High level expression of C-terminal truncated recombinant chicken interferon-gamma in baculovirus vector system

Chicken interferon-gamma (ChIFN-gamma) was expressed by baculovirus in a C-terminal truncated form, namely ChIFN-gammaT, to accelerate the secretion of the expressed protein. It is also expressed as ChIFN-gammaT bearing poly His tag, ChIFN-gammaTHis, for easy purification. The expressed proteins were detected by SDS-PAGE analysis with Coomassie brilliant blue staining. The purified ChIFN-gammaTHis with nickel chelated column showed anti-viral activity in vitro and stimulation of the secretion of nitrogen intermediates such as nitric oxide in chicken peripheral blood mononuclear cells. Antiserum against ChIFN-gammaTHis recognized the 15 kDa, 16 kDa, and 32 kDa bands that seemed to be an unglycosylated monomer, a glycosylated monomer, and a homodimer of ChIFN-gammaTHis in the culture supernatant, respectively. The anti-serum also recognized around 14 kDa and 28 kDa bands in the sera of chickens or concanavalin A stimulated spleen cell culture supernatants that seemed to be monomeric and dimeric forms of a natural ChIFN-gamma, respectively.     

Identification of a bovine enteric syncytial virus as a nongroup A rotavirus

An atypical or nongroup A rotavirus was identified in feces obtained from gnotobiotic calves in which fecal preparations originally derived during an epizootic of neonatal calf diarrhea had been serially passaged. The epizootic was previously reported to be caused by a noncharacterized viral agent that induced the formation of epithelial syncytia on small intestinal villi of experimentally infected calves. This bovine, nongroup A rotavirus was found to be antigenically related to a described atypical rotavirus of rats by immunofluorescence and by enzyme immunoassay. Complementary DNA derived from the atypical rat rotavirus cross hybridized with RNA obtained from the bovine virus, but not with RNA extracted from group A rotaviruses. Complementary DNA derived from SA-11 group A rotavirus cross hybridized with other group A rotavirus RNA, but not with RNA obtained from either the rat or bovine nongroup A isolates. Additionally, another similar, if not identical, bovine atypical rotavirus was identified in a second epizootic of neonatal calf diarrhea that occurred several hundred kilometers and 8 months apart from the original epizootic.     

Surface proteins of Breda virus

The serotypes 1 and 2 of Breda virus from feces of experimentally infected gnotobiotic calves were studied with respect to their sedimentation and density properties in sucrose gradients and their structural polypeptides; Berne virus, the proposed prototype of the new family Toroviridae, was included for comparison. After Breda-1 virus had been stored at 4 C for a prolonged period, it showed a heterogeneous sedimentation behavior (480 to 520 Svedberg units [S]) and density (1.18 to 1.21 g/ml) indicative of its poor state of preservation. In contrast, freshly prepared Breda-2 virus sedimented at 350 S and showed a buoyant density of 1.18 g/ml; these values compare well with those of Berne virus (400 S and 1.16 g/ml, respectively). Efficient purification of the Breda viruses could be achieved by a 2-step method, involving pelleting by ultracentrifugation followed by isokinetic and isopyknic sucrose gradient centrifugation. Radioiodinated purified virus showed polypeptides with apparent molecular weights of 105,000, 85,000 37,000, and 20,000; another labeled protein of 65,000 D is of doubtful virus specificity. Mouse immune serum raised against Breda-2 virus recognized the polypeptides of the homologous virus and the 2 highest molecular weight proteins of Breda 1 virus in radioimmune precipitation. The same serum inhibited hemagglutination of the heterologous serotype to a low, but significant, degree and efficiently neutralized the infectivity of Berne virus. These observations are taken as indications that the 105,000- and 85,000-D polypeptides represent surface structures of torovirions, probably peplomeric proteins.     

