致病性猪伪狂犬病病毒PRV-JF株的分离与鉴定

从临床疑似猪伪狂犬病发病仔猪的脑组织病料中，经聚合酶链式反应（PCR）证实为猪伪狂犬病病毒（PRV）野毒感染，采用无PCV1污染的猪肾细胞系（PK-15）分离培养，经蚀斑克隆纯化，培育1株细胞培养适应毒，命名为PRV-JF株。该分离株经细胞培养传代，能够产生典型的细胞病变，病毒滴度随代次显著增加，第24代毒价达10^8.5 TCID50／mL。免疫过氧化物酶单层细胞试验（IPMA）检测病毒抗原分布在细胞核及细胞质内。病毒感染细胞可被已知PRV阳性血清中和。电镜负染观察到病毒粒子呈椭圆或圆形外观，无囊膜的病毒粒子直径约110nm～150nm，有囊膜的成熟病毒粒子直径约150nm～180nm。PCR鉴定该毒株含有gE基因，其序列与GenBank登录的7株PRV同源性为97.7％～100％。用不同剂量病毒培养物接种家兔于24h～72h内全部死亡。研究表明，PRV-JF分离株对易感动物具有高致病性，为进一步开展该病毒流行病学、致病机理、疫苗免疫与诊断研究奠定了基础。     

猪圆环病毒2型灭活疫苗最小免疫保护剂量研究

猪圆环病毒2型(PCV2)是引起断奶仔猪多系统衰竭综合征(PMWS)的主要病原,疫苗是防制该病的主要手段.为研究PCV2灭活疫苗的免疫效果,本研究将PCV2 SH株灭活,用生理盐水稀释成5×105.0TCID50/mL(高剂量)、2.5×1050 TCID50/mL(中等剂量)、1.25×105.0 TCID50/mL(低剂量)、6.3×104.0 TCID50/mL(超低剂量)4种剂量,与等量白油佐剂乳化后,通过断乳仔猪的免疫攻毒试验观察该灭活疫苗的免疫特性.结果表明,PCV2疫苗抗原含量大于1.25×105.0 TCID50/mL时免疫仔猪后均可以产生特异性抗体,能够明显减轻攻毒后仔猪的临床症状、缓解组织病变、降低病毒血症,对免疫猪有一定的保护效果,可以作为预防PCV2感染的疫苗使用.本研究为研制商品化的PCV2灭活疫苗提供实验依据.     

猪细小病毒细胞适应株的培育及鉴定

从临床表现为皮炎消瘦症状的仔猪肝脏中分离到1株病毒,经聚合酶链式反应（PCR）证实为猪细小病毒（PPV）,采用仔猪原代肾细胞和传代ST细胞分离培养,经蚀斑克隆纯化,培育1株ST传代细胞培养适应毒株,命名为PPV-BQ2007株。免疫过氧化物酶单层细胞试验（IPMA）检测病毒抗原主要分布在细胞核及细胞质内。病毒感染细胞可被已知PPV阳性血清中和。免疫电镜可清晰见到聚集成团的大小不一的病毒粒子,近似圆形,无囊膜,直径大小约20nm-22nm。该分离株经ST细胞培养传代,能够产生典型的细胞病变,病毒滴度随代次显著增加,第30代后毒价达10^7 TCID50/mL以上,且毒价和血凝价稳定。测序结果表明PPV-BQ2007株VP2基因与NCBI公布的皮炎型毒株Kresse株的同源性最高,达99.8％,在系统进化分支上处于同一个分支。PPV-BQ2007株传代细胞培养适应株的成功培育和鉴定,为进一步开展该病毒流行病学、致病机理、疫苗免疫与诊断研究等奠定了良好基础。     

