Detection of Pseudorabies Virus Antibodies in Human Encephalitis Cases

Pseudorabies virus(PRV),a veterinary pathogen that infects domestic animals as well as wild animals such as wild boar and feral swine,was recently reported to infect human and led to endophthalmitis and encephalitis.A retrospective seroepidemiologic survey was conducted using 1,335 serum samples collected from patients with encephalitis and ELISA positive rates were 12.16%,14.25%,and 6.52%in 2012,2013,and 2017,respectively.

Pathogenicity of a currently circulating Chinese variant pseudorabies virus in pigs

AIM:To test the pathogenicity of pseudorabies virus(PRV)variant HN1201 and compare its pathogenicity with a classical PRV Fa strain.METHODS:The pathogenicity of the newly-emerging PRV variant HN1201 was evaluated by different inoculating routes,virus loads,and ages of pigs.The classical PRV Fa strain was then used to compare with HN1201 to determine pathogenicity.Clinical symptoms after virus infection were recorded daily and average daily body weight was used to measure the growth performance of pigs.At necropsy,gross pathology and histopathology were used to evaluate the severity of tissue damage caused by virus infection.RESULTS:The results showed that the efficient infection method of RPV HN1201 was via intranasal inoculation at 107 TCID50,and that the virus has high pathogenicity to 35-to 127-d old pigs.Compared with Fa strain,pigs infected with HN1201 showed more severe clinical symptoms and pathological lesions.Immunochemistry results revealed HN1201 had more abundant antigen distribution in extensive organs.CONCLUSION:All of the above results suggest that PRV variant HN1201 was more pathogenic to pigs than the classical Fa strain.

Subculturing cells have no effect on CRISPR/Cas9-mediated cleavage of UL30 gene in pseudorabies virus

CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9-mediated inhibition.In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.

Kaempferol inhibits Pseudorabies virus replication in vitro through regulation of MAPKs and NF-κB signaling pathways

Pseudorabies virus(PRV),in the family Herpesviridae,is a pathogen of Aujeszky’s disease,which causes great economic losses to the pig industry.Recent outbreaks of Pseudorabies imply that new control measures are urgently needed.The present study shows that kaempferol is a candidate drug for controlling PRV infection,as it possesses the ability to inhibit PRV replication in a dose-dependent manner in vitro.Kaempferol at a concentration of 52.40μmol L^(-1) could decrease PRV-induced cell death by 90%.With an 50%inhibitory concentration(IC50)value of 25.57μmol L^(-1),kaempferol was more effective than acyclovir(positive control)which has an IC50 value of 54.97μmol L^(-1).A mode of action study indicated that kaempferol inhibited viral penetration and replication stages,decreasing viral loads by 4-and 30-fold,respectively.Addition of kaempferol within 16 h post infection(hpi)could significantly inhibit virus replication,and viral genome copies were decreased by almost 15-fold when kaempferol was added at 2 hpi.Kaempferol regulated the NF-κB and MAPKs signaling pathways involved in PRV infection and changed the levels of the target genes of the MAPKs(ATF-2 and c-Jun)and NF-κB(IL-1α,IL-1βand IL-2)signaling pathways.The findings of the current study suggest that kaempferol could be an alternative measure to control PRV infection.

Research Progress on Pseudorabies Gene-deleted Vaccine

Pseudorabies is caused by pseudorabies virus (PrV), which is a member of family Herpesviridae, subfamily Alphaherpesvirinae and is the agent of acute infectious disease in many domestic and wild animals. Swine was the natural host and reservior of PRV, which inflicts major economic loss in pig industries world wide. Immunization with safe, effective vaccine is main measurements to prevent the disease.In this assay, research progress on PRV gene-deleted vaccine used extensively today was discussed.

Design of live-attenuated animal vaccines based on pseudorabies virus platform

Pseudorabies virus(PRV)is a double-stranded DNA virus with a genome approximating 150 kb in size.PRV contains many non-essential genes that can be replaced with genes encoding heterogenous antigens without affecting viral propagation.With the ability to induce cellular,humoral and mucosal immune responses in the host,PRV is considered to be an ideal and potential live vector for generation of animal vaccines.In this review,we summarize the advances in attenuated recombinant PRVs and design of PRV-based live vaccines as well as the challenge of vaccine application.

