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David A. Brian - One of the best experts on this subject based on the ideXlab platform.
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Bovine Coronavirus 5′-Proximal Genomic Acceptor Hotspot for Discontinuous Transcription Is 65 Nucleotides Wide
Journal of virology, 2006Co-Authors: Aykut Ozdarendeli, David A. BrianAbstract:Coronaviruses are positive-strand, RNA-dependent RNA polymerase-utilizing viruses that require a polymerase template switch, characterized as discontinuous transcription, to place a 5′-terminal genomic leader onto subgenomic mRNAs (sgmRNAs). The usually precise switch is thought to occur during the synthesis of negative-strand templates for sgmRNA production and to be directed by heptameric core donor sequences within the genome that match an acceptor core (UCUAAAC in the case of Bovine Coronavirus) near the 3′ end of the 5′-terminal genomic leader. Here it is shown that a 22-nucleotide (nt) donor sequence engineered into a packageable Bovine Coronavirus defective interfering (DI) RNA and made to match a sequence within the 65-nt virus genomic leader caused a template switch yielding an sgmRNA with only a 33-nt minileader. By changing the donor sequence, acceptor sites between genomic nt 33 and 97 (identical between the DI RNA and the viral genome) could be used to generate sgmRNAs detectable by Northern analysis (∼2 to 32 molecules per cell) by 24 h postinfection. Whether the switch was intramolecular only was not determined since a potentially distinguishing acceptor region in the DI RNA rapidly conformed to that in the helper virus genome through a previously described template switch known as leader switching. These results show that crossover acceptor sites for discontinuous transcription (i) need not include the UCUAAAC core and (ii) rest within a surprisingly wide 5′-proximal “hotspot.” Overlap of this hotspot with that for leader switching and with elements required for RNA replication suggests that it is part of a larger 5′-proximal multifunctional structure.
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Bovine Coronavirus 5 proximal genomic acceptor hotspot for discontinuous transcription is 65 nucleotides wide
Journal of Virology, 2006Co-Authors: Aykut Ozdarendeli, David A. BrianAbstract:Coronaviruses are positive-strand, RNA-dependent RNA polymerase-utilizing viruses that require a polymerase template switch, characterized as discontinuous transcription, to place a 5′-terminal genomic leader onto subgenomic mRNAs (sgmRNAs). The usually precise switch is thought to occur during the synthesis of negative-strand templates for sgmRNA production and to be directed by heptameric core donor sequences within the genome that match an acceptor core (UCUAAAC in the case of Bovine Coronavirus) near the 3′ end of the 5′-terminal genomic leader. Here it is shown that a 22-nucleotide (nt) donor sequence engineered into a packageable Bovine Coronavirus defective interfering (DI) RNA and made to match a sequence within the 65-nt virus genomic leader caused a template switch yielding an sgmRNA with only a 33-nt minileader. By changing the donor sequence, acceptor sites between genomic nt 33 and 97 (identical between the DI RNA and the viral genome) could be used to generate sgmRNAs detectable by Northern analysis (∼2 to 32 molecules per cell) by 24 h postinfection. Whether the switch was intramolecular only was not determined since a potentially distinguishing acceptor region in the DI RNA rapidly conformed to that in the helper virus genome through a previously described template switch known as leader switching. These results show that crossover acceptor sites for discontinuous transcription (i) need not include the UCUAAAC core and (ii) rest within a surprisingly wide 5′-proximal “hotspot.” Overlap of this hotspot with that for leader switching and with elements required for RNA replication suggests that it is part of a larger 5′-proximal multifunctional structure.
