猪繁殖与呼吸综合征基因免疫的研究
|
摘要
本实验选取猪繁殖与呼吸综合征病毒CH-1a株的保护性抗原基因及细胞因子IFN-γ、IL-2基因构建了7种重组真核表达质粒：1. pIRESorf5/4（表达全长ORF5、ORF4基因）；2. pIRESorf6/5（表达全长ORF6、ORF5基因）；3. pIRESorf5/7（表达全长ORF5、ORF7基因）；4. pIRESorf7/IFN-γ（表达全长ORF7、IFN-γ基因）；5. pIRESorf5/IFN-γ（表达全长ORF5、IFN-γ基因）；6. pIRESorf5/IL-2（表达全长ORF5、IL-2基因）；7. pIRESorf7/IL-2（表达全长ORF7、IL-2基因）。获得这7种表达质粒后，利用IIF检测在体外的表达情况，转染7种质粒的细胞及接毒细胞均可看到细胞胞浆有特异性荧光，而未转染细胞及转染pIRES1neo空载体的细胞在细胞胞浆中无特异性荧光。任意选择构建好的2种质粒pIRESorf5/4、pIRESorf5/7免疫BALB/c小鼠，结果表明pIRESorf5/4、pIRESorf5/7都能发生表达。分别用这7种重组质粒和空载体pIRES1neo以及猪繁殖与呼吸综合征油乳剂灭活苗肌注30日龄的断奶仔猪，采用股四头肌多点注射的方式进行了四次免疫，间隔为两周。第四次免疫后两周，所有猪均用2.0ml的CH-1a株强毒用滴鼻的方法进行攻毒，攻毒后每天观察并详细记录猪的发病和死亡情况，免疫及攻毒前后均采血检测抗体和外周血CD4~+、CD8~+ T细胞的变化。结果表明，构建的7种真核表达质粒都可以诱导免疫猪产生体液免疫应答和细胞免疫应答。真核表达质粒诱导的免疫应答可以使免疫猪在一定程度上抵抗强毒的致病性攻击，但不能阻止病毒的感染。免疫前各组均检测不到抗体，第一次免疫后两周开始出现抗体，第三次和第四次免疫后抗体逐步升高。用流式细胞术检测猪外周血淋巴细胞CD4~+、CD8~+ T细胞的变化，结果表明我们构建的7种真核表达质粒免疫动物后可诱导机体产生细胞免疫应答。第四次免疫后两周用PRRSV同型强毒进行攻毒，空载体对照组、阴性对照组的猪在攻毒后20天内各有一头猪出现体温升高，体温达40℃，其余大部分猪体温维持在38-39℃。免疫组和对照组均有猪组织脏器出现不同程度的病理变化，表现出个体差异，免疫组获得了部分保护。本实验对猪繁殖与呼吸综合征基因免疫进行了初步研究，为以后进一步研究奠定了基础。
Seven plasmids against PRRSV were constructed.pIRESorf5/4 contained the ORF4 and ORF5 genes of PRRSV CH-1a.pIRESorf6/5 contained the ORF6 and ORF5 genes of PRRSV CH-1a.pIRESorf5/7 contained the ORF5 and ORF7 genes of PRRSV CH-1a.pIRESorf7/IFN-γ contained the ORF7 gene of PRRSV and porcine IFN-γ gene.pIRESorf5/IFN-γ contained the ORF5 gene of PRRSV and porcine IFN-γ gene.pIRESorf7/IL-2 contained the ORF7 gene of PRRSV and porcine IL-2 gene. pIRESorf5/IL-2 contained the ORF5 gene of PRRSV and porcine IL-2 gene.The foreign genes of these plasmids were expressed by IIF test of the Marc-145 cells transfected with the plasmids.Ten BALB/c mice were injected with plasmids pIRESorf5/4 and pIRESorf5/7.The antibodies against PRRSV were identified after the second inoculation.Thirty-day old, PRRSV-negative piglets were divided into 10 groups (three piglets per group)，The piglets of each group were received the seven types of plasmids，pIRES1neo and inactivated vaccine respectiveiy.The inoculated pigs were injected with 400μg of recombinant of unaltered plasmid DNA,or with 2.0ml of inactivated vaccine by intramuscular injection every 2 weeks for a total of four inoculations.The recombinant plasmids and inactivated vaccine could induce humoral and cellular immunity of the pigs.The percentage of CD4~+ and CD8~+ increased at different extents.Two weeks later,every pig was challenged in nasally with 2.0ml of PRRSV cultures (~106TCID50/ml).One pig of pIRES1neo group and negative control group had a fever,and the body temperature exceed 40℃ in 20 days after challenge.The temperature of other pigs retain 38-39℃，Viscera of some pigs of immunized group and negative control group had special pathologic change，especially in pneumonia.The contribution of PRRSV in tissues of pigs of different groups was various. The recombinant plasmids could protect the pigs from PRRS disease at different extents, but could not prevent the infection of PRRSV.
