Microarray-Based Detection and Differentiation of Virulent and Attenuated Pseudorabies Virus

DNA microchip used in this study was formed from miniature arrays of pseudorabies virus （PrV） gene-specific probes immobilized on a glass surface. Hybridization using DNA microchip （microarrays） was used for differentiation between virulent and attenuated PrV. The presence of four gene segments （gB, gD, gE~, and gE ） encoding conservative glycoprotein B （gB）, D （gD）, and E （gE） of PrV was monitored using multiplex PCR. The amplicons were labeled with Cy5 or Cy3 dyes followed by hybridization to the gene-specific capture probes on the microchip. The presence of gD and gB, gE~ gene fragments was shown in virulent （gE~ genotype） and attenuated PrV （gE genotype）, whereas gE- gene （deleted domain in gE gene） was demonstrated only in virulent, not in attenuated, virus. No cross-hybridization was observed when fluorescence labeled-PCR products of PrV were hybridized using capture probes of related viruses, such as porcine respiratory and reproductive syndrome virus （PRRSV）, porcine parvovirus （PPV）, Japanese encephalitis virus （JEV）, and porcine circovirus type 2 （PCV-2）. The assay was 10 times sensitive than gD gene-specific PCR. Overall, the results of this study suggested that the microarray might be very useful for detection and differentiation of virulent PrV from attenuated one.

Expression of Pseudorabies Virus gE Core Epitopes in Escherichia coli Strain BL21 and Utilization of Indirect PRV gE-ELISA

Pseudorabies virus glycoprotein E （PRV gE） has been recognized as a suitable diagnostic antigen for pseudorabies. In order to produce gE antigen in large quantities and at low cost, a gene fragment encoding PRV gE core epitopes was expressed in E. coli BL21 expression system. SDS-PAGE and Western Blotting revealed that the expression product in culture supematant of E. coli BL21 was a recombinant protein, approximately 51.8 Kd. At 5 h post-induction, protein concentration assay showed that the expression product amounted to 1.65 mg/ml, accounting for 24. 17% of total proteins in the culture supematant. An indirect PRV gE-ELISA was established by using the recombinant expression product as a coating antigen. Cross-reactivity assay showed that this antigen was PRV specific. In addition, the assay was consistently reproducible. Comparison of detection results of 240 serum samples between PRV gE-ELISA and a commercially available PRV diagnostic kit showed that there was no significant difference between these two methods （P 〉 0.05 ）.

Establishment and Application of a SYBR Green I Real-time Quantitative PCR Assay for the Detection of LAT Gene of Pseudorabies Virus in Bama Miniature Pigs

[ Objective] This study aimed to establish a rapid, sensitive and specific SYBR Green I real-time quantitative PCR assay for the detection of latent pseudorabies virus （PRV） infection. [ Method ] SYBR Green I real-time quantitative PCR conditions and system for early detection of latent pseudorabies virus in- fection were optimized and compared with conventional PCR to investigate the sensitivity and specificity. Subsequently, the established assay was applied to detect different clinical samples. [ Result] The sensitivity of SYBR Green I real-time quantitative PCR assay （52 copies/μl） was 1 000 times higher than that of conven- tional PCR （5.2×1^04 copies/μl） and the detection time was shortened by 1/2. The established assay could be used to detect PRV but could not be used to detect PCV2, PPV, CSFV or PRRSV. Various tissues were collected from Bama miniature pigs with latent PRV infection under sterile conditions for real-time PCR detection. Results showed that viral copy number in the brain, nasal swab, inguinal lymph node, liver, lung and spleen was above 20, while PRV was not detected in the kidney and heart tissues. [ Conclusion] The established SYBR Green I real-time quantitative PCR assay for PRV/.AT detection was specific, sensitive and rapid, which could be used for pathogen monitoring, epidemiological investigation and quantitative study of PRV.

Development of PPA-ELISA for Detecting Antibodies against Porcine Pseudorabies Virus Using Truncated Recombinant Glycoprotein gD Expressed in E.coli

