Main Contents Genetic analysis of PRRSV (Porcine Reproductive & - - PDF document

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Võ Khánh Hưng

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

NÔNG LÂM UNIVERSITY – VIET NAM DEPARTMENT OF BIOTECHNOLOGY

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Main Contents

 Genetic analysis of PRRSV (Porcine Reproductive &

Respiratory Syndrome) in Vietnam

 Genetic analysis of PCV (Porcine Circovirus) in the South

• f 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)

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Wh Why PRRS y PRRSV, PCV and CFS PCV and CFSV are are the the emerging diseases

emerging diseases????

????

 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.

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Gene Genetic tic anal analysis of PRRS ysis of PRRSV in Vietna V in Vietnam

PRRSV in Vietnam???

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RT-PCR RT-PCR

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Methode

RNA extraction RT-PCR Sequencing Genetic analysis

PRRSV infected samples RNA extraction RT-PCR

Vaccine

+ BSL-PS 100 + Porcillis + AmerVac + IngelVac

Sequencing Phylogenetic

NSP 2 F : ’-AAA GAC CAG ATG GAG GAG GA-’ NSP 2 R : ’-GAG CTG AGT ATT TTG GGC GTG-’

Amplifying NSP2 gene

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Phylogenetic of PRRSV based on NSP2 gene

(Vo KH. And Nguyen NH., 2011) Deletion of 87 nucleotide NA EU

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(93.9 – 96.1%) (91.4 – 99.1%)

Conclusion

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 PRRSV

isolates in Vietnam had deletion

• f

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.

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PMWS

wasting palpable lymphadenopathy

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Hội chứng gầy còm sau cai sữa

Post Weaning Multisystemic Wasting Syndrome

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Gene Genetic tic analysis analysis of PCV in Vie

• f PCV in Vietnam

nam

PMWS

Intestina juice in breast cativy Intestina juice in abdominal cativy Stuffing spleen+ hepatitis

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Hội chứng gầy còm sau cai sữa

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Gene Genetic tic anal analysis of PCV in Vietna ysis of PCV in Vietnam PCV in Vietnam?????

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(Long JG. et al., 2010)

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

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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.

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???? 1.1, 1.2, 1.3 2.2, 3.3 ?????? 2.1 1.1, 2.1 2.2, 2.3 2.1 2.2, 2.3

Distribution of CSFV in Asian

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Phylogenetic of CSFV based on E gene

Classical swine fever (CSF) caused by a pestivirus (CSFV)

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Phylogenetic of CSFV based on E gene

(Tran TD., 2008)

95%-98% 92%-93%

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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.

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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

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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)

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Methods f Methods for det r detecting and q cting and quatifying atifying PRRS PRRSV

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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)

specific test Sensitivity test Melt curve analysis Tandard curve

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Conclusion

 The sensibility of the assay was 101 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 83.4oC and that of US PRRSV amplicon was 84.4oC.  EvaGreen-based real time RT-PCR quick, sensitive and accurate results for quantifying and genotyping PRRSV , which was used to determined genotypes.

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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)

specific test sensitivity test Tandard curve

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Conclusion

 The developed assay is specific for both NA type and Chinese

type.

 Sensibility assay met 101 copies/ml of sample from infected

pigs.

 The developed method for detecting and quantifing PRRSV

NA type is a realized method that is usefμl for preventing and controlling disease caused by PRRSV .

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Metho Methods f s for det r detecting and q cting and quatify atifying ng PRRS PRRSV

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LAMP (loop-mediated isothermal amplification) Notomi, 2000)

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The mechanism of LAMP reaction

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Methods f Methods for det r detecting and q cting and quatifying PRRS atifying PRRSV

 LAMP (loop-mediated isothermal amplification)

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Methods f Methods for det r detecting and q cting and quatifying atifying PRRS PRRSV

 LAMP (loop-mediated isothermal amplification)

Gel assay

Eye assay

specific test

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Methods f Methods for det r detecting and q cting and quatifying atifying PRRS PRRSV

 LAMP (loop-mediated isothermal amplification) sensitivity test

Gel assay

UV assay Eye

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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 .

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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 Hưng, NguyễnVăn Chí, Lê Nguyễn Ngọc Hạnh, TrầnThị Dân, Trần

Thị Bích Liên, Nguyễn Đình Quát. quatifing and genotyping of porcine reproductive and respiratory syndrome virus by real-time rt-pcr using evagreen. Accepted by Journal

• f veterinary sciences and technology 2011.

