Global summit on
August 27-28, 2015, Dubai, UAE

Scientific Programme(Day 2 : Aug-28-2015)

Virology Others

Session Introduction

Gheyath K. Nasrallah
Qatar University college of Arts and Scinces, Qatar
Title: Seroprevalence of anti-HEV antibodies among blood donors in Qatar and evaluation of performance of four enzyme immunoassay kits for detection of these antibodies.
Nasrallah has completed his Ph.D four years ago from Dalhousie Univesity, and postdoctoral studies from Jason’s Zebrafish Lab at The IWK Health and Recerch Center, Halifax, Canada. Currently, he is serving as an Asssitant professor of immunology & Microbiology and Head of Conference and Research Committee at the Department of Health Sciences in Qatar University. In addition to his interest in studying Legionella pneumophila pathogeneis, part of Dr. Nasrallah’s Lab studies the molecular and seroprevelance of blood-borne viruses among blood donors in Qatar. He recently has published more 16 papers in reputed journals.

HEV is the etiologic agent of acute hepatitis E. Although fecal-oral is the known route of transmission, recently, a transmission through blood transfusion was documented. Thus, it remains questionable whether there is a need for HEV screening prior transfusion. Studies carried out on HEV infection in the Middle East are scarce. Our goals are (i) to investigate the seroprevalence of HEV among blood donors in Qatar and (ii) to evaluate performance of some commercially available anti-HEV IgG ELISA kits (Wantai Biological Pharmacy, China; Diagnostic Automation, USA; and Euroimmun; Germany) and an immunoblot kit (Mikrogen Diagnostik; Germany). Out of total 2536 (2436 males, 100 females) analyzed samples, 515 (20.3%) and 11 (0.43%) blood samples tested positive for anti-HEV IgG and IgM, respectively. There was a significant association between anti-HEV IgG seropositive and nationality (p= 0.00001). For kit evaluations, only the first 926 specimens were tested by the above four assays. 177/926 samples tested positive for anti-HEV IgG by one or more assays. However, only 77 (77/177; 43.5%) samples tested positive by all assays, suggesting a fairly high degree of variation in performance between these kits. The accordance of each kit with the rest of the kits was as follows: Diagnostic Automation (169/177; 95.5%), Wantai (166/177; 93.8%), Mikrogen (164/177; 92.7%), and Euroimmun (78/177; 44.1%). This is the first study of its kind showing a high seroprevalence of anti-HEV among blood donors in Qatar. Because of test results discrepancies of the serology kits, a confirmatory test for detection of HEV RNA is warranted.

Aboul-Ata E. Aboul-ata
Plant Pathology Research Institute, Egypt
Title: Gene Expression and Gene Suppression Protocols for Human and Plant Virus-Infection Control in Specific Reference to HSV-2 and PVX-Eg2
Prof. Aboul-Ata is ArSV president (Arab Society for Virology). He is Editor and reviewer for different Global scientific journal. He is P.I. and consultant for some MERC projects

Gene expression protocols have been started nine years ago. More simplification was manipulated in vaccine development for HSV-2 subunits i.e. HSV-2gD and HSV-2VP16 via Nicotiana benthamiana. Three chimeric viruses i.e. TMV CP/HSV-2 gD/TMGMV CP SP, TMV CP/HSV-2 VP16/TMGMV CP SP and TMV CP/GFP/TMGMV CP SP were engineered and transfected into tobacco seedlings. The GFP construct was done to confirm positive insertion. Expressed protein was confirmed by bioassay, DAS-ELISA using MAb of HSV-2 gD, MAb of HSV-2 VP16 and PAb of TMV, RT-PCR and Western Blot Hybridization. Mice were immunized by expressed protein. Gene expression protocols were modified and simplified gradually in developing countries to increase vaccine novelty. Gene suppression protocols were manipulated lately to control PVX-Eg2 strain in potatoes. RNA constructs of Sense (PVX-Eg2cpVs), antisense (PVX-Eg2cpCs) were designed, cloned and sub- cloned for gene transfection using Agrobacterium inoculation technique. Seedlings of potato (Solanum tuberosum L. cv. Spunta) and tobacco (Nicotiana benthamiana) were inoculated with the three previous constructs. Construct-treated plants were mechanically inoculated with the PVX-Eg2 isolate. Bioassay and PCR amplification have been able to evaluate transfected-plant resistance against PVX-Eg2 that is caused by siRNA of PVX-Eg2cp. Using Bioassay and PCR analysis, we were able to detect PVX viral genome in all challenged plants those infiltrated with either pFGC5491 vector. Sense/antisense-transfected plants were resistant to PVX-Eg2. Sense construct-treated plants and those treated with antisense construct have no positive reaction with both tests.

