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1.  E6-Specific Detection and Typing of Human Papillomaviruses in Oral Cavity Specimens from Iranian Patients 
Iranian Biomedical Journal  2017;21(6):411-416.
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.
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.
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.
The PCR-based assay, developed in the current study, could be used for HPV detection, quantification, and genotyping in epidemiological and clinical studies.
PMCID: PMC5572438
Real-time PCR; Genotyping; Iran
2.  Effect of TGF-β/smad signaling pathway blocking on expression profiles of miR-335, miR-150, miR-194, miR-27a, and miR-199a of hepatic stellate cells (HSCs)  
The aim of this study was to determine the effect of inhibition of TGF-β/smad signaling on the expression profiles of miR-335, miR-150, miR-194, miR-27a, miR-199a of hepatic stellate cells (HSCs).
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.
In this study, for inhibition of TGF-β 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.
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).
The results of this study suggest that blocking TGF-β smad-signaling can also differentially modulate microRNA expression in support of activation and fibrogenesis of HSCs.
PMCID: PMC5495898  PMID: 28702135
Fibrosis; microRNAs; HSCs; TGF-β; shRNA
3.  Epidemiology of Rotavirus-Norovirus Co-Infection and Determination of Norovirus Genogrouping among Children with Acute Gastroenteritis in Tehran, Iran 
Iranian Biomedical Journal  2016;20(5):280-286.
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.
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.
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.
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
PMCID: PMC5075141  PMID: 27137790
Gastroenteritis; Rotavirus; Norovirus; Coinfection; Epidemiology
4.  A Molecular Approach to Nested RT-PCR Using a New Set of Primers for the Detection of the Human Immunodeficiency Virus Protease Gene 
The human immunodeficiency virus (HIV-1) is the etiologic agent of AIDS. The disease can be transmitted via blood in the window period prior to the development of antibodies to the disease. Thus, an appropriate method for the detection of HIV-1 during this window period is very important.
This descriptive study proposes a sensitive, efficient, inexpensive, and easy method to detect HIV-1.
Patients and Methods
In this study 25 serum samples of patients under treatment and also 10 positive and 10 negative control samples were studied. Twenty-five blood samples were obtained from HIV-1-infected individuals who were receiving treatment at the acquired immune deficiency syndrome (AIDS) research center of Imam Khomeini hospital in Tehran. The identification of HIV-1-positive samples was done by using reverse transcription to produce copy deoxyribonucleic acid (cDNA) and then optimizing the nested polymerase chain reaction (PCR) method. Two pairs of primers were then designed specifically for the protease gene fragment of the nested real time-PCR (RT-PCR) samples. Electrophoresis was used to examine the PCR products. The results were analyzed using statistical tests, including Fisher’s exact test, and SPSS17 software.
The 325 bp band of the protease gene was observed in all the positive control samples and in none of the negative control samples. The proposed method correctly identified HIV-1 in 23 of the 25 samples.
These results suggest that, in comparison with viral cultures, antibody detection by enzyme linked immunosorbent assay (ELISAs), and conventional PCR methods, the proposed method has high sensitivity and specificity for the detection of HIV-1.
PMCID: PMC5035394  PMID: 27679699
HIV-1; Nested PCR; HIV Protease
5.  Construction of AAV-rat-IL4 and Evaluation of its Modulating Effect on Aβ (1-42)-Induced Proinflammatory Cytokines in Primary Microglia and the B92 Cell Line by Quantitative PCR Assay 
Interleukin-4 (IL-4), as the most prominent anti-inflammatory cytokine, plays an important role in modulating microglial activation and inflammatory responses in Alzheimer’s disease (AD), a chronic inflammatory disorder.
The current study aimed to develop a new recombinant Adeno-associated viral (rAAV) vector that delivers IL-4 and then assess the counterbalancing effect of the new construct along with recombinant IL-4 (rIL-4) protein in in-vitro models of AD.
