INRS-Institut Armand-Frappier, Centre de Microbiologie et Biotechnologie, Université du Québec, 531 boul. des Prairies, Laval, Québec, Canada H7V 1B71
Author for correspondence: Serge Dea. Fax +1 450 686 5627. e-mail serge.dea{at}inrsiaf.uquebec.ca
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HEV, which specifically infects pigs, has a strong tropism for epithelial cells of the upper respiratory tract and for the central nervous system (CNS) (Andries & Pensaert, 1980 ). The virus causes two clinical syndromes: the vomiting and wasting disease, and an encephalomyelitis (Pensaert & Andries, 1993
). The mode of transmission is through nasal secretions and the virus is spread via the peripheral nervous system to the CNS. This virus infection is believed to be widespread in many pig-producing countries as a subclinical condition. However, in the autumn of 1998 and 1999, several clinical manifestations suggestive of HEV infections were reported in Québec and Ontario, Canada. A cytopathogenic coronavirus strain, IAF-404, was isolated from the brain of a paretic weaned piglet and cultivated in the established human rectal tumour cell lines, HRT-18 and/or HRT-18G cells (Storz et al., 1996
). This field HEV isolate, as well as the reference HEV-67N (ATCC VR741) strain, induced the production of syncytia in infected cell monolayers, which did not appear to depend on the presence of trypsin in the culture medium. The IAF-404 HEV strain displayed HA and high AE and RDE activities, and displayed cross-antigenic relatedness with the reference strain by haemagglutination inhibition (HI) tests (data not shown).
As the HEV genomic sequence was still unknown, sequencing analysis of the 3'-terminal end (8·1 kb) of the genome of both HEV strains was undertaken to allow comparison with other haemagglutinating coronaviruses. This paper describes the molecular characteristics of the deduced non-structural (ns) and structural proteins encoded by the ORF2bORF7 genes of the prototype 67N strain (HEV-67N) and the IAF-404 field isolate of HEV.
RTPCR using the Taq (Invitrogen) or Pwo (Roche Diagnostic) DNA polymerase was carried out to amplify the HE, S, ORF4/5 and MN regions of the viral genomes. The viruses were propagated for less than five passages in HRT-18 and/or HRT-18G cells. Genomic RNA was extracted from concentrated virus or lysates of virus-infected cells by Tripure (Boehringer Mannheim). The oligonucleotide primers were designed according to the cDNA nucleotide sequence of the Mebus strain of BCoV (Kienzle et al., 1990 ; Lapps et al., 1987
) or the partial cDNA nucleotide sequence of the prototype HEV-67N strain available in the literature (Vieler et al., 1995
, 1996
), and that of the specific IAF-404 sequence reported here. The location of the primers on the HEV genome is illustrated in Fig. 1
. For sequencing of the cDNA clones, RTPCR-amplified cDNA fragments with A overhangs were ligated into a TA cloning vector (plasmid pCR2.1; Invitrogen) providing single 5' T overhangs at the insertion sites. As an alternative, PCR-amplified gene fragments were extracted from the PCR-amplified mixtures and sequenced directly, following purification of the amplified cDNAs using the QIAquick PCR Purification Kit (Qiagen), according to the manufacturers instructions. Sequencing was performed with an Automated Laser Fluorescent DNA sequencer (Pharmacia LKB). To overcome the error rate of the RT and Taq DNA polymerase, clones from at least two different RTPCR events were sequenced. The sequences were computer-analysed with the MacVector 7.0 program (Oxford Molecular Group).
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Sequencing analysis of the 3' end of the genome of both the prototype HEV-67N strain and the Québec HEV IAF-404 field isolate revealed only one, two, three and seven amino acid changes among their ns 12·7 kDa, M, N and HE proteins, respectively, but 28 amino acid changes for their S protein. These observations suggested that this porcine coronavirus has remained genetically stable since its first isolation in North America in 1962 (Greig & Bouillant, 1972 ). The minor differences revealed between the amino acid sequences of the ns and structural proteins of the HEV-67N and HEV IAF-404 strains are probably not sufficient to explain the divergence observed in their virulence for pigs.