Seven plasmids against PRRSV were constructed.pIRESorf5/4 contained the ORF4 and ORF5 genes of PRRSV CH-1a.pIRESorf6/5 contained the ORF6 and ORF5 genes of PRRSV CH-1a.pIRESorf5/7 contained the ORF5 and ORF7 genes of PRRSV CH-1a.pIRESorf7/IFN-γ contained the ORF7 gene of PRRSV and porcine IFN-γ gene.pIRESorf5/IFN-γ contained the ORF5 gene of PRRSV and porcine IFN-γ gene.pIRESorf7/IL-2 contained the ORF7 gene of PRRSV and porcine IL-2 gene. pIRESorf5/IL-2 contained the ORF5 gene of PRRSV and porcine IL-2 gene.The foreign genes of these plasmids were expressed by IIF test of the Marc-145 cells transfected with the plasmids.Ten BALB/c mice were injected with plasmids pIRESorf5/4 and pIRESorf5/7.The antibodies against PRRSV were identified after the second inoculation.Thirty-day old, PRRSV-negative piglets were divided into 10 groups (three piglets per group)，The piglets of each group were received the seven types of plasmids，pIRES1neo and inactivated vaccine respectiveiy.The inoculated pigs were injected with 400μg of recombinant of unaltered plasmid DNA,or with 2.0ml of inactivated vaccine by intramuscular injection every 2 weeks for a total of four inoculations.The recombinant plasmids and inactivated vaccine could induce humoral and cellular immunity of the pigs.The percentage of CD4~+ and CD8~+ increased at different extents.Two weeks later,every pig was challenged in nasally with 2.0ml of PRRSV cultures (~106TCID50/ml).One pig of pIRES1neo group and negative control group had a fever,and the body temperature exceed 40℃ in 20 days after challenge.The temperature of other pigs retain 38-39℃，Viscera of some pigs of immunized group and negative control group had special pathologic change，especially in pneumonia.The contribution of PRRSV in tissues of pigs of different groups was various. The recombinant plasmids could protect the pigs from PRRS disease at different extents, but could not prevent the infection of PRRSV.
引文
1郭宝清,陈章水.从疑似PRRS流产胎儿分离PRRSV的研究.[J]中国畜禽传染病，1998,20(4):193-196
2陈博文,孙颖杰.猪繁殖和呼吸系统综合征的血清学检测及病毒的分离和鉴定.（初报）[J]中国兽医杂志,1996,22(5):6-8
3杨汉春,管山红.猪繁殖和呼吸系统病毒的分离与初步鉴定.[J]中国兽医杂志,1997,23(10):9-10
4刘文兴,蔡雪辉,郭宝清,猪生殖-呼吸道综合征病毒（PRRSV）分离鉴定及其与欧美PRRSV抗原的比较.[J]中国畜禽传染病,1998,20(4):193-196
5严亚贤,孙建和,王恒安,等.猪生殖-呼吸道综合征病毒上海株的分离鉴定.[J]中国预防兽医学报,2000,22(5):161-163
6张鹤晓,王刚,王彩兰.等.应用间接免疫荧光试验从猪群中检测出PRRS抗体.[J]中国兽医杂志,1996,22(11):7-8
7王刚,张鹤晓,甘孟侯,等.IPMA检测猪生殖和呼吸道综合征病毒抗体的研究.[J]中国兽医杂志,1996,22(12):3-5
8 王刚,张鹤晓,甘孟侯,等.Dot-ELISA检测猪生殖和呼吸综合征抗体的研究.[J]中国兽医杂志,1996,22(13):3-5
9郭宝清,陈章水,刘文兴.应用间接免疫荧光法从国内生殖障碍猪群中检测生殖和呼吸综合征阳性抗体的研究.[J]中国兽医科技,1996,26(3):3-5
10杨汉春,黄芳芳,张旭,等.用单克隆抗体鉴定猪繁殖和呼吸道综合征病毒分离株.[J]中国兽医科技,1997,23(4):3-5
11陆光进,冯加明,俸玉维.应用间接免疫荧光试验猪生殖-呼吸道综合征抗体.[J]中国畜禽传染病,1997,5:36-37
12马志永,简中友,姜平.斑点酶联免疫吸附试验检测猪生殖与呼吸综合征病毒抗体方法的建立.[J]中国兽医科技,1997,27(4):3-4
13查红波,陈西钊,张鹤晓,等.应用间接酶联免疫吸附试验检测猪生殖与呼吸综合征病毒抗体的研究.[J]中国兽医科技,1997,27(9):20-21
14梁杏娴,卢洪芬,董振光.猪繁殖与呼吸综合征的诊断.[J]中国兽医杂志1998,24(4):28-29
15娄高明,林绍荣,黄茂,等.仔猪暴发繁殖与呼吸综合征.[J]中国兽医学报,1998，18(5):445-447
16晁学元,刘得胜，崔海龙,等.猪繁殖与呼吸综合征血清学调查.[J]中国兽医杂志,1999,25(7):17-18
17张晓梅,杨汉春,任慧英,等.用LSAB免疫组化染色法检测猪生殖与呼吸综合征病毒抗原的研究.[J]中国预防兽医学报,1999,21(6):427-429
18蔡雪辉,郭宝清,柴文君,等.检测猪生殖与呼吸综合征病毒抗体ELISA方法的建立.[J]中国预防兽医学报,2000,22(2):122-125
19娄高明,杜伟贤,谢明权,等.猪生殖与呼吸综合征RT-PCR诊断方法的建立.[J]中国兽医学
报,2000,20(2):141-144
20李军,林继煌,姜平,等.用免疫金技术检测猪生殖与呼吸综合征病毒.[J]畜牧与兽医,2001,33(2):1-2
21王云峰,周艳君,仇华吉,等.应用重组N-蛋白- ELISA检测猪生殖与呼吸综合征抗体的研究.[J]中国兽医杂志,2001,37(3):3-5
22杨小燕,李晓华,沈绍新,等.猪繁殖与呼吸综合征的血清学调查.[J]中国兽医杂志,2001,37(4):17-18
23卫秀余,姚龙涛,金爱华.猪生殖－呼吸道综合征的血清学调查. [J]中国畜禽传染病,
1998,20(增刊):87-88
24 Wensvoort G,Terpstra C,Pol JMA,et al.Immunomodulation of host immune responses following porcine reproductive and respiratory syndrome Virus infection.[J] Vet Q,1991,13:121-190
25 Bilodeau R,Dea S,Sauvageau RA, et al.Examination of virus shedding in semen from vaccinated and from previously infected boars after experimntal challenge with porcine reproductive and respiratory syndrome virus.[J]Vet.Rel.,1991,129:102-103
