摘要
猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)归属于套式病毒目动脉炎病毒科属,由该病毒引起的猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)已成为世界养猪业生产中主要疫病之一。该病主要引起妊娠母猪早产、流产、死胎、木乃伊胎等繁殖障碍以及各年龄段猪的呼吸道症状和高死亡率。目前PRRS已经遍及全球主要养猪的国家和地区,给世界养猪业造成了巨大的经济损失。
PRRSV侵入宿主细胞首先通过与细胞膜表面上的特异性受体结合,利用细胞内吞作用而感染易感细胞。现已报道的PRRSV感染Marc-145细胞的受体有CD163和Vimentin。CD163属于富含半胱氨酸超家族的清道夫受体蛋白,不但能够结合PRRSV,还能介导病毒在细胞内的复制。Vimentin是一个普遍存在于细胞内的波形蛋白,单克隆抗波形蛋白抗体可以阻断PRRSV对Marc-145细胞的感染,对PRRSV非易感的BHK-21和CRFK细胞在表达了猴的波形蛋白之后变成了PRRSV易感细胞,同时波形蛋白与其它中间细丝蛋白组成的多聚体可能参与PRRSV在细胞内的复制和转移。
我的导师周恩民教授利用所制备的针对PRRSV GP5蛋白的单克隆抗独特型抗体(Mab2-5G2),从PRRSV易感细胞上发现了一个可能的PRRSV细胞受体蛋白。该蛋白为非肌肉肌球蛋白II型重链A(non-muscle myosin heavy chain II-A,NMHC II-A)。NMHC II-A广泛存在于自然界。在哺乳动物中,它是非肌肉肌球蛋白II型(NM II)重链的一个亚型,在整个细胞周期,包括细胞迁移、胞质分裂、细胞吸附和细胞形态变化过程中发挥重要的作用。已有研究证明NMHC II-A与T细胞上的趋化因子受体CXCR4结合,从而介导趋化因子的信号传导,可溶性CD163蛋白通过与T细胞上NMHC II-A的协同作用而结合T细胞,这些都说明了NMHC II-A在免疫反应中的作用。
本文主要研究了NMHC II-A对PRRSV感染Marc-145细胞的阻断作用。首先根据NMHC II-A羧基端基因序列人工合成七条多肽,通过病毒中和实验证明其中三条多肽对PRRSV感染Marc-145细胞有阻断作用。为探求NMHC II-A羧基端的生物学功能,从Marc-145细胞中提取总RNA,利用特异性引物,经RT-PCR克隆得到NMHC II-A羧基端基因(命名为PRA)。将PRA连接至pET-28a(+)原核表达载体,经诱导表达得到重组的NMHC II-A羧基端蛋白(命名为PRA蛋白)。经分段表达蛋白,Western-blot和I-ELISA实验证明了PRA蛋白能与Mab2-5G2特异性结合,并且找到了比较准确的结合位点。通过间接免疫荧光实验证明了PRA蛋白能够与Marc-145细胞的细胞膜特异性结合。更重要的是,通过病毒中和实验,荧光病毒中和实验,荧光定量PCR实验证明了PRA蛋白能够在一定程度上阻断PRRSV对Marc-145细胞的感染。将重组质粒pEGFP-N1-PRA转染至Marc-145细胞,与未转染的Marc-145细胞相比,PRRSV感染转染pEGFP-N1-PRA的Marc-145细胞的能力降低了50%。另外,还证明了NMHC II-A的阻断剂(R)-(+)-Blebbistatin对Marc-145细胞的胞质分裂没有影响,但对PRRSV感染Marc-145细胞有阻断作用。这些实验结果充分证明了PRA蛋白在PRRSV感染易感细胞过程中的作用,为进一步研究PRRSV感染细胞的分子机制提供了一定的依据,也为进一步探求PRRS的防控提供了新的思路。
Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by PRRS virus (PRRSV) which belongs to the family Arteriviridae. PRRS mainly causes reproductive disorder, including premature, abortion, fetal death, mummy and porcine respiratory syndrome at variety of ages with high mortality. At present, PRRS has prevailed in the main world while pig-raising country and districts. Nowadays the study to pathogenic and immunologic mechanism of PRRSV is not clear. There is also no proper way for controling of PRRSV infection.
