Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe.

A plasmid, pG3BS, containing a cDNA clone from the 5' coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.     

Differentiation of feline coronavirus type I and II infections by virus neutralization test

Feline coronavirus (FCoV) is divided into two types I and II, based on their growth in vitro and antigenicity. In this study, virus neutralization (VN) test was applied for type differentiation of FCoV infections. Sera of cats which were clinically and serologically diagnosed as feline infectious peritonitis (FIP) possessed significantly higher VN titers to type I FCoV, and sera from cats experimentally infected with FIPV type II had high VN titers to type II but not type I viruses. A total of 79 cat sera collected in the years between 2004 and 2005 were examined to evaluate seroprevalence by the VN test, showing the following results: (1) 50 cats (63.3%) were sero-positive to FCoV; (2) of the 50 FCoV positive cat serum samples, 49 (98%) showed significantly higher titers to type I virus and only one (2%) for type II virus. These results indicate that the VN test described here can be used for serological differentiation of FCoV infections of cats, and that FCoV type I is a dominant type in recent years of Japan.     

Immune response in sows given transmissible gastroenteritis virus or canine coronavirus

Twelve pregnant sows were inoculated oral-nasally 8 weeks before farrowing with attenuated transmissible gastroenteritis virus (TGEV), tissue culture-adapted canine coronavirus (CCV), or fluids from mock-infected culture (controls). A 2nd dose of the same inoculum, one-half oral-nasally and one-half intramammarily, was given 6 weeks later. Neutralizing antibodies for TGEV and CCV were demonstrated in sera, colostrum, and milk whey from the virus-vaccinated sows. Homologous geometric mean neutralizing titers were generally 4-fold higher than were heterologous titers. After challenge exposure of the nursing pigs with virulent TGEV, average morbidity and mortality for the pigs were 81% and 34% (mortality range = 11% to 63%), respectively, in the TGEV-vaccinated group; 83% and 39% (mortality range = 15% to 83%), respectively, in the CCV-vaccinated group; and 97% and 84% (mortality range = 78% to 100%), respectively, in the controls. Seemingly, sera from swine exposed to CCV could test serologically positive for TGEV-neutralizing antibody, and TGEV and CCV share at least 1 common neutralizing determinant that may be involved in protection.     

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.     

Infection with porcine respiratory coronavirus does not fully protect pigs against intestinal transmissible gastroenteritis virus

Eight nine-week-old specific-pathogen-free pigs which had been infected with the transmissible gastroenteritis virus (TGEV)-related porcine respiratory coronavirus (PRCV) and four uninfected littermates were challenged with TGEV. The previous PRCV infection failed to protect them against the enteric TGEV infection. Virus excretion in faeces was detected by an ELISA in all the pigs for three to six consecutive days after inoculation. Although little diarrhoea was observed, the infection extended through much of the small intestine of one of the previously infected pigs four days after inoculation. Challenge with TGEV caused a secondary neutralising antibody response. By using a peroxidase conjugate of a monoclonal antibody which recognises a specific antigenic site on TGEV, antibodies against TGEV could be distinguished from antibodies against PRCV in an ELISA blocking test.     

Isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis

A porcine respiratory, non-enteric virus which is related to the coronavirus transmissible gastroenteritis virus (TGEV) has been isolated in pigs and in cell culture. The isolate was designated TLM 83. It has become very widespread and enzootic among the swine population in Belgium and in other swine raising countries. It causes an infection of the lungs and appears to spread by aerogenic route. It does not replicate in the enteric tract. The experimental infection in conventional and gnotobiotic pigs in isolation remains subclinical. The infection, either experimental or in the field, results in the formation of antibodies which neutralise the classical enteric TGEV. Based on this relationship, this virus is assumed to be a new TGEV-related porcine respiratory coronavirus or TGEV itself which has totally lost its tropism for the enteric tract.     

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.     

Cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses

Cross-protection studies between the feline infectious peritonitis (FIP) and the porcine transmissible gastroenteritis (TGE) viruses were conducted in cats, pigs and pregnant gilts. Cats vaccinated with TGE virus developed neutralizing antibodies against TGE virus and low titer antibody against FIP virus detected by an indirect fluorescent antibody technique but were not protected against a virulent FIP virus challenge. Baby pigs and pregnant gilts vaccinated with FIP virus did not develop detectable antibodies to TGE virus. Nevertheless, it appeared that vaccination of swine with FIP virus conferred some immunity against TGE virus infection. Seventeen-day-old pigs vaccinated with two doses of FIP virus had a 67% survival rate following a virulent TGE virus challenge, and 75% of the 3-day-old pigs suckling either FIP or TGE-virus-vaccinated gilts survived virulent TGE virus infection in contrast to 0% survival of baby pigs suckling unvaccinated gilts.     

猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用

猪Delta冠状病毒(PDCoV)、猪流行性腹泻病毒(PEDV)和猪传染性胃肠炎病毒(TGEV)均是能引起仔猪腹泻的冠状病毒,通过临床症状和剖检很难鉴别诊断,建立同时检测且能够鉴别诊断这3种疾病的快速检测方法对于临床诊断具有重要的意义。参照GenBank中登录的PDCoV,NPEDV M和TGEV N基因的基因序列,利用Primer5.0设计合成3对能扩增特异性目的片段的引物,构建重组质粒,并对RT-PCR反应条件进行优化,建立了检测PDCoV,PEDV和TGEV的多重RT-PCR诊断方法,并对所建立的方法进行敏感性、特异性和重复性分析。利用该方法对河南176份临床样品进行检测,并与普通RT-PCR检测方法对该检测方法进行比较分析。结果表明,所建立的三重RT-PCR方法对PDCoV的最低检测量是3.14×103拷贝/μL;PEDV的最低检测量是3.88×104拷贝/μL和TGEV的最低检测量是3.68×104拷贝/μL。所建立的方法具有较好的特异性,对猪博卡病毒(PboV)、猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟(CSFV)和猪圆环病毒(PCV)等猪常见病毒的扩增结果均为阴性。应用所建立的三重RT-PCR方法对176份临床收集的仔猪腹泻样品进行PEDV,PDCoV和TGEV检测,并将检测结果和单重RT-PCR检测方法比较,结果显示,多重RT-PCR检测结果与常规单一RT-PCR的结果符合率均为100%。综上,本试验建立了能同时检测PDCoV,PEDV和TGEV的三重RT-PCR方法,为临床上这3种疫病的快速检测和流行病学调查奠定了基础。     

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.     

Evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the United States

A respiratory variant of transmissible gastroenteritis virus (TGEV), designated PRCV-Ind/89, was isolated from a swine breeding stock herd in Indiana. The virus was readily isolated from nasal swabs of pigs of different ages and induced cytopathology on primary porcine kidney cells and and on a swine testicular (ST) cell line. An 8-week-old pig infected oral/nasally with the respiratory variant and a contact pig showed no signs of respiratory or enteric disease. These pigs did not shed virus in feces but did shed the agent from the upper respiratory tract for approximately 2 weeks. Baby pigs from 2 separate litters (2 and 3 days old) also showed no clinical signs following oral/nasal inoculation with PRCV-Ind/89. In a third litter, 5 of 7 piglets (5 days old) infected either oral/nasally or by stomach tube developed a transient mild diarrhea with villous atrophy. However, virus was not isolated from rectal swabs or ileal homogenates of these piglets, and viral antigen was not detected in the ileum by fluorescent antibody staining even though the virus was easily recovered from nasal swabs and lung tissue homogenates. Swine antisera produced against PRCV-Ind/89 or enteric TGEV cross-neutralized either virus. In addition, an anti-peplomer monoclonal antibody, 4F6, that neutralizes TGEV also neutralized the PRCV-Ind/89 isolate. Radioimmunoassays with a panel of monoclonal antibodies indicated that the Indiana respiratory variant and the European PRCV are antigenically similar.     

