Isolation And Identification Of Japanese Encephalitis Virus And The Reseach Of Interaction With PK-15 Cells At The MicroRNA Level

Japanese encephalitis virus (JEV), a member of flaviviridae, is a severe pathogen of zoonotic infectious diseases. The virus could cause serious damage to human being through invading central nervous system after infection of human being, causing the patient dead or sufferring from serious neural sequelae. At the same time, JEV can infect swine, resulting in breeding obstacle, leading to seriously restricting the development of swine industry and causing significant economic losses. As a small non-coding RNA of the post-transcription gene regulatory mechanism, miRNA takes part in a wide range of life processes, including growth, differentiation, apoptosis and metabolism. miRNA also plays an important role in viral entry and the antiviral response in the host cells. But no report is available about the interaction reseach between JEV and PK-15 cells at the microRNA Level, related research is very important for a deep understanding of the pathogenic mechanism of JEV. The specific content of papers is as follows:Three strains of JEV (JEV-SC-1, JEV-SC-2 and JEV-SC-3) were isolated from pig abortion stillbirth samples in Sichuan Province through observing cytopathic effect (CPE) and PCR detection. All of the three strains belonged to genotype Ⅲ. The three strains could both cause CPE in BHK-21 cells with titers of 10-581 TCID50/0.025ml.10-512 TCID50/0.025ml and 10-442 TCIDso/0.025ml, respectively. The JEV-SC-1 strain was used to infect BHK-21、Vero and PK-15 cells, respectively; supernatant were collected at different times points post-infection. The established JEV quantitative real-time PCR in the text was used to detect the virus contents of different samples, and the JEV one-step growth curve of different groups was drew respectively. At the same time, observing CPE after JEV infected these three kinds of cells. In result, JEV-SC-1 strain could proliferate in BHK-21、Vero and PK-15 cells and cause different levels of CPE:the CPE began to apper at 36h after JEV infected BHK-21 cells, while at 48h the CPE began to apper after JEV infected Vero and PK-15 cells; at 0-24h after JEV infected BHK-21 cells, virus RNA maintained at a low level, at 24-48h post-infection, virus RNA was undertaken a logarithmic growth, at 48-72h post-infection, the growth rate slowed down, and virus RNA maintained at a high level, then the virus content gradually decreased; at 0-12h after JEV infected Vero cells, virus RNA maintained at a low level, at 12-48h post-infection, virus RNA was undertaken a logarithmic growth, at 48-72h post-infection, the growth rate slowed down, and virus RNA maintained at a high level, then the virus content was falling fast after 72h; at 0-24h after JEV infected PK-15 cells, virus RNA maintained at a low level, at 24-60h post-infection, virus RNA was undertaken a logarithmic growth, at 60-84h post-infection, the growth rate slowed down, and virus RNA maintained at a high level, then the virus content gradually decreased; At the same time, comparing the three one-step growth curves showed that the titer of JEV harvested from Vero cells is higher than BHK-21 and PK-15 cells. The results show that the time of CPE began to apper in JEV infected BHK-21 cells is earlier than Vero and PK-15 cells; while the time of entering the logarithmic period in JEV infected Vero cells is faster than BHK-21 and PK-15 cells, and the virus titer is also higher. This research provided the oretical support for the large scale production of vaccines, but also laid the foundation for further study on the biological characteristics of JEV-SC-1 strain.In this study, we used Illumina deep sequencing to identify and differentially expressed analysis of miRNA in JEV-infected and uninfected PK-15 cells. We identified 522 and 427 miRNA in the infected and uninfected cells, respectively. Overall,132 miRNA were expressed significantly differently after challenge with JEV: 78 were upregulated and 54 downregulated. The sequencing results for selected miRNA were confirmed with RT-qPCR. The potential target genes of the differentially expressed miRNA were predicted with two miRNA target prediction algorithms, miRanda and TargetScan. Gene Ontology (GO) analysis of the host target genes revealed that these dysregulated miRNA are involved in complex cellular pathways, including metabolic pathway, inflammatory response, and immune response. Our findings will underpin further studies of miRNA roles in JEV infection and identify potential candidates for antiviral therapies against JEV.7 differentially expressed miRNA after JEV infected PK-15 cells, which bioinformatically predicted to target viral genome, were analyzed in this study to filter the miRNA which can suppress JEV replication, including upregulation of mir-145-5p、downregulation of mir-744、mir-450a the most significant upregulation、 mir-18a the most significant downregulation、mir-499-5p the strongest ability of prediction combined with viral genome、mir-lOb and mir-181a which have potential antiviral ability. Transfecting the above seven miRNA mimics and negative control mir-NC into PK-15 cells,6h later infecting JEV (MOI=0.05), and the only JEV-infected cells (NT) as control group. Supernatant were collected at 12h、36h post-infection respectively, total RNA was extracted unified and detected by qPCR. Results showed that after 12h、36h post-infection, the difference of virus content between mir-NC group and NT group were not large, indicating that miRNA mimics and transfection reagent have no toxic effects on cells. Among them, mir-145-5p、 mir-499-5p、mir-18a and mir-450a can inhibit the gene expression level of JEV, and mir-499-5p has the strongest role to inhibit JEV replication. These results will lay the foundation for further studies to verify the effect of mir-499-5p in inhibition of JEV replication, and also provide theoretical basis for host miRNA as antiviral drugs.