The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines

Journal of Virological Methods
Volume 193, Issue 2, November 2013, Pages 639-646

Jose A. Regla-Nava, Jose M. Jimenez-Guardeño, Jose L. Nieto- Torres, Thomas M. Gallagher, Luis Enjuanes, Marta L. DeDiego

Abstract

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis.

Keywords

SARS, Coronavirus, Mouse adapted, Stably transformed murine cells, SARS-CoV receptor ACE2, Proinflammatory cytokines

Middle East respiratory syndrome coronavirus (MERS-CoV): challenges in identifying its source and controlling its spread

Microbes and Infection
Volume 15, Issues 8–9, July–August 2013, Pages 625-629

Lu Lu, Qi Liu, Lanying Du, Shibo Jiang

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV), a novel human coronavirus that caused outbreaks of a SARS-like illness in the Middle East, is now considered a threat to global public health. This review discusses the challenges in identifying the source of this fatal virus and developing effective and safe anti-MERS-CoV vaccines and therapeutics in order to control its spread and to combat any future pandemic.

Keywords

Coronavirus, MERS-CoV, SARS-CoV, Source, Vaccine, Therapeutics

An immunosuppressed Syrian golden hamster model for SARS-CoV infection

Virology
Volume 380, Issue 2, 25 October 2008, Pages 312-321

Scott R. Schaecher, Jennifer Stabenow, Christina Oberle, Jill Schriewer, R. Mark Buller, John E. Sagartz, Andrew Pekosz.

Abstract

Several small animal models have been developed for the study of severe acute respiratory syndrome coronavirus (SARS-CoV) replication and pathogenesis. Syrian golden hamsters are among the best small animal models, though little clinical illness and no mortality are observed after virus infection. Cyclophosphamide was used to immunosuppress hamsters leading to a prolonged disease course and higher mortality after SARS-CoV infection. In addition, there was a significant weight loss, expanded tissue tropism, and increased viral pathology in the lung, heart, kidney, and nasal turbinate tissues. Infection with recombinant SARS-CoV viruses bearing disruptions in the gene 7 coding region showed no significant change in replication kinetics, tissue tropism, morbidity, or mortality suggesting that the ORF7a (7a) and ORF7b (7b) proteins are not required for virus replication in immunosuppressed hamsters. This modified hamster model may provide a useful tool for SARS-CoV pathogenesis studies, evaluation of antiviral therapy, and analysis of additional SARS-CoV mutants.

Keywords

SARS-CoV, Coronavirus, Cyclophosphamide, ORF7a, ORF7b, Hamster, Accessory gene, Pathogenesis

The design and application of DNA chips for early detection of SARS-CoV from clinical samples

Journal of Clinical Virology
Volume 33, Issue 2, June 2005, Pages 123-131

Yi-ming Zhou, Ren-quan Yang, Sheng-ce Tao, Ze Li, Qiong Zhang, Hua-fang Gao, Zhi-wei Zhang, Jian-yu Du, Pei-xuan Zhu, Li-li Ren, Liang Zhang, Dong Wang, Lan Guo, Yan-bin Wang, Yong Guo, Yan Zhang, Chuan-zan Zhao, Can Wang…Jing Cheng

Abstract

Background:

SARS coronavirus has been identified as the cause of severe acute respiratory syndrome (SARS). Few tests allow confirmation or exclusion of SARS within the first few days of infection. A gene chip is a useful tool for the study of microbial infections mainly for its capability of performing multi-target analysis in a single test.

Objectives:

Investigate the possibility of early detection of SARS virus from clinical samples using the gene chip-based method.

Study design:

We purified RNA from SARS-CoV obtained from routinely collected peripheral blood and sputum samples of 34 patients who had been identified as probable SARS patients by following the interim U.S. case definition. Four segments of the SARS-CoV were amplified using reverse transcription-nested PCR and the products examined using the 70-mer gene chips for SARS-CoV detection.