Detection of Serum Antibodies of Porcine Pseudorabies Virus(PRV)in Binzhou City in 2014

To understand immunization effect of pseudorabies vaccine and infection status of porcine pseudorabies( PR) in pig farms and guide prevention and control against PR,453 copies of blood samples collected from 43 different scale pig farms in four counties( districts) of Binzhou City were detected with ELISA to investigate PRV g B antibody and g E antibody. Detection results showed that the g B antibody positive rate of sows was 75. 58%,and that of fattening pigs was68. 67%; the pig farms with positive rate higher than 70% accounted for 74. 42% of total survey pig farms. The PRV g E antibody positive rates of sows and fattening pigs were 25. 41% and 26. 67%,and the positive rates of pig farms were 46. 51% and 44. 33%,respectively. There were regional differences among counties( districts).

Global asymptotic stability in a pseudorabies virus model with age structure

Pseudorabies is a highly contagious disease caused by pseudorabies virus(PRV)or suid herpesvirus 1(SuHV1),causing significant economic losses to the swine industry in countries where the disease exists.In this paper,we formulate an age structure model of pseudorabies virus that takes into account disease-related mortality and vertical trans-mission.We find a threshold to determine the stability and existence of the disease.We show that there is always a globally asymptotically stable boundary equilibrium if and only if R_(02)<1+q,which means that the disease always exists in piglets and will die out in adult pigs.When R_(02)>1+q,the boundary equilibrium is unstable and there exists a unique disease-endemic equilibrium,which is globally asymptotically stable.We give detailed proofs of our theoretical results and numerical examples.Brief concluding re-marks are also provided.

Current Status and Challenge of Pseudorabies Virus Infection in China

Pseudorabies(PR),also called Aujeszky's disease,is a highly infectious disease caused by pseudorabies virus(PRV).Without specific host tropism,PRV can infect a wide variety of mammals,including pig,sheep,cattle,etc.,thereby causing severe clinical symptoms and acute death.PRV was firstly reported in China in 1950s,while outbreaks of emerging PRV variants have been documented in partial regions since 2011,leading to significant economic losses in swine industry.Although scientists have been devoting to the design of diagnostic approaches and the development of vaccines during the past years,PR remains a vital infectious disease widely prevalent in Chinese pig industry.Especially,its potential threat to human health has also attracted the worldwide attention.In this review,we will provide a summary of current understanding of PRV in China,mainly focusing on PRV history,the existing diagnosis methods,PRV prevalence in pig population and other susceptible mammals,molecular characteristics,and the available vaccines against its infection.Additionally,promising agents including traditional Chinese herbal medicines and novel inhibitors that may be employed to treat this viral infection,are also discussed.

Genome Characteristics and Evolution of Pseudorabies Virus Strains in Eastern China from 2017 to 2019

Since late 2011,outbreaks of pseudorabies virus(PRV)have occurred in southern China causing major economic losses to the pig industry.We previously reported that variant PRV forms and recombination in China could be the source of continued epidemics.Here,we analyzed samples from intensive pig farms in eastern China between 2017 and 2019,and sequenced the main glycoproteins(gB,gC,gD,and gE)to study the evolution characteristics of PRV.Based on the gC gene,we found that PRV variants belong to clade 2 and detected a founder effect during by the PRV epidemic.In addition,we detected inter-and intra-clade recombination;in particular,inter-clade recombination in the gB genes of strains FJ-ZXF and FJ-W2,which were recombinant with clade 1 strains.We also found specific amino-acid changes and positively selected sites,possibly associated with functional changes.This analysis of the emergence of PRV in China illustrates the need for continuous monitoring and the development of vaccines against specific variants of PRV.

Host Interferon-Stimulated Gene 20 Inhibits Pseudorabies Virus Proliferation

Host interferon-stimulated gene 20(ISG20)exerts antiviral effects on viruses by degrading viral RNA or by enhancing IFN signaling.Here,we examined the role of ISG20 during pseudorabies virus(PRV)proliferation.We found that ISG20 modulates PRV replication by enhancing IFN signaling.Further,ISG20 expression was upregulated following PRV infection and poly(I:C)treatment.Ectopic expression of ISG20 inhibited PRV proliferation in PK15 cells,whereas knockdown of ISG20 promoted PRV proliferation.In addition,ISG20 expression upregulated IFN-βexpression and enhanced IFN downstream signaling during PRV infection.Notably,PRV UL24 suppressed the transcription of ISG20,thus antagonizing its antiviral effect.Further domain mapping analysis showed that the N terminus(amino acids 1-90)of UL24 was responsible for the inhibition of ISG20 transcription.Collectively,these findings characterize the role of ISG20 in suppressing PRV replication and increase the understanding of host-PRV interplay.