PRRSV has a highly specific cell tropism, both in PAM and in the monkey kidney-derived cell lines; the virus enters through a mechanism of receptor-mediated endocytosis. Until now, two PRRSV receptors (CD163 and Vimentin) have been described on Marc-145 cells. CD163 is a cellular protein in the scavenger receptor cysteine-rich superfamily, functioned as interacting with PRRSV and may determine the replication levels of PRRSV. Vimentin is an intermediate-filament protein, which is widely expressed in various cell types. Anti-vimentin Abs block PRRSV infection of Marc-145 cells. When simian vimentin was delivered to BHK-21cells and CRFK cells, they became susceptible to PRRSV. Vimentin is also thought to be involved in the replication and transportation of PRRSV inside the cell by forming a complex with the other components of the intermediate filaments.
An internal image Mab2-5G2 which was generated against idiotypic antibodies specific for GP5 antigen of PRRSV identified a soluable protein prepared from Marc-145 cells. The protein was identified to be non-muscle myosin heavy chain II-A (NMHC II-A). NMHC II-A is distributed ubiquitously throughout nature. It is one isoform of NM II heavy chain and it plays a role in a variety of cellular processes including cell migration, cytokinesis, cell adhesion and cell shape change both during development and in the adult organism. An association between the C-termininal of CXCR4 and CCR5 and the motor protein NMHC II-A has been found and this biochemical association may have a key role in lymphocyte migration. Soluble CD163 binds with T lymphocytes coeffected with NMHC II-A. These have proved the function of NMHC II-A in immuno-reaction.
In this article, we mainly study the block function of C-terminus NMHC II-A in Marc-145 cells to PRRSV infection. At first, seven peptides which located on the C-terminal of NMHC II-A were synthesized. Virus neutralization experiment showed that three peptides among the seven ones can block PRRSV infection of Marc-145 cells. In order to study the biological function of C-terminus NMHC II-A, total RNA was extracted from Marc-145 cells and specific primes were synthesized. After RT-PCR, the C-terminus NMHC II-A named PRA was cloned and sequenced, the Gene bank number was HM490008. PRA was then cloned to pET-28a (+) vector, after expression, PRA protein was obtained. Expression of truncated proteins, western-blot and I-ELISA experiments showed that PRA protein can specifically bind with Mab2-5G2; also the specific binding site was identified. IFA experiment proved PRA protein can specifically bind the membrane of Marc-145 cells. What was more important was that VN, FFN and Real Time PCR experiments identified that PRA protein can block PRRSV infection of Marc-145 cells in some degree. PRA was also cloned to pEGFP-N1 vector; the ability of PRRSV infecting the transfected Marc-145 cells was decreased by 50% in comparison with non-transfected cells. Otherwise, (R)-(+)-Blebbistatin which was the inhibitor of NMHC II-A not only can not affect the cytokines of Marc-145 cells but also block PRRSV infection by VN experiment. These results showed that C-terminus NMHC II-A can block PRRSV infection of Marc-145 cells. They provide a noval approach for further understanding of PRRSV infection and control of PRRS.
引文
安同庆,田志军,肖燕等.高致病性猪繁殖与呼吸综合征病毒变异株主要囊膜糖蛋白GP5的遗传变异分析[J].中国预防兽医学报, 2008, 30 (11):851-856.
陈勇军,苏鑫铭,高晓飞等.猪繁殖与呼吸综合征病毒ORF5基因的融合表达[J].农业生物技术学报, 2006, 14 (3):319-322.
仇华吉,童光志.猪繁殖与呼吸综合征[M].吉林:科学技术出版社, 2000:5.
杜文金,刘卫.应用重组猪繁殖与呼吸综合征病毒核衣壳蛋白为抗原建立ELISA检测方法的研究[J].中国兽医科技, 2000, 30 (6):7-9.
高云英,贺玉胜,刘秀琴等.猪繁殖与呼吸综合症研究进展[J].甘肃农业大学学报, 2002, 37 (6):139-144.
高志强,郭鑫,杨汉春.猪繁殖与呼吸综合征病毒基因组遗传变异研究进展[J].中国农业科技导报, 2002, 4 (6):64-67.
高志强.猪繁殖与呼吸综合征病毒全基因组分子遗传特征分析[D].北京:中国农业大学, 2003.
高志强,郭鑫,杨汉春等.猪繁殖与呼吸综合征病毒缺失变异株的基因组特征[J].畜牧兽医学报, 2005, 36:578-584.
郭宝清,陈章水,刘文兴等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国预防兽医学报, 1996, 12 (2):1-5.
李华,杨汉春,郭玉璞.猪繁殖与呼吸综合征病毒(PRRSV)感染的免疫学研究进展[J]. 中国兽医杂志, 1999, 25 (9):40-42.
