| Japanese encephalitis(JE),a disease that usually threat to the both human and animal.It was caused by Japanese encephalitis virus(JEV)and spread by mosquitoes.The swine are usually considered as the most susceptible animals to the infection. Generally appearing as sporadic and latent, this infection can result in sow reproduction disorder and thus, great economic losses in the development of pig raising industry. Therefore, the control of JEV can not only minimize damages for pig raising industry, but also be of significance for human health.To better understand the genetic evolutionary relationship of JEV, this study focuses on the complete genome sequence of JEV wild strains JEV/sw/HN-2/2009 amplified and determined by RT-PCR. The length of the total genome sequence measured is 10977 bp. Comparing the whole genome and E genome of JEV wild strains JEV/sw/HN-2/2009 and 34 virus sequences taken from Gen Bank as reference, the nucleotide and amino acid sequence homology can be analyzed by Meg Align software. The results revealed that the isolated strain JEV/sw/HN-2/2009 and the attenuated live vaccine strain SA14-14-2 share the highest sequence identity of their entire gene nucleotide and amino acid sequence, 99.7% and 99.6% respectively. The 35 JEV strains belonging to 5 different genotypes, the isolates and vaccine strains SA14-14-2 are on the branches of genotype Ⅲ, and they have a close genetic relationship. Meanwhile, comparing the sequence of the isolates and the vaccine strain, we analyzed the differences in nucleotide and amino acid sites by Meg Align software. It is detected that the 10701 st bits of the fragment 3′-UTR in JEV/sw/HN-2/2009 was inserted a Base G. Also, there are differences between these two strains, for instance, 31 nucleotide and 13 amino acid sites, which are mainly distributed in the NS2 b and NS5 protein area.To develop a JE vaccine which is more effective and suitable for large-scale production.In this study,porcine isolate JEV/sw/GZ/09/2004 was proliferated in sysceptible strains of Vero cells.To proliferating strain JEV/sw/GZ/09/2004 on Vero cell while carrying out a series of optimization on virus multiplication condition and expand culture, a large amount of high titer virus solution with up to 107.6/m L of TCID50 cultured in spinner flask can be harvested, which lays the foundation for the preparation of JE vaccine. After optimizing inactivation and emulsification of JEV virus solution, good preparation for oil emulsion inactivated vaccine with good stability of strain JEV/sw/GZ/09/2004 were successfully made. The JEV on the body of animals(pigs) model of infection were initially established. After infected by JEV, the symptoms were obvious including a continuous fever rising to 41 degrees Celsius, depression and occasionally accompanied by neurological symptoms. The infected pig dissection showed that the mainly changes were hyperemia of brain, meninges and spinal cord membrane, different degrees swelling of Lymph node and testis, the slight hyperemia of inflammation of testis and epididymis. Viremia mainly appeared in 3~7 days after infection, some of the infected piglets antibody become positive, which establishment of the model lays a foundation for the evaluation of the immune effect of the vaccine.Experiments on immunization efficacy of vaccines were performed,the pigs tested were still keeping its higher antibody level until the 7 weeks later, and the antibody level is better than the commercially available live attenuated vaccine,which showed that JE vaccine of pigs prepared before had higer immunogenicity and security, as well as considerable application prospect. |
PDF Full Text Request