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

Blood–retinal barrier breakdown in experimental coronavirus retinopathy: association with viral antigen, inflammation, and VEGF in sensitive and resistant strains

Journal of Neuroimmunology
Volume 119, Issue 2, 1 October 2001, Pages 175-182

Stanley A. Vinores, Yun Wang, Melissa A. Vinores, Nancy L. Derevjanik, Albert Shi, Diane A. Klein, Barbara Detrick, John J. Hooks

Abstract

Intraocular coronavirus inoculation results in a biphasic retinal disease in susceptible mice (BALB/c) characterized by an acute inflammatory response, followed by retinal degeneration associated with autoimmune reactivity. Resistant mice (CD-1), when similarly inoculated, only develop the early phase of the disease. Blood–retinal barrier (BRB) breakdown occurs in the early phase in both strains, coincident with the onset of inflammation. As the inflammation subsides, the extent of retinal vascular leakage is decreased, indicating that BRB breakdown in experimental coronavirus retinopathy (ECOR) is primarily due to inflammation rather than to retinal cell destruction. Vascular endothelial growth factor (VEGF) is upregulated only in susceptible mice during the secondary (retinal degeneration) phase.

Keywords

Coronavirus, Blood–retinal barrier, Vascular endothelial growth factor, Retinopathy

Reduced Macrophage Infiltration and Demyelination in Mice Lacking the Chemokine Receptor CCR5 Following Infection with a Neurotropic Coronavirus

Virology
Volume 288, Issue 1, 15 September 2001, Pages 8-17

William G. Glass, Michael T. Liu, William A. Kuziel, and Thomas E. Lane

Abstract

Studies were performed to investigate the contributions of the CC chemokine receptor CCR5 in host defense and disease development following intracranial infection with mouse hepatitis virus (MHV). T cell recruitment was impaired in MHV-infected CCR5−/− mice at day 7 postinfection (pi), which correlated with increased (P ≤ 0.03) titers within the brain. However, by day 12 pi, T cell infiltration into the CNS of infected CCR5−/− and CCR5+/+ mice was similar and both strains exhibited comparable viral titers, indicating that CCR5 expression is not essential for host defense. Following MHV infection of CCR5+/+ mice, greater than 50% of cells expressing CCR5 antigen were activated macrophage/microglia (determined by F4/80 antigen expression). In addition, infected CCR5−/− mice exhibited reduced (P ≤ 0.02) macrophage (CD45highF4/80+) infiltration, which correlated with a significant reduction (P ≤ 0.001) in the severity of demyelination compared to CCR5+/+ mice. These data indicate that CCR5 contributes to MHV-induced demyelination by allowing macrophages to traffic into the CNS.

Keywords

chemokine, chemokine receptor, demyelination, multiple sclerosis, macrophage, neuroimmunology

Regular Articles Variation of the sequence in the gene encoding for transmembrane protein M of canine coronavirus (CCV)

Molecular and Cellular Probes
Volume 15, Issue 4, August 2001, Pages 229-233

A. Pratelli, V. Martella, G. Elia, N. Decaro, A. Aliberti, D. Buonavoglia, M. Tempesta and C. Buonavoglia

Abstract

A nucleotide variability in the sequence of the gene encoding for the transmembrane protein M of canine coronavirus (CCV) is described. A total of 177 faecal samples from pups with enteritis were analysed by a PCR and n-PCR specific for CCV. Four samples, collected from a dog presenting a long-duration shedding of CCV, and a sample from another diarrhoeic dog, were found positive by PCR but negative by n-PCR. Sequence analysis of the samples revealed silent nucleotide substitutions in the binding site of the internal primer used for the n-PCR. Moreover, the nucleotide substitutions occurring over the whole fragment of the five samples analysed were similar.

Keywords

canine coronavirus, M protein, variation

Comparison of the sialic acid binding activity of transmissible gastroenteritis coronavirus and E. coli K99

Virus Research
Volume 75, Issue 1, May 2001, Pages 69-73

Christel Schwegmann, Gert Zimmer, Teruo Yoshino, Marie-Luise Enss, Georg Herrler

Abstract

Transmissible gastroenteritis coronavirus (TGEV) and Escherichia coli K99 are both enteropathogenic for pigs with infections being most severe in neonate animals. For both microorganisms, a sialic acid binding activity has been shown to be an essential pathogenicity factor. Here we demonstrate with haemagglutination and haemagglutination-inhibition assays that TGEV and E. coli K99 differ in their sialic acid binding activities with respect to the type and amount of sialic acid residues required on the erythrocytes surface as well as with respect to the type of sialoglycoconjugate preferentially recognized. Intestinal mucins from piglets (12–14 days old) and adult animals were shown to inhibit TGEV to the same extent. From our results we conclude that E. coli K99 and TGEV interact with different sialoglycoconjugates to establish an intestinal infection. The implications for the enteropathogenicity of TGEV are discussed.

