5^th World Congress on Virology December 07-09, 2015 Atlanta, USA

Type A influenza viruses are unique among agents of infections in both humans and a variety of mammals (horses, swine, whales, seals, etc.) and birds. Due to the fact that the swine are susceptible to influenza A viruses of human and birds, they are considered intermediate host in which the genetic reassortment takes place between swine, human and avian viruses. Reassortment may lead to the emergence of new antigenic variants of influenza virus with the potential to cause epidemics in humans. However, the level of virulence and epidemiological activity of different virus variants seems far from being identical and depends on both the molecular biological features of the strains, and characteristics of their ecology. This determines the need for monitoring of circulating influenza viruses among swine in the key points located on the major migratory channels of birds. North and South regions of the Kazakhstan are historically established places for migration and aggregations of migrating birds. Collection of biological materials was carried out during the spring and autumn migration of birds, as well as in the epidemic season, when the probability of detecting swine influenza virus increases. 508 biosamples (337 nasopharyngeal swabs and 171 blood sera) have been collected from swine of the small and large pig farms of Kostanai, Karaganda, Aktobe and Almaty oblasts in 2014. Primary screening of 256 biosamples (nasopharyngeal swabs), carried out in RT-PCR using AmliSens PCR test system produced by the Institute for Epidemiology (Moscow), showed the presence of genetic material of the influenza virus in 86 samples (33.6% of the total number of examined samples). Influenza A/H1 virus RNA was detected in 35 samples (13.7%), A/H3 virus RNA - in five samples (1.95%). RNA of simultaneously both influenza virus subtypes (Нsw1+ Н3N2) was also detected in one sample (0.4%). These data indicate circulating influenza virus of mixed etiology with a predominance of influenza А/Нsw1 virus in the swine population. As a result of primary infection of chick embryos with 256 biosamples collected from swine in various regions of Kazakhstan, 12 GAA have been isolated with the hemagglutination titres of 1:2-1:16 and infective activity of 2.67-3.75 lgEID50/0.2ml. Identification in HAI and NAI assays of five isolates from Almaty (10/14), Karaganda (04/14 and 16/14) and Kostanai (12/14 and 24/14) oblasts enabled to attribute them to influenza A virus with the H1N1 antigenic formula. Serological analysis of 171 blood sera collected from healthy and sick swine in the pig farms of Karaganda oblast to detect antibodies against influenza A/H1N1 and A/H3N2 virus was carried out in IEA and HAI assay. The greatest number of specific antibodies both in IEA and HAI was revealed against the A/H1N1subtype of the influenza virus. Thereby, the findings confirm the circulation of influenza А/Н1N1 virus in the swine population on the territory of the Northern and Southern Kazakhstan. All this dictates the need for molecular epizootological monitoring of swines for the earliest detection of potential pandemic strain of influenza virus.

Tatiana Betakova has completed her Doctor of Sciences (D.Sc.) in 2015, PhD at the age of 29 years from Institute of Virology and postdoctoral studies from NIH, Bethesda, USA and NIMR, London, UK. She is principal investigator and scientific secretary of Institute of Virology, Slovak Academy of sciences. She has published more than 40 papers in reputed journals and has been serving as an editorial board member of repute

Non-structural protein 1 (NS1) is an important virulence factor encoded by influenza A virus and exerts its function through interacting with viral/cellular RNAs and proteins. NS1 interferes with the host innate immune response and promotes effective viral replication. In this study, we demonstrated the effect of shRNAs targeting the NS1 gene to treat an established infection of IAV. The level of mRNAs of NS1, IFN-α, IFN-β, IP-10, IFN-λ, and RIG-1 were compare in cells and in the lungs of infected mice. In cells treated with shRNAs was significantly reduced NS1 mRNA while the levels of RIG-1, IFN-α, IFN-β and IP-10 mRNAs were not altered. NS1 mRNA was significantly reduced in the lungs treated with shRNAs, and prolonged the survival of infected mice. IFN-α, IP-10 and IFN-λ mRNAs were radically increased on the third day after infection. On the other hand, levels of IFN- β mRNAs were reduced by half. Injection of transfection reagent insignificantly influenced the level of corresponding mRNAs. Application of shRNAs resulted in reduction of IFN-α and IP-10 mRNAs compared to positive controls. Levels of IFN-β mRNAs ranged from 36% to 74% and were lower than mRNAs in controls. IFN-λ mRNA was repeatedly increased in the lungs treated with some shRNAs. The level of RIG-1 mRNA decreased after infection with IAV. However, RIG-1 was strongly induced in infected mice injected with transfection reagent or control shRNA. Percentage changes of all tested shRNAs were lower and did not exceed the level reached in positive control.

