Neil R. Blacklow

Astroviruses as a cause of gastroenteritis in children.

BACKGROUNDnInfection with astroviruses has been associated with gastroenteritis in children, and serologic surveys indicate that this infection may be frequent. The importance of astroviruses as agents of gastroenteritis has not been shown in a controlled study, however.nnnMETHODSnWe used monoclonal antibody-based enzyme immunoassays to detect astroviruses, enteric adenoviruses, and rotaviruses in stool samples obtained from age-matched children with and children without gastroenteritis. The samples were obtained in two studies, three years apart, among patients attending an outpatient clinic in Bangkok, Thailand.nnnRESULTSnIn the first study, astroviruses were detected in 8.6 percent (96 of 1111) of the children with gastroenteritis and in 2.0 percent (19 of 947) of the children without gastroenteritis. In the second study the rates were 8.6 percent (50 of 580) and 2.1 percent (11 of 512), respectively. For both studies combined, enteric adenoviruses were detected in 2.6 percent of those with gastroenteritis and in 0.5 percent of the controls, whereas rotaviruses were detected in 19 percent of those with gastroenteritis and in 1.0 percent of the controls. The clinical findings associated with astrovirus infection were similar to those associated with rotavirus infection, except for a trend toward greater dehydration in the children infected with rotaviruses.nnnCONCLUSIONSnThese two controlled studies involving a total of 3150 Thai children provide evidence that astroviruses are a common cause of viral gastroenteritis. Astroviruses were found in association with gastroenteritis more frequently than were enteric adenoviruses, and with nearly half the frequency of rotaviruses.

Human astrovirus isolation and propagation in multiple cell lines.

Summary.u2002Laboratory adapted human astrovirus serotypes 1 through 7 were tested for growth in 15 human, 7 simian, and 10 other non-primate mammalian cell lines. Propagation of all seven serotypes was successful in the human cell lines Caco-2, T84, HT-29, and in the African green monkey kidney cell line MA-104. Both primary and secondary African green monkey kidney cells were more effective than Rhesus monkey kidney cells for cultivation of astrovirus. Except for human foreskin cells, all of the other human and simian cell lines supported growth of at least one astrovirus serotype. The only non-primate cell line that permitted sustained passage of astroviruses was the BHK-21 (C13) cell line for astrovirus serotype 2. Seventeen human stool specimens that had previously been shown to be astrovirus positive by ELISA were cultured in Caco-2, T84, HT-29, SK-CO-1, PLC/PRF/5, MA-104, and VERO cells. Caco-2 cells (13 isolates), T84 cells (12 isolates) and PLC/PRF/5 cells (12 isolates) were the cell lines most effective for isolation of human astroviruses from clinical stool specimens. By immunofluorescent staining of infected cells, culturing of the same 17 specimens in shell vials for 18u2009h was positive for astroviruses in all 17 specimens in Caco-2 cells, 12 in T84 cells, and 7 in PLC/PRF/5 cells. Shell vial assay is suitable as a rapid and sensitive culture technique for detection of astroviruses in clinical specimens.

Plaque quantitation and virus neutralization assays for human astroviruses.

SummaryAstroviruses, 28 nm-diameter, RNA-containing viruses which have been implicated in gastroenteritis can be cultivated in cell cultures containing trypsin, but do not show distinguishable cytopathic effects. However, with the 5 known astrovirus serotypes which we have been able to cultivate, 3 (types 1, 2, and 5) formed well-defined plaques in LLCMK2 cell cultures under an agar overlay containing trypsin. A virus neutralization assay based on plaque reduction was applied to these 3 serotypes. It was found that rabbit antisera prepared against individual serotypes neutralized virus type-specifically, and no cross-neutralization titers were obtained with any of the antisera to the 5 astrovirus serotypes. The type-specific neutralization observed agreed with the specificities seen by immunofluorescence (IF), whereas ELISA tests with the same antisera show cross-reactivity among all 5 serotypes. There was no virus neutralization detected with astrovirus monoclonal antibodies which were reactive with 5 serotypes by ELISA and IF. The results we have obtained permit quantitative techniques to be applied to epidemiological and biological studies of the human astroviruses.

Preparation and characterization of monoclonal antibodies to enteric adenovirus types 40 and 41

SummaryWe have prepared monoclonal antibodies to each of the enteric adenoviruses types 40 and 41. Three different hybridoma cell lines were selected which produced antibody found to react by radioimmunoprecipitation with adenovirus (Ad) hexon antigens. One was specific for Ad 40, another for Ad 41, and a third one reacted with both types. When tested in an enzyme immunoassay against all 41 known human Ad types, the type-specific monoclonal antibody against Ad 40 reacted homotypically, as did the monoclonal antibody against Ad 41. In addition, these monoclonal antibodies neutralized the homologous enteric Ad type. The monoclonal antibody which reacted with both enteric Ad types also showed lower levels of reactivity with the group C adenoviruses types 2, 5, and 6. The monoclonal antibodies produced will provide a definitive means for rapid identification of specific Ad types, and will be useful in defining the relationship of enteric adenoviruses to other types.

Immunological characterization of the Marin County strain of astrovirus

SummaryMarin County virus (MCV) was isolated from a stool suspension and serially propagated in human embryonic kidney cell cultures. MCV particles in stool and cell-propagated virus stocks showed reactivity by immune electron microscopy (IEM) with rabbit antiserum to astrovirus type 5. MCV antigen was also detected in two MCV stool samples by enzyme immunoassay (EIA) with an astrovirus group-specific monoclonal antibody. Acute and convalescent sera from 3 of 3 MCV-infected patients showed seroconversion to cell-propagated MCV by EIA. Immunofluorescence of MCV propagated in cell culture showed positive reactivity with an astrovirus group specific monoclonal antibody and astrovirus type 5 antiserum, with some cross-reactivity with astrovirus type 1. Similar results were obtained with the prototype strain of astrovirus type 5. However, in plaque-reduction assays, both the prototype astrovirus type 5 and MCV were neutralized by type 5 antiserum only. We conclude that MCV can be serially propagated by techniques used for previously described astroviruses and is serotypically an astrovirus type 5.

Medical Virology of Small Round Gastroenteritis Viruses

Despite the medical importance of viral diarrheas, the responsible etiologic agents were totally unknown until the discovery of Norwalk virus in 1972 (Kapikian et al. 1972). The main reason for this delay in their recognition was the inability to cultivate in cell culture etiologic agents from stool specimens. This was in marked contrast to the success in cultivating the many viruses causing respiratory tract disease, measles, mumps and other common viral syndromes. It was only when electron microscopic (EM) and antigen detection techniques were applied to the study of stool specimens that viral gastroenteritis agents were discovered and their medical importance subsequently ascertained.