key: cord-315780-uhi66unn
authors: Paton, David; Ibata, Georgina; Sands, Jenny; McGoldrick, Adrian
title: Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus
date: 1997-07-31
journal: Journal of Virological Methods
DOI: 10.1016/s0166-0934(97)00055-4
sha: 
doc_id: 315780
cord_uid: uhi66unn

Abstract An RT-PCR method was developed that amplified genetic material from the 5′ end of the S protein gene of both transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), but discriminated between the two by the size of the product generated. A number of restriction endonuclease enzymes were assessed for recognition of the amplicons so produced. The assay was shown to detect viral RNA from all of the 26 different TGEV and PRCV isolates examined, covering a period from 1946 to 1996. Detection of TGEV in clinical specimens was possible using a spin column method to extract RNA and sensitivity was compared to virus isolation and antigen detection ELISA. The method could provide a means of confirming positive results from immunological screening tests such as FAT and ELISA, reducing the need for virus isolation and convalescent serology.

(TGE) is a highly contagious pig disease that has been reported from many parts of the world including America, Europe and Asia. The causative virus, TGEV, is a member of the coronaviridae, and has a large, single-strand, positive sense RNA genome. Since the mid 198Os, a variant respiratory form of the TGE virus known as porcine respiratory coronavirus (PRO, has become common in pigs in Europe and has more recently been reported from North America and Asia (Pensaert et al., 1986; Wesley et al., 1990) . It generally causes mild disease under experimental conditions. In Europe, the emergence of PRCV has been associated with a reduction in the incidence and severity of cases 0166-0934/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PZI SO166-0934(97)00055-4 of TGE. Compared to TGEV, PRCV has a deletion of between 672 and 681 nucleotides near the 5' end of the S gene, resulting in the loss of some antigenic sites on the S protein (Laude et al., 1993) , and of sialic acid binding activity (Schultze et al., 1996) .

Rapid methods for the detection of TGEV are important because of the highly contagious nature of the disease. Since the virus is often difficult to adapt to growth in cell cultures, the most widely used techniques are immunological, principally a fluorescent antibody test (FAT) for cryostat sections of intestine and an antigen ELISA for detection of virus in faeces (Paton, 1992) . Differentiation of TGEV from the closely related PRCV is possible using anti-S protein monoclonal antibodies (mAbs) directed against non-neutralising antigenic sites that are lacking in PRCV (Garwes et al., 1988; Callebaut et al., 1988) . There are also reports of the use of DNA probes and of RT-PCR as detectors of TGEV RNA (Bae et al., 1991; Vaughn et al., 1996; Wesley et al., 1991; Jackwood et al., 1995) but the methods were not shown to be suitable for the direct detection of virus in clinical samples. An in situ hybridization method for detection of TGEV and PRCV has been reported, but requires tissues rather than faeces (Sirinarumitr et al., 1996) . Here we report the development of an RT-PCR method for the detection and differentiation of TGEV/PRCV. A total of 25 archived viruses were examined in this study, mainly European, but also including isolates from North America and Japan (Table 1) . The viruses had been passaged to varying extents ( > 60 in some cases) in a variety of cell cultures. To prepare stocks for this study, viruses were passaged in a pig kidney cell line (LLCPKl, Flow), maintained in modified Eagle's medium with 10% foetal calf serum.

Samples used for RT-PCR were extracted from either cell lysates or cell culture supernatants. Uninfected cell controls were always processed concomitantly. In the case of cell lysates, cultures were processed at 7-36 h post infection or mockinfection, depending on the extent of virus growth, and total RNA was extracted by the acid-phenol method (Stallcup and Washington, 1983) . For cell culture supernatants, RNA was recovered using commercially available spin columns (QIAamp HCV kits, Qiagen).

