Roles Of WSSV-miR66and WSSV-miR68Encoded By Shrimp White Spot Syndrome Virus (WSSV) In Virus Infection

MiRNAs are small non-coding RNAs that regulate targets posttranscriptionally and are involved in diverse cellular processes. Up to now, many viruses have been found to encode miRNAs, which play an important role in viral lifecycles by performing infection-associated functions, such as avoiding host defenses and maintaining latent infection. White spot syndrome virus (WSSV) is a fatal pathogen that infects most crustacean. Previous studies in our laboratory have shown that WSSV can encode40distinct miRNAs. These miRNAs (such as WSSV-miR211and WSSV-miR212) have been verified to participate in virus infection. In this study, the roles of WSSV-encoded WSSV-miR66and WSSV-miR68in viral infection were analyzed on the basis of previous studies in our laboratory.Results of Northern Blot analysis showed that WSSV-miR68was expressed in the hemocytes of WSSV-infected Penaeus japonicas from6h to72h post-infection. Anti-miRNA oligonucleotides (AMOs) were employed to inhibt the expression of WSSV-miR68in order to study its role in viral infection. The results showed that when WSSV-miR68-specific AMO was injected into shrimp, WSSV-miR68expression would be inhibited, which could last to36h post-infection. Meanwhile, WSSV replication in Penaeus japonicas hemocytes would also be inhibited before36h post-infection. When AMO was injected three times, inhibition of WSSV replication in Penaeus japonicas hemocytes could still last to36h post-infection, which is not longer than that when AMO was injected twice. Mortality analysis showed that WSSV-induced Penaeus japonicas death could be inhibited at12h and24h post-infection whenever AMO was injected once or twice.The research showed that WSSV-miR66was expressed in hemocytes of WSSV-infected Penaeus japonicas from6h to72h post-infection. When WSSV-miR66-specific AMO was injected into shrimp, WSSV-miR66expression would be inhibited, which could last to36h post-infection. Meanwhile, WSSV replication in Penaeus japonicas hemocytes would also be inhibited before36h post-infection. Inhibition of WSSV-miR66expression resulted in decrease in WSSV-induced shrimp mortality before24h post-infection. Results of target prediction showed that WSSV-miR66could interact with target genes from both host and virus.These results above demonstrate that WSSV-encoded WSSV-miR66and WSSV-miR68play an important role in WSSV infection.