J. Med. Microbiol. Ð Vol. 49 2000), 1119±1127 # 2000 The Pathological Society of Great Britain and Ireland ISSN 0022-2615

VIROLOGY

Temperature-sensitive acetylesterase activity of haemagglutinin- speci®ed by respiratory bovine coronaviruses

X.Q.LIN,V.N.CHOULJENKO,K.G.KOUSOULASandJ.STORZ

Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA

Numerous respiratory bovine coronaviruses RBCV) were isolated recently from nasal swab samples and lung tissues of feedlot cattle with acute respiratory tract disease. These newly emerging RBCV isolates exhibited distinct phenotypic features that differentiated them from enteropathogenic bovine coronaviruses EBCV). The RBCV strains had a receptor-destroying function mediated by acetylesterase AE) activity of the haemagglutinin-esterase HE) glycoprotein. The HE genes of wild-type EBCV strain LY138 and RBCV strains OK-0514 OK) and LSU-94LSS-051 LSU) were cloned, sequenced and transiently expressed in COS-7 cells. The enzymic properties of HEproteins in COS-7 cellular extracts and in puri®ed virus preparations were assayed at room temperature, 378C and 398C by two different assays. One assay used rr- nitrophenyl PNPA) as and detected serine-esterase activity; the second assay monitored AEfunction with bovine submaxillary mucin BSM) as substrate. The PNPA tests con®rmed that HEproteins of EBCVand RBCV were functionally expressed in transfected COS-7 cells. Time-dependent determination of the AEactivity of puri®ed RBCV OK and LSU particles showed lower AEactivity at 39 8C than at 378C, whereas the puri®ed EBCV LY particles retained full AE activity at both 378C and 398C. Transiently expressed RBCV HEexhibited a marked reduction of AEactivity after 40 min of assay time at 378C. In contrast, the AEactivity of the transiently expressed EBCV HE remained stable beyond 40 min. The deduced amino-acid sequences of the HEproteins speci®ed by the RBCV strains OK and LSU contained speci®c amino-acid changes in comparison with the EBCV LY strain, which may be responsible for the observed enzymic differences. These results are consistent with the hypothesis that RBCV strains have evolved to selectively replicate in respiratory tissues and that HE may play a role in this tissue tropism.

