Correlation between ebv co-infection and HPV16 genome integrity in Tunisian cervical cancer patients

Infection with high risk Human papillomavirus (HR-HPV) is necessary but not sufficient to cause cervical carcinoma. This study explored whether multiple HR-HPV or coinfection with Epstein-Barr virus (EBV) influence the integration status of HPV16 genome. The presence and typing of HPV in a series of 125 cervical specimens were assessed by polymerase chain reaction (PCR) using the specific primers for the HPV L1 region. As for EBV infection, the viral EBNA1 gene was used for its detection through PCR amplification. Disruption of the HPV E2 gene was assessed by amplification of the entire E2 gene with single set of primers, while E2 transcripts were evaluated by a reverse transcription PCR method (RT-PCR). The overall prevalence of HPVDNA was of 81.8% in cervical cancers versus 26.9% in benign lesions. In HPV positive cases, HPV16 and HPV18 were the most prevalent types, followed by HPV types 33, 31. EBV EBNA1 prevalence was statistically more frequent in cervical carcinomas than in benign lesions (29.5%, vs 9.6%; P=0.01). No viral infection was detected in healthy control women. The uninterrupted E2 gene was correlated with the presence of E2 transcripts originating from the HPV episomal forms. It was observed that integration was more common in HPV18 and EBV coinfection. The presence of EBV caused a five-fold [OR= 5; CI= 1.15-21.8; P = 0.04] increase in the risk of HPV16 genome integration in the host genome. This study indicates that EBV infection is acting as a cofactor for induction of cervical cancer by favoring HPVDNA integration.

Molecular characterization of Chikungunya virus during an outbreak in South India.

Introduction: Re-emergence of Chikungunya is a major public health problem in the southern states of India. Objectives: This study was undertaken to investigate an outbreak of Chikungunya, in June-August 2008 using PCR and determine the prevalent genotypes of Chikungunya virus (CHIKV) associated with the outbreak. Materials and Methods: Samples of blood were collected (in heparinized vacutainer tubes) from suspected patients of CHIKV infection from both Government Taluk Hospital in Kerala and a tertiary care hospital in Chennai, Tamil Nadu. A one-step RT-PCR was carried out on a block thermo-cycler targeting the E2 gene that codes for the viral envelope protein. The amplicons were verified for 305 bp size by standard agarose gel electrophoresis. The PCR products were purified, sequenced, and compared with other CHIKV strains reported from different geographical regions. A phylogenetic tree was constructed using MEGA 4. Results: Altogether 118 samples were collected from patients who presented with sudden onset of fever and/or joint pain, myalgia, and headache. CHIKV infection was confirmed by RT-PCR in 14 patients and all these cases were from Kerala. The positivity correlated with the early stage of the disease as all these patients had fever of less than seven days duration. The study isolates have been allotted the GenBank accession nos. GQ272368-GQ272381. Phylogenetic analysis of recent CHIKV isolates by partial sequencing of E2 region shows that isolates are closely related to strains from neighboring states and the African type. Conclusion: RT-PCR is a useful technique for the early detection of CHIKV infection during outbreaks. Molecular characterization of the strains indicates that majority of the strains have originated from the Central/East African strains of CHIKV.

Expressing of bovine viral diarrhea-mucosal disease virus Changchun184 strain of E2 gene in BCG / 中国兽医学报

E2 gene of BVDV Changchun 184 strain was cloned and inserted into the shuttle expression plasmid vector pMV261,the recombinant shuttle plasmid pMV261-E2 was constructed.Then pMV261-E2 was transformed into BCG successfully and obtained recombinant BCG which was resistive to kanamyein.The recombinant BCG were identifieated by PCR.E2 gene expression in recombinant BCG was induced in 45℃,then the SDS-PAGE and western blotting was used to analyze the expression product.The results indicated the BVDV E2 gene was expressd in BCG successfully.

Expression of Major Antigen Domains of Gene of E2 CSFV and Analysis of its Immunological Activity / 中国病毒学·英文版

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.

Secretory Expression of E2 Main Antigen Domain of CSFV C Strain and the Establishment of Indirect ELISA Assay / 中国病毒学·英文版

The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21-CodonPlus (DE3)-RIL using the pGEX-4T-1 expression vector and the soluble recombinant product was purified with Glutathione Sepharose TM<'4B> by centrifugation. The soluble recombinant protein showed good immune reactions and was confirmed by Western blot using anti-CSFV-specific antibodies. Then an indirect ELISA with the purified E2 protein as the coating antigen was established to detect antibody against CSFV. The result revealed that the optimal concentration of coated antigen was 0.6 μg/well and the optimal dilution of serum was 1:80. The positive cut-off value of this ELISA assay was OD<,tested serum>/OD<,negative serum>≥2.1- The E2-ELISA method was evaluated by comparison with the indirect hemagglutination test (IHAT). When a total of 100 field serum samples were tested the sensitivity and specificity were 90.3% and 94.7% respectively. Specificity analysis showed that there were no cross-reactions between BVD serum and the purified E2 protein in the E2-ELISA.

Generation of a Recombinant Baculovirus Expressing The E2 Protein of Classical Swine Fever Virus and Its Immunogenicity in a Mouse Model / 生物化学与生物物理进展

Baculovirus-mediated gene transfer into mammalian cells has been used to develop non-replicative vector vaccines against a number of diseases in several animal models.A baculovirus pseudotyped with the glycoprotein of vesicular stomatitis virus was used as vector to construct the recombinant baculovirus expressing classical swine fever virus(CSFV) E2 protein under the control of ie1 promoter from white spot syndrome virus.The E2 gene was shown to be efficiently expressed in both insect and mammalian cells.Intramuscular injection of mice with the recombinant baculovirus resulted in the production of high-level CSFV-specific antibodies.Specific lymphoproliferative responses to the CSFV stimulation were induced in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay.The results indicates that the pseudotyped baculovirus-delivered gene can be a potential non-replicative vaccine against CSFV infection.

Construction of recombinant plasmid PIRES2-HPV18-EGFP and its expression in HeLa cells / 第三军医大学学报

Objective To construct a recombinant plasmid for expressing the HPV18 E2 gene and develop some materials for HPV18 related disease.Methods The E2 gene of HPV18 was amplified by PCR from PBR322-HPV18 and cloned into PIRES2-EGFP.The sequence of cloned HPV18E2 was confirmed by restriction analysis and DNA sequencing,then transfected into HeLa cells.E2 gene was detected by RT-PCR.Results HPV18 E2 gene fragment was amplified by PCR and ligated to PIRES2-EGFP,then transfected into HeLa cells successfully.The expression of green fluorescence cells was observed by fluorescence microscopy.Specific band could be seen in transfected cells by RT-PCR.Conclusion Recombinant plasmid PIRES2-EGFP is effectively expressed after being transfected into HeLa cells in vitro.