| Pseudorabies virus(PRV)is infectious to almost all the mammals,among which pigs are its natural host.PRV can infect pigs of different ages,mainly causing neurological symptoms and high mortality in piglets,reproductive failure in breeding pigs,thus causing significant economic losses to global pig industry.At present,porcine pseudorabies is still prevalent worldwide,and it is also a major animal disease that needs to be effectively controlled and eradicated in China.In addition to pigs,PRV infection can happen in many other non-natural hosts,which are characterized by fever,pruritus,and encephalomyelitis with a mortality of 100%.It should be noted that 25 cases of PRV infection in humans have been reported in China since 2017,although PRV was previously thought to be non-infectious to humans.The infected patients were almost engaged in pig breeding,slaughtering,and selling.They suffered from severe nervous system damage and sequelae,which endangered their life and health.Therefore,PRV is one of the important infectious pathogens that threatens the global pig industry and public health.And exploration about the pathogenesis of pseudorabies from perspectives of both pathogen and hosts is essential for effective prevention and control of PRV infection.In late 2011,though vaccination has been gradually nationwide carried out,porcine pseudorabies caused by the PRV variant strains broke out and became epizootic in China.Compared with the previous classical PRV strains,the variants showed enhanced virulence and neurotropism.Meanwhile,the PRV strains,responsible for infections in humans,have been reported to be closely related to these variant strains in the phylogenetic trees.Therefore,it is critical to investigate the biological characteristics of PRV variant strains for revealing its pathogenic mechanism.Although there have been researches on virulence comparison between classical and variant strains,the etiological differences between variant strains have been rarely explored,and the information on PRV genome diversity and evolution is still limited.So far,researches on PRV pathogenicity has mainly focused on the infection in pigs,while only few studies reported the cases about PRV cross-species infection in humans.And to the best of our knowledge,PRV has never been isolated from these human cases before,making it difficult to etiologically support the diagnosis of PRV infection in humans.Characteristics of the disease,especially infection characteristics,which are needed for the diagnosis and treatment,are also lacked.Furthermore,since no virus has been isolated from the patients,there has been no reports on the pathogenic mechanism of PRV infection in humans.This study here expounded the biological characteristic diversity and genetic evolution mechanism of PRV strains in pigs in China through etiological tests and genome sequencing.Through the systematic study in 4 cases of human infection with PRV,the characteristics of human infection with PRV were summarized.And the PRV strain was isolated from the patient,which confirmed PRV infectivity to humans at the level of live virus isolation.Furthermore,genomic analysis of PRV strains,transcriptome analysis of neurons infected with PRV,and mechanism analysis of the cytopathic differences induced by different PRV strains in human cells were conducted to preliminarily clarify the pathogenic mechanism of PRV infection in humans.The research results could provide a basis for the treatment and prevention of human pseudorabies encephalitis.The research details are as follows:1.The biological diversity and genetic evolution mechanism of PRV strains prevalent in ChinaIn this study,43 PRV variant strains isolated from 15 provinces in China from 2012 to 2017 were tested in virus titer,plaque size,immunogenicity,and virulence.54 PRV variant strains were high-throughput sequenced,and their genomic diversity,genetic relationship,and recombination were analyzed.The results revealed considerable differences in immunogenicity,virulence,and virus spread ability between the epizootic strains in China after 2012.JX2/CHN2015 was screened and proved to be a potential strain for vaccine development.Phylogenetic analysis divided the PRV strains into two genotypes,indicating that the PRV strains evolved independently in Asia and Europe.23 recombination events were identified by recombination analysis,which might greatly contribute to the evolution of PRV.Of the 23 events,the classical vaccine strain BarthaK61 participated in the 16 recombination events.Additionally,37 unique mutations were identified in 19 ORFs,which distinguish the novel variants from classic strains.These unique mutations will be helpful for further genotype-phenotype studies and for revealing the detailed mechanism of virulence enhancement in the variant strains.2.The first human-originated PRV strain was isolated to provide etiological evidence for PRV infection in humansFour acute encephalitis cases in humans were confirmed as PRV infection based on clinical symptoms,laboratory diagnosis,and metagenomic next-generation sequencing(m NGS).The infection characteristics were summarized,providing basis for clinical diagnosis of PRV infection.The cerebrospinal fluid samples of the patients were collected for virus isolation.For the first time,a PRV strain,designated h SD-1/2019,was isolated and identified.Phylogenetic analysis and biological characteristic tests showed that h SD-1/2019 was similar to the Chinese PRV variant strains.Moreover,h SD-1/2019 showed high pathogenicity to mice and pigs,and it induced acute neurological symptoms in pigs.The isolation of h SD-1/2019 provided direct evidence for the cross-species transmission of PRV to humans for the first time,implying the great risk of PRV transmission from pigs to humans.3.Human-originated PRV strain induced a high-level inflammation response in human neuronsBased on the isolate h SD-1/2019,SK-N-SH was applied as a model in vitro to characterize the transcriptomes of neurons infected by PRV.The comparative analysis of cells infected with variant strain h SD-1/2019 and classical strain Ea,respectively,was conducted.The results indicated that h SD-1/2019 infection induced a stronger response in the neurons than Ea.The differentially expressed genes in SK-N-SH cells infected with h SD-1/2019 were mainly enriched in the inflammation-related biological processes and signaling pathways.Furthermore,during h SD-1/2019 infection,MAPK/ERK signaling pathway were confirmed to be specifically activated and the inflammatory molecules LIF and GDF15 were specifically up-regulated.The expression levels of LIF and GDF15 were related with MAPK/ERK phosphorylation.4.PRV variant strains specifically induced syncytia to escape immunity in human cellsThe cytopathic effects induced by different PRV strains were observed in different cell lines.The variant and classical strains could result similar cytopathic effects in porcine and mouse cells.However,PRV variant strains,including h SD-1/2019,induced significant syncytia in the human cells,while the classical strain Ea showed completely different cytopathic effects.Therefore,this specific cytopathic effects caused by the variant strains in human cell lines might be important for PRV infection in humans.To reveal the underlying mechanism,cell staining,live-cell monitoring,inhibitor,and Western Blot were conducted based on PRV infections in SK-N-SH cells.It was concluded that h SD-1/2019 infection activated intracellular MAPK/ERK,PI3K/Akt,and Rho A/ROCK2 signaling pathways,leading to cytoskeleton rearrangement,thus inducing syncytia formation.And h SD-1/2019 could utilize syncytia to escape the effects of neutralizing antibodies and interferon alpha. |
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