Introduction of membrane proteins [3]. It forms the short peplomer on the viral envelope [4±6]. Bovine coronavirus BCV) is a member of the Coronaviridae family and possesses a single, positive- The BCV HE serves as a receptor-binding and stranded RNA genome of 31kb in length [1,2]. receptor-destroying glycoprotein similar to the haemag- Similar to coronaviruses of antigenic group II, this glutinin-esterase-fusion HEF) glycoprotein of in¯uen- virus contains a ®fth structural protein, the haemag- za C virus [5, 7±13]. It binds to the N-acetyl-9-O- glutinin-esterase HE), in addition to the nucleocapsid acetylneuraminic acid residues of glycoproteins or N), membrane M), envelope E) and spike S) glycolipids on the surfaces of erythrocytes and proteins. This 140-kDa glycoprotein is a disulphide- susceptible cells, which is considered to be the major linked dimer of two identical 65-kDa glycosylated receptor determinant of BCV [11, 14, 15]. The recep- subunits and thus belongs to the 65-kDa class I group tor-destroying enzyme RDE) activity of BCV HE is the ®rst example of such an enzyme identi®ed on positive-stranded RNA viruses [11, 12]. with Received 13 March 2000; revised version accepted 8 May receptor-destroying functions have been identi®ed as 2000. major structural components of negative-stranded RNA Corresponding author: Dr J. Storz. viruses such as orthomyxoviruses and paramyxoviruses 1120 X. Q. LIN ET AL. [16]. The receptor-destroying functions are mediated by of trypsin enhancement. Trypsin activation was re- neuraminidases NA) of in¯uenza A and B viruses as quired for the isolation of EBCV [32]. 2) The RBCV well as paramyxoviruses which remove terminal N- have unusually high cell-fusing activities for the G acetylneuraminate from glycoconjugates [17]. In con- clone cells. 3) The RBCV have a restricted haemag- trast, RDE of both in¯uenza C virus and BCV have glutination pattern and agglutinate only mouse and rat, acetylesterase AE) activities that hydrolyse an but not chicken red blood cells RBC). The EBCV linkage to release the acetyl group from position C-9 of agglutinates both rodent and chicken RBC [33]. 4) N-acetyl-9-O-acetylneuraminic acid, potentially eluting Comparative analysis of wild-type RBCV and EBCV adsorbed virions [11, 12, 14]. The 9-O-acetyl residue is nucleotide and amino-acid sequences at the 39 genomic important for in¯uenza C virus recognition of a portion 9.5 kb) revealed that RBCV-speci®c nucleotide glycoprotein receptor, a major determinant for the cell and amino-acid changes were disproportionally con- tropism [18]. centrated within the HE gene, S gene and the genomic region between the S and E genes [31]. Most of these Inhibition of the AE activity of the BCV HE peplomer RBCV isolates have RDE activities for rat RBC [34]. by diisopropyl¯uorophosphate resulted in some reduc- tion of virus infectivity, indicating that binding of HE It was hypothesised that differences in structural and to the N-acetyl-9-O-acetylneuraminic acid residue is functional properties of HE and other BCV structural involved in the infective process [11]. Enzymic proteins may cause altered tissue tropism and virus removal of the N-acetyl-9-O-acetylneuraminic acid pathogenicity. The objectives of this investigation were residue from cell membranes or treatment with HE- to identify the enzymic reaction associated with the speci®c monoclonal antibodies MAbs) inhibits BCV RDE function of HE for RBCV, to characterise the AE infections [19, 20]. Furthermore, cells infected with a activities of the HE glycoproteins from wild-type recombinant baculovirus expressing the HE of BCV strains of RBCV and EBCV in puri®ed virus prepara- exhibited haemadsorption and esterase activities, both tions and as transiently expressed gene products at of which could be blocked by MAbs with infectivity speci®c body temperatures and to compare their HE neutralisation activity [21, 22]. Consequently, it cDNA-predicted amino-acid sequences. appears that the HE of BCV is important in virus infectivity. In addition to the S glycoprotein which has strong receptor-binding properties [20, 23, 24], binding of the HE glycoprotein of the short peplomers to N- Materials and methods acetyl-9-O-acetylneuraminic acid residues on the cell Virus strains and virus puri®cation surface may function as a pre-receptor interaction for BCV. However, the HE of mouse hepatitis virus strains The wild-type strains of RBCV, OK-0514 OK) and A59 and JHM is not expressed in productive infections, LSU-94LSS-051 LSU), were used at their second to and thus is not essential for replication of these strains fourth passages. The EBCV wild-type strain LY138 [25]. LY) was used at its second and third passages. All viruses were propagated in the G clone cell line. Virus Numerous coronaviruses were isolated recently from puri®cation was performed according to Zhang et al. nasal swab samples and lung tissues of feedlot cattle [35]. Brie¯y, the media of infected G clone cell with signs of acute respiratory tract disease including a cultures were clari®ed by low speed centrifugation after severe form of shipping fever pneumonia [26±30]. three freezing and thawing cycles. Supernatant ¯uids These virus isolates multiplied only in the G clone of were collected and precipitated by polyethylene glycol human rectal tumour-18 HRT-18) cells, and not in 10% w/v. Suspensions of precipitates were loaded on to cultured bovine cells, such as Georgia bovine kidney a sucrose 20% cushion and further puri®ed by a GBK) and bovine turbinate BT) cells which are sucrose 20±60% gradient. The puri®ed virus prepara- permissive for the known respiratory bovine viruses tions were resuspended in TNE buffer 100 mM Tris- including bovine herpes virus-1 BHV-1),bovine HCl, pH 7.4, 10 mM NaCl, 1m M EDTA) and stored at parain¯uenza type-3 virus PI-3), bovine respiratory À708C. Virus infective titres in the puri®ed prepara- syncytial virus BRSV) and bovine viral diarrhoea virus tions for RBCV OK and LSU, and EBCV LY ranged BVDV). These viruses were identi®ed as respiratory from 3:0 3 109 to 4:0 3 109 plaque-forming units bovine coronaviruses RBCV) on the basis of their pfu)/ml. morphological features as determined by electron microscopy, their ability to induce cell-to-cell fusion after infection of G clone cells, and the nucleotide sequence of structural genes. Phenotypic and genotypic Assays for haemagglutinin HA) and RDE properties of RBCV differentiated them from the previously reported enteropathogenic bovine corona- The assays for HA and RDE were performed as viruses EBCV) [26±31]. Distinguishing features are as reported previously [27±29] with washed rat RBCs follows. 1) The RBCV were isolated in the ®rst G prepared as suspensions of 0.5% with PBS pH 7.4) clone cell passage at neutral pH ranges without the use containing bovine serum albumin 0.05%. HAEMAGGLUTININ-ESTERASE PROTEINS OF RBCV AND EBCV 1121 USA), counter-stained with Evans blue, and examined Cloning and sequencing of the cDNA specifying by ¯uorescence microscopy Olympus AH-2, Olympus the HE glycoprotein and alignment of predicted amino-acid sequences of different HE genes Optical, Japan). Viral RNA extraction, cDNA synthesis and DNA sequencing were performed as described previously [31], with TRI reagent Molecular Research Center, Acetylesterase assay for acetate release from Cincinnati, OH, USA), Ready-To-Go You-Prime First- bovine submaxillary mucin BSM) Strand Beads Pharmacia Biotech, Uppsala, Sweden), PCR reagents, AmpliTaq and Gene-Amp PCR system Cells in six 35-mm wells were transfected with each 9600 Perkin-Elmer, Norwalk, CT, USA), TA-cloning plasmid DNA as described above, and used to kit Invitrogen, San Diego, CA, USA), restriction determine AE activity according to Herrler et al. New England Biolabs, Beverly, MA, [14]. Brie¯y, the cells were scraped off 69 h after USA) and SequenaseTM Amersham Life Science, transfection, washed with phosphate-buffered saline Arlington Heights, IL, USA). A cDNA HE fragment PBS) and resuspended in 0.6 ml of PBS. The cell was ampli®ed by PCR with a speci®c primer pair suspension was treated with sonication at a power 5F16/3B10 which corresponded to the sequences setting of 5.5 for 10 pulses Branson Soni®er Cell upstream and downstream of HE gene, respectively. Disruptor 200, Branson Ultrasonics, Danbury, CT, The actual primer sequences used were: 5F16, 59 USA) and soluble proteins were collected from the AGAATGGCAGTTGCTTATG 39; 3B10, 59 GAT- supernate after low speed centrifugation. The supernate TATGGTCTAAGCATCATG 39. The 1272-bp DNA was mixed in 30-ìl amount with 50 ìl of BSM type fragment obtained by RT-PCR was ®rst cloned into I-S, Sigma; 25 mg/ml in PBS), and incubated at 378C the pCRTM II vector and then cloned into the unique or 398C; 10 ìl of puri®ed virus preparation were used. NotI and HindIII restriction sites of the The mixtures were assayed at speci®c time points for eukaryotic expression plasmid vector pcDNA3.1/ the presence of acetate with a commercial test kit Zeo ‡) Invitrogen). The predicted amino-acid se- Boehringer-Mannheim, Indianapolis, IN, USA). Reac- quences of the HE genes from EBCV LY, and RBCV tion controls consisted of a sample of BSM and the OK and LSU were aligned with the aid of the same amount of the supernate from transfected COS-7 Sequence Analysis Software Package version 6.1of cell lysate or puri®ed virus preparation incubated at the Genetics Computer Group of the University of 48C. The values of the control samples were subtracted Wisconsin and the MacVector Software IBI, New from those of the test samples. Haven, CT, USA).

