20/09/2011 NÔNG LÂM UNIVERSITY – VIET NAM DEPARTMENT OF BIOTECHNOLOGY Diagnosis and Gene Diagnosis and Genetic analysis of some tic analysis of some viruses caused emerging d viruses caused emerging diseases in seases in pig pig in Vietnam in Vietnam Võ Khánh H ư ng 1 Main Contents Genetic analysis of PRRSV (Porcine Reproductive & Respiratory Syndrome) in Vietnam Genetic analysis of PCV (Porcine Circovirus) in the South of Vietnam Phylogenetic analysis of Classical swine fever virus (CSFV) in a Mekong Delta provinces Somes methods for detecting and quantifying PRRSV , CSFV and PCV PCR and RT-PCR Realtime PCR LAMP (loop-mediated isothermal amplification) 2 1
20/09/2011 Wh Why PRRS y PRRSV, PCV and CFS PCV and CFSV are are the emerging diseases emerging diseases ???? the ???? They are a highly contagious viral disease and have immunosuppressive effects in pigs, it may increase their susceptibility to other agents causing diseases. Be become the chronic disease Hard to detecting by clinical fidings. BUT, It is a implicit threat . Cause damage in some important organs serious illness Swine industry is very important in Vietnam economy also in the world. 3 Gene Genetic tic anal analysis of PRRS ysis of PRRSV in Vietna V in Vietnam PRRSV in Vietnam??? 4 2
20/09/2011 RT-PCR RT-PCR 5 Methode Vaccine + BSL-PS 100 RNA extraction PRRSV infected samples + Porcillis RT-PCR + AmerVac Sequencing + IngelVac Genetic analysis RNA extraction RT-PCR Amplifying NSP2 gene NSP 2 F : ’-AAA GAC CAG ATG GAG GAG GA-’ NSP 2 R : ’-GAG CTG AGT ATT TTG GGC GTG-’ Sequencing Phylogenetic 6 3
20/09/2011 Phylogenetic of PRRSV based on NSP2 gene (Vo KH. And Nguyen NH., 2011) (91.4 – 99.1%) Deletion of 87 nucleotide NA (93.9 – 96.1%) EU 7 Conclusion PRRSV isolates in Vietnam had deletion of 87 nucleotides and were the same to PRRSVs isolated from China in 2007. Homology between studied PRRSVs in Vietnam and chinese type PRRSV isolated at china were high (93.9 – 96.1%) in the nucleotide sequence. PRRSV isolates from North Vietnam and PRRSV isolates from SouthVietnam were high identity (91.4 – 99.1%). This study was undertaken to assess the regional variation of prevalent PRRSV and to establish a sequence database for PRRSV molecular epidemiological studies. 8 4
20/09/2011 H ộ i ch ứ ng g ầ y còm 1 Gene Genetic tic analysis analysis of PCV in Vie of PCV in Vietnam nam sau cai s ữ a wasting PMWS Post Weaning Multisystemic Wasting Syndrome palpable lymphadenopathy 9 H ộ i ch ứ ng g ầ y còm 1 Intestina sau cai s ữ a juice in breast cativy PMWS Intestina Stuffing juice in spleen+ abdominal hepatitis 10 cativy 5
20/09/2011 Gene Genetic tic anal analysis of PCV in Vietna ysis of PCV in Vietnam PCV in Vietnam????? (Long JG. et al., 2010) 11 Phylogenetic of PCV based on ORF2 (Vo KH. and Nguyen NH., 2011) 98.8 – 99.5 %. 99.1-99,3% The result show that all of the studied PCV isolates in Vietnam belong to PCV type 2b and 2d 12 6
20/09/2011 Conclusion Conclusion All of the PCV isolates in Vietnam belong to PCV type 2b and 2d. The similarity between PCV2d isolates from studied regions and some regions of China is high (99.1-99,3%). Specially, PCV2 type b and PCV2 type d are simultaneously present at the same farm. The situation of PCV2 infection at some farm in Vietnam is complicated. Subgenotype PCV2 typd d viruses are predominate genotype circulating in studied regions. More knowledge we get for prevention and treatment PMWS epidemic caused by PCV in Vietnam. 13 Phylogenetic of CSFV based on E gene 1.1, 2.1 2.1 2.1 2.2, 2.3 2.2, 2.3 ?????? Classical swine fever (CSF) caused by a pestivirus (CSFV) 1.1, 1.2, 1.3 2.2, 3.3 ???? 14 Distribution of CSFV in Asian 7
20/09/2011 Phylogenetic of CSFV based on E gene (Tran TD., 2008) 95%-98% 92%-93% 15 Conclusion Conclusion All the studied CSFV isolates belonged to genogroup 2 and were clustered in subgroup 2.1 and 2.2. The isolates have high identity of nucleotide (97%-99%) compared to CSFV isolates from other regions in Vietnam (DN-VN, LD-VN, QN-VN). It is possible that the presence of these strains in the studied province may be the result of CSFV circulation among provinces in Vietnam territory. The attenuated live Thiveral vaccine and C vaccine strains were clustered in subgroup 1.1 and were very distantly related to the CSFV isolates found. 16 8
