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SUMMARYVirus structural antigens were localized within a line of monkey kidney (MA104) cells infected with the simian rotavirus SA11 using electron microscopic immunoperoxidase techniques. When hyperimmune guinea-pig anti-SA11 serum was used, virus particles, membranes of virus-associated endoplasmic reticulum, and viroplasmic inclusions were most heavily labelled. A general cytoplasmic reaction (ribosomes, intracytoplasmic membranes, etc.) with anti-SA 11 serum was also observed, but nuclei were unstained. In addition, several other virus-induced structures were found to contain rotavirus proteins, including convoluted smooth membrane within the endoplasmic reticulum, aberrant virus-like particles, and 15 to 20 nm diam. cytoplasmic tubules. Monospecific antiserum to VP7 (outer capsid glycoprotein, mol. wt. 38 000) reacted strongly with virus particles and the virus-associated endoplasmic reticulum, but reacted poorly with viroplasmic inclusions. The nucleus and general cytoplasm were unstained with anti-VP7. In contrast, monospecific antisera to VP2 and VP6 (inner capsid proteins, tool. wt. 94000 and 41000 respectively) reacted very strongly with viroplasmic inclusions. Virus particles, endoplasmic reticulum and cytoplasmic ribosomes were also labelled with these sera. These results indicate that rotavirus inner capsid proteins are synthesized throughout the cytoplasm and become concentrated in viroplasmic inclusions, while the outer capsid glycoprotein is synthesized primarily on ribosomes of the rough endoplasmic reticulum. Thus, the outer capsid layer appears to be acquired during virus budding into cisternae of the endoplasmic reticulum.

Rotaviruses are the major pathogens that cause life-threatening diarrhea in young children and animals. We inserted a simian rotavirus SAll gene 6 cDNA into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. The major capsid antigen (VP6) was expressed in high yields (20 to 150 eug/106 cells) when Spodopterafrugiperda cells were infected with baculovirus recombinants containing SAll gene 6 inserts. Reactivity with monospecific polyclonal and monoclonal antibodies suggested that VP6, expressed intraceliularly or found in the media, maintained native antigenic determinants. VP6 purified from the media from infected cells also possessed a native oligomeric structure, was immunogenic in guinea pigs, and was able to spontaneously assemble into morphologic subunits. Antisera from immunized guinea pigs failed to neutralize virus in plaque reduction assays, but detected homologous and heterologous rotavirus strains when tested by immunofluorescence, immunoprecipitation, and enzyme-linked immunosorbent assays.