DNA transfection and transient expression of Serine-esterase assay for nitrophenol release from BCV HE genes in COS-7 cells r-nitrophenyl acetate PNPA) The HE genes were transfected into COS-7 cells with The serine-esterase activity was determined according LipofectamineTM Life Technologies, Gaithersburg, to Vlasak et al. [11]; 35 ìl of the soluble protein MD, USA). At 18±24 h before transfection, COS-7 preparation or 5 ìl of puri®ed virion preparation were cells were plated at an appropriate cell density to incubated with 1m M PNPA in PBS at room tempera- obtain c. 50±80% con¯uency the following day. ture. The release of nitrophenol was measured at Plasmid DNA 2 ìg) and 10 ìl of Lipofectamine 405 nm for 10 min at intervals of 1 min. A sample of reagent were mixed for each transfection and incubated the PNPA incubated with untransfected or uninfected for 30 min at room temperature to form DNA± cell preparation was used as the background level, liposome complexes which were placed on to the cells which was subtracted from the test samples. grown in a 35-mm well with antibiotic-free NTCT medium Sigma). After incubation at 378C for 7±9 h, the transfection mixture was replaced with DMEM Sigma) with fetal bovine serum 10% and the cells Results were incubated for another 60 h. When transient Analysis of HA and RDE activities of RBCV and expression of HE was assessed by indirect immuno- EBCV ¯uorescence assay IFA), 0.5 ìg of plasmid DNA and 2.5 ìl of Lipofectamine were applied to COS-7 cells The HA and RDE activities of RBCV and EBCV were grown on a 12-mm coverslip Fisher Scienti®c, analysed before and after virus puri®cation Table 1). Pittsburgh, PA, USA). The transfected cells were ®xed Virus preparations of RBCV and EBCV agglutinated rat with methanol 60 h after transfection, incubated with RBC with HA titres of 64±128/50 ìl, and their polyclonal antiserum from BCV-infected cattle [36, 37] associated RDE titres were 4±8 before puri®cation. and then with ¯uorescein isothiocyanate FITC)- The speci®c HA and RDE titres of the virus labelled af®nity puri®ed goat anti-bovine IgG Kirke- preparations increased to 4096 and 1024, respectively, gaard and Perry Laboratories, Gaithersburg, MD, after virus puri®cation. 1122 X. Q. LIN ET AL. Table. 1. HA and RDE activities of respiratory RBCV) activities. At 378C, release of acetate from BSM by and enteropathogenic bovine coronaviruses EBVC) these COS-7 cell extracts containing the HE of EBCV HA RDE LY increased for up to 60 min Fig. 3f). In contrast, the Stage Virus titre titre ability of expressed HE of RBCV OK and LSU to Before puri®cation hydrolyse 9-O-acetylated sialic acids in BSM at this RBCV-OK-0514-3 64 4 reaction temperature increased over time for 40 min, RBCV-LSU-94LSS-051-2 128 8 and subsequently declined sharply to minimal levels of EBCV-LY138-3 128 8 activity at 60 min Fig. 3d and e). The expressed HE of After puri®cation EBCV LY rapidly decreased acetate liberation after 30- RBCV-OK-0514-3 2048 512 min reaction time at 398C, whereas the AE activity of RBCV-LSU-94LSS-051-2 4096 1024 EBCV-LY138-3 4096 1024 the expressed HE of RBCV OK and LSU was further reduced after 40 min Fig. 3d, e and f). The AE HA titres were expressed as haemagglutination units HAU)/50 ìl, re¯ecting the highest titre with complete agglutination; RDE titres activities of uninfected or untransfected cell prepara- were indicated as the highest dilutions with complete elution of tions were also tested, and the results were similar to adsorbed virions, that is the de-aggregation of previous aggregation. those obtained with puri®ed virus preparations or transfected cell lysate at 48C data not shown). Transient expression of HE on transfected COS-7 cells Comparison of HE cDNA-predicted amino-acid sequences speci®ed by EBCV and RBCV The HE genes from RBCV OK and LSU, and EBCV LY were cloned in front of the bovine growth hormone Comparison of the deduced amino-acid sequences of poly-adenylation signal sequence BGH pA) of the the RBCV OK and LSU, and EBCV LY strains transient expression vector pcDNA3.1/Zeo ‡) under revealed speci®c amino-acid differences in HE Fig. the control of the cytomegalovirus immediate early 4). The HE amino-acid sequence of EBCV LY differed promoter Pcmv) and transiently expressed in COS-7 from those of both RBCV OK and RBCV LSU at cells Fig. 1A). All three HE proteins were expressed in positions 49, 379 and 392. Both amino-acid substitu- similar amounts as indicated by IFA with BCV-speci®c tions at positions 49 and 379 were from Asn LY) to polyclonal antibody. Fluorescent signals were detected Thr OK, LSU), while the amino-acid substitution at in the cytoplasm and perinuclear sites of c.5%of position 392 was from Leu LY) to Ile OK, LSU). COS-7 cells at 69 h after transfection Fig. 1B).

