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

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 59 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 From the Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691 (Kim, Chang, Parwani, Saif), and the School of Veterinary Medicine, Tufts University, North Grafton, MA 01536-1895 (Sestak). Received for publication May 20, 1999. herd tested only PRCV positive, whereas others were TGEV positive (inconsistent herd immune status). These new TGEV strains may represent naturally occurring recombinants with reduced virulence between TGEV and PRCV strains, or the presence of PRCV antibodies in these herds may have complicated the diagnosis and modulated the severity of conventional TGEV infections. TGEV is a major cause of neonatal diarrhea and also causes enzootic diarrhea in older pigs.7 It costs the swine industry in the US nearly $200 million a year.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.5 These investigators used restriction endonuclease analysis to confirm the identity of their RT-PCR products. Use of the RT-nested PCR assay for detection and differentiation of TGEV/PRCV directly from nasal swabs or feces has not been reported. Therefore, the objective of this study was to develop and use RT-nested PCR assays to detect and differentiate TGEV/PRCV directly from fecal and nasal swab specimens from experimentally infected pigs and from field outbreak specimens. Four field samples were obtained from a midwest swine herd with sporadic diarrhea cases in nursery pigs. The BW 021898B sample consisted of intestinal contents from a nursery pig with mild diarrhea (clinically suspect for transmissible gastroenteritis). Three nasal swab samples (BW126, BW154, and BW155) were obtained from normal TGEVseronegative sentinel nursery pigs placed in contact with the diarrheic pigs in the same nursery. Swine testicular (ST) cells were used for virus isolation, growth, and cell culture 386 Brief communications Table 1. Reference and field TGEV and PRCV strains. Isolate Isolation date Location P no. (PP)* Source TGEV reference strains M5C Miller M6 Miller P115 Purdue 1965 1965 1952 Ohio Ohio Indiana 2 (2) 6 (2) 115 E. Bohl, OARDC,† Wooster, OH L. J. Saif, OARDC, Wooster, OH E. Bohl, OARDC, Wooster, OH PRCV reference strains ISU-1 ISU-3 199