Results:

A blind-test of both peripheral blood and sputum specimens lead to the positive detection of SARS-CoV in 31 out of 34 patients. SARS-CoV was not found in peripheral blood or sputum specimens from three patients. Two of the 34 patients were only 3 days post-onset of symptoms and were subsequently confirmed to be SARS positive. Our results indicate that the gene chip-based molecular test is specific for SARS-CoV and allows early detection of patients with SARS with detection rate about 8% higher than the single PCR test when the sputum sample is available.

Keywords

SARS, Coronavirus, SARS-CoV, Early detection, Gene chip

Interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

Virology
Volume 329, Issue 1, 10 November 2004, Pages 11-17

Bruno Sainz Jr. , Eric C. Mossel, C. J. Peters, Robert F. Garry

Abstract

Recent studies have shown that interferon-gamma (IFN-γ) synergizes with IFN-α/β to inhibit the replication of both RNA and DNA viruses. We investigated the effects of IFNs on the replication of two strains of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). While treatment of Vero E6 cells with 100 U/ml of either IFN-β or IFN-γ marginally reduced viral replication, treatment with both IFN-β and IFN-γ inhibited SARS-CoV plaque formation by 30-fold and replication by 3000-fold at 24 h and by > 1 × 105-fold at 48 and 72 h post-infection. These studies suggest that combination IFN treatment warrants further investigation as a treatment for SARS.

Keywords

Coronavirus, Interferon-gamma, Interferon-beta

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

Journal of Virological Methods
Volume 121, Issue 1, October 2004, Pages 85-91

Miriam E.R. Darnella, Kanta Subbaraob, Stephen M. Feinstonea, Deborah R. Taylora

Abstract

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a novel coronavirus termed SARS-CoV. Due to the severity of this disease, the World Health Organization (WHO) recommends that manipulation of active viral cultures of SARS-CoV be performed in containment laboratories at biosafety level 3 (BSL3). The virus was inactivated by ultraviolet light (UV) at 254 nm, heat treatment of 65 °C or greater, alkaline (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments. We describe the kinetics of these efficient viral inactivation methods, which will allow research with SARS-CoV containing materials, that are rendered non-infectious, to be conducted at reduced safety levels.

Keywords

SARS, Coronavirus, Virus inactivation, Tissue culture

ZCURVE_CoV: a new system to recognize protein coding genes in coronavirus genomes, and its applications in analyzing SARS-CoV genomes

Biochemical and Biophysical Research Communications
Volume 307 (2003), Pages 382–388

Ling-Ling Chen, Hong-Yu Ou, Ren Zhang, and Chun-Ting Zhanga.

Abstract

A new system to recognize protein coding genes in the coronavirus genomes, specially suitable for the SARS-CoV genomes, has been proposed in this paper. Compared with some existing systems, the new program package has the merits of simplicity, high accuracy, reliability, and quickness. The system ZCURVE_CoV has been run for each of the 11 newly sequenced SARS-CoV genomes. Consequently, six genomes not annotated previously have been annotated, and some problems of previous annotations in the remaining five genomes have been pointed out and discussed. In addition to the polyprotein chain ORFs 1a and 1b and the four genes coding for the major structural proteins, spike (S), small envelop (E), membrane (M), and nuleocaspid (N), respectively, ZCURVE_CoV also predicts 5–6 putative proteins in length between 39 and 274 amino acids with unknown functions. Some single nucleotide mutations within these putative coding sequences have been detected and their biological implications are discussed. A web service is provided, by which a user can obtain the annotated result immediately by pasting the SARS-CoV genome sequences into the input window on the web site (http://tubic.tju.edu.cn/sars/). The software ZCURVE_CoV can also be downloaded freely from the web address mentioned above and run in computers under the platforms of Windows or Linux.
2003 Elsevier Inc. All rights reserved.