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Bovine Coronavirus I protein synthesis follows ribosomal scanning on the bicistronic N mRNA
Virus research, 1997Co-Authors: Savithra D. Senanayake, David A. BrianAbstract:Abstract The mRNA encoding the 49-kDa nucleocapsid protein (N) of the Bovine Coronavirus is bicistronic. A 23-kDa protein, termed the I protein for the ‘internal’ open reading frame (ORF), is also synthesized but in the +1 reading frame beginning 61 nt downstream of the N start codon. Sequences flanking the N and I start codons suggest that the I ORF might be accessed by scanning ribosomes passing over the N start codon. Here we test this idea and demonstrate with translation studies both in vitro and in vivo that the I protein is synthesized according to the leaky scanning model for initiation of translation on the subgenomic N mRNA molecule.
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the ucuaaac promoter motif is not required for high frequency leader recombination in Bovine Coronavirus defective interfering rna
Journal of Virology, 1996Co-Authors: Ruey-yi Chang, Rajesh Krishnan, David A. BrianAbstract:The 65-nucleotide leader on the cloned Bovine Coronavirus defective interfering (DI) RNA, when marked by mutations, has been shown to rapidly convert to the wild-type leader of the helper virus following DI RNA transfection into helper virus-infected cells. A model of leader-primed transcription in which free leader supplied in trans by the helper virus interacts by way of its flanking 5'UCUAAAC3' sequence element with the 3'-proximal 3'AGAUUUG5' promoter on the DI RNA minus strand to prime RNA replication has been used to explain this phenomenon. To test this model, the UCUAAAC element which occurs only once in the BCV 5' untranslated region was either deleted or completely substituted in input DI RNA template, and evidence of leader conversion was sought. In both cases, leader conversion occurred rapidly, indicating that this element is not required on input RNA for the conversion event. Substitution mutations mapped the crossover region to a 24-nucleotide segment that begins within the UCUAAAC sequence and extends downstream. Although structure probing of the Bovine Coronavirus 5' untranslated region indicated that the UCUAAAC element is in the loop of a prominent stem and thus theoretically available for base pair-directed priming, no evidence of an unattached leader early in infection that might have served as a primer for transcription was found by RNase protection studies. These results together suggest that leader conversion on the DI RNA 5' terminus is not guided by the UCUAAAC element and might arise instead from a high-frequency, region-specific, homologous recombination event perhaps during minus-strand synthesis rather than by leader priming during plus-strand synthesis.
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Evidence for a Pseudoknot in the 3′ Untranslated Region of the Bovine Coronavirus Genome
Advances in Experimental Medicine and Biology, 1995Co-Authors: Gwyn D. Williams, Ruey-yi Chang, David A. BrianAbstract:A potential pseudoknot was found in the 3′ untranslated region of the Bovine Coronavirus genome beginning 63 nt downstream from the stop codon of the N gene. Mutation analysis of the pseudoknot in a cloned defective interfering RNA indicated that this structural element is necessary for defective interfering RNA replication.
Dongwan Yoo - One of the best experts on this subject based on the ideXlab platform.
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Targeted RNA recombination of the membrane and nucleocapsid protein genes between mouse hepatitis virus and Bovine Coronavirus.
Journal of Veterinary Science, 2001Co-Authors: Gabriella Koljesar, Dongwan YooAbstract:Abstract The targeted RNA recombination was attempted to substitute the membrane (M) protein gene and part of the nucleocapsid (N) protein gene of mouse hepatitis virus with the corresponding sequences from Bovine Coronavirus. Using a defective interfering (DI) RNA-like cDNA construct derived from pMH54, 690 nucleotides representing the entire M gene and the 5' most 915 nucleotides of the N gene of the mouse hepatitis virus Albany 4 mutant were attempted to be replaced. Upon infection of cells with Albany 4 followed by transfection with synthetic RNA transcribed from the DI-like cDNA construct, recombinant mouse hepatitis viruses as the large plaque forming phenotype were isolated by plaque assays at the non-permissive temperature of 391 degrees C. By RT-PCR and sequencing, those large plaque phenotypes were confirmed to have contained the thermostable phenotype marker derived from the transfected RNA, demonstrating that recombination occurred between the Albany 4 genomic RNA and the in vitro RNA transcripts. Further analysis of the recombinant viruses indicated that there combination had taken place within the region of 222 nucleotides between positions 916 and 1,137 of the N gene. This is the region immediately downstream of the replacement sequence and the start of the temperature resistant phenotype marker. The results suggest that the M and part of the N genes of Bovine Coronavirus may not be able to complement the function of those of mouse hepatitis virus. This study redirects our current approach of utilizing the MHV targeted RNA recombination as a means to study Bovine Coronavirus genetics towards the construction of an infectious cDNA clone.