Keywords

TGEV, E. coli K99, Sialic acid, Mucins, Glycolipids

Orchitis in a Cat Associated with Coronavirus Infection

Journal of Comparative Pathology
Volume 124, Issues 2–3, February 2001, Pages 219-222

O. G. Sigurdardottir, Ø. Kolbjørnsen and H. Lutz

Abstract

A case of severe, pyogranulomatous and necrotizing orchitis in a cat, which later succumbed to systemic feline infectious peritonitis (FIP), is described. The 3·5-year-old cat, positive for feline immunodeficiency virus infection, presented with a left testicular enlargement. A few months after castration the animal was humanely destroyed due to declining health. Post-mortem examination revealed inflammatory lesions in abdominal organs and in the brain compatible with FIP. Infection was confirmed with a reverse transcriptase–polymerase chain reaction test and by immunohistochemical demonstration of coronavirus antigen in the affected tissues, including the left testicle. FIP is usually a systemic disease. However, lesions and presenting clinical signs in a single organ system such as the brain are not uncommon. The results of this case study indicate that orchitis, although rare, should be on the list of lesions of FIP.

Keywords

coronavirus antigen, orchitis, feline infectious peritonitis,

Identification of Nucleocapsid Binding Sites within Coronavirus-Defective Genomes

Virology
Volume 277, Issue 2, 25 November 2000, Pages 235-249

Raymond Cologna, Jeannie F. Spagnolo, and Brenda G. Hogue

Abstract

The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N–viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3′ end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949–4524), as well as a region at the 3′ end of the MHV N ORF (nts 4837–5197) within MIDI-C. Binding to the full-length MIDI-C transcript (∼5500 nts) and to an ∼1-kb transcript from the gene 1a region (nts 935–1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.

Keywords

nonviral RNAs, coronavirus nucleocapsid (N) protein, in vivo, in vitro, hepatitis

Lactogenic immunity following vaccination of cattle with bovine coronavirus

Vaccine
Volume 19, Issues 2–3, 15 September 2000, Pages 189-196

C.F Crouch, S Oliver, D.C Hearle, A Buckley, A.J Chapman, M.J Francis

Abstract

In order to investigate the ability of an oil adjuvanted vaccine containing bovine coronavirus antigen to enhance lactogenic immunity in the calf, pregnant cows and heifers were vaccinated and specific virus neutralising antibody levels determined in serum, colostrum and milk. Pre-existing antibody titres (as a result of natural infection) in the serum of these animals were found to be significantly increased as a result of a single shot vaccination carried out between 2 and 12 weeks before calving. This was reflected in a similar increase in the titre and duration of specific antibody in milk and colostrum that was passed on to the calves. The overall response observed was highly dependent on an adequate antigen payload being incorporated within the single dose vaccine. No abnormal local or systemic reactions were observed as a result of vaccination. It is hoped that this approach will lead to the production of a superior commercial vaccine for the protection of neonatal calves against enteric coronavirus infection.

Keywords

Bovine coronavirus, Cattle, Lactogenic immunity

Downstream Ribosomal Entry for Translation of Coronavirus TGEV Gene 3b

Virology
Volume 269, Issue 1, 30 March 2000, Pages 172-182

Jennifer Black O'Connor, David A.Brian

Abstract

Gene 3b (ORF 3b) in porcine transmissible gastroenteritis coronavirus (TGEV) encodes a putative nonstructural polypeptide of 27.7 kDa with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of 31 kDa. In the virulent Miller strain of TGEV, ORF 3b is 5′-terminal on mRNA 3–1 and is presumably translated following 5′ cap-dependent ribosomal entry. For three other strains of TGEV, the virulent British FS772/70 and Taiwanese TFI and avirulent Purdue-116, mRNA species 3–1 is not made and ORF 3b is present as a non-overlapping second ORF on mRNA 3. ORF 3b begins at base 432 on mRNA 3 in Purdue strain. In vitro expression of ORF 3b from Purdue mRNA 3-like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. With mRNA 3-like transcripts modified to carry large ORFs upstream of ORF 3a, it was demonstrated that ribosomes can reach ORF 3b by entering at a distant downstream site in a manner resembling ribosomal shunting. Deletion analysis failed to identify a postulated internal ribosomal entry structure (IRES) within ORF 3a. The results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of ORF 3b from TGEV mRNA 3. One possible consequence of this feature is that ORF 3b might also be expressed from mRNAs 1 and 2.