A pair of oligonucleotide primers was designed to amplify the 5' end of the gene encoding the S protein of TGEV/PRCV. The target region straddles a large deletion (672-681 nucleotides) found exclusively in isolates of PRCV, but not TGEV. The forward primer (F1121, 5'-TATTTGTG-GTYTTGGTYGTAATGC)

is equivalent to nucleotide 1 l-34 of the S protein gene of TGEV, and the reverse primer (R1122, 5'-GGCTGT'ITG-GTAACTAATTTRCCA)

is complementary to nucleotides 896-873. The primers were chosen by analysis of an alignment of TGEV/PRCV sequences available in GenBank. The predicted size of the amplified product is 886 bp for TGEV and 205-214 bp for PRCV.

Reverse transcription was in 20 ~1 volumes, using 2 ~1 of sample, 0.5 ~1 of random hexamers (Pharmacia, 50 pmol), 1 ~1 of dNTPs (40 mM), 4 ,ul of 5X RT buffer (Promega), 0.5 ,ul of RNAsin (20 U, Promega) and 0.5 ,ul of M-MLV reverse transcriptase (100 U, Promega). Samples were incubated at 37°C for 30 min and then at 95°C for 5 min, before cooling to 4°C. PCR was in 100,ul volumes, using 10 ~1 of 10 x buffer (500 mM KCl, 100 mM Tris-Cl pH 8.3, 15 mM MgCl,, 0.1% gelatin), 1 ~1 dNTPs (40 mM), 1 ~1 Triton Xl00 (lo%), 2 ~1 cDNA, 0.5 ~1 of each primer (50 PM) and 0.5 ~1 of TAq Polymerase (2.5 U, Promega). The temperature profile was 32 cycles of 45 s at 95°C 1 min at 50°C and 2 min at 72°C. There was then a final extension time of 5 min at 72°C.

A 5 ~1 aliquot of each PCR product was visualised by agarose gel electrophoresis (1% agarose, 100 volts for 30 min, 0.8 pug/ml ethidium bromide included in gel) and subsequent U.V. transillumination. An RT-PCR product of the size expected for TGEV or PRCV was amplified from total RNA extracted from cells infected with each of the viruses examined in this way (Fig. 1 ). The large difference in the size of amplicon for TGEV and PRCV is obvious. The slightly larger size of amplicon obtained with American PRCV isolates, compared to European ones was also evident if the amplicons were run on a polyacrylamide gel (data not shown). (Wesley et al., 1990) *Accession numbers at Bmo Collection of Animal Pathogenic Microorganisms (original strain designation in parenthesis). tCVL, Central Veterinary Laboratory (the authors' laboratory). @not known.

The sensitivity of the RT-PCR for virus detection in culture fluids was compared with cell culture virus isolation. Virus titres were calculated as 50% tissue culture infectious doses (TCID,,) by titrating virus stock:s in tenfold steps, and assaying for cell culture infectivity by viral cytopathic effect (cpe) and immunostaining of cultures fixed in 20% acteone (Holnn Jensen, 1981) . Immunostaining used a polyclonal antiserum against TGEV 64-216, a rabbit anti-porcine peroxidase conjugate (Dako) and the substrate 3-amino 9-ethyl carbazole. The supernatant fluids from cell cultures infected with TGEV strains 70-772 and Slagharen had cell culture infectivity titres of 104.75 and 102.25 per 50 ~1, whilst with RT-PCR a visible band was detectable from samples diluted to 10W4 and 10 -* respectively.

The technique's application to clinical samples was evaluated on specimens obtained following investigation of a pig herd that showed typical signs of epizootic TGE (Jones and Paton, 1996) . The TGE virus isolated from the case was designated 96-1933. Two faecal samples and three samples of small intestine were obtained from three-week-old pigs with diarrhoea. A sample of intestine was homogenised in PBS and fed to four five-day-old piglets held in isolation and fed on milk replacer. Profuse diarrhoea began within 18 h in all pigs and 8 faeces samples were collected over the next 6 h, after which the pigs were euthanased. Faeces samples from the field case and the experimentally infected pigs were all TGEV positive in a routinely used ELISA (Paton, 1992) . The dilution at which TGEV could be detected in ELISA was between 10W2 and 10e4. FAT on cryostat sections of intestine, using a commercial source of TGEV antiserum (VMRD, Inc.), confirmed TGEV antigen in two out of three of the field samples and in all four pigs inoculated experimentally. Clarified, 10% homogenates of intestine and faeces from both the field outbreak and the subsequent experimentallyinduced cases were used to inoculate cell cultures for attempted virus isolation as previously described (Paton, 1992) . Cell cultures used included secondary pig kidney, LLCPKl , secondary canine kidney and a canine rectal tumour cell line (A72). In each case, except for the secondary pig kidney cultures, virus isolation was attempted with and without added trypsin (10 pg/ml, with daily medium renewal). The 96-1933 isolate grew very poorly in the cell types assessed. No cpe was evident in any culture after four blind passages, with or without added trypsin, but immunostaining revealed a small number of infected plaques in cells within the A72 and LLCPKl cultures. Tenfold dilution of the inoculum from the third cell culture pass lead to complete loss of detectable infectivity at the fourth passage.

For RT-PCR, samples of faeces were diluted 1 in 10 in a disruption buffer containing 500 mM Tris-Cl, pH 8.3, with 2% PVP-40, 1% PEG 6000, 140 mM NaCl, and 0.05% Tween 20 (Rowhani et al., 1995) , vortexed and left to stand at room temperature for 10 min. Earlier experiments had shown an increase in sensitivity of up to ten-fold, when disruption buffer was used instead of PBS (data not shown). The suspensions were clarified by centrifugation at 2000 g for 2 min and 140 ~1 of supernatant was used in QIAamp spin-column kits, according to the manufacturer's recommendation. Ten per cent intestinal homogenates in PBS plus antibiotics were clarified by centrifugation at 3000 g for 10 min. Thereafter, 140 ~1 aliquots were processed in QIAamp kits as above. All of the faeces samples were positive by RT-PCR, and the same two out of three pigs from the source outbreak for 96-1933 were positive for TGEV using both FAT on cryostat sections of intestine and RT-PCR on gut homogenates. To assess the sensitivity of the RT-PCR, three positive faeces samples were diluted in ten fold steps in disruption buffer, prior to extraction of RNA. The dilutions at which TGEV could be detected in the three samples were lo-', 10W5, and 10W6 for RT-PCR, compared to 10W2, 10e4, and 10e2 respectively for ELISA.

To confirm the authenticity of the amplified products restriction endonuclease (RE) analysis was assessed using each of three enzymes (HueIII, HinjI and Suu3AI) for TGEV and the enzyme Sau3AI for PRCV. These enzymes were predicted to cut the amplified region of the Purdue strain of TGEV. The DNA was extracted from a 50-~1 aliquot of each PCR reaction using Wizard prep columns (Promega) and a one tenth volume of this was digested for 1 h in a 20-~1 volume using the manufacturer's recommended digestion buffer (Promega). Half of the digest was then examined by agarose electrop:horesis, as above, except using 2% high resolution agarose (Metaphor, Flowgen) and electrophoresis at 80 volts for 45 min. The restriction enzyme AYaeIII produced the predicted cut into two fragments of approximately 520 and 380 bp for all TGElV isolates (Fig. 2) . The other enzymes did not g:ve a consistent pattern (data not shown). HinJl gave extra bands with two isolates (83-3289 and 96-1933) , whilst Sau3AI gave extra bands with 96-1933 and did not cut 84-3658, nor the PRCV isolate Indiana 89.

Isolation of TGEV in cell cultures is a slow and unreliable diagnostic method. The process may require multiple blind passages to be made before characteristic cpe is evident, although this period may be somewhat shortened by immunostaining of the cell monolayer with TGEV-specific reagents. It has been observed that some isolates are more readily adapted to cell culture than others and in a large proportion of cases, success is very elusive (Vaughn and Paul, 1993 tious virus was present. Attempts to improve yield by trypsin treatment, using a method modified from that of Honda et al. (1990) had no beneficial effect.