Madhu Khanna
University of Delhi, India
Title: Efficacy of Influenza Vaccination in Elderly Population
Madhu Khanna is currently working as an Associate Professor in the Department of Virology at University of Delhi, India

Background: Vaccination has been regarded as the primary and most effective method for prevention of influenza virus infection in the elderly. The individuals aged 60 years or above are at greater risk of acquiring serious infections and the related complications. However, there are reports indicating lower influenza vaccine effectiveness in elderly individuals as compared to the young adults. The age-related deterioration of immune system in the elderly (immuno-senescence) leads to lower antibody responses. This makes them more vulnerable towards influenza virus infection even after vaccination. Therefore, assessment of vaccine efficacy becomes necessary to manage the viral infections in the elderly individuals.
Methodology: In the present study, 76 individuals, aged 60 years or older, were enrolled with informed consent from the pulmonary Out-Patient-Department (OPD) of AIIMS and Viswanathan Chest Hospital, VPCI, Delhi, India for intramuscular injection of inactivated trivalent split virion influenza vaccine, VAXIGRIP (Sanofi Pasteur SA, Lyon, France). The study was carried out from January to June, 2014 and the enrolled individuals had no history of influenza vaccination for atleast a year before from the date of vaccination. The existing antibody titers against the respective antigens of the vaccine strains (A/California/7/2009 (H1N1)pdm09, A/Texas/50/2012 (H3N2), B/Massachusetts/2/2012) were assessed in the pre-vaccination serum samples by hemagglutination inhibition (HAI) assay. The post-vaccination serum samples of 31 individuals were collected after 21 days, 3 months and 6 months of vaccination and analyzed by HAI.
Results: Of the 76 individuals enrolled, 31 came for follow-up analysis. The study observed participation of more male individuals (74.19%) than the females (25.8%). The mean age of the 31 participants, whose blood samples were collected both pre- and post-vaccination, was about 66 years. There was no evidence of fever, pain/tenderness/redness at the site of injection, or other systemic symptoms, such as, headache, malaise, chills, muscle aches, or any other adverse events post-vaccination. The pre-vaccination antibody titer and post-vaccination sero-protection were higher for the influenza A virus strains (A/H3N2 ˃ A/H1N1) in comparison to the influenza B virus vaccine strain. The post-vaccination sero-conversion rate was observed to be higher for the A/California/7/2009 (H1N1)pdm09 strain. Both sero-protection and sero-conversion rates for all the three strains remained almost consistent till 3 months but dropped significantly after 6 months from the day of vaccination.
Conclusions: Inspite of proven effectiveness of influenza vaccines, the elderly tend to have lower protective antibody levels. In our study, the participants reported no local or adverse effects after administration of VAXIGRIP. The relatively higher pre-vaccination antibody levels and post-vaccination sero-protection against the A/H3N2 may be due to continuous circulation and thus, exposure towards the viral strain. The initial higher sero-conversion rate for A/California/7/2009 (H1N1)pdm09 strain may be due to the presence of some pre-existing cross-reactive antibodies. The lower immunogenicity of the influenza B strain may be attributed to its low incidence in the population during the study period. Our study demonstrates a protective immune response by the vaccine analyzed in the study (VAXIGRIP) and thus, advisable for use in the elderly for better prevention and control of the viral infection.

Sabah Bidawid
Food Directorate, Health Canada, Canada
Title: Lab-on-A-Chip microfluidics-based technology for the rapid detection of foodborne viruses
Sabah Bidawid has completed his Ph.D. from The University of Ottawa, Canada. Following more than 25 years conducting research in food virology, Dr. Bidawid is currently the Chief of the Microbiology Research Division in Health Canada. He has more than 29 publications, in reputed journals, serves on the editorial board of journals, Expert member on WHO/FAO Virus Committees, expert reviewer for ESFA, Coordinator of Food & Water Safety Genomic Initiative, a member of the European Virology Network (COST929), and past president of the Virus & Parasite Development Group at the International Association for Food Protection –IAFP- (USA).

Food- and water-borne viruses, such as norovirus (NoV), hepatitis A virus (HAV), and hepatitis E virus (HEV), have been increasingly incriminated in causing sporadic, as well as major outbreaks world-wide. While most efforts have been focused on NoV, since it is the leading non-bacterial causative agents of gastroenteritis, there is increasing evidence that HEV which is predominantly prevalent in the eastern part of the world, maybe more prevalent in the western hemisphere than initially thought as shown by population seroprevalence reaching close to 40% in some regions. While working on, and leading a major Genomic Research Development Initiative project, we developed an innovative microfluidics-based device that would enable us to detect virulent E. coli O157 and non-O157 within few hours as compared to few days by the currently available methods. Taking advantage of acquiring considerable knowledge and expertise in developing this technology, we have embarked on adapting and modifying it for the rapid isolation and detection of foodborne viruses in a matter of hours rather than many days of labor-intensive and harsh methodologycurrently used for virus detection. While the principles aresimilar, the Lab-on-A-Chip microfluidics-based platform for virus detection is quite different from the bacterial platform. Modifications are required to accommodate isolation of a smaller size virus particlefrom food samples for subsequent viral RNA release, PCR-amplification, and microarray characterization, all conducted on a platform almost the size of a CD or DVD. Trials are underway to develop this platform to achieve detection capability comparable to the bacterial chip.

Maged Gomaa Hemida
King Faisal University, Saudi Arabia
Title: Dromedary camels and the transmission of MERS coronavirus (MERS-CoV)
Hemidareceived his Ph.D from University of Guelph, 2009. He pursued hisPDF trainingat theUniversityof British Columbia (James Hogg iCapture Centre). His research area of interest is “One Health Concept” with special emphasis on emerging viruses/host interaction. Currently, studying the molecular evolution and pathogenesis of MERSCoV in the Middle East. He published more than 40 original Research papers on high impacted journals. Meanwhile, he received several Research grants, prestigious honors and scholarship throughout his academic carrier. Currently, he is a reviewer of many granting agencies as well as editorial board member of many international journals.

