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1、Virus Genes 17:1, 3342, 1998 # 1998 Kluwer Academic Publishers, Boston. Manufactured in The Netherlands. Nucleotide and Predicted Amino Acid Sequences of All Genes Encoded by the 30Genomic Portion (9.5kb) of Respiratory Bovine Coronaviruses and Comparisons Among Respiratory and Enteric Coronaviruses
2、 VLADIMIR N. CHOULJENKO, KONSTANTIN G. KOUSOULAS,* XIAOQING LIN, Accepted February 2, 1998 Abstract. The 30-ends of the genomes (9538bp) of two wild-type respiratory bovine coronavirus (RBCV) isolates LSU and OK were obtained by cDNA sequencing. In addition, the 30-end of the genome (9545) of the wi
3、ld-type enteric bovine coronavirus (EBCV) strain LY-138 was assembled from available sequences and by cDNA sequencing of unknown genomic regions. Comparative analyses of RBCV and EBCV nucleotide and deduced amino acid sequences revealed that RBCV-specifi c nucleotide and amino acid differences were
4、disproportionally concentrated within the S gene and the genomic region between the S and E genes. Comparisons among virulent and avirulent BCV strains revealed that virulence-specifi c nucleotide and amino acid changes were located within the S and E genes, and the 32kDa open reading frame. Key wor
5、ds: cDNA, S, non-structural, structural, respiratory, coronavirus Introduction Coronaviruses are important etiological agents of human and animal diseases including respiratory infection, gastroenteritis, hepatic and neurological disorders as well as immune-mediated disease such as feline infectious
6、 peritonitis, and other persistent infections(1,2).Entericbovinecoronaviruses (EBCV) are generally associated with enteric disease of newborn calves and winter dysentery of adult cattle (2). Recently, numerous respiratory bovine corona- viruses (RBCV) were isolated in our laboratory from cattle arri
7、ving with fever and respiratory disease in feedlots or livestock shows of 8 different states in the USA. The cytopathogenic, cell fusion, and other phenotypic properties of these viruses were different from the known EBCV (3). Coronaviruses contain a single stranded, capped, and polyadenylated posit
8、ive-sense (infectious) RNA molecule of approximately 30kb length,which directs the synthesis of a nested set of subgenomic mRNAs (4,5). The 30-end of the genomic RNA consists of approximately 9.5kb and contains the spike (S) glycoprotein, the hemagglutinin-esterase (HE) glyco- protein, the integral
9、membrane (M) protein, the small membrane protein (E) and the phosphorylated nucleo- capsid (N) protein and a number of ORFs potentially encoding non-structural proteins (Ns) (5). The 32kDa non-structuralproteinisaphosphoproteinthat accumulates in the cytoplasm of infected cells (6,7). It is notknown
10、 whetherthe 12.7 and 4.8kDa ORFs are expressed in infected cells, while the 4.9kDa putative protein, most likely, is not translated (8). BCV uses N-acetyl-9-O acetyl neuraminic acid as receptor determinant to initiate infection (9). Although the HE glycoprotein also has an affi nity for 9-O-acetylat
11、ed sialic acid, the S glycoprotein was identifi ed as the major sialic acid binding protein of BCV (10). The S glycoprotein facilitates viral *Corresponding Author. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assig
12、ned the accession number AF058942, AF058943, AF058944. attachment to susceptible cells, causes cell fusion after cell-surface expression (fusion from within), and induces viral infectivity neutralizing antibodies (1). Porcine transmissible gastroenteritis virus (TGEV) strains were isolated which exh
13、ibited respiratory tissue tropism. These viruses contained point muta- tions or deletions within the fi rst 250aa of TGEV S1 which were associated with reduced enteropathogeni- city and loss of hemagglutinating activity (1113). To examine the genetic basis for the phenotypic differences between RBCV
14、and EBCV, we cloned and sequenced the 30-end of the viral genomes of two virus strains RBCV-LSU-94LSS-051-2(LSU) and RBCV- OK-0514-3(OK) that originated from Louisiana and Oklahoma cattle, respectively. We report here, the nucleotide and predicted amino acid sequences of all genes encoded by the 30g
15、enomic portion (9.5kb) of two wild-type RBCV strains and comparisons among respiratory and enteric coronaviruses. Materials and Methods Viruses and cell line.All RBCV and EBCV strains were propagated in the G clone of human rectal tumor cells (HRT-18G) developed recently through selec- tion and medi
16、um modulation (3). Supernatant fl uids from infected HRT-18G cells were collected and viruses were purifi ed as described (14). RBCV OK and LSU virus stocks were tested at the third and fourth passages, respectively. EBCV LY-138(LY) virus stocks were prepared at the second passage, while the EBCV-L9 virus strain, derived from the EBCV-Mebus strain, had been propagated 80 times in cell cultures. Strategy for cDNA construction and assembly of the 9.5Kb cDNA sequence representing the 30-end of dif