伪狂犬病病毒弱毒株LY株的分离鉴定

从辽阳某猪场的10日龄仔猪中分离到1株病毒，经纯化后测得其毒价为10^7．29TCID50／mL。细胞中和试验表明，该病毒能被猪伪狂犬病病毒标准阳性血清所中和。电镜下可见到典型的疱疹病毒粒子，具有囊膜及外周纤突。所分离的病毒对氯仿、胰蛋白酶、乙醚敏感，在pH5．0-9．0下稳定，56℃ 30min可以灭活。应用特异性引物，通过PCR能扩增出伪狂犬病病毒1240bp的gD基因。分离病毒对3日龄乳鼠有一定的致病力，但对家兔、3—5日龄仔猪及妊娠母猪都有很高的安全性。用不同剂量的病毒培养液肌肉注射于3—5日龄仔猪，14d后用10^5.7TCID50伪狂犬病病毒强毒攻击，所有试验仔猪均可得到有效保护。用分离毒免疫母猪，其后代可获高滴度的母源抗体，15日龄的仔猪能抵抗10^5.7TCID50强毒的攻击。试验的结果初步说明，所分离的病毒为伪狂犬病病毒(命名为PRVLY株)，并可能是一株弱毒株，而且具有很好的免疫保护作用。     

嵌合型猪圆环病毒（PCV1—2）及PCV2 ORF2真核表达质粒对商品猪的免疫保护

应用本实验室构建的嵌合型猪圆环病毒（PCV1—2）及真核表达质粒pcDNA3．1／V5-His-ORF2作为免疫原免疫母源抗体ELISA效价在0．07～0．60不等的商品猪,9头猪随机分为4组,1组（3头）肌肉注射免疫10^3.5 TCID50的PCV1-2／头,2组（2头）肌肉注射真核表达质粒200μg／头,3组（2头）肌肉注射空载体（pcDNA3．1）200μg／头,4组（2头）不免疫作为攻毒对照组。于免疫后42d,PCV1—2组及真核表达质粒组产生了PCV2抗体。免疫后42d所有组攻毒PCV2和PRRSV,剂量分别为2×10^4.5 TCID50／头和10^6 TCID50／头。攻毒后21d,攻毒对照组猪淋巴结比免疫组显著肿大,免疫组猪血清、淋巴结中PCV2病毒栽量低于对照组,攻毒对照组猪淋巴结中PCV2抗原含量高于免疫组。这些结果表明,嵌合型PCV1-2及真核表达质粒肌肉注射免疫商品猪后,对PCV2感染能产生保护性免疫应答,有可能成为候选疫苗。     

猪细小病毒体外培养适应毒株的培育及动物感染试验

从初产母猪流产胎儿的肠系膜淋巴结中分离到一株猪细小病毒(PPV),采用无污染的猪睾丸传代细胞系(ST)对该毒株传代,培育成一株细胞适应毒株,命名为PPV-HJ毒株。该毒株经ST细胞连续传50代,于第25代起毒价显著提升,第40代毒价维持稳定(107.2 TCID50/mL)。用免疫过氧化物酶单层细胞染色试验(IPMA)检测表明,病毒感染的阳性细胞染成棕红色,病毒抗原主要分布在细胞核中。免疫电镜观察到PPV粒子呈球状,直径约21nm。基因克隆测序证实,该毒株的NS1基因序列与GenBank中PPV-ZJ毒株同源性最高,达98.41%。用该毒株第50代培养物(1×107.2 TCID50/mL)接种35日龄仔猪,未表现出明显的临床症状,剖检未观察到明显的病理学变化,PCR法在猪的肺脏、扁桃体、腹股沟淋巴结、下颌淋巴结、小肠中检测到病毒核酸,表明该毒株对易感动物具有一定的感染性。本研究成功地培育出一株PPV体外细胞培养适应毒株,其繁殖性能稳定,为今后开展PPV与猪圆环病毒2型(PCV-2)混合感染的协同致病性研究,以及联合疫苗的研制奠定了基础。     