Middle East Severe Acute Coronavirus (MERS-CoV) is an existential threat to global public health. The virus has been repeatedly detected in dromedary camels (Camelus dromedaries). Adult animals in many countries in the Middle East as well as in North and East Africa have high (>90%) sero-prevalence to the virus and dromedaries are a natural host for this virus. MERS-CoV isolated from dromedaries is genetically and phenotypically similar to viruses from humans. Our goal is to summarise relevant aspects of dromedary camel husbandry, animal movements, trade and the use and consumption of camel dairy and meat products in the Middle East that may be relevant to the ecology and epidemiology of MERS. It is important to understand the ecology and epidemiology of MERS so that zoonotic disease can be prevented and epidemic or pandemic threats mitigated. To understand the modes and risk factors of human MERS, it is important to exclude cases that have been acquired from other humans or affected health care facilities and to focus on index cases of the disease. Such cases are presumed to be zoonotic in origin and livestock exposure has been reported in some, but not all or even most cases. In conclusion, transmission of MERSCoV is complicated and further studies are undergoing to explore this to improve our understanding of the role of the dromedary as a source of human infection.

Maged Gomaa Hemida
King Faisal University, Saudi Arabia
Title: Paucity of infection of humans and other livestock in direct contact or in housed in proximity of a dromedary camel herd with documented MERS coronavirus infection
Hemidareceived his Ph.D from University of Guelph, 2009. He pursued hisPDF trainingat thethe Universityof British Columbia (James Hogg iCapture Centre). His research area of interest is “One Health Concept” with special emphasis on emerging viruses/host interaction. Currently, studying the molecular evolution and pathogenesis of MERSCoV in the Middle East. He published more than 40 original Research papers on high impacted journals. Meanwhile, he received several Research grants, prestigious honors and scholarship throughout his academic carrier. Currently, he is a reviewer of many granting agencies as well as editorial board member of many international journals.

Middle East Respiratory syndrome coronavirus (MERS) is one of themajor health concerns worldwide. Dromedary camels are likely to be a natural host of MERS.Transmission between camels is clearly documented. However, it is still unclear whether this is the only natural host for this virus. Clearly, transmission from camels to humans is not efficient and infection is not directly proportional to exposure. Heterogeneity of human susceptibility to MERS infection may be one possible explanation. On the other hand, many human cases of MERS do not have an obvious history of direct exposure to camels or their products. Thus, the possibility of another intermediate host in MERS transmission to humans remains possible. To test this hypothesis, different samples were collected from positive MERS camel populations.Serum samples were also collected from people in close contact with these animals. Samples from birds shared the habitat with these animals such as doves, sparrow as well as from sheep and goat flocks in close proximity of these animals were collected. Samples from mites, ticks, mosquitoes and flies were also collected from these flocks. Detection of the virus was done by Real time PCR while detection of the viral antibodies was done by the Pseudovirus particle neutralization Assay. Absence of antibodies in sera of close contact people, sheep, goat, and birds was reported. Onlycamels showed neutralizing antibodies. Meanwhile, swabs from tested birds, flies and mosquitoes were negative. Thus, transmission of MERS is too complicated. Further studies are needed to study the human/MERS/camels interactions.

Eman Sh. Al-Obeidy
Medical City Hospital-Virology Unit, Iran
Title: Herpes Simplex Virus Meningitis in Iraqi Children’s under 5 Years of Age

Background: Herpes simplex virus (HSV) is a member of Herpesviridae and a leading cause of human viral diseases. Meningitis occurs as a complication of HSV-1 or HSV-2 primary infection.
Objectives: We aimed to evaluate HSV meningitis in Iraqi children less than 5 years of age.
Patients and Methods: Sixty eight cerebrospinal fluid samples were taken from children referred with meningitis symptoms. Samples with negative bacterial infection results were tested for viral, biochemical and cytological assays. DNA extraction and PCR were performed.
Results: HSV-1 detected in 10 (14.7%) samples whereas HSV-2 infections were detected in 2 cases (2.9%). Cases with positive results had fever and CSF pleocytosis. Vomiting, headache and higher count of WBC were observed in 3, 2 and 3 cases respectively. The cerebrospinal fluid (CSF) glucose and protein levels were normal and 9 cases (of both viral infection showed positive C-reactive protein (CRP) results. Also erythrocyte sedimentation rate (ESR) was higher than normal in all positive cases.
Conclusions: Distribution of HSV types in children with meningitis in our area predominantly was type 1 compared with type 2, which has been reported more in other area.

Reem Kamal Elias
University of medical science and technology, Sudan
Title: Risk factors for HPV DNA detection in Middle-aged Women
Reem kamal Elias has completed his M.Sc. from university of medical science and technology, Sudan. She is the microbiologist of jarash international hospital.