Marcela Kudelova has completed her postdoctoral studies from Comenius University and ScD thesis from Slovak Academy of Sciences. She is the head of Department of Molecular Pathogenesis of Virology, Institute of Virology, Slovak Academy of Sciences, Slovak Republic. She has published more than 55 papers in reputed journals and has been serving as an editorial board member of repute.

The murine γ-herpesvirus 68 (MHV-68) known naturally infect rodents still actually provides a unique experimental model for dissecting important topics of human immunity to large DNA viruses that persist in B lymphocytes. Three scopes of interest related to MHV-68 are addressed by our laboratory and produce new knowledge about this virus. The first addresses the hypothesis that ticks, known to transmit multiple pathogens which can cause diseases in humans and animals, have a role in MHV-68 circulation in nature. The second is whether it is possible that MHV-68 might transform mouse cells in vitro. And the third addresses to the hypothesis that MHV-68 might induce the production of growth factor related compounds described for some herpesviruses. Recently we were the first to report the detection of MHV-68 in Dermacentor reticulatus ticks and of a live virus in their organs. Along with previous finding of MHV-68 in Ixodes ricinus ticks these results allow suggest MHV-68 as a potential arbovirus, the second one known as its own DNA genome. More, we prepared MHV-68 transformed mouse fibroblast cell line that acquired transformed phenotype after infection with UV irradiated MHV-68. In transformed cell line, viral antigen and DNA could be detected indicating that it might be oncogenic. In medium of these cells we identified compounds resembling growth factors which have displayed transforming and transformed phenotype suppressing activity in normal and tumor cells. Bivalent properties of compounds have been blocked entirely by antisera against MHV-68 and monoclonal antibodies against glycoprotein B suggesting their viral origin .

Dr. Ramon-Luing completed her PhD at the age of 27 years in the field of Engineering/biochemistry with focus in Biotechnology (Research Center and Advanced Studies, CINVESTAV). She did a postdoctoral trainning in the field of trichomonosis (CINVESTAV) and tuberculosis (Institute of Biomedical Research, UNAM). Since 2013 she has been a researcher in medical sciences at the National Institute of Respiratory Diseases; also she is member of the National Research System of Mexico. To date she has published 8 papers; currently she began in the study of HIV and respiratory diseases.

Immune reconstitution inflammatory syndrome (IRIS) refers to the paradoxical deterioration or unmasking of infections after antiretroviral therapy (ART). Although a high proportion of IRIS events are secondary to M. tuberculosis infection, a variety of other opportunistic infections have also been implicated. Our goal is to study highly specific molecules related to infection and systemic inflammation and to elucidate their potential predictive and diagnostic value as IRIS biomarkers. This is a prospective cohort study, currently, forty-eight HIV+ patients naïve to treatment have been enrolled. We evaluated markers of T cell activation (HLA DR, CD38 and Glut-1) and exhaustion (Tim-3 and PD-1) on CD4+ and CD8+ T cells, CD4 count and plasma viral load at baseline (pre-ART), 8 weeks after ART and around the IRIS events. Gene expression of Tim-3, Galectin-9, Glut-1, CXCL10 and Granulysin was analyzed by qPCR. Among the 48 HIV+ patients, 13 developed herpes associated-IRIS. At baseline HIV+ patients who lately developed IRIS showed an increased expression of Tim-3, PD-1 and Glut-1 receptors on T cells compared with those IRIS- (P=0.001). A highly activated phenotype (CD38, Glut-1, HLA-DR, Tim-3 and PD-1 expression) was also identified in HIV+IRIS+ patients. Early ART initiation resulted in a decline in CD38, PD-1, and Tim-3 expression on HIV+IRIS- patients at the 8 weeks (P=0.01). Tim-3 and Glut-1 mRNA relative expression levels were elevated after 8 weeks of ART and significantly more elevated during the IRIS events. Relative mRNA level of CXCL10, was reduced in HIV+IRIS- patients. Granulysin relative expression did not have significant changes among patients. Our data suggest that longitudinal measurements of T cell activation and exhaustion markers should be included alongside HIV-1 mRNA levels. This type of analysis is important for understanding the immune mechanisms underlying IRIS development, which must be explored in larger cohorts.