Materials and Methods
The rAAV-IL4 was originally prepared and then employed along with rIL-4 protein to counter Amyloid β (1-42)-induced proinflammatory cytokines in a primary microglia cell culture and the B92 rat microglia continuous cell line, using relative Real-Time PCR assay.
Aβ (1-42) stimulated the production of the proinflammatory cytokines IL6, IL1β, TNFα, and IL18 in both the primary microglia cell culture and the B92 cell line. Both the rAAV-IL4 construct and the rIL-4 protein were found to inhibit production of the most important Aβ (1-42)-induced proinflammatory cytokine mRNAs in the two types of cells with different patterns.
It seems that the new construct can serve as an appropriate option in the modulation of Aβ-induced proinflammatory cytokine gene expression and microglia activation in patients affected by AD.
PMCID: PMC4870549  PMID: 27217922
Alzheimer Disease; Adeno-Associated Viral Vector; Amyloid β (1-42); Proinflammatory Cytokines; IL-4; Primary Microglia Cell; B-92 Cell Line
6.  Blocking of SMAD4 expression by shRNA effectively inhibits fibrogenesis of human hepatic stellate cells  
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-β pathways, but the extent of SMAD4 as a co-SMAD protein remained less clear.
Patients and methods:
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, α-SMA, TGF-β 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 intoLX-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-β pathway blocking.
PMCID: PMC4600516  PMID: 26468346
SMAD4; Liver fibrosis; shRNA; Hepatic stellate cell
7.  Prevalence of GBV-C among Iranian HBV positive patients using PCR-RFLP technique 
The aim of this study was to investigate the prevalence of GBV-C among Iranian HBV positive patients using PCR-RFLP technique.
GBV-C was a member of flaviviridae family and recently propose to classify as members of a fourth genus in this family, named Pegivirus and suggest that at least one quarter of the world's population has been infected with this virus. GBV-C can be transmitted via the blood-borne route, although vertical and sexual transmission is very well documented.
Patients and methods
100 serum samples were collected from HBsAg positive patients in 2011–2012. RNA was extracted with Qiagene mini kit. cDNA was synthesized by Reverse Transcriptase method and amplified by Semi- nested PCR method. After designing specific primers, the semi nested PCR was optimized, then sequences of PCR products were analyzed with software such as neb cutter, and sites of restriction enzymes were determined and suitable enzymes were selected for RFLP (Restriction Fragment Length Polymorphism).
PCR products were analyzed in 2% agarose gels containing ethidium bromide and were visualized with ultraviolet (UV) light. A 230 bp band was observed in comparison with 100 kb ladder; this band indicates our target gene of GBV-C genome have been isolate from serum samples.
It seems that Co-infection of GBV-C and HBV are common and This method had acceptable sensitivity for detecting GBV-C and determining its genotype, and more affordable than the other techniques; so the results of this study showed the prevalence of GBV-C were 12 serums of 100 serums HBsAg positive in goal population and one sample from 12 GBV-C positive serums was genotype 3 and the others were genotype 2.
PMCID: PMC4017541  PMID: 24834291
GBV-C; HBsAg positive; Semi nested –PCR; Genotype
8.  Polymorphisms within the Promoter Region of the Gamma Interferon (IFN-γ) Receptor1 Gene are Associated with the Susceptibility to Chronic HBV Infection in an Iranian Population 
Hepatitis Monthly  2012;12(11):e7283.
chronic hepatitis B virus (HBV) infection is a multifactorial disease that can result in serious clinical complications. Host genetic background especially the genes that encode immunologic factors like INF-γ and its receptor (IFN-γ R) are critical in the pathogenesis of infection.
The current study aimed to investigate the association between two single nucleotide polymorphisms (SNPs) at positions -611 and -56 within the promoter region of gamma interferon receptor1 gene (IFN-γ R1) and chronic HBV infection.