Background and Objectives: Strong epidemiologic evidence indicates that human papillomavirus (HPV) is the main etiologic factor of cervical cancer. A few cohort studies suggest that most HPV infections are transient in young women and that persistent HPV infections are more common in older women. Little is known about the determinants of persistent HPV infections. The present study was aimed at increasing our knowledge about these determinants.
Goals: To identify risk factors for genital HPV DNA detection among cytologically normal middle-aged women.
Study Design: Eight hundred ten women who participated as control subjects in three case control studies on cervical cancer in Spain, Colombia, and Brazil were included in this study. After an interview, women underwent a gynecologic examination with collection of exfoliated cells for a Papanicolaou smear and HPV DNA detection. Human papilloma virus DNA was detected by polymerase chain reaction (PCR) based hybridization techniques.
Results: The HPV positivity rate was 10.5% in the whole population, but was higher in the areas with high incidence of cervical cancer (17% in Brazil and 13% in Colombia) than in Spain (4.9%), which is a low‐risk area for cervical cancer. Age was related to the prevalence of HPV DNA in Brazil, but not in Spain and Colombia. In univariate analyses in all three countries, the prevalence of HPV DNA was positively associated with the number of lifetime sexual partners and inversely associated with the levels of family income and with age at first sexual intercourse. There was four times increase in the odds ratio (OR) of HPV infection in women who had six or more lifetime sexual partners compared with those with one or less. The use of any kind of contraceptive tended to decrease the OR for HPV detection. Their ORs ranged from 0.44 (barrier methods) to 0.48 (oral contraceptives). In Spain and Colombia, antibodies against Chlamydia trachomatis were positively associated with the prevalence of HPV DNA. In a final multivariate model, the Positive associations with lifetime number of sexual partners, socioeconomic status, and C. trachomatis persisted.
Conclusions: These results support the sexual transmission of HPV and suggest that socioeconomic status and antibodies to C.trachomatis are independent predictors of HPV detection in middle-aged cytologically normal women.

Veterinary Virology
Viral epidemiology and diagnostics

Session Introduction

Sandra Helena Alves Bonon
State University of Campinas, Brazil
Title: Immunocompetent Brazilian Patients with Nervous Systems Disorders and Herpesvirus detected in Cerebral Spinal Fluid: the importance of molecular detection methods
Sandra Helena Alves Bonon has completed her Ph.D from State University of Campinas, and postdoctoral studies from Chiba University/Japan in Molecular Biology of Infectious Diseases.She is the head of Laboratory of Virology Research, Faculty of Medical Sciences, State University of Campinas since 1997. She has published more than 30 papers in high impact journals and has been serving as apaper revisor.

Herpesvirus (HHV)reactivation may occur in the nervous system (NS) and the lack of suitable treatment may cause sequelae. Standard test for confirmation of HHV-DNA is PCR in the cerebrospinal fluid (CSF). The high sensitivity of the nested PCR in the detection of HHV (1 to 8) provides advantages in the diagnosis of active infection caused by these viruses in the NS. Clinical data were obtained from the information contained in the medical records. Results. In 51.9%, the HHV-DNA was detected at least once in CSF samples by nested PCR technique. In these positive samples, 55.5% were from patients with diagnostic hyposhesis of encephalitis. The occurrence of monoinfection occurred in 37% positive patients. The occurrence of coinfection occurred in 63% positive patients. The most common viruses found in the samples were EBV and HCMV, with 44.4% and 55.5%, respectively. A patient with initial hypothesis of encephalitis that was positive for these two viruses died due to complications such as acute respiratory failure, epilepsy and encephalitis. HSV-1 was observed 18.5%; HSV-2 in 7.4%; VZV, 11.1%; HHV-6, 37% and HHV-7, 33.3%.HHV-8 was not found.PCRin the CSF has revolutionized the diagnosis of viral infections of the NS, particularly those caused by HHV. Therefore, this study verified the role HHV infections play in the NS in immunocompetent patients, focusing on diagnoses of viral infection in the NS. With the detection of viral DNA, early antiviral therapy may be instituted for viruses which there are antivirals available.

Konstantin Chumakov
FDA Center for Biologics Evaluation and Research, USA
Title: Metagenomics-based molecular epidemiology of viral diseases

Molecular epidemiology and environmental surveillance are parts of a rational strategy to control infectious diseases. They have been widely used in the worldwide campaign to eradicate poliomyelitis, which otherwise would be complicated by the inability to rapidly respond to outbreaks and determine sources of the infection. The conventional scheme involves isolation of viruses from patients and the environment, followed by their identification by nucleotide sequences analysisto determine phylogenetic relationships. This is a tedious and time-consuming process that yields definitive results when it may be too late to implement countermeasures. Because of the difficulty of high-throughput full-genome sequencing, most such studies are conducted by sequencing only capsid genes or their parts. Therefore the important information about the contribution of other parts of the genome and inter- and intra-species recombination to viral evolution is not captured. Here we propose a new approach based on rapid concentration of sewage samples with tangential flow filtration followed by deep sequencing and reconstruction of nucleotide sequences of viruses present in the samples. The entire nucleic acids content of each sample is sequenced, thus preserving in digital format the complete spectrum of viruses. A set of rapid algorithms was developed to separate deep sequence reads into discrete populations corresponding to each virus, and assemble them into full-length consensus contigs, as well as to generate a complete profile of sequence heterogeneities in each of them. This provides an effective approach to study molecular epidemiology and evolution of natural viral populations.

