In situ hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in formalin-fixed paraffin-embedded tissues

Sirinarumitr, Theerapol; Paul, P. S.; Kluge, John P.; Halbur, Patrick G.

doi:10.1016/0166-0934(95)01901-4

Cited by 29 publications

(29 citation statements)

References 24 publications

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“…PRCV was detected in several types of lung cells (bronchial and bronchiolar epithelial cells, type I and II pneumocytes, alveolar and septal macrophages) using antigen detection methods [35,36,92,114] and different experimental studies yielded conflicting data as to the main virus target cell. In studies with Belgian PRCV isolates, cells of the alveolar epithelia and septa were predominantly infected [35,36], while US isolates were shown to replicate mainly in bronchiolar epithelial cells [114]. Nasal virus excretion continues until 8-9 DPI [21,72,138,139] suggesting a similar duration of virus replication in the lungs.…”

Section: Porcine Respiratory Coronavirusmentioning

confidence: 99%

“…High amounts of virus are isolated from the lungs (10 7.5 -10 8.3 50% tissue culture infectious doses (TCID 50 )/g tissue) and from nasal swabs (10 6.5 -10 7.3 TCID 50 /100 mg secretions) during the first week after inoculation [35,36,139]. PRCV was detected in several types of lung cells (bronchial and bronchiolar epithelial cells, type I and II pneumocytes, alveolar and septal macrophages) using antigen detection methods [35,36,92,114] and different experimental studies yielded conflicting data as to the main virus target cell. In studies with Belgian PRCV isolates, cells of the alveolar epithelia and septa were predominantly infected [35,36], while US isolates were shown to replicate mainly in bronchiolar epithelial cells [114].…”

Section: Porcine Respiratory Coronavirusmentioning

confidence: 99%

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Proinflammatory cytokines and viral respiratory disease in pigs

2000

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-Swine influenza virus (SIV), porcine respiratory coronavirus (PRCV) and porcine reproductive and respiratory syndrome virus (PRRSV) are enzootic viruses causing pulmonary infections in pigs. The first part of this review concentrates on known clinical and pathogenetic features of these infections. SIV is a primary respiratory pathogen; PRCV and PRRSV, on the contrary, tend to cause subclinical infections if uncomplicated but they appear to be important contributors to multifactorial respiratory diseases. The exact mechanisms whereby these viruses cause symptoms and pathology, however, remain unresolved. Classical studies of pathogenesis have revealed different lung cell tropisms and replication kinetics for each of these viruses and they suggest the involvement of different lung inflammatory responses or mediators. The proinflammatory cytokines interferon-α (IFN-α), tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1) have been shown to play key roles in several respiratory disease conditions. The biological effects of these cytokines and their involvement in human viral respiratory disease are discussed in the second part of this review. The third part summarises studies that were recently undertaken in the authors' laboratory to investigate the relationship between respiratory disease in pigs and bioactive lung lavage levels of IFN-α, TNF-α and IL-1 during single and combined infections with the above viruses. In single SIV infections, typical signs of swine "flu" were tightly correlated with an excessive and coordinate production of the 3 cytokines examined. PRCV or PRRSV infections, in contrast, were subclinical and did not induce production of all 3 cytokines. Combined infections with these 2 subclinical respiratory viruses failed to potentiate disease or cytokine production. After combined inoculation with PRCV followed by bacterial lipopolysaccharide, both clinical respiratory disease and TNF-α/IL-1 production were markedly more severe than those associated with the respective single inoculations. Taken together, these data are the first to demonstrate that proinflammatory cytokines can be important mediators of viral respiratory diseases in pigs. respiratory coronavirus, PRCV), et le virus du syndrome dysgénésique et respiratoire porcin (porcine reproductive and respiratory syndrome virus, PRRSV) sont des virus enzootiques provoquant des infections pulmonaires chez le porc. Les caractéristiques cliniques et pathogénétiques de ces infections sont données dans la première partie de cette revue. Le SIV est un agent pathogène respiratoire primaire ; les PRCV et PRRSV, en revanche, ne causent que des infections subcliniques en l'absence de complications, mais ils contribuent de manière importante aux maladies respiratoires multifactorielles. Cependant, les mécanismes précis par lesquels ces virus provoquent des symptômes et une pathologie demeurent inconnus. Les études classiques de pathogenèse ont révélé un tropisme pour les cellules pulmonaires et des cinétiques de réplication différents pour...

