Study On Antigenicity And Animal Model Of Nipah Virus(NiV) Epidemic Strain Based On Pseudovirus

Nipah virus?NiV?is a zoonotic virus with wide host range and high fatality rate^[1-4],it belongs to Paramyxovirinae Paramyxovirinae Henipavirus with Hendra virus?He V?^[5].In late 1998,NiV broke out for the first time in a pig farm in Malaysia^[6].In1999,105 out of 265 human infections were fatal,with a mortality rate of 40%^[7].It was named Nipah virus because it was isolated in nipah town^[8-10].NiV infection leads to Nipah virus disease?NVD?,which can lead to death in severe cases^[11-13].Nipah epidemic was detected in Bangladesh in 2001,followed by outbreaks almost every year^[14,15].In addition,the disease has also been found in India,Cambodia,the Philippines and other places^[16].Sequence analysis showed that Nipah virus was divided into at least two evolutionary branches,the Bangladesh and Malaysia isolates^[17].In Malaysia,the virus passed from fruit bats to pigs,which then to humans,while in Bangladesh,the virus passed directly from fruit bats to humans,followed by human-to-human transmission^[18,19].As there is currently no effective vaccine or drug against this virus,NiV is listed as a priority infectious disease and urgently needs to accelerate research on this virus in 2018^[20].The Centers for Disease Control and Prevention?CDC?classified NiV as a Biosafety Level 4?BLS-4?pathogen due to its high virulence and high fatality rate^[21-27].Therefore,the study of this virus has strict criteria for experimental conditions and experimenters.The design idea of the project is to select and construct all the nipah virus epidemic strains that can be downloaded from the National Center for Biotechnology Information?NCBI?website up to now on the basis of the NiV pseudovirus with high titer constructed in the department.The amino acids of fusion protein?Fprotein?and attachment protein?G protein?of virus strains from different isolation time,different isolation sites and different species sources were analyzed.NiV two proteins are required to infect cells,namely,the F protein and the G protein.In this study,on the basis of the established reference strain,namely the Malaysian strain?Genbank:AF212302?,the gene sequences of F protein and G protein of the following nipah virus strains were synthesized after mutation or codon optimization.The Genbank Numbers of the epidemic strains were FJ513087.1?JN808864.1?AJ627196.1?AY988601.1?MK801755?FN869553.1?AF376747.1?JN808863.1?MH396625.1.The eukaryotic expression plasmids pc DNA3.1-NiV-F and pc DNA3.1-NiV-G of each epidemic strain were constructed,and the HEK293T cells were co-transfected with the HIV skeleton plasmid,and the epidemic strain NiV pseudovirus was packaged.The transfection reagent,skeleton plasmid,mass ratio of membrane plasmid to skeleton plasmid,and the time of virus collection were all consistent with the conditions of the constructed reference pseudovirus.Finally,five pseudoviruses of nipah epidemic strain were successfully constructed,namely:FJ513087.1,JN808864.1,MK801755,JN808863.1,MH396625.1.The neutralization and wide spectrum of DNA vaccines and monoclonal antibodies against the reference strain were evaluated by using the pseudovirus of each epidemic strain of NiV successfully constructed.The results showed that the DNA vaccine designed for the reference strain had a good protective effect on the epidemic strain of NiV,and was broad-spectrum.Among the monoclonal antibodies against the reference strains,F monoclonal antibodies were neutralizing against all the endemic strains.Among G protein monoclonal antibodies,NIG02-1G9 was used against virus strain 3,NIG05-4F1 against virus strain 10,NIG05-3C8 against virus strains 2,9 and10 decreased by more than 4 times compared with the reference strain.Sequence analysis and mutation of the unsuccessfully constructed virus strains were carried out to explore the sites affecting the packaging of the pseudovirus.The results showed that the amino acid sites affecting the virus strain FN869553.1 were 63and 460 respectively.The amino acid site affecting the virus strain AF376747.1 was 348of F protein.The amino acid sites affecting the virus strain AJ627196.1 were 20 and 272G proteins,respectively.The amino acid sites affecting virus strain AY988601.1 were sites 207 and 252 of F protein,respectively.A imaging hamster model of NiV pseudovirus was established based on the high titer pseudovirus.Five?six weeks hamsters were infected by intraperitoneal?IP?.The distribution of the virus in hamsters were observed dynamically by using in vivo imaging technique,main infection viscera are spleen,pancreas,liver,heart.