李兴玉.猪生殖呼吸综合征病毒分子生物学研究进展[J].四川畜牧兽医, 2003, 30 (9):45-48.
刘光清,蔡雪晖,仇华吉等.猪繁殖与呼吸综合征的研究进展[J].中国预防兽医学报, 2001, 23 (1):72-76.
刘国信.高致病性猪蓝耳病的综合防治[J].北京农业, 2007, 8:34-35.
刘金霞,周艳君,安同庆,仇华吉,王云峰,童光志.猪繁殖与呼吸综合征病毒ORF2基因的表达及其单克隆抗体的制备[J].中国预防兽医学报, 2006, 28 (6):636-639.
刘书亮,王红宁. PRRS的病原学和免疫学研究进展[J].四川畜牧兽医, 2000, 27 (7):79-83.
萨姆布鲁克等. 2002.分子克隆实验指南(第二版).黄培堂,等译.北京:科学出版社.
舒展,魏润生,高生智等猪生殖与呼吸综合征甘肃株的分离鉴定[J].中国兽医科技, 2001,31 (10):17-18.
童光志,周艳君,郝晓芳等.高致病性猪繁殖与呼吸综合征病毒的分离鉴定及其分子流
行病学分析[J].中国预防兽医学报, 2007, 29 (5):323-327.
王刚,张鹤晓,甘孟侯等. IPMA检测猪生殖和呼吸综合征病毒抗体的研究[J].中国兽医杂志, 1996, 22 (12):3-5.
王伯法,等主编.病理学技术[M].北京:人民卫生出版社, 2000, 822-834.
王旭荣,马红艳,祝卫国等.高热病猪群中PRRSV的调查及其流行毒株的分离[J].中国兽医杂志, 2008, 44 (3):39-40.
翁伟平,张鸳,郭挺伟等. GST-LRF融合蛋白的表达及其单克隆抗体的制备[J].西北农业学报, 2001, (1):40-43.
吴国平,郭川,曹敏杰等.原核高效表达著繁殖与呼吸综合征病毒核衣壳蛋白的特性及应用[J].中国兽医学报, 2007, 27 (2):150-154.
吴锦艳,田宏,尚佑军等.高致病性猪蓝耳病病毒GS/LZh/07株的分离、鉴定及其非结构蛋白Nsp2基因特性分析[J].畜牧兽医学报, 2009, 40 (3):483-444.
吴延功,徐天刚,王志亮等.重组N蛋白抗原检测PRRS抗体ELISA的研究[J].中国兽医学报, 2005, 25 (4):339-342.
夏平安,尹彦涛,李素萍等.猪繁殖与呼吸综合征病毒重组N蛋白的高效表达及间接ELISA方法的建立[J].中国兽医学报,2009, 29 (5):537-541.
颜世敢,曹洪敬,许学孟.猪繁殖与呼吸综合征的研究进展[J].山东畜牧兽医, 2000 (5): 37-39.
杨汉春,管山红,尹晓敏等.猪繁殖与呼吸综合征病毒的分离与初步鉴定[J].中国兽医杂志, 1997, 23(10): 9-10.
杨汉春,黄芳芳,张旭等.用单克隆抗体鉴定猪繁殖与呼吸综合征病毒分离株.中国兽医杂志, 1998, 23 (4):3-5.
杨汉春.猪免疫抑制性疾病的流行特点与控制对策[J].中国畜牧兽医, 2004, 31 (5):41-43.
姚龙涛.当前国内外猪的重要疾病与控制[J].养猪, 2004, 4:33-35.
殷震,刘景华. 1997.动物病毒学.第二版,北京:科学出版社, 613-628.
于丹,韩宗玺,邵昱昊,李慧昕.抗鸡传染性支气管炎病毒N蛋白单克隆抗体制备与鉴定[J].中国预防兽医学报, 2009, 31 (8):654-656.
张春玲,张婉华,臧克伟等.猪繁殖与呼吸综合征病毒S-1株ORF5和ORF7基因的克隆及变异分析[J].上海农业学报, 2008, 24 (2):40-45.
赵耘,罗长宝,陈茹等.猪生殖和呼吸综合征病毒B13株ORF7基因的克隆及在杆状病毒系统中的表达[J].病毒学报, 2001, 3 (17)1:75-79.
周宗安译. 1991.单克隆抗体技术手册.南京:南京大学出版社, 48-87.
Albina E. Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview[J]. Vet Microbiol, 1997, 55:309-316.
