rologie i - European Congress of Virology

5 th European Congress of VirologyPPR viral RNA were compared in terms of performance. During JanuaryOctober 2012, 32 sheep, each from different flocks, submitted to KonyaVeterinary Control Institute with PPR suspect. One step RT PCR, whichamplified a 448 bp fragment in the F gene, detected PPRV in 9 samples.Twelve of these 32 samples were found to be positive by real time RT PCR,and most infected animals (9/12) showed a high viral load with individualcycle threshold (Ct) values are less than 30. The present study suggeststhat N gene based one step real time RT PCR was more suitable for therapid and specific detection of PPRV in clinical samples.Key words: Peste des petits ruminants virus, RT PCR, Real time RT PCR,F gene, N genecoated with VP7 monoclonal antibody was added to form immuno complex.Signal DNA annealed to DNA strands covalently bound to the GNPwere released by heating and characterized by PCR and real time PCR. Adetection limit of 0.1fg/ml was measured for VP7. Further, a simple GNPAmodified from BCA was developed. GNP was prepared through coatedwith designed single stranded signal DNA and VP7 monoclonal antibody.After immuno complex was formed (GNP VP7 MMP) and purified, thesignal DNA present in the immuno complex was detected by PCR and realtime PCR. The GNPA has a VP7 detection limit of 0.01fg/ml which is 1order of magnitude more sensitive than that of BCA. These approachescan reliably detect BTV, and it is important to prevent bluetongue.REF 339Comparative study of liquid phase blocking ELISA (LPBE) and solidphase competition ELISA (SPCE) methods for the detection of antibodiesto the structural proteins of foot and mouth disease types Oand A virusesMurat SEVIK 1 , Feridun ÖZTÜRK 21 Veterinary Control Institute/Molecular Microbiology, Konya, TURKEY;2 Department of Virology/Faculty of Veterinary Medicine/Selcuk University,Konya, TURKEYTwo foot and mouth disease virus (FMDV) structural protein antibodydetection methods, liquid phase blocking ELISA (LPBE) and solid phasecompetition ELISA (SPCE), were compared in the study. These methodswere compared using sera collected from cattle with no history of exposureto FMDV (n=30), cattle (n=180) vaccinated with oil adjuvanted bivalentvaccine (containing O1 Manisa, A22 Iraq FMDV strains) and internationalreference sera (positive, weak positive and negative) for FMDV serotypesO and A. The results showed that the SPCE had a better specificity (96.6%for serotype O and 100% for serotype A) than the LPBE (90% for serotypeO and 93.3% for serotype A). Sensitivity of LPBE (97.2% for serotypeO and 98.3% for serotype A) was almost equivalent to that of the SPCE(98.3% for serotype O and 98.8% for serotype A). It can be concludedthat SPCE is more suitable than LPBE for use as a screening test for thedetection of antibodies against structural proteins of FMDV.This study was summarized from a PhD thesis and supported by ScientificResearch Projects Coordination Center of Selcuk University, Konya,Turkey (Project No: 09202025).Key words: Cattle, foot and mouth disease, structural protein, LPBE,SPCEREF 341Baculovirus expressed type specific spike proteins in the differentiationof type 1 and 2 feline coronavirus infectionYing Ting WANG 1 , Cho Hua WAN 2 , Ling Ling CHUEH 11 Graduate Institute of Veterinary Medicine, School of Veterinary Medicine,National Taiwan University, Taipei, TAIWAN; 2 Graduate Instituteof Molecular and Comparative Pathology, School of Veterinary Medicine,National Taiwan University, Taipei, TAIWANFeline infectious peritonitis (FIP), is a fatal disease caused by feline coronavirus(FCoV). FCoVs are divided into serotypes I and II. Despite thehigher prevalence of type I FCoV infection in epidemiological survey usingboth molecular or serological assay, infection of type II FCoV appears tobe highly significantly correlated to FIP. This study aim to develop a novelserological assay to differentiate two types of FCoV infection throughthe baculovirus expressed type specific spike proteins from type I and IIFCoV. Two type specific regions in spike gene corresponding to the putativereceptor binding domain of spike protein from type II virus and therelated region from type I virus were selected, amplified and cloned intoa baculovirus expression system to produce two type specific recombinantviruses. A novel indirect immunofluorescence assay (IFA) using S.frugiperda (Sf 9) insect cells infected with the recombinant baculovirusexpressing two FCoV type specific spike proteins was developed. TheIFA test was first characterized using two type confirmed FCoV positivesera and two negative sera from SPF cats. Clinical sera from 42 FIP suspectedanimals were subjected for further test, twenty four (24/42, 57%)and five (5/42, 11%) sera were type I and II seropositive respectively.Thirteen (13/42, 30%) sera were positive to both types of recombinantproteins. These type specific IFA test is capable to distinguish two typesof virus infection. The relationship between serotypes and the diseasemanifestation will be evaluated further.REF 340Assays for detection of bluetongue virusHuiqiong YIN, Minxian JIA, Shu YANG, Rui WANG, Jianguo WANG,Jingang ZHANGViral Safety Laboratory of the National Center of Biomedical Analysis,Institute of Transfusion Medicine, the Academy of Militar, Beijing, CHINABluetongue caused by bluetongue virus (BTV) has been included in theWorld Organization for Animal Health (OIE) list of notifiable diseases.Reliable assays of BTV detection are essential for fighting against bluetongue.A series of techniques were developed for BTV detection includingRT PCR, real time RT PCR, ELISA, bio barcode assay (BCA) and goldnanoparticle probe based assay (GNPA). BTV RT PCR and real time RTPCR diagnostic kits detect BTV NS1 gene through a pair of primers anda TaqMan probe respectively. The kits had been approved by Ministry ofAgriculture of the People‘s Republic of China. The ELISA kit detects BTVVP7 through anti VP7 monoclonal antibody. After preclinical study, clinicaltrial is being applied. In BCA, VP7 was captured by gold nanoparticle(GNP) coated with polyclonal antibody. Magnetic microparticle (MMP)REF 342Comparative investigation of border disease virus infection in sheepflocks in Konya Province, TurkeyOguzhan AVCI, Sibel YAVRUUniversity of Selcuk, Faculty of Veterinary Medicine, Department of Virology,Konya, TURKEYThe aim of the present study was to determine the serological and virologicalstatus of animals persistently infected (PI) with border disease virus(BDV), with focus on the prevalence of pestiviruses in sheep and lambs.This study is the first to our knowledge, to investigate the role of viralinfections in abortions in the sheep population of Konya Province, Turkey.The prevalence of antibodies against BDV was as follows: sheep,79% (n=1000); lambs, 43.4% (n=500); and rams, 6% (n=50). Of 1000leukocyte samples examined with a direct enzyme linked immunosorbentassay (ELISA), 11 (1.1%) (first sampling) were antibody positive. Thirtysheep were determined to be PI and were sacrificed. Of 1011 sheep leukocytesamples, 14 were examined using a direct immunoperoxidase (IP)S214 Virologie, Vol 17, supplément 2, septembre 2013