26 Keffaber KK.Reproductive failure of unknown etiology.[J] Am Assoc. Swine Pract. Newslett,1989,1:1-9
27 Shimizu M,Yamada S,MuRakaMi Y,et al.Isolation of swine infertility and respiratory syndrome Virus from Heko-heko disease of pigs.[J] J. vet. MedSci,1994,6:389-391
28 Tetpstra C,Wensvoort G, Poi JMA.Expremental reproduction of porcine epidemic abortion and respiratory syndrome (Mystery Swine Disease) by infection with Lelystad virus.[J] Vet. Q,1991,13:131-136
29 Wensvoort G,Terpstra IC,Pol JMA, et al.Mystery swine disease in the Netherlands:the isolation of Lelystad virus.[J] vet Q, 1991,13:121 -130
30 Benfield DA,Nelson E,Collins JE,et al.Characterization of swine infertility and respiratory syndrome virus(isolate ATCC VR-2332).[J]J.Vet Diagn Invest,1992,4:127-133
31 Collins JE,Benfield DA,Christianson WT,et al.Isolation of swine infertility and respiratory syndrome virus(isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs.[J]J.Vet.Diagn.Tnvest,l992,4:117-126
32 Dea S, Bilodeau R,Athanassious R,et al.Swine infertility and respiratory syndrome In Quebec:isolation and characterization of an enveloped virus serologically related to Lelystad virus.[J]J.Can.Vet.,1992,33:891-908
33 Baron T, Albina E,Leforban Y,et al.Report on the first outbreak of the porcine reproductive and respiratory syndrome(PRRS) In France.[J]Ann. Recl Vet.,1992,23:
161-166
34 Meulenberg JJM, Hulst MM,Meijer D,et al.Lelystad virus,the causative agent of porcine epidemic abortion and respiratory syndrome(PRRS) is related to LDV and EAV. [J] Virology,1993,1(92):62-72.
35 Meullenberg JJ,Peltrsen-DEN Besten ADE et al.Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus.[J]Virology,1997,206:155-183
36 Kjm HS,Kwang J,Yoon.et al.Enhanced reproduction of the porcine reproductive and respiratory syndrome(PRRS) in a homogenous subpopulation of MA-104 cell line.[J] Atchives of Virology,1993,133:477-453
37 Moos B,Elroy-Stein O,Mizukami T,et al.New mammalian expression vectors.[J]. Nature, 348:
91-92
38 Molitor TW,Dautista EM,Choi CS.Immunity to PRRSV: double-edged sword.[J]Vet Microbiol,l997,55(1-4):265-276
39 Molitor T,LeitnerG,ChoiC,et al.Modulation of host immune response by SIRS virus.[J].Am. Assoc.Swine Prac. Newslett,1992,4:27-28
40李华,杨汉春,郭玉璞.猪繁殖与呼吸综合症病毒(PRRSV)感染的免疫学研究进展.[J]中国兽医杂志,1999,25 (9):40-42
41 Shimizu M,Pan IC.Hess WRT and B lymphocyte in porcine blood.[J]AM. J. Vet. Res., 1976,31:309-317
42 Molitor TW,Bautista EM,Choi CS.Immunity to PRRSV: Double-edged sword.[J]Vet Microbiol,1997,55(1/4):265-276
43 Lopez FL,Domenech N,Alvarez B,et al. Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection.[J]Virus Res, l999,64(1):38-42
44 Saalmuller. A,Ilirt W,Maurer S, et al.Discrimination between two subsets of porcine CD8+ cytolytic T lymphocytes by the expression of CD5 antigen.[J] Immunology，1994,81:578-583
45 Albina E,Piriou L,Hutet E, et al.Immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). [J]Vet Immunol Immunopathol 1998,6 (l):49-66
46 Ummerfield A,Rziha HJ,Saalmuller A.Functional characterization of porcine CD4+、CD8+ extrathymicT lymphocytes.[J]Celluar lmmunology 1996,168:291-296
47 Roland RR,Rpbinson B,Ste FJ,et al.Inhibition of porcine reproductive and respiratory
syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine.[J]Arch Virology,2001,146(3):539-555
48 Zhou Y,Barghusen S,Choi C, et al.Effects of SIRS virus infection in leukocyte populations in the peripheral blood and on cytokine expression in alveolar macrophages of growing pigs.[J].Am. Assoc. Swine pract. Newslett,4:28
49 Butista EM,Molitor TW.Cell-mediated immunity to porcine reproductive and respiratory syndrome virus in swine.[J]Viral Immunol,1997,2:83-94
50 Drew TW.Does PRRSV cause immunosuppression? A review of the published evidence. PRRS & Aujeszky's Disease Proceedings.Ploufragan, France, 21th -24th,June 1999,59
