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Identification of Nonstructural Protein 8 as the N-Terminus of the RNA-Dependent RNA Polymerase of Porcine Reproductive and Respiratory Syndrome Virus

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Virologica Sinica 2018年第5期33卷 429-439页

作者： Yuanyuan Liu Yunhao Hu Yue Chai Liping Liu Jiangwei Song Shaochuan Zhou Jia Su Lei Zhou Xinna Ge Xin Guo Jun Han Hanchun Yang key laboratory of animal epidemiology of the ministry of agriculture College of Veterinary Medicine and State Key Laboratory of Agrobiotechnology China Agricultural University

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expression of viral replicase proteins encoded by two ORFs, namely ORF1 a and ORF1 b. In particular, translation of ORF1 b depends on a-1-ribosomal frameshift strategy. Thus, nonstructural protein 9 (nsp9), the first protein within ORF1 b that specifies the function of the viral RNA-dependent RNA polymerase, is expressed as the C-terminal extension of nsp8, a small nsp that is encoded by ORF1 a. However, it has remained unclear whether the mature form of nsp9 in virus-infected cells still retains nsp8,addressing which is clearly critical to understand the biological function of nsp9. By taking advantage of specific antibodies to both nsp8 and nsp9, we report the following findings. (1) In infected cells, PRRSV nsp9 was identified as a major product with a size between 72 and 95 k Da (72–95 KDa form), which exhibited the similar mobility on the gel to the in vitro expressed nsp8–9 ORF1 b, but not the ORF1 b-coded portion (nsp9 ORF1 b). (2) The antibodies to nsp8, but not to nsp7 or nsp10, could detect a major product that had the similar mobility to the 72–95 KDa form of nsp9. Moreover, nsp9 could be co-immunoprecipitated by antibodies to nsp8, and vice versa. (3) Neither nsp4 nor nsp2 PLP2 was able to cleave nsp8–nsp9 in vitro. Together, our studies provide experimental evidence to suggest that nsp8 is an N-terminal extension of *** findings here paves way for further charactering the biological function of PRRSV nsp9.

关键词： Porcine reproductive and respiratory syndrome virus (PRRSV) Nsp8 Nsp9

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Nsp2 and GP5-M of Porcine Reproductive and Respiratory Syndrome Virus Contribute to Targets for Neutralizing Antibodies

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Virologica Sinica 2019年第6期34卷 631-640页

作者： Jia Su Lei Zhou Bicheng He Xinhui Zhang Xinna Ge Jun Han Xin Guo Hanchun Yang key laboratory of animal epidemiology of the ministry of agriculture College of Veterinary Medicine and State Key Laboratory of Agrobiotechnology China Agricultural University

Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous *** though several viral structural proteins have been regarded as inducers of neutralizing antibodies(NAs)against PRRSV,the mechanism underlying limited cross-neutralization among heterologous strains is still *** the present study,examinations of NA cross reaction between a highly pathogenic PRRSV(HP-PRRSV)strain,JXwn06,and a low pathogenic PRRSV(LP-PRRSV)strain,HB-1/3.9,were conducted with viral neutralization assays in MARC-145 *** of the JXwn06-hyperimmuned pigs’sera could neutralize HB-1/3.9 in vitro and vice *** address the genetic variation between these two viruses that are associated with limited crossneutralization,chimeric viruses with coding regions swapped between these two strains were *** neutralization assays indicated that variations in nonstructural protein 2(nsp2)and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/***,we substituted the nsp2-,glycoprotein2(GP2)-,GP3-,and GP4-coding regions together,or nsp2-,GP5-,and membrane(M)protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental *** results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 *** together,these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.

关键词： Porcine reproductive and respiratory syndrome virus(PRRSV) Neutralizing antibody(NA) Non-structural protein 2(nsp2) Structural proteins(SPs)

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Anti-Recombinant Gametocyte 56 Protein IgY Protected Chickens from Homologous Coccidian Infection

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Journal of Integrative agriculture 2012年第10期11卷 1721-1728页

作者： DING Jun LIU Qiao-rong HAN Jin-peng QIAN Wei-feng LIU Qun key laboratory of animal epidemiology and Zoonosis Ministry of Agriculture/College of Veterinary Medicine China Agricultural University

