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DNA vaccines encoding for murine rotavirus proteins VP4, VP6, or VP7 were tested in adult BALB/c mice for their ability to induce immune responses and protect against rotavirus challenge. A gene gun was used to inoculate vaccines into the epidermis. Rotavirus-specific serum antibodies, as measured by ELISA, and virus-specific cytotoxic T lymphocyte responses were generated by each of the three vaccines, but virus-neutralizing antibodies were detected only in mice that were inoculated with DNA vaccines encoding for VP4 and VP7. Efficacy of the vaccines was determined by challenge with 100 ID50 of homotypic rotavirus. Each of the three vaccines was effective in protecting mice against infection after rotavirus challenge as determined by reduction (P < .001) in virus excretion in mice receiving the DNA vaccines. These results demonstrate that DNA vaccination has potential as a new approach for control of rotavirus infections.

The relative importance of astrovirus and adenoviruses as etiologic agents of diarrhea among children in day care was examined. Stool specimens from this prospective study were screened for both astrovirus and adenovirus hexon with two new indirect double-antibody assays and for enteric adenoviruses with an EIA specific for serotypes 40 and 41. Astrovirus was detected in a significantly greater percentage of children with diarrhea (4%, 21/524) than of those without (less than 1%, 1/138) (P less than .05); however, no difference between such such children with adenovirus infections was found (8%, 43/565, and 8%, 10/129, respectively). Overall, 30% (13/43) of all adenovirus hexon-positive specimens were enteric serotypes, and by extrapolation, enteric adenoviruses were identified in an equal percentage of children (2%) with and without diarrhea. This study documents the presence of astrovirus and enteric adenoviruses among children in day care in the United States, associates astrovirus with diarrhea in this setting, and suggests that viral agents may be the most common enteric pathogens among children with diarrhea in day care.

Administration of rimantadine to mice via drinking water, following a prophylactic dose, reduced lung virus titers by greater than 3 log10 plague-forming units (pfu)/ml but caused only marginal reductions in lung virus titers when therapy was started 8 h after exposure to virus. Mice given rimantadine prophylactically plus therapeutically were resistant to rechallenge with virus at a dose equivalent to that used for the primary infection (50 pfu/mouse) but not to a high dose (1 x 10(5) pfu/mouse). Virus-neutralizing-antibody titers were reduced only by rimantadine treatment, which included prophylaxis, whereas the cytotoxic T lymphocyte (CTL) response was depressed by treatment given with or without prophylaxis. Mice infected with rimantadine-resistant virus had no decrease in CTL or antibody responses when treated with rimantadine. Therefore, the depression in CTL and antibody responses associated with rimantadine treatment appears to be due to a decrease in the amount of viral antigen available or interference with viral antigen processing and not to nonspecific immunosuppressive effects.

An enzyme-linked immunosorbent assay (ELISA), based on monoclonal antibodies to the astrovirus group antigen, was designed for the detection of astroviruses in stools of patients with gastroenteritis. Compared to immune electron microscopy used as the standard test, the sensitivity of the astrovirus ELISA was 91% (31/34) and the specificity was 96% (54/56). All five of the known astrovirus serotypes could be detected in 16 samples on which serotyping was done. In tests on 155 stools containing other enteric viruses, including adenoviruses, rotaviruses, caliciviruses, Hawaii virus, Snow Mountain virus, and Norwalk virus (30, 20, 70, 24, 4, and 7 samples, respectively), only 3 were positive in the astrovirus ELISA. The combined specificity for all astrovirus immune electron microscopy-negative samples was 98% (206/211). The results demonstrate that the new ELISA provides a sensitive and specific means for the diagnosis of astrovirus gastroenteritis.