The purpose of this study was to develop a method for detecting antibodies against porcine pseudorabies virus （PRV）. According to the published genomic sequence of PRV SA strain, an approximately 1 070-bp gD gene fragment was amplified by PCR. The PCR products were cloned into the prokaryotic expression vector pET30a and the positive recombinant plasmid was transformed into E. coli BL21. Through induction with IPTG, the recombinant gD protein was expressed as inclusion bodies. As analyzed by western blot assay, the purified recombinant gD protein had good antigenicity and high specificity. Using the purified gD protein as coating antigen and horseradish peroxidase labeled staphylococcal protein A （PPA） as secondary antibody, we developed a PPA-ELISA for detecting antibodies against porcine PPV. No cross-reaction with the positive sera against seven common pathogens in pigs including classical swine fever virus, porcine parvovirus, porcine reproductive and respiratory syndrome, Japanese encephalitis virus, porcine circovirus type 2, porcine epidemic diarrhea virus, transmissible gastroenteritis virus was observed. The repeatability test showed that the intra- and inter-assay coefficients of variation were lower than 5% and 10%, respectively. Compared with the ELISA gD antibody test kit produced by IDEXX, the coincidence, sensitivity and specificity of the developed PPA-ELISA were 92.0%, 95.1% and 88.1%, respectively. The developed PPA-ELISA had good repeatability, sensitivity and specificity and was a rapid and simple serological method for surveillance of PRV antibodies in pig herds as well as for rapid diagnosis and epidemiological investigation of PRV infection.

Isolation and Identification of a Porcine Pseudorabies Virus Strain in Taizhou City

In this study, the liver, kidney and spleen tissues were collected from pigs with suspected PR in a pig farm in Jiangyan District, Taizhou City for virus isolation and identification. The isolated virus was inoculated onto PK15 monolayer cells. The virus culture was collected to extract genomic DNA for PCR assay and indirect immunoinfluscent assay. The results showed that the isolated virus was porcine pseudorabies virus, which was named TAIZ130417. The growth titer of the isolated virus reached 10 8.12 TCID 50 /ml on PK15 cells. Rabbits inoculated with the isolated virus soon exhibited pseudorabies symptoms such as itching and eventually died. The results provided reference for in-depth research and scientific prevention and control of pseudorabies.

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.

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.

Isolation and Identification of Sheep Pseudorabies Virus

In a sheep farm with mixed culture of pig and sheep in Shandong Province,sheep were attacked by a disease featured by foaming at the mouth,neurological symptoms and partial hair slip of legs,and the mortality of the disease was as high as 100%.In order to determine the pathogen,dead sheep were analyzed through pathogen isolation,PCR assay and direct immunofluorescence identification,and the pathogen was confirmed as pseudorabies virus（PRV）.Sequencing results showed that the g E gene of the isolated strain shared the homology of 97%-99% with the nucleotide sequence of known PRV genome in the NCBI databases,suggesting the isolate was PRV.The virus had obvious cytopathic effect through BHK cell line passage till the seventh generation,and the amount of half virus tissue cell infection（TCID50） was 1×107.5/m L following ReedMuench method.Two healthy sheep with the body weight of 20 kg were injected with the viral fluid of the isolate,and typical symptoms of pseu-dorabies（PR） were observed after 4 d.According to clinical symptoms and PCR diagnosis results,the epidemic situation of sheep farm was effec-tively controlled through comprehensive measures such as eliminating swinery in the farm,strengthening disinfection of pigsty,injecting sick sheep with pseudorabies serum,supplementing healthy sheep herb with antivirus traditional medicine Qiqing Baidu granule.

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.

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.

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.

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.

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.

Comparison on Detection Results of PRV Wild Virus Antibody Provided by Different Institutions

The detection results from different institutions were performed at the first stage of PRV wild virus antibody supervision in swine breeding. The serum samples were collected from 71 individuals and each individual was sampled twice at one week interval. The results showed that the positive coincidences for gE antibody between two institutions were 35.71% and 45.45 % ：espectively, with the total detection coincidences of 87.32% and 91.55% correspondingly. The positive coincidences for gE antibody between the two detections of each institution were 40.00% and 75.00% respectively, with the total detection coincidences of 87.32% and 97.18% correspondingly. It indicates that it is very necessary to screen the detection institution at the first supervision stage of PRV wild vires for swine population.

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.

建立多重PCR方法快速鉴别猪伪狂犬疫苗毒和野毒(英文)

Three pairs of primer were designed for amplification of porcine pseudorabies virus （PRV） gB, gE, and TK gene by multiplex PCR （multi-PCR） in order to differentiate vaccine strains from field isolates. Three specific bands were obtained respectively at the expected size, 427 bp （gB gene）, 298 bp （gEgene）, and 208 bp （TKgene）, Then four different gene-deleted vaccines of PRV were detected by multi-PCR. One ex- pected specific band was observed in one of samples, while two bands in the others. As shown by the detection results, the multi-PCR has high sensitivity and specificity and should be applied in pathogen diagnosis and epidemiological investigation in the future.