51 Duan X,Nauwynck HJ,Pensaert MB.Virus quantification and identification of celluar targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproctive and respiratory syndrome virus (PRRSV).[J]Vet Microbiol,1997,56 (1/2):9-19
52 Thanawongnuwech R,Thacker EL,Halbur PG.Effect of porcine reproductive and respiratory syndrome virus (PRRSV) (isolate ATCC VR-2385) infection on bactericidal activity of porcine intravascullar macrophages (PIMs) in vitro comparisons with pulmonary alveolar macrophages (PAMs).[J]Vet Immunol Immunopathol,1997,59: 323-335
53 Rossow KD,Morrison RB,Goyal SM, et al.Lymph node lesions in neonatal pigs congenitally exposed to porcine reproductive and respiratory syndrome virus.[J]J Vet Diagn Invest,1994,6:368-371
54 Zhou Y,Barghusen S,Choi C, et al.Effect of SIRS Virus infection in leukocyte populations in the peripheral blood and on cytokine expression in alveolar macrophages of growing pigs.[J]Am Assoc Swine Pract Newsl,1992,4:28
55 Thanawongiuwech R,Halbur PG,Ackermann MR, et al.Effects of low (modified-live virus vaccine) and high (VR-2385) virulence strains of porcine reproductive and respiratory syndrome virus (PRRSV) on pulmonary clearance of copper particles in pigs.[J]Vet Pathol,1998
56 Ohlinger VF,Haas B,Sallmuller A, et al.In vivo and in vitro studies on the immunobiology of PRRS.[J] Am Assoc Swine Pract Newsl,1992,4:16
57 Thacker EL,Halbur PG,Paul PS, et al.Detection of intracellular porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein in porcine macrophages by flow cytometry.[J]J Vet Diagn Invest,1998
58 Molitor TW,Choi CS,Leitner G, et al.Immunomodulation of host immune responses following porcine reproductive and respiratory syndrome Virus infection.[J]Vet Immunol
Immunopathol,1995
59 Plana-Daran J,Bastons M,Urniza A, et al.Efficacy of an inactivated Vaccine for prevention of reproductive failure induced by porcine reproductive and respiratory syndrome virus.[J]Vet Microbiol,1997,55(1/4):361-370
60 Nielsen TL,Nielsen J,Have P, et al.Examination of Virus shedding in semen from Vaccinated and from previously infected boars after experimental challenge with porcine reprodrctive and respiratory syndrome virus.Vet Microbiol,1997,54 (2):101-112
61 Scortti M,Jimenez R,Prieto C, et al.Efficacy in gilts of an inactivated PRRS Vaccine against PRRS-induced reproductive disease.PRRS ( Aujeszky's Disease,Ploufragan France,21th-24th June,1999,289
62 Rogan D,Gagnon CA,Therrien D, et al.Evaluation of the immuncyenicety of an inactivated porcine reproductive and respiratory syndrome (PRRS) virus vaccine. PRRS ( Aujeszky's Disease,Ploufragan France,21th-24th,June 1999,283
63 Kritas SK,Mavromatis J,Kostoylou P, et al.Field experience with the use of porcine reproductive and respiratory syndrome.Abstr 2nd Inter Symp on porcine reproductive and respiratory syndrome,Copenhagen, Denmark,August 9-10,1995.
64 Gdrcyca D,Spiess D,Harris L, et al.The effect porcine reproductive and respiratory syndrome (PRRS) virus challenge in pregnant sows after subsequent exposure to virulent virus.24h Annu Meeting,Am Assoc Swine Pract,Missouri,1993,51-52
65 Shin J-H,Torrison J,Choi CS,et al.Monitoring of PRRS virus infections in boars. Abstracts Second Inter Symp on Poucine Reproductive and Respiratory Syndrome (PRRS),Copenhagen,1995,55
66 Nielsen TL,Nielsen J,Have P, et al.Examination of virus shedding in semen from vaccinated and from previously infected boars after experimntal challenge with porcine reproductive and respiratory syndrome virus.[J]Vet Microbiol,1997,54(2):101-112
67 Schagemann G,Wilms-Schulze KA.Safety and efficacy of Ingelvac PRRS MLV (Resp PRRS/Repro) in sows at all stages of reproduction.PRRS ( Aujeszky's Disease, Ploufragan France,21th-24th June,1999,287-288
68 Botner A,Strandbygaard B,Sorensen KJ,et al.Appearance of acute PRRS-like symptoms in sow herds after vaccination with a modified live PRRS vaccine.[J]Vet Rec,1997,141: 497-499