 Võ Khánh Hưng, NguyễnVăn Chí, LêVinhThắng, Lê Nguyễn Ngọc Hạnh,

TrầnThị 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

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Phylogenetic of PRRSV based on ORF5 gene

(Vo KH. And Nguyen NH., 2010)

01NP1.2 BJ-4 S1 RespPRRS MLV BSL-100 VR2385 PA8 PL97-1 HN1 VR-2332 F1 Kitasato 93-1 PrimePac SP P129 Ingelvac PRRS JA142 CH-1a HB-2 GD-1 HB-1(sh) 90/HCM/VN 91/HCM/VN T2/HCM/VN T5HCMVN 3428/DN/VN 82/DN/VN 07QN SY0608 07HEN 07BJ 07HEBTJ EuroPRRSV Amervac PRRSV Porcilis LV B13 Lelystad

60 80 86 76 99 99 99 96 88 71 31 60 20 30 32 93 62 93 54 44 40 39 62 97 26 35 51 27 33 44 99 0.02

2.1 2.3 2.2 1

89.7-99.7 % 89.4 – 100 % 95.5 – 100 % 95.7 % 98.5 - 99.8 % 98.5 – 99.7 % 87.9 - 88.6 %

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 Porcine Reproduction and Respiratory Syndrome (PRRS) caused by a

Arterivirus is a highly contagious viral disease of pigs. The gene encoding glycoprotein 5 (ORF5) of porcine reproductive and respiratory syndrome virus (PRRSV) isolates from Ho Chi Minh city, Dong Nai and Ba Ria Vung Tau provinces of Viet Nam and a North American- type vaccine, BSL-PS100 (currently available in this country) were analysed by using RT-PCR and sequencing. Sequences were then compared with other PRRSV sequences available through GenBank. Results showed that, All the studied PRRSV isolates belonged to subgroup 2.2 of North American (NA) type and has high similarity to high virulent china isolates of PRRSV (98,5 - 99,7 %). Mealwhile, the currently available vaccine in Viet Nam, BSL-PS100, were clustered in subgroup 2.1 and was distantly related to the PSSRV isolates from the studied provinces in Viet Nam (87,9 – 88,6 %). These results may contribute to the knowledge of PRRSV epidemiology in HCMC, Dong Nai, Ba Ria Vung Tau

• f Viet Nam and may help to take into consideration for control and

preventive measures by vaccinating to protect pigs in HCMC, Ba RiaVungTau and Dong Nai from PRRSV infection.

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Phylogenetic of PRRSV based on ORF7 gene

(Vo KH. And Nguyen NH., 2010)

4/BRVT/VN 82DNVN 3428DNVN VD/BRVT/VN 07BJ 07HEBTJ 07/QN/VN SY0608 T2HCMVN T5HCMVN 07HEN CH-1a HB-2 P129 Ingelvac PRRS ATP JA142 1/BRVT/VN 6/BRVT/VN PrimePac SP F1 HN1 BS-PS100 PA8 BJ-4 RespPRRS MLV PL97-1 01NP1.2 S1 VR-2332 EuroPRRSV Amervac-PRRS LV LV4.2.1 Porcilis PRRS

56 46 99 99 98 95 79 78 67 54 20 38 92 40 26 17 30 34 61 80 74 58 64 25 11 0.02

94.6 – 100 % 1 2.1 90.6 – 100 % 92.7-100 %

2.2 2.2

100 % 89.5 – 91.9 % 90.6 – 91.7 % 98.7 – 100 % 100 % 98.7 - 99.2 % 91.1 % 93 – 93.5 % 93.8 %

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Nucleocapsid protein N (ORF7) protein bearing a RNA binding domain is suggested to play a role in viral replication, is highly antigenic and is the abundant protein in virions, and has been useful for the diagnosis of PRRS. To more fully understand the genetic diversity

• f PRRSV in south Viet Nam, the ORF7 sequences of two PRRSVs isolated from HCMC, two

PRRSVs isolated from Dong Nai province and four PRRSVs isolated from Ba Ria Vung Tau was analyzed, compared and used for buiding their phylogenetic tree. The results showed that almost studied Dong Nai, Ba Ria Vung Tau and HCMC isolates belong to subgroup 2.2 of the North American (NA) type and has high identity with PRRSVs isolated in mainland China from 2006 and 2007. Especially, there are two Ba Ria Vung Tau isolates (1 BRVT VN and 6 BRVT VN) separated from other studied isolates and other virus PRRSV on over the world and have the trend of seting up a new group in phylogenetic tree of PRRSVs. These isolates have low identify with other stuied isloats (89,5 – 90,3 %) and BSL-PS vaccine (91,1 % ). Moreover, the BSL-PS100 (Besta, Singapor) vaccine, were popularly used in Viet Nam for PRRSV vaccination, were clustered in subgroup 2.1 and was distantly related to the PRRSV isolates from studied provinces. These results may contribute to the knowledge of PRRSV epidemiology in HCMC, Dong Nai, Ba Ria Vung Tau of Viet Nam and may help to take into consideration for control and preventive measures by vaccine to protect pigs in Dong Nai, Ba Ria Vung Tau and HCMC from PRRSV infection.