Materials and Methods
Genomic DNA from peripheral blood samples of 200 chronically HBV infected patients and 200 healthy blood donors, as controls, were collected and genomic DNA was extracted by phenol-chloroform method and DNA analysis genotype identification was performed by PCR-RFLP.
The results indicated that both SNP’s frequency had a significant difference in the patient and control groups. At position -56, TT genotype was associated with patient group and P value was 0.002 and at position -611, GG genotype was further observed in control group and P value was 0.006.
Presence of G allele at position -611 within promoter of IFN-γ R1 gene in the enrolled population for the study was related to lower risk of disease, and presence of T allele at position -56 was also related to susceptibility to chronic HBV infection. Men had higher frequency of chronic HBV infection, which might be the result of high risk behavior.
PMCID: PMC3539059  PMID: 23300496
Hepatitis B Virus; Single Nucleotide Polymorphism; Interferon Gamma Receptor; Polymorphism, Restriction Fragment Length
9.  Construction and Preparation of Three Recombinant Adenoviruses Expressing Truncated NS3 and Core Genes of Hepatitis C Virus for Vaccine Purposes 
Hepatitis Monthly  2012;12(8):e6130.
In spite of dozens of clinical trials to establish effective therapeutic and/or preventive vaccine to resolve HCV infection, no real vaccine has been proved to date. Genetic vaccines based on replication-defective adenoviruses have proved to elicit strong and long lasting T-cell responses against a number of viral antigens and are even currently being used for vaccine trials in humans. According to the controversy in the immune modulatory effects of both core and NS3 full length genes, it seemed more practical to employ some parts of these HCV proteins for vaccine design.
To generate recombinant Adenoviral vectors containing new overlapping-truncated region of NS3 gene or both the N- and C-terminal deleted parts of core gene, as well as a fusion fragment derived from both of them.
Materials and Methods
The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared. The integrity and sequence of the transfer vectors were confirmed, and followed by experiments involving homologous recombination between them and the adenovirus backbone plasmid in the bacterial host. Recombinant Ad-pNS3, Ad-pCore and Ad-pNS3pCore viruses were prepared by transfection of these new recombined constructs into 293 packaging cell lines. The virus titer was then calculated by an immunohistochemistry based method. The RT-PCR, Real-Time PCR and western blotting were used to evaluate gene expression by all recombinant constructs. The production of complete virion particles was evaluated by detailed electron microscopy in addition to the appearance of typical cytopathic effects (CPE) and GFP expression patterns in 293 cells. The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells.
RT-PCR, Real-Time PCR or western blotting confirmed expression of truncated fragment of NS3, core or a fusion fragment of theirs by newly constructed Ad-pNS3, Ad-pCore, Ad- pNS3pCore particles. Electron microscopy, which revealed many adenovirus-like particles and characteristics of CPE in infected cells in addition to GFP detection, confirmed the infectivity, potency and integrity of recombinant adenoviral particles.
These adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.
PMCID: PMC3475015  PMID: 23087750
Vaccines; Genetic Vector; Genes
10.  Phylogenetic Analysis of Torque Teno Virus in Hepatitis C Virus Infected Patients in Shiraz 
Hepatitis Monthly  2012;12(7):437-441.
Torque teno virus (TTV) was the first human Circoviridae detected in a Japanese patient with unknown hepatitis in 1997. Subsequently, several studies performed to evaluate different aspects of Torque teno virus pathogenesis.
The present study aimed to determine dominant genotype of Torque teno virus in chronic hepatitis disease using 5΄-UTR sequence among patients infected by hepatitis C virus in Shiraz – Iran.
Patients and Methods
The study conducted in 240 patients with chronic hepatitis C from Prof. Alborzi Clinical Microbiology Research Center. The presence of Torque teno virus DNA and its genotype in plasma was assessed by nested polymerase chain reaction using two primer sets for 5΄-UTR and N22 regions. Phylogenetic analysis was performed based on 5΄-UTR region.