Thymidine kinase gene mutation leads to reduced virulence of pseudorabies virus

To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK- mutant were sequenced. Analysis of the tk gene sequence of TK mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All

Research and development of a novel subunit vaccine for the currently circulating pseudorabies virus variant in China

Pseudorabies(PR)is a devastating viral disease which leads to fatal encephalitis and respiratory disorders in pigs.Commercial gE-deleted live pseudorabies virus(PRV)vaccine has been widely used to control this disease in China.However,the new-emerging variants of PRV compromises the protection provided by current vaccines and lead to the outbreak of PR in vaccinated pig herds.Several killed and live vaccine candidates based on current PRV variants have been reported to be effective to control the disease.A subunit vaccine based on gB protein,one major PRV glycoprotein which elicits strong humoral and cellular immune responses,however,was never evaluated for protection against the current circulating PRV variants.In this study,full-length PRV gB protein was successfully expressed in baculovirus/insect cells in the soluble format and was tested on 3-week-old piglets as a subunit vaccine.Compared with unvaccinated pigs,the gB-vaccinated pigs developed specific antibody-mediated responses and were protected from the virulent PRV HN1201 challenge.All vaccinated pigs survived without showing any PRV-specific respiratory and neurological signs,but all unvaccinated pigs died within 7 days after HN1201 challenge.Hence,this novel gB-based vaccine could be applied as an effective subunit vaccine to control PRV variant in China.

Phospho-proteomics identifies a critical role of ATF2 in pseudorabies virus replication

Pseudorabies virus(PRV),an etiological agent of pseudorabies in livestock,has negatively affected the porcine industry all over the world.Epithelial cells are reported as the first site of PRV infection.However,the role of host proteins and its related signaling pathways in PRV replication is largely unclear.In this study,we performed a quantitative phosphoproteomics screening on PRV-infected porcine kidney(PK-15)epithelial cells.Totally 5723phosphopeptides,corresponding to 2180 proteins,were obtained,and the phosphorylated states of 810 proteins were significantly different in PRV-infected cells compared with mock-infected cells(P<0.05).GO and KEGG analysis revealed that these differentially expressed phosphorylated proteins were predominantly related to RNA transport and MAPK signaling pathways.Further functional studies of NF-κB,transcription activator factor-2(ATF2),MAX and SOS genes in MAPK signaling pathway were analyzed using RNA interference(RNAi)knockdown.It showed that only ATF2-knockdown reduces both PRV titer and viral genome copy number.JNK pathway inhibition and CRISPR/Cas9 gene knockout showed that ATF2 was required for the effective replication of PRV,especially during the biogenesis of viral genome DNA.Subsequently,by overexpression of the ATF2 gene and point mutation of the amino acid positions 69/71 of ATF2,it was further demonstrated that the phosphorylation of ATF2 promoted PRV replication.These findings suggest that ATF2 may provide potential therapeutic target for inhibiting PRV infection.

Differential CircRNA Expression Profiles in PK-15 Cells Infected with Pseudorabies Virus Type Ⅱ

Circular RNAs(circ RNAs)belong to a class of non-coding RNAs with diverse biological functions.However,little is known about their roles in case of pseudorabies virus(Pr V)infection.Here,we analyzed the expression profile of host circ RNAs from a virulent Pr V type II strain DX(Pr V-DX)infected and an attenuated g E/TK deficient(g E-TK-Pr V)strain of Pr V infected PK-15 cells.Circ RNAs were identified by findcirc and analyzed with DESeq 2.Compared with the mock cells,449 differentially expressed(DE)circ RNAs(233 down-regulated and 216 up-regulated)from Pr V-DX infected and578 DE circ RNAs(331 down-regulated and 247 up-regulated)from g E-TK-Pr V infected PK-15 cells were identified.In addition,459 DE circ RNAs(164 down-regulated and 295 up-regulated)between the Pr V-DX and g E-TK-Pr V infected cells were identified.The expression patterns of 13 circ RNAs were validated by reverse transcription quantitative real-time PCR(RT-q PCR)and results were similar as of RNA-seq.The putative target mi RNA binding sites of DE circ RNAs were predicted by using mi Randa and ps Robot.The circ RNA-mi RNA-m RNA network was constructed and certain mi RNAs that have possible roles in antiviral immune response,such as mi R-210 and mi R-340,were predicted.GO and KEGG pathway analysis demonstrated that DE circ RNAs were enriched in the processes such as cellular metabolism,protein binding,RNA degradation and regulation of actin cytoskeleton.Collectively,these findings might provide the useful information for a better understanding of mechanisms underlying the interaction between Pr V-II and host cells.