20/09/2011 Methods f Methods for det r detecting and q cting and quatifying PRRS atifying PRRSV RT-PCR and nested RT-PCR Distinguishing EU and US PRRSV (Vo KH. And Nguen NH., 2009) T5 26 Lad DC (+) O EU US O EU US O EU US T5 26 Lad DC (+) O EU US O EU US O EU US 17 Methods f Methods for det r detecting and q cting and quatifying atifying PRRS PRRSV Distin Distinguishing ishing v vacccin cccine viru virus and f s and field viru eld virus of s of PRRS PRRSV b by RLFP assa RLFP assay (Ngu (Nguyen NH.,20 n NH.,2010) 18 9
20/09/2011 Methods f Methods for det r detecting and q cting and quatifying PRRS atifying PRRSV Realtime RT-PCR QUATIFING AND GENOTYPING OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS BY REAL-TIME RT-PCR USING EVAGREEN (Vo Khanh Hung et al, 2011) Sensitivity test Tandard curve 19 Melt curve analysis specific test Conclusion The sensibility of the assay was 10 1 copies/ul. The specificity of the assay was confirmed by using PCV (porcine circovirus) and PRRSV-free blood of pig. Genotyping of EU PRRSV and US PRRSV was based on the difference of melt temperature (Tm) in melt curve analysis. The Tm of EU PRRSV amplicon was 83.4 o C and that of US PRRSV amplicon was 84.4 o C. EvaGreen-based real time RT-PCR quick, sensitive and accurate results for quantifying and genotyping PRRSV , which was used to determined genotypes. 20 10
20/09/2011 Methods f Methods for det r detecting and q cting and quatifying PRRS atifying PRRSV Realtime RT-PCR DEVELOPMENT OF ONE-STEP REALTIME QUANTITATIVE RT-PCR ASSAYBASED ON TAQMAN PROBE FOR DETECTION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS N NA TYPE (Vo Khanh Hung et al, 2011) sensitivity test Tandard curve 21 specific test Conclusion The developed assay is specific for both NA type and Chinese type. Sensibility assay met 10 1 copies/ml of sample from infected pigs. The developed method for detecting and quantifing PRRSV NA type is a realized method that is usef μ l for preventing and controlling disease caused by PRRSV . 22 11
20/09/2011 Metho Methods f s for det r detecting and q cting and quatify atifying ng PRRS PRRSV LAMP (loop-mediated isothermal amplification) Notomi, 2000) 23 The mechanism of LAMP reaction 24 12
20/09/2011 Methods f Methods for det r detecting and q cting and quatifying PRRS atifying PRRSV LAMP (loop-mediated isothermal amplification) 25 Methods for det Methods f r detecting and q cting and quatifying atifying PRRS PRRSV LAMP (loop-mediated isothermal amplification) Gel assay Eye assay specific test 26 13
20/09/2011 Methods f Methods for det r detecting and q cting and quatifying atifying PRRS PRRSV LAMP (loop-mediated isothermal amplification) Gel assay UV assay Eye sensitivity test 27 The targets of my group focused in Genetic analysis of PRRSV , PCV and CFSV all regions of Vietnam also in the world. Developing LAMP technique for detecting PRRSV , PCV and CFSV and producing LAMP kit for PRRSV , PCV , CFSV . Developing PCR assay for distinguishing vacccine virus and field virus of PRRSV , PCV also CFSV . 28 14
20/09/2011 Re Reference Vo Khanh Hung, Nguyen Ngoc Hai, Genetic analysis of porcine reproduction and respiratory virus in Dong Nai and Ho Chi Minh city . Journal of agricultural sciences and technology, 1, 57 – 63 (2010) Vo Khanh Hung, Nguyen Ngoc Hai, Genetic analysis of porcine reproduction and respiratory virus in Dong Nai and Ho Chi Minh city based on ORF7 . Journal of veterinary sciences and technology, 17, 25-33 (2010) Võ Khánh H ư ng, Nguy ễ nV ă n Chí, Lê Nguy ễ n Ng ọ c H ạ nh, Tr ầ nTh ị Dân, Tr ầ n Th ị Bích Liên, Nguy ễ n Đ ình Quát. quatifing and genotyping of porcine reproductive and respiratory syndrome virus by real-time rt-pcr using evagreen . Accepted by Journal of veterinary sciences and technology 2011. Võ Khánh H ư ng, Nguy ễ nV ă n Chí, LêVinhTh ắ ng, Lê Nguy ễ n Ng ọ c H ạ nh, Tr ầ nTh ị Dân. quatifing and genotyping of porcine reproductive and respiratory syndrome virus by real-time rt-pcr using evagreen. Accepted by Journal of agricultural sciences and technology 2011. NotomiT, Okayama H, Masubuchi H, 2000. Loop mediated isothermal amplification of DNA. Nucleic Acids Res 2000, 28-63 29 30 15
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