Transmissible gastroenteritis virus: plaques and a plaque neutralization test.

A plaquing system and plaque neutralization test in porcine thyroid cells were used to study different transmissible gastroenteritis isolates and hemagglutinating encephalomyelitis virus. Among transmissible gastroenteritis virus isolates, plaque size varied considerably and mixed size ranges sometimes occurred. The most recently isolated viruses produced smaller plaques than the laboratory viruses or hemagglutinating encephalomyelitis virus. All transmissible gastroenteritis virus isolates reacted in the plaque neutralization test with a transmissible gastroenteritis virus antiserum which showed no activity against hemagglutinating encephalomyelitis virus. Plaque neutralization results both from experimentally infected pigs and following a field outbreak demonstrated the reliability of this test and its greater sensitivity than the conventional tube test.     

Antiviral activity against transmissible gastroenteritis virus, and cytotoxicity, of natural porcine interferons alpha and beta.

Porcine interferon (POIFN)-alpha prepared in primed peripheral blood leukocyte cultures induced with Newcastle disease virus and POIFN-beta from PK-15 cell cultures induced with polyinosinic:polycytidylic acid were partially purified by precipitation with potassium thiocyanate and anion exchange chromatography. Mean purification factors in terms of units of POIFN per mg of protein, of 37 and 12 were obtained for POIFN-alpha and POIFN-beta respectively. In yield reduction assays in swine testis and pig kidney cell cultures, POIFN-alpha and POIFN-beta had greater antiviral activity against vesicular stomatitis virus than against transmissible gastroenteritis virus (TGEV). The antiviral effects were greater at higher concentrations of interferon (IFN), and when the IFN treatments were continued postinfection. Porcine interferon-beta showed greater antiviral activity against TGEV than POIFN-alpha, but this may have been partly due to cytotoxicity. There were no major differences in the antiviral activities of crude and partially purified IFN preparations. Both types of IFN showed antiviral activity against TGEV in yield reduction assays in porcine intestinal explant and intestinal epithelial cell cultures. Crude POIFN-beta was found to be rapidly cytotoxic, especially in porcine cells, and some fractions of partially purified POIFN-beta were also cytotoxic. The cytotoxicity of POIFN-beta was partially neutralized by antibodies against human IFN-beta, but human IFN-beta was not cytotoxic for porcine or bovine cells.     

Inner structures of some coronaviruses.

When treated with formaldehyde, Tween 80, sodium oleate and Nonidet P-40, avian infectious bronchitis virus, porcine transmissible gastroenteritis virus, neonatal calf diarrhea coronavirus, porcine hemagglutinating encephalomyelitis virus as well as the human coronavirus show similar inner structures by negative staining. The first one is an inner membranous bag. This structure could be evaginated following treatments used and does not show the characteristic projections of coronaviruses. Subsequently, the inner fold could be separated from the outer membrane at the point of junction between these two membranes. Each virus does not react in the same way to the action of the different products. The transmissible gastroenteritis virus appears more sensitive to treatments than other viruses. On the other hand, the hemagglutinating encephalomyelitis virus is the most resistant. The variable sensitivities of these viruses are not related to the type of host-cells. Also, a second internal structure, which is more dense than the viral particle, encircles partially the aperture of the internal tongue-shaped structure and seems to emerge from the viral particle through the aperture of the inner bag.     

Case-control study on the association of porcine circovirus type 2 and other swine viral pathogens with postweaning multisystemic wasting syndrome.