猪流行性腹泻病毒S1D蛋白的优化表达及其单克隆抗体的制备

为进一步探究猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)S蛋白的抗原表位及其功能,本试验通过优化S1D基因的密码子,构建了S1D基因未优化的重组原核表达质粒pET-S1D和已优化的pET-ΔS1D,并进行了诱导表达和纯化。使用SDS-PAGE和Western blotting方法验证S1D、ΔS1D蛋白在大肠杆菌内得到正确表达,利用Image J软件对S1D、ΔS1D蛋白表达量进行灰度扫描,通过t检验分析两者差异性。将纯化的ΔS1D 蛋白免疫 BALB/c小鼠,通过细胞融合、筛选及亚克隆,获得单克隆细胞株。利用体内诱生法制备抗PEDV S1D蛋白的单克隆抗体腹水,使用ELISA、Western blotting、间接免疫荧光试验3种方法对腹水效价及特异性进行检测和验证。SDS-PAGE和Western blotting结果显示,表达S1D、ΔS1D蛋白的样品均在34 ku处出现正确的目的条带。t检验结果表明, S1D、ΔS1D两者蛋白表达量差异极显著(P<0.01)。ELISA结果显示, 腹水的抗体效价达到了1∶1 000 000,腹水与PEDV病毒粒子和纯化后的ΔS1D蛋白反应均呈阳性,与PEDV N蛋白、pET-32a(+)空载体蛋白和猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪瘟病毒(Classical swine fever virus,CSFV)、猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)和猪急性腹泻综合征冠状病毒(Swine acute diarrhea syndrome coronavirus,SADS-CoV) 5种病毒反应均呈阴性。Western blotting结果显示,腹水与ΔS1D蛋白和PEDV S蛋白分别在34和180 ku处有特异性条带出现,与pET-32a(+)空载体蛋白、正常Vero细胞蛋白均无特异性条带出现。间接免疫荧光试验结果显示,腹水及阳性对照组均能使细胞出现特异性绿色荧光信号,而空白及阴性对照组均未见绿色荧光信号。密码子优化可在原核表达系统中显著提高重组蛋白的表达水平,本研究基于高效表达的ΔS1D蛋白,成功制备了1株能稳定分泌与 PEDV S蛋白特异性结合的单克隆抗体的细胞株,为进一步探究 PEDV S蛋白抗原表位及蛋白功能的研究奠定了基础。     

Economic impact of an epizootic of pseudorabies in a commercial swine herd in Ohio, achieving test negative status and quarantine release by use of vaccination and test and removal.

The effect of pseudorabies in a commercial farrow-to-finish operation on selected production and economic values was estimated. Pseudorabies was first diagnosed in this herd by circle testing done in March 1988, as a required part of follow up from another herd that had been diagnosed with pseudorabies in the area. A pseudorabies virus vaccination program was initiated in the herd at that time. The mean litter size of pigs born alive varied from 9.26 to 10.02 pigs/litter throughout the study period; however, there was a twofold increase in suckling pig mortality and a 2.6-fold increase in nursery pig mortality when the months of the epizootic were compared with pre-epizootic months. In the 6-month period following the epizootic, suckling pig mortality was three-fold higher than that reported in the preepizootic months. Total net loss for this operation was estimated at $99,700 from when the epizootic started until eradication, when calculating losses directly. The major economic losses (76.5% of total loss) were related to suckling pig mortality, which was $16,240 during the epizootic or $24/inventoried sow/week; $19,395 in the 6 months following the epizootic or $3.8/inventoried sow/week; and $40,628 thereafter until eradication 26 months later or $0.37/inventoried sow/week. Nursery pig mortality losses were 12.6% of total net losses; $754 during the epizootic, $357 in the 6 months after the enzootic, and $11,444 thereafter until eradication 26 months later. Sow culling and deaths accounted for 9.4% of net losses that took place from 6 months after the epizootic until eradication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study on Relationship between Intestinal Epithelium Tight Junction Proteins mRNA Expression and Piglets Weaning Diarrhea