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Section: Porcine Respiratory Coronavirusmentioning

confidence: 99%

Section: Porcine Respiratory Coronavirusmentioning

confidence: 99%

Proinflammatory cytokines and viral respiratory disease in pigs

2000

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“…7 PRCV causes infected swine to be diagnosed as TGEV positive in conventional serologic tests. 8 Several investigators have described the use of molecular assays to detect and differentiate TGEV/PRCV strains including reverse transcription-polymerase chain reaction (RT-PCR), 5 cDNA probes, 12,13 in situ hybridization, 10 and RT-PCR/restriction fragment length polymorphism. 2 To differentiate TGEV/PRCV with reference virus strains from tissue culture, an RT-PCR assay was developed with primers targeted to the S gene deletion.…”

mentioning

confidence: 99%

Development of a Reverse Transcription-Nested Polymerase Chain Reaction Assay for Differential Diagnosis of Transmissible Gastroenteritis Virus and Porcine Respiratory Coronavirus from Feces and Nasal Swabs of Infected Pigs

et al. 2000

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Abstract. Transmissible gastroenteritis virus (TGEV), a coronavirus, replicates in intestinal enterocytes and causes diarrhea in young pigs. Porcine respiratory coronavirus (PRCV), a spike (S) gene natural deletion mutant of TGEV, has a respiratory tissue tropism and causes mild or subclinical respiratory infections. Conventional antigen-based diagnostic tests fail to differentiate TGEV and PRCV, and a blocking ELISA test to serologically differentiate TGEV/PRCV-infected pigs is conducted on convalescent serum retrospectively after disease outbreaks. A reverse transcription (RT)-nested polymerase chain reaction (PCR) with primers targeted to the S gene deletion region to differentiate TGEV/PRCV was developed. The specificity of the RT-nested PCR was confirmed with reference and recent field strains of TGEV/PRCV, and its sensitivity was analyzed by testing nasal and fecal samples collected from pigs at various days postinoculation (DPI) with TGEV or PRCV. Specific PCR products for TGEV/PRCV were detected only with the homologous reference or field coronaviruses and for 10-14 DPI of pigs with TGEV (feces) or PRCV (nasal samples). The RT-nested PCR assay was more sensitive than antigen-based assays on the basis of duration of virus detection in experimentally infected pigs and was directly applicable to nasal as well as fecal specimens from the field.Transmissible gastroenteritis virus (TGEV) is a member of the Coronaviridae family and is enveloped with a positive-stranded RNA genome. 3,7 Porcine respiratory coronavirus (PRCV) represents a natural deletion mutant of TGEV that appeared in 1983-1984 in Europe and in 1988 in the US. 3 Coronaviruses have 3 major structural proteins: the spike (S), the integral membrane glycoprotein, and the nucleocapsid protein. 3 TGEV replicates primarily in small intestinal enterocytes, whereas PRCV replicates predominantly in the respiratory tract. 3,7 According to sequence comparisons of PRCV and TGEV, PRCV has a large deletion in the 5Ј region of the S gene and minor deletions in genes 3 and 3-1. 3,11 These deletions are thought to influence the viral tissue tropism and virulence. The deletion size in the S gene ranges from 621 to 681 bp depending on the origin of the strain. 11 Recently, strains of TGEV with reduced enteropathogenicity were reported in the field. 6 A similar suspect TGEV outbreak of reduced virulence (mild diarrhea and intestinal lesions, slow disease spread among pigs) in nursery pigs from a swine herd in the US Midwest was investigated. Diagnosis of TGEV in these pigs was sporadic and inconsistent and presumably complicated by the presence of antibodies to PRCV confirmed by a blocking differential ELISA test on sera from a number of pigs in this herd (L. J. Saif and P. Lewis, unpublished). However, this latter test showed inconsistent results for TGEV/PRCV differentiation with serially collected samples from the same pigs within the herd (inconsistent individual immune status), and some pigs in the Received for publication May 20, 1999. herd tested only PRCV...

