用LSAB免疫组化染色法检测猪生殖—呼吸道综合征病毒抗原？…

本研究在国内首次成功建立了辣根过氧化物酶标记的链霉亲和素－生物系（ＬＳＡＢ）免疫组化染色法检测猪生殖－呼吸道综合征病毒（ＰＲＲＳＶ）抗原。应用ＬＳＡＢ染色技术检测１２头人工感染ＰＲＲＳＶ美洲株（ＡＴＣＣ ＶＲ－２３３２）或国内分离株（Ｂ９６－４，Ｂ９６－５）的ＳＰＦ仔猪组织细胞内的ＰＲＲＳＶ抗原，阳性检出率为１００％。     

用猪感染肺泡巨噬细胞诊断生殖—呼吸道综合征

用灌洗肺脏收集的肺泡巨噬细胞建立了检测活猪生殖－－呼吸道综合征病毒（ＰＲＲＳＶ）感染的高效敏感方法。５头１０周龄（１头）和１４周龄（４）的猪，口鼻接种口接触感洒ＰＲＲＳＶ。感染ＰＲＲＳＶ前收集每头仔猪的诊断样品在以后９周内，每周采集一次血清。第２和第４－９周收集肺居噬细胞，血清和肺泡巨噬细胞均适于早期感染的检测，但时间稍长后肺泡巨噬细胞为更好。４时仅从１头仔猪血清中分离到病毒，而从４头仔狸的肺泡巨     

猪繁殖呼吸综合征病毒(PRRSV)S毒株接种本动物的回归试验

猪繁殖呼吸综合征 (ＰＲＲＳ)是由ＰＲＲＳＶ引起的传染病。 1 997年我们从上海郊县发病猪场的死胎和死仔中分离到ＰＲＲＳＶＳ1毒株 ,经鉴定属美洲型毒株〔1〕。ＰＲＲＳＶ对母猪和仔猪易感 ,感染后在临床症状方面可分二大类 ,一类是母猪表现为繁殖障碍 ;另一类是仔猪和育成猪表现出呼吸道症状。本试验将ＰＲＲＳＶ上海分离株接种易感仔猪 ,以复制出ＰＲＲＳ病症 ,从而进一步确证分离到的Ｓ1毒株属ＰＲＲＳＶ。1　材料和方法1 .1　试验用仔猪 ,从上海远郊一个ＰＲＲＳ阴性猪场购买 ,隔离饲养 ,经检测抗体阴性 ,作为本试验用猪。1 .2　病毒 …     

猪繁殖与呼吸综合征病毒对受精卵发育的影响

１４头同期发情青年母猪排卵后３２小时，取出受精卵进行微注射或在（Ｂｅｌｔｓｖｉｌｅ）胚胎培养液－３（加或不加猪繁殖与呼吸综合征病毒，ＰＲＲＳＶ）中培养７２小时。使用猪肺泡巨噬细胞分离病毒，反转录酶聚合酶链式反应和荧光抗体技术检测样品中的病毒。结果与对照组相比，微注射或加有ＰＲＲＳＶ的胚胎培养均未明显抑制体外猪胚胎发育（分别为Ｐ＝０．７５和Ｐ＝０．１４）。用１０～２０ＴＣＩＤ５０的病毒微注射或大浓度病毒处理胚胎进行培养，在胚胎中均检测不出ＰＲＲＳＶ。本试验得出结论是：４～１０－细胞期猪胚胎对ＰＲＲＳＶ体外感染不敏感。     

PRRS病毒BJ-4株全基因组序列测定与分析

采用分段设计引物,分段扩增、克隆、序列测定与拼接的方法,对猪繁殖与呼吸综合征（ＰＲＲＳ）病毒ＢＪ－４株进行了全基因组序列测定和分析。设计合成２２对引物,利用ＲＴ－ＰＣＲ技术扩增得到 ２２条片段,分别与 ｐＧＥＭ－Ｔ－ｅａｓｙ载体连接,转化大肠杆菌ＪＭ１０９。筛选阳性重组子进行测序,然后将测得的序列顺次拼接得到ＰＲＲＳ病毒ＢＪ－４株全基因组序列。序列测定结果表明ＰＲＲＳ病毒ＢＪ－４株基因组全序列共长１５５０４个核苷酸,含有８个开放阅读框,５’端１９０ｂｐ先导序列,３’端有 ２５６ｂｐ ＵＴＲ,其中包括…     