The results of pairwise comparisons with BCoV and HCoV-OC43 of the predicted amino acid residues of viral proteins encoded by genes located at the 3'-terminal end of the genome of both HEV strains studied, representing approximately one-third of the complete size of the viral genome, are summarized in Table 1. Multiple alignments (not shown) of the sequences of both HEV strains with those of BCoV (Mebus strain) and HCoV-OC43 allowed the demonstration of identical amino acid residues for both HEV strains that differed from those of the two other haemagglutinating coronaviruses. In addition to the ns proteins described above, the predicted amino acid sequences of the S proteins of both HEV strains were apparently four and ten amino acid residues shorter than the homologous proteins of HCoV-OC43 and BCoV, respectively. Sequencing analysis of both North American HEV isolates revealed the presence of 34, 192, 11, 9 and 14 specific amino acid substitutions for HEV in the HE, S, 12·7 kDa ns, M and N proteins, respectively. Consequently, percentages of amino acid identity of 90, 80, 86, 93 and 95% were demonstrated for the previously mentioned proteins of HEV and BCoV, with less than 16% amino acid identity for the ns 4·9 kDa protein. Approximately the same percentages of amino acid identity were obtained with the homologous proteins of HCoV-OC43.
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The significance of specific amino acid residues for HEV strains, in view of their virulence for pigs and their respiratory and neurological tropism, is difficult to interpret, considering that they are apparently distributed randomly among the previously mentioned proteins, except for the S protein. The S1 portion of the envelope-associated S glycoprotein is known to carry major antigenic determinants of coronaviruses that trigger the host immune response to induce the production of virus-neutralizing (VN) antibodies (Dea & Tijssen, 1989 ; Deregt & Babiuk, 1987
; Vautherot et al., 1992
). Consequently, sequence differences in S1 are thought to be responsible for many of the antigenic differences between coronaviruses (Spaan et al., 1988
). As expected, the variability of the predicted amino acid sequence of the HEV S protein appeared to be concentrated in the S1 region, with only 73/71% identities compared with that of the enteropathogenic Mebus strain of BCoV and of the human HCoV-OC43 respiratory coronavirus, respectively, whereas a 97% amino acid identity was demonstrated for the S1 portion of the S glycoproteins of both HEV strains. As displayed in Fig. 2(b)
, this variability included amino acid changes in the predicted N-terminal signal peptide (aa 117) (Von Heijne, 1986
) and a few clusters of amino acid substitutions within the N-terminal portion and the hypervariable region of S1 (aa 452593) (Parker et al., 1990a
, b
; Yoo et al., 1991
). Furthermore, in comparison with BCoV and HCoV-OC43, three specific gaps within this hypervariable region of S1 (aa 464469, 494495 and 502511) were identified for both HEV strains. The putative N-linked glycosylation sites (NXS and NXT), identified in the S1 sequence of BCoV strains associated with variable pathologies (Gelinas et al., 2001
), appeared to be mostly conserved in the S1 region of the HCoV-OC43 peplomeric glycoprotein, but this was not the case for either of the HEV strains studied.
Overall, the data from sequencing analysis presented here indicates that the 3'-terminal end of the genome of the Québec field isolate IAF-404 of HEV appears relatively well conserved in comparison with the reference HEV-67N strain. However, sequences of genes encoding the four major structural proteins revealed species specificity between haemagglutinating coronaviruses, which, for diagnostic purposes, will be useful to distinguish among these viruses. With the potential risk of recombination between members of the subgroup of haemagglutinating coronaviruses, notably at the level of subgenomic mRNAs synthesized in the infected cells during the replication cycle, specific and group-specific immunologic or genomic probes are required for a definitive diagnosis and control of possible interspecies infections. Recently, group-specific or species-specific single and multiplex RTPCR approaches have been designed for the detection and differentiation of haemagglutinating coronaviruses (Boutin et al., 2001 ). The propagation of the Québec IAF-404 field isolate of HEV in porcine primary lung cells (data not shown) may allow us to study the pathogenesis of HEV in swine with a better efficacy than with the prototype HEV-67N strain, which has been propagated for several passages on human and porcine primary and continuous cell cultures, resulting in the attenuation of its virulence for pigs.
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References |
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Received 11 March 2002;
accepted 21 May 2002.