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Genomic relationship of porcine hemagglutinating encephalomyelitis virus to bovine coronavirus and human coronavirus OC43 as studied by the use of bovine coronavirus S gene-specific probes

Summary The genomic relationship of porcine hemagglutinating encephalomyelitis virus (HEV) to bovine coronavirus (BCV) and human coronavirus (HCV) strain OC43 was examined by dot blot hybridization assays. Two BCV S gene-specific probes were generated by polymerase chain reaction from the avirulent L9-strain of BCV. Probes were located in the S1 and the S2 region of the peplomeric (S) glycoprotein gene. The S1 probe (726 bp) hybridized with BCV and HCV-OC43, but not with HEV under moderate stringency hybridization conditions (50 °C). Only slight signals were present with mouse hepatitis virus (MHV) and no signals were observed with feline infectious peritonitis virus (FIPV) or canine coronavirus (CCV). At high stringency conditions (60 °C) the S1 probe hybridized with BCV only. Using the S2 probe (680 bp) under moderate strin-gency conditions, hybridization signals were obtained with BCV, HCV-OC43 and HEV (strains 67N, NT9, VW572). The signals obtained by the three HEV strains were altogether weaker than with BCV and HCV-OC43. The S2 probe did not react with MHV, FIPV and CCV. At high stringency the S2-specific probe hybridized with BCV and HCV-OC43 but did not hybridize with HEV. Nucleotide sequence analysis of the region covering the S2 probe in HEV revealed 92.6% nucleotide sequence homology to BCV and 91.9% to HCV-OC43. In contrast, the region covering the S1 probe in HEV could not be amplified using the BCV S1-specific primers. The hybridization and sequencing results thus indicate a closer genomic relationship between BCV and HCV-OC43 than there is between HEV and BCV or HCV-OC43, respectively.


Introduction
Coronaviruses are important pathogens in animals and man. Two major antigenic groups of mammalian coronaviruses are distinguished.  [13].
The coronavirus genome consists of a single-stranded, nonsegmented RNA with positive polarity. Three to four major structural proteins are encoded: the nucleocapsid protein (N), the membrane protein (M) and the spike or peplomer protein (S). Members of the second antigenic group have an additional glycoprotein, the hemagglutinin esterase protein (HE). The S proteins of BCV and MHV are cleaved by host-cell proteases into two subunits of similar size, S 1 and $2, respectively [1, 3,17].
To date, studies on the HEV genome have not been conducted. In order to study the relationship of HEV to BCV and HCV-OC43 two probes were generated from the BCV S gene and investigated for their reactivities with HEV and HCV-OC43 by dot-blot hybridization assays.

Indirect immunofluorescence assay
An indirect immunofluorescence assay was performed with monoclonal antibodies (Mab) I7, F7 and 116 which were shown to differentiate between BCV, HCV-OCA3 and HEV [16]. Virus infected cells were fixed with formaldehyde for 10 rain, permeabilized with 0.1% Triton X in PBS for 6 rain and incubated each with Mab F7, I7 and I16 at 37 °C for 1 h. After rinsing two times with PBS the slides were stained with an anti mouse IgG-FITC (Sigma, Deisenhofen, Germany) at 37 °C forl h.

RNA isolation
RNA was isolated from virus-infected HRT-18 cells by the guanidinium-thiocyanate method using a RNA-isolation-kit (Stratagene, Heidelberg, Germany) according to the manufacturers' instructions.

eDNA synthesis andprobepreparation
Single-stranded cDNA was synthesized with SuperScript Plus RNase H-reverse transcriptase (GIBCO BRL, Eggenstein, Germany) and primer 3' S 1 or 3' S2a, respectively, at 47.5 °C for BCV and 42 °C for HEV according to the manufacturers' instructions.
Probes were amplified in a reaction mixture adjusted to 1 x PCR buffer, 200 gM dATP, dGTP, and dCTP each, 160 g M dTTP and 40 la M DIG-11-dUTP (Boehringer, Mannheim, Germany), 400 nM of each primer and 0.5 U Tfl-polymerase (Biozym, Hameln, Germany) in a final volume of 50 gl with annealing temperatures of 58 °C.

Dot blot hybridization
To determine the detection limit of each probe log 2-dilutions ofa BCV-L9 suspension as well as a dilution series of homologous DNA in amounts ranging from 100 ng to 40 pg were directly applied to the nylon membrane.
For dot blot hybridization assays log-2 dilutions of each virus starting with 5 x 105 TCIDs0were adjusted to 1 x SSC (0.015 M NaCitrat, 0.15 M Nacl, pH 7.0) and directly applied to a positively charged nylon membrane (Boehringer, Mannhein, Germany) presoaked with H20 and equilibrated with 20 x SSC, by means of a dot-blot apparatus (Biorad, Mttnchen, Germany). Blots were air dried and viral RNA was fixed and denatured by UV-illumination for 5 min (TFL-20M, Biometra, G6ttingen, Germany). Prehybridization and hybridization were basically done according to the manufacturers instructions (DIG luminescent detection kit for nucleic acids, Boehringer, Mannheim, Germany). Prehybridization was performed at 42 °C for 4 h followed by an incubation with hybridization solution containing 30 ng/mI of freshly denatured Digoxigeninlabeled probe at 42 °C overnight. Membranes were washed twice at room temperature for 5 min in 2 x SSC, 0.1% SDS and then twice for 15 min at moderate (50 °C) or high (60 °C) stringency conditions in 0.1 x SSC, 0.1% SDS. The hybridization signals were immunologically detected according to the manufacturers directions. The blots were exposed to a X-Ray film for 7 h.

