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Detection of Group 2a Coronaviruses with Emphasis on Bovine and Wild Ruminant Strains

Group 2a of the Coronaviridae family contains human and animal pathogens that include mouse hepatitis virus, rat coronavirus, human respiratory coronaviruses OC43 and the recently identified HKU1 strain, a newly recognized canine respiratory coronavirus, porcine hemagglutinating encephalomyelitis virus, equine coronavirus, bovine coronavirus (BCoV), and wild-ruminant coronaviruses. The presence of a hemagglutinin-esterase (HE) surface glycoprotein in addition to the viral spike protein is a distinguishing characteristic of most group 2a coronaviruses. BCoV is ubiquitous in cattle worldwide and is an economically significant cause of calf diarrhea, winter dysentery of adult cattle, and respiratory disease in calves and feedlot cattle. We have developed and optimized laboratory diagnostic techniques, including virus isolation in HRT-18 cell cultures, antibody and antigen ELISA, and RT-PCR, for rapid, sensitive, and reliable diagnosis of BCoV and related wild ruminant coronaviruses.


Introduction
Coronaviruses cause a broad spectrum of diseases in domestic and wild animals, poultry, and rodents, ranging from mild to severe enteric, respiratory, and systemic disease, as well as the common cold or pneumonia in humans (1-3). From Recently SARS-CoV emerged, likely from a wildlife reservoir, as a new CoV (group 2b) causing severe respiratory disease in humans (4-10). Bats are a suspect reservoir for SARS-like CoVs with civet cats possibly playing a role as an intermediate host (6,(8)(9)(10)(11)(12)(13).
The widespread prevalence of infections caused by group 2 coronaviruses, their extensive host range, the various disease manifestations, a high frequency of genomic recombination events, and the potential for interspecies transmission (BCoV, SARS-CoV) are characteristics that require continuous monitoring and improvement of diagnostic tests for these CoVs (8,9). A summary of infections and standardized diagnostic tests available for group 2a CoVs is shown in Table 1.
Our laboratory has focused extensively on the study of bovine coronavirus (BCoV) and bovine-like CoVs for many years. Bovine coronavirus causes acute diarrhea in neonatal calves and winter dysentery in adult cattle causing large economic losses owing to diarrheal morbidity and mortality costs and decreased milk production (2, [27][28][29][30][31]. The enteropathogenic coronaviruses were initially identified and isolated from neonatal calves with severe diarrhea (31). Subsequently, coronaviruses were revealed to be a cause of winter dysentery in adult cattle (2,32-35) as well as respiratory disease (shipping fever pneumonia) (29, [36][37][38][39][40], and antigenic, biologic, and genetic variation among these strains was demonstrated (28,37,(41)(42)(43). Repeated upper respiratory BCoV infections occur frequently in calves, and subclinically infected animals may be reservoirs for BCoV (28,30 Detailed methods for cell culture propagation of the enteric BCoVs were described by Saif et al. in 1988 (48). General procedures for the isolation of winter dysentery (49) and respiratory strains of BCoV in HRT-18 cell cultures (using cloned HRT-18 cells from the L. J. Saif laboratory) (37), antibody and antigen ELISA, and RT-PCR have been optimized and standardized in our laboratory (2,28,50,51) and are described in detail in the following sections. For the antigen (Ag) ELISA tests, monoclonal antibodies (MAb) were produced and characterized by our laboratory and standardized for routine use in ELISA, immunoblotting, and immunohistochemistry (41,50-52).

Preparation of Fecal and Nasal Samples
Fecal and nasal samples are used for virus isolation, antigen capture ELISA, and RNA extraction for RT-PCR. Feces from animals (domestic cattle or wild ruminants), with or without clinical signs, should be collected in sterile fecal cups, put on ice, and transported. Then, 0.5 g of fecal sample is diluted in 4 ml of Dil. #5, vortexed, and centrifuged at 2000 × g for 30 min. The supernatant is aspirated and stored at -70 • C until use. Samples should be filtered (using 0.22-nm filters) before inoculation onto the HRT-18 cell culture monolayers.
Paired sterile polyester-tipped swabs are used to collect nasal secretions from each nostril of domestic cattle or wild ruminants, put on ice and transported. observed approximately 72 h after inoculation, after five to seven initial blind passages (Fig. 1). 4. Immunofluorescence, Ag-ELISA, and RT-PCR tests can be used to confirm virus presence.

Antibody-Capture ELISA (Serological Test) for Detection of Virus-Specific Antibodies in Bovine or Wild-Ruminant Serum (51)
1. Blood is collected and allowed to clot. Then serum is aspirated, heat inactivated (56 • C, 30 min), and stored at -20 • C until use.

RNA Extraction
TRIzol LS reagent is used for viral RNA extraction from fecal and nasal samples following the manufacturer's protocol (see Note 7). Briefly: 1. A 250-l sample is mixed with 750 l TRIzol LS in an Eppendorf tube, vortexed, and incubated for 5 min at room temperature. 2. Then 200 l chloroform is added to each tube, vortexed for a short time, and incubated for 10 min at room temperature. 3. After centrifugation, at 12,000 × g for 15 min at 4 • C, the supernatant (400 l) is transferred to new Eppendorf tubes and an equal volume of 100% isopropyl alcohol is added to each tube. Tubes are vortexed and incubated for 10 min at room temperature.
4. Then tubes are centrifuged at 12,000 × g for 10 min at 4 • C, the supernatant is discarded, and 800 l of 75% EtOH is added to each tube. 5. Samples are vortexed and centrifuged at 7500 × g for 5 min at 4 • C. 6. Finally, the supernatants are removed gently, the pellet is dried using a DNA Speed Vac dry machine (model DNA110, Savant INC, NY) low spin for 10 min and 40 l RNase-free (DEPC-treated) water is added for elution. 7. After incubation at 55 • -60 • C for 10 min, RNA samples should be stored at -20 • C or applied directly for one-step or nested RT-PCR.

One-Step RT-PCR
1. Each primer pair listed in Table 2

Nested PCR (28)
The nested-PCR protocol is as described for the RT-PCR procedure above (Section 3.4.2), but with minor modifications. For the nested-PCR master mix, 5 l of undiluted RT-PCR product (cDNA) is used as a template instead of the RNA sample. The PCR master mix should not contain AMV reverse transcriptase and RNasin. For precautions against cross-contamination (see Note 11).

Conclusions
In summary, multiple diagnostic tests have been developed and standardized for the routine diagnosis of BCoV and the closely related ruminant Group 2a CoVs. These include antigen or antibody detection by ELISA, virus isolation in cell culture (HRT-18 cells), and, often the most sensitive method, detection of viral RNA by RT-PCR. These methods have provided evidence for the presence of CoVs closely related genetically or antigenically to BCoV, not only from wild ruminants, but also from dogs (21) and humans (53). These findings and experimental transmission studies (8,44,54) confirm a broad host range for bovine-like group 2a CoVs and support a need to monitor their presence in other host species, including humans, by applying the diagnostic assays outlined in this chapter and the sequencing of new isolates.  . (4) was not optimal in RT-PCR with some CoV samples from wild ruminants. Thus, based on the consensus sequence (for all available RdRp sequences of coronaviruses from different groups) in all polymorphic positions, degenerative nucleotides were introduced. After these primers were tested with positive samples (validated in other assays), they demonstrated consistently positive results. 9. The coronavirus nucleoprotein gene was chosen for group-specific primer development because it has been shown to be variable in amino acid and nucleotide composition among the viruses that comprise the three coronavirus antigenic groups, but highly conserved within these groups. 10 The optimal melting temperature for group 2a-specific primers and nested-PCR is 56 • C; for pancorona-and BCoV-specific primers, the optimal melting temperature is 50 • C. 11. If RT-PCR sensitivity is low, a nested PCR method can increase PCR product yield. However, this will also increase the risk of cross-contamination. Standard precautions to avoid cross-contamination should be implemented. These include: use of gloves with frequent changing; employing of aerosol resistant pipettors and pipette tips; preparing of PCR master mixes in UV cabinets that should never be used for work with any sample materials or PCR DNA products; regular decontamination of work surfaces and all equipment with 10% chlorine or another commercially available disinfectant; separate working areas for each step of the PCR analysis as well as separate storage areas for PCR reagents and contaminated samples (47).