Isolation, Identification Of Porcine Hemagglutinating Encephalomyelitis Virus And Screening Of Virus Receptors

Porcine hemagglutinating encephalomyelitis causes by hemagglutinating encephalomyelitis virus (HEV). HEV causes two distinct types of symptoms in sucking piglets either encephalomyelitis, or the vomiting and wasting disease. The mortality rate for piglets under 3 week old is 20%-100%. Some serological surveys on of HEV infection showed the positive rate for antibodies to HEV was very widespread, probably global. It was reported that HEV infection outbroke in mainland and Taiwan province of China. More morbidity and mortality resulted in severe economic expenses in pig industry.In order to determine the cause of death of the suspected HEV case in JiuTai county of Changcun district, Virus isolation and systematic identification were carried out. The results showed one HEV strain was obtained and it was named HEV-JT06. The sero-epidemiological investigations had been conducted in order to determine the prevalence of HEV infection in Jilin province. The antibodies to HEV-67N in the sera of 1117 pigs from 36 herds in 5 districts of Jilin province were detected by using the haemagglutination/ haemagglutination inhibition (HA/HI) test. In the 5 districts, 52.4% sera were positive. Especially in Changchun district, the positive rate of the sera was highest, 81.6%. These data provide an important reference for prevention and cure of porcine diseases in Jilin Province.For understanding HEV-JT06 strain in molecular biology, cloning, sequencing stepwise, spliceing and evolutionary analysis were conducted for five complete genes of HEV-JT06. The results were analyzed by using DNAStar software, DNAMAN 4.0 software and ExPASy Proteomics Server. The data showed amino acids were changed in different degree in five structral genes of HEV-JT06 strain. More homology appeared between HEV-JT06 and HEV-67N (GenBank accession number: AY078417). And the potential N-glycosylation sites in deduced HE and S protein for strain JT06 were changed, respectively. But these resultsdidn't infer that how high or low immunogenicity HEV-JT06 strain had. Phylogenetic tree built by UPMGA algorithm showed there were most closely evolutionary relationship between HEV-JT06 strain and HEV-67N, and HEV-JT06 strain was evolved from HEV-67N strain of North America germ line.Some studies indicated S protein in coronavirus played an key effect during the process of infection. So, S1 protein of HEV was expressed in Pichia pastoris yeast and purified by using the Ni-NTA affinity columnin. PK cytomembrane proteins were extracted and transferred to NC membrane. After coloration, there appeared a limpid protein band about 90kDa on the NC membrane, which inferred that it maybe the receptor of HEV S1 protein.T7 phage display library of PK cells was constructed for sieving the overall cytomembrane receptors of HEV. The data showed that the library contained 1.02×10⁷ clones, and approximately 96% of the library were recombinant. The titer of the amplied library was 7×10¹⁰ pfu/mL. The cDNA fragments longer than 300bp in length were 90% of 100 plaques by PCR identification. The HEV that were purified by sucrose gradient centrifugation were used to screen the library. The cDNA inserts in these plaques were amplified by PCR using primers flanking the inserts. The phage DNA were sequenced and nucleotide sequences were compared by BLAST searches with the GenBank database. Amino acid sequences were analyed through ExPASy Proteomics Server and DNAStar software. About 100 clones were then randomly picked from individual plaques, and their DNA sequences were PCR amplified and analyzed on agarose gel to determine the size of the inserts. PCR analysis showed that 60% of the phage clones had an insert size of 400bp. The sequence showed that this insert was 430 bp in length and contained a 363bp open reading frame (ORF), which was predicted to encode 121 amino acids. This protein was designated PKCP1 and high homology appears between PKCP1 and Sus scrofa mRNA. Analytic result of PKCP1 protein showed the protein had the characteristic of hydrophilicity, immunogenicity and good antigenicity. This study will provide the materials for developing vaccine of HEV infection. Moreover, these results establish a foundation of studying the infectious mechanism mediated by receptors.