Man Xing has completed her bachelor’s degree from Jilin University in 2013 and she is studying for the Master degree in Institut Pasteur of Shanghai, Chinese Academy of Sciences now. She has published a paper in Emerging Microbes and Infections in 2014 and applied for a patent in 2015

Cetuximab is a mouse-human chimeric monoclonal antibody, approved for marketing to treat patients with advanced colorectal cancer and head and neck squamous cell carcinoma in several countries around the world. It works by blocking the epidermal growth factor receptor (EGFR) from receiving signals or interacting with other proteins to promote its normal function. The demand of Cetuximab is increasing in clinic to treat patients with certain cancers due to its specificity and sufficient effect, however, the complicated techonlogies and high cost limit its wide applications. Here, we generated an adenovirus-mediated Cetuximab for cancer gene therapy, results in rapid, high-level antidody expression after one single intramuscular injection while at a low cost. The full-length gene Cetuximab was cloned into two serotypes of adenoviral vector, respectively, termed AdC68-CTB and Hu5-CTB. Indirect fluorescence assay and binding ELISA were performed to verify binding specificity and affinity, data suggested that cetuximab expressed by both recombinant adenoviruses carries the same bioactive as commercial Erbitux. After infected with AdC68-CTB or Hu5-CTB, the viability of colorectal cancer cells NCI-H508 and DiFi were inhibited, and the cells displayed the phosphorylation status of EGFR and its downstream effectors mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) down-regulated. Our results further showed that a single administration of AdC68-CTB or Hu5-CTB induced sustained experssion of cetuximab at a high serum level, and tumor volume were dramatically reduced in NCI-H508 or DiFi-inoculated nude mice. In conclusion, adenovirus expressing full-length cetuximab could be a novel alternative method to effectively treat colorectal cancer.

Xinying Tang has completed her bachelor degree from Nanjing Normal University in 2012 and she is a Ph.D candidate in Institut Pasteur of Shanghai. She has published three papers in reputed journals, including Gene therapy, Journal of Virology and Emerging Microbes and infections

Influenza virus (IV) is a continuously evolving virus that widely spreads in humans and contributes to substantial morbidity and mortality. Re-emergence of human infection with avian influenza virus poses extra challenge in IV control. Artificial microRNA (amiRNA) mediated RNA interference has become a powerful antiviral approach due to its high specificity and rapid effect. Here we designed several amiRNAs targeting conserved regions among different clades of H5N1 hemagglutinin. Expression and delivery efficiency were enhanced by presenting functional amiRNA with chimpanzee adenoviruses serotype 68 (AdC68). One amiRNA, HA-1405, significantly limited H5N1 replication in vitro and the inhibition efficiency was up to 95% against clade2.3.2 H5N1. Treatment with AdC68 conjugated HA-1405, termed as AdC68(HA-1405), almost abolished viral structural protein expression as well as virus growth in A549 cell line. Moreover, prophylactic administration with AdC68(HA-1405) markedly alleviated clinical symptoms and reduced five to ten fold of lung viral titers against four clades of H5N1 in ICR mice. Our results further showed that AdC68(HA-1405) conferred 70% and 40% immediate protection against lethal clade2.3.2 and clade2.3.4 H5N1 challenge respectively. Taken together, these data provided information that HA-targeting amiRNA delivered by AdC68 could be pursued as a potential agent for highly pathogenic avian influenza viruses prevention and treatment.

Abdulsalam Ibrahim has studied Science Laboratory Technology and Biochemistry at Lagos State polytechnic and Ladoke Akintola University respectively with excellent grades in Nigeria. He moved on to the Arctic University of Norway, to study a Master’s degree program in Biomedicine and is presently a member of the Molecular Inflammation Research Group. His research focuses on signal transduction, inflammation and the novel Merkel cell polyomavirus. He co-authorized a publication “The Role of Merkel Cell Polyomavirus and Other Human Polyomaviruses in Emerging Hallmarks of Cancer” (Viruses 2015). Abdulsalam’s current study investigates the Reciprocity of Inflammation and Merkel Cell Polyomavirus in Tumorigenesis.