A field-based case-control study was conducted to assess the strength of association of porcine circovirus type 2 (PCV2) and some major swine viruses with postweaning multisystemic wasting syndrome (PMWS). Cases were defined as individual pigs with a clinical history of progressive weight loss and histopathological lesions characteristic of PMWS. Controls were pigs without clinical signs and histopathological lesions typical of PMWS. A total of 31 cases and 56 controls was identified from diagnostic submissions. Serum and various tissues were collected from all animals and assayed for PCV, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus, porcine enterovirus types 1-3, swine influenza virus, porcine respiratory coronavirus, transmissible gastroenteritis virus, porcine endogenous retrovirus, porcine lymphotropic herpesvirus type 1, and bovine viral diarrhea virus. The proportion of case and control pigs positive for each virus was determined and statistically compared for determining the strength of the association that each virus had with PMWS individually or in combinations. Porcine circovirus type 2 had the strongest association (OR = 9.3, P = 0.006) with PMWS among the viruses tested for. Risk for PWMS was much higher (OR = 31.2, P = 0.0009) if the animal was concurrently infected with PCV2 and PRRSV, suggesting that development of PMWS may be enhanced by cofactor(s). Because PCV2 was also found in 62.5% of the controls, PCV2 from 5 cases and 4 controls were selected and genetically compared. No significant genetic difference was observed between PCV2 from PMWS and control pigs.     

Antiviral and antigenic properties of recombinant porcine interferon gamma

Recombinant porcine interferon gamma (rPoIFN gamma) induced a dose-dependent inhibition of the cytopathic effect produced by vesicular stomatitis virus (VSV) challenge of both homologous and heterologous (bovine) cell lines. In addition, an antiviral effect of rPoIFN gamma was demonstrable against the coronavirus transmissible gastroenteritis virus (TGEV) infection of porcine epithelial cells and of pulmonary macrophages. A rabbit anti-PoIFN gamma antiserum was prepared and shown to specifically neutralize the antiviral effects of natural and recombinant porcine IFN gamma preparations. This antiserum could also neutralize recombinant bovine IFN gamma but not recombinant human IFN gamma. These results suggest antigenic homology of porcine and bovine IFN gamma but antigenic differences between these molecules and human IFN gamma.     

Outbreaks in Quebec pig farms of respiratory and reproductive problems associated with encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) was isolated from tissues of aborted fetuses and weaned and suckling piglets from 4 different pig farms in Quebec. The farms were experiencing reproductive failure in sows of different parities concomitant to respiratory problems in suckling and postweaning piglets. At necropsy, gross lesions were confined to the lung and consisted of pulmonary congestion and edema of various degrees. Lesions of multifocal interstitial to proliferative pneumonia were found in the lungs of these piglets. Bacteriologic examination of various tissues from necropsied pigs yielded no pathogens in most cases. No significant antibody titers against 3 swine viruses (transmissible gastroenteritis virus, porcine parvovirus, and swine influenza virus) and two bovine viruses (bovine viral diarrhea and infectious bovine rhinotracheitis viruses) were detected in the sera of convalescent pigs. The Quebec EMCV isolates were antigenically related to the reference ATCC-VR129 strain of EMCV, as demonstrated by indirect immunofluorescence, serum neutralization (SN), and Western immunoblotting. However, one of the Quebec isolates could be distinguish by SN. EMCV-specific SN antibody titers up to 1:12,800 were detected in thoracic and ascitis fluids of aborted fetuses and in sera of convalescent pigs. A possible pneumotropic EMCV variant in swine may exist.     

A serological survey of selected pathogens in wild boar in Slovenia

Serum samples collected from 178 shot wild boars (Sus scrofa) were tested for the presence of antibodies against classical swine fever virus, Aujeszky's disease virus (ADV), porcine reproductive and respiratory syndrome virus, porcine respiratory coronavirus (PRCV), transmissible gastroenteritis virus, swine influenza virus, porcine parvovirus (PPV), swine vesicular disease virus, Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae, Salmonella spp., Brucella spp. and Haemophilus parasuis (HPS) throughout Slovenia during the hunting season 2003/2004. The number of samples corresponds to 3% of the total hunting bag. By enzyme-linked immunosorbent assay (ELISA) antibodies against ADV were detected in 55 sera (31%), against PRCV in five sera (3%), PPV in 87 sera (49%), APP in 93 sera (52%), M. hyopneumoniae in 38 sera (21%), Salmonella spp. in 85 sera (47%) and HPS in 33 sera (18%).     

