Study On The Function Of MicroRNA Cluster Encoded By Pseudorabies Virus LLT Gene

Pseudorabies virus(PRV),the etiological pathogen of Aujeszky’s disease(AD),belongs to alphaherpesvirus subfamily with distinctive features such as neuroinvasion and neurovirulence in natural host and non-natural host animals.China is considered as the biggest pork consumption country in the world and PRV is one of the most important pathogens in pig industry.AD was first documented in China in 1948,which became epidemic in late 1980,and cost huge economicloss.Several virus strains including Ea were isolated from aborted fetus.PRV Ea strain was extensively studied due to its high pathogenicity and immunogenicity.PRV Ea was used as live virus vector to express foreign genes from other viruses,as reference strain to study the genetic relationship for PRV isolates,and as model pathogen to study virus-host interactionA live attenuated vaccine strain PRV Bartha,invented in 1960 in Hungary,was introduced to China to control the spread of PRV in pigs in the 1960 s.And sone live attenuated marker vaccines based on PRV local isolates were developed since the isolation of PRV local strains.AD was effectively controlled under the widely application of both inactivated and gene-deleted live marker vaccines in pig farms However,AD reoccurred in Bartha vaccinated pig farms and spread quickly to more than 23 provinces in China since late 2011.A large number of novel PRV strains were isolated.To better understand the cause of AD outbreak,we compared the biological difference between Ea and newly emerged variants by growth kinetics,full-length genome sequencing and experimental confirmation.Full-length genome sequence of Ea disclosed by PacBio SMRT combined with Illumina reveals that Ea is clustered with HNX in same subgroup.PRV china reference strain is genetically closer to newly emerged variant strains,indicating that domestic PRV strain evolves independently of foreign strains,leading to the emergence of variant strain.Growth analysis showed that PRV HNX forms bigger plaque than Ea.Although amino acid sequences are conserved between Ea and variant strain,protein composition in virions and protein expression level in infected cells were different between Ea andvariant strain.The difference of protein composition and expression level may be the reason different pathogenicity of Ea and variant strains.PRV can establish lifelong latency in nature nervous system.The most abundant transcript,large latency transcript(LLT),is the only detectablegene expression product during lateny in trigeminal ganglion(TG).PRV LLT harbors a ~4.6 Kb stable Intron,which primarily encodes a microRNA cluster containing 11 miRNAs.Previous study predicted that these miRNAs may be involved in regulating viral and host gene expression.In our previous study,LLT miRNA cluster has been demonstrated to be associated with virus replication,apoptotic inducing ability and gene expression of IE180 and EP0 in vitro.However,the interection between LLT miRNA cluster and virus virulence is still unknown.Here,to fully uncover the function ofmiRNA cluster during the life cycle of PRV,piglets,nature host of PRV,were inoculated with PRV mi RNA cluster deleted virus.The physical parameters,virus shedding assays and SN50 titer in piglets reveals that LLT miRNA cluster can enhance PRV virulence in pigs.The over expression plasmid pEGFP-C1-Intron can express miRNAs in vitro and,surprisingly,LLT Intron induces protection to mice as an antiviral virulence factor against lethal challenge.Collectively,our findings suggest that PRV LLT Intron is involved in viral virulence,and plays an important role in the modulation of host response for full pathogenesis.Specific PRV miRNA mutant virus is needed to further study the function of each single miRNA.As it will take a long time to obtain a mutant virus by tranditional construction method based on homologous recombination,we constructed a PRV Ea BAC clone to improve the efficiency of mutant virus construction in this study.PRV mi RNA7 deleted virus PRV-ΔmiR7 and revertant virus PRV-ΔmiR7-R were generated based on two-step Red recombination.Growth analysis showed that PRV-ΔmiR7 forms smaller plaque than PRV Ea,indicating that miRNA7 is associated with PRV replication.