Ahmed Mohammed Ashshi
Umm Al-Qura University, Saudi Arabia
Title: Detection of dengue virus and its antibodies among Saudi blood donors: The first findings

Dengue is the most important mosquito-borne viral disease with an estimated 100 million infected cases and 25,000 deaths per year worldwide. It is caused by Dengue virus (DENV), which is a single-stranded RNA virus with five serotypes (DENV-1, DENV-2, DENV-3, DENV-4 and DENV-5). Transfusion safety is of paramount importance to the recipient of blood products. In recent decades, the safety of blood products with regards to the risk of HIV, HBV and HCV infections has increased dramatically; however, emerging infectious diseases still pose new threats and risks to the safety of blood products and transfusions.In this concept, a special attention has recently been paid to the significant role of blood transfusion in the transmission of DENV and/or its antibodies from asymptomatic infected blood donors to recipients. Given the absence of an approved blood screening test for DENV in Saudi Arabia and in response to the new epidemiological situation, the current study was designed to determine the seroprevalence of DENV infection and/or its antibodies among Saudi blood donors.In this study, healthy adult male Saudi blood donors, negative for HIV, HCV and HBV infections and accepted according to the KSA's blood donation policy, were screened for DENV-NS1 antigen and IgM and IgG anti-DENV antibodies. The results showed that among the tested donors, 1% showed positivity for DENV-NS1 antigen, 6% were positive for anti-DENV IgM antibody and 7% were positive for anti-DENV IgG antibody.
Conclusions:This study provides the first data on seropositivity for DENV and its antibodies among Saudi Arabian blood donors and highlights the importance of establishing quantitative or molecular serological methods for screening for DENV and/or its antibodies inblood donors, so that the quality of blood transfusions is guaranteed and the endemicity of DENV is reduced.

Chris D. Kalogeropoulos
University of Ioannina, Greece
Title: Viral ocular disease: Global epidemiology, classic and new clinical entities and management

Purpose: To assess the impact of various viruses affecting the eye and the concepts and principles of management of the ocular inflammation.
Material and Methods: Epidemiology of viral ocular disease throughout the world with emphasis of new viruses and new clinical entities concerning the eye (either as keratitis or uveitis etc.) was investigated taking into consideration literature and World Health Organization (WHO) reports. Areas of epidemic outbreak shared a special focus and investigation with regard to virus contamination and spread. Management regarding prevention (including vaccination), elimination of epidemic outbreaks (including WHO surveillance and initiatives) and clinical approach (both, in time diagnosis and appropriate treatment with emphasis to therapeutic innovations) is presented, based on collected data from the literature.
Results: Beside the classic global expansion of herpesviruses, common causes of keratitis, scleritis, uveitis and retinal vasculitis, emphasis must be given in the herpetic origin optic neuritis with concomitant or not central nervous system (CNS) involvement and in the increase of cytomegalovirus uveitis in immunocompetent patients as well (and not only in patients infected by Human Immunodeficiency Virus). Keratitis, conjunctivitis and uveitis due to adenoviruses and enteroviruses and other common viruses are universally found. Regarding emerging and reemerging infectious diseases there were during the recent years outbreaks of West Nile virus with signs of meningitis – encephalitis and uveitis along with severe retinal vasculitis in United States, Canada, Middle East, other countries of Asia and Africa and Greece. Chinkungunya virus endemic areas are regions of Asia, Africa and areas of Indian and Pacific oceans and uveitis is one of the virus manifestations. Ebola virus can also be the cause of severe uveitis in affected survivors, especially in West Africa due to recent outbreaks. Human Papillomavirus (HPV) can affect also the eye considered as cause of conjunctival papillomas and is rather more common than in the past. It is expected that HPV vaccination will achieve a decrease of ocular disease incidence as well. Other viruses affecting the eye are bunyaviruses in Africa (retinal vasculitis that can lead to blindness) and arenaviruses in South America and West Africa mainly (hemorrhagic conjunctivitis and high mortality). According literature, it is well established that vaccination, travelers control, new diagnostic techniques, medications and hygiene level can reduce the incidence of viral infection and consequently the eye morbidity.
Conclusions: Keratitis, uveitis, retinal vasculitis, scleritis, optic neuritis and also conjunctival inflammation and neoplasia are common manifestations of viruses. In addition, ocular involvement is a not uncommon manifestation of viral CNS affection. Care concerning endemic areas, WHO initiatives, vaccination and new therapies performed after a timely diagnosis will decrease the outbreaks and spread of dangerous and life threatening viruses along with a dramatic decrease of ocular morbidity, potentially leading to blindness.

Agronomy and Veterinary Institute, Morocco
Title: Emergence of a new genotype in Moroccan broiler chicken flocks : Phylogenetic analysis of avian infectious bronchitis virus in poultry flocks from 2010 through 2014.
Siham FELLAHI has completed his Veterinary Doctor at the age of 24 years from Agronomy and Veterinary Institute Hassan II, Morocco (2002-2008) and Master on “Biotechnology and Engineering of Microorganisms” from University Moulay Ismail (2009-2011). Currently, she registered in last years of the PhD program within the UFR “avian Pathology” in the Institute Agronomy and Veterinary Hassan II, under topic “Molecular characterization of Avian Infectious Bronchitis Virus in Morocco”. She has 5 oral communications in the international congress and 4 publications under examination in reputed journals. She is member of the European cooperation in science and technology (COST Action).

