key: cord-0689192-uv056oxq authors: Saif, Linda J.; Heckert, Robert A.; Miller, Kathy L.; Tarek, Mohamed M. title: Cell culture propagation of bovine coronavirus date: 1988 journal: J Tissue Cult Methods DOI: 10.1007/bf01404267 sha: 8dbaa4e3335c02bc77a8b2e729bbaa88a1768c97 doc_id: 689192 cord_uid: uv056oxq Although most field strains of bovine coronavirus (BCV) grow poorly in cell culture and fail to produce cytopathic effects (CPE) until after blind passage, primary calf kidney (PCK) and Vero cells have permitted primary isolation of virus. Cell culture-adapted strains of BCV replicate in PCK, bovine embryonic lung, bovine fetal thyroid, bovine fetal brain, bovine skin cells, ovine fetal kidney cells, and the cell lines pig kidney K3 and 15, Vero, human embryonic lung fibroblasts, HRT-18, MDBK and BEK-1, with trypsin useful for enhancing replication. Organ culture as well as suckling mouse, rat, and hamster brains also support the growth of cell culture-adapted BCV strains. Viral growth is most commonly detected by CPE, immunofluorescence, hemagglutination, and hemadsorption assays or electron microscopy of supernatants from infected cells. In this report, the optimal conditions for the growth and plaque assay of the NCDV strain of BCV in MDBK cells are described. The coronaviruses were first recognized and morphologically defined as a group by Tyrrell et al., (39-41 j but have have been known by other names for almost 5 decades (33) . The Coronaviridae is a monogeneric family comprising 11 viruses that infect vertebrates (29) . II. Stair et al. (30) first described the isolation and partial characterization of a eoronavirus-like agent from calves A. with diarrhea. The viral particles were 107 to 160 nm, polymorphic, and had an ll-nm petal-shaped fringe. Sharpee et al. {28) further characterized this coronavirus-like agent and showed that it possessed all six major properties common to members of the Coronaviridae family. The virion is composed of four proteins and possesses a lipid bilayer. The genome consists of a single-stranded polyadeylated RNA which is infectious and of positive polarity. B. Bovine coronavirus (BCV) causes severe diarrhea in calves 3 to 30-d old which can result in death due to dehydration and acidosis (18, 24, 30) . In addition to enteric replication of BCV, several workers have reported respiratory replication and clinical signs of upper respiratory infection. Thomas Journal of Tissue Culture Methods Vol. 11, No. 3, 1988 139 In this report, the optimal cell culture conditions for the growth and plaque assay of the NCDV {Mebus) strain of BCV in MDBK cells are described. Also included is a review of the literature pertaining to the cultivation of BCVs in cell and organ cultures. if. Autoclave at 15 lb pressure for 15 min. If a precipitate occurs when autoclaving this can be subsequently prevented by the addition of 0.6 ml of 1 N HCI to reduce the pH to about 6.5 before autoclaving. Amount depends on color. iii. The solution is stored at 4 ° C. iv. Before use, the pH of the HBSS is increased to the desired point by the addition of sterile NaHCO3 solution IpH 7.2). In our laboratory a 7% stock solution is used which has been sterilized by filtration through a Seitz filter using positive pressure. 11. Invert plates gently with agar side up and place plates in an incubator at 37 ° C in 7% CO2 and at least 85% humidity. Plaques will appear within 3 or4 d. plaque assay. Plaques appeared within 2 to 3 d as opalescent areas, which remained colourless after neutral red or crystal violet staining. {42) Tektoff et al. (35, 36) also proved that HRT-18 (RPMI 1640, 20% FBS), Vero (Medium 199, 5% FBSL and MDBK (Eagle-Earle medium, 5% FBS, 0.25 g/liter casein hydrolysate) cell lines were permissive to cell culture-adapted strains of BCV. Infectivity and viral morphogenesis were followed by transmission electron microscopy, hemadsorption and scanning electron microscopy, immunofluorescence, and hemagglutination (HA) assays. The Mebus cell culture-adapted strain of BCV has also been propagated in the MA-321 strain of human embryonic lung fibroblasts (Eagle's minimum essential medium, 10% FBSL HA titers with rat or chicken erythocytes reached a peak at 72 h and remained stable until 144 h postinfection. At 4 to 5 d CPE appeared as refractile, oval or