猪生殖—呼吸道综合征病毒对妊娠中期母猪和和胎儿的致病性

通过两个实验评价了猪生殖－呼吸道综合征病毒（ＰＲＲＳ）经鼻腔接种母猪和经胎盘感染妊娠中期胎儿以及经子宫接种ＰＲＲＳ毒病直接感染胎儿的可能性。实验１对８头妊娠４５－５０天的母猪经鼻腔接种ＰＲＲＳ病毒（ＡＴＣＣ ＶＲ－２３３２）、４头对照母猪用未感染的细胞培养物接种，接毒后１、３、５、７和９天，接毒母猪出现毒血症，由粪便和鼻分泌物排出病毒并出现白细胞减少症，接毒后７、１４或２１天剖杀实验母猪的７１头胎     

猪繁殖和呼吸综合征（PRRS）血清流行病学调查初报

用美国Ｈｅｒｄｃｈｅｋ猪繁殖和呼吸综合征病毒（ＰＲＲＳ）ＥＬＩＳＡ抗体检测试剂盒对来自华北地区和华东地区８个ＰＲＲＳ可疑猪场共４７份血清进行检测。结果是：华北地区６个猪场，华东地区２个猪场ＰＲＲＳＶ抗体阳性检出率分别为５／６和０／２。８个猪场流产母猪、新生弱仔猪，有呼吸症状的育成猪及种公猪阳性检出率分别为１４／２０，５／１６，４／１０和１／１。表明ＰＲＲＳ在我国已经存在，并正在流行     

规模化猪场猪繁殖与呼吸综合征病毒和猪圆环病毒2型混合感染的控制

2022年2月1日，陕西省某规模化母猪场产房仔猪断尾去势液检测猪繁殖与呼吸综合征病毒核酸阳性，该批仔猪于2022年2月23日断奶至下游育肥猪场，猪群断奶后3 d出现咳嗽、少食、发热等临床症状，根据临床症状、剖检大体病理变化、兽医实验室检测结果诊断为猪繁殖与呼吸综合征病毒和圆环病毒2型混合感染。为迅速控制猪群病情，对该猪场免疫程序及保健方案进行调整，并对猪群进行为期18周的猪繁殖与呼吸综合征和圆环病毒2型病毒血症期和抗体水平的持续跟踪发现，紧急防控方案实施后2周大群趋于稳定，猪繁殖与呼吸综合征疫苗二免后10周猪群的病毒血症消失，圆环病毒2型疫苗二免后猪群的病毒血症虽未消失，但阳性率有所下降，猪繁殖与呼吸综合征的抗体水平在猪群二免后8周趋于稳定，猪群受圆环病毒2型疫苗毒株及野毒的双重刺激，抗体衰减规律较为复杂，从持续的跟踪检测分析发现圆环病毒2型疫苗二免后6周抗体水平有所下降，8周后抗体水平上升并持续至该批猪出栏。     

猪繁殖和呼吸综合征血清流行病学调查初报

用美国Ｈｃｒｄｃｈｃｋ猪繁殖和呼吸综合征病毒ＥＬＩＳＡ抗体检测试剂盒坚来自华北地区和华东地区８个ＰＲＲＳ可疑猪场共４７份血清进行检测。结果是；华北地区６个猪场，华东地区２个猪场ＰＲＲＳＶ抗体阳性检出率分别为５／６和０／２。８个猪场流产母猪，新生弱仔猪，有呼吸 状的育成猪及种公猪阳性栓率分别为１４／２０，５／１６，４／１０和１／１。表明ＰＲＲＳ在我国已经存在，并正在流行。     

