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A Rapid and sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

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Virologica Sinica 2012年第1期27卷 1-9页

作者： Vinayagamurthy Balamurugan arnab sen Gnanavel Venkatesan Vinita Yadav Vandna Bhanot Veerakyathappa Bhanuprakash Raj Kumar Singh Division of Virology Indian Veterinary Research InstituteMukteswar 263138UttarakhandIndia Project Directorate on Animal Disease Monitoring And Surveillance(PD-ADMAS) Bangalore 560024KarnatakaIndia Animal Health Division ICAR-NEH RegionMeghalaya 793103India Indian Veterinary Research Institute Bangalore 560024KarnatakaIndia National Research Centre on Equines Hisar 125001HaryanaIndia

A sensitive and rapid single step real time （rt） RT-PCR was standardized using one-step Brilliant SYBR Green kit for detection and semi-quantitation ofpeste des petitis ruminants virus （PPRV） using the virus RNA and matrix （M） protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit （sensitivity） of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was -0.0001 cell culture infectious dose 50% units （TCID50）. Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection （dpi） and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

关键词： PPR M gene SYBR green RT-PCR Early diagnosis Clinical samples

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Study on Passive Immunity:time of Vaccination in Kids Born to Goats Vaccinated Against Peste des petits ruminants

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Virologica Sinica 2012年第4期27卷 228-233页

作者： Vinayagamurthy Balamurugan arnab sen Gnanavel Venkatesan Kaushal Kishor Rajak Veerakyathappa Bhanuprakash Raj Kumar Singh Division of Virology Indian Veterinary Research InstituteMukteswar 263138Nainital DistrictUttarakhandIndia Project Directorate on Animal Disease Monitoring and Surveillance(PD_ADMAS) HebbalBangalore 560024India Animal Health Division Indian Council of Agricultural Research-North Eastern HillUmiam 793103India Indian Veterinary Research Institute Bangalore 560024India National Research Centre on Equines Hisar 125001India

In this study,the decay of maternal peste des petits ruminants virus(PPRV) antibodies in kids born to goats vaccinated with Asian lineage IV PPR vaccine and the efficacy of passive immunity against PPRV was assessed to determine the appropriate period for vaccination in *** samples collected from kids born to vaccinated,unvaccinated and infected goats at different time intervals were tested by PPR competitive ELISA and serum neutralization test(SNT).Maternal antibodies in kids were detectable up to 6 months with a decline trend from the third month onwards and receded below the protective level by the fourth *** kid with an SN titre of 1:8 at the time of immunization showed significant PPRV specific antibody response(percentage inhibition of 76;SN titers >1:16),when tested on 21 day post-vaccination and was completely protected from infection upon virulent PPRV ***,the kid with 1:8 SN titers was completely protected from PPR infection on active ***,PPR vaccination is recommended in kids,aged 4 months and born to immunized or exposed *** could be a suitable period to avoid window of susceptibility in kids to PPRV and the effort to eliminate PPR infection from susceptible populations.

关键词： Peste des petitis ruminants Goats Vaccination Passive immunity Evaluation Time of vaccination in kids

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Evaluation of Efficacy of Stabilizers on the Thermostability of Live Attenuated Thermo-adapted Peste des petits ruminants Vaccines

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Virologica Sinica 2011年第5期26卷 324-337页

作者： Thachamvally Riyesh Vinayagamurthy Balamurugan arnab sen Veerakyathappa Bhanuprakash Gnanavel Venkatesan Vinita Yadav Raj Kumar Singh Division of Virology Indian Veterinary Research InstituteCampus Mukteswar 263138Nainital DistrictUttarakhandIndia Veterinary Type cultures Centre National Research Centre on EquinesSirsa RoadHisar 125001HaryanaIndia Project Directorate on Animal Disease Monitoring And Surveillance (PD-ADMAS) HebbalBangalore 560024India National Research Centre on Equines Sirsa RoadHisar 125001HaryanaIndia

In this study, thermo-adapted （Ta） PPR vaccines were assessed for their stability at 25, 37, 40, 42 and 45℃ in lyophilized form using two extrinsic stabilizers {lactalbumin hydrolysate-sucrose （LS） and stabilizer E} and in reconstituted form with the diluents （1 mol/L MgSO4 or 0.85% NaC1）. The lyophilized vaccines showed an expiry period of 24-26 days at 25℃, 7-8 days at 37℃ and 3-4 days at 40℃. LS stabilizer was superior at 42℃ with a shelf-life of 44 h, whereas in stabilizer E, a 40 h shelf-life with a comparable half-life was observed. At 45 ℃, the half-life in stabilizer E was better than LS and lasted for 1 day. Furthermore, the reconstituted vaccine maintained the titre for 48 h both at 4℃ and 25℃ and for 24-30 h at 37℃. As both the stabilizers performed equally well with regard to shelf-life and half-life, the present study suggests LS as stabilizer as a choice for lyophilization with 0.85% NaCldiluent, because it has better performance at higher temperature. These Ta vaccines can be used as alternatives to existing vaccines for the control of the disease in tropical countries as they are effective in avoiding vaccination failure due to the breakdown in cold-chain maintenance, as this vaccine is considerably more stable at ambient temperatures.

关键词： PPR Thermo-adapted Vaccines Stabilizers Diluents Thermo-stability

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Cytokines Expression Profile and Kinetics of Peste des petits ruminants Virus Antigen and Antibody in Infected and Vaccinated Goats

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Virologica Sinica 2012年第4期27卷 265-271页

作者： Arun Patel Kaushal Kishor Rajak Vinayagamurthy Balamurugan arnab sen Shashi Bhusan Sudhakar Veerakyathappa Bhanuprakash Raj Kumar Singh Awadh Bihari Pandey Division of Virology Indian Veterinary Research InstituteMukteswar 263138Nainital DistrictUttarakhandIndia Project Directorate on Animal Disease Monitoring And Surveillance(PD_ADMAS) Bangalore 560024KarnatakaIndia Animal Health Division Indian Council of Agricultural Research-North Eastern Hill RegionMeghalaya 793103India Indian Veterinary Research Institute Bangalore 560024KarnatakaIndia National Research Centre on Equines Hisar 125001HaryanaIndia

The present study deals with the co-ordination of cytokine （IL-4 and IFN-7） expression and kinetics of peste des petits ruminants （PPR） virus antigen and antibody in PPRV infected and vaccinated goats. The infected animals exhibited mixed cytokine （both TH1 and TH2） responses in the initial phase of the disease. The infected and dead goats had increased IFN-T response before their death; while IL-4 remained at the base level. The cytokine expression in recovered animals was almost similar to that of vaccinated ones, where a unique biphasic response of IL-4 expression was observed with an up-regulation of IFN-T on 7th days post vaccination （dpv）. Analysis of PPR virus antigen and antibody kinetics in different components of blood from infected and vaccinated animais revealed that the PPR virus antigen load was highest in plasma followed by serum and blood of the infected animals, whereas vaccinated animals showed only marginal positivity on 9th dpv. The antibody titer was high in serum followed by plasma and blood in both vaccinated and infected animals. Therefore, it is inferred that the presence of antigen and antibody were significant with the expression of cytokine, and that a decreased response of IL-4 was noticed during intermediate phase of the disease i.e., 7 to 12m days post infection （dpi）. This indicates the ability to mount a functional TH2 response after 14th dpi could be a critical determinant in deciding the survival of the PPR infected animal.

关键词： Peste despetits ruminants (PPR) Goat Cytokines Real time RT-PCR ELISA

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