Purification of turkey coronavirus by Sephacryl size-exclusion chromatography

Journal of Virological Methods
Volume 104, Issue 2, July 2002, Pages 187-194

C.C. Loa, T.L. Lin, C.C. Wu, T.A. Bryan, H.L. Thacker, T. Hooper, D. Schrader

Abstract

Sephacryl S-1000 size-exclusion chromatography was used to purify turkey coronavirus (TCoV) from infected turkey embryo. TCoV was propagated in the 22-day-old turkey embryos. Intestines and intestinal contents of infected embryos were harvested and homogenized. After low speed centrifugation, the supernatant was concentrated by ultracentrifugation through a cushion of 30 or 60% sucrose solution, or by ammonium sulfate precipitation. The purification methods included sucrose gradient and Sephacryl S-1000 size-exclusion chromatography. Ultracentrifugation through a cushion of 60% sucrose solution was better than the other two methods for concentration of TCoV from intestinal homogenate. The most effective method for purifying TCoV and removing extraneous materials was size-exclusion chromatography as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More spike-rich particles were observed in the sample purified by chromatography than those purified by sucrose gradient as examined by electron microscopy. Differentiation of turkey anti-TCoV antiserum from normal turkey serum was better achieved by ELISA plates coated with TCoV preparation purified by size-exclusion chromatography than that purified by sucrose density gradient. The results indicated that Sephacryl S-1000 chromatography was useful for purification of TCoV.

Keywords

Turkey coronavirus, Virus propagation, Virus purification

Coronavirus-related nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study

Journal of Hospital Infection
Volume 51, Issue 1, May 2002, Pages 59-64

A. Gagneur, J. Sizun, S. Vallet, M. C. Legrand, B. Picard, and P. J. Talbot

Abstract

The incidence of nosocomial viral respiratory infections (NVRI) in neonates and children hospitalized in paediatric and neonatal intensive care units (PNICU) is unknown. Human coronaviruses (HCoV) have been implicated in NVRI in hospitalized preterm neonates. The objectives of this study were to determine the incidence of HCoV-related NVRI in neonates and children hospitalized in a PNICU and the prevalence of viral respiratory tract infections in staff. All neonates (age≤28 days) and children (age>28 days) hospitalized between November 1997 and April 1998 were included. Nasal samples were obtained by cytological brush at admission and weekly thereafter. Nasal samples were taken monthly from staff. Virological studies were performed, using indirect immunofluorescence, for HCoV strains 229E and OC43, respiratory syncytial virus (RSV), influenza virus types A and B, paramyxoviruses types 1, 2 and 3 and adenovirus. A total of 120 patients were enrolled (64 neonates and 56 children). Twenty-two samples from 20 patients were positive (incidence 16.7%). In neonates, seven positive samples, all for HCoV, were detected (incidence 11%). Risk factors for NVRI in neonates were: duration of hospitalization, antibiotic treatment and duration of parenteral nutrition (P<0.01). Monthly prevalence of viral infections in staff was between 0% and 10.5%, mainly with HCoV. In children, 15 samples were positive in 13 children at admission (seven RSV, five influenza and three adenovirus) but no NVRI were observed. In spite of a high rate of community-acquired infection in hospitalized children, the incidence of NVRI with common respiratory viruses appears low in neonates, HCoV being the most important pathogen of NRVI in neonates during this study period. Further research is needed to evaluate the long-term impact on pulmonary function.

