MiRNAome Of Pseudorabies Virus Variant Infected Cells And Analysis Of Viral Antigenicity

Pseudorabies virus(PRV) is the causative agent of pseudorabies. PRV causes neurological and respiratory system disorders in newborn piglets and reproductive disease in pregnant sows. PR has been effectively controled through the use of gene deleted vaccine. However, in recent years, the PRV variant causing higher mortality is widespread popularity in the swine industry in China, which has caused significant economic losses to the domestic swine industry. The PRV variant JS-2012 strain was isolated in a Bartha-K61-vaccinated pig farm in Jiangsu province in 2012. Genome sequence analysis and infection assay of PRV JS-2012 showed PRV has a lot of variation and more virulence for pig at different age. The existing vaccine does not provide complete protection. This paper systemically studied the changing of miRNAome and post-transcriptional regulatory responses to the process of PRV variant JS-2012 infection in porcine kidney epithelial(PK-15) cells. In order to lay the foundation for further study the PRV reproduction, immune and persistent infection after the miRNA expression.A total of 109,595,539 and 112,037,904 clean reads were obtained from PRV-infected cell sample and mock libraries using Illumina deep sequencing. 36 PRV miRNAs were predicted derived from the PRV infected sample. All 11 known mature miRNA sequences were highly expressed and comparatively conserved from variant JS-2012 strain. In addition, we found 25 novel viral miRNAs and 20 of these miRNAs were confirmed through stem-loop RT-qPCR with the exception of prv-miR-1, prv-mi R-6, prv-miR-16, prv-miR-21 and prv-miR-25. Unlike the usual miRNAs encoded by other α-herpesviruses, which are found clustered in the large latency transcript(LLT). In our study, three novel miRNAs, prv-miR-11-5p, prv-miR-12a-3p, prv-mi R-17a-5p were located in the LLT region, while the other miRNAs were distributed throughout the PRV genome like β-herpesviruses. This phenomenon was reported for the frist time. Some miRNAs located in IRS or TRS were presented in two copies, including miR-LLT10a/b-3p, prv-miR-LLT11a/b-5p, prv-miR-12a/b-3p, prv-miR-13a/b-5p, prv-miR-14a/b-5p and prv-miR-17a/b-5p. Moreover, the prv-miR-18 through prv-miR-25, prv-miR-12 b, prv-miR-13 b, prv-miR-14 b and prv-miR-17 a were encoded on the opposite DNA strand to the latency associated transcript.vmiRNAs are predicted to target multiple genes involved in lytic or latency. We use dual luciferase report system to detect the function of mainly PRV miRNA. The results showed prv-miR-LLT1, prv-miR-LLT7 and prv-miR-LLT9 can target the transcription activator IE180, prv-miR-LLT7 can target the VP16 involved in viral DNA replication. These vmiRNAs may involved in regulating the PRV replication. However, overexpressed vmiRNA and then incubated JS-2012 to PK-15 cell, which couldn’t significant change the level of protein expression for IE180 and VP16.Virus can affect host miRNA expression profiles to facilitate their replication. We compared the expression of the host miRNA between the PRV JS-2012 infected and uninfected PK-15 cells. 303 and 295 mature host miRNA were detected in PRV-infected PK-15 cells and uninfected cells, respectively. The 283 known and 239 novel miRNAs were found in both libraries. Eight miRNAs were up-regulated after infection with PRV and 5 miRNAs were down-regulated. GO analysis of the host target genes of dysregulated miRNAs were involved in complex cellular processes including the metabolic pathway, biological regulation and immune response. We artificially synthesized miRNAs mimics and miRNA inhibitor or NC of these dysregulated host miRNA. Overexpressed these miRNA and then PRV JS-2012 strain were inoculated to PK-15 cells. The results showed these dysregulated host miRNA has no direct effect on viral replication.This is obviously the PRV JS-2012 mutant virulent strain can infected the pig-vaccined by break through immune defenses of vaccine. To identify the antigenic genetic variations of PRV JS-2012. We prepared the rabbit and mice polyclonal antibody against PRV JS-2012 mutant virulent strain, SC classical virulent strain and Bartha-K61 vaccine strains. The results of cross neutralization test showed that the antiserum against Bartha-K61 strain not neutralized the JS-2012 strain very well, which may indicate that antigenicity of JS-2012 has changed. In addition, we used Bartha-K61 and Bucharest vaccine immuned piglets. Using the vaccine polyclonal antibody crosses neutralized JS-2012, SC, Bartha-K61 and Bucharest strains. Both vaccines produced neutralizing antibody after immuned 14 d. Both of the two polyclonal antibody against Bartha-K61 and Bucharest vaccines had the highest neutralization titer against parent strain, and had the lowest neutralization titer against JS-2012 strain.The results further illustrated the antigenicity of JS-2012 has changed.In summary, This paper frist provided novel insight into the role of virus-encoded miRNAs and host dysregulated miRNAs in response to PRV variant JS-2012 infection. This study analyzed the vmiRNA conservative and identified more new vmiRNA from the PK-15 cells infected with variant PRV strain. We also have preliminarily studied the functions of vmiRNA. In addition, This study identified the differently expressed host mi RNA from the infected and uninfected PK 15 cells. It is significant for further understanding of the regulation from miRNA in cells infected with PRV. At the same time, this study established foundation for study genetic variation of the PRV prevalent strains.