Small RNA sequencing analysis reveals regulation of microRNA expression in Madin-Darby canine kidney epithelial cells infected with Canid alphaherpesvirus 1.

Abstract

Canid alphaherpesvirus 1 (CHV-1) infection can cause spontaneous abortions in pregnant dams, and in young puppies, fatal systemic infections are common. MicroRNAs (miRNAs) affect viral infection by binding to messenger RNAs, and inhibiting expression of host and/or viral genes. We conducted deep sequencing of small RNAs in CHV-1-infected and mock-infected Madin-Darby Canine Kidney (MDCK) epithelial cells, and detected sequences corresponding to 282 cellular miRNAs. Of these, 18 were significantly upregulated at 12h post-infection, most of which were encoded on the X chromosome. We next quantified the mature forms of several of the miRNAs using stem loop RT-qPCR. Our results revealed a discordance between the levels of small RNAs corresponding to canine miRNAs, and levels of the corresponding mature miRNAs, which suggests a block in miRNA biogenesis in infected cells. Nevertheless, we identified several mature miRNAs that exhibited a statistically significant increase upon infection. These included cfa-miR-8908b, a miRNA of unknown function, and cfa-miR-146a, homologs of which target innate immune pathways and are known to play a role in other viral infections. Interestingly, ontology analysis predicted that cfa-miR-8908b targets factors involved in the ubiquitin-like protein conjugation pathway and peroxisome biogenesis among other cellular functions. This is the first study to evaluate changes in miRNA levels upon CHV-1 infection. Based on our findings, we developed a model whereby CHV-1 infection results in changes in levels of a limited number of cellular miRNAs that target elements of the host immune response, which may provide clues regarding novel therapeutic targets.