Identification and Characterization of Cyprinid Herpesvirus-3 (CyHV-3) Encoded MicroRNAs

Data Availability: Relevant data are contained within the paper and its Supporting Information files. All unique sequence tags obtained following virus infections are available for download via the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad.3c7m4).MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional gene regulation. Some viruses encode their own miRNAs and these are increasingly being recognized as important modulators of viral and host gene expression. Cyprinid herpesvirus 3 (CyHV-3) is a highly pathogenic agent that causes acute mass mortalities in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) worldwide. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. Deep sequencing of small RNA fractions prepared from in vitro CyHV-3 infections led to the identification of potential miRNAs and miRNA–offset RNAs (moRNAs) derived from some bioinformatically predicted pre-miRNAs. DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. The evidence also suggested the presence of an additional four high-probability and two putative viral pre-miRNAs. MiRNAs from the two confirmed pre-miRNAs were also detected in gill tissue from CyHV-3-infected carp. We also present evidence that one confirmed miRNA can regulate the expression of a putative CyHV-3-encoded dUTPase. Candidate homologues of some CyHV-3 pre-miRNAs were identified in CyHV-1 and CyHV-2. This is the first report of miRNA and moRNA genes encoded by members of the Alloherpesviridae family, a group distantly related to the Herpesviridae family. The discovery of these novel CyHV-3 genes may help further our understanding of the biology of this economically important virus and their encoded miRNAs may have potential as biomarkers for the diagnosis of latent CyHV-3.Funding: This work was supported by the Marine Institute Research Fellowship programme (https://www.marine.ie; OHD), Defra https://www.gov.uk/government/organisati​ons/department-for-environment-food-rura​l-affairs; research contracts FC1193 and FC1202; KW), the Programme for Research in Third Level Institutions (PRTLI) (http://www.hea.ie/en/prtli,official; RH, DW) Cycle 5 [The Bioanalysis and Therapeutics (BioAT) programme at DCU]. The PRTLI is co-funded through the European Regional Development Fund (ERDF), part of the European Union Structural Funds Programme 2007–2013 (http://eustructuralfunds.gov.ie). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript