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

Abstract

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...

Pathology of US Porcine Epidemic Diarrhea Virus Strain PC21A in Gnotobiotic Pigs

“…Only coronavirus-like particles were observed in the fecal sample by electron microscopy (Figure 1). The sample was negative for rotavirus groups A and C and for transmissible gastroenteritis virus/porcine respiratory coronavirus by RT-PCR ( 7 , 8 ). …”

“…For the pig-passaged PC21A strain, RT-PCR/PCR results were negative for transmissible gastroenteritis virus/porcine respiratory coronavirus ( 7 ), rotavirus groups A–C ( 8 ), caliciviruses ( 13 , 14 ), astroviruses ( 15 ), circoviruses, enterovirus, kobuvirus, and bocavirus. For pigs 1 and 2, the detection of fecal virus shedding 24–48 h after inoculation coincided with the onset of clinical signs; for pigs 3 and 4, fecal shedding occurred before the onset of clinical signs (Table 1).…”

Development of Reverse Transcriptase PCR and Nested PCR to Detect Porcine Hemagglutinating Encephalomyelitis Virus

“…PCR is a sensitive, specific and rapid diagnostic tool to detect pathogens of diseases. PCR can detect noninfectious pathogens [13,14]. For example, formalin-fixed, paraffin-embedded tissue samples can be used for PCR [5].…”

Replication of coronavirus

“…Örneğin TGEV domuzlarda ishale sebep olmaktadır (107)(108)(109). Bunun yanı sıra, FCoV kedilerde semptomik enfeksiyonlara neden olurken az miktarda da ishale neden olmaktadır (110)(111)(112).…”