Avian infection bronchitis virus (IBV) remains a major cause of losses for poultry production around the word. The aim of this study is to investigate the molecular analyses of IBV genotypes in poultry flocks in sixteen areas of Morocco between 2010 and 2014. Molecular characterization of IBV strains was carried out on 50 RT-PCR positives samples by sequencing partial S1 gene containing hypervariable regions (nt 705-1097). Two predominant genotypes were detected, with a dominance of Massachusetts type (66%), among which 25% of the samples were identical to the H120 vaccine. The second most common genotype (present in 32% of the flocks) was surprisingly Italy 02, revealing the first detection of this genotype in Morocco and in Africa. 793/B, the predominant genotype in the late nineteen-nineties in Morocco, was only detected in one occasion and was identical to the 4/91 vaccine strain. Among Fifty isolates sequenced, three isolates were found genetically highly distant from known avian IBV based on their S1 genes: IBV/Morocco/01, IBV/Morocco/30, and IBV/Morocco/38, nucleotide sequence identities reached 89.5% to 90.9% among the three isolates. The deduced protein sequence identities ranged from 29.7% (between IBV/Morocco/38 and Egypt SCU-14/2013-1) to 78.2% (between IBV/Morocco/01 and Spain/05/866). Amino acid sequence comparison and phylogenetic analysis indicated the emergence of a new Moroccan genotype, clustering with regionally related isolates from Spain (Spain/05/866) and belonging to a new sub-genotype. These results justify permanent monitoring of circulating strains in order to rationally modify vaccination strategies to make them appropriate to the evolving field situation.

Islamic Azad University, Iran
Title: The new insight about Interspecies transmission of Iranian H9N2 influenza viruses from avian to human
Masoud soltanialvar graduated as Doctor in Veterinary Medicine from the Veterinary School of Ahwa, Iran, in 2005.He worked a few years in veterinary practice, and then joined , Islamic Azad University ,Shoushtar Branch.where he worked for 9 years as assistance professor for Division of microbiology . He has many years of experience with the poultry viral disease of all continents as well as with the prevention strategy of diseases using vaccines. In 2010 he graduated as Doctor in Veterinary Virology (PH.D).

Documented cases of human infection with H9N2 avian influenza viruses, first detected in 1999 in Hong Kong and China, indicate that these viruses can be directly transmitted from birds to humans. In this study, we characterized the mutation in the Hemagglutinin (HA) genes and proteins that correlates with a shift in affinity of the Hemagglutinin (HA) protein from the “avian” type sialic receptors to the “human” type in 10 Iranian isolates. We delineated the genomes and receptor binding profile of HA gene of some field isolates and established their phylogenetic relationship to the other Asian H9N2 sublineages.A total of 1200 tissue samples collected from 40 farms located in various states of Iran during 2008–2010 as part of a program to monitor Avian Influenza Viruses(AIV) infection. To determine the genetic relationship of Iranian viruses, the Hemagglutinin (HA) genes from ten isolates were amplified and sequenced (by RT-PCR method). Nucleotide sequences(orf) of the (HA) genes were used for phylogenetic tree construction.Deduced amino acid sequences showed the presence of L226(234 in H9 numbering) in all ten Iranian isolates which indicates a preference to binding of α (2–6) sialic acid receptors, So these Iranian H9N2 viruses have the potential to infect human beings .These isolates showed high degree of homology with 2 human H9N2 isolates A/HK/1073/99 , A/HK/1074/99 . Phylogenetic analysis of showed that all the HA genes of the Iranian H9N2 viruses fall into a single group within a G1-like sublineage which had contributed as donor of six internal genes to H5N1 highly pathogenic avian influenza. The results of this study indicated that all Iranian viruses have the potential to emerge as highly pathogenic influenza virus, and considering the homology of these isolates with human H9N2 strains, it seems that the potential of these avian influenza isolates to infect human should not be overlooked.

Viral Hepatitis- Virus-host interactions
Organ Specific Cancers and Human Tumour Virology
Viral Immunology

Session Introduction

Ramona Gabriela Ursu
University of Medicine and Pharmacy “Grigore T. Popa”, Romania
Title: HPV - a risk factor in special Romanian populations: head&neck cancers patients, women requiring conization and HIV positive women
Ramona Gabriela Ursu has completed his Ph.D from University of Medicine and Pharmacy “Grigore T. Popa” (PhD thesis: “Asymptomatic sexually transmitted diseases. Clinical utility of high risk HPV type viral load quantification by Real Time PCR”), Iasi, Romania and now she is doing her postdoctoral studies (“HPV infections - risk factor for special populations”) at the same university. She is Lecturer, Microbiology Department, since 02.2014, Consultant MD in Laboratory Medicine. She is involved Europen studies regarding HPV associations with HNC, and also in oher projects, like N. meningitidis detection by RT PCR, VHB genotyping/INNOLiPA, braf600E-quasa/RT PCR for malignant melanoma.

Background: HPV (Human Papilloma Virus) infection is associated with many cancers, including head, neck and cervical cancers. Romania is rated by GLOBOCAN 2014 on the first places in Europe for cervical cancer and head&neck cancers also.
Aim: to assess the burden of HPV infection in case of different cases of head & neck cancers (HNC), in case of HR HPV women positive wich required conization and in case of HIV positive women.
Method: from HNC patiens we selected 15 fresh tumor samples (lips, tongue, tonsil, hypopharynx) and cervical cells in case of 55 HR HPV positive women requiring conozation and 40 HIV positive women. We used Linear Array HPV Genotyping Test for detection of 37 HPV types.
Results: all HNC cancers samples we negative for any 37 HPV tested. The HR HPV positive women were positive for HPV 16, 18, 31 and 52 at 6 month after conization. The HIV positive women were positive for HPV in 40% of cases.
Conclusions: these results are pilot studies of further prospective studies. For cervical cancer we think that HPV vaccination should be implemented in our country. For HNC we will extend our tested cases.