DNA of Torque teno virus was detected in 220 out of 240 (92 %) patients with chronic hepatitis C by the use of 5΄-UTR primer based PCR method and in 12 out of 240 (5%) by the use of N22 primer. Based on phylogenetic analysis it was shown that the Dominant genotype in this study was 11. Genotypes 1, 3, 17, and 22 were also detected. Some sequences could not be classified to a specific genotype.
The prevalence of Torque teno virus DNA in patients with chronic hepatitis C disease by the use of 5΄-UTR primer appeared to be higher compared to that revealed by N22 primer. We observed five genotypes among hepatitis C chronic patients in our study.
PMCID: PMC3437454  PMID: 23008723
Hepatitis C; Prevalence; Shiraz
11.  Genotyping and Infection Rate of GBV-C among Iranian HCV- Infected Patients 
Hepatitis Monthly  2010;10(2):80-87.
Background and Aims
Hepatitis G virus/GB virus-C (HGV/GBV-C) is a newly identified member of the Flaviviridae family. Its clinical significance in chronic hepatitis C infection remains controversial. There is a geographical difference in the distribution of GBV-C in the world. The frequency of GBV-C infection among hepatitis C virus (HCV) infected patients varies. The aim of the current study was to determine the prevalence and genotypes of GBV-C among Iranian patients infected with chronic HCV.
Infection with GBV-C was surveyed in 71 chronic confirmed hepatitis C infected patients. These samples were collected at the Digestive Disease Research Center (DDRC) of Shariati Hospital, Tehran, Iran from January to October 2007. The 5’-UTR region of GBV-C RNA was detected using a novel in-house touchdown nested reverse transcription polymerase chain reaction (RT-PCR), the products were sequenced and the results were aligned and phylogenically analyzed.
Of the 71 HCV-infected patients, 31 (43.6%) were found positive for GBV-C RNA. Sequencing and phylogenic analysis showed that the samples were Genotype 2 of GBV-C.
It seems that there is a high rate of GBV-C infection among Iranian patients infected with chronic HCV. In comparison with the six reference genotypes, it was observed that all the samples were categorized in Genotype 2 of GBV-C, prevalent in North America, Africa and in European countries.
PMCID: PMC3270361  PMID: 22312378
GBV-C; HCV; Prevalence; Genotyping; Iranian; 5’-UTR
12.  An Accurate Confirmation of Human Immunodeficiency Virus Type 1 (HIV-1) and 2 (HIV-2) Infections with a Dot blot assay Using Recombinant p24, gp41, gp120 and gp36 Antigens 
An immunoblot assay using four recombinant proteins corresponding to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene products was developed to confirm the presence of antibodies to HIV-1 and 2 in sera reactive in screening ELISAs. Serum samples for testing were obtained from healthy seronegative blood donors and from different categories of HIV-infected individuals (asymptomatic HIV-infected, and AIDS). A positive reaction was defined as reactivity against gag (p24) and at least one other env (either gp41 or gp120) HIV gene products; negative result was defined as no reaction with any antigen; and indeterminate result was defined as reactivity with gag (p24) or with env (gp41 or gp120) alone. None of the 180 serum samples from healthy seronegative blood donors gave a positive result, and only 4 of these samples (2.2%) gave indeterminate results. The recombinant HIV Dot blotting assay identified seropositive individuals with a high degree of accuracy; none of the 125 HIV-seropositive subjects had a negative test result. Reactivity with these antigens, demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. All seronegative and seropositive samples were tested both with the commercially available ELISA and by Western blot. The recombinant in-house HIV Dot blot assay accurately identified more seropositive and seronegative samples and had fewer indeterminate results than did commercial Western blot (as interpreted by CDC criteria).
PMCID: PMC1074714  PMID: 15912198
Human Immunodeficiency Virus; Recombinant p24; gp41; gp120; gp36 Proteins; Dot Blot assay

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