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Journal of Integrative Agriculture 2021年第8期20卷 2227-2239页

作者： CHEN Xu CHEN Ya-qin YIN Zhong-qiong WANG Rui HU Huai-yue LIANG Xiao-xia HE Changliang YIN Li-zi YE Gang ZOU Yuan-feng LI Li-xia TANG Hua-qiao JIA Ren-yong SONG Xu Natural Medicine Research Center College of Veterinary MedicineSichuan Agricultural UniversityChengdu 611100P.R.China

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.

关键词： kaempferol antiviral activity pseudorabies virus

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

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Frontiers of Agricultural Science and Engineering 2016年第1期3卷 81-86页

作者： Shijun YAN He YAN Chaolin ZHANG Tongyan WANG Qingyuan YANG Zhe SUN Yan XIAO Feifei TAN Xiangdong LI Kegong TIAN National Research Center for Veterinary Medicine College of Animal Science and Veterinary Medicine Henan Agricultural University

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.

关键词： pseudorabies virus RR1 and RR2 ribonucleotide reductase vaccine candidate

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

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Frontiers of Agricultural Science and Engineering 2015年第3期2卷 216-222页

作者： Yuzhou WANG Tongyan WANG He YAN Fanli YANG Linghua GUO Qingyuan YANG Xule HU Feifei TAN Yan XIAO Xiangdong LI Kegong TIAN National Research Center for Veterinary Medicine Luoyang 471003China College of Animal Science and Veterinary Medicine Henan Agricultural UniversityZhengzhou 450002China

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.

关键词： pseudorabies virus glycoprotein B protein subunit vaccine

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

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Agricultural Biotechnology 2014年第4期3卷 39-44页

作者： Guangjun GUO Sufang LU Guanggang QU Feng LI Lin DONG Yanli BI Jinliang WANG Feng WEI Na TANG Chunling ZHANG Zhuang DING Zhiqiang SHEN Shandong Binzhou Animal Science & Veterinary Medicine Academy College of Veterinary Medicine Jinlin University Shandong Lvdu Bio-Science & Technology Co. Ltd.

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 ）.

关键词： pseudorabies virus Glycoprotein E PRV strain SA Gene expression ELISA

Cross-priming amplification-based strip as a new tool for on-site detection of pseudorabies virus

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Cross-priming amplification-based strip as a new tool for on...

中国畜牧兽医学会动物传染病学分会第九次全国会员代表大会...

作者： Xing-Yu Meng Yao Gao Huawei Zhang Yuzi Luo Yuan Sun Hua-Ji Qiu State Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural Sciences

pseudorabies(PR),also called as Aujeszky’s disease,is an important infectious di sease of pigs and other mammals caused by pseudorabies virus(PRV).This paper pre sents a novel assay based on cross-priming amplification in combination with gold nan oparticle immunochromatographic strip for rapid and on-site detection of wild-type PR V.In the assay,FITC and biotin were added at the 5’-end of the two primers and bo th 5’-ends of amplification products were also labeled with FITC and biotin respective ly.Then the amplification products(biotin-dsDNA-FITC),as novel targets,were recog nized by standardized strips.The assay had an excellent visual results from 2.0×10 to 2.0×10 copies of PRV and the detection limit was determined to be 2.0×10 copies.No cross-reactivity was observed with several common porcine viruses.When testing a total of 293 field swine samples,the agreement between the CPA-strip assa y and a triplex real-time PCR or virus isolation were 100%(293/293) or 97.3%(285/293) for wild-type PRV strains.Collectively,the assay is specific,sensitive and conve nient that enables rapid,on-site diagnosis of PRV infections.

关键词： Cross-priming amplification Immunochromatographic strip On-site detection pseudorabies virus

Microarray-Based Detection and Differentiation of Virulent and Attenuated pseudorabies virus

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Agricultural Sciences in China 2007年第2期6卷 234-243页

作者： LIU Li-na LIU Zheng-fei RUAN Li WANG Zhao-xiong LI Ren-feng ZHANG Song-lin CHEN Huan-chun HE Qi-gai Key Laboratory of Preventive Veterinary Medicine of Hubei Province~National Key Laboratory of Agricultural Microbiology~College ofVeterinary Medicine Huazhong Agricultural University Wuhan 430070 P.R.China

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.