rounded cells, with a cytoplasm rich in microvacuoles. Complete degeneration of cell monolayers was observed 6 to 10 d postinfection (12 L A porcine renal cell culture-adapted strain of BCV (SC-1) was grown in Passages 2 to 5 of primary fetal (3 to 4 mo. gestation) ovine renal cells {Eagle's minimum essential medium, 10 to 15% FBSL CPE was observed at 48 to 72 h postinoculation as formation of syncytia. Virus replication was detected by hemadsorption of rat erythrocytes and cell culture supernatant HA titers of 1/64 to 1/128 (3L BCV has also been shown to 'replicate in calf testicle cells (23L and D2 bovine fetal spleen cells (7) . Organ culture of BCV has been achieved in two organ systems. Stott et al. {32) showed that the Mebus cell culture-adapted strain would produce hemagglutinating activity and replicated in 5-to 6-too. gestation fetal bovine tracheal organ culture {Eagle's basal medium, 0.14% sodium bicarbonate, 0.09% bovine plasma albumin, 5% tryptose phosphate broth, antibiotics, HEPES buffer, pH 7.2). Hemagglutinin titers increased with viral passage. Positive immunoftuorescence was first observed at 7 d and peaked Conditions for enhancing growth of cell cultureadapted strains of BCV in certain cell culture systems have been described. Dea et al. {81 showed that the factors important in increasing yield and appearance of CPE in Vero cells were a) a slightly acidic inoculum (pH 6.5 to 7.0k b) growth in a basic medium ipH 8.0 to 8.5k c) exposure to hypertonic medium; d) polycation DEAE-dextran {25 #g) treatment of cells; e) washing of cells with medium containing trypsin (5 #g/ml); and f} incubation of cells with dactinomycin (0.01 to 0.05 t~g). Storz et al. (311 also showed the importance of trypsin in the replication and cytopathogenicity of the cell culture-adapted L9 strain of BCV. Trypsin treatment (10 ~g/mt) accelerated CPE and size of plaques, facilitated cell fusion, improved the amount of cellreleased hemagglutinin, and increased the infectivity yields in bovine fetal thyroid {BFTy) and bovine fetal brain cells {BFB) {Eagle minimum essential medium, antibiotics, 10% heat-inactivated fetal calf or lamb serumL When virus was pretreated with trypsin or trypsin was present only during viral adsorption, plaque enhancement was not observed. Trypsin was also important for replication of the Mebus strain of BCV in bovine embryonic lung cells (Eagle's minimal essential medium, 0.22% sodium bicarbonate, 20 mM HEPES buffer, 0.01% pyruvic acid, 0.029% L-glutamine, 10% FBS, antibiotics)(38L Toth showed that increased amounts of trypsin (1,5,or 10 #g/ml) increased the susceptibility of bovine embryonic lung cells to BCV a millionfold. Hirano et al. (13) found that when trypsin, at a final concentration of 5 gg/ml, was added to both the viral diluent and the overlay medium, plaques increased in size and numbers on BEK-1 cells infected with the Kakegawa strain of BCV. This did not occur if 1% trypsin was added to either the viral diluent or the overlay medium alone. Cyr-Coats and Storz (71 demonstrated that the celladapted BCV L9 strain could be induced to replicate noncytopathically in D2 bovine fetal spleen ceils only in the presence of trypsin. The bovine fetal spleen cells were however nonpermissive for all wild-type BCV, even in the presence of trypsin. The mechanism(s) whereby trypsin enhances coronavirus replication is not yet fully understood. Tryspin treatment of cells might promote attachment of virions by uncovering otherwise unavailable receptor sites. Trypsin treatment of virus might modify configurations of protein molecules in the virus envelope to render them more compatible with cellular receptor sites. Alternatively, trypsin may destroy a broadly active viral inhibitor produced by cells in culture, thereby allowing multiple rounds of viral replication (4, 9, 14 ,38}. Bovine coronavirus can also be propagated by animal inoculation. This may be a useful, if not necessary, method of producing large pools of field BCV strains which may not otherwise propagate well in cell culture systems. The LY-138 strain of BCV, first isolated in 143 1965, was maintained by oral inoculation of conventional, colostrum-deprived calves for many years by this method (11 ). Akashi et al. (2) serially passaged the Kakegawa strain of BCV in suckling mice, rats, and hamsters by inoculation with brain emulsions from infected laboratory animals. The Kakegawa strain, at Passage 10 in primary bovine kidney cell cultures, was inoculated intracerebraUy and subcutaneously into the laboratory animals. Infected animals showed nervous symptoms, and died. The recovered virus could be clearly differentiated from mouse hepatitis virus by cross-neutralization tests. The Mebus strain of BCV was also adapted to suckling mouse brain by Kaye In our laboratory we have successfully propagated NCDV BCV to high titers (107 pfu/ml) in roller bottles of MDBK cells using pancreatin in the maintenance medium. We have also developed a plaque assay for BCV in these cells by adding pancreatin and DEAE to the agar overlay medium. V. Properties of a coronavirus isolated from a cow with epizootic diarrhea Propagation of the Kakegawa strain of bovine coronavirus in suckling mice, rats and hamsters Le test d'inhibition de l'hemaggintination {IHA) pour le diagnostic de l'infection a coronavirus chez les bovins Broadly active inhibitor of viruses spontaneously produced by many cell types in culture Pneumonic a eoronavirus ehez les bovine: A propos de deux observations Replication of an enteric bovine coronavirus in intestinal organ cultures Bovine coronavirus cytopathic expression and plaque formation: host cell and virus strain determine trypsin dependence. 68th Annual Conference of Research Workers in Animal Disease Bovine coronavirus isolation and cultivation in continuous cell lines Phosphatidybserine inhibition of OC43 and NCDCV coronavlrus infectivity Preliminary studies of a bovine coronavirus (BCV) antigen responsible for neutralization Morphology and morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves Antigenic and biological relationships between human coronavirus OC43 and neonatal calf diarrhoea coronavirus Plaque assay of bovine coronavirus in BEK-1 cells Cell-produced viral inhibitor: possible mechanism of action and chemical composition Replication of bovine coronavirus in cell line BEK~I culture Calf diarrhoea coronavlrus. Lancet I h 5~ Adaptation d'un isolement de eoronavirus enterlque bovine a la culture eelluiaire et caracterisation de la souche obtenue Coronaviral enteritis of young calves: virologic and pathologic findings in naturally occurring infections Enterites virales chex l'bomme et l'animal; viral Enteritis in humans and animals Une tignee cellulaire particulierement sensible a la replication du coronavirus enteritique bovin: les ceUules HRT 18 Coronavirus infection of the bovine respiratory tract Neonatal calf diarrhea: propagation, attenuation, and characteristics of a c~ronavirus-like agent Incurved disc from particles associated with a bovine coronavirus growing in Vero and call testicle cell cultures Altered metabolism, acute shock, and therapeutic response in a calf with severe coronavirus-induced diarrhea Coronavlrus replication in the intestinal and respiratory tracts during infection of calves Experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts Physico-chemlcal properties of call diarrhea coronavirus Characterization of a calf diarrheal coronavirus The biology of coronavirnses Neonatal calf diarrhea: purification and electron microscopy of a coronavirus-like agent Enhancement of plaque formation and ceil fusion of an enteropathogenic coronavirus by trypsin treatment Replication of a bovine coronavirus in organ cultures of foetal trachea The molecular biology of coronaviruses Epizootic diarrhoea of adult cattle associated with a coronavirus-like agent Propagation of bovine coronavirus on Veto cell line: electron microscopic studies Propagation of bovine enteritic coronavirus in three cell systems: electron microscopic studies. Proceeding of the Fourth International Symposium on Neonatal Diarrhea. Veterinary Infectious Disease Organization A search for new microorganisms in calf pneumonia by the inoculation of gnotobiotic calves Trypsin-enhanced replication of neonatal calf diarrhea eoronavirus in bovine embryonic lung cells Plaque assay for titration of bovine enteric coronavirus Studies on the cultivation of poliomyelitis viruses in tissue culture. I. The propagation of poliomyelitis viruses in suspended cell culture of various human tissues OR Queue Systems Grand Island, NY '~ Fisher Scientific Co