Merkel cell polyomavirus (MCPyV) is common in the human population with seropositivity >50%. Infection occurs in childhood and the virus is part of the normal human skin flora. Its genome is functionally divided into: (i) early region encoding the oncoproteins large T- and small t- antigens, 57 kT, and the ALTO protein, (ii) late region encoding the capsid proteins, and (iii) the non-coding control region (NCCR) encompassing the origin of replication and the promoters controlling the expression of the early and late genes. MCPyV is involved in the pathogenesis of Merkel cell carcinoma (MCC) as observed that approximately 80% of all examined MCC specimens are virus-positive. MCPyV-positive MCCs contain integrated viral genome and express truncated large T-antigen, whereas viral DNA is episomal and encodes full-length large T-antigen in other cell types. The NCCRs of most MCPyV isolates are 459-469 bp and differ by few point mutations. However, the NCCR of a strain isolated from healthy skin (strain 16b), feces (strain HB039C), and a Kaposi’s sarcoma sample (strain TKS) is ~20 bp longer due to a repeated sequence. The early and late promoters of isolate MCC350 (464 bp) and 16b were cloned in a luciferase reporter plasmid and their promoter activities were compared in HEK293 cells. Our preliminary results demonstrate that the early promoters are stronger than the late promoters and the early 16b is stronger than the early MCC350, while their late promoters have similar activity. The effects of full-length as well as truncated large T-antigen are being examined

Dr. Shirley Komninakis has a Ph.D. in Sciences from the School of Medicine, University of São Paulo (FMUSP) and postdoctoral by the Federal University of São Paulo (UNIFESP), Brazil. She is post-graduate advisor and associate professor at the UNIFESP and researcher on Retrovirology Laboratory. Its main research line involving molecular epidemiology studies of HIV and viral hepatitis, epigenetic study and transcriptome of HIV + patients genomes, as well as metagenomic studies. Currently, Dr. Komninakis is also senior researcher at the Laboratory of Clinical ImmunologySchool of Medicine of ABC, where will perform a project to analyze the exome of individuals with rare diseases.

With the advent of Highly Active Therapy (HAART) for the treatment of HIV-infected individuals was the emergence of resistant viral majority and minority populations selected by selective pressure. Despite the monitoring of resistance mutations ARVs, there is insufficient data related to transmitted resistance and polymorphisms caused by drugs used to treat rescue. The aim of this study was to evaluate through large-scale sequencing the occurrence of transmitted resistance mutations (TDRM). Of the 19 sequenced samples were evaluated 10 individuals with late diagnosis and four recent infection of HIV-1. Polymorphisms were found in all sequences evaluated for the protease gene (PR), reverse transcriptase (RT) and integrase (IN). The frequency of mutations can be seen in the chart below. Our results showed that in patients with recent infection (infected at least 180 days) there are viral populations HIV who carry mutations to all classes of drugs, particularly the NNRTIs, in contrast to those with late diagnosis.

Middle East Respiratory syndrome coronavirus (MERSCoV) is one of the major health concerns not only in Saudi Arabia but also worldwide. This is due to the high morbidity, and mortality among the affected people up to 60 %. Others and we has been recently reported the presence of neutralizing antibodies in sera of dromedary camels in different regions of Saudi Arabia. We also reported for the first time the complete genome sequencing of MERSCoV from camels and deposited these sequences in the gene bank. Viruses isolated from camels are quiet identical to that of human moreover, camel viruses are able to replicate in human cells of respiratory origins both in vitro and ex vivo model. However, the exact mechanism of transmission from camels to human still unidentified. There might be other options such as presence of unidentified intermediate host or insects that completing the circuit from dromedary camels to human. To test this hypothesis, we selected one farm previously showed the presence of the virus last year. Samples have been collected from different birds live close by this dromedary camel herd such as doves, sparrow as well as from mites, ticks, ticks, mosquitoes, flies. Swabs and sera were collected from the indicated birds as well as from camels. Total RNAs were collected from the indicated birds as well as from flies, mites, ticks and mosquitoes. Detection of the virus was done by the standard Real time PCR kits using the RDRP primers and probes. Detection of the viral antibodies was done by the Pseudovirus particle neutralization Assay. Our results showed that the only animal showing neutralizing antibodies to MERSCoV is dromedary camels whereas, none of the tested sera from other species showed any titre for MERS. Meanwhile, swabs from tested birds, flies and mosquitoes were negative as well. These results suggested that transmission of MERS is much more complicated than expected. We suggest the continuous search for the intermediate host of MERs among other species of animals and birds or even rodents and bats. Further studies are currently in progress to explore the potential roles of camels in the transmission of MERS as well as the possibility of other intermediate hosts if any.

Talita Cristina Dellariva completed her degree in Biological Sciences from State University of Campinas (UNICAMP) in 2013 and nowadays she has been developing her master's degree in Clinical Medicine in the Faculty of Medical Sciences, State University of Campinas, São Paulo, Brazil.