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Full-Length Genomic Sequence of Bovine Coronavirus (31kb)
Advances in experimental medicine and biology, 2001Co-Authors: Dongwan Yoo, Yanlong PeiAbstract:Bovine Coronavirus (BCV) is an important veterinary pathogen which causes neonatal diarrhea in newborn calves and winter dysentery in adult cattle. Recent studies indicate that BCV also infects the respiratory tract of cattle producing severe respiratory problems, especially in feedlot cattle. BCV belongs to the antigenic group II of Coronaviruses and shares the antigenic and genetic similarities with mouse hepatitis virus (MHV), human Coronavirus strain OC43, turkey Coronavirus, and hemagglutinating encephalomyelitis virus of pigs. Approximately 10 kb of the 3’ most BCV genome has been sequenced, and this region is known to encode all the structural proteins plus the 32k nonstructural protein which resides immediately upstream of the hemagglutinin-esterase (HE) glycoprotein gene. The comparative studies indicate that the genome organization of BCV is similar to but distinct from MHV. In BCV, the HE gene is functional coding for a major envelop protein while it is an optional gene in MHV. Similarly, the 32k protein gene is absent in some of the MHV variants. The region between the spike (S) protein gene and the small membrane (E) protein gene also shows a significant sequence divergence among Coronaviruses. Two thirds of the BCV genome remain largely undetermined.
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Phosphorylation of the Nucleocapsid Protein of Bovine Coronavirus Expressed with a Recombinant Baculovirus Vector
Journal of Microbiology and Biotechnology, 1992Co-Authors: Dongwan Yoo, G J Cox, Ick Dong YooAbstract:Post-translational modifications of the nucleocapsid protein of Bovine Coronavirus (Quebec strain) were investigated. Coronavirions were radiolabelled in vivo with inorganic orthophosphate and analysed by SDS-PAGE, followed by autoradiography. A single polypeptide with a migration rate of 55 KDa was identified by metabolic phosphate labelling, demonstrating that the nucleocapsid protein of Bovine Coronavirus was a phosphoprotein. A gene encoding the nucleocapsid protein was inserted immediately downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis baculovirus. Spodoptera frugiperda cells infected with this recombinant baculovirus synthesized a 55 KDa polypeptide, as demonstrated by immunoprecipitation with anti-nucleocapsid monoclonal antibody. The recombinant nucleocapsid protein synthesized in Spodoptera cells could also be labelled by orthophosphate. Phosphoamino acid analysis showed that both serine and threonine residues were phosphorylated in authentic, as well as in recombinant nucleocapsid proteins, with a relative phosphorylation ratio of 7:3. Our studies demonstrated that the nucleocapsid protein of Bovine Coronavirus was a serine and threonine-phosphorylated protein and that Spodoptera insect cells were able to properly phosphorylate the relevant foreign proteins.
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The S2 subunit of the spike glycoprotein of Bovine Coronavirus mediates membrane fusion in insect cells.
Virology, 1991Co-Authors: Dongwan Yoo, Michael D. Parker, Lorne A. BabiukAbstract:The hemagglutinin/esterase (HE), spike precursor (S) and the S1 and S2 subunits of the spike precursor protein of Bovine Coronavirus were expressed in Spodoptera frugiperda (Sf9) cells, and the cell-fusing activity of each recombinant glycoprotein was examined. Extensive syncytia formation was observed in cells infected with the S2 recombinant but not with the HE or S1 recombinant baculoviruses. Fusion of Sf9 cells expressing the intact S protein precursor was evident after trypsin treatment. These results demonstrate that proteolytic cleavage of the S spike precursor is required for fusion induction and that the fusion is mediated by the S2 subunit. These observations may reflect the biological role of the S2 subunit in fusion-penetration during Bovine Coronavirus infection.
Sanjay Kapil - One of the best experts on this subject based on the ideXlab platform.
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Bovine Coronavirus associated syndromes.
Veterinary Clinics of North America-food Animal Practice, 2010Co-Authors: Melanie J. Boileau, Sanjay KapilAbstract:Bovine Coronaviruses, like other animal Coronaviruses, have a predilection for intestinal and respiratory tracts. The viruses responsible for enteric and respiratory symptoms are closely related antigenically and genetically. Only 4 Bovine Coronavirus isolates have been completely sequenced and thus, the information about the genetics of the virus is still limited. This article reviews the clinical syndromes associated with Bovine Coronavirus, including pneumonia in calves and adult cattle, calf diarrhea, and winter dysentery; diagnostic methods; prevention using vaccination; and treatment, with adjunctive immunotherapy.
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Characterization of Bovine Coronavirus isolates/from eight different states in the USA
Veterinary microbiology, 1999Co-Authors: Sanjay Kapil, Kay L. Richardson, Trisha R Maag, Sagar M. GoyalAbstract:Bovine Coronavirus isolates from eight different states of the USA were compared for their antigenic properties and susceptibility to hygromycin B. Antigenic differences were observed among the isolates in a one-way hemagglutination-inhibition (HI) test using a polyclonal antiserum against the Mebus Bovine Coronavirus isolate. Differences were observed on isoelectric focusing among viral proteins with isoelectric points between 4.45-4.65. Most of the BCV isolates were susceptible to hygromycin B (0.5 mM) whereas a few hygromycin B resistant isolates were also found.
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characterization of Bovine Coronavirus isolates from eight different states in the usa
Veterinary Microbiology, 1999Co-Authors: Sanjay Kapil, Kay L. Richardson, Trisha R Maag, Sagar M. GoyalAbstract:Bovine Coronavirus isolates from eight different states of the USA were compared for their antigenic properties and susceptibility to hygromycin B. Antigenic differences were observed among the isolates in a one-way hemagglutination-inhibition (HI) test using a polyclonal antiserum against the Mebus Bovine Coronavirus isolate. Differences were observed on isoelectric focusing among viral proteins with isoelectric points between 4.45-4.65. Most of the BCV isolates were susceptible to hygromycin B (0.5 mM) whereas a few hygromycin B resistant isolates were also found.
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Development and Applications of a Bovine Coronavirus Antigen Detection Enzyme-Linked Immunosorbent Assay
Clinical and diagnostic laboratory immunology, 1999Co-Authors: S. L. Schoenthaler, Sanjay KapilAbstract:We developed a monoclonal antibody-based, antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for Bovine Coronavirus. We compared the ELISA with electron microscopy and the hemagglutination test and found a close correlation between them. The sensitivity of the ELISA was 104 Bovine Coronavirus particles per ml of 10% fecal suspension. Compared with electron microscopy, Bovine Coronavirus ELISA had 96% specificity.
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Application of immunohistochemistry and in situ hybridization for detection of Bovine Coronavirus in paraffin-embedded, formalin-fixed intestines.
Journal of clinical microbiology, 1997Co-Authors: Z Zhang, G A Andrews, C Chard-bergstrom, H C Minocha, Sanjay KapilAbstract:A monoclonal antibody (MAb) (Z3A5) against spike protein subunit of Bovine Coronavirus (BCV) reacted with the virus in formalin-fixed intestines in an immunoperoxidase test. We found an 88% correlation between immunohistochemistry with Z3A5 and in situ hybridization with a BCV nucleoprotein cDNA probe. MAb Z3A5 reacted with 90 BCV isolates from the United States and was an effective reagent for the diagnosis of BCV.