The experiment was carried out to compare diarrhea status between Min pig and Landrace pig in a week after weaning, detect two varieties piglets jejunum, ileum and colon tight proteins Zonula occludens-1(ZO-1)and Occludin mRNA expression before and after weaning. The intention was to discuss the relationship between tight proteins expression and piglets weaning diarrhea. The results showed that Min piglets diarrhea rate, diarrhea frequency were very significantly lower than Landrace pig within one week after weaning (P<0.01), diarrhea days was significantly lower than Landrace pig within one week after weaning (P<0.05);Min piglets not weaning group ileum and colon ZO-1 and Occludin mRNA expression was very significantly higher than Landrace pig not weaning group (P<0.01);Min piglets weaning diarrhea group ileum and colon ZO-1 and Occludin mRNA expression was significantly higher than weaning health group (P<0.05). The results indicated that Min piglets intestinal tight junction proteins mRNA high expression before weaning might be had relation with guarantee piglets intestinal health and decrease occurrence diarrhea;Min pig fast recover intestinal mucosa function and rehabilitation by increased tight junction proteins mRNA expression after weaning. So tight junction proteins mRNA high expression before and after weaning had an important significance for piglets to resist weaning diarrhea.     

Aujeszky's disease: sanitation of swine herds in enzootically infected areas using labelled vaccines

Both in the Federal Republic of Germany and in some neighbouring countries the epizootic situation of Aujeszky's disease has been unsatisfactory for a long time, especially in areas with a high pig density. New findings on vaccines with certain protein deletions have recently indicated the possibility that the disease might be eradicated even in vaccinated herds. By using labelled vaccines it seems possible to distinguish the carriers of vaccine virus antibodies from the carriers of field virus antibodies and to eliminate the animals infected with field virus, while leaving the other animals in the herd and protecting them from infection by vaccination. This procedure would also be practicable in epizootically infected areas and would be less expensive than eradicating the disease on the basis of serological tests without using vaccines. A skeleton of an eradication programme, which was discussed and agreed with scientific experts and with the Federal Laender, as well as rough estimates of costs are being presented.     

猪嵴病毒病原特性及检测技术研究进展

猪嵴病毒（porcine kobuvirus,PKV）是近年来在健康猪和腹泻猪粪便中新检测到的小核糖核酸病毒科嵴病毒属成员,可能引起猪的腹泻,对养猪业造成重大经济损失。研究发现,PKV广泛分布在猪中,在腹泻和临床健康猪中均已被检测到,阳性率从3.9%~100.0%各不相同。一个典型的PKV病毒粒子直径为30 nm,基因组全长为8 120 bp,包括1个含2 488个氨基酸的开放性阅读框（ORF）;PKV是典型的小RNA病毒科的基因组结构：1个5'非编码区,1个L蛋白,结构蛋白P1（VP0、VP3和VP1）,非结构蛋白P2（2A、2B和2C）和P3（3A、3B、3C和3D）,1个3'非编码区和1个Poly（A）尾巴。PKV的2B编码区存在30个氨基酸的缺失,VP1蛋白是小RNA病毒科变异最频繁的结构蛋白,其含有主要的抗原表位,可促进机体产生中和抗体。检测PKV的方法有反转录聚合酶链式反应（RT-PCR）、TaqMan探针实时荧光定量RT-PCR和逆转录环介导扩增（RT-LAMP）方法。作者对PKV的分类学、流行概况、基因组结构、遗传特性及检测技术等研究现状做一简要概述,以期为进一步研究及了解PKV提供参考。     

Detection of transmissible gastroenteritis coronavirus antigens by a sandwich enzyme-linked immunosorbent assay technique

A new sandwich enzyme-linked immunosorbent assay, using monoclonal and polyclonal antibodies, was developed to detect transmissible gastroenteritis virus antigens from cell culture and from intestinal wash or feces obtained from experimentally infected pigs. This technique was shown to be suitable for the detection of virulent field strain unadapted to cell culture. Cross reactions had not been observed with other enteric pathogens, rotavirus, porcine epizootic diarrhea virus, and Escherichia coli.     

Characterization of parainfluenza type 3 virus isolated from the lung of a lamb with pneumonia

A virus designated DH-1 was isolated from the lung of a lamb that died during the course of an epizootic of acute respiratory tract disease. The virus contained ribonucleic acid and was sensitive to heat (56 C), lipid solvent, and acid (pH 3.0). Inoculated cell cultures absorbed and culture fluids agglutinated guinea pig erythrocytes. Morphogenic and morphologic characteristics of the isolate were compatible with those of the genus Paramyxovirus of the family Paramyxoviridae. Serologic results indicated that the virus was indistinguishable from prototype parainfluenza virus type 3.     

