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