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1、Virus Research 76 (2001) 4357 Bovine coronaviruses associated with enteric and respiratory diseases in Canadian dairy cattle display different reactivities to anti-HE monoclonal antibodies and distinct amino acid changes in their HE, S and ns4.9 protein Anne-Marie Ge linas, Martine Boutin, A. Marie-
2、Jose e Sasseville, Serge Dea * Centre de Microbiologie and Biotechnologie, INRS-Institut Armand Frappier, Uni?ersite du Que bec,531boule?ard des Prairies, La?al, Que bec, Canada, H7V1B7 Received 28 January 2001; received in revised form 23 February 2001; accepted 23 February 2001 Abstract Bovine cor
3、onavirus isolates associated with recent outbreaks of respiratory disease in Ontario and Quebec dairy farms were compared to reference strains known to be responsible for neonatal calf diarrhea (NCD) or winter dysentery (WD) of adult cattle. In respect to their hemagglutinating properties and their
4、higher RDE activities with rat erythrocytes, WDBCoV strains differed from NCDBCoV strains and respiratory bovine coronaviruses RBCoV strains. Serologically, three MAbs directed to the HE glycoprotein of the WDBCoV strain BCQ.2590 recognized two serogroups amongst NCDBCoV strains by hemagglutination
5、inhibition, whereas only one of the MAbs failed to react toward three of the four RBCoV isolates tested. Sequencing analysis of the S (S1 portion), HE, ORF4 and ORF5 genes of BCoV isolates associated with different clinical syndromes indicated that neither insertions or deletions could explain their
6、 distinct tropism. For the HE glycoprotein, a total of 15 amino acids (aa) substitutions were identifi ed by comparing fi eld isolates to the prototype Mebus strain. Two specifi c proline substitutions were identifi ed for virulent strains being located in the signal peptides (aa 5) and aa position
7、367; one specifi c aa change was revealed at position 66 for RBCoV fi eld isolates. Analysis of the S1 portion of the S glycoprotein revealed a total of eight aa changes specifi c to enteropathogenic (EBCoV) strains and eight aa changes specifi c to RBCoV strains. For all BCoV isolates studied, the
8、region located between the S and M genes (ORF4) apparently encodes for two non-structural (ns) proteins of 4.9 and 4.8 kDa. A specifi c non-sense mutation was identifi ed for the nucleotide at position 88 of the putative 4.9 kDa protein gene of RBCoV isolates resulting in 29 rather that 43 aa residu
9、es. The ORF5, which encodes a 12.7 ns protein and the 9.5 kDa E protein, was highly conserved amongst the BCoV fi eld isolates. 2001 Elsevier Science B.V. All rights reserved. Keywords:Bovine enteropathogenic and respiratory coronaviruses; Winter dysentery; Antigenic and genomic variability; Esteras
10、e; Hemagglutinin; Monoclonal antibodies; Structural proteins * Corresponding author. Fax: +1-450-6865627. E-mail address:serge.deainrs-iaf.uquebec.ca (S. Dea). 0168-1702/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1702(01)00243-X A.-M. Ge linas et al./Virus Res
11、earch76 (2001) 435744 Bovine coronavirus (BCoV) is a member of the familyCoronaviridae,orderNidovirales(De Vries et al., 1997). The genome of coronaviruses consistsinasinglestranded,capped,and polyadenylated positive-sense RNA molecule of approximately 26 to 30 kb in length, that yields a 3?-cotermi
12、nal nested set of subgenomic mRNAs in the infected cells (Spaan et al., 1981; Lai, 1986). Coronaviruses are mostly spherical in shape, en- veloped with a diameter ranging between 120 and 150 nm, and possess an helical nucleocapsid. The virion contains four structural proteins: the spike (S) glycopro
13、tein of 160200 kDa which forms the long club-shaped surface projections, the matrix glycoprotein (M) of 2038 kDa, the phosphory- lated nucleocapsid protein (N) of 5060 kDa and the small membrane protein (E) of about 9.5 kDa (Spaan et al., 1988 (a review). Based on serologi- cal cross-reactivities an
14、d genomic relatedness, the coronaviruses have been separated into three dis- tinct subgroups (Schockley et al., 1987; Dea et al., 1990). The BCoV belongs to the second subgroup, with hemagglutinating encephalomyelitis virus of swine(HEV),humanrespiratorycoronavirus (HCoV-OC43),turkeyentericcoronavir
15、us (TCoV)andmurinehepatitiscoronaviruses (MHVs). Members of this subgroup possess an additional 140 kDa envelope-associated glyco- protein, the hemagglutininesterase (HE), corre- sponding to a second fringe of granular surface projections (King et al., 1985; Hogue and Brian, 1986; Dea et al., 1990).
16、 Both the S and HE glycoproteins are able to hemagglutinate red blood cells by binding to N-acetyl-9-O neuraminic acid (Vlasak et al., 1988; Schultze et al., 1991b), elicit the production of virus neutralizing antibod- ies (Dea et al., 1990; Vautherot et al., 1992), and are probably involved in determining the viral tissue and host tropism (Spaan et al., 1988). The HE is further associated to acetyl esterase (AE) and receptor destroying enzyme (RDE) activities (Vlasak et al., 1988; Parker