Allende, R., Kutish, G.F., Laegreid, W., et al. Mutation in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype[J]. Arch. Virol., 2000, 145:1149-1161.
Benfield D.A., Nelson E., Collins J.E., et al. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332)[J]. J Vet Diagn Invest., 1992, 4:127-133.
Calvert, J. G., Slade, D. E., Shields, S. L.,et al. CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses[J]. J Virol., 2007, 81:7371-7379.
Charerntantanakul W., Platt, R., Roth, J.A., Effects of porcine reproductive and respiratory syndromevirus-infected antigen-presenting cells on T cell activation and antiviral cytokine production[J]. Viral Immunol., 2006, 19:646-661.
Choi C. S., Christianson W. T., Gustafson K. V., et al. Antibody- dependent enhancement of PRRS virus replication[J]. Am.Assoc. Swine.Pratt. News, 1992, 4:19-21.
Conti MA, Kawamoto S, Adelstein RS. Non-muscle myosin II. 2008. In Myosin: A Superfamily of Molecular Motors. Coluccio LM ed., pp223-264.
Conzelmann Visser K. N., Thiel H., et al. Molecular characterization of Procine reproductive and respiratory syndrome virus, a member of the artervirus group[J]. Virology, 1993, 193:329-339.
Dea S., Gagnon C.A., Mardassi H.,et al. Antigenic variability among North-American and European strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein[J]. J Clin Microbiol, 1996, 34:1488-1493.
Dee S.A., Torremorell M., Rossow K., et al. Identification of genetically diverse sequences (ORF5) of porcine reproductive and respiratory syndrome virus in a swine herd[J]. Can J Vet Res., 2001, 65:254-260.
Delputte P. L., and Nauwynck H. J. Porcine arterivirus infection of alveolar macropHages is mediated by sialic acid on the virus[J]. J. Virol., 2004, 78:8094-8101.
Delputte P. L., and Nauwynck H. J. Porcine arterivirus entry in macrophages: heparan sμlfate-mediated attachment, sialoadhesin-mediated internalization, and a cell-specific factor mediating virus disassembly and genome release[J]. Adv. Exp. Med. Biol., 2006, 581:247-252.
Delputte P. L., Vanderheijden N., Nauwynck H. J., and Pensaert M. B. Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages[J]. J. Virol., 2002, 76:4312-4320.
Delputte P. L., Costers S., and Nauwynck H. J. Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulpHate and sialoadhesin[J]. J. Gen. Virol., 2005, 86:1441-1445.
Delputte P. L., Van Breedam W., Barbe F., et al. IFN-a treatment enhances porcine arterivirus infection of monocytes via upregulation of the porcine arterivirus receptor sialoadhesin. J Interferon Cytokine Res., 2007a, 27:757-766.
Delputte P. L., Van Breedam W., Delrue I., et al. Porcine arterivirus attachment to the macrophage specific receptor sialoadhesin is dependent on the sialic acid-binding activity of the N-terminal immunoglobulin domain of sialoadhesin[J]. Virol., 2007b, 81:9546-9550.
Desrosiers R. Transmission of pathogens: We, veterinarians, should change our tune[J]. International Pigletter, 2004, 2 (4): No. 2c.
Duan X., Nauwynck H. J., Pensaert M. B. Effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) [J]. Arch. Virol., 1997, 142:2483-2497.
Duan X., Nauwynck H. J., Pensaert M. B. Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV)[J]. Vet. Microbiol., 1997, 56:9-19.
Duan X., Nauwynck H. J., Favoreel H. W., et al. Identification of a putative receptor for porcine reproductive and respiratory syndrome virus on porcine alveolar macrophages[J]. J. Virol., 1998, 72:4520-4523.
Fabriek B. O., Dijkstra C. D., van den Berg T. K. The macrophage scavenger receptor CD163[J]. Immunobiology, 2005, 210:153-160.
Geoffrey G, Labarque Hans J,et al.Effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs[J]. J.Gen. Virol., 2000, 81:1327-1334.
Gorp HV, Breedam WV, Delputte PL,Nauwynck HJ. Sialoadhesin and CD163 join forcesduring entry of the porcine reproductive and respiratory syndrome virus[J]. 2008. J. Gen. Virol. 89:2943-2953.
Hall W V. Porcine reproductive and respiratory syndrome (PRRS) virus: a significant disease of pigs[J]. Aust Vet J, 2005, 83 (5): 260-261.