Porcine parvovirus: frequency of naturally occurring transplacental infection and viral contamination of fetal porcine kidney cell cultures.

The frequency of naturally occurring transplacental infection of swine with porcine parvovirus (PPV) and one of the possible consequences of such infection--the presence of PPV in cell cultures prepared from fetal tissues--were investigated. Transplacental infection was indicated by the presence of high titers of hemagglutination inhibiting (HI) antibody for PPV in serums of 0-day-old, hysterectomy-derived, colostrum-deprived pigs of 3 of 82 litters. All letters were farm-raised dams. Moreover, cell cultures prepared from 3 of 49 lots of fetal porcine kidneys (FPK) collected from an abattoir during an interval of 14 months were found contaminated with PPV. Because each lot was usually comprised of kidneys from 2 litters, the latter finding suggests that 3 of approximately 98 litters were infected. Prior infection of FPK cell cultures with PPV resulted in only slight interference of replication of other selected viruses; i.e., porcine enterovirus (PEV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), and hemagglutinating encephalomyelitis virus (HEV). Moreover, PPV and HEV were propagated in the same cell cultures during 5 serial passages of the viruses. In contrast, when copropagation of PPV and VSV was attempted, PPV was not detected after the 2nd serial passage.     

Effect of recombinant DNA-derived bovine alpha-1 interferon on transmissible gastroenteritis virus infection in swine

Recombinant DNA-derived bovine interferon alpha 1-1 (BoIFN) inhibited replication of both vesicular stomatitis virus and transmissible gastroenteritis virus in cultures of swine testicular cells. Newborn pigs were orally inoculated with BoIFN and subsequently had interferon in their gastric contents and serum; however, interferon was found only occasionally in intestinal washings. Incubation of BoIFN with gastric contents from a newborn suckling pig did not affect antiviral activity, whereas intestinal (small intestine) contents from the same animal inactivated BoIFN within 1 minute. Beginning at 6 hours of age, newborn, colostrum-deprived pigs were given 1 mg of BoIFN orally every 12 hours. These pigs were not protected against challenge exposure to virulent transmissible gastroenteritis virus at 48 hours of age; disease and mortality were similar for these pigs and for control pigs not given BoIFN prior to challenge exposure. The BoIFN did not impair growth rate of pigs and did not cause obvious disease or lesions.     

Swine Interferon I. Induction in Porcine Cell Cultures with Viral and Synthetic Inducers

The production of interferon by porcine kidney (PK₁₅) cell culture in response to viral and synthetic inducers was studied. The inducers used included a synthetic double-stranded polyribonucleotide, polyriboinosinic-polyribocytidylic acid (Poly I:C), swine influenza virus and three strains of pseudorabies virus. Following exposure to these inducers cell culture fluids were examined for interferon by the plaque-reduction method.

The Poly I:C and the swine influenza virus induced production of interferon by PK₁₅ cell cultures, whereas, all three strains of pseudorabies virus at the two concentrations tested failed to induce production of interferon in vitro.

The antiviral substance produced in PK₁₅ cells was identified as an interferon because it was pH stable, non-dialyzable, sensitive to trypsin, non-sedimentable, relatively heat stable, host-species specific and it possessed broad-spectrum antiviral activity. The latter was demonstrated by inhibition of vesicular stomatitis, vaccinia and pseudorabies viruses. Differences in interferon activity against the different viruses were observed.