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.     

Intestinal replication of a porcine respiratory coronavirus closely related antigenically to the enteric transmissible gastroenteritis virus

One-week-old piglets were inoculated with the porcine respiratory coronavirus (PRCV) either intravenously or directly into the lumen of the gastrointestinal tract. Both inoculation routes resulted in the isolation of virus from the caudal small intestine. Viral replication, however, was only observed upon inoculation into the digestive tract in quantities of greater than or equal to 10(3) TCID50. Replication remained limited to a few unidentified cells located in or underneath the epithelial layer at villus- or crypt-sites. Virus was excreted in the faeces for several days but infection of the respiratory tract occurred rarely in the same pigs. The results of this study indicate that small changes in molecular structure between PRCV and transmissible gastroenteritis virus have resulted in important changes in host cell tropism.     

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.     

Development of PCR-based techniques to identify porcine transmissible gastroenteritis coronavirus isolates.

Sixteen isolates of transmissible gastroenteritis virus and one isolate of porcine respiratory coronavirus were characterized using RT-PCR amplification of 4 antigenic subsites in the site A epitope on the TGEV spike gene. The PCR products were digested with restriction enzymes Sau3AI and SspI and the sizes of the fragments were determined. Three different digestion patterns were observed with each enzyme. The recognition site for Sau3AI was missing in 1 isolate, was present in 13 isolates and 3 isolates had 2 sites. PCR-products with a single site had 3 different fragment sizes and the other isolates produced 2 fragments with different sizes. The SspI recognition site was not present in 5 isolates and 12 isolates had a single site that produced 2 fragments of different sizes. Based on the restriction fragment sizes, the 17 isolates were separated into 7 groups. Direct sequencing of the 455 bp nested set fragments demonstrated greater than 96% sequence homology among the 16 isolates and 100% homology in the 4 antigenic subsites in the conserved site A epitope. The groups are discussed in relation to their sequence homology and virulence. In vitro procedures have been developed to identify several porcine enteric coronavirus isolates at the strain level.     

An ELISA for the detection of serum antibodies to both transmissible gastroenteritis virus and porcine respiratory coronavirus.

A competition ELISA utilizing a mAb directed towards a peplomer protein epitope common to TGEV, PRCV and related feline and canine coronaviruses is described.     

猪源益生菌体外抗猪传染性胃肠炎病毒作用的研究

为研究猪源益生菌抗猪传染性胃肠炎病毒(TGEV)的作用,并分析其可能的抗病毒机制,本研究在体外细胞感染猪睾丸细胞(ST)模型上,采用MTT比色法、TCID50法、间接免疫荧光法以及半定量RT-PCR法评价益生菌抗TGEV的作用.MTT试验中,在3种作用方式(益生菌预处理细胞,再接入病毒;益生菌与病毒同时接入细胞;病毒先感染细胞,再接入益生菌)下,所选益生菌对TGEV感染细胞均有抑制作用:益生菌预处理细胞后,再感染病毒,细胞存活率升高;益生菌与病毒体外相互作用后接入细胞,病毒对细胞的侵害能力下降;病毒感染细胞后再接入益生菌,益生菌抵抗TGEV感染细胞的能力相对较弱.间接免疫荧光试验和半定量RT-PCR试验分别采用益生菌预处理细胞,再接入病毒以及益生菌和病毒体外作用后同时接入细胞两种方式来检测益生菌对TGEV感染细胞的抑制作用,结果与MTT结果一致.实验结果表明所选的3株益生菌及其代谢产物均能够抑制TGEV感染细胞,但菌株间存在差异.不同的作用方式对TGEV感染细胞的抑制作用也有所差异,其中益生菌预处理组的抑制作用最强,病毒感染细胞后再用益生菌进行处理组的抑制作用最弱.