The Study Of Peste Des Petits Ruminants Virus Hemagglutinin Protein And Its Receptor

Peste des petits ruminants (PPR) is an acute, highly contagious and economically important viraldisease of small ruminants, especially goats and sheep. PPR virus (PPRV) is a member of the genusMorbillivirus, family Paramyxoviridae. PPR has been widely spreading since it first occurred in Africa.PPRV infection was officially reported in Tibet, China, in July2007. Like other viruses, the coremechanism of PPRV infection is the binding of virus to specific receptors on the cell surface.Distribution, content and structure of these receptors in animals determine the susceptibility to virusesand tissue tropism. At present, the PPRV receptor characteristics and its interaction with virus remainunclear. Therefore, this study aimed at the characterization of PPRV hemagglutinin (H) protein andvirus receptor-signaling lymphocyte activation factor (SLAM).First of all, we cloned the extracellular fragment of H gene (tH gene) of PPRV Nigeria75/1strainand expressed the tH protein in E.coli BL21(DE3) and yeast GS115, respectively. The exogenous tHproteins were obtained successfully, of which the expression levels were4.68g/L and0.5～1g/L,respectively. Western blotting showed that the recombinant proteins were of good immunogenicity.Caprine SLAM gene was cloned from peripheral blood lymphocyte by RT-PCR. Sequence analysisshowed that the caprine SLAM ORF had1017bp, encoding338amino acids, and that the slamsequences from goat, sheep, cattle, water buffalo, killer whale and dolphin shared high homologies atthe nucleotide and amino acid levels.8amino acid residues in the V region of caprine SLAM possiblyinvolved in determining host-virus specificity were completely conserved in sheep, cattle and waterbuffalo. There were three conserved leucine-rich motifs (TXXYXXV/I/A) in SLAM cytoplasmicdomain. In addition, the recombinant SLAM proteins produced in prokaryotic and eukaryoticexpression system also had good immunogenicity.The V region of caprine SLAM is the functional domain combining with viral proteins, and the keyamino acid residues determine the specificity of host-virus interaction. Therefore, the gene of tH,SLAM and its several deletion mutants which lacked transmembrane and intracellular fragments, asignal peptide fragment or C-terminal fragment (amino acids29-136) or N-terminal fragment (aminoacids137-240), were directionally cloned into eukaryotic expression vectors pcDNA3.1and pEGFP-N1,respectively. The recombinant plasmids were co-transfected into CHO-K1cells, and then the key aminoacid residues of viral protein and SLAM protein interactions was identified initially byco-immunoprecipitation. The results indicated that the N-terminal fragment (amino acids29-136) wasessential to interact with PPRV H protein.In order to determine the relationship between viral tissue tropism and distribution of receptor inhost, in this study the tissue distribution and expression analysis of PPRV and SLAM were analyzed byTaqMan fluorescence quantitative PCR quantified as a fold change (2-ΔΔCtvalues) compared to that ofblood at3days post inoculation (dpi) and immunohistochemical method. The results showed thatSLAM mRNA was detected in all the samples investigated. SLAM antigen positive siginals presentedin lymphoid tissues, digestive system, heart, liver, kidney and cerebrum. With the exception of the pancreas, gluteus, dorsal muscle, or abdominal muscle at different days post inoculation, PPRV nucleicacid was detected in other samples. PPRV levels in thymus, hilar lymph node, tonsil and turbinate bonewere higher than that of other samples. PPRV levels at3,5and28dpi were higher than those at1,7and14dpi, especially at5dpi. PPRV antigen was distributed extensively in respiratory system,digestive system, liver, kidney, heart and cerebellum. In addition, a real-time PCR based on TaqMan technology was established to detect the nucleic acid of PPRV. Blood samples were the most valuablediagnostic material for use in an epidemic situation. The higher levels of RNA detected at28dpi maysuggest some level of persistent infection.In view of the difference between the expression of viral load and SLAM expression, we speculatePPRV may also interact with other receptors. PPRV receptors in peripheral blood lymphocyte (PBL)cell membrane were identified by virus overlay protein binding assay (VOPBA). The result showed thattwo proteins with molecular weight of around38ku and100ku, respectively, likely to combine withPPRV. The characteristics of these two proteins remain to be studied further.