Interferon induction in porcine leukocytes with transmissible gastroenteritis virus

Leukocytes were harvested from the peripheral blood, mesenteric lymph node and small intestinal lamina propria from groups of three piglets before, and 1,2 and 3 weeks after infection with virulent transmissible gastroenteritis virus (TGEV) at 2 weeks of age. The donor piglets developed clinical signs of transmissible gastroenteritis which persisted for up to 3 days, and they developed peak serum titres of TGEV-neutralizing antibodies 2 weeks post-infection. The leukocytes were cultured in the presence of pokeweed mitogen (PWM), various dilutions of purified TGEV, or control media for 3 or 5 days, and the culture supernatants were tested for antiviral activity in MDBK cells challenged with vesicular stomatitis virus. The antiviral activity was characterized as porcine interferon (IFN)- or porcine IFN-τ on the basis of its stability at pH 2.0 and neutralization by anti-human IFN- antibodies. Viability of the leukocytes in culture, determined by trypan blue exclusion, was highest for the peripheral blood leukocytes and lowest for the mesenteric lymph node leukocytes. There were no consistent differences in antiviral activity between cultures incubated for 3 or 5 days. Porcine IFN- was found in the supernatants of the leukocyte cultures stimulated with TGEV antigen, harvested before or after infection of the donor piglets with TGEV. Porcine IFN-τ was demonstrated in the supernatants of the leukocyte cultures stimulated with PWM, more frequently when the leukocytes were harvested post-infection. This was the first demonstration of IFN induction in vitro in leukocytes from porcine gut-associated lymphoid tissue.     

Antigenicity of structural components from porcine transmissible gastroenteritis virus

Pregnant sows were inoculated with inactivated transmissible gastroenteritis virus and with preparations of virus surface projections and subviral particles derived by detergent treatment of the virus. Neutralising antibody was demonstrated in serum and colostrum from animals that received whole virus or preparations of surface projections whereas subviral particles failed to stimulate neutralising antibody formation. Similar results were obtained with serum from rabbits inoculated with whole virus and structural components. All three preparations stimulated the formation of agglutinating antibodies, as demonstrated by sedimentation analysis and filtration studies with radiolabelled virus.The immunoglobulin classes responsible for neutralising antibody activity in sows inoculated by the intramammary route were examined. In each case where the immunoglobulin class was determined, IgG was found. One sow that received surface projections also had IgA with neutralising activity in her colostrum. In contrast, infection of sows with live whole virus resulted in neutralising antibody of the IgG, IgM and IgA classes.     

Neutralization of porcine transmissible gastroenteritis virus by complement-dependent monoclonal antibodies

Monoclonal antibodies (MAB) to each of the 3 major structural proteins of transmissible gastroenteritis virus (TGEV) of swine were compared, using their virus-neutralizing (VN) activity in the presence or absence of guinea pig, rabbit, or swine complement. The MAB against the peplomer protein had similar VN titers for TGEV in the presence or absence of complement from any source. The MAB against the matrix protein had VN activity for TGEV only in the presence of complement, whereas MAB specific for the nucleocapsid protein did not possess VN activity in the presence or absence of complement. Serum from sows containing antibodies against TGEV peplomer and matrix proteins had slightly higher VN titers in the presence of complement than in the absence of complement. High concentrations of complement from swine serum (128 U) had little effect on the infectivity of TGEV for swine testes cells, whereas 32 U of complement from rabbit serum and 64 U of complement from guinea pig serum were able to neutralize virulent and attenuated TGEV in the absence of known antibodies for TGEV. Complement (less than or equal to 8 U) from any source did not decrease the infectivity of TGEV by greater than 0.5 log10 units.     

猪传染性胃肠炎疫苗的研究进展

猪传染性胃肠炎是由猪传染性胃肠炎病毒(TGEV)引起的一种以严重腹泻、呕吐和脱水为临床特征的高度接触性传染病。可侵害各年龄的猪,已成为猪的一种世界性疾病,给养猪业造成巨大经济损失,有效的疫苗接种是目前大多数国家控制本病流行的重要措施。在过去的十年中,人们重点构建基因工程疫苗,但效果不一。灭活苗或弱毒活苗虽然仍显示着良好的优势,但一种安全、有效新型疫苗的研制已成为必然趋势。     

Antigenic variation of porcine transmissible gastroenteritis virus detected by monoclonal antibodies