Discussion Serine-esterase and AE enzymic activities of puri®ed virus preparations and extracts of transfected COS-7 cells This study examined and compared the structure and function of HE glycoproteins speci®ed by RBCV and High levels of serine-esterase activity were detected in EBCV strains. It demonstrated that the HE of RBCV puri®ed virus preparations at room temperature with has AE activity, which mediates the RDE function, a the synthetic serine-esterase substrate PNPA Fig. 2a). characteristic of BCV. Moreover, it has shown that the The RBCV OK and LSU, and EBCV LY virus enzymic activities of HE could be measured by preparations with identical pfu titres released equiva- transient expression of HE genes in COS-7 cells. This lent amounts of nitrophenol from PNPA. The COS-7 transient expression system allows the functional cell extracts containing HE speci®ed by RBCV OK and characterisation of HE proteins in the absence of any LSU, and EBCV LY strains hydrolysed PNPA effec- other BCV components. It was found that the HE of tively Fig. 2b). The COS-7 cell extracts containing HE RBCV can be differentiated from that of EBCV on the of RBCV OK exhibited a lower rate of time-dependent basis of temperature sensitivity, and unique RBCV- serine-esterase activity than those of RBCV LSU and speci®c amino-acid changes which may cause the EBCV LY. observed AE enzymic activities were identi®ed.