Survey of desert bighorn sheep in California for exposure to selected infectious diseases

From February 1983 to June 1985, 188 desert bighorn sheep (Ovis canadensis nelsoni, = 161 and Oc cremnobates, = 27) from 18 herds in 17 mountain ranges and one captive herd were caught, marked, and had blood, fecal, and nasal mucus samples collected. Nasal swab specimens were cultured bacteriologically and virologically specifically for parainfluenza-3 (PI-3) virus. Bacterial flora differed from herd to herd. Pathogenic pneumophilic bacteria (eg, Pasteurella sp) seldom were found. Parainfluenza-3 virus was isolated from 6 bighorn sheep in 3 herds. Fecal specimens were examined for parasite ova and low numbers of lungworm (Protostrongylus sp) larvae were found in feces from 2 herds. Sera were evaluated for antibodies against respiratory syncytial virus, ovine progressive pneumonia, infectious bovine rhinotracheitis, PI-3, bovine viral diarrhea, brucellosis, leptospirosis, contagious ecthyma, bluetongue, and epizootic hemorrhagic disease. Blood clots were cultured virologically for bluetongue and epizootic hemorrhagic disease. Serologic evidence of bluetongue and/or epizootic hemorrhagic disease was found in 9 herds, and bluetongue virus (serotypes 10,11,13 and 17) was isolated from 3 herds. Antibody titers against PI-3 and respiratory syncytial virus were found in 9 and 13 herds, respectively. Evidence of bovine viral diarrhea infection was found in 6 herds, whereas infectious bovine rhinotracheitis was found in only 1 herd. Antibody titers against contagious ecthyma were found in 9 of 18 herds in California, and active lesions were seen occasionally. Evidence of ovine progressive pneumonia, leptospirosis, or brucellosis was not found.     

Bovine parainfluenza-3 virus proteins recognized by antibodies of aerosol-exposed calves

Bovine parainfluenza-3 (BPI-3) virus proteins were radiolabeled with [35S]methionine. Control serum containing antibodies to BPI-3 virus obtained from calves that had been immunized by aerosol exposure and by IM inoculation and sera of cattle that were exposed to BPI-3 virus by aerosol only were used to immunoprecipitate BPI-3 virus proteins. The immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral proteins with estimated molecular weights of 72,000, 70,000, and 58,000 were recognized by the control serum and the sera from calves exposed to BPI-3 virus only by aerosol. These proteins were the hemagglutinin/neuraminidase, nucleoprotein, and fusion protein. The results indicated that a single aerosol exposure to BPI-3 virus induces antibodies to the same BPI-3 virus proteins as do combined aerosol and parenteral inoculations with the virus.     

The gp135 of caprine arthritis encephalitis virus affords greater sensitivity than the p28 in immunodiffusion serology

The 135,000 mw glycoprotein (gp135) and the 28,000 mw internal protein (p28) of caprine arthritis encephalitis virus are major viral constituents in precipitin lines formed between crude antigen preparations and sera from infected goats. In testing 307 goat and sheep sera, 118 samples were positive in a gp135 assay and only 82 were positive in a p28 assay. However, some goat sera were found which reacted only with the p28 and therefore testing for antibody against both proteins may be necessary to identify a maximum number of virus infected goats by immunodiffusion.     

河南省南阳市某猪场一例猪流行性腹泻的实验室诊断

为对南阳市某猪场发生腹泻的病原进行鉴定,结合猪场发病情况、临床症状、剖检病理变化进行分析,并运用HE染色、RT-PCR等实验室技术综合判定。结果显示,某场新生仔猪发病率100%,临床表现为严重呕吐、腹泻且病死率较高,肠道组织切片发现空肠绒毛脱落、回肠细胞碱性粒子变多,RT-PCR结果显示猪流行性腹泻病毒(PEDV)阳性,提示本次某猪场腹泻主要是由猪流行性腹泻病毒感染造成的。     