Ghazi Jamjoom
King Fahd Center for Medical Research, Saudi Arabia
Title: Hepatitis B Virus Hepatocarcinogenesis: A study on chronically-infected and hepatocellular carcinoma patients from Saudi Arabia

Hepatitis B virus (HBV) is a global health problem with over two billion people in the world exposed to infection, and over 350 million persistently infected. Chronic HBV infection is a major cause of liver cirrhosis and hepatocellular carcinoma (HCC). The mechanism of HBV carcinogenesis may be dependent on virus integration in the host genome with activation of proto-oncogenes (insertional mutagenesis) or it may be directed by transcriptional activation by viral oncogene products such as HBx or Pre S2. There are 8 genotypes (A-H) of HBV which exhibit different potential for tumorgenesis. Sequence variation of HBV genome with emergence of variants during chronic infection may also contribute to HCC development. HBV mutations in Pre-S region, basal core promoter (BCP) region, etc. may precede or predict tumor formation. Certain BCP mutations such as A1762T/G1764A have been extensively studied. Several studies have provided evidence that mutations in the preS2 region are correlated with more progressive liver disease including cirrhosis and HCC. We are attempting to assess the prevalence of different HBV mutations by sequencing of specific regions of the virus. Thus far we have identified several pre-S2 deletions and pre-S2 start codon deletion mutations among 100 chronic HBV patients of different HBV genotypes in Saudi Arabia. The pre-S1, pre-S2, and S- regions of the HBV genome were sequenced and phylogenetically analyzed to elucidate the genotype and detect the mutations in these regions. Details of the results will be shown and discussed together with other findings that may shed light on HBV hepatocarcinogenesis.

Mohamed Labib Salem
Tanta University, Egypt
Title: High numbers of myeloid-derived suppressor cells associates with low levels of dendritic and T cells in Egyptian patients with chronic HCV infection who failed IFN-α therapy
Mohamed L. Salem (PhD, 1995) is the Professor of Immunology at Tanta University, Egypt and Visiting Professor at Hollings Cancer Center and Department of Microbiology and Immunology, Medical University of South Carolina, USA. Dr. Salemis the Director of Delta Development Research Center which is a regional branch of Academy of Scientific Research and Technology (ASRT), Egypt; Director of the Grant, Innovation & Technology Transfer Center, and Founder and Director of Center of Excellence in Cancer Research, Tanta University, Egypt.Dr. Salem obtained his PhD in 1995 through a channel systembetween Tanta University, Egypt and Kyushu University, Japan. From 1997 -2001, he was a Postdoctoral Fellow at Kyushu University, Japan and from 2002-2010 as an Assistant Professor at Medical University of South Carolina, USA. Dr. Salem is member of the National Committee for Professorship Promotion (Zoology) and Member of the National Committee for the Biological Sciences (ASRT). He is also a founder member of the Egyptian Association of Cancer Research and Middle Eastern Association for Cancer Research, Canada. Dr Salem won the State Incentive Award in 2003 and the State Excellent Award in 2009 in Basic Sciences from the Academy of Scientific Research and Technology, Egypt based on his research on cancer immunotherapy. His h-index is 24 and published about 111 articles in peer-reviewed journals and attended more than 70 national and international workshops and conferences in Immunology. He supervised more than 55 Master and PhD students. He is a member of several editorial boards of peer-reviewed journals as well as ad hoc reviewer for more than 80 journals. He has been invited to give talks in cancer immunotherapy in Egypt, UAE, Jordan, Saudi Arabia, USA, and Canada.Dr Salem’s research focuses on understanding the cellular and molecular mechanisms underlying how cancer and HCV escape from host immunity, in particular the rules of immunoregulatory cells including lymphoid and myeloid lineages. Additionally his research has been focusing on developing effective anticancer immunotherapies strategies, in particular the use of adoptive cell therapy.His research projects have been funded by National Institute of Health, USA, Science and Technology Development Fund (STDF), Egypt, USA National Academy of Sciences, Tanta University, Egypt, private foundationand industry.

Hepatitis C Virus (HCV), with genotype 4, is epidemic in Egypt and causes chronic hepatitis, where anti-HCV therapy (Ribavirin and IFN-α) is not effective in 60% of these patients. We have recently reported accumulation of myeloid derived suppressor cells (MDSCs) and suppressed immunity in cancer patients. The aim of this study was to assess the frequency of myeloid cells, including cells with the phenotype of MDSCs and dendritic cells (DCs), in chronic HCV patients and correlates it with the responses of the patients to IFN-α-based therapy. Peripheral blood waswithdrawn from 154 patients with chronic HCV infection and from 25 healthy volunteers. The patients were categorized into responders and non-responders based on viral titer upon IFN-α treatment. The relative and absolute numbers of MDSCdefined as Lin-/HLA-DR-/CD33+/CD11b+ was increased in all HCV patients coincided with increases in the frequency of DCs and T cells. The frequencies of MDSC and DCs in non-responders were higher than those in responders who had higher levels of IL-2 than non-responders. The levels of MDSC after 4-6 months of IFN-α treatment in the responders was lower than during the treatment. The high numbers of MDSC coincided with decreasedlevels of bilirubin and hemoglobin but with no AST and ALT. Our data showed that, regardless of IFN-α therapy, the Egyptianchronic HCV patients harbor high levels of MDSCs with higher levels in non-responders than responders. Opening a new avenue of the use of the cells as biomarker of responsiveness to IFN-based therapy and implicate to the use of drugs that are known to target these cells to treat HCV patients.

