The replication of Nodamura virus, type species of the family Nodaviridae, takes place principally into the interfibrillar spaces of the sarcoplasm in close association with mitochondria in the infected muscles of both G. mellonella and suckling mice. The most striking event in infection of G. mellonella muscle cells was the aggregation and shape modification of numerous mitochondria (elongation, interdigitation, and vesiculation) at an early stage of infection. These clusters of mitochondria were cemented by a thick electron-dense material at the periphery of which viral particles undergo maturation process. In a later stage, degenerated and dilated mitochondria showed a clear assembling of virus particles on their outer membrane and occasionally on some inner membranes. These facts and the localization of RNA by RNase-gold technique in the assembling sites of viruses around and inside the mitochondria allowed us to postulate a role for the mitochondria as supports and/or energy suppliers for viral RNA synthesis and translation.

The tegument of bovine herpesvirus-1 (BHV-1) carries an abundant protein of 96 kDa, termed VP8. Immunolabeling using VP8-specific antiserum and colloidal gold-labeled protein A as the electron-dense marker was used to identify VP8 in the virions and virus-infected cells. VP8 was confirmed to be a tegument protein that, like the herpes simplex virus-1 homologue VP13/14, contains O-linked carbohydrates. VP8 was found in the nucleus of virus-infected cells as early as 2 hr postinfection. Since VP8 is a gamma2 protein, this protein cannot be newly synthesized at this time and must be acquired from the inoculum. This supports the hypothesis that early during infection, VP8 has a function in modulation of alpha gene expression. Later during infection, VP8 was observed in the cytoplasm around nucleocapsids and in dense inclusions, which accumulated in the cisternae of the Golgi. In addition, de novo-synthesized VP8 continued to accumulate in the nucleus in dense areas and around nucleocapsids. In calves, VP8 stimulated T cell proliferation and antibody production, both after BHV-1 challenge and after immunization with purified VP8. These results suggest a role for VP8 in the induction of humoral and specifically cell-mediated immunity to BHV-1.

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...

We combined the protein G-gold complex with several polyclonal and monoclonal antibodies for localization of various antigenic sites. The labelings were compared with those obtained using the protein A-gold complex. The results from either the immunodot experiment or immunoelectron microscopy have demonstrated that, for rabbit and guinea pig antibodies, both protein G-gold and protein A-gold complexes label several different specific antibodies with similar efficiency. However, with antibodies raised in goats or in mice, and particularly with mouse monoclonal antibodies, protein G-gold yielded intense and specific labeling, whereas protein A-gold yielded intense and specific labeling, whereas protein A-gold was very variable; it either gave weaker signals or failed to reveal any specific site or, as with one monoclonal, both protein G and protein A gave similar results. The higher affinity and versatility of protein G over protein A, established by the immunochemical approach, was confirmed by immunocytochemistry. Because of its enhanced reactivity with monoclonal antibodies and its broader affinity for polyclonal antibodies, protein G-gold complex appears to be a better and more versatile probe for high-resolution immunocytochemistry.

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