The AE functions of HE proteins were further tested in Overall, the serine-esterase activity assayed by the a 60-min assay by monitoring the release of acetate PNPA method indicated that transiently expressed HE from a natural esterase substrate, BSM, at 378C and was enzymically active. However, this assay did not 398C Fig. 3). Puri®ed preparations of RBCV OK and detect any differences between HE of RBCV and LSU, and EBCV LY released similar amounts of EBCV. In contrast, the AE activity tested with BSM as acetate over time at 378C Fig. 3a, b and c). Puri®ed substrate, which mimics the natural enzymic function EBCV LY preparation hydrolysed 9-O-acetylated sialic of HE, indicated that the AE activity of RBCV- acids in BSM at a higher level at 398C than it did at speci®ed HE obtained from either puri®ed virus 378C. In contrast, puri®ed RBCV OK and LSU preparations or through transient expression in COS-7 preparations had reduced AE activities at 398C than cells was more temperature-labile than that of EBCV. at 378C. Cell extracts from COS-7 cells transfected Speci®cally, the AE activities of puri®ed EBCV LY with HE genes were also assayed for their AE preparation were stable at both 378C and 398C, whereas HAEMAGGLUTININ-ESTERASE PROTEINS OF RBCV AND EBCV 1123

(A)

Hind III HE Gene Not I a T7

BGH pA Pcmv f1 ori

SV40 ori

b pcDNA 3.1/Zeo (ϩ) 5.0 kb

Amplicillin Zeocin

SV40 pA

ColE1

Fig. 1. Cloning and expression of HE gene in COS-7 cells. A) The PCR-ampli®ed HE gene was cloned into pcDNA3.1/Zeo ‡)vector. B) Expression of HE genes from RBCV-OK-0514-3 a), RBCV-LSU-94LSS-051-4 b)and EBCV-LY138-2 c) was detected after transfection into COS-7 cells by indirect immuno¯uorescence assay. The G clone cells infected with RBCV-OK-0514-3 were used as positive control d). reduced AE activity of puri®ed RBCV OK and LSU was temperature-labile at 398C, whereas HE of RBCV was observed at 398C as compared with that at 378C. In OK and LSU was not stable at either 378Cor398C. contrast, transient expression of HE in COS-7 cells This property indicates that HE interaction with other indicated that HE of EBCV LY was stable at 378C, but viral structural components may stabilise its structure 1124 X. Q. LIN ET AL. a from the predicted intramembrane-anchoring sequence. 1.5 The third RBCV-speci®c amino-acid change involved the conservative amino-acid substitution of Ile392 RBCV) from Leu EBCV). These speci®c amino- 1.2 392 acid changes may be responsible for the altered AE activities of RBCV. Future experiments with site- 0.9 directed mutagenesis should determine whether these amino-acid changes are speci®cally associated with the function of RBCV HE. 0.6 The coronavirus HE genes have 30% amino-acid sequence homology with HEF glycoprotein of in¯uenza 0.3 C virus and may have been derived by a recombination between an HEF mRNA of in¯uenza C virus and the genomic RNA of an ancestral coronavirus during a 0 mixed infection [40]. Importantly, coronavirus HE has 01234567891011 both receptor-binding [5, 8, 9] and AE-mediated recep- tor-destroying functions which are similar to the HEF 405 of in¯uenza C virus [11, 12, 14, 15]. The in¯uenza C OD b virus HEF glycoprotein mediates viral attachment to 0.4 cells through its receptor-binding activity for 9-O- acetyl-N-acetylneuraminic acid and viral penetration 0.32 through its fusion activity [7, 10, 13]. The AE of this viral protein, which hydrolyses an ester linkage to release acetate from the viral receptor determinant, 9- 0.24 O-acetyl-N-acetylneuraminic acid, allows release of mature virus from infected cells or from agglutinated erythrocytes. 0.16 The receptor-destroying activity of in¯uenza A and B viruses is mediated by a neuraminidase NA) that 0.08 catalyses the cleavage of the á-ketosidic linkage between a terminal N-acetylneuraminate and an adjacent D-galactose or D-galactosamine, and releases 0 N-acetylneuraminate from the glycoconjugates on the 01234567891011 cell surface [41, 42]. Another potential role of NA was Time (min) suggested in that it may permit transport of the virus through the mucin layer present in the respiratory tract, Fig. 2. Serine-esterase activities associated with puri®ed facilitating the virus interaction with the target RBCV and EBCV, and expressed HE proteins from epithelial cells. Some NA N1and N9 subtypes) have RBCV and EBCV. r-Nitrophenyl acetate was incubated at room temperature with a) puri®ed RBCV-OK-0514-3 both a receptor- and a NA ±h±), RBCV-LSU-94LSS-051-2 ´ ´ ´e´ ´ ´) and EBCV- [43, 44]. However, the receptor speci®city and function LY138-3 - -s--); and b) expressed HE proteins from of this receptor-binding activity is still unknown. The RBCV-OK-0514-3 ±h±), RBCV-LSU-94LSS-051-4 precise role of HE glycoprotein in coronavirus evolu- ´´´e´ ´ ´), and EBCV-LY138-2 - -s--). Data are means tion, replication and pathogenesis remains a mystery. It and SEM n ˆ 3). has been suggested that the coronavirus HE serves as a second virus attachment protein in addition to the S and enzymic functions. Multiprotein interaction be- glycoprotein peplomer [12, 45]. Theoretically, a re- tween S and HE glycoproteins was ®rst suggested by markable selective advantage is provided by the HE to experiments with MAbs against HE of EBCV-L9 which coronaviruses in infections of mucous membrane. The inhibited both haemagglutination and AE activities HE could facilitate virus release and spread, virus [38, 39]. migration through the mucus layer, and ultimately virus infection of epithelial cells through its speci®c and The HE cDNA-predicted amino-acid sequences speci- reversible binding to the substrate, cleavage and ®ed by RBCV contained three amino-acid substitutions. rebinding. Two substitutions constituted non-conservative changes of the small polar Asn to the small non-polar Thr. The EBCV primarily causes severe diarrhoea in These substitutions were located nine amino acids neonatal calves and winter dysentery of adult cattle immediately downstream from the conserved esterase by infecting absorptive epithelial cells of the small active site FGDS40) and 18 amino acids upstream intestine [46±48]. The temperature at this site equals HAEMAGGLUTININ-ESTERASE PROTEINS OF RBCV AND EBCV 1125

