8^th World Congress on Virology November 28-29, 2016 San Antonio,USA

To investigate the construction and expression of rBCG combining human granulocytemacrophage colony stimulating factor(GM-CSF) and EB virus (EBV) encoding immediate early gene(BZLF1).Then the antitumor activity and immunological mechanisms of rBCG including fusion genes GM-CSF and BZLF1 were researched. Methods The purified GM-CSF and BZLF1 were amplified by PCR and inserted into plasmid pMV261, then transformed them intoEscherichia coli DH5α(E.coli . DH5α). In LB culture medium containing kanamycin, the positive colony was selected, the correct sequence of the GM- CSF and BZLF1 were connected by the polypeptide (Gly4Ser)3 series with the splicing overlap extension technology. The fusion gene GCBF was constructed and cloned into pMV261, transformed competent bacteria and selected on LB culture medium flat containing kanamycin. Plasmid pMVGCBF extracted from positive clones were transformed into competent BCG. Western- blot was employed for determination of expression of GCBF. C57BL/6 mice were used to build animal models of EB virus-positive tumors. Antitumor activity was analyzed by the formation time of tumors, survival time and tumor weight. The specific antibodies of mice stimulated with rBCG was detected by ELISA, lactate dehydrogenase assay was used to detect the mouse cellular immunity, HE staining of tumor tissue to assay lymphocyte infiltration in tumor of mice,so the rBCG immune effect was researched. At last,we used statistical methods to analized the immunization of rBCG.Results The RT-PCR product sizes of objective gene GM-CSF and BZLF1 were 461bp and 788bp, consistented with expected values. The recombinant plasmid was confirmed by restriction double enzymes，amplification and sequencing ,then the fusion gene (1209bp ) was correctly inserted into the vector.pMVGCBF were correctly transformed into competent BCG.The expression of fusion protein GCBF was detected in rBCG.Tumor formation time was delayed in mice immunized by rBCG.Tumor growth was slow and survived time of mice was prolonged.The mice immunized by rBCG could produce specific IgG antibodies of GM-CSF and BZLF1.At the same time,specific CTL activity was detected in mice and tumor tissue infiltration of lymphocytes was found by microscopy. Conclusion rBCG encoding GCBF fusion gene was successfully constructed to have provided basis for further study of the function of rBCG.The mice immunized by rBCG could produce humoral and cellular immune responses.The mice produced a strong antitumor effect. The EB virus-positive tumors was significantly inhibited in mice.

Yepin Yu has completed his Bachelor degree from Xiamen University and now doctorates in Marine Biology in South China Sea Institute of Oceanology, Chinese Academy of Sciences. He is a Ph D. student of Prof. Qin, the finder of Singapore Grouper Iridovirus (SGIV). Yepin’s academic interests include viral immune evasion and virus-host reation. He has published 3 papers in reputed journalspi

It has been demonstrated that tumour necrosis factor receptor (TNFR) homologues encoded by viruses are usually involved in virus immune evasion by regulating the host immune response or mediating apoptotic cell death. Here, a novel TNFR-like protein encoded by Singapore grouper iridovirus (SGIV VP51) was cloned and characterized. Amino acid analysis showed that VP51 contained three cysteine-rich domains (CRDs) and a transmembrane domain at its C terminus. The expression of VP51 in vitro enhanced cell proliferation, and affected cell cycle progression via altering the G1/S transition. Furthermore, VP51 overexpression improved cell viability during SGIV infection via inhibiting virus-induced apoptosis, evidenced by the reduction of apoptotic bodies and the decrease of caspase-3 activation. In addition, overexpression of VP51 increased viral titre and the expression of viral structural protein gene MCP and cell proliferation promoting gene ICP-18. In contrast, the expression of the viral apoptosis inducing gene, LITAF, was significantly decreased. Although all three CRDs were essential for the action of VP51, CRD2 and CRD3 exerted more crucial roles on virus-induced apoptosis, viral gene transcription and virus production, while CRD1 was more crucial for cell proliferation. Together, SGIV TNFR-like products not only affected cell cycle progression and enhanced cell growth by increasing the expression of the virus encoded cell proliferation gene, but also inhibited virus-induced apoptotic cell death by decreasing the expression of the viral apoptosis inducing gene. Our results provided new insights into understanding the underlying mechanism by which iridovirus regulated the apoptotic pathway to complete its life cycle.

