Inhibition of SARS-CoV replication by siRNA

Antiviral Research
Volume 65, Issue 1, January 2005, Pages 45-48

Chang-Jer Wu, Hui-Wen Huang, Chiu-Yi Liu, Cheng-Fong Hong, Yi-LinChan

Abstract

Serious outbreaks of severe acute respiratory syndrome (SARS), caused by the newly discovered coronavirus SARS-CoV, occurred between late 2002 and early 2003 and there is an urgent need for effective antiviral agents. RNA interference in animals and post-transcriptional gene silencing plants is mediated by small double-stranded RNA molecules named small interfering RNA (siRNA). Recently, siRNA-induced RNA interference(RNAi) may provide a new approach to therapy for pathogenic viruses, e.g. HIV and HCV. In this study, the silencing potential of seven synthetic siRNAs against SARS-CoV leader, TRS, 3′-UTR and Spike coding sequence have been applied to explore the possibility for prevention of SARS-CoV infection. We demonstrate that siRNAs directed against Spike sequences and the 3′-UTR can inhibit the replication of SARS-CoV in Vero-E6 cells, and holds out promise for the development of an effective antiviral agent against SARS-CoV.

Keywords

SARS-CoV, siRNA, Spike protein, Antiviral agent

Intranasal immunization with inactivated SARS-CoV (SARS-associated coronavirus) induced local and serum antibodies in mice

Vaccine
Volume 23, Issue 7, 4 January 2005, Pages 924-931

Di Qu, Bojian Zheng, Xin Yao, Yi Guan, Zheng-Hong Yuan, Nan-Shan Zhong, Li-Wei Lu, Jian-Ping Xie, Yu-Mei Wen

Abstract

SARS-CoV (severe acute respiratory syndrome-associated coronavirus) strain GZ50 was partially purified and inactivated with 1:2000 formaldehyde. In cell culture the inactivated virus blocked the replication of live virus by decreasing the TCID5.0 of the live virus 103.6 to 104.6 times. Inactivated GZ50 was used to immunize mice intranasally either alone, or after precipitation with polyethylene glycol (PEG), or with CpG, or CTB as an adjuvant. The titer of serum neutralizing antibodies was up to 1:640. In mice immunized with adjuvants or PEG precipitated GZ50, specific IgA was detected in tracheal-lung wash fluid by immunofluorescence. Though serum antibodies were detected, no anti-SARS-IgA could be detected in mice immunized only with inactivated GZ50. The roles of adjuvants in intranasal immunization with inactivated. SARS-CoV is discussed.

Keywords

SARS-CoV, Intranasal immunization, Inactivated vaccine

Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry

Biochemical and Biophysical Research Communications
Volume 325, Issue 2, 10 December 2004, Pages 445-452

Yuxian He, Yusen Zhou, Pamela Siddiqui, Shibo Jiang

Abstract

The only severe acute respiratory syndrome (SARS) vaccine currently being tested in clinical trial consists of inactivated severe acute respiratory syndrome-associate coronavirus (SARS-CoV). However, limited information is available about host immune responses induced by the inactivated SARS vaccine. In this study, we demonstrated that SARS-CoV inactivated by β-propiolactone elicited high titers of antibodies in the immunized mice and rabbits that recognize the spike (S) protein, especially the receptor-binding domain (RBD) in the S1 region. The antisera from the immunized animals efficiently bound to the RBD and blocked binding of RBD to angiotensin-converting enzyme 2, the functional receptor on the susceptible cells for SARS-CoV. With a sensitive and quantitative single-cycle infection assay using pseudovirus bearing the SARS-CoV S protein, we demonstrated that mouse and rabbit antisera significantly inhibited S protein-mediated virus entry with mean 50% inhibitory titers of 1:7393 and 1:2060, respectively. These data suggest that the RBD of S protein is a major neutralization determinant in the inactivated SARS vaccine which can induce potent neutralizing antibodies to block SARS-CoV entry. However, caution should be taken in using the inactivated SARS-CoV as a vaccine since it may also cause harmful immune and/or inflammatory responses.