猪链球菌与猪生殖—呼吸道综合征病毒的相互作用

经鼻内接种猪生殖－呼吸道综合征病毒ＡＴＣＣ ＶＲ－２３３２株后接种猪链球菌血清２型８７５５５株的无特定病原二代仔猪，呈现临床症状和化脓性脑膜炎的病变，并从组织、脑和脑脊髓膜培养出大量ＳＳ２；但单独接种ＰＲＲＳＶ或ＳＳ，或联合接种ＰＲＲＳＶ和ＳＳ２型ＤＨ５株的猪却都不出现临床症状，病变和组织内细胞，这表明ＰＲＲＳＶ可激化仔猪对有毒性蛋白的ＳＳ株的易感性。     

抗独特型抗体对猪繁殖与呼吸综合征病毒感染的免疫作用

用PRRSV感染SPF猪，血清检测结果显示，机体不仅产生抗PRRSV抗原的各种抗体(Ab1)，而且产生针对这些抗体的抗独特型抗体(Ab2)。根据各种蛋白质的等电点不同，应用IEF技术分离纯化出PRRSV感染猪血清中的不同IgG。分别以纯化的抗PRRSV—GP5蛋白、抗PRRSV-M蛋白的Ab2免疫SPF猪各5头，7d后经鼻腔感染PRRSV，定期采集血样进行病毒分离或鉴定试验。抗PRRSV-GP5蛋白的Ab2免疫的猪，其血样自感染后3～7d均检出PRRSV；3头猪在感染后14～63d未检出PRRSV；2头猪在感染后14～35d检出PRRSV，从42～56d转为阴性，其中1头猪在63d时检出PRRSV。抗PRRSV—M蛋白的Ab2免疫的猪，其血样自感染后3～7d均检出PRRSV；2头猪在感染后14～63d未检出PRRSV；3头猪在感染后14～35d检出PRRSV，从42～56d转为阴性，其中1头猪在63d时检出PRRSV。抗PRRSV—GP5和抗PRRSV—M蛋白的Ab2免疫作用显著，可作为PRRSV-GP5和PRRSV—M蛋白的替代抗原产生具有中和效应的抗体，保护机体免受PRRSV的感染。     

Polyclonal activation of B cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (PRRSV)-infected pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection associated with an inefficient humoral immune response. A study of lymphoid B cells and specific humoral immune response was performed in blood and several lymphoid organs collected from PRRSV experimentally-infected pigs. Groups of specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate of PRRSV, and blood, tonsils, spleen and mediastinal lymph nodes (MLN) were collected at various times postinfection (p.i.) (3-60 days). Lymphoid cells were isolated, immunolabeled for cytofluorometric determination of B cell percentages, used for counting specific anti-PRRSV antibody secreting B cells by an ELISPOT assay, or cultured for metabolic activity. The presence of anti-PRRSV antibodies in the serum of infected pigs was determined using a commercial ELISA assay. Virus detection was performed in all tissues, including lungs, by virus isolation and RT-PCR. The results show that percentages of B cells increased in tonsils as soon as 3 days until 17 days p.i. in PRRSV-infected pigs while they increased in spleen at 3 days p.i. only, due to an increase of larger Ig(high)-producing B cells. Metabolic activity of lymphoid cells from blood and spleen increased at 3 days p.i. only while lymphoid cells from tonsils and MLN transiently decreased at that time and increased thereafter up to 60 days p.i. Anti-PRRSV antibody-secreting B cells occurred in tonsils after 10 days p.i. and strongly increased up to 60 days p.i. However, specific anti-PRRSV-secreting B cells were detected in blood and spleen after 17 days p.i and in MLN only after 45 days p.i. Specific antibodies were detectable in serum at 10 days p.i., reached the maximum level at 45 days and remained high up to 60 days p.i. Infectious virus was detected in lungs and MLN as soon as 3 days p.i., and remained detectable up to 45 days p.i. in tonsils of one pig while viral RNA was detected in most organs up to 60 days p.i. In vitro experiments revealed that inactivated virus induced a stimulation of lymphoid cells isolated from PRRSV-infected pigs while it was cytotoxic for lymphoid cells from control pigs. Taken together, these results indicate that viral infection induced simultaneously a polyclonal activation of B cells, mainly in tonsils, and an exaggerated and prolonged specific humoral immune response due to persistent viral infection in lymphoid organs.     