Keywords

Coronavirus, Severe acute respiratory syndrome, SARS-CoV, Genome, Gene-finding, Mutation

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

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

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

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

Effects of the common cold on mood and performance

Psychoneuroendocrinology
Volume 23, Issue 7, October 1998, Pages 733-739

Andrew Smith, Marie Thomas, Julie Kent, Karl Nicholson

Abstract

Previous research has shown that both experimentally-induced and naturally occurring upper respiratory tract illnesses (URTIs) influence mood and mental functioning. None of the previous studies of naturally occurring colds has conducted appropriate virological assays to determine the nature of the infecting agent. This is an essential methodological step in studies of malaise associated with URTIs. The aim of this research was to investigate the effects of naturally occurring colds on mood and objective measures of performance. This was done by first conducting a cross-sectional comparison of 37 healthy people and 158 volunteers with colds and then a longitudinal study in which 100 volunteers developed colds and 87 remained healthy. Virological techniques were used to identify infecting agents and comparisons made across the different groups. The results showed that having a cold was associated with reduced alertness and slowed reaction times. These effects were observed both for colds where the infecting virus was identified and those where it was not. Similar effects were obtained for both rhinovirus and coronavirus colds. One may conclude that upper respiratory tract illnesses lead to a reduction in subjective alertness and impaired psychomotor functioning. This was true for both illnesses where the infecting agent was identified and for those clinical illnesses where no virus was detected. It is now important to identify the mechanisms linking infection and illness with the behavioural changes. Similarly, the impact of these effects on real-life activities such as driving needs examining. Finally, methods of treatment need to be developed which not only treat the local symptoms of the illnesses but remove the negative mood and the performance impairments.

Keywords

Common cold, Rhinovirus, Coronavirus, Reaction time, Alertness

Comparison of immunofluorescence with monoclonal antibodies and RT-PCR for the detection of human coronaviruses 229E and OC43 in cell culture

Journal of Virological Methods
Volume 72, Issue 2, June 1998, Pages 145-152

Jacques Sizun, Nathalie Arbour, Pierre J Talbot

Abstract

Human coronaviruses, with two known serogroups named 229E and OC43, cause up to one third of common colds and may be associated with serious diseases such as nosocomial respiratory infections, enterocolitis, pericarditis and neurological disorders. Reliable methods of detection in clinical samples are needed for a better understanding of their role in pathology. As a first step in the design of such diagnostic procedures, the sensitivities and specificities of two viral diagnostic assays were compared in an experimental cell culture model: an indirect immuno-fluorescence assay using monoclonal antibodies and reverse transcriptase-polymerase chain reaction amplification of viral RNA from infected cells. Immunofluorescence detected human coronaviruses in cells infected at a MOI as low as 10−2 (log TCID50/ml=4.25 for HCV-229E and 2.0 for HCV-OC43; log PFU/ml=4.83 for HCV-229E and 1.84 for HCV-OC43) versus 10−3 (HCV-OC43) or 10−4 (HCV-229E) for reverse transcriptase-polymerase chain reaction amplification (log TCID50/ml=1.75 for HCV-229E and 1.5 for HCV-OC43; log PFU/ml=2.3 for HCV-229E and 1.34 for HCV-OC43). There were no false positive signals with other human respiratory pathogens: influenza virus, respiratory syncytial virus and adenovirus. Moreover, each assay was coronavirus serogroup-specific. These results demonstrate the potential usefulness of immunofluorescence with monoclonal antibodies and reverse transcriptase-polymerase chain reaction RNA amplification for the rapid detection of human coronaviruses in infected cell cultures. Both methods could be applied to clinical specimens for the diagnosis of human infections.

Keywords

Coronavirus, 229E, OC43, Immunofluorescence, RT-PCR, Diagnostic

The Molecular Biology of Coronaviruses

Advances in Virus Research
Volume 48, 1997, Pages 1-100

Michael M.C. Lai, David Cavanagh

Abstract

This chapter discusses the manipulation of clones of coronavirus and of complementary DNAs (cDNAs) of defective-interfering (DI) RNAs to study coronavirus RNA replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. The nature of the coronavirus genome is nonsegmented, single-stranded, and positive-sense RNA. Its size ranges from 27 to 32 kb, which is significantly larger when compared with other RNA viruses. The gene encoding the large surface glycoprotein is up to 4.4 kb, encoding an imposing trimeric, highly glycosylated protein. This soars some 20 nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet. Coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of RNA synthesis, translational control, and protein transport and processing. It remains a treasure capable of generating unexpected insights.