Aissa Larousse Jameleddine
Bordeaux University Hospital, France
Title: Natural prevalence of hepatitis C virus (HCV) variants resistant to protease and polymerase inhibitors in HCV genotype 1-infected patients in Tunisia

Background: Hepatitis C virus (HCV) protease inhibitors (PIs) and polymerase inhibitors nucleos(t)ide inhibitors (NS5B-NIs) and non-nucleos(t)ide inhibitors (NS5B-NNIs) have recently been developed to inhibit protease (NS3) or polymerase (NS5B) activities. The drawback of antivirals treatment is the emergence of resistance mutations to the drugs. The prevalence of such mutations conferring resistance to PIs, NS5A-NIs and NS5B-NNIs before treatment is not been investigated so far in the Tunisian population.
Objectives: The aim of this study was to detect HCV variants bearing resistance mutations to NS3 and NS5B inhibitors in hepatitis C patients infected with HCV genotype 1.
Study design: We investigated the prevalence of known substitutions conferring resistance to HCV-PIs, NS5B-NIs and NS5B-NNIs in 149 untreated patients, infected with HCV genotype 1 (genotype 1a=7; genotype 1b=142).
Results: Twelve patients (9.2%) of the 131/149 HCV NS3 sequences analyzed showed amino-acid substitutions associated with HCV PIs resistance mutations (T54S, n=4 (3.05%); V55A, n=2 (1.52%); Q80K, n=4 (3.05%); R155K, n=1 (0.76%); A156V, n=1 (0.76%). One (1%) of the 95/149 HCV NS5B sequences analyzed showed the substitution V321I conferring resistance to NS5B-NIs, while 34 of 95 (36.8%) showed substitutions conferring resistance to NS5B-NNIs (C316N, n=2 (2%); M414L, n=1 (1%); A421V, n=8 (8.5%); M423A, n=1 (1%); M423T, n=2 (2%); I424V, n=5 (5.25%); C445F, n=1 (1%); I482T, n=2 (2%); V494A, n=1 (1%); P496A, n=1 (1%); V499A, n=15 (16%); S556G, n=5 (5.25%)).
Conclusions: Naturally occurring substitutions conferring resistance to NS3 or NS5B inhibitors exist in a substantial proportion of Tunisian treatment-naïve patients with HCV genotype 1. Their influence on treatment outcome should be assessed.

Shadab Perveen
University of Karachi, Pakistan
Title: Rare genetic variation in Hepatitis Delta Virus (HDV) that influence genotype determination

Background: Hepatitis delta virus (HDV) is a satellite virus that only infects the hepatic cells already infected with Hepatitis B virus. The delta virus infection leads to a clinical outcome, which is more severe than HBV infection alone with severity of infection varying with the delta virus genotype. During sampling of delta virus in Karachi, Pakistan, a variant of Genotype I delta virus was encountered that was initially misdiagnosed as genotype II, based on PCR-RFLP analysis using SmaI restriction enzyme. Correct genotype determination of the newly found strains was carried out using phylogenetic analysis and an attempt was made to analyze its origin and propagation in our population.
Methods: Blood samples were collected from HBsAg +ve and HDAg +ve patients diagnosed with chronic hepatitis delta infection. Viral RNA was extracted, reverse transcribed and used to amplify HDV R0 region by RT-Nested PCR. The PCR products were screened with RFLP using SmaI restriction enzymes for determining the genotype of the delta virus. Nucleotide sequencing of PCR-amplified R0 region was also carried out and used for phylogenetic analysis for clade determination. In silico analysis for evidence of recombination was also carried out in order to determine the origin of the delta virus isolates of interest.
Results: PCR-RFLP analysis using SmaI enzyme showed that three of our delta virus isolates belonged to genotype II. Upon nucleotide sequencing and subsequent phylogenetic analysis, the so-called genotype II clustered with genotype I R0 sequences reflecting their true identity. A deeper nucleotide analysis exposed a single base pair selectively neutral mutation at the single SmaI restriction site within the B cell epitope, which caused these three strains to be falsely categorized as genotype II. Sequences analyzed in silico for recombination showed putative exchange of genetic material within and across genotype I and II.
Conclusions: Phylogenetic analysis of our sequences have shown a clear misdiagnosis of genotype in our isolates and proved the short-comings of SmaI PCR-RFLP based diagnosis of delta virus genotype. Results of recombination analyses have suggested a possible reason for the high frequency of occurrence of this mutation in our sampling.

Wafa Irahim Elhag
Al Neelain University – Khartoum, Sudan
Title: Molecular Detection of Human Papillomavirus Type-16 DNA in Cervical Cancer Tissue Biopsies

Objective: The aim of the study is to detect the frequency of human papillomavirus type-16 among patients with cervical carcinoma.
Method: Fifty specimens of treated cervical biopsy sections (Paraffin embedded) were included in the study from April to October 2012. DNA was extracted followed by the detection of E6 gene of human papillomavirus type-16 using non-probed SYBER green real-time PCR.
Result: Thirty (60%) showed positive results as compared with the sigmoid curve of the positive control for HPV type-16; while 20 (40%) were negative. Most of the positive results were among the age group 31-50 years.
Conclusion: Human papillomavirus type-16 was detected in 60% of women with cervical cancer, which seems to have a strong association with cancer development.