of patients. The results showed that there is a high seroPrevalence of HCMV IgM than IgG among pregnant women in the process of abortion in Ilam; correlation between Age and IgG anti body seroPrevalence was same as other reported. Based on the current studies, it seems that more sensitive and specific method such as NAT method is needed for determination of CMV and abortion procces
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Polymerase chain reaction [PCR] is one of the most important progress in the field of molecular biology and diagnosis. Despite simplicity in concept, the reaction needs complex interaction between target sequence, primers, dNTPs and DNA polymerase for a successful amplification and diagnosis. For the detection of RNA viruses highly sensitive and specific technique is required. Hence amplification based on Nested PCR would improve sensitivity, and also use of one step reaction would decrease probable contaminations as reported previously in several studies. The aim of the current study was to develop a new, rapid and sensitive one step-one tube Nested PCR in a closed system, by using two novel coherent primers. In this study, a novel and special primer development method was used for one step-one tube reaction. After development and optimization, the assay was evaluated with known positive and negative controls. The developed assay was performed on 50 HCV positive samples and 10 negative controls and 5 samples from each HIV, HBV, TTV [Torque Teno Virus] and GBV-C [Hepatitis G Virus: HGV]. Based on the obtained results, sensitivity and specifity was calculated. 48 out of 50 HCV positive samples showed expected band while none of the negative controls gave any band. Based on the specific primer design system which has been used in the current study; the inner primer was synthesized as complementary of routine PCR primers and was bound to the outer primers. Therefore, there is no probability for false priming in the both rounds of the reaction, hence there would be no nonspecific amplifications. Other advantages of this assay system were prevention of contamination which was due to one step-one tube reaction, decrease in duration and the cost of the reaction
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Despite sensitive antibody based blood donor screening, infection can be transmitted during window period. Therefore sensitive methods based on nucleic acid test [NATs] have been considered. The aim of this project was to design a more sensitive method for detection of PCR products and diagnosis of HIV-1/HCV co- infection accurately.After designing specific primers and probes, the Multiplex RT-PCR method was optimized and the PCR products were labeled with Digoxigenin. The PCR product was denatured under alkaline condition and was hybridized with the specific probe that had a biotin at 5' end, and then was added to streptavidin coated wells. After washing an antibody against DIG, conjugated with alkaline phosphates enzyme was used, following second washing, the substrate [ABTS] solution was added to each reaction well. Development of green color shows the positive where as no color shows negative results. 35 samples were tested with the developed method including 27 positive samples, 8 confirmed negative and 4 standard panels. False negative or positive reactions were not observed. This method had acceptable sensitivity and specificity for detecting HCV and HIV-1 infections during window period, also the method can be quantified which can be used for the flow-up and treatment of patients. In addition to the very high sensitivity of the test, it is cost effective and takes less time to perform
Subject(s)
HIV-1 , Reverse Transcriptase Polymerase Chain Reaction/methods , Enzyme-Linked Immunosorbent Assay , HIV InfectionsABSTRACT
Objectives:A sensitive and accurate dot blot assay using recombinant p24 [gag], gp41 and gp120 [env] proteins of HIV-1 and also recombinant gp36, the specific HIV-2 antigen was developed to confirm the presence of antibodies in sera reactive in screening enzyme-linked immunosorbent assays.Methods:We collected sera from Iranian 125 confirmed HIV positive Iranian samples [seropositive group] from AIDS patients, asymptomatic HIV-infected subjects, HIV-infected intravenous drug users and also hemophilic infected subjects. The samples were obtained from the AIDS Specimen Bank, Pasture Institute, Iran during 2002 to 2003. We also obtained 180 samples [seronegative group] from healthy blood donors. Recombinant antigens were expressed in Escherichia coli. By use of highly purified antigens, the dot blot procedure was developed. Analysis of the results was accomplished by capturing the dot blot images.Results:We established and interpreted the results using Centers for Disease Control criteria. We defined the positive test result as the presence of antibody against at least 2 different HIV gene products, one of which had to be an env gene product while a negative test result was defined as no antibodies against any of the HIV gene products and an indeterminate result was defined as antibodies reacting with only one HIV env gene product or against gag gene product only.Conclusion:The recombinant HIV dot blotting assay identified seropositive individuals with a high degree of accuracy; none of the HIV-seropositive subjects had a negative test result. Reactivity with these antigens, demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. The different sets of Western blot interpretative accepted criteria did not make a difference in interpretation of the seronegative and seropositive samples
Subject(s)
Humans , HIV Antibodies/analysis , HIV Antibodies/immunology , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Evaluation Study , HIV-1 , HIV-2ABSTRACT
The Western blot [WB] assay is the most widely accepted confirmatory assay for the detection and confirmation of antibodies to human immunodeficiency virus type 1 [HIV-1] and 2 [HIV-2]. However, indeterminate WB reactivity to HIV-1 and HIV-2 proteins may occur in individuals who do not appear to be infected with HIV. In this study, we describe the results of indeterminate WB reactivity in Iranian patients with discordant screening assays. The samples were obtained from the Iranian Blood Transfusion Center, Tehran, Iran and evaluated in the Biotechnology Process Development Center, Pasteur Institute of Iran, Tehran, Iran between 2003 and 2004. A total of 4707 were tested for the presence of HIV-1 antibodies. Six hundred and four [12.8%] patients tested for HIV were positive for HIV-1 antibody. Nine [1.49%] have discordant results among screening assays and indeterminate WB results as interpreted by Centers for Disease Control and Prevention [CDC] criteria. Most [66.7%] of these indeterminate WB results were due to p24 reactivity. However, 2 [22.2%] display reactivity to both gp41 and gp120 proteins [Positive by World Health Organization [WHO] criteria]. Of 9 WB assays initially indeterminate by the CDC criteria and with follow-up samples, 8 [88.8%] became negative when retested subsequently while one [11.1%] remained indeterminate for more than a year and were thus considered negative. In addition, all the indeterminate samples were negative when assessed by polymerase chain reaction assay. In general, there was an 88.8% concordance between the CDC and WHO criteria for an indeterminate WB result. The CDC II criteria best met the specified objectives for diagnosis in our setting