Hirose O, Kudo H, Yoshizawa S, Hiroike T, Nakane T. Prevalence of porcine reproductive and respiratory syndrome virus in Chibaprefecture[J]. J Jpn Vet Med Assoc., 1995, 48:650-653.
Hopper SA, White MEC, Twiddy W. An outbreak of blue-eared pig disease (Procine reproductive and respiratory syndrome) in four pig herds in Great Britain[J]. Vet Re., 1992, 1331:140-144.
Jeong-Ki Kim,Al-Majhdi Fahad,Kumar Shanmukhappa,Sanjay Kapil. Defining the Cellular Target(s) of Porcine Reproductive and Respiratory Syndrome Virus Blocking Monoclonal Antibody 7G10[J]. J. Virol., 2006, 80:689–696.
Keffaber K K.Reproductive failure of unkown etiology[J].American Association of Swine Pratitioners Newsletter., 1989, 1:1-10.
Kim H.S., Kwang J., Yoon I.J., et al. Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line[J]. Arch. Virol. 1993, 133:477-483.
Le Potier M-F., Blanquefort P., Morvan E., et al. Results of a control programmer for the porcine reproductive and respiratory syndrome in the French“Pays de la Loire”region[J]. Vet Microbiol, 1997, 55:355-360.
Lee C., Jay G., Calver et al. A DNA-launched reverse genetics system for procine reproductive and respiratory syndrome virus reveals that homodimerization of nucleocapsid protein is essential for virus infectivity[J]. Virology, 2005, 331:47-62.
Loemba H.D., Mounir S., Mardassi H., et al. Kinetics of humoral immune response to the majorstructural proteins of the procine reproductive and respiratory syndrome virus[J]. Arch Virol, 1996, 141:751-761.
Limouze J, Straight AF, Mitchison T, Sellers JR.2004.Specificity of blebbistatin, an inhibitor of myosin II.J Muscle Res Cell Motil. 25:337-41.
Magar R., Larochelle R., Nelson E.A., et al. Differential reactivity of a monoclonal antibody to the membrane protein of porcine reproductive and respiratory syndrome virus[J]. Can J Vet Res, 1997, 61:69-71.
Magar R., Larochelle S., Dea S., et al. Antigenic comparison of Canadian and US isolates of porcine reproductive and respiratory syndrome virus using monoclonal antibodies to thenucleocapsid protein[J]. Canadian Journal of Veterinary Reseach, 1995, 59:232-234.
Mardassi H., Mounir S., Dea S. Identification of major difference in the nucleocapsid protein genes of a Quebec strain and European strains of procine reproductive and respiratory syndrome virus[J]. Journal of General Virology, 1994, 75:681-685.
Mardassi H., Mounir S., Dea S..Molecular analysis of the ORFs 3 to 7 of porcine reproductive and respiratory syndrome virus, Québec reference strain[J]. Arch. Virol., 1995, 140:1405-1418.
Meng X. J., Paul P. S., Halbur P. G., et al. pHylogenetic analysis of the putative M (ORF6) and N (ORF7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the US and Europe[J]. Archives of Virology, 1995, 140:745-755.
Meng X.-J., Paul P. S., Halbur P. G., et al.Sequence comparison of open reading frame 2 to 5of low and high virulence United States isolates of porcine reproductive and respiratory syndrome virus[J]. J Gen Virol., 1995, 76:3181-3188.
Meng X.J., Paul P.S., Halbur P.G., et al. phylogenetic analysis of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe[J]. Arch. Virol., 1995, 140:745-755.
Meulenberg J.J.M., Hulst M.M., De Meijer E.J., et al. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEAPS), is related to LPV and EAV[J]. Virology, 1993, 192:62-72.
Molitor T. W., Bautista E. M., Choi C. S. Immunity to PRRSV: double -edged sword[J]. Vet Microbiol., 1997, 55:265-276.
Nelsen C.J., Murtaugh M.P., Faaberg K., et al. Porcine reproductive and respiratory syndrome virus comparison; divergent evolution on two continents[J]. J Virol., 1999, 73:270-280.
Phani B. Das, Phat X. Dinh, Israrul H. Ansari, Marcelo de Lima, Fernando A. Osorio, and Asit K. Pattnaik. The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor CD163[J]. Journal of virology, 2010, 84:1731-1740.
Pringle C.R. Virus Taxonomy A bulletin from the Xth International Congress of Virology in Jerusalem[J]. Arch Virol. 1996, 141:2251-2256.