Sagar M. Goyal - One of the best experts on this subject based on the ideXlab platform.
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characterization of Bovine Coronavirus isolates from eight different states in the usa
Veterinary Microbiology, 1999Co-Authors: Sanjay Kapil, Kay L. Richardson, Trisha R Maag, Sagar M. GoyalAbstract:Bovine Coronavirus isolates from eight different states of the USA were compared for their antigenic properties and susceptibility to hygromycin B. Antigenic differences were observed among the isolates in a one-way hemagglutination-inhibition (HI) test using a polyclonal antiserum against the Mebus Bovine Coronavirus isolate. Differences were observed on isoelectric focusing among viral proteins with isoelectric points between 4.45-4.65. Most of the BCV isolates were susceptible to hygromycin B (0.5 mM) whereas a few hygromycin B resistant isolates were also found.
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Characterization of Bovine Coronavirus isolates/from eight different states in the USA
Veterinary microbiology, 1999Co-Authors: Sanjay Kapil, Kay L. Richardson, Trisha R Maag, Sagar M. GoyalAbstract:Bovine Coronavirus isolates from eight different states of the USA were compared for their antigenic properties and susceptibility to hygromycin B. Antigenic differences were observed among the isolates in a one-way hemagglutination-inhibition (HI) test using a polyclonal antiserum against the Mebus Bovine Coronavirus isolate. Differences were observed on isoelectric focusing among viral proteins with isoelectric points between 4.45-4.65. Most of the BCV isolates were susceptible to hygromycin B (0.5 mM) whereas a few hygromycin B resistant isolates were also found.
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Evaluation of shell vial cell culture technique for the detection of Bovine Coronavirus.
Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians Inc, 1995Co-Authors: Rafique A. Tahir, Kern A. Pomeroy, Sagar M. GoyalAbstract:The effect of blind passage and centrifugation on the isolation of Bovine Coronavirus in human rectal tumor cells cultured in shell vials was investigated. A total of 68 fecal samples known to be positive for Bovine Coronavirus by transmission electron microscopic (TEM) examination were used. The samples were centrifuged onto human rectal tumor cell monolayers and incubated in the presence of trypsin. The growth of Bovine Coronavirus in infected cells was demonstrated by fluorescent antibody staining, and the extracellular virus was detected and confirmed by hemagglutination and hemagglutination-inhibition tests, respectively. Of the 68 TEM-positive samples, 51 (75%), 58 (85%), and 61 (90%) grew in shell vial cell cultures at first, second, and third passages, respectively. Of the 51 cultures positive on first passage, 19 were examined by TEM; 18 of these were positive for Bovine Coronavirus. The shell vial technique was also compared with direct detection of Bovine Coronavirus by staining cryostat sections of infected tissues in a direct fluorescent antibody assay. The results of direct fluorescent antibody assay were available for 54 of the 68 samples, of which 53 (98%) and 43 (80%) were positive by shell vial technique and direct fluorescent antibody assay, respectively. For identification of Bovine Coronavirus, shell vials using human rectal tumor cells in the presence of trypsin is more sensitive than direct fluorescent antibody assay but is relatively less sensitive than transmission electron microscopy.
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Experimental infection with a virulent pneumoenteric isolate of Bovine Coronavirus.
Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians Inc, 1991Co-Authors: Sanjay Kapil, Kern A. Pomeroy, Sagar M. Goyal, Ava M. TrentAbstract:Bovine Coronavirus (BCV) is 1 of the important causes of scours in young calves. Although the role of BCV in respiratory tract infections has not been clearly defined, there is increasing evidence that it causes upper respiratory tract infection in addition to the intestinal disease. Focal involvement of lungs was recently reported in 2 of 16 calves experimentally infected with BCV, but the sequence of events including clinical signs has not been clearly described for pneumotropic BCV. The present report describes a consistent, diffuse lung involvement in calves experimentally infected with a field isolate of BCV. The details of the experimental design have been described. Briefly, newborn, colostrum-deprived, unvaccinated calves were infected orally at 5 days of age with either a virulent pneumoenteric isolate of BCV (Minnesota isolate, calves 2, 6, and 7) or with an attenuated strain of BCV (Mebus strain, calves 1 and 9). The virus suspension was fed slowly to the calves with a 20-ml syringe. Although direct inoculation of the virus into the nasal cavity was unlikely, it was not possible to control the carryover of virus to the nasal cavity via insertion of the tongue into the nostril. Four calves (calves 3, 4, 5, and 8), housed in separate rooms were included as uninfected controls. One of the uninfected controls (calf 3) developed a natural infection with BCV. Nasal cells were collected by inserting cotton swabs into the anterior nasal cavity of the calves. The swabs were rotated gently to dislodge the cells of the nasal cavity. Sufficient number of cells were collected without difficulty. The swabs were placed in Hanks’ balanced salt solution (HBSS) and immediately transported to the laboratory where they were vortexed for 1 minute to separate nasal cells from the swab and the mucus. The swab was removed from the tube and the resulting suspension was centrifuged at 650 x g for 5 minutes. The pellet (approx. 0.05 ml packed cells) was mixed with equal volume of HBSS and vortexed again. The cell suspension was placed on 8-well slides with 30 μ1/well and allowed to dry at room temperature. The slides were washed with phosphate-buffered saline (PBS), pH 7.2, dried, and then fixed in acetone for 10 minutes. After drying again, the wells were covered with a drop of FITC-labeled Bovine anti-BCV conjugate and incubated in a humid chamber for 1 hour. The conjugate was found to be specific for Bovine Coronavirus and did not react with uninfected Madin-Darby Bovine kidney cells or with uninfected tissues from control calves (gut and lungs). The slides were washed with PBS (pH 8.5) and counterstained with Evan’s blue. The calves were kept under strict quarantine and were
Lorne A. Babiuk - One of the best experts on this subject based on the ideXlab platform.
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Bovine Coronavirus nonstructural protein ns2 is a phosphoprotein.
Virology, 1991Co-Authors: Michael D. Parker, Lorne A. BabiukAbstract:Abstract To investigate the nature of the Bovine Coronavirus (BCV) ns2 protein, the gene encoding this protein was cloned and was expressed as a β-galactosidase fusion protein. Antiserum raised against this protein reacted specifically with BCV-infected fixed cells in indirect immunofluorescence microscopy and precipitated an in vitro synthesized product approximately 32-kDa in molecular weight and an equivalent protein from BCV-infected cells. The synthesis of ns2 was found to be similar to the structural proteins of BCV and pulse-chase experiments indicated that ns2 protein was stable and that it accumulated in BCV-infected cells. Synthesis of ns2 in the presence of [31P] orthophosphate revealed that it is a phosphoprotein. Phosphoamino acid analysis confirmed the phosphorylated nature of ns2 and identified serine and threonine as its phosphorylated amino acid residues. This is the first demonstration of a phosphorylated nonstructural protein in Coronavirus-infected cells.
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The S2 subunit of the spike glycoprotein of Bovine Coronavirus mediates membrane fusion in insect cells.
Virology, 1991Co-Authors: Dongwan Yoo, Michael D. Parker, Lorne A. BabiukAbstract:The hemagglutinin/esterase (HE), spike precursor (S) and the S1 and S2 subunits of the spike precursor protein of Bovine Coronavirus were expressed in Spodoptera frugiperda (Sf9) cells, and the cell-fusing activity of each recombinant glycoprotein was examined. Extensive syncytia formation was observed in cells infected with the S2 recombinant but not with the HE or S1 recombinant baculoviruses. Fusion of Sf9 cells expressing the intact S protein precursor was evident after trypsin treatment. These results demonstrate that proteolytic cleavage of the S spike precursor is required for fusion induction and that the fusion is mediated by the S2 subunit. These observations may reflect the biological role of the S2 subunit in fusion-penetration during Bovine Coronavirus infection.