     

猪6种常见病毒多重PCR检测方法的建立及应用

旨在建立一种可同时检测猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)、猪细小病毒(PPV)和猪巨细胞病毒(PCMV)的多重PCR检测方法。参考相关文献及序列比对结果设计6对特异性引物,建立了可同时检测以上6种病毒的多重PCR方法并应用此方法对临床病例进行了检测。结果表明,所建立的多重PCR方法灵敏度高,对6种病毒的最低核酸检测量分别为12.8pg(PRRSV)、46pg(CSFV)、16pg(PRV)、23.5pg(PCV-2)、72pg(PPV)、8.6pg(PCMV),对猪流感病毒(SIV)、猪乙型脑炎病毒(JEV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)无特异性扩增。应用该方法对临床145份样品进行检测,总阳性率为83.45%,2种以上病毒混合感染阳性率为69.66%。说明所建立的多重PCR检测方法敏感,可用于猪群中上述6种猪病病原的单一感染或混合感染的鉴别诊断。     

Swine Interferon II. Induction in Pigs with Viral and Synthetic Inducers

The production of interferon by pigs in response to viral and synthetic inducers was studied. The inducers used included polyriboinosinic-polyribocytidylic acid (Poly I:C), swine influenza virus and pseudorabies virus. Following intravenous inoculation of pigs with the inducers, sera were examined for interferon by the plaque-reduction method in porcine kidney (PK₁₅) cell cultures using vesicular stomatitis virus as the challenge inoculum. It was shown that pigs can produce interferon in response to each of these inducers. The pseudorabies virus used in this investigation was found to be a better interferon inducer than the swine influenza virus.

The interferon produced in pigs was identified as an interferon because it was pH stable, non-dialyzable, sensitive to trypsin, non-sedimentable and possessed broad-spectrum antiviral activity as well as host-species specificity.

     

Enzyme linked immunoassay and fluorescent antibody techniques in the diagnosis of viral diseases using staphylococcal protein-A instead of anti-gamma-globulins

Staphylococcal protein-A (SpA) is known to interact with the crystallizable fragment (Fc) of IgG molecules from several species. In the present study, SpA coupled to either fluorescein isothiocyanate (FITC) or peroxidase was used in place of antisera to IgG for the fluorescent antibody (FA) techniques and the enzyme linked immunoassay (ELISA). The SpA conjugates produced low background staining when applied in these techniques, and provide a rapid, highly specific and sensitive means for the identification of viral isolates and the detection of serum antibodies. Moreover, SpA is a single reagent that replaces various preparations of anti-gamma globulin against many species. SpA-FITC conjugate was successfully applied for the identification of pseudorabies virus, hog cholera virus, swine vesicular disease virus, transmissible gastroenteritis virus, porcine parvovirus and porcine enteroviruses. Antibody titers against the mentioned viruses could be determined semi-quantitatively in the indirect FA test with SpA-FITC. In our laboratory the ELISA became a routinely practicable serological test for the detection of antibodies only after we introduced SpA-peroxidase as a marker for the IgG.     

基于SYBR Ⅰ实时荧光定量PCR对猪流行性腹泻病毒野毒和疫苗弱毒鉴别诊断方法的建立

本研究参考GenBank中登录的猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)野毒株和弱毒疫苗株(CV777弱毒疫苗株)在高度保守ORF3基因核苷酸序列的差异,设计一对特异性荧光定量引物,分别建立基于SYBRⅠ实时荧光定量RT-PCR方法。结合熔解曲线分析可见,其野毒株和弱毒疫苗株熔解温度(Tm)分别为(81.84±0.17)℃和(83.16±0.14)℃,扩增产物的熔解曲线分析均只出现1个单特异峰,对传染性胃肠炎病毒、猪轮状病毒、猪细小病毒、猪流感病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病毒、猪瘟病毒均检测不到荧光信号。结合熔解曲线可直接鉴别猪群中PEDV的感染情况和程度,可对免疫猪群PEDV野毒感染和疫苗免疫做出快速准确的鉴别诊断,尤其是对PEDV弱毒疫苗免疫后仍爆发PEDV野毒感染的研究更有临床意义。     

Detection of turkey enteric coronavirus by enzyme-linked immunosorbent assay and differentiation from other coronaviruses