猪两种腹泻病毒混合感染的病原诊断

对我省流行的疑似猪病毒性腹泻进行了病原诊断，通过细菌培养、ＲＮＡ电泳和电镜形态观察等辅助诊断和免疫萤光特异性诊断，确诊该病病原为猪传染性胃肠炎病毒（ＴＧＥＶ）与猪流行性腹泻病毒（ＰＥＤＶ）的混合感染，混合毒代号为ＴＰＶ３。     

猪伪狂犬病毒PCR检测方法的建立及应用

根据猪伪狂犬病毒（PRV）gE基因序列保守区段,设计一对特异性引物,通过优化反应条件,建立了可区分猪伪狂犬病毒野毒株与基因缺失疫苗株的PCR检测方法,并对该方法的敏感性、特异性和重复性进行了验证。结果显示,该PCR方法可扩增出388 bp的目的片段;对模板的最低检测量为1.1 pg;与猪圆环病毒Ⅱ型、猪细小病毒、猪支原体、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪乙型脑炎病毒、猪流行性腹泻病毒无交叉反应,具有高特异性。采用建立的PCR方法对2014年以来全国不同地区81个猪场421份疑似病料进行检测,发现PRV猪场平均阳性率为35.80%,样品平均阳性率为 25.42%。该方法灵敏度高、特异性强、重复性好,可用于PRV的临床诊断和流行病学调查。     

PEDV、TGEV、RV多重RT-PCR检测方法的建立与应用

为了快速准确诊断猪流行性腹泻病毒 (PEDV)、猪传染性胃肠炎病毒 (TGEV)、猪轮状病毒(RV)引起的猪腹泻病,通过设计三对引物,建立了扩增PEDV、TGEV、RV的多重RT-PCR方法,用于临床上大量腹泻病料的鉴定。该方法特异敏感,能同时检测PEDV、TGEV、RV,而对猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病毒、猪圆环病毒2型病毒、乙型脑炎病毒、猪细小病毒等无扩增,检测的抗原稀释极限为107。用于35个猪场120份临床样品的检测,PEDV阳性率占37.5%,TGEV阳性率占0.75%,RV阳性率占1.25%。PEDV阳性病料测序结果分析表明,与近几年分离毒株亲缘关系较近,与经典毒株和疫苗株亲缘关系较远。结果表明建立的多重PCR方法特异性强、敏感度高,能用于临床诊断及流行病学调查。     

猪流行性腹泻病毒细胞受体研究进展

猪流行性腹泻病毒主要引起仔猪腹泻,发病率及病死率极高,严重影响养猪业的发展。而猪流行性腹泻病毒要感染仔猪,必须与宿主细胞表面的受体结合。目前已有研究证实,猪流行性腹泻病毒的细胞表面受体为猪氨基肽酶N(pAPN)。为了能更好的揭示pAPN在病毒感染过程中的机制,学者针对pAPN的结构、功能以及与病毒的感染结合区域进行了系统的研究,并开展了筛选pAPN结合短肽的工作,以此来探索病毒受体的阻断剂。为进一步研究猪流行性腹泻病毒感染机制及其细胞受体的作用提供参考,论文就目前pAPN最新研究进展进行了综述。     

猪轮状病毒Rotavirus A pig/China/NMTL/2009/G9P[23]株的分离与鉴定

A群轮状病毒(GAR)是引起人类和多种动物腹泻的主要病原体,病原的分离与鉴定是轮状病毒(RV)流行病学、病原学等研究的基础.本研究采用接种MA 104细胞的方法,从内蒙古某猪场患病猪腹泻粪便中分离一株病毒,经3次蚀斑克隆纯化后,采用电镜观察、PCR鉴定和序列测定进行分析,结果表明该分离株为猪轮状病毒(PoRV).致病性试验表明该分离株能够引起仔猪急性腹泻,RT-PCR扩增其VP4、VP6和VP7的基因节段并进行序列测定,按照A群RV的最新分类方法,确定该分离株的VP6、VP7和VP4基因型分别为I5型、G9型和P[23]型.因此,将该分离株命名为Rotavirus A pig/China/NMTL/2009/Q9P[23].