猪圆环病毒2型遗传标记毒株的构建及生物学特性鉴定

以临床分离的猪圆环病毒2型（PCV2）为材料，利用PCR法扩增病毒基因组，将2个基因组顺式连接插入到质粒载体中构建感染性分子克隆。通过引物设计替换碱基，在病毒基因组内插入Sal Ⅰ限制性内切酶位点作为遗传标记，经重组质粒转染细胞获得带有遗传标记的新毒株。采用免疫过氧化物酶单层细胞试验、免疫电镜、核酸序列分析表明，在病毒感染细胞中检出病毒特异抗原，其抗原性、病毒形态学及基因序列与亲本毒株一致。鉴于新病毒基因组内插入一个SalⅠ酶切住点，用PCR与限制性片段长度多态性分析法可与野生型病毒相鉴别。新毒株经细胞连续传60代，体外培养增殖性能稳定，毒价可达10^6.6CID50/mL。取病毒培养物经静脉和滴鼻途径接种35日龄抗体阴性仔猪4头，接种后表现出体温升高、进行性消瘦、被毛粗糙及体表淋巴结肿胀症状，迫杀后多种脏器中均能检测到病毒抗原与核酸。研究表明，利用感染性分子克隆手段构建带有遗传标记的PCV2新毒株，为进一步开展该病毒的致病性、疫苗免疫、分子诊断等研究奠定了基础。     

伪狂犬病病毒弱毒株LY株的分离鉴定

从辽阳某猪场的10日龄仔猪中分离到1株病毒,经纯化后测得其毒价为107.29TCID50/mL.细胞中和试验表明,该病毒能被猪伪狂犬病病毒标准阳性血清所中和.电镜下可见到典型的疱疹病毒粒子,具有囊膜及外周纤突.所分离的病毒对氯仿、胰蛋白酶、乙醚敏感,在pH5.0～9.0下稳定,56℃ 30 min可以灭活.应用特异性引物,通过PCR能扩增出伪狂犬病病毒1 240 bp的gD基因.分离病毒对3日龄乳鼠有一定的致病力,但对家兔、3～5日龄仔猪及妊娠母猪都有很高的安全性.用不同剂量的病毒培养液肌肉注射于3～5日龄仔猪,14 d后用105.7TCID50伪狂犬病病毒强毒攻击,所有试验仔猪均可得到有效保护.用分离毒免疫母猪,其后代可获高滴度的母源抗体,15日龄的仔猪能抵抗105.7TCID50强毒的攻击.试验的结果初步说明,所分离的病毒为伪狂犬病病毒(命名为PRV LY株),并可能是一株弱毒株,而且具有很好的免疫保护作用.     

猪瘟病毒流行株与疫苗株主要抗原编码基因差异研究

从全国7个省市1200多份可疑猪瘟病料中分离出9个猪瘟病毒（HCV）野毒株，编号分别为HCV－01－09。将9株野毒分别通过PK15细胞分别通过PK15细胞传6代，测其毒价，并提纯做电镜观察，结果表明：毒价范围在10^2-10^7TCID50，电镜观察均可清晰地见到直径为25－70nm，略呈圆形的病毒颗粒，具有较完整的囊膜和纤突结构。从9株野毒中选取5株经猪瘟阴性猪各传3－4代，测其毒力、病原性、致死性，结果表明：各毒株在传代中其上述生物学特性上有变化和差别。用猪瘟兔化弱毒疫苗分别对这5株野毒做免疫保护相关性试验，结果证明：攻毒后的疫苗接种猪100％保护，而攻毒对照猪100％死亡，且对照猪在攻毒1周后便出现猪瘟野毒感染，而免疫猪在整个观察期均未见到野毒感染。用不同剂量的猪瘟兔化弱毒，表明猪瘟兔化弱毒不通过胎盘垂直感染仔猪。将猪瘟野毒株、石门系强毒株、兔化弱毒株及1982年分离的郑州野毒等毒株进行主要抗原编码基因差异研究，结果表明：猪瘟病毒可分2个基因组6个基因亚组，HCV－02、03、06、07等4个野毒株与国内的C株、石门强毒株、国外的C株、日本GPE株、ALD株、意大利Brescia株均属同一基困组，而HCV－08株及郑州株等2个野毒与法国的Alfor株同属另一个基因组。     