Keywords

Coronavirus, DNAs, RNAs, clones

In vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero TGEV coronavirus injection

Research in Immunology
Volume 148, Issue 4, May 1997, Pages 247-256

I.Splíchal, Z.Řeháková, M.Šinkora, J.Šinkora, I.Trebichavský, H.Laude, B.Charley

Abstract

Non-infectious UV-inactivated transmissible gastroenteritis virus (TGEV) was previously shown to induce interferon alpha (IFIMα) secretion following in vitro incubation with blood mononuclear cells. In this study, pig foetuses at different stages of gestation were injected in utero with (a) partially UV-inactivated wild TGEV or (b) fully UV-inactivated wild or dm49-4 mutant TGEV Coronavirus. Nucleated cells from foetal liver, bone marrow, spleen and blood were isolated 10 or 20 h after injection and assayed ex vivo for IFNα secretion by ELISPOT and ELISA techniques. The administration of TGEV induced IFNα-secreting cells in foetal lymphohaematopoietic organs at mid-gestation. In contrast, IFNα was not detected in control sham-operated foetuses. A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M of TGEV mutant dm49-4 was associated with lower or absent IFNα in utero inducibility by mutant virus as compared with wild virus. Row cytometry analysis did not show differences in leukocyte surface marker expression between control and TGEV- or between dm49-4 and wild virus-treated foetus cells, with the exception of a reduction in percentages of polymorphonuclear cells in TGEV-treated lymphohaematopoietic tissues, which is probably due to IFNα secretion. The present data provided in vivo evidence of IFNα secretion at the cell level in foetal lymphohaematopoietic organs. Such IFNα-secreting cells in lymphohaematopoietic tissues may be the source of IFNα detected during foetal infections.

Keywords

Coronavirus, Transmissible gastroenteritis virus, IFNα, ELISA, ELISPOT, Foetus, Pig

Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM

Virus Research
Volume 45, Issue 2, December 1996, Pages 101-109

Hong-QiangGao, Jennifer J.Schiller, Susan C.Baker

Abstract

The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5′-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.

Keywords

Coronavirus, Polymerase polyprotein, Proteolytic processing

In situ hybridization technique for the detection of swine enteric and respiratory coronaviruses, TGEV and PRCV, in formalin-fixed paraffin-embedded tissues

Journal of Virological Methods
Volume 56, Issue 2, February 1996, Pages 149-160

Theerapol Sirinarumitr, Prem S. Paul, John P. Kluge, Patrick G. Halbur

Abstract

The in situ hybridization (ISH) technique was developed to detect the swine coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in cell culture and tissue sections from TGEV-or PRCV-infected pigs. The 35S-labeled RNA probes were generated from two plasmids pPSP.FP1 and pPSP.FP2 containing part of the S gene of TGEV. The procedure was first standardized in cell cultures. The radiolabeled pPSP.FP2 probe detected both TGEV and PRCV in virus-inoculated cell cultures, whereas pPSP.FP1 probe detected TGEV but not PRCV. The probe was then used to detect TGEV or PRCV in tissues of pigs experimentally infected with TGEV or PRCV or naturally infected with TGEV. Again, the probes detected TGEV in intestines of experimentally and naturally infected pigs and PRCV in the lungs of experimentally infected pigs. TGEV RNA was detected mainly within the enterocytes at the tips of villi and, less often, within some crypt epithelial cells. PRCV was shown to replicate mainly in the bronchiolar epithelial cells and in lesser amount in type II pneumocytes, type I pneumocytes, alveolar macrophages and bronchial epithelial cells, respectively. ISH has potential applications as a diagnostic test for the detection and differentiation of TGEV and PRCV in tissues and in studies to gain a better understanding of the mechanism of pathogenesis of enteric and respiratory coronavirus infections.

Keywords

Single stranded RNA probe, In situ hybridization, Transmissible gastroenteritis virus (TGEV), Porcine respiratory coronavirus (PRCV), Coronavirus