69 Christopher-Hennings J,Nelson EA,Nelson JK, et al.Effects of a modified-live virus vaccine against porcine reprductive and respiratory syndrome in boars.[J]Am J Vet Res, 1997,58(1):40-49
70 Mortensen Ｓ,Thomsen LK,Buch HV,et al.Adverse effects of the INGELAC PRRS Modifred Live Vaccine on sow productivity in hevds infected with porcine reproductive and respiratory syndrome virus.Proceedings of the 15th IPVS Congress, Birmingham, England,5-9 July 1998,128
71 Mortewsen S,Sugaard R.Within-herd transmission of a modified live PRRS vaccine strain from vaccinated pigs to non-vaccinated sow populations.PRRS & Aujeszky's Disease,
Ploufragan France,21th-24th June,1999,269~270
72 Meier W,Wheeler J,Husmann RJ, et al.Characteristics of the immune response of pigs to PRRS virus.PRRS & Aujeszky's Disease,Ploufragan France,21th-24th June,1999, 71-74
73 Dee SA.The decision on using PRRS vaccine in the breeding herd:When and how to use it.In Proc 23d Annu Meet Allan D Leman Swine Conf,1996,143-146
74 Mengeling WL,Lager KM,Vorwald AC,et al.Comparison among strains of porcine reproducive and respiratory syndrme virus for their ability to cause reproductive failure.[J]Am J Vet Res,1996,57:834-839
75 Mengeling WL,Lager KM,Vorwald AC.1996a.An overview on vaccination for porcine reproductive and respiratory syndrome.In Proc 23d Allen D Lenan Swine Conf, 139-142
76 Wills RW,Zimmerman JJ,Yoon K, et al.Porcine reproductive and respiratory syndrome virus:Isolation from chronically infected swine.In Proc 26th Annu Meet Am Assoc Swine Pract,1995,387-389
77 Torrison J,Knoll M,Weisman B.Evidence of pig-to-pig transmission of a modified-live PRRS virus vaccine.In Proc 27th Annu Meet Am Assoc Swine Pract,1996,89-91
78 Mengeling WL,Lager KM,Vorwald AC.Clincical effects of porcine reproductive and respiratory syndrome virus on pigs during the early postnatal interval.[J] Am J Vet Res, 1998,59:52-55
79 Plana Duran J,Climent I, Sarraseca J,et al.Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91.Involvement of ORF3 and ORF5 proteins in protection.[J] Virus Genes,1997,14:19
80 Gonin P,Pirzadeh B,Carl A,et al.Seroneutra-lization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the GP5 major envelope glycoprotein.[J] J Vet Diagn Invest,1999,11:20~26
81 Pirzadeh B,Dea S.Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants. [J]J Gen Virol,1997, 78(8):
1867-1873
82 Meulenberg JJM,Van Njeuwstadt AP,Van Essen-Zandbergen A,et al.Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus. [J]J Virol,1997,71:6061-6067
83 Duran JP,Climent I,Sarraseca J,et al.Baculovirus expression of porcine reproductive and respiratory syndrome virus strain Olot/91.Involvement of ORF3 and ORF5 proteins in protection.[J]Virus Genes,1997,14 (1):19-29
84 Bohm W,Mertens T,Schirmbeck R, et al.Routes of plasmid DNA vaccination that prime murine humoral and cellular immune responses. [J]Vaccine,1998,16 (9-10):949-954
85 Cohen J.Naked DNA points way to vaccines.[J]Science,1993,259:1691-1692
86 Davis HL,Whalen RG,Demeneix BA.Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression.[J]Hum.Gene Ther., 1993,4:151-159
87 Fynan EF,Webster RG,Fuller DH, et al.DNA vaccines: Protective immunization by parenteral mucosa and gene-gun inoculation. [J]Proc Natl Acad Sci USA,1992,90(8): 11478-11482
88 Hariharan MJ,Driver DA,Townsend K,et al. DNA immunization against herpes simplex virus:enhanced efficacy using a sindbis virus-based vector.[J] J.Virol, 1998, 72:950-958
89 Montagomery DL,Shiver JW,Leander KR,et al.Heterologous and homologous protection against influenza A by DNA vaccination:Optimization of DNA vectors.[J] DNA Cell Biol.,1993,12:777-783
90 Okada E,Sasaki S,Ishii N, et al. Intranasal immunization of a DNA vaccine with IL-12 and granulocyte-macrophage clony-stimulating factors (GM-CSF)-expressing plasmids in liposomes induces strong mucosal and cell-mediated immune responses against HIV-1 antigens. [J] J. Immunol.,1997,159 (4):3678-3687
91 Shi Z,Curicl DT,Tang D.DNA-based non-invasive vaccination onto the skin.Vaccine, 1999,17:2136-2141