Keywords

SARS-CoV, Vaccine, Spike protein, Receptor-binding domain, Antibodies

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

Development and evaluation of an efficient 3′-noncoding region based SARS coronavirus (SARS-CoV) RT-PCR assay for detection of SARS-CoV infections

Journal of Virological Methods
Volume 120, Issue 1, 1 September 2004, Pages 33-40

Huo-Shu H Houng, David Norwood, George V Ludwig, Wellington Sun, Minta Lin, David W Vaughn

Abstract

The severe acute respiratory syndrome (SARS) epidemic originating from China in 2002 was caused by a previously uncharacterized coronavirus that could be identified by specific RT-PCR amplification. Efforts to control future SARS outbreaks depend on the accurate and early identification of SARS-CoV infected patients. A real-time fluorogenic RT-PCR assay based on the 3′-noncoding region (3′-NCR) of SARS-CoV genome was developed as a quantitative SARS diagnostic tool. The ideal amplification efficiency of a sensitive SARS-CoV RT-PCR assay should yield an E value (PCR product concentration increase per amplification cycle) equal to 2.0. It was demonstrated that the 3′-NCR SARS-CoV based RT-PCR reactions could be formulated to reach excellent E values of 1.81, or 91% amplification efficacy. The SARS-CoV cDNA preparations derived from viral RNA extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same PCR formulation developed in this study. The viral genomic copy (or genomic equivalences, GE) per infectious unit (GE/pfu) of SARS-CoV used in this study was also established to be approximate 1200–1600:1. The assay’s detection sensitivity could reach 0.005 pfu or 6–8 GE per assay. It was preliminarily demonstrated that the assay could efficiently detect SARS-CoV from clinical specimens of SARS probable and suspected patients identified in Taiwan. The 3′-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region.

Keywords

Severe acute respiratory syndrome, SARS coronavirus, Real-time RT-PCR, Quantitative RT-PCR, 3′-Noncoding region.

The 3D structure analysis of SARS-CoV S1 protein reveals a link to influenza virus neuraminidase and implications for drug and antibody discovery

Journal of Molecular Structure: THEOCHEM
Volume 681, Issues 1–3, 26 July 2004, Pages 137-141

Xue Wu Zhang, Yee Leng Yap

Abstract

The spike protein of SARS-associated coronavirus (SARS-CoV) is an important target for anti-SARS drug discovery. Its S1 domain is responsible for receptor binding and SARS-CoV entry into cells. In this study, we constructed a rational 3D model for S1 domain of SARS-CoV spike protein by fold recognition and molecular modeling techniques. We found that there is a structure similarity between S1 protein and influenza virus neuraminidase. Our analyses suggest that the existing anti-influenza virus inhibitors and anti-neuraminidase antibody could be used as a starting point for designing anti-SARS drugs, vaccines and antibodies. Interestingly, our prediction for antibody is consistent with a recently experimental discovery of anti-SARS antibody.

Keywords

SARS-CoV, S1 protein, Structure, Influenza virus, Inhibitor, Antibody

Evolution and Variation of the SARS-CoV Genome

Genomics, Proteomics & Bioinformatics
Volume 1, Issue 3, August 2003, Pages 216-225

Jianfei Hu, Jing Wang, Jing Xu, Wei Li, Yujun Han, Yan Li, Jia Ji, Jia Ye, Zhao Xu, Zizhang Zhang, Wei Wei, Songgang Li, Jun Wang, Jian Wang, Jun Yu, Huanming Yang.

Abstract

Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARS-CoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us.

Key words

SARS, SARS-CoV, motif frequency profile, genomic signature, Chaos Game Representation, PUP

Complete Genome Sequences of the SARS-CoV: the BJ Group (Isolates BJ01-BJ04)

Genomics, Proteomics & Bioinformatics
Volume 1, Issue 3, August 2003, Pages 180-192

Shengli Bi, E’de Qin, Zuyuan Xu, Wei Li, Jing Wang, Yongwu Hu, Yong Liu, Shumin Duan, Jianfei Hu, Yujun Han, Jing Xu, Yan Li, Yao Yi, Yongdong Zhou, Wei Lin, Jie Wen, Hong Xu, Ruan Li, …Huanming Yang.

Abstract

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city’s hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.

Key words

SARS, SARS-CoV, haplotype, substitution, phylogeny

Genome Organization of the SARS-CoV

Genomics, Proteomics & Bioinformatics
Volume 1, Issue 3, August 2003, Pages 226-235

Jing Xu, Jianfei Hu, Jing Wang, Yujun Han, Yongwu Hu, Jie Wen, Yan Li, Jia Ji, Jia Ye, Zizhang Zhang, Wei Wei, Songgang Li, Jun Wang, Jian Wang, Jun Yu, Huanming Yang.

Abstract

Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves. Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.

Key words

SARS-CoV, genome annotation, transcription, ORF, PUP, TRS

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

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

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