Rey M, Vicente-Manzanares M, Viedma F, Yá?ez-MóM, Urzainqui A, Barreiro O, Vázquez J, Sánchez-Madrid F. Cutting edge: association of the motor protein nonmuscle myosin heavy chain-IIA with the C terminus of the chemokine receptor CXCR4 in T lymphocytes[J]. J.Immunol., 2002, 169:5410-5414.
Rossow K.D. Porcine reproductive and respiratory syndrome Vet Pathol, 1998, 35(1): 1-20.
Rottier P.J.M., Welling G.W., Welling-Wester S., et al. Predicted membrane to pology of the corona virus Protein EI[J]. Proc Natl Acad Sci USA, 1986, 81:1421-1425.
Sellers JR. Myosin: a diverse superfamily. Biochem. Biophy. Acta, 2000, 1496:3-22.
Snijder E.J., Meulenberg J.J.M., The molecular biology of retroviruses[J]. J Gen Virol, 1998, 79:961-979.
Straight AF, Cheung A, Limouze J, Chen I, Westwood NJ, Sellers JR, Mitchison TJ. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science, 2003, 299:1743-7.
Suarez P., Zardoya R., Martin M. J., et al. phylogenetic relationships of European strains of porcine reproductive and respiratory syndrome virus (PRRSV) inferred from DNA sequences of putative ORF5 and ORF7 genes[J]. Virus Research, 1996, 42:159-165.
Terpstra C., Wensvoort G., Pol J. M. A. Expeimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with Lelystad virus: Koch’s postulates fulfilled[J]. Vet Q, 1991, 13:131-136.
Timmermann M, Buck F, Sorg C, H?gger P. Interaction of soluble CD163 with activated T lymphocytes involves its association with non-muscle myosin heavy chain type A. Immunol. Cell. Biol., 2004, 82:479-87.
Vanderheijden N., Delputte P. L., Favoreel H. W., et al. Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macropHages[J]. J. Virol., 2003, 77:8207-8215.
Weiland E., Wieczork-Krohmer, Kohl D., et al. Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4[J]. Vet Microbiol, 1999, 66:171-186.
Wensvoort G., Terpstra C., Pol J.M.A., et al. Mystery Swine Disease in the Netherlands: the isolation of lelystad virus[J]. Vet Q. 1991, 13:121-130.
William L. Mengeling, Kelly M. Lager, Ann C. Vorwald., et al. Comparative safety and efficacy of attenuated single-strain and multi-strain vaccines for porcine reproductive and respiratory syndrome[J]. Veterinary Microbiology, 2003, 93:25-38.
Wills, R.W., Doster, A.R., Galeota, J.A., et al. Duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus[J]. Clin. Microbiol. 2003, 41:58-62.
Wootton S.K., Yoo D., Rogan D., Full-length sequence of a Canadian porcine reproductiveand respiratory syndrome virus (PRRSV) isolate[J]. Arch. Virol., 2000, 145:2297-2323.
Wai-Hong Wu, Ying Fang, Raymond R.R. Rowland, Steven R. et al., The 2b protein as a minor structural component of PRRSV. Virus Research, 2005, 114:177-181.
Xiao Z., Batista L., Dee S., Halbur P., et al. The level of virus-specific T-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of the virus load[J]. J Virol., 2004, 78:5923-5933.
Yoon K.J., Wu L.L., Zimmerman J.J., et al. Antibody-dependent enhancement (ADE) of porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs[J]. Viral Immunal., 1996, 9(1):51-63.
Yoon K. J., Wu L.L., Zimmerman J.J., et al.Field isolates of porcine reproductive and respiratory syndrome virus (PRRSV) vary in their susceptibility to antibody dependent enhancement (ADE) of infection[J]. Vet Microbiol., 1997, 55(1-4):277-287.
Zhang Y.,Sharma R.D.,Paul P.S. Monoclonal antibodies against conformationally dependent epitopes on porcine reproductive and respiratory syndrome virus[J]. Vet Microbiol, 1998, 63:125-136.
Zhou E-M., Clavijo A., Jiang Z. J., et al. Induction of auto-anti-idiotypic antibodies specific for antibodies to matrix and envelope glycoprotein from pigs experimentally infected with porcine reproductive and respiratory syndrome virus[J]. Vet Immunol Immunopathol. 2004, 101:49-59.
Ziebuhr J., Snijder E. J., Gorbalenya A. E. Virus-encoded proteinases and proteolytic processing in the Nidovirales[J]. J.Gen.Virol., 2000, 81:853 -879.