To avoid the difficulties of virus isolation in routine diagnosis, immunoassays such as FAT and ELISA have been developed for the detection of TGEV antigens in gut sections or faeces (Pensaert et al., 1968; Bernard et al., 1986; Van Nieuwstadt et al., 1988) . However, in many European countries, TGEV has been either absent or present at very low levels in the last ten years. In such circumstances, a positive diagnosis of TGEV becomes more significant, and it may be necessary to confirm diagnoses made by antigen detection tests using an independent assay procedure. Serology is an option, but suffers from two disadvantages. Firstly, rapid diagnosis may not be possible, due to the need to allow time for convalescence and antibody development. Secondly, it usually requires a repeat visit to the farm to be made, for the appropriate samples to be collected. There are reports of the development of nucleic acid probes for detection of TGEV RNA (Bae et al., 1991; Vaughn et al., 1996; Wesley et al., 1991) , but the methods did not describe direct use on clinical samples and may lack sensitivity for such work.

It would be desirable to assess the RT-PCR method on a larger number of clinical samples, including ones from older pigs. From a small number of titrations, the RT-PCR method appeared more sensitive than ELISA, but slightly less sensitive than virus isolations. The sensitivity of the RT-PCR may have been reduced by using only two of 50 ~1 of extracted RNA and again only two of 20 ~1 of the RT reaction mix. Nevertheless, a recent field isolate (96-1933) that was not readily grown in cell culture was detected with a strong RT-PCR signal, using faeces or intestine submitted from the field case. Another TGEV isolate (85-45210) could not be recovered in cell culture from an archived frozen stock of liquid culture supernatant, but an RT-PCR product was amplified. Furthermore, sequencing revealed that the amplicons obtained from each of these two viruses were unique (data not shown).

Differentiation of transmissible gastroenteritis virus from porcine respiratory coronavirus and other antigenically related coronaviruses by using cDNA probes specific for the 5' region of the S glycoprotein gene

Detection of transmissible gastroenteritis coronavirus antigens by a sandwich enzyme-linked immunosorbent assay technique

Antigenic differentiation between transmissible gastroenteritis of swine and a related porcine respiratory coronavirus

A cytopathic virus causing a transmissible gastroenteritis in swine. I. Isolation and properties

Identification of epitopes of immunological importance on the peplomer of porcine transmissible gastroenteritis virus

Differentiation of porcine coronavirus from transmissible gastroenteritis virus

Cytopathogenicity of transmissible gastroenteritis virus in pigs

Detection of antibodies against hog cholera virus and bovine viral diarrhoea virus in porcine serum. A comparative examination using CF, PLA and NPLA assays

The multiplication of transmissible gastroenteritis viruses in several cell lines originated from porcine kidney and effects of trypsin on the growth of the viruses

Molecular differentiation of transmissible gastroenteritis virus and porcine respiratory coronavirus strains

Classical transmissible gastroenteritis returns

Porcine respiratory coronavirus: molecular features and virus-host interactions

Pathogenicity of experimental infection with "pneumotropic" porcine coronavirus

Transmissible gastroenteritis

Diagnosis of transmissible gastroenteritis in pigs by means of immunofluorescence

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

Development of a detection system for woody plants based on PCR analysis of immobilized virions

Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuramic acid) binding activity

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

Region-specific initiation of mouse mammary tumour virus RNA synthesis by endogenous RNA polymerase II in preparations of cell nuclei

The isolation of the cytopathogenous strains of the originator of transmissible gastroenteritis of pigs in tissue cultures

Comparison of the antibody response to transmissible gastroenteritis virus and porcine respiratory coronavirus, using monoclonal antibodies to antigenic sites A and X of the S glycoprotein

Comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection

Antigenic and biological diversity among transmissible gastroenetritis virus isolates of swine

Three new isolates of porcine respiratory coronavirus with various pathogenicities and qike (S) gene deletions

Use of non-radioactive DNA probes to differentiate porcine respiratory coronavirus and transmissible gastroenteritis virus isolates

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

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

We are very grateful to all those who supplied us with viruses for use in this study. Viruses were given by L. Valicek, Brno, Czech Republic; A. van Nieuwstadt, Lelystad, The Netherlands; K. van Reeth, Gent, Belgium and M. Frey, Ames, USA. Our thanks to G. Wibberley for TGE Antigen ELISA testing, and to P. Lowings for helpful discussions. This work was supported by the Ministry of Agriculture Fisheries and Food, UK.