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Porcine Reproduction and Respiratory Syndrome caused by a Arterivirus (PRRSV) is a highly contagious viral disease of pigs. Most of detected Reproductive and Respiratory Syndrome virus in Vietnam belong to North American type and chinese type. To gain a better understanding of the genetic diversity and evolution of PRRSV in Vietnam, the nsp2 genes of 18 PRRSV strains collected in the North Vietnam (Ha Noi capital and Bac Giang province) and South of Vietnam (Ho Chi Minh city, Dong Nai province and Binh Duong province) in 2009 and 2010 were partially

• sequenced. These sequences were then analyzed along with some PRRSV strains that is isolated all
• ver the world. The result indicated that Nsp2 gene of all 18 PRRSVs isolated in our studies had

deletion of 87 nucleotides and were the same to PRRSVs isolated from China in 2007. Sequence analysis indicated that the homology between studied PRRSVs in Vietnam and chinese type PRRSV isolated at china in 2007 were high (93.9 – 96.1%) in the nucleotide sequence. On other hand, The identity of nsp2 genes between five isolates from North Vietnam (4 isolates from Bac Giang, 1 isolates from Ha Noi) and thirteen isolates from South Vietnam (8 isolates from Ho Chi Minh city, 4 isolates from Binh Duong and 1 isolates from Dong Nai) were high (91.4 – 99.1%). Beside that, the result show that almost isolates from each province is different in comparision with another

• province. Also, the result indicates that there were the genetic evolution of PRRSV along the time in

2009 and 2010. This study was undertaken to assess the regional variation of prevalent PRRSV and to establish a sequence database for PRRSV molecular epidemiological studies.  Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine.

Increasing evidence indicates that a variant strain of porcine circovirus (PCV), designated type 2 PCV (PCV-2), is responsible for PMWS. So, detecting, typing and analysing phylogenetic tree of PCV2 is important for diagnosis, typing and molecular epidemiological study on PCV2. A part of ORF2 that encoded capsid protein of PCV2 was sequenced and used for genetic analysis. In this study, 13 isolates from Dong Nai province and Ho Chi Minh city (HCMC) was sequenced and used to build phylogenetic tree with some reference isolates in over the world from genebank. The result show that all of the isolates belong to PCV type 2b and 2d. Specifically, 1 isolate from HCMC and 4 isolates from Dong Nai province belong to PCV2 type b ( with the similar is 98.8 – 99.5 % in nucleotid sequence); 1 PCV isolate from HCMC and 7 isolates from Dong Nai province belong to PCV2 type d which is new subgenotype prevalent in china. 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. This result show that the situation of PCV2 infection at some farm in Dong Nai province and HCMC is complicated. Of 13 isolates in this study, the majority belonged to PCV2 type d (61.54%), indicating that subgenotype PCV2 typd d viruses are predominate genotype circulating in studied regions. The more studies in molecular epidemiologic of PCV2 in other regions in Vietnam the more knowledge we get for prevention and treatment PMWS epidemic caused by PCV in Vietnam.

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 Porcine reproductive and respiratory syndrome (PRRS) caused by PRRSV is

detrimental to economy. There are two types of PRRSV , being North American (NA) type and Europe (EU) type. EvaGreen-based real time RT-PCR protocol was established for quantifying and genotyping PRRS virus, in which F7 and R7 primers were designed on ORF7 of the virus. A standard curve used in the assay was created by a plasmid DNA template. The whole ORF7 of PRRSV genome was inserted into pGEMT Easy plasmid, then, cloned into E.coli DH5α. The sensibility of the assay was 101 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 83.4oC and that of US PRRSV amplicon was 84.4oC. The results showed that EvaGreen-based real time RT-PCR assay could offer quick, sensitive and accurate results for quantifying and genotyping PRRSV , which was used to determined genotypes and viral load of blood samples and swab from post weaning pigs.

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