Electron Microscopy of Coronavirus-Like Particles Characteristic of Turkey Bluecomb Disease

Ritchie, A. E.; Deshmukh, D. R.; Larsen, C. T.; Pomeroy, B. S.

doi:10.2307/1589153

Cited by 61 publications

(40 citation statements)

References 10 publications

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“…3,5 Fringed particles that could be mistaken for coronaviruses were frequently observed, particularly in cases of specimens obtained from diarrheic turkey poults. 5,23 In the present study, PAG-IEM coupled to direct airfuge ultracentrifugation of samples on EM grids proved to be a rapid, simple, and sensitive test for detection, identification, and ultrastructural studies of coronavirus particles or their subunits. As previously reported, 1,15 direct ultracentrifugation of samples on an EM specimen grid increased the sensitivity and quality of preparations.…”

Section: Discussionmentioning

confidence: 64%

“…Turkey and bovine enteric coronaviruses (TCV, BCV), porcine hemagglutinating encephalomyelitis virus (HEV), porcine transmissible gastroenteritis virus (TGEV), human HCV-OC43 and HCV-229E respiratory coronaviruses, mouse hepatitis virus (MHV), and 3 serotypes of avian infectious bronchitis virus (IBV) were used in the study. (The prototype egg-adapted Minnesota strain 23 of TCV was kindly supplied to us by B. S. Pomeroy. a ) The virus was serially propagated in the presence of trypsin b in HRT-18 cells, a cell line derived from human rectal adenocarcinoma, as described previously.…”

Section: Methodsmentioning

confidence: 99%

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Identification of Coronaviruses by the Use of Indirect Protein A-Gold Immunoelectron Microscopy

Dea

Garzón

1991

J VET Diagn Invest

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Abstract. Concentration by airfuge and protein A-colloidal gold immunoelectron microscopy (PAG-IEM) offered a rapid and sensitive method for detection and identification of coronaviruses from various species. The method was applied to partially purified tissue culture-adapted or egg-adapted mammalian and avian coronaviruses and to clarified fecal samples from diarrheic calves and turkey poults for detection of enteric coronaviruses. Aggregates of virus coated with specific antibody were seen in virus samples mixed with homologous antiserum but not in control samples containing preexposure serum. At least a 10-50-fold enhancement of the sensitivity of direct EM for virus detection was obtained using protein A-colloidal gold complex as an electron-dense marker. The PAG-IEM method demonstrated low nonspecific background labeling and permitted detection of soluble and particle-associated antigen. Reciprocal cross-reactivity was detected among the subgroup of mammalian hemagglutinating coronaviruses, and antisera to 4 members of other subgroups only recognized their homologous virus.Coronaviruses, particularly enteropathogenic strains, from avian and mammalian species are difficult to grow in cell cultures. 2 6 Direct electron microscopy (DEM) of viral particles in clarified fecal specimens and fluorescent antibody techniques for detection of coronavirus antigens in the cells of infected gut are generally used for the diagnosis of enteric coronaviral infections. 11,14,18,19,21 The detection of coronavirus antigens by indirect immunofluorescence (IFF) is dependent on tissue being removed very shortly after death, and most antigen is present early in the course of disease. 19,21 The lack of highly specific antisera can also compromise the specificity of the IFF technique. Coronaviruses are difficult to identify in negatively stained preparations because they are very pleomorphic and the peplomers are lost during processing of clinical specimens. 10,11,14 It is difficult to distinguish coronavirus particles from cell artifacts such as parts of nuclear or cytoplasmic membranes and other fringed bodies 10 Several immunoelectron microscopy (IEM) . methods for detecting enteric coronaviruses are more sensitive than conventionnal DEM. techniques; 17,24 however, the sensitivity of these methods is still dependent on the morphologic identification of virus particles or immune complexes on the EM grids. The recently described colloidal gold immunoelectron mi- Presented at the 33rd Annual Meeting of the AAVLD, Denver, CO, October 6-11, 1990.Received for publication February 13, 1991. croscopy technique improves detection of bovine enteric coronavirus (BCV) in fecal samples. 9 The purpose of the present study was to evaluate the efficiency of combining the concentration of viral suspensions by airfuge and the indirect protein A-colloidal gold immunoelectron microscopy labeling (PAG-IEM) techniques for the detection and differentiation of avian and mammalian enteric and respiratory coronaviruses. Materials and methodsViruses an...