Mouse myeloma cells (SP2/O) were fused with spleen cells from BALB/c mice immunized with detergent-solubilized antigen of purified virus, and 21 monoclonal (MC) antibodies reactive in enzyme-linked immunosorbent assay with the TO-163 strain of porcine transmissible gastroenteritis (TGE) virus were obtained. Of these MC antibodies, 14, 6 and 1 were IgG1, IgG2a and IgM, respectively. All of the MC antibodies contained light chains of the kappa type. Of these MC antibodies, 8 were found to have neutralization (NT) activity against the TO-163 strain. Comparison of 7 strains of TGE virus by NT tests using our panel of MC antibodies confirmed their close antigenic relationships, but also revealed the occurrence of distinct antigenic differences. These results suggest that there may be at least 6 different epitopes involved in NT reaction on the virion of the TO-163 strain. This notion was confirmed by the competitive binding assay.     

猪传染性胃肠炎病毒感染相关猪源miRNA的筛选及表达差异分析

经生物信息学预测发现猪传染性胃肠炎病毒(TGEV)3′UTR可能是ssc-miR-182的潜在作用位点,本研究以猪传染性胃肠炎病毒为研究对象,构建TGEV 3′UTR双荧光素酶报告基因载体,并将其与合成的ssc-miR-182模拟体瞬时共转染至猪小肠上皮细胞,通过荧光监测仪测定荧光素酶活性;同时通过荧光定量PCR方法检测TGEV感染猪小肠上皮细胞后在不同时间段ssc-miR-182的表达变化情况。研究结果显示,ssc-miR-182对TGEV 3′UTR有一定的抑制作用,并且TGEV在感染小肠上皮细胞以后ssc-miR-182的表达明显上调。结果表明,宿主ssc-miR-182与TGEV的感染和复制增殖相关。     

猪传染性胃肠炎病毒单克隆抗体的制备与鉴定

以纯化的猪传染性胃肠炎病毒(TGEV)作为抗原免疫BALB/c小鼠,经3次免疫后,通过聚乙二醇方法进行融合,利用有限稀释法在HAT培养基上筛选杂交瘤细胞,共获得12株既能稳定生长并可以分泌特异性抗TGEV的单克隆抗体的杂交瘤细胞系,分别命名为8D2、4H4、5D6、7H10、4C3、9G1、3A2、8G1、5G6、2D7、4D6、6B10。间接ELISA、Western blot、间接免疫荧光结果表明,获得的12株单抗能特异性识别TGEV,通过间接ELISA做病原检测,显示12株单抗与猪流行性腹泻病毒(PEDV)和猪轮状病毒(PrV)不发生交叉反应。经抗体亚类鉴定,该12株单克隆抗体均为IgG2b。12株抗TGEV的单抗制备成功,为猪传染性胃肠炎病原特性研究和病原快速检测提供了物质基础。     

Sites of replication of a porcine respiratory coronavirus related to transmissible gastroenteritis virus

A porcine respiratory coronavirus (PRCV) was inoculated by aerosol into nine hysterectomy-derived and colostrum-deprived pigs at the age of one week. They were killed at different times after inoculation and tissues were sampled for virus isolation and immunofluorescence. Results indicate that virus replicated to high titres in the respiratory tract. Replication mainly occurred in alveolar cells but also in epithelial cells of nasal mucosa, trachea, bronchi, bronchioli, in alveolar macrophages and in tonsils. After primary replication in the respiratory tract, viraemia occurred. Virus also reached the gastrointestinal tract after swallowing. Subsequently, PRCV was observed to replicate in the ileum. The infection spread within a few days from the ileum to the duodenum. Replication in the small intestine remained limited to a few cells located in or underneath the epithelial layer of villi and, or, crypts. The cell type could not be identified. Virus was isolated from mesenteric lymph nodes in all pigs, but immunofluorescence was not observed. Results show that small changes in molecular structure between transmissible gastroenteritis virus and PRCV resulted in important changes in host cell tropism.     