寡核苷酸探针原位检测石蜡切片中PRRSV核酸

根据GenBank收录的美洲型猪繁殖与呼吸综合征病毒（PRRSV）ATCCVR-2332株ORF6和ORF7基因序列，用O1igo软件设计并合成大小为37bp的寡核苷酸探针，经生物素标记后，成功建立了原住检测石蜡组织切片中PRRSV核酸的方法。该探针能检测到56PgPRRSV核酸的RT—PCR产物DNA，能特异检测出PRRSV核酸及其PCR产物，而对猪瘟病毒（HCV）、猪细小病毒（PPV）、猪伪狂犬病病毒（PRV）、猪乙脑病毒（JEV）的核酸呈阴性反应。应用该方法检测PRRSVSC-1株人工感染的28日龄仔猪，在感染后7d即可在肺脏、肾脏、扁桃体、胸腺、肺门淋巴结、十二指肠和大脑检测到PRRSV核酸。该法可用于仔猪PRRSV感染的诊断和组织中核酸的定位及分布研究，也可用于甲醛固定组织的回顾性诊断。     

Attempts to transmit porcine reproductive and respiratory syndrome virus by aerosols under controlled field conditions

An experimental infection with porcine reproductive and respiratory syndrome virus (PRRSV) was established in 150 five-month-old pigs housed in a fan-ventilated finishing facility, the infected barn. To determine whether air exhausted from the wall fans contained infectious PRRSV, a trailer containing 10 four-week-old PRRSV-naive sentinel pigs was placed 10 m from the building from day 3 after the 150 pigs were infected until day 10. To connect the two airspaces, one end of an opaque plastic tube, 15 m in length and 5 cm in diameter, was fastened to the wall fan of the infected barn, and the other end was placed inside the trailer. Air from the building was exhausted into the trailer 24 hours a day for seven consecutive days and PRRSV infection was monitored in the infected pigs and the sentinel pigs. Air samples were collected from the infected barn and the trailer. PRRSV infection was detected in the infected pigs three and seven days after they were infected, but not in the sentinel pigs. All the air samples were negative for PRRSV by PCR, virus isolation and a pig bioassay.     

The effect of a killed porcine reproductive and respiratory syndrome virus (PRRSV) vaccine treatment on virus shedding in previously PRRSV infected pigs

Two experiments were conducted to investigate if virus shedding could be reduced following a killed porcine reproductive and respiratory syndrome virus (PRRSV) vaccination (KV) of PRRSV infected pigs. In experiment 1, PRRSV infected pigs were vaccinated with KV on days 14 and 28 following infection. Viremia and serum neutralizing (SN) antibody were compared to infected pigs with no KV. The second experiment was conducted in an identical manner. In addition to viremia and SN antibody, virus in oropharyngeal scrapings and interferon gamma (IFN-gamma) producing cells were monitored. Magnitude and duration of viremia were not different between KV vaccinated and non-vaccinated groups. No virus was detected in oropharyngeal scraping from any pig, nor was there a difference in the detection of viral RNA. In both experiments, however, increases in SN titer and number of IFN-gamma producing cells were observed. The SN titer was significantly higher in KV vaccinated groups than in non-vaccinated group on days 42 and 42-56 following infection in experiments 1 and 2, respectively. The number of IFN-gamma producing cells was slightly higher in KV vaccinated groups than in non-vaccinated group on days 42 and 63. These observations suggest that KV had no effect on virus shedding. However, previously infected pigs responded immunologically to KV, as demonstrated by increases in SN antibody titers and IFN-gamma producing cells.     

