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.

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.

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.

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 ）.

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.

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.

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 gB antibody and gE antibody. Detection results showed that the gB antibody positive rate of sows was 75.58%, and that of fattening pigs was 68.67% ; the pig farms with positive rate higher than 70% accounted for 74.42% of total survey pig farms. The PRV gE antibody positive rates of sows and fatte- ning 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）.

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.

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.

Pseudorabies virus manipulates mitochondrial tryptophanyl-tRNA synthetase 2 for viral replication

The pseudorabies virus(PRV)is identified as a double-helical DNA virus responsible for causing Aujeszky's disease,which results in considerable economic impacts globally.The enzyme tryptophanyl-tRNA synthetase 2(WARS2),a mitochondrial protein involved in protein synthesis,is recognized for its broad expression and vital role in the translation process.The findings of our study showed an increase in both mRNA and protein levels of WARS2 following PRV infection in both cell cultures and animal models.Suppressing WARS2 expression via RNA interference in PK-15 cells led to a reduction in PRV infection rates,whereas enhancing WARS2 expression resulted in increased infection rates.Furthermore,the activation of WARS2 in response to PRV was found to be reliant on the cGAS/STING/TBK1/IRF3 signaling pathway and the interferon-alpha receptor-1,highlighting its regulation via the type I interferon signaling pathway.Further analysis revealed that reducing WARS2 levels hindered PRV's ability to promote protein and lipid synthesis.Our research provides novel evidence that WARS2 facilitates PRV infection through its management of protein and lipid levels,presenting new avenues for developing preventative and therapeutic measures against PRV infections.

Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation

Herpesviruses antagonize host antiviral responses through a myriad of molecular strategies culminating in the death of the host cells.Pseudorabies virus(PRV)is a significant veterinary pathogen in pigs,causing neurological sequalae that ultimately lead to the animal's demise.PRV is known to trigger apoptotic cell death during the late stages of infection.The virion host shutdown protein(VHS)encoded by UL41 plays a crucial role in the PRV infection process.In this study,we demonstrate that UL41 inhibits PRV-induced activation of inflammatory cytokine and negatively regulates the cGAS-STING-mediated antiviral activity by targeting IRF3,thereby inhibiting the translocation and phosphorylation of IRF3.Notably,mutating the conserved amino acid sites(E192,D194,and D195)in the RNase domain of UL41 or knocking down UL41 inhibits the immune evasion of PRV,suggesting that UL41 may play a crucial role in PRV's evasion of the host immune response during infection.These results enhance our understanding of how PRV structural proteins assist the virus in evading the host immune response.

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