Molecular biology of transmissible gastroenteritis virus

The causative agent (TGEV) of porcine transmissible gastroenteritis belongs to the Coronaviridae, a family of enveloped viruses with a positive, single-stranded RNA genome. Important progress has recently been made concerning the molecular biology of TGEV. The research work of our group has been focused on two main aspects: genome structure and functional domains of the envelope proteins. TGEV genomic RNA is organised into seven regions. The sequence of six of them, i.e. the 3' most 8300 nucleotides, has been established from cDNA clones. Three genes encoding the structural proteins, the peplomer protein E2, the transmembrane protein E1 and the nucleoprotein, have been identified. Additional open reading frames allowed for the prediction of four non-structural polypeptides, the role of which remains to be discovered. The remaining part of the genome (estimated length 20 kb) is thought to encode the polymerase. Expression of TGEV genes involves the production of six subgenomic mRNAs, which together with the virion RNA, form a 3' terminal nested set. The peplomer glycoprotein E2 (220 kDa) is 1431 residues long and highly glycosylated. Several domains were identified, including a C-terminal anchoring region and at least four major antigenic sites, which cluster in the amino half part of the molecule. Two sites containing most of the critical neutralisation determinants are highly conserved among TGEV strains. The glycoprotein E1 (29kDa) is mostly embedded in the membrane and plays a crucial role in the virion architecture. However, a short N-terminal domain protruding out of the particle mediates complement-dependent neutralisation, and induces alpha interferon synthesis, likely through a direct interaction with the lymphocyte membrane.     

猪传染性胃肠炎病毒(TGEV)研究进展

猪传染性胃肠炎 (TGE)是由猪传染性胃肠炎病毒 (TGEV)引起的一种以严重腹泻、呕吐和脱水为临床特征的高度接触性传染病。 TGE首次由Doyle和 Hutchings1 946年在美国报道 ,日本 (1 95 6年 )和英国 (1 95 7年 )先后报道该病 ,这之后欧洲、北美、亚洲等多个国家相继报道发生了 TGE,现已成为一种世界性猪的疾病。我国从 6 0年代起就有TGE的报道 ,近些年来有进一步流行的趋势 ,尤其是冬季和早春寒冷季节常呈地方性暴发流行 ,给养猪业造成极大危害。在 1 984年和 1 986年间 ,欧洲北美等地又报道了一种特别的 TGEV自然缺失变异株 -猪呼吸…     

The survey of porcine teschoviruses, porcine circovirus and porcine transmissible gastroenteritis virus infecting piglets in clinical specimens in China

A multiplex PCR assay was developed and evaluated for its ability to simultaneously detect three viral infections of swine. Specific primers were carefully selected from articles published for each of the following three viruses: porcine circovirus type II (PCV2), porcine teschovirus (PTV) and porcine transmissible gastroenteritis virus (TGEV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 168 bp (PTV) and 499 bp (TGEV). The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 6.60?×?10², 8.43?×?10² and 7.30?×?10² copies for PCV2, PTV and TGEV, respectively. Among 127 samples which were collected from Heilongjiang, Jilin, Henan and Guangxi provinces, the single infection of PCV2, PTV and TGEV was 99.21, 46.88 and 65.35 %, respectively, and co-infection of the three viruses was 26.77 %. In conclusion, the multiplex PCR has the potential to be useful for routine molecular diagnosis and epidemiology.     

猪传染性胃肠炎病毒S1基因在食品级乳酸菌中的表达

猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)纤突蛋白S携带主要的B淋巴细胞抗原表位,是诱导机体产生中和抗体和提供免疫保护作用的主要结构蛋白,本试验通过PCR扩增,获得TGEV S蛋白上约1 218bp的保护性抗原基因S1,将其克隆到pMD18-T载体并进行核酸序列测定。通过双酶切连接成功获得重组质粒pNZ8149-S1,电转化至乳酸乳球菌NZ3900中,经乳链菌肽诱导,SDS-PAGE和Western-Blot分析结果显示,TGEV S1基因获得了表达,表达产物具有反应原性。间接免疫荧光试验结果表明,重组乳酸菌表达的蛋白位于菌体表面。     

猪乳汁中抗猪传染性胃肠炎病毒IgA抗体间接ELISA检测方法的建立

为建立检测猪乳汁中抗猪染性胃肠炎病毒(TGEV)Ig A抗体的间接ELISA方法,本研究以TGEV重组N蛋白为包被抗原,以辣根过氧化物酶标记羊抗猪Ig A为检测抗体,并采用方阵法确定包被抗原和待检乳汁的最佳工作浓度,对各种反应条件进行优化,建立了猪乳汁中TGEV Ig A抗体的间接ELISA检测方法。该方法检测稀释160倍的阳性乳清仍呈阳性;与猪流行腹泻和猪轮状病毒感染阳性乳清无交叉反应;批内和批间变异系数均小于10%。利用建立的ELISA方法与间接免疫荧光方法分别对134份临床样品进行检测,阳性检出率分别为58.2%(78/134)和59.7%(80/134),总符合率为85.6%。本研究建立的检测方法能够有效评估乳汁中TGEV Ig A抗体的水平。