关键词： pseudorabies virus microarray diagnosis differentiation test

Retrograde tracer systems based on pseudorabies virus

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Retrograde tracer systems based on Pseudorabies virus

中国神经科学学会第十二届全国学术会议

作者： Jia Fan Miu Huan Xu Xiaoqin Lv Pei Xu Fuqiang Brain research center Wuhan Institute of Physics and Mathematics Chinese Academy of Sciences University of Chinese Academy of Sciences Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences

Objective Neural circuit is the foundation of the brain function. Tracers based on neurotropic viruses, such as pseudorabies virus, rabies virus, vesicular stomatitis virus, herpes simplex virus, have been developed for displaying the full morphology of a neuron and mapping the neural circuit. In these systems, the pseudorabies virus is a DNA virus, it can infect swine, mouse, rat and other animals. However, it cannot infect human. The tracer based on the Bartha strain, when it enters a neuron, it can transport in the neuron and spread from a neuron to the connected neuron with retrograde pattern. The tool could be used to determine the input circuit. The virus can be modified to develop various retrograde tools to resolve different questions. Methods Two tracer systems based on pseudorabies virus could be developed, i) transmonosynaptic tracer（specific cell type and non-specific cell type）: the TK gene is a switch, which could be deleted to prepare TK-deleted pseudorabies virus. When the helper virus expressing TK gene was injected into the target region, the TK was expressed and it can induce the replication of TK-deleted pseudorabies virus in neuron and assemble new viruses, then these new viruses can trans-monosynapse. ii) transmultisynaptic tracer（specific cell type and non-specific cell type）: the cre and flp sitespecific recombinases are excellent tools, and many transgenic animal models have been developed, such as PV-Cre, Thy1-Cre. Based on these advantages, the pseudorabies virus could be modified to determine the cell type specific neural circuit. Results and Conclusion The recombinant viruses and the wild type viruses are similar in viral growth, and they can label neuron and depict the neural circuit with retrograde pattern.

关键词： Retrograde pseudorabies virus Trans-monosynapse Trans-multisynapse

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

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Agricultural Biotechnology 2015年第3期4卷 62-66页

作者： Sufang LU Guangjun GUO Ling MO Feng WEI Bingmei DONG Wentong ZHANG Zhiqiang SHEN Shandong Binzhou Animal Science & Veterinary Medicine Academy/Shandong Research & Development Center of Veterinary Propolis Vaccines Shandong Lvdu Bio-Sciences & Technology Co. Ltd.

[ 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.

关键词： SYBR Green I pseudorabies virus gE deleted virus LAT gene Latent infection

Preparation and characterization of an antiserum against truncated UL31 protein of pseudorabies virus

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Preparation and characterization of an antiserum against tru...

第七届中国畜牧科技论坛

作者： Li mei-li Cui wei Mo chuncong Wang jinlin mingsheng Cai Department of Pathogenic Biology and Immunology Guangzhou Medical University Institute of Immunology Sino-French Hoffmann InstituteGuangzhou Medical University

pseudorabies virus(PRV) early protein UL31 is a homologue of herpes simplex virus 1(HSV-1) UL31,which is a multifunctional protein and important for HSV-1 infection.However,the precise roles of PRV UL31 in virus life cycle are still poorly understood.Arelatively crucial tool for uncovering the function of UL31 is an antiserum specifically detects UL31 in the PRV-infected cells.For this purpose,a recombinant truncated UL31 protein consisting of N-terminal 27 aa of UL31 fused to EYFP and His-tag was expressed,purified and used for the preparation of antiserum.Western blot analysis and immunofluorescent assay showed that this antiserum specifically detected purified truncated UL31 as well as fulllength UL31 in the PRV infected cells.These results demonstrate that the prepared antiserum could serve as avaluable tool for further studies of UL31 functions.

关键词： pseudorabies virus UL31 protein truncated protein antiserum

Comprehensive diagnosis of variant pseudorabies virus epidemic in central Shandong province, China

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Comprehensive diagnosis of variant pseudorabies virus epidem...

中国畜牧兽医学会兽医病理学分会第二十一次学术研讨会暨中...

作者： Dongfang Hu Zhendong Zhang Lin Lv Sidang Liu College of Animal Science and Technology of Shandong Agricultural University

＜正＞Objective An epidemic that mainly endangered 3–7-day-old piglets struck many farms in Shandong province in2013 and caused heavy losses.To identify the pathogenesis,the type of lesions,and the causative agent,systemic examinations were performed.Materials and methods The study was conducted on a representative farm,and 10 of the dead piglets were necropsied in a

关键词： Bartha-K61 glycoprotein E variant pathology pseudorabies virus serology