D NA sequencing
Non-radioactive sequencing reactions were performed using the prism ready reaction dye-deoxy terminator cycle sequencing kit (ABI, Applied Biosystems Inc., Weiterstadt, Germany) according to the manufacturers instructions. Further analysis was done on the AB1373A sequencer using standard protocols from ABI. The PCR products were purified for sequencing with the Quiaquick-spin PCR purification kit (Quiagen, Chatsworth, CA). 300 ng purified DNA were used as template for each sequencing reaction. The primers used for sequencing were 3' S2b and 5' S2b as well as two additional primers (3' $3: 5' -TATAGTTAGCAGCACAGGTA-3' ; 5' $3: 5' -GCT-GAACATTTCAACACCC-3' ). For detailed DNA sequence analysis the DNASTAR software package (DNASTAR Inc., London, U.K.) was used.

Indirect immunofluorescence assay
The indirect immunofluorescence assay results gained by the use of Mabs, previously published by Vautherot et al. [16], revealed different reaction patterns between the bovine, human and porcine coronavirus strains (Table 1). Mab J7 only  [16] bSpecific for the BCV-S gtycoprotein, no antigenic cross reactivity with other hemagglutinating coronaviruses ~Specific for the BCV-S glycoprotein, antigenic cross reactivity with HCV-OC43 dSpecific for the BCV-S glycoprotein, antigenic cross reactivity with HCV-OC43 and HEV reacted with BCV. Mab F7 recognized BCV as well as HCV-OC43 and Mab I16 recognized HEV-67N, -NT9 and -VW572, additionally.

Detection limit of the BCVspecific S1 and S2 probe
Using the $1 probe, the minimal detectable amounts of the BCV-L9 virus suspension were 7.8 x 103 and 3.1 x 104 TCIDs0 and the minimal detectable amounts of homologous DNA were 781 pg and 3.12 ng at washing temperatures of 50 °C and 60 °C, respectively. By hybridization with the $2 probe the detection limit was lower. With this probe 3.9 x 103 TCIDs0 of BCV-L9 or 390 pg DNA at 50 °C and 1.5 x l04 TCIDs0 or 781 pg DNA at 60 °C could be detected.

Cross hybridization
The hybridization results are shown in Fig. 2. In general both probes did not react with viruses belonging to the antigenic unrelated group (CCV, FIPV) or with mock infected cells (data not shown). The S 1 probe hybridized with BCV and HCV-OC43

Discussion
Hybridization and sequencing results presented in this paper indicate a high degree of genomic relationship of HEV to BCV and HCV-OC43. However, the two BCV S gene-specific probes used for cross hybridization experiments were able to differentiate these three hemagglutinating coronaviruses.
The S 1 probe failed to detect HCV-OC43 by hybridization under high stringency conditions. This result reflects the nucleotide sequence data. Alignment of the published genome sequences of the region corresponding to the $2 probe in BCV and HCV-OC43 revealed 24 mismatches. In contrast, 41 mismatches as well as 24 insertions into the HCV-OC43 genome occured in the sequence corresponding to the S 1 probe [7,19].
The failure of the S 1 probe to hybridize to HEV even under moderate stringency hybridization conditions and the failure of the $2 probe to detect HEV under high stringent hybridization conditions indicated that the S genes of HEV and BCV or HCV-OC43, respectively, were less related. This hypothesis was further confirmed by PCR results utilizing the BCV Sl-and S2-specific primers. Both primer pairs produced amplicons of the expected length from HCV-OC43 cDNA. In contrast, a specific amplicon from HEV cDNA could be synthesized only with the $2 primers. The nucleotide sequence analysis of this 680 bp-amplicon (HEV strain 67N) and the alignment with the corresponding sequence in BCV and HCV-OC43 revealed a lesser degree of homology between HEV-67N and BCV-L9 (92.6%) or HCV-OCA3 (91.9%) than exists between BCV-L9 and HCV-OC43 (96.3%).
Sequence data of the S genes of BCV and HCV-OC43 revealed a high degree of homology between their deduced aminoacid sequences (91.2% identity). As compared to the $2 protein (95.3% homology) greater sequence divergence occured in the S 1 protein (88% homology) [7,11,19]. The hybridization results presented in this paper indicate that the $2 region of HEV-67N is similarly conserved, as it is reported for BCV and HCV-OC43 [7,11,19].
The failure of the S 1 probe to hybridize with HEV and the obvious lack of the BCV S 1-specific primers during HEV-PCR are indicative of a higher degree of genotypic divergence between HEV and BCV or HCV-OC43, respectively, than there is between BCV and HCV-OC43.
Similar differences were demonstrated on the phenotypic level. HEV, BCV and HCV-OC43 share colm~on antigenic determinants but they can be differentiated by their reactivities with certain anti-BCV Mabs [15,16]. Identical results were obtained for the coronavirus strains used in this study by immunofluorescence assays with Mabs directed against different S 1 epitopes, which were published by Vautherot et al. [16].
HEV was recently shown to have a strong tropism to neurons in experimentally infected young mice [18]. In naturally infected newborn pigs the virus spreads to the central nervous system via peripheral nerves. BCV and HCV-OC43, in contrast, have a tropism to the respiratory and enteric tract [17]. It could be hypothesized that divergences in the putative bulbous part (SI) of the S protein are related to tissue tropism. The complete amino acid sequence of the HEV S protein is needed to provide the knowledge for studying its possible role in tissue tropism.