Human Herpesviruses (HHVs) are an important cause of infections in transplantation and immunocompromised patients in general. This group of virus consists in three subfamilies: α-herpesvirinae, β–herpesvirinae and γ–herpesvirinae which are included the eight Human Herpesviruses species. Currently, the diagnosis of infectious diseases have been a considerable advance after molecular techniques implantation, especially the Polymerase Chain Reation (PCR), once it presents high sensitivity and specificity to detect low quantities of nucleic acids. The use of consensus-degenerate primers in PCR has been used as an efficient alternative for the detection and identification of Herpesviridae family members in clinical samples. The aim of this study was to improve the Nested-PCR (N-PCR) technique using consensus-degenerate primers to detect human herpesvirus subfamilies. Forty plasma-DNA samples from HSCT patients were analyzed using N-PCR technique with degenerate primers in both reactions. These primers were designed from highly conserved amino acid sequences of herpesviral DNA polymerase gene. The results show that 8/40 (20%) were positive to α-herpesvirinae and/or γ–herpesvirinae; 19/40 (47.5%) were positive to β–herpesvirinae and 5/40 (12.5%) presented coinfection with the three subfamilies. The development and improvement of N-PCR using consensus-degenerate primers have been fundamentally important in helping the clinical diagnosis, therapy, classification and epidemiological studies of viruses and to discover new herpesvirus species.

Marcin Skoreński is PhD student at Wroclaw University of Technology, Faculty of Chemistry, Division of Medicinal Chemistry and Microbiology. His research is focused on the development of low molecular weight compounds designed to target proteases involved in the pathogenesis of v viral infections (hepatitis C virus (HCV)n West Nile virus (WNV), herpes viruses, influenza). He is a co-author of 5papers, 14 patents and more than 10 pending patent applications.

The HAT protease also known as TMPRSS11D or airway trypsin-like protease is a member of the serine protease family. In humans HAT is responsible for several biologically important processes including modulation of urokinase-type plasminogen activator receptor and fibrinogen processing. Moreover elevated level of HAT has been reported for patients suffering from chronic skin diseases (such as psoriasis) and respiratory tract dysfunction. Additionaly HAT was found to cleave the surface glycoprotein hemagglutinin of the influenza virus. Without this proces virus particles can not enter the host cel and start replication procesc, thus enzyme responsible for this cleavage are consider as promising drug target for influenza treatment. The main objective of our research was the design and synthesis of selective, irreversible, active site-directed inhibitors of HAT protease. The target compounds belong to the group of phosphonic analogues of amino acids containing various, structurally diverse ester groups. These compounds are known as highly specific and irreversible inhibitors of serine proteases and have been successfully used as inhibitors of many physiologically important serine proteases such as thrombin, human neutrophil elastase, cathepsin G, proteinase 3, and dipeptidyl peptidase IV. Since the complete biological and physiological functions of HAT protease are yet to be discovered, the development of potent and specific inhibitors might provide very useful tools to study the HAT. Some recent reports consider HAT protease as a promising therapeutic target which inactivation could potentially limit the influenza virus infection hence the development of specific inhibitors might lead to a new class of antiviral agents.

Glen R. Gallagher completed undergraduate degrees in Microbiology and Clinical Laboratory Science at The Ohio State University. After graduation he extended his training into the Public Health sector as an Emerging Infectious Disease fellow through the Association of Public Health Laboratories at the Centers of Disease control Dengue branch lab in San Juan, Puerto Rico. Currently he is a graduate student at The University of Massachusetts Medical School under the guidance of Dr. Robert Finberg and Dr. Jennifer Wang. Broadly he is interested in viral replication and viral pathogenesis with current projects on influenza replication and evolution as well as the contributions of coxsackievirus on the development of type 1 diabetes.

Viral replication is dependent on entry into host cells, and is often mediated by membrane-associated proteins. These interactions can take place on the cell surface or in endosomes to mediate fusion. We sought to identify membrane-associated proteins that are potentially important for the replication of multiple viruses as these proteins could be targets for broad-spectrum viral replication suppression. Based on a previous genome wide siRNA screen for proteins involved in influenza replication, we identified membrane-associated protein targets and performed a focused screen to identify proteins involved in replication of other viruses. The screen included 33 siRNA targets, which were knocked down in A549 human lung adenocarcinoma cells and in U2OS human osteosarcoma cells. Following knockdown, cells were infected with various GFP expressing viruses including influenza and vesicular stomatitis virus. Genes identified in this screen that inhibit viral replication could potentially provide insights into mechanisms of attachment or entry utilized by multiple virus families.