Keywords

Human coronavirus, virus, nosocomial infection, PNICU, infant, neonate

The Use of Bovine Serum Protein as an Oral Support Therapy Following Coronavirus Challenge in Calves

Journal of Dairy Science
Volume 85, Issue 5, May 2002, Pages 1249-1254

J. D. Arthington, C. A. Jaynes, H. D. Tyler, S. Kapil, and J. D. Quigley

Abstract

The objective of this experiment was to investigate the therapeutic efficacy of a supplemental bovine serum protein blend fed to calves challenged with virulent coronavirus. Twelve Holstein bull calves (approximately 3 wk of age) were allocated by initial body weight to Control (n = 5) and treated (n = 7) groups. On d 0, all calves were orally challenged with 1 × 107 plaque forming units of virulent coronavirus isolate. Infection was allowed to progress for 24 h before treatment was started. On d 1, treated calves began receiving 160 g of dry bovine serum powder (16 g IgG) mixed into milk replacer powder (67 g) at both an a.m. and p.m. feeding. Control calves received only milk replacer powder (227 g) at both feedings. Response to coronavirus challenge and dietary treatment was monitored prior to a.m. and p.m. feeding by the collection of multiple clinical measures. Fecal consistency was decreased by coronavirus challenge but was not affected by dietary treatment. Mean daily rectal temperature and heart rate were not affected by dietary treatment. Average packed cell volume was higher in treated calves than in control (35.0 and 27.0%). Coronavirus challenge resulted in an immediate increase in respiration rate, decreasing by d 7. Control calves tended to have a greater average respiration rate compared with treated (28.7 vs. 26.8 breaths/min). Treated calves had a higher average feed intake than control (0.57 vs. 0.44 kg/d). These data suggest that bovine-serum supplemented milk replacer may decrease the severity of disease in young calves exposed to coronavirus.

Keywords

bovine serum, coronavirus, calf

Prevalence of canine coronavirus antibodies by an enzyme-linked immunosorbent assay in dogs in the south of Italy

Journal of Virological Methods
Volume 102, Issues 1–2, April 2002, Pages 67-71

Annamaria Pratelli, Gabriella Elia, Vito Martella, Alessandra Palmieri, Francesco Cirone, Antonella Tinelli, Marialaura Corrente, Canio Buonavoglia

Abstract

An enzyme-linked immunosorbent assay (Elisa), using as antigen canine coronavirus-infected CrFK cell supernatant, was developed to detect antibodies against canine coronavirus (CCoV). Out of a total of 109 dog serum samples, 80 which were positive by routine virus neutralisation test were also Elisa positive. Seventeen samples which were negative by the virus neutralisation test, were positive by Elisa and by the confirmatory Western blotting test. The Elisa was substantially more sensitive than the virus neutralisation test in detecting antibodies to CCoV and may be used as an alternative technique to virus neutralisation.

Keywords

Dog, Coronavirus, Antibodies, Elisa

The effect of immunosuppression on protective immunity of turkey poults against infection with turkey coronavirus

Comparative Immunology, Microbiology and Infectious Diseases
Volume 25, Issue 2, March 2002, Pages 127-138

Chien Chang Loa, Tsang Long Lin, Ching Ching Wu, Thomas Bryan, Tom Hooper, Donna Schrader

Abstract

The objective of the present study was to evaluate the protective effect of humoral and cellular immunities on turkeys infected with turkey coronavirus (TCV). Two trials were conducted with two separate hatches of turkey poults. Turkeys were experimentally immunosuppressed with cyclosporin A (CsA) or cyclophosphamide (CY) and infected with TCV. Prior to infection, treatment with CsA selectively suppressed T cell activity as revealed by 2–3 fold decreased (p<0.1) lymphocyte proliferation responses to a T cell mitogen, concanavalin A (Con A). Treatment with CY mainly induced B cell deficiency as indicated by significant reductions (p<0.05) in antibody responses to sheep erythrocytes 7 days after injection. Body weight gain of turkeys treated with CY was significantly lower (p<0.05) than that of untreated turkeys at 9 days post-infection (PI). Turkeys treated with CY had 1–2 fold higher immunofluorescent antibody assay (IFA) scores for TCV antigens (p<0.05) in the intestine than untreated turkeys at 9 or 14 days PI. These results suggested that humoral immunity against TCV infection may be important in turkeys.

Keywords

Cyclophosphamide, Cyclosporin A, Enteritis, Immunity, Immunosuppression, Turkey coronavirus