Keywords

porcine transmissible gastroenteritis coronavirus, gene 3b, ribosomal scanning, ribosomal shunting

Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum

Veterinary Microbiology
Volume 72, Issues 3–4, 15 March 2000, Pages 183-206

K. Naslund, M. Traven, B. Larsson, A. Silvan, N. Linde

Abstract

Isotype-capture ELISAs for BCV-specific IgA and IgM were developed and tested on milk and serum samples from Swedish cattle. The capture ELISAs showed higher sensitivity than indirect ELISAs for detection of BCV-specific IgA and IgM. In the capture ELISAs the agreement between detection in milk and serum samples was 94% for IgA and 86% for IgM. The correlation between log10 titres in milk and serum was r=0.82 (P<0.001) for IgA and 0.84 (P<0.001) for IgM. Milk seemed a better target than serum for diagnosing specific IgA at low levels. There was no variation in the isotype-specific BCV antibody titres between healthy quarters of the same udder, but subclinical mastitis was associated with higher levels of IgA antibodies and weak false IgM positive reactions in undiluted milk. Bovine IgA and IgM antibodies in milk and serum showed high stability towards freezing and thawing and storage at room temperature.

The antibody responses to BCV were followed in milk and serum from six dairy cows and in serum from four calves for a period of 1 year after an outbreak of winter dysentery (WD). In this outbreak some animals became reinfected with BCV. The IgA and IgM capture ELISAs differentiated between primarily BCV infected and reinfected animals. In the primarily infected cattle, IgM antibodies were first detected in milk and serum four to nine days after the first WD symptoms observed, and were subsequently detected for at least 2–3 weeks. IgM was also detected in the reinfected cows, but mostly at lower levels and for a shorter period of time than in the primarily infected animals. In milk, however, the IgM response of the reinfected cows was detected for a longer period of time than in serum. Six months after the outbreak, IgA was still detected in both serum and milk of all six cows and also in serum of one calf. The reinfected cows showed higher and more long-lasting peak levels of IgA in milk and serum than the primarily infected cows, indicating boosting of the IgA response.

Keywords

Cattle, IgA, IgM, Isotype-capture, Bovine coronavirus, Milk, Reinfection

Unique N-linked glycosylation of murine coronavirus MHV-2 membrane protein at the conserved O-linked glycosylation site

Virus Research
Volume 66, Issue 2, February 2000, Pages 149-154

Yasuko K. Yamada, Mikiko Yabe, Takahiro Ohtsuki, Fumihiro Taguchi

Abstract

The membrane (M) proteins of murine coronavirus (MHV) strains have been reported to contain only O-linked oligosaccharides. The predicted O-glycosylation site consisting of four amino acid residues of Ser–Ser–Thr–Thr is located immediately adjacent to the initiator Met and is well conserved among MHV strains investigated so far. We analyzed the nucleotide sequence of a highly virulent strain MHV-2 M-coding region and demonstrated that MHV-2 had a unique amino acid, Asn, at position 2 at the conserved O-glycosylation site. We also demonstrated that this substitution added N-linked glycans to MHV-2 M protein resulting in increment of molecular mass of MHV-2 M protein compared with JHM strain having only O-linked glycans.

Keywords

Murine coronavirus, MHV, M protein, N-glycosylation, O-glycosylation

Release of Coronavirus E Protein in Membrane Vesicles from Virus-Infected Cells and E Protein-Expressing Cells

Virology
Volume 263, Issue 2, 25 October 1999, Pages 265-272

Junko Maeda, Akihiko Maeda, Shinji Makino

Abstract

Coronavirus E protein is a small viral envelope protein that plays an essential role in coronavirus assembly; coexpression of coronavirus M and E proteins results in the production of virus-like particles. The present study demonstrated that mouse hepatitis virus (MHV) E protein was released as an integral membrane protein in lipid vesicles from E-protein-expressing mammalian cells, in the absence of other MHV proteins. Furthermore, our data indicated that the E-protein-containing vesicles, which had a slightly lighter buoyant density than that of MHV, were released from MHV-infected cells. These data implied that E protein alone can drive the production and release of coronavirus envelope in the absence of M protein.

Keywords

Coronavirus, E protein, mouse hepatitis virus

Development of a nested PCR assay for the detection of canine coronavirus

Journal of Virological Methods
Volume 80, Issue 1, June 1999, Pages 11-15

A Pratelli, M Tempesta, G Greco, V Martella, C Buonavoglia

Abstract

A diagnostic test for canine coronavirus (CCV) infection based on a nested polymerase chain reaction (n-PCR) assay was developed and tested using the following coronavirus strains: CCV (USDA strain), CCV (45/93, field strain), feline infectious peritonitis virus (FIPV, field strain), trasmissible gastroenteritis virus (TGEV, Purdue strain), bovine coronavirus (BCV, 9WBL-77 strain), infectious bronchitis virus (IBV, M-41 strain) and fecal samples of dogs with CCV enteritis. A 230-bp segment of the gene encoding for transmembrane protein M of CCV is the target sequence of the primer. The test described in the present study was able to amplify both CCV and TGEV strains and also gave positive results on fecal samples from CCV infected dogs. n-PCR has a sensitivity as high as isolation on cell cultures, and can therefore be used for the diagnosis of CCV infection in dogs.

