Global Transcriptional Analysis Of FMDV Persistently Infected BHK-21Cells

Foot-and-mouth disease is a highly contagious viral disease of cloven-hoofed livestock. Its causative agent, foot-and-mouth disease virus (FMDV) contains an unpaired positive-sense-strand RNA genome of about8,300nucleotides. In domestic livestock, the disease tends to cause more acute and short-lived infections, but often the immune system fails to clear virus, which can linger as a persistent infection from which transmission is thought to be relatively rare. Under certain conditions persistent infection is possible lead to the large-scale acute infections. Therefore, It is of great significance for disease control to study the mechanism of foot-and-mouth disease virus persistent infection.By the help of ammonium chloride, our lab have previously established a persistence of FMDV O naming PI (persistent infection) in cell culture. The following experiments had figured out that ammonium chloride exerted its primary effect on the host cell but not on the virus itself in the process of viral persistence establishment. Thus, further investigations were needed to attempt to search host cell proteins of factors pertaining to FMDV persistence in BHK-21cell. And we need to focus on host cells for the research of the mechanisms of establishing persistent infection. We firstly performed systematic functional genomics studies using cross-species hybridization (Bessen et al.) microarray technology on differences in gene expression of BHK-21during acute and persistent FMDV infections (relatively to normal BHK-21cells).We analyzed the differentially expressed genes (DEGs) between the mock infected and the FMDV infected BHK-21(acute and persistent respectively). Of the two infection patterns, numbers and expressional values of the differentially expressed genes (relative to normal BHK-21cells) in host cells are of great difference. It seemed that in persistent infection, genes expression in host cells were more inclined to down-regulated (the number of genes down-regulated is almost10time of the up-regulated ones), while in acute infection genes expression regulation distributed equally on both sides. These DEGs were uploaded to DAVID bioinformatics resources, and the resulting1676DEGs in acute infection BHK-21and808DEGs in persistent BHK-21were mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps provided by the website. Pathways unique to persistent infection showed obvious difference with ones unique to acute infection. Regulation pathways such as GnRH signaling, gap junction and VEGF signaling, are implicated in cell proliferation and growth, which seem to help host cells to survive lytic infection. The different pathway analysis output between acute and persistent infected BHK-21indicate a unique mechanism taken by the establishment of FMDV persistent infection. Of all the genes expression changed in FMDV infected cells versus mock infected cells, the ones involved in both acute and persistent infections with significantly expression changed genes were particularly interesting.33genes were found on the fold-change gene lists of both acute and persistent infections. Among the33genes, the12genes up-regulated in acute infection while down-regulated in persistent infection were key genes that help to depend which infection pattern, acute or persistent, taken by virus-host symbiotic system. To corroborate the results obtained with the microarray analysis, we performed qRT-PCR on a selected group of DEGs. As for the three genes APEX2, EBP, and P4HA2, the fold changes tested by qRT-PCR, no matter in acute infection or in persistent infection, were almost consistent with the results in microarray. And it can be concluded that APEX2, EBP, and P4HA2play significant roles in the establishment of persistence.Due to the deficiency of Syrian hamsters genome database, it is important for us to choose proper candidate microarray platforms. In this research, we hybridized RNA from syrian hamsters to whole mouse genome array. We prelimilary analyzed the result datas of mouse microarray according to the way of analyzing human’s. Then we focused on the difference of microarray results between human and mouse. We found that no matter in the circurmstance of acute infections or persist infections, the number of hybridization signals of human genome microarray was much more than that of mouse genome microarray (There were tens of thousands of the number of genes in human genome microarray results, while there only were two thousand genes in mouse’s). Therefore, phylogenetic distance dose not necessarily represent a main criterion for assessing the compatibility level required between the target (syrian hamsters) and reference species (human or mouse). Further analysis suggestted that in persistent infections, there were5DEGs (differentially expressed genes) and regulated towards the same side on both gene lists of human and mouse arrays. These genes may have potential function of contrubiton to maintaining persistent infection, and the detail mechanisms require further research.