Establishment And Evaluation Of New,rapid And Practical Assays For Peste Des Petits Ruminats (PPR)

Peste des petits ruminants(PPR)is an acute and contagious disease in ruminants.PPR is characterized by fever,stomatitis,oculo and nasal secretion,pneumonia and diarrhea.The causative agent of PPR is the peste des petits ruminants virus(PPRV).It is a negative,single-stranded RNA virus of the family Paramyxoviridae,genus MorbillivirusPPRV primarily affects sheep,especially goats with extremely high morbidity,several wild ruminants can also be infected experimentally.PPR is one of the notifiable disease of the World Animal Health Organization(OIE)and classified into list A of the List of Quarantine Diseases for the Animals Imported to the People’s Republic of China.The first case of PPR was reported in Cote d’Ivoire in 1942.To date,the PPRV epidemic has been found in more than 70 countries,ranging from the Middle East,Africa to Asia.As an animal infectious disease crossing geographical borders,PPR has caused severe consequences,especially on the development of animal husbandry in the developing countries.Given the severe situation of the widespread disease,the World Organization for Animal Health(OIE)and the Food and Agriculture Organization of the United Nations(FAO)launched the Global Strategy for the Control and Eradication of PPR(GCES)in 2015.In China,the disease was first officially reported in Tibet in July,2007.Since March of 2014,a series of outbreaks were reported in several provinces of China.Due to its rapid speed of spread,huge span and high risk of spread,the Ministry of Agriculture of China has lunched the eradication program of PPRV(2016-2020),relgulated new measures of the prevention and eradication of PPRV.Based on the successful experiences of the eradication of the Rinderpest around the globe,it is vital for PPRV control and eradication to establish the rapid and sensitive laboratory and on-site detection assays.Characterized by time-consuming,effort-taking or low specificity and sensitivity,conventional laboratory methods such as virus isolation test,neutralization test,agar gel diffusion test cannot meet the requirements of rapid and sensitive detection,timely vaccination and control.While for the new methods such as RT-PCR and the real-time RT-PCR,the professional detection instruments and staff were necessary,which are usually not available in remote areas for an onsite test.Meanwhile,due to the widely use of PPRV live vaccine,it is difficult to distinguish the vaccine strain of PPRV from the wild strain of PPRV.To achieve the goal of freedom of PPRV of the zone wih vaccination,three practical assays were established and evaluated in this research targeting at timely on-site post vaccination evaluation(PVE)of PPR,serological test in laboratory for PPR quarantine and eradication and differentiation of of infected and vaccinated animals(DIVA).The established assays provide a set of methods from pen side to the laboratory and differentiation of diagnosis to achieve the goal of control and eradication of PPRV.The summaries of the specific research are as follows:1.Establishment and evaluation of QDs-LFIAS of PPRV.The highly luminescent water-soluble carboxyl-functionalized quantum dots(QDs)were used as the signal output and were conjugated to streptococcal protein G(SPG),which was capable of binding to immunoglobulin G(IgG)from many species through an amide bond to capture the target peste des petits ruminants virus(PPRV)IgGs.When exposed to PPRV IgG,QD-SPG bound to AcNPV-PPRV-N protein,which was expressed by the the recombinant baculovirus,resulting in the formation of a complex that subsequently produced a bright fluorescent band in response to 365 nm ultraviolet excitation.A lateral-flow immunoassay strip(LFIAS)combining with QDs for detection of PPRV antibody was established.The analytical sensitivity evaluation showed that the QD-LFIAS limit of detection(LOD)for PPRV antibody was superior to competitive enzyme-linked immunosorbent assay(C-ELISA)and the immunochromatographic strip(ICS).For the analytical specificity,no cross reaction was observed when the positive sera of bluetongue virus,canine distemper virus,goat pox virus,and foot-and-mouth disease virus were tested.Further evaluation using field samples indicated that the diagnostic specificity and sensitivity of the QD-LFIAS was 99.47% and 97.67%,respectively,with excellent agreement between QD-LFIAS and C-ELISA.The developed QDs-LFIAS is a fast and ultrasensitive test-strip system especially for on-site,point-of-care diagnosis and post vaccination evaluation(PVE).2.Establishment and evaluation of DAS-ELISA against PPRV antigen in serumHybridising the spleen cells of immuned mice with myeloma cells(SP2/0),filtering to obtain the hybridoma cell(5B11)which was able to secrete stable PPRV monoclonal Antibody(MAb),with this MAb and specific egg yolk immunoglobulin(IgY)against PPRV,a double antibody-based sandwich enzyme linked immunosorbent assay(DAS-ELISA)for the detectioin of the serum antigen of PPRV was developed and evaluated.Specific tests indicate that PPRV positive samples were confirmed while other virus such as BTV,EHDV,VSV,FMDV samples confirmed negative.Repetitive tests reveals that the CV of intra-assay lies between 2.14%~6.18% and CV of the interexperiment lies between 4.82%~8.37% with favourable results.Clinical samples tested by DAS-ELISA and RT-PCR simultaneously indicate that the diagonostic specificity and sensitivity are 99.2% and 93.9% respectively.The agreement of both methods is 98.1%.This assay is especially designed for the purpose of rapid serological diagnosis of PPRV inffected animal with or without vaccination and for the epidemiology investigation.3.Establishment and evaluation of real-time RT-PCR assays intended for the differentiation of infected and vaccinated animals(DIVA)of PPRBased on the whole sequences of the genome of attenuated vaccine strain of PPRV(Nigerai 75/1)used in Tibet,China,two sets of optimized primers and probes were designed for the development of the real-time RT-PCR assay intended for DIVA.The specificity test showed that the attenuated vaccine strain of PPRV could only be amplified by YM real-time RT-PCR,while viruses of control group and wild strain of PPRV failed to show an amplification signal,and the wild strain of PPRV could only be amplified by XZ real-time RT-PCR,while viruses of control group and the vaccine strain of PPRV failed to show an amplification signal.The limit of detection(LOD)of the YM real-time RT-PCR and the XZ real-time RT-PCR was 1.38pg/μL and 0.16pg/μL respectively.The developed YM real-time RT-PCR and XZ real-time RT-PCR assays are suitable for DIVA of PPR in China and PPRV surveillance,prevention and furher more eradication by the end of 2020.