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“…2,3,5,[10][11][12]16,24,25,29,31 In general, these methods are based on antigenic or genomic differences between TGEV and PRCV S proteins/genes. Blocking or competition ELISAs have been used by several authors, with TGEV as the coating antigen.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of the Baculovirus-Expressed S Glycoprotein of Transmissible Gastroenteritis Virus (TGEV) as Antigen in a Competition ELISA to Differentiate Porcine Respiratory Coronavirus from TGEV Antibodies in Pigs

Sestak¹,

Zhou²,

Shoup³

et al. 1999

J VET Diagn Invest

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Abstract. The spike (S) glycoprotein of the Miller strain of transmissible gastroenteritis virus (TGEV) was recently cloned and expressed in baculovirus. The recombinant S protein was used as the coating antigen in a competition (blocking) enzyme-linked immunosorbent assay (ELISA) in combination with monoclonal antibodies to the S protein epitope A (conserved on TGEV and porcine respiratory coronavirus [PRCV]) or epitope D (present on TGEV only) to differentiate PRCV-from TGEV-induced antibodies. One set (set A) of 125 serum samples were collected at different times after inoculation of caesarean-derived, colostrum-deprived (n ϭ 52) and conventional young pigs (n ϭ 73) with 1 of the 2 porcine coronaviruses or uninoculated negative controls (TGEV/PRCV/negative ϭ 75/30/20). A second set (set B) of 63 serum samples originated from adult sows inoculated with PRCV and the recombinant TGEV S protein or with mock-protein control and then exposed to virulent TGEV after challenge of their litters. Sera from set A were used to assess the accuracy indicators (sensitivity, specificity, accuracy) of the fixed-cell blocking ELISA, which uses swine testicular cells infected with the M6 strain of TGEV as the antigen source (ELISA 1) and the newly developed ELISA based on the recombinant S protein as antigen (ELISA 2). The sera from set B (adults) were tested for comparison. The plaque reduction virus neutralization test was used as a confirmatory test for the presence of antibodies to TGEV/PRCV in the test sera. The accuracy indicators for both ELISAs suggest that differential diagnosis can be of practical use at least 3 weeks after inoculation by testing the dual (acute/convalescent) samples from each individual in conjunction with another confirmatory (virus neutralization) antibody assay to provide valid and complete differentiation information. Moreover, whereas ELISA 1 had 10-20% false positive results to epitope D for PRCV-infected pigs (set A samples), no false-positive results to epitope D occurred using ELISA 2, indicating its greater specificity. The progression of seroresponses to the TGEV S protein epitopes A or D, as measured by the 2 ELISAs, was similar for both sets (A and B) of samples. Differentiation between TGEV and PRCV antibodies (based on seroresponses to epitope D) was consistently measured after the third week of inoculation.Transmissible gastroenteritis virus (TGEV), a prominent cause of neonatal diarrhea, causes death in 90-100% of seronegative pigs under 2 weeks of age and costs the US swine industry nearly $200 million/ year. 15,19 Surveys of TGEV antibodies in swine sera collected prior to the detection of porcine respiratory coronavirus (PRCV) indicated that 19-54% of swine herds in North America have antibodies, 17 but few differential serosurveys have been done since the detection of PRCV. 32 A variant of TGEV, referred to as PRCV, was isolated from pigs in Europe in 1986 and identified in the USA in 1989. 15,18,19 As reported from Iowa swine herds, a recent increase observed in TGEV/PRCV seropre...

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In situ hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in formalin-fixed paraffin-embedded tissues

Cited by 29 publications

References 24 publications

Proinflammatory cytokines and viral respiratory disease in pigs

Proinflammatory cytokines and viral respiratory disease in pigs

Development of a Reverse Transcription-Nested Polymerase Chain Reaction Assay for Differential Diagnosis of Transmissible Gastroenteritis Virus and Porcine Respiratory Coronavirus from Feces and Nasal Swabs of Infected Pigs

Evaluation of the Baculovirus-Expressed S Glycoprotein of Transmissible Gastroenteritis Virus (TGEV) as Antigen in a Competition ELISA to Differentiate Porcine Respiratory Coronavirus from TGEV Antibodies in Pigs

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