92 Wolff JA,Malone RW,Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science,1990,247:1465-1468
93 Wu Y,Kipps TJ.Deoxyribonucleic acid vaccines encoding antigens with rapid proteasome-dependant degradation are highly efficient inducers of cytolytic T lyphocytes. [J] J. Immunol.,1997,159:6037-6043
94 Zhou X,berglund P,Rhodes G, et al.Self-replicating Semlik Forest virus RNA as recombinant vaccine.[J]Vaccine,1994,12:1510-1514
2陈博文,孙颖杰.猪繁殖和呼吸系统综合征的血清学检测及病毒的分离和鉴定.（初报）[J]中国兽医杂志,1996,22(5):6-8
3杨汉春,管山红.猪繁殖和呼吸系统病毒的分离与初步鉴定.[J]中国兽医杂志,1997,23(10):9-10
4刘文兴,蔡雪辉,郭宝清,猪生殖-呼吸道综合征病毒（PRRSV）分离鉴定及其与欧美PRRSV抗原的比较.[J]中国畜禽传染病,1998,20(4):193-196
5严亚贤,孙建和,王恒安,等.猪生殖-呼吸道综合征病毒上海株的分离鉴定.[J]中国预防兽医学报,2000,22(5):161-163
6张鹤晓,王刚,王彩兰.等.应用间接免疫荧光试验从猪群中检测出PRRS抗体.[J]中国兽医杂志,1996,22(11):7-8
7王刚,张鹤晓,甘孟侯,等.IPMA检测猪生殖和呼吸道综合征病毒抗体的研究.[J]中国兽医杂志,1996,22(12):3-5
8 王刚,张鹤晓,甘孟侯,等.Dot-ELISA检测猪生殖和呼吸综合征抗体的研究.[J]中国兽医杂志,1996,22(13):3-5
9郭宝清,陈章水,刘文兴.应用间接免疫荧光法从国内生殖障碍猪群中检测生殖和呼吸综合征阳性抗体的研究.[J]中国兽医科技,1996,26(3):3-5
10杨汉春,黄芳芳,张旭,等.用单克隆抗体鉴定猪繁殖和呼吸道综合征病毒分离株.[J]中国兽医科技,1997,23(4):3-5
11陆光进,冯加明,俸玉维.应用间接免疫荧光试验猪生殖-呼吸道综合征抗体.[J]中国畜禽传染病,1997,5:36-37
12马志永,简中友,姜平.斑点酶联免疫吸附试验检测猪生殖与呼吸综合征病毒抗体方法的建立.[J]中国兽医科技,1997,27(4):3-4
13查红波,陈西钊,张鹤晓,等.应用间接酶联免疫吸附试验检测猪生殖与呼吸综合征病毒抗体的研究.[J]中国兽医科技,1997,27(9):20-21
14梁杏娴,卢洪芬,董振光.猪繁殖与呼吸综合征的诊断.[J]中国兽医杂志1998,24(4):28-29
15娄高明,林绍荣,黄茂,等.仔猪暴发繁殖与呼吸综合征.[J]中国兽医学报,1998，18(5):445-447
16晁学元,刘得胜，崔海龙,等.猪繁殖与呼吸综合征血清学调查.[J]中国兽医杂志,1999,25(7):17-18
17张晓梅,杨汉春,任慧英,等.用LSAB免疫组化染色法检测猪生殖与呼吸综合征病毒抗原的研究.[J]中国预防兽医学报,1999,21(6):427-429
18蔡雪辉,郭宝清,柴文君,等.检测猪生殖与呼吸综合征病毒抗体ELISA方法的建立.[J]中国预防兽医学报,2000,22(2):122-125
19娄高明,杜伟贤,谢明权,等.猪生殖与呼吸综合征RT-PCR诊断方法的建立.[J]中国兽医学
报,2000,20(2):141-144
20李军,林继煌,姜平,等.用免疫金技术检测猪生殖与呼吸综合征病毒.[J]畜牧与兽医,2001,33(2):1-2
21王云峰,周艳君,仇华吉,等.应用重组N-蛋白- ELISA检测猪生殖与呼吸综合征抗体的研究.[J]中国兽医杂志,2001,37(3):3-5
22杨小燕,李晓华,沈绍新,等.猪繁殖与呼吸综合征的血清学调查.[J]中国兽医杂志,2001,37(4):17-18
23卫秀余,姚龙涛,金爱华.猪生殖－呼吸道综合征的血清学调查. [J]中国畜禽传染病,
1998,20(增刊):87-88
24 Wensvoort G,Terpstra C,Pol JMA,et al.Immunomodulation of host immune responses following porcine reproductive and respiratory syndrome Virus infection.[J] Vet Q,1991,13:121-190
25 Bilodeau R,Dea S,Sauvageau RA, et al.Examination of virus shedding in semen from vaccinated and from previously infected boars after experimntal challenge with porcine reproductive and respiratory syndrome virus.[J]Vet.Rel.,1991,129:102-103
26 Keffaber KK.Reproductive failure of unknown etiology.[J] Am Assoc. Swine Pract. Newslett,1989,1:1-9
27 Shimizu M,Yamada S,MuRakaMi Y,et al.Isolation of swine infertility and respiratory syndrome Virus from Heko-heko disease of pigs.[J] J. vet. MedSci,1994,6:389-391
28 Tetpstra C,Wensvoort G, Poi JMA.Expremental reproduction of porcine epidemic abortion and respiratory syndrome (Mystery Swine Disease) by infection with Lelystad virus.[J] Vet. Q,1991,13:131-136
29 Wensvoort G,Terpstra IC,Pol JMA, et al.Mystery swine disease in the Netherlands:the isolation of Lelystad virus.[J] vet Q, 1991,13:121 -130
30 Benfield DA,Nelson E,Collins JE,et al.Characterization of swine infertility and respiratory syndrome virus(isolate ATCC VR-2332).[J]J.Vet Diagn Invest,1992,4:127-133
31 Collins JE,Benfield DA,Christianson WT,et al.Isolation of swine infertility and respiratory syndrome virus(isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs.[J]J.Vet.Diagn.Tnvest,l992,4:117-126
32 Dea S, Bilodeau R,Athanassious R,et al.Swine infertility and respiratory syndrome In Quebec:isolation and characterization of an enveloped virus serologically related to Lelystad virus.[J]J.Can.Vet.,1992,33:891-908
33 Baron T, Albina E,Leforban Y,et al.Report on the first outbreak of the porcine reproductive and respiratory syndrome(PRRS) In France.[J]Ann. Recl Vet.,1992,23:
161-166
34 Meulenberg JJM, Hulst MM,Meijer D,et al.Lelystad virus,the causative agent of porcine epidemic abortion and respiratory syndrome(PRRS) is related to LDV and EAV. [J] Virology,1993,1(92):62-72.
