Study On The Infection Of Rhesus Monkey CD11c+ Cells In KM Strain Of Human Norovirus

[Objective]Noroviruses(NOVs)is a single-stranded RNA virus.Viral infection mainly causes acute viral gastroenteritis in humans.It often causes a violent epidemic.However,because its genome is easy to mutate,it is difficult to culture in vitro,no effective norovirus vaccine has been developed at present.In this study,norovirus was isolated and identified from clinical specimens.Through human B cells and rhesus monkey PBMC infection experiments,flow sorting and Real-Time PCR techniques were used to explore the best cell infection model of norovirus,so as to provide experimental basis for further study of norovirus animal model and development of effective vaccine.[Methods]The main contents of the study include two parts,First of all,The nucleic acid were extracted from the clinical samples obtained from CDC in Kunming and then amplified by PCR reaction.The sequences of 694 to 1020 loci between Norovirus ORF1 and ORF2 were obtained by sequencing with specific primers.After online BLAST,sequence analysis and phylogenetic tree analysis showed that it was highly homologous to norovirus G?.15.The second part is mainly about the experiment of virus infection.Firstly,human norovirus KM strain was inoculated into human B cell culture,and the pathological changes of cells after passage and the changes of virus expansion at different infection time were observed.Further understanding of norovirus particles by electron microscopy and Western Blot.To further study the value-added of norovirus on human B cells,the standard curve was constructed by synthesizing standard samples,and the genome amplification in passage culture was analyzed by Real-Time PCR to complete the amplification passage of virus.The genome amplification in subculture was analyzed by Real-Time PCR,and the preservation of virus was completed,and then the whole gene sequence of the virus was obtained by the second generation sequencing of the virus genome,and the source of the human norovirus strain was further determined by constructing the evolutionary tree analysis.Then the human norovirus KM strain infected rhesus monkey PBMC was analyzed,first isolated from fresh monkey blood PBMC,then inoculated with human norovirus KM strain in PBMC culture to observe the occurrence of cell lesions after passage.Finally,different cell groups were selected by FACS,and the amplification of human norovirus in different cell groups was analyzed by Real-Time PCR analysis.[Results]By the first part of the experiment,we identified and named the human norovirus KM strain,obtained the whole gene sequence of the virus strain.Part of the short gene fragment between norovirus ORF1 and ORF2 was obtained by PCR sequencing with specific primers.After online BLAST,sequence alignment and phylogenetic tree analysis showed that it was highly homologous to norovirus G ?.15.The second part of the experiment,by constructing the standard curve of human norovirus,using the Real-Time PCR to quantitatively detect the virus,found that human norovirus KM strain can infect human B cells and carry out the value of passage in the cells,and 48 h after infection,the maximum copy value can be obtained when the virus is transmitted to the fifth generation.Norovirus G?/H u/JP/2007/GII.P15_G?.15/Sappo-ro/HK299 is highly homologous;At the same time,it can be seen by flow sorting that the cells isolated from different CD groups rhesus monkey blood have different sensitivity to norovirus,among which CD 11c cells have the highest sensitivity to norovirus.[Conclusions]The human norovirus KM strain is highly homologous to the G?.15 type norovirus.It can infect human B cells and carry on the passage increment in the cells,and is highly susceptible to the CD11c+cells isolated from the whole rhesus monkey blood,which further advances the research progress on animal models of norovirus infection.