E6-specific detection and typing of human papillomaviruses in oral cavity specimens from Iranian patients

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

Construction of an eGFP expression plasmid under control of T7 promoter and IRES sequence for assay of T7 RNA polymerase activity in mammalian cell lines

Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site [IRES] sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells. For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity. Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient. Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics

Design, construction and expression of recombinant vector containing the rabies virus nucleoprotein gene

Rabies is an acute encephalitis that causes more than 60,000 deaths worldwide. The only way to save individuals bitten by a rabies-infected animal is the timely use of effective vaccines. Treatment with new generation vaccines is expensive. Therefore, there is a global movement towards the production of less expensive vaccines which retain and improve upon the quality and effectiveness of the vaccine. Production and evaluation of non-classical vaccines is one of the approaches taken in this regard. In this study, we describe a new eukaryotic expression system to express the nucleoprotein N gene of rabies virus which, if suitable, may be evaluated for anti-rabies vaccine production. The complete sequence of the N gene of rabies virus PV subtype was amplified by real-time polymerase chain reaction and cloned into the pCDNA3.1[+] vector. The cloned gene was excised from the vector by restriction enzyme digestion and sequenced. Due to mutations detected in the N gene, the gene coding sequence was purchased as a recombinant pGH/N vector. Vector pGH/N was amplified and following enzymatic digestion, the excised N gene was once again cloned into vector pCDNA3.1[+]. Successful cloning was confirmed using restriction digests and quick check. The recombinant vector pCDNA3.1[+]/N was transformed into cultured BSR cells and protein N expression was analyzed using fluorescent antibody test [FAT]. Electrophoresis confirmed amplification of the nucleoprotein N gene and subsequent restriction enzyme digestion showed that the N gene had been successfully cloned into the recombinant pCDNA3.1[+]/N vector. However, DNA sequencing revealed the presence of mutations within the N gene. Restriction digest of the commercial pGH/N vector showed that the N gene had been excised from the vector. Successful cloning of the N gene into the pCDNA3.1[+] expression vector was confirmed using restriction digests and quick check. Protein expression in BSR cells was assayed by immunostaining with anti-ribonucleocapsid FITC-conjugated antibody and visually confirmed by fluorescence microscopy. This study showed that the protein N of rabies virus subtype PV can be expressed in a eukaryotic expression system using the pCDNA3.1[+] expression vector

[In vitro expansion of antigen-specific natural regulatory T cells in hepatitis C virus infection mediated by Core antigen]

Hepatitis C virus [HCV] is one of the most relevant persistent infections afflicting the human population. Control of viral replication in HCV infection has been associated with the cellular component of the host immune response. Several mechanisms have been proposed to explain this abnormal immune response, among them an altered activity of regulatory T cells [Tregs] being the most recently postulated. As the first report, in the present study the ability of HCV-core antigen in increase the frequency of natural Tregs [nTregs] in the mixed population of PBMCs was evaluated. Peripheral blood mononuclear cells [PBMCs] from chronic HCV infected patients [n=19] and normal controls [n=6] were analyzed to study the effect of HCV-core antigen in frequency of HCV specific nTregs. For this, PBMCs of different groups were isolated, cultured and stimulated with core antigen. Then an in-house triple-stain flowcytometric method was used to investigate the frequency of nTregs. The results showed that, following incubation with HCV-core Ag, a population of nTregs was increased but, in negative controls the number of nTregs did not increase. The present data supporting the idea that nTregs are able to respond specifically to HCV antigen suggests that Tregs could contribute to an inadequate response against the HCV infection, leading to chronic infection and supports the view that specific natural Tregs may be implicated in host immune tolerance during HCV infection. It is reasonable that HCV vaccine candidates avoid epitopes that lead to strong nTregs stimulation