35 Meullenberg JJ,Peltrsen-DEN Besten ADE et al.Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus.[J]Virology,1997,206:155-183
36 Kjm HS,Kwang J,Yoon.et al.Enhanced reproduction of the porcine reproductive and respiratory syndrome(PRRS) in a homogenous subpopulation of MA-104 cell line.[J] Atchives of Virology,1993,133:477-453
37 Moos B,Elroy-Stein O,Mizukami T,et al.New mammalian expression vectors.[J]. Nature, 348:
91-92
38 Molitor TW,Dautista EM,Choi CS.Immunity to PRRSV: double-edged sword.[J]Vet Microbiol,l997,55(1-4):265-276
39 Molitor T,LeitnerG,ChoiC,et al.Modulation of host immune response by SIRS virus.[J].Am. Assoc.Swine Prac. Newslett,1992,4:27-28
40李华,杨汉春,郭玉璞.猪繁殖与呼吸综合症病毒(PRRSV)感染的免疫学研究进展.[J]中国兽医杂志,1999,25 (9):40-42
41 Shimizu M,Pan IC.Hess WRT and B lymphocyte in porcine blood.[J]AM. J. Vet. Res., 1976,31:309-317
42 Molitor TW,Bautista EM,Choi CS.Immunity to PRRSV: Double-edged sword.[J]Vet Microbiol,1997,55(1/4):265-276
43 Lopez FL,Domenech N,Alvarez B,et al. Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection.[J]Virus Res, l999,64(1):38-42
44 Saalmuller. A,Ilirt W,Maurer S, et al.Discrimination between two subsets of porcine CD8+ cytolytic T lymphocytes by the expression of CD5 antigen.[J] Immunology，1994,81:578-583
45 Albina E,Piriou L,Hutet E, et al.Immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). [J]Vet Immunol Immunopathol 1998,6 (l):49-66
46 Ummerfield A,Rziha HJ,Saalmuller A.Functional characterization of porcine CD4+、CD8+ extrathymicT lymphocytes.[J]Celluar lmmunology 1996,168:291-296
47 Roland RR,Rpbinson B,Ste FJ,et al.Inhibition of porcine reproductive and respiratory
syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine.[J]Arch Virology,2001,146(3):539-555
48 Zhou Y,Barghusen S,Choi C, et al.Effects of SIRS virus infection in leukocyte populations in the peripheral blood and on cytokine expression in alveolar macrophages of growing pigs.[J].Am. Assoc. Swine pract. Newslett,4:28
49 Butista EM,Molitor TW.Cell-mediated immunity to porcine reproductive and respiratory syndrome virus in swine.[J]Viral Immunol,1997,2:83-94
50 Drew TW.Does PRRSV cause immunosuppression? A review of the published evidence. PRRS & Aujeszky's Disease Proceedings.Ploufragan, France, 21th -24th,June 1999,59
51 Duan X,Nauwynck HJ,Pensaert MB.Virus quantification and identification of celluar targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproctive and respiratory syndrome virus (PRRSV).[J]Vet Microbiol,1997,56 (1/2):9-19
52 Thanawongnuwech R,Thacker EL,Halbur PG.Effect of porcine reproductive and respiratory syndrome virus (PRRSV) (isolate ATCC VR-2385) infection on bactericidal activity of porcine intravascullar macrophages (PIMs) in vitro comparisons with pulmonary alveolar macrophages (PAMs).[J]Vet Immunol Immunopathol,1997,59: 323-335
53 Rossow KD,Morrison RB,Goyal SM, et al.Lymph node lesions in neonatal pigs congenitally exposed to porcine reproductive and respiratory syndrome virus.[J]J Vet Diagn Invest,1994,6:368-371
54 Zhou Y,Barghusen S,Choi C, et al.Effect of SIRS Virus infection in leukocyte populations in the peripheral blood and on cytokine expression in alveolar macrophages of growing pigs.[J]Am Assoc Swine Pract Newsl,1992,4:28
55 Thanawongiuwech R,Halbur PG,Ackermann MR, et al.Effects of low (modified-live virus vaccine) and high (VR-2385) virulence strains of porcine reproductive and respiratory syndrome virus (PRRSV) on pulmonary clearance of copper particles in pigs.[J]Vet Pathol,1998
56 Ohlinger VF,Haas B,Sallmuller A, et al.In vivo and in vitro studies on the immunobiology of PRRS.[J] Am Assoc Swine Pract Newsl,1992,4:16
57 Thacker EL,Halbur PG,Paul PS, et al.Detection of intracellular porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein in porcine macrophages by flow cytometry.[J]J Vet Diagn Invest,1998
58 Molitor TW,Choi CS,Leitner G, et al.Immunomodulation of host immune responses following porcine reproductive and respiratory syndrome Virus infection.[J]Vet Immunol
Immunopathol,1995
59 Plana-Daran J,Bastons M,Urniza A, et al.Efficacy of an inactivated Vaccine for prevention of reproductive failure induced by porcine reproductive and respiratory syndrome virus.[J]Vet Microbiol,1997,55(1/4):361-370
60 Nielsen TL,Nielsen J,Have P, et al.Examination of Virus shedding in semen from Vaccinated and from previously infected boars after experimental challenge with porcine reprodrctive and respiratory syndrome virus.Vet Microbiol,1997,54 (2):101-112
61 Scortti M,Jimenez R,Prieto C, et al.Efficacy in gilts of an inactivated PRRS Vaccine against PRRS-induced reproductive disease.PRRS ( Aujeszky's Disease,Ploufragan France,21th-24th June,1999,289
62 Rogan D,Gagnon CA,Therrien D, et al.Evaluation of the immuncyenicety of an inactivated porcine reproductive and respiratory syndrome (PRRS) virus vaccine. PRRS ( Aujeszky's Disease,Ploufragan France,21th-24th,June 1999,283
63 Kritas SK,Mavromatis J,Kostoylou P, et al.Field experience with the use of porcine reproductive and respiratory syndrome.Abstr 2nd Inter Symp on porcine reproductive and respiratory syndrome,Copenhagen, Denmark,August 9-10,1995.