Coccidiosis is caused by intra-cellular infection of Eimeria spp., which goes through a complex life cycle in the intestinal mucosa of infected hosts. Specific immunoglobulins （IgY） could be produced in egg yolk by immunizing hens with specific antigens. In the present study, we cloned the E. maxima gain56 gene, expressed the GST-GAM56 fusion protein and raised IgY to GST-GAM56 in hens. The anti-GST-GAM56 IgY antibody was isolated and used to treat chickens infected with E. maxima oocysts. Intramuscular injection of the antibodies provided minimal protection against parasite infection. However, oral dosing of the IgY 3 or 5 d after oocyst inoculation significantly improved body weight gain, reduced oocyst output and intestinal lesion score were reduced at 3 or 5 d after oocyst challenging, compared to the untreated control group. Our findings suggest that the IgY to gam56 could be an effective prophylactic or therapeutic agent against E. maxima infection in chickens and should have a practical application value.

关键词： Eimeria maxima recombinant GST-GAM56 IgY therapeutic agent

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A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses

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Journal of Integrative agriculture 2016年第9期15卷 2105-2113页

作者： WEI Yan-di GAO Wei-hua SUN Hong-lei YU Chen-fang PEI Xing-yao SUN Yi-peng LIU Jin-hua PU Juan key laboratory of animal epidemiology and Zoonosis Ministry of Agriculture/College of Veterinary Medicine/State Key Laboratory of Agrobiotechnology China Agricultural University

H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.

关键词： avian influenza viruses H9 subtype infectious bronchitis viruses duplex RT-PCR

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The cellular interactome for glycoprotein 5 of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus

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Journal of Integrative agriculture 2016年第8期15卷 1833-1845页

作者： DU Ji-ge GE Xin-na DONG Hong ZHANG Ning ZHOU Lei GUO Xin YANG Han-chun key laboratory of animal epidemiology and Zoonosis Ministry of Agriculture/College of Veterinary Medicine and State Key Laboratory of Agrobiotechnology China Agricultural University

supported by the Earmarked Fund for Modern Agro-industry Technology Research System of China （CARS-36） from the ministry of agriculture of China;the National Natural Science Foundation of China （31572549）;the Nati... 详细信息

关键词： PRRSV glycoprotein 5(GP5) interactome profile mitofilin calnexin

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RNA-seq reveals the critical role of Csp A in regulating Brucella melitensis metabolism and virulence

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Science China(Life Sciences) 2016年第4期59卷 417-424页

作者： Zhen Wang Wenxiao Liu Tonglei Wu Pengfei Bie Qingmin Wu animal Science and Technology College Beijing University of Agriculture key laboratory of animal epidemiology and Zoonosis of the ministry of agriculture College of Veterinary Medicine China Agricultural University

Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of Brucella to survive and multiply in the hostile environment of host macrophages is essential for its virulence. The cold shock protein Csp A plays an important role in the virulence of B. melitensis. To analyze the genes regulated by Csp A, the whole transcriptomes of B. melitensis NI?csp A and its parental wild-type strain, B. melitensis NI, were sequenced and analyzed using the Solexa/Illumina sequencing platform. A total of 446 differentially expressed genes were identified, including 324 up-regulated and 122 down-regulated genes. Numerous genes identified are involved in amino acid, fatty acid, nitrogen, and energy metabolism. Interestingly, all genes involved in the type IV secretion system and Lux R-type regulatory protein Vjb R were significantly down-regulated in NI?csp A. In addition, an effector translocation assay confirmed that the function of T4 SS in NI?csp A is influenced by deletion of the csp A gene. These results revealed the differential phenomena associated with virulence and metabolism in NI?csp A and NI, providing important information for understanding detailed Csp A-regulated interaction networks and Brucella pathogenesis.

关键词： Brucella melitensis cold shock protein transcriptome virulence

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Identification of the strain-specifically truncated nonstructural protein 10 of porcine reproductive and respiratory syndrome virus in infected cells

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Journal of Integrative agriculture 2018年第5期17卷 1171-1180页

作者： ZHANG Zhi-bang XU Lei WEN Xue-xia DONG Jian-guo ZHOU Lei GE Xin-na YANG Han-chun GUO Xin key laboratory of animal epidemiology and Zoonosis Ministry of Agriculture/College of Veterinary MedicineChina Agricultural University/State Key Laboratory of AgrobiotechnologyChina Agricultural University

The nonstructural protein 10（nsp10） of porcine reproductive and respiratory syndrome virus（PRRSV） encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10 a, was found in PRRSV-infected cells and the production of nsp10 a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10 a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10 a production. Finally, we demonstrated that nsp10 a exerted little influence on the growth kinetics of PRRSV in vitro.