A double-antibody ELISA for the detection of coronaviruses in intestinal contents from turkey poults with diarrhea was developed. Antibodies were raised in rabbits and guinea pigs against a Minnesota isolate of turkey enteric coronavirus (TCV) propagated in embryonating turkey eggs and were purified by density-gradient centrifugation. The specificity of antisera was confirmed by hemagglutination-inhibition and immunoelectron microscopy. Absorption of anti-TCV hyperimmune sera with egg extracts or egg ovalbumin and the use of different dilution and blocking buffers influenced the sensitivity and specificity of the ELISA. Reciprocal cross-reactivity was detected among turkey, chicken, bovine, and murine coronaviruses. Antisera to the transmissible gastroenteritis virus of swine, the rabbit enteric coronavirus, or the human coronavirus strain 299E failed to react with TCV. The TCV cross-reacted only moderately with the avian infectious bronchitis virus and the hemagglutinating encephalomyelitis virus of swine. Investigations with samples from 47 commercial turkey flocks in Quebec with episodes of transmissible enteritis revealed that the ELISA was more sensitive than was electron microscopy for detection of TCV.     

Serologic survey of viral antibodies in the Peruvian alpaca (Lama pacos)

Sera from more than 100 alpacas (Lama pacos) from the Peruvian southern sierra were examined for antibodies to 8 viruses known to infect other domestic animals. On the basis of these serologic findings and previously published serologic or clinical data, it is now known that the alpaca can be infected with the following viruses: parainfluenza-3, bovine respiratory syncytial virus, bovine herpesvirus-1, bluetongue virus, border disease virus, influenza A virus, rotavirus, rabies virus, vesicular stomatitis virus, foot-and-mouth disease virus, and contagious ecthyma virus.     

Viral susceptibility of a cell line derived from the pig oviduct.

Seventeen of 24 RNA viruses and eight of nine DNA viruses replicated in a cell line derived from a pig fallopian tube. The following RNA viruses grew poorly in it: the virus of transmissible gastroenteritis of pig and the swine-influenza, Sendai and bovine para-influenza type 3 viruses. Among other RNA viruses an untyped swine para-myxovirus and some picornaviruses, rhabdoviruses and togaviruses attained high titers and produced an extensive cytopathic effect. Among the DNA viruses a porcine adeno, equine rhinopneumonitis, infectious bovine rhinotraceheitis, pseudorabies and porcine cytomegalo viruses replicated in pig fallopian tube cells as well as in other cells generally used to grow them.     

A serodiagnostic ELISA using recombinant antigen of swine transmissible gastroenteritis virus nucleoprotein.

A serodiagnostic ELISA utilizing the recombinant nucleoprotein (rN protein) of transmissible gastroenteritis virus (TGEV) was developed, and evaluated by examining a panel of 141 virus neutralization (VN) positive and 101 negative sera. The rN protein-based ELISA (rnELISA) appeared to be highly sensitive and specific (98.6% and 98.0%, respectively) when it was compared to the VN test. The result was similar to that of an ELISA based on purified viral antigens with showing good correlation (R=0.829). No cross-reaction was detected with antisera against porcine epidemic diarrhea virus, hog cholera virus, type A rotavirus, pseudorabies virus and swine vesicular disease virus in this ELISA. The rnELISA can be an alternative for the diagnosis of TGE with a great advantage in antigen preparation.     

Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals

Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.     

猪腹泻相关肠道病毒分离研究进展

猪腹泻是全球养猪生产中最常见的疾病之一,病毒性腹泻具有传播快、致死率高和防控难的特点。常见的引起仔猪腹泻的病毒包括猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV),近几年新发现的猪δ冠状病毒(PDCoV)、猪嵴病毒(PKV)和猪急性腹泻综合征冠状病毒(SADS-CoV)也与仔猪腹泻相关。病毒分离是病毒感染诊断的金标准,也是进行病毒研究的前提。对上述6种常见猪肠道病毒的分离进展进行综述,可为掌握这些病毒的分离情况、疾病发展动向监测、疫苗研发以及猪腹泻病的防控提供参考。