64 Gdrcyca D,Spiess D,Harris L, et al.The effect porcine reproductive and respiratory syndrome (PRRS) virus challenge in pregnant sows after subsequent exposure to virulent virus.24h Annu Meeting,Am Assoc Swine Pract,Missouri,1993,51-52
65 Shin J-H,Torrison J,Choi CS,et al.Monitoring of PRRS virus infections in boars. Abstracts Second Inter Symp on Poucine Reproductive and Respiratory Syndrome (PRRS),Copenhagen,1995,55
66 Nielsen TL,Nielsen J,Have P, et al.Examination of virus shedding in semen from vaccinated and from previously infected boars after experimntal challenge with porcine reproductive and respiratory syndrome virus.[J]Vet Microbiol,1997,54(2):101-112
67 Schagemann G,Wilms-Schulze KA.Safety and efficacy of Ingelvac PRRS MLV (Resp PRRS/Repro) in sows at all stages of reproduction.PRRS ( Aujeszky's Disease, Ploufragan France,21th-24th June,1999,287-288
68 Botner A,Strandbygaard B,Sorensen KJ,et al.Appearance of acute PRRS-like symptoms in sow herds after vaccination with a modified live PRRS vaccine.[J]Vet Rec,1997,141: 497-499
69 Christopher-Hennings J,Nelson EA,Nelson JK, et al.Effects of a modified-live virus vaccine against porcine reprductive and respiratory syndrome in boars.[J]Am J Vet Res, 1997,58(1):40-49
70 Mortensen Ｓ,Thomsen LK,Buch HV,et al.Adverse effects of the INGELAC PRRS Modifred Live Vaccine on sow productivity in hevds infected with porcine reproductive and respiratory syndrome virus.Proceedings of the 15th IPVS Congress, Birmingham, England,5-9 July 1998,128
71 Mortewsen S,Sugaard R.Within-herd transmission of a modified live PRRS vaccine strain from vaccinated pigs to non-vaccinated sow populations.PRRS & Aujeszky's Disease,
Ploufragan France,21th-24th June,1999,269~270
72 Meier W,Wheeler J,Husmann RJ, et al.Characteristics of the immune response of pigs to PRRS virus.PRRS & Aujeszky's Disease,Ploufragan France,21th-24th June,1999, 71-74
73 Dee SA.The decision on using PRRS vaccine in the breeding herd:When and how to use it.In Proc 23d Annu Meet Allan D Leman Swine Conf,1996,143-146
74 Mengeling WL,Lager KM,Vorwald AC,et al.Comparison among strains of porcine reproducive and respiratory syndrme virus for their ability to cause reproductive failure.[J]Am J Vet Res,1996,57:834-839
75 Mengeling WL,Lager KM,Vorwald AC.1996a.An overview on vaccination for porcine reproductive and respiratory syndrome.In Proc 23d Allen D Lenan Swine Conf, 139-142
76 Wills RW,Zimmerman JJ,Yoon K, et al.Porcine reproductive and respiratory syndrome virus:Isolation from chronically infected swine.In Proc 26th Annu Meet Am Assoc Swine Pract,1995,387-389
77 Torrison J,Knoll M,Weisman B.Evidence of pig-to-pig transmission of a modified-live PRRS virus vaccine.In Proc 27th Annu Meet Am Assoc Swine Pract,1996,89-91
78 Mengeling WL,Lager KM,Vorwald AC.Clincical effects of porcine reproductive and respiratory syndrome virus on pigs during the early postnatal interval.[J] Am J Vet Res, 1998,59:52-55
79 Plana Duran J,Climent I, Sarraseca J,et al.Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91.Involvement of ORF3 and ORF5 proteins in protection.[J] Virus Genes,1997,14:19
80 Gonin P,Pirzadeh B,Carl A,et al.Seroneutra-lization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the GP5 major envelope glycoprotein.[J] J Vet Diagn Invest,1999,11:20~26
81 Pirzadeh B,Dea S.Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants. [J]J Gen Virol,1997, 78(8):
1867-1873
82 Meulenberg JJM,Van Njeuwstadt AP,Van Essen-Zandbergen A,et al.Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus. [J]J Virol,1997,71:6061-6067
83 Duran JP,Climent I,Sarraseca J,et al.Baculovirus expression of porcine reproductive and respiratory syndrome virus strain Olot/91.Involvement of ORF3 and ORF5 proteins in protection.[J]Virus Genes,1997,14 (1):19-29
84 Bohm W,Mertens T,Schirmbeck R, et al.Routes of plasmid DNA vaccination that prime murine humoral and cellular immune responses. [J]Vaccine,1998,16 (9-10):949-954
85 Cohen J.Naked DNA points way to vaccines.[J]Science,1993,259:1691-1692
86 Davis HL,Whalen RG,Demeneix BA.Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression.[J]Hum.Gene Ther., 1993,4:151-159
87 Fynan EF,Webster RG,Fuller DH, et al.DNA vaccines: Protective immunization by parenteral mucosa and gene-gun inoculation. [J]Proc Natl Acad Sci USA,1992,90(8): 11478-11482
88 Hariharan MJ,Driver DA,Townsend K,et al. DNA immunization against herpes simplex virus:enhanced efficacy using a sindbis virus-based vector.[J] J.Virol, 1998, 72:950-958
89 Montagomery DL,Shiver JW,Leander KR,et al.Heterologous and homologous protection against influenza A by DNA vaccination:Optimization of DNA vectors.[J] DNA Cell Biol.,1993,12:777-783
90 Okada E,Sasaki S,Ishii N, et al. Intranasal immunization of a DNA vaccine with IL-12 and granulocyte-macrophage clony-stimulating factors (GM-CSF)-expressing plasmids in liposomes induces strong mucosal and cell-mediated immune responses against HIV-1 antigens. [J] J. Immunol.,1997,159 (4):3678-3687
91 Shi Z,Curicl DT,Tang D.DNA-based non-invasive vaccination onto the skin.Vaccine, 1999,17:2136-2141
92 Wolff JA,Malone RW,Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science,1990,247:1465-1468
93 Wu Y,Kipps TJ.Deoxyribonucleic acid vaccines encoding antigens with rapid proteasome-dependant degradation are highly efficient inducers of cytolytic T lyphocytes. [J] J. Immunol.,1997,159:6037-6043
94 Zhou X,berglund P,Rhodes G, et al.Self-replicating Semlik Forest virus RNA as recombinant vaccine.[J]Vaccine,1994,12:1510-1514