A monoclonal antibody-based enzyme immunoassay (EIA) was developed for direct detection of enteric adenoviruses in stool specimens from individuals with gastroenteritis. Tests specific for each of the enteric adenoviruses, adenovirus type 40 (Ad 40) and type 41 (Ad 41), were designed. The sensitivity of the assay was determined by comparing the results of the EIA with isolation of virus in Graham 293 cells from stools that contained particles having adenovirus morphology. The standard for specificity was analysis of adenovirus genome profiles after digestion with SmaI endonuclease. The sensitivity was 95.8% (23 of 24) for Ad 40 and 97.1% (34 of 35) for Ad 41. The specificity was 95.7% (45 of 47) and 97.2% (35 of 36), respectively. The two type-specific monoclonal antibodies could be mixed in an EIA for identification of enteric adenoviruses in stools without loss of reactivity in either type. The EIA permits rapid diagnosis and type-specific identification of enteric adenoviruses in gastroenteritis.

Cultivation of human astroviruses in human embryonic kidney or LLCMK2 cell cultures was corroborated for four of the five serotypes originally reported (types 1, 2, 4, and 5). By using type-specific rabbit antisera and immunofluorescence of virus-infected cells, we readily distinguished between serotypes of astrovirus; however, these serotypes showed a high degree of cross-reactivity by enzyme-linked immunoassay, a result indicating the presence of a group antigen. We prepared monoclonal antibodies to astrovirus type 2 antigen and selected them on the basis of group antigen reactivity. The antibodies were reactive with the four astrovirus serotypes that we could cultivate, as well as with the Marin County strain of astrovirus. A previously reported cell-cultivated astrovirus type 3 also reacted with the monoclonal antibodies. These monoclonal antibodies, and the finding of group reactivity among the human astroviruses, should facilitate studies on the importance of these viruses as agents of viral gastroenteritis.

The outer membrane complex of Rochalimaea quintana was isolated by use of ethylene-eiaminetetraacetate and was compared biochemically and biologically both with lipopolysaccharide (LPS) isolated by phenol-water extraction of whole organisms and with lipids isolated by chloroform-methanol extractions of the phenol-water insoluble residues. The outer membrane consisted of protein and LPS components, as distinguished by precipitin tests with sera from patients with trench fever or tests with hyperimmune animal sera. The outer membrane protein component, but not LPS, also reacted with sera from infections with Rickettsia tsutsugamushi. The LPS contained 2-keto-3-deoxy-octonate and heptose. The outer membrane and phenol-extracted LPS were reactive in the chick embryo lethality test, limulus assay, and complement activation. Outer-membrane activity was confined to the LPS component. The lipid extracts were reactive in chick embryo lethality and limulus assays, but did not activate complement.

Enzyme immunoassay (EIA) tests were used to diagnose trench fever and to determine cross-reactions of Rochalimaea quintana with other rickettsiae. The results were compared with those obtained by counterimmunoelectrophoresis (CIE). All sera from cases of primary or relapsed forms of trench fever were positive both in EIA, with serum antibody titers of 1:20-1:640, and in CIE, giving one to three precipitin lines. Sera from patients with other rickettsial infections were also tested for reactivity with R. quintana antigen: typhus group (Rickettsia prowazekii, Ricketsia mooseri), 15 sera; spotted fever group (Ricketsia ricketsii, Rickettsia akari), eight sera; and scrub typhus (Rickettsia tsutsugamushi), six sera. Strong reactions occurred with four sera from patients with scrub typhus, giving one or two lines in CIE and EIA titers of 1:40-1:160; these results were extended to guinea pig antisera to R. tsutsugamushi. About 50% of typhus group sera reacted with a single line in CIE and had antibody titers of 1:20-1:80 by EIA. The results show that EIA is accurate for the diagnosis of trench fever and, with the results obtained by CIE, suggest that R. quintana is antigenically related to R. tsutsugamushi and possibly to rickettsiae in the typhus group as well.

We investigated antigenic relationships between human calicivirus (HCV) strains and Norwalk virus by using immune electron microscopy (IEM) and radioimmunoassay (RIA). Three serologically distinct HCV strains, UK1, UK2, and Japan, were demonