Isolation And Identification Of PPRV GZL-14 And Establishment Of Sandwich ELISA For Viral Antigen Detection And Its Application

Peste des petits ruminants?PPR?is a highly contagious and economically important viral diseases affecting sheep,goats and wild small ruminants.PPR is listed as one of the communicable diseases by OIE and a category infectious disease in China,which pose a severethreat to sheep industry.In this study,we isolate and characterize a PPRV strain named PPRV GZL-14 froman outbreak of an unknown epidemic in a goat/sheep herds characterized by high fever,stomatitis,diarrhea,pneumonia and enteritis with a morbidity and mortality up to 34%,50%expression respectively.Electron microscope observation revealed a large number of virus particles with a diameter of 130-150 nm in the samples collected and the tissue cultures.Sequencing the PCR-amplified fragment revealed it contained the sequence that shared a high sequence identity with PPRV,and isolated virus was named as GZL-14 strain.Complete genome sequence of GZL14 was revealed to contain 16058 bp.The recombinant protein N was expressed,purified and used as antigen to generate antibodies.A sandwich ELISA for detection of peste des petits ruminants virus antigens was developed using antibodies generated against the recombinant proteins N derived from PPRV,and used to investigate the potential PPRV infections in sheep/goat population in Jilin province.The concentrations for antibody coating and antibody conjugated with HRP were optimized using square matrix titrimetry.The criterion for sandwich ELISA was expressed as the average of OD₄₉₀plus 3 standard deviation from 48 PPRV-negative feces samples and determined as positive when sample's OD₄₉₀?0.221.The established sandwich ELISA was demonstrated to have a high specificity and sensitivity with a well repeatability.Detection of the feces samples from Jilin province and Inner Mongolia autonomous regions discovered a various subclinical infection in the clinically healthy sheep/goat herds.The findings in this study provide a solid evidence for the PPRV subclinical infections,thus laying a basis for the prevention and control of this emerging disease in China.