Lingli Zhou is a Ph.D. Student from University of Chinese Academy of Sciences with particular direction in marine virology. She was interested in antiviral research of virology, and aptamer selection using SELEX. She holds a master’s degree in China University of Geosciences in biochemistry and molecular biology

Generation and characterization of novel DNA aptamers against coat protein of grouper nervous necrosis virus (GNNV): Nervous necrosis virus (NNV) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. However, there is no efficient method to control NNV infections. Aptamers generated by selective evolution of ligands by exponential enrichment (SELEX) are short, single-stranded nucleic acid oligomers. They display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. In this study, three novel DNA aptamers (A5, A10, and B11) that specifically target the coat protein (CP) of grouper nervous necrosis virus (GNNV) were selected using SELEX. Secondary structures and minimum free energy (ΔG) predictions indicated that these aptamers could form stable, secondary stem-loop structures. Electrophoretic mobility shift assays, enzyme-linked immunosorbent assays, Kd measurements, the co-localization of tetramethylrhodamine (TAMRA) labeled-aptamers with the CP and flow cytometry analysis revealed that these aptamers could specifically bind the CP with high (nanomolar) affinities. Moreover, all three aptamers did not show any cytotoxic effects in vitro or in vivo, and anti-viral analysis indicated the selected aptamers could inhibit NNV infection in vitro and in vivo. TAMRA-labeled aptamers could bind to NNV virions and directly enter NNV-infected cells, suggesting they could be used as tracers to study the mechanism of viral infection, as well as for targeted therapy. This is the first time that aptamers targeting a viral protein of marine fish have been generated and characterized. These aptamers hold promise as diagnostic, therapeutic, and targeted drug delivery agents for controlling NNV infections

Vegetarian diet and their effect on human beings have been studied world wide.There are evidences that vegetarians have lower rates of coronary heart diseases because of low LDL cholesterol , lower prevalence of obesity, lower rates of hypertension and diabetes mellitus. Cancer rates of vegetarians are lower and life expectancy are greater.The risk of colorectal cancer is lower in vegetarians .There are different categories Of vegetarians . Vegans who eat no animal products Lactoovovegetarians who eat no meat but eggs and dairy foods or bothPescovegetarians who eat fish, but other meats less than 1 time Semivegetarians who eat meat aside from fish occasionally but less than weekly non vegetarians who eat meats aside from fish more than 1 time per week.Since Rajasthan tops the list of states with highest vegetarians ( more than 98% population )in India. While Telangana has highest proportions of meat eaters in India.And the persons fall in Lactovovegetarian group.Vegetarian diet includes wheat , rice, vegetables, buckwheat, Amaranthus and fruits.Buckwheat (Fagopyrum esculentum ) ( family Polygonaceae ) is healthiest food and is rich in essential amino acids, Magnese, Magnesium, Cupper, Fiber, Phosphorus, and Protein.Vegetarians have low rates of viral diseases. Vegetarians have less HPV( human Papiloma Virus).Nutrition facts Volume 13 July 10th 2013 Michael Greager M.D.

Cheol-Hee Yoon completed his PhD at the age of 34 years from Sungkyunkwan University in 2008, and studied from Sothern California University School of Medicine as postdoctoral fellow. He is studying in a field of chronic viral disease as scientific researcher of Division of AIDS, Korea National Institute of Health. He has published more than 25 papers in peer reviewed journals

Despite the successful inhibition of human immunodeficiency virus type 1 (HIV-1) replication by combination antiretroviral therapy, cells latently infected with HIV-1 remaining in patients are a major obstacle for eradication of HIV-1 infection. The tumor suppressor factor p53 is activated by HIV-1 infection, and inhibits HIV-1 replication. However, a therapeutic strategy based on p53 activity has not been considered for elimination of latently infected cells. Herein, p53-linked apoptosis of cells latently infected with HIV-1 and uninfected cells was compared. Upon treatment with 5-fluorouracil (5-FU), apoptosis was increased in latently infected ACH2 cells encoding competent p53 compared with uninfected parent A3.01 cells, while the apoptosis of latently infected p53 null J1.1 cells was less than that of uninfected cells in analysis with flow cytometry and Western blotting using antibodies for cleaved caspase-3 and PARP. The levels of expression and activation of p53 were higher in both latently infected ACH2 and NCHA2 cells than in uninfected cells. Furthermore, the levels of p53 in both cells were further increased upon 5-FU treatment. Consistent with p53 status, apoptosis of ACH2 and NCHA2 cells were markedly increased compared with uninfected and J1.1 cells upon treatment with other anticancer drugs such as doxorubicin and etoposide. Knockdown of p53 in cells with latent HIV-1 infection diminished apoptosis upon 5-FU treatment. Our results indicate that when treated with anticancer drugs, apoptosis of cells with latent HIV-1 infection was increased via the p53 activation pathway and may provide information for application of anticancer drugs to selectively eliminate HIV-1 reservoirs