a d 5 4

4 3

3

2 2

1 1

0 0

b e 5 4

4 3

3 g) µ 2 2 Acetate (

1 1

0 0

f 5 c 4

4 3

3 2 2

1 1

0 0 010203040506070 010203040506070

Time (min)

Fig. 3. Acetate release from bovine submaxillary mucin BSM) by puri®ed RBCV and EBCV, and expressed HE proteins from RBCV and EBCV. Puri®ed RBCV-OK-0514-3 a), RBCV-LSU-94LSS-051-2 b), EBCV-LY138-3 c), and expressed HE proteins from RBCV-OK-0514-3 d), RBCV-LSU-94LSS-051-4 e) and EBCV-LY138-2 f)were incubated with BSM at 378C h) and 398C e). Data are means and SEM n ˆ 3). the body temperature of around 398C of cattle, contribute to RBCV infections of respiratory tracts of typically higher than the temperature of the epithelial cattle. The AE of the RBCV strains may have lost their surface of air passages, which is about 378C. It is stability at 398C because these viruses evolved through hypothesised that the observed AE activities of HE replication in respiratory tissues. Conversely, it is described in this paper may directly or indirectly possible that conformational changes leading to less 1126 X. Q. LIN ET AL.

103 13366534937–40182085A.A. 1 103 13366534937–40182085A.A.

RBCV OK P V D FGDS T R G* V V

RBCV LSU P V D FGDS T R D V L*

EBCV LY P V D FGDS N R D V V

0 1 36939534527..367400 415396392379357344350297A.A. 424

RBCV OK R D Q D P T I R*

RBCV LSU M* D Q D P T I G

EBCV LY R D Q D P N L G

Fig. 4. Comparison of the predicted amino-acid sequences of the HE proteins speci®ed by different bovine coronavirus strains. The compared strains included newly isolated RBCV strains OK and LSU, and prototype EBCV wild-type strain LY. Amino acids A.A.) 1±18 are the predicted signal peptide and amino acids 396±415 are the predicted intramembrane-anchoring sequence. ÃRBCV strain-speci®c amino-acid changes in comparison with EBCV LY. Boxed amino acids are unique to RBCV.

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