Research Of Replication Of PPRV N75-1 Strain In Goat Endometrial Epithelial Cells

Peste des Petits Ruminants(PPR), caused by Peste des Petits Ruminants Virus(PPRV), is a acute and highly contagious disease that mainly infects ruminants such as sheep and goats. PPR was identified as infectious disease by Office International Des Epizooties(OIE), and it is the First Class contagion in China. In recent years, PPR has broke out or been spreading in many countries which has a huge threat to sheep industry and public health in those countries. It is very important to intensify research on the analysis of detection and replication of PPRV. In this study, according to the sequences of N gene of PPRV, we designed a couple of primer for N gene and construct vector. In this study, we develop a quantitative RT-PCR method by diluting gradient standard curve of qRT-PCR. We detect and analysis the replication regulation of PPRV N75-1vaccine strain in the goat endometrial epithelial cells(EEC) by using qRT-PCR established above, and expression pattern of N protein of PPRV N75-1vaccine strain in EEC was detected by Western blot assay. The results obtained in this study were as follows:1. The method of quantitative RT-PCR detecting PPRV is a sensitive, specific, and reproducible way to make a quantitative detection of PPRV. The results showed that the established of correlation coefficient of standard curve R2 =0.973, the amplification efficiency is 126.0%.2. PPRV could cause cytopathic effect(CPE) on EEC: slight CPE began to show up at the 48 h post inoculated with PPRV, and it was obvious after 72 h. when came to 96 h, EEC started to lysis. Collecting EEC inoculated with PPRV at MOI 10 for 0h, 1h, 3h, 6h, 9h, 12 h, 24 h, 48 h, 72 h, the transcription level of N gene of PPRV was detected by qPCR. The relative high levels of PPRV were detected at 3 h post inoculation(p.i.), and marked increased virus levels were detected at 6 h p.i., and peaked at 9 h p.i. A decreased level of PPRV was detected at 12 h p.i., and the viral levels do not changed until 72 h p.i. The expression of PPRV N protein detected by Western Blot showed similar results. The expression levels of PPRV N protein were detected as early as at 1 h p.i., and an increased level was shown at 6 h p.i., and decreased at 12 h p.i.In this study, we established a method of qRT-PCR detecting N gene of PPRV, and make analysis on the replication regulation of PPRV N75-1vaccine strain in EEC. The study can make an experimental basic for the further research of mechanism that PPRV damage the reproductive system of goat, and it also has theoretical significance and potential application value to control PPR.