Keywords

Canine coronavirus, Dogs, Nested-polymerase chain reaction

Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus

Virus Research
Volume 60, Issue 2, April 1999, Pages 137-145

K.W Tibbles, D Cavanagh, T.D.K Brown

Abstract

Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.

Keywords

Coronavirus, 3CLproteinase, His-tagged, Bacterial expression, Trans processing

ECG changes after rabbit coronavirus infection

Journal of Electrocardiology
Volume 32, Issue 1, January 1999, Pages 21-32

Lorraine K.Alexander DRPH, Bruce W.Keene DVM, Boyd L.Yount BS, Joachim Dieter Geratz MD, J.David Small DVM, MPH, Ralph S.Baric PhD

Abstract

This study examines the electrocardiographic (ECG) changes following rabbit coronavirus (RbCV) infection. We have shown that infection with RbCV results in the development of myocarditis and congestive heart failure and that some survivors of RbCV infection go on to develop dilated cardiomyopathy in the chronic phase. Serial ECGs were recorded on 31 RbCV-infected rabbits. Measurements of heart rate; P-R interval; QRS duration; QTc interval; and P-, QRS-, and T-wave voltages were taken. The recordings were also examined for disturbances of conduction, rhythm, and repolarization. The acute and subacute phases were characterized by sinus tachycardia with depressed R- and T-wave voltages as well as disturbances of conduction, rhythm, and repolarization. In most animals in the chronic phase, the sinus rate returned to near-baseline values with resolution of the QRS voltage changes. The ECG changes observed during RbCV infection are similar to the spectrum of interval/segment abnormalities, rhythm disturbances, conduction defects, and myocardial pathology seen in human myocarditis, heart failure, and dilated cardiomyopathy. Because animals often died suddenly in the absence of severe clinical signs of congestive heart failure during the acute phase, RbCV infection may increase ventricular vulnerability, resulting in sudden cardiac death. RbCV infection may provide a rare opportunity to study sudden cardiac death in an animal model in which the ventricle is capable of supporting ventricular fibrillation, and invasive techniques monitoring cardiac function can be performed.

Keywords

coronavirus, myocarditis, heart failure, ECG

Adaptation of human enteric coronavirus to growth in cell lines

Journal of Clinical Virology
Volume 12, Issue 1, January 1999, Pages 43-51

J.ames P.Luby, R.ebecca Clinton, S.tanleyKurtz

Abstract

Background: The existence of human enteric coronavirus (HEC) has been debated since its first description in stool by electron microscopy (EM) in 1975. Needed to resolve the issue is its cultivation in readily available cell lines.

Objectives: To grow HEC in cell lines. To describe its characteristics and to differentiate it from other human and animal coronaviruses.

Study design: Originally grown in human fetal intestinal organ culture, HEC was passed in J774 cells (a mouse macrophage cell line) and C6/36 cells (a mosquito cell line). Its cytopathic effect (CPE) and pattern of immunofluorescence were described. Its appearance was ascertained by negative staining and transmission EM. Its structural proteins were delineated by polyacrylamide gel electrophoresis (PAGE) and Western blotting (WB). The antigenic character of the virus was determined by immunofluorescence and WB. Agglutination with mouse erythrocytes was performed.

Results: In J774 cells, HEC induced the formation of giant cells and small syncytia. Immunofluorescence in both J774 and C6/36 cells was limited to the cytoplasm. Studies with transmission EM revealed the virus to have the typical appearance of other coronaviruses, to be 80–120 nm in diameter, and to bud into cysternae of the endoplasmic reticulum. By PAGE and WB, its major protein has an average molecular weight (MW) of 41 kilodaltons (kDa). Two other proteins had MWs of 190 and 24 kDa. By immunofluorescence and WB, HEC is antigenically distinct from human coronaviruses 0C43 and 229E and mouse hepatitis virus (A59 strain). Preparations of HEC did not agglutinate mouse erythrocytes.

Conclusion: We conclude that HEC is a human coronavirus that is antigenically unrelated to 0C43 and 229E viruses. Growth of HEC in readily available cell lines should aid in elucidating its role as a pathogen in human diarrheal illnesses.

Keywords

Human enteric coronavirus, J774 cells, C6/36 cells, Adaptation, Growth in cell lines