Cloning and Characterization of UL34 Gene of Pseudorabies Virus HB Isolate

In this study, China isolate HB of pseudorabies virus(PRV) was confirmed and genotypically characterized by amplifying and sequencing of partial UL34, a conservative gene involved in the egress of nucleocapsids from the nucleus, for phylogenetic analysis. The open reading frame(orf) of UL34 of PRV HB isolate is composed of 786 nucleotides, which encoded 262 amino acids. In addition, a potential transmembrane domain(241-260 aa) and 11 potential phosphorylation sites were also found in the UL34 of PRV HB isolate. Multiple amino acids alignment indicated that UL34 proteins of PRV strains derived from different geographic origins were highly conservative, but some mutations were also found. Phylogenetic analysis based on UL34 protein indicated that PRV HB strain was evolutionarily distinct from other recent China strains sequenced so far, forming a single clade within the phylogeny. Moreover, PRV HB isolate had close evolutionary relationship with Bo HV-1 and Bo HV-5 within the Alphaherpesvirinae. Taken together, these results indicated that PRV strains were in the progress of evolution. This study has expanded the knowledge of genetic profiles of PRV strains.

RR1 and RR2 gene deletion affects the immunogenicity of a live attenuated pseudorabies virus vaccine candidate in natural pig host

As virulence-determining genes, RR1 and RR2 encode the small subunit and large subunit of viral ribonucleotide reductase(RR) in pseudorabies virus which have been extensively studied in mice. However,their role in pigs has not been adequately investigated. In this study, we deleted RR1 and RR2 genes based on a TK/g E/g I triple gene-deleted pseudorabies virus and tested its efficacy in pigs as a vaccine candidate. The rescued virus showed similar growth properties and plaque size in vitro as its parent strain. In an animal study, the virus could elicit humoral immune responses shown by generation of g B-specific antibodies and virus neutralizing antibodies.However, vaccination could not provide protection against virulent pseudorabies virus challenge since vaccinated pigs showed clinical pseudorabies-specific syndromes. The deficiency in protection may due to the generation of late and low levels of gB antibodies and virus neutralizing antibodies.

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus(PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK^(-)/gE^(-)/GP5^(+)expressing GP5 of porcine reproductive and respiratory syn-drome virus(PRRSV),based on the PRV genetically depleted vaccine strain TK^(-)/gE^(-)/LacZ^(+),scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene(the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK^(-)/gE^(-)/LacZ^(+),resulting in a PRV recombinant named TK^(-)/gE^(-)/GP5m^(+),which expressed the modified GP5m protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK^(-)/gE^(-)/GP5m^(+)and TK^(-)/gE^(-)/GP5^(+)expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies(the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK^(-)/gE^(-)/GP5m^(+)against PRRSV were higher than that induced by TK^(-)/gE^(-)/GP5^(+).Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.

Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush

Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration.However,morphological assessments of retinorecipient regions are limited by the opacity of brain tissue.In this study,we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury.Specifically,we performed light-sheet imaging of whole brain tissue after a clearing process.We found that pseudorabies virus 724(PRV724)mostly infected retinal ganglion cells,and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains.Unexpectedly,PRV724-traced neurons were more widely distributed compared with data from previous studies.We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus,intergeniculate leaflet,ventral lateral geniculate nucleus,central amygdala,basolateral amygdala,Edinger-Westphal nucleus,and oculomotor nucleus,but not the superior vestibular nucleus,red nucleus,locus coeruleus,gigantocellular reticular nucleus,or facial nerve nucleus.Our findings demonstrate that the tissue clearing technique,combined with retrograde trans-synaptic viral tracing,can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury.Thus,our approach may be useful for future estimations of optic nerve injury and regeneration.

猪伪狂犬病野毒抗体不同检测单位的比对试验

在种猪群伪狂犬病野毒(gE)抗体监测的第一阶段,进行了不同检测单位的比对试验。对来自71个个体间隔1周的两次血清样本进行检测,2个检测机构之间的gE抗体阳性符合率分别为35.71%、45.45%,总体检测符合率分别为87.32%、91.55%;2个检测机构各自的gE抗体阳性符合率分别为40.00%、75.00%,总体检测符合率分别为87.32%、97.18%。表明在猪群进行伪狂犬病野毒感染鉴定的初期,有必要对检测单位进行筛选。