关键词： porcine reproductive and respiratory syndrome virus(PRRSV) nonstructural protein 10(nsp10) viral replication

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H9N2 influenza virus in China： a cause of concern

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Protein & Cell 2015年第1期6卷 18-25页

作者： Yipeng Sun Jinhua Liu key laboratory of animal epidemiology and Zoonosis Ministry of Agriculture College of Veterinary MedicineChina Agricultural University Beijing 100083 China

The recent human infection with avian influenza virus revealed that H9N2 influenza virus is the gene donor for H7N9 and H10N8 viruses infecting humans. The crucial role of H9N2 viruses at the animal-human interface might be due to the wide host range, adaptation in both poultry and mammalian, and extensive gene reassortment. As the most prevalent subtype of influenza viruses in chickens in China, H9N2 also causes a great economic loss for the poultry industry, even under the long-term vaccination programs. The history, epidemiology, bio- logical characteristics, and molecular determinants of H9N2 influenza virus are reviewed in this paper. The contribution of H9N2 genes, especially RNP genes, to the infection of humans needs to be investigated in the future.

关键词： Influenza Poultry H9N2 Evolution,Virulence Antigenic drift

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Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4

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Journal of Integrative agriculture 2017年第11期16卷 2573-2585页

作者： ZHANG Feng GAO Peng GE Xin-na ZHOU Lei GUO Xin YANG Han-chun key laboratory of animal epidemiology of the ministry of agriculture College of Veterinary Medicine State Key Laboratory ofAgrobiotechnology China Agricultural University Beijing 100193 P.R.China

Porcine reproductive and respiratory syndrome virus.（PRRSV） actively induces cell apoptosis both in vitro and in vivo, which can contribute critically to viral pathogenesis. Previous studies have shown that the PRRSV nonstructural protein 4 （nsp4） is an important mediator of this process, but the underlying molecular details remain poorly understood. In this study, we found that the PRRSV nsp4 interacted with the mitochondrial inner membrane protein cytochrome cl （***） and induced its proteolytic cleavage. Interestingly, the cleaved N-terminal fragment of *** was found to exert apoptotic activity, which could cause mitochondrial fragmentation, resulting in apoptotic cell death. And RNA interference （RNAi） silencing experiments further confirmed the crucial role which *** played in nsp4- and PRRSV-induced cell apoptosis. Thus, our data provide an important piece of mechanistic clues for PRRSV-induced cell apoptosis and also elucidate a novel mechanism for the 3C-like proteases in this finding.

关键词： PRRSV nonstructural protein4 (nsp4) cytochrome cl (cyto.cl) interaction cleavage apoptosis

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Detection and Characterization of β-Lactam Resistance in Haemophilus parasuis Strains from Pigs in South China

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Journal of Integrative agriculture 2012年第1期11卷 116-121页

作者： GUO Li-li ZHANG Jian-min XU Cheng-gang REN Tao ZHANG Bin CHEN Ji-dang LIAO Ming key laboratory of animal Disease Control and Prevention Ministry of Agriculture/Key Laboratory of Zoonoses Prevention and Control of GuangdongCollege of Veterinary MedicineSouth China Agricultural UniversityGuangzhou 510642P.R.China

To characterize the β-lactam resistance in veterinary clinical isolates of Haemophilus parasuis, 115 isolates were examined for the β-lactam resistance, the possession of β-lactamase, and the presence of β-lactamase genes. The genetic relationship among isolates was evaluated by pulsed-field gel electrophoresis （PFGE）. Overall, the commonly detected resistance phenotypes were resistant to ampicillin （26.09%）, penicillin （22.61%）, amoxicillin （21.74%）, cefazolin （14.78%）, cefaclor （12.17%）, and cefotaxime （6.96%）. These strains showed high minimal inhibitory concentration （MICs） to oxacillin. 20.87% strains produced β-lactamase, and 4.35% strains showed extended-spectrum b-lactamase （ESBL） phenotype. Moreover, 19 strains harboured bla genes including TEM-1 （n=5）, TEM-116 （n=10）, and ROB-1 （n=5）. Significantly, one strain possessed both TEM-1 and ROB-1, and displayed resistance to cefotaxime （MIC=8 mg L-1）. The epidemiological analysis of PFGE revealed high genetic diversity among bla-positive isolates. This work shows that TEM- and ROB-type β-lactamases are prevalent in H. parasuis isolates in China.

关键词： Haemophilus parasuis β-lactamase genes resistance pulsed-field gel electrophoresis

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