氧化应激对PRRSV致PAM细胞TLR3/NF-κB分子转录的影响

为探讨氧化应激对猪繁殖与呼吸综合征病毒(PRRSV)感染猪肺泡巨噬细胞(PAM)TLR3/NF-κB信号分子转录的影响,体外分离培养PAM,分为对照组、PRRSV感染组、抗氧化剂NAC+PRRSV组,促氧化剂H2O2+RRRSV组。分别在培养6、12、24、48、72h收集细胞,观察各组的细胞病变、real-time PCR检测PRRSV、TLR3、TRIF和NF-κB mRNA转录量的变化。结果显示,PRRSV感染组PRRSV、TLR3、TRIF及NF-κB mRNA的转录量与对照组相比随感染时间的延长显著升高(P0.05),48h达到最大值;NAC处理接毒组各信号分子mRNA的转录量比PRRSV感染组同时间点略低;H_2O_2处理接毒组比PRRSV感染组的略高。结果表明,氧化应激可增强PRRSV致PAM细胞TLR3/NF-κB分子mRNA的转录量,NF-κB的活化可能是PRRSV导致细胞损伤的机制之一。     

Assessing the duration of persistence and shedding of porcine reproductive and respiratory syndrome virus in a large population of breeding-age gilts

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus in the order Nidovirales, family Arteriviridae, genus Arterivirus. The virus induces a prolonged viremia, replicates in macrophages, and produces persistent infection. The purpose of this study was to determine if PRRSV could persist for 90 d or more in a large population of breeding-age gilts housed under environmental conditions typical of commercial swine production and to determine if experimentally infected gilts could shed virus to naïve sentinel gilts beyond 90 d postinfection. Using the intranasal route, we inoculated 120 PRRSV-naïve gilts, 4 mo of age, with 5 mL of cell culture fluid containing a total dose of 10^2.4 TCID₅₀ of a field isolate (MN-30100) of PRRSV. The index gilts were organized into 3 groups (A, B, and C), 40 gilts per group. To assess the dynamics of the experimental infection, a monitor group of 30 index gilts was blood-tested on days 0, 3, 7, 14, 30, 60, 90, 120, 150, and 180 postinfection. PRRSV viremia was detected with the polymerase chain reaction (PCR) on days 3, 7, and 14 and by virus isolation (VI) on days 7 and 14. PRRSV antibodies were detected from day 14 by enzyme-linked immunosorbent assay (ELISA). To assess shedding, 30 PRRSV-naïve sentinel gilts were commingled with the index gilts on day 90 postinfection and tested by PCR, VI, and ELISA every 15 d until 180 d postinfection; all samples were negative. To assess persistence, 40 index and 10 sentinel gilts were slaughtered at 120 (group A), 150 (group B), or 180 (group C) d postinfection. Evidence of PRRSV was not detected by PCR or VI in any tissue samples from the 120 index gilts. These results indicate that persistence and shedding of PRRSV are of short duration in breeding-age gilts.     

Investigation of T-cell responses and viral mRNA persistence in lymph nodes of pigs infected with porcine rubulavirus

Selected lymphocyte subpopulations were studied and the distribution of viral mRNA were investigated during acute and persistent porcine rubulavirus (PoRV-LPMV) infection in Vietnamese pot-bellied pigs. Six pigs infected with PoRV-LPMV at 17 days of age exhibited clinical signs 7-10 days post-inoculation (pi). One infected piglet died 11 days pi while the other five recovered around day 13 pi and survived until euthanasia on day 277 pi. Increased numbers of CD8+, CD4+ and CD2+ T cells were detected during the acute phase of infection while CD8+ cells were elevated throughout the infection, including during the persistent stage. Specific antibodies against the haemagglutinin-neuraminidase protein of PoRV-LPMV were detected during persistent infection. Although infectious virus could not be recovered from tissues from any of the infected pigs at necropsy 277 days pi, PoRV-LPMV mRNA was detected in lymph nodes, pancreas and central nervous system using a nested polymerase chain reaction technique. Continued lymphocyte interaction with viral RNA may be an important factor in promoting cellular and humoral responses during persistent PoRV-LPMV infection.