Dr Sherif El-Kafrawy has completed his PhD in 1999 from Menoufiya University, Egypt. He was appointed as an Assistant Professor of molecular biology at the National Liver Institute, and is now working as an Assistant Professor of molecular virology in King Abdulaziz University. He has published more than 25 papers in reputed journals

Hepatitis B virus (HBV) is a member of the family Hepadnaviridae and is classified into ten genotypes (A-J) and 34 sub-genotypes. These genotypes differ in geographic distribution, HBeAg seroconversion rate, clinical outcome, prognosis, and response to antiviral treatment. HBV is transmitted through percutaneous or parenteral routes and is affecting human health allover the world. The prognosis of chronic HBV (CHB) infection includes hepatic failure, cirrhosis, and hepatocellular carcinoma (HCC). In the last two decades several studies have been published concerning the epidemiology and prevalence of HBV in Saudi Arabia. However, studies exploring the evolutionary aspect and molecular variations in in HBV are very limited. The introduction of mandatory vaccination have reduced the prevalence in young Saudis, <25 years old, but the prevalence of in individuals more than 25 years is about 4%. Expatriates working in Saudi Arabia, 33% of the population, contribute to the burden of HBV infection in the country. Genotype D is the most prevalent HBV genotype in Saudi Arabia. The molecular characterization of HBV in Saudi Arabia is an urgent need to develop appropriate diagnosis and treatment strategies. The presence of drug resistant mutations in treatment naïve patients have been documented and is shown to be one of the factors affecting the response to antiviral treatment. The best reported approach to investigate the prevalence of these mutations is deep sequencing using next generation sequencing techniques. To approach this point of research, we performed deep sequencing of the virus from chronically infected HBV patients from Saudi Arabia. The results of the Next generation sequencing and the rare mutations found in the samples of the recruited subjects as well as their clinical data will be presented in the presentation.

Kyung-Chang Kim has completed his PhD at the age of 39 years from Korea University and postdoctoral studies from Northwestern University. He works as researcher in the Division of AIDS, Korea National Institute of Health. He has published several papers in peer reviewed journals and has been serving as a board member of the Korean Society for AIDS since 2016

We found 70 candidate genes in association with the establishment and maintenance of HIV-1 latency through integrative analysis with DNA methylation, histone modification and expression profile in HIV-1 latently infected cell lines. To select more effective genes on HIV-1 latency among 70 genes, we performed the functional enrichment analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The p-value < 0.1 was selected as cut off criterion. The significantly enriched Gene Ontology (GO) categories were identified as cell cycle, cell death, and signal transduction. 24 genes from the significantly enriched categories included 13 up-regulated and 11 down-regulated genes. To identify the expression level of 24 genes between normal cells (A3.01, Jurkat) and HIV-1 latently infected cells (ACH2, NCHA1, NCHA2, NCHA3, J1.1), we analyzed microarray data and found that gene expression level of 24 genes was very different according to cell lines. Among them, 10 genes showed similar patterns in more than three HIV latently infected cells and were selected as candidate genes: TNFSF13B, APBB2, ANK1, MSRA, AVEN, NQO1, FOXN4, RASAL1, JAG2, and PLOD1. To evaluate how the expression level of 10 genes are in vivo, we measured the quantitative RNA expression in HIV-1 infected patients using Real time-PCR. TNFSF13B and APBB2 were highly expressed as shown in HIV-1 latently infected cell lines. Therefore, these results suggest that selected genes may be potential biomarkers and play a key role in HIV-1 latency