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Section: Discussionmentioning

confidence: 64%

Section: Methodsmentioning

confidence: 99%

Identification of Coronaviruses by the Use of Indirect Protein A-Gold Immunoelectron Microscopy

Dea

Garzón

1991

J VET Diagn Invest

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“…In the natural host, it infects and destroys the mature absorptive epithelial cells of the small and large intestines leading to an often severe diarrhoea in poults under 6 weeks of age, but only mild clinical signs in adults (Deshmukh et al, 1974;Pomeroy et al, 1978). The morphological and physicochemical characteristics of TCV resemble those of other members of the Coronaviridae (Ritchie et al, 1973;Panigrahy et al, 1973;Dea et al, 1986;Dea & Tijssen, 1988b). Little is known with respect to the molecular and antigenic structure of the virion, because of the difficulties in propagating TCV strains in conventional tissue culture systems and the lack of highly specific immunological probes (Deshmukh et al, 1973;Pomeroy, 1984).…”

Section: Introductionmentioning

confidence: 99%

“…Little is known with respect to the molecular and antigenic structure of the virion, because of the difficulties in propagating TCV strains in conventional tissue culture systems and the lack of highly specific immunological probes (Deshmukh et al, 1973;Pomeroy, 1984). Ritchie et al (1973) did not observe cross-reactivity, in immunoelectron microscopy studies, 0000-8789 © 1989 SGM between the prototype Minnesota strain of TCV and coronaviruses from other species, using hyperimmune serum obtained by experimental inoculation of turkeys with coronavirus-positive caecal suspensions. Different isolates of TCV are considered to be antigenically identical or closely related (Pomeroy et al, 1975;Patel et al, 1977).…”

Section: Introductionmentioning

confidence: 99%

“…Different isolates of TCV are considered to be antigenically identical or closely related (Pomeroy et al, 1975;Patel et al, 1977). Field isolates can be propagated by oral inoculation and intestinal infections of young turkey poults, or by inoculation into embryonating turkey eggs (Deshmukh et al, 1973;Ritchie et al, 1973). Our previous studies on the morphology and some of the biological activities of egg-adapted TCV strains have revealed that the virus possesses a haemagglutinating activity which may be associated with short granular projections located near the base of the characteristic large bulbous peplomers (Dea & Tijssen, 1988b).…”

Section: Introductionmentioning

confidence: 99%

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Antigenic and Polypeptide Structure of Turkey Enteric Coronaviruses as Defined by Monoclonal Antibodies

Dea

Tijssen

1989

Journal of General Virology

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SUMMARYTwenty-nine hybridoma cell lines, producing monoclonal antibodies (MAbs) to the Minnesota strain of turkey enteric coronavirus (TCV), have been established by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of the egg-adapted or tissue culture-adapted virus. The hybridomas produced mainly IgG2a or IgG1 antibodies. Western immunoblotting experiments with purified virus, and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts, allowed assessment of the polypeptide specificity of the MAbs. Sixteen hybridomas secreted antibodies directed to the peplomeric protein (E2, gp200/gpl00) and putative intracellular precursors of apparent Mr 170K to 180K and 90K. Four hybridomas produced antibodies that selectively reacted with a glycoprotein with an Mr of 140K (E3). This polypeptide species corresponded to the major structural component of small granular projections, located near the base of the larger bulbous peplomers, and was found to be responsible for haemagglutination. The major neutralization-mediating determinants were found to be carried by both E2 and E3 glycoproteins. Eight hybridomas produced MAbs directed to the major nucleocapsid protein (N, 52K), and only one MAb reacted with a low Mr structural glycoprotein (24K), corresponding to the matrix (El) protein. By indirect immunofluorescence, MAbs of different specificity also revealed distinct patterns of distribution of the viral antigens within the cells. The location on the virion of the antigenic determinants recognized by MAbs of different specificity was determined by the use of an immunogold electron microscopy technique. Comparison of nine TCV Quebec strains, using MAbs directed to peplomer and haemagglutinin proteins of the prototype Minnesota strain, confirmed their close antigenic relationship, but also revealed the occurrence of at least two distinct antigenic groups.

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Viral Enteric Infections

Saif¹,

Guy²,

Day³

et al. 2019

Diseases of Poultry

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Electron Microscopy of Coronavirus-Like Particles Characteristic of Turkey Bluecomb Disease

Cited by 61 publications

References 10 publications

Identification of Coronaviruses by the Use of Indirect Protein A-Gold Immunoelectron Microscopy

Identification of Coronaviruses by the Use of Indirect Protein A-Gold Immunoelectron Microscopy

Antigenic and Polypeptide Structure of Turkey Enteric Coronaviruses as Defined by Monoclonal Antibodies

Viral Enteric Infections

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