伪狂犬病弱毒株的分离鉴定及生物学特性的研究

在流行病学调查中分离到1株病毒,经鉴定为伪狂犬病弱毒株,定名为F971株。分离病毒经克隆纯化后测得其毒价为10^7.59TCID50/ml,通过细胞中和试验表明分离病毒能也有效地被猪伪狂犬病毒闽A株阳性血清中和。病毒在电镜下可以清楚地观察到囊膜及外周纤突。分离株对3日龄乳鼠有一定的致病力,但对家兔、3日龄乳猪及妊娠母猪都有很高的安全性。用不同的剂量10^0、10^-1、10^-2肌肉注射3日龄乳猪后14天用10^5.7TCID50伪狂犬病强毒攻击,所有试验仔猪均得到保护。用分离株免疫母猪,其后代可获高滴度的母源抗体,15日龄的仔猪能抵御10^5.7TCID50强毒的攻击。用ELISA普查试剂盒测定免疫猪抗体,结果均为阳性,而用g^1-ELISA试剂盒测定抗体时,结果均为阴性。证明分离株具有缺损g^1糖蛋白的特性。综合上述特性,确定F971为1株g^1糖蛋白缺损的猪伪狂犬病弱毒株。     

The effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets

The effects of transplacental porcine circovirus type 2 (PCV2) infection on porcine epidemic diarrhoea virus (PEDV)-induced enteritis were examined in neonatal piglets. Six pregnant sows were randomly allocated to an infected (n=3) or control group (n=3). Three pregnant sows were inoculated intranasally with 6 mL of tissue culture fluid containing 1.2 x 10(5) tissue culture infective doses 50% (TCID(50))/mL of PCV2 strain SNUVR000470 three weeks before the expected farrowing date. Three control pregnant sows were similarly exposed to uninfected cell culture supernatants. Thirty piglets from PCV2-infected sows were randomly assigned to two groups (A and B) of 15 piglets each. Another 30 piglets from noninfected sows were randomly assigned to two groups (C and D) of 15 piglets each. The piglets in groups A and C were dosed orally at three days of age with 2mL of virus stock (1 x 10(6.5) TCID(50)/mL) of the PEDV strain, SNUVR971496, at the third passage. The mean villous height and crypt depth (VH:CD) ratio in PEDV-infected piglets from PCV2-infected sows (group A) were significantly different from those of the PEDV-infected piglets from PCV2 negative sows (group C) at 36, 48, and 72 h post-inoculation (hpi) (P<0.05). In PEDV-infected piglets from PCV2-infected sows (group A), significantly more PEDV nucleic acid was detected in the jejunal tissues (P<0.05) at 24 hpi than in the same tissues of the PEDV-infected piglets from PCV2 negative sows (group C). Thereafter, at 36, 48, 60, and 70 hpi significantly more PEDV nucleic acid (P<0.05) was detected in the jejunal tissues of the PEDV-infected piglets from PCV2 negative sows (group C) than those of the PEDV-infected piglets from the PCV2-infected sows (group A). It is concluded that the clinical course of PEDV disease was markedly affected by transplacental infection of PCV2.     

PCV2a和PCV2b感染性克隆的体外拯救

 研究旨在获得PCV2a和PCV2b的拯救毒株。对筛选得到的PCV2a和PCV2b感染性克隆分别进行酶切，使酶切得到的全长基因组DNA自身环化并分别转染PK15细胞。经IFA和PCR验证，确认成功拯救出两亚型PCV2病毒。病毒传至第9代时对PCV2a和PCV2b的毒价分别进行测定，结果显示PCV2a的毒价为103.5TCID50/mL， PCV2b的毒价为104.6TCID50/mL。本试验为分型特异性序列与病毒致病力相关性的研究打下了基础.     

Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2)

Postweaning multisystemic wasting syndrome (PMWS) in swine is causally associated with the newly recognised pathogen, porcine circovirus type 2 (PCV2). In this study, 3-week-old SPF PCV2-seronegative piglets were inoculated intranasally with PCV2. The effect of immunostimulation on the induction of PMWS was investigated by immunisation with keyhole limpet hemocyanin (KLH) emulsified in incomplete Freunds adjuvant. The study was terminated 5 weeks after inoculation. While disease was not observed in the age-matched controls, two out of five non-immunised PCV2-infected piglets died on postinoculation day (PID) 21, and one was euthanized on PID 25 in moribund condition. These animals had appeared lethargic with persistent fever from PID 12 onwards. The euthanized pig appeared smaller than littermates and suffered from jaundice. At postmortem examination, gastric ulceration, icterus, and liver and thymus atrophy were observed. Furthermore, histological lesions of degenerating hepatocytes and hepatitis in combination with lymphoid depletion and syncytial cells in lymph nodes were consistent with the diagnosis of PMWS. One out of five immunostimulated PCV2-infected piglets was euthanized on PID 22 with convulsions after a period with wasting. This pig was lethargic from PID 14 onwards with persistent fever from PID 8 and transient dyspnoea. No differences in clinical signs, gross pathologic or histological findings were observed for the remaining non-immunostimulated and immunostimulated PCV2-infected piglets. All 10 PCV2-inoculated piglets seroconverted to PCV2 within 14 days after inoculation. By virus isolation, quantitative polymerase chain reaction (Q-PCR), and immunostaining of cryostat sections, it was demonstrated that lymphoid tissue contained abundant PCV2 antigen. Viral DNA load in serum samples was assessed by Q-PCR. All four PMWS-affected piglets had high levels of PCV2 DNA in serum, suggesting that there was a correlation between high levels of viral DNA in serum and the development of PMWS. In conclusion, infection with PCV2 caused PMWS in SPF piglets, however, the immunostimulation did not seem to play a critical role.     

猪圆环病毒2型灭活疫苗效力检验中参考疫苗的应用

制备了4批不同抗原含量的猪圆环病毒2型（PCV2）灭活疫苗,灭活前PCV2病毒含量分别为107.0、106.5、105.5和104.5TCID50/mL。对此4批PCV2灭活疫苗,利用本实验室建立的小白鼠效力检验标准进行效力检验,即以PCV2参考疫苗作为对照品,测定待检疫苗免疫小白鼠后血清中PCV2抗体效价的高低来判定疫苗效力。3次重复检测结果都显示灭活前病毒含量为107.0、106.5和105.5TCID50/mL批次的疫苗合格,而灭活前病毒含量为104.5TCID50/mL批次的疫苗不合格,这与猪体内的效力检验结果一致,表明建立的针对猪圆环病毒2型灭活疫苗的小白鼠效力检验方法具有良好的准确性和重复性。     

Porcine circovirus 2 replication in colostrum-deprived piglets following experimental infection and immune stimulation using a modified live vaccine against porcine respiratory and reproductive syndrome virus

Porcine circovirus type 2 (PCV2) infection is now recognized as the major factor in the development of post-weaning multisystemic wasting syndrome (PMWS). Although Koch's postulates have been fulfilled for PCV2 and PMWS, the severe clinical expression of the disease observed in field cases has been difficult to reproduce experimentally. Some studies have demonstrated that immune stimulation associated with the use of some commercially available swine vaccines may trigger progression of PCV2 infection to disease and lesions characteristic of PMWS. Here we describe the effects on PCV2 infection in an experimental model following the use of a commercially available modified live vaccine to porcine respiratory and reproductive syndrome virus (PRRSV). Although none of the piglets infected with PCV2 developed clinical PMWS, the severity of microscopical lesions and the PCV2 antigen load associated with these lesions were higher in the PRRSV-vaccinated piglets compared with those detected in the PCV2 only infected animals.     

猪流行性腹泻弱毒株的培育

用ＰＥＤＶＣＶ７７７株强毒适应Ｖｅｒｏ细胞系并传至１４７代。以ＰＥＤＶ沪株做效检用强毒。传代毒８３代之后适应了仔猪肾原代细胞。自９０代起进行了５次克隆纯化,克隆是在２次群斑（由５～７个单斑组成）的基础上,再进行３次单斑挑选（均是１ｍｍ以内小空斑）。以８３７斑５株继续传代并做系列试验。传代毒的毒价为１０７．０～１０７．５ＴＣＩＤ５０／０３ｍｌ。免疫接种途径为后海穴位。克隆后５代（总代次１０４代）～２５代的５批次主动免疫试验的总保护率为９５９２％（４７／４９）,对照组８８８％（１６／１８）发病。克隆后１７代～４０代的８批次被动免疫试验的总保护率为９６２％（７６／７９）,对照组１００％（１０／１０）发病。以克隆后２５代（总代次１２５代）进行稳定性试验,经６代次返祖传代毒力未返强,仍是稳定的,已符合弱毒株的标准。在与ＴＧＥ弱毒株组合制备的二联弱毒苗的初步田间试验中取得良好效果。     

Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus

Postweaning multisystemic wasting syndrome (PMWS) is a recently emerged disease affecting pigs. Type 2 porcine circovirus (PCV2) has been associated with this syndrome although other factors are required in association with this virus for PMWS expression. The aim of this study was to investigate whether general immunostimulation (injections of keyhole limpet hemocyanin emulsified in incomplete Freund adjuvant and of thioglycollate medium) could strengthen the severity of PMWS in six-week-old specific-pathogen-free (SPF) piglets transfected with pure tandem-cloned PCV2 DNA by the intramuscular route. Non-immunostimulated piglets transfected with the viral clone did not present clinical signs but only mild pathological microlesions characteristic of PMWS. These piglets seroconverted and high viral genome loads and infectious titers were detected in the lymphoid organs at the end of the trial. Mild-to-moderate forms of PMWS were generally observed in the immunostimulated transfected piglets, as well as one severe form for a piglet (8003) which died. These piglets with mild-to-moderate forms had higher DNA loads than the transfected-only animals. Thus, viral replication was enhanced by immunostimulation. This is the first time that clinical PMWS has been reported in an SPF immunostimulated piglet infected with a pure inoculum consisting of tandem-cloned PCV2 DNA. This result confirms that PCV2 is the agent of PMWS and that immunostimulation could enhance PMWS in SPF piglets transfected with a PCV2 DNA clone.     

Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2)

Porcine circovirus (PCV)-2, a newly described single-stranded circular DNA virus pathogen of swine is the cause of postweaning multisystemic wasting syndrome (PMWS). In gnotobiotic piglets, PCV-2 infection alone produces asymptomatic infection without evidence of overt PMWS. Gnotobiotic piglets infected with PCV-2 were injected with keyhole limpet hemocyanin in incomplete Freund's adjuvant (KLH/ICFA), and the effects on virus production and development of PMWS were determined. In the first experiment, piglets were injected subcutaneously on the left hip and shoulder, and viral burden was assessed in regional lymph nodes draining the injection sites and in contralateral lymph nodes 13-14 days after infection. Immune activation increased the number of virus antigen-positive cells in draining lymph nodes and increased the amount of infectious virus recovered by 1-4 log10. In a second experiment, the effects of injections of KLH/ICFA with or without concurrent stimulation of peritoneal macrophages by intraperitoneal injections of thioglycollate broth on induction of PMWS was assessed. All immunized piglets developed moderate to severe PMWS, whereas none of the piglets infected with PCV-2 alone developed PMWS. In PMWS-affected piglets, extensive replication of PCV-2 was documented by both immunocytochemistry and quantitative viral titrations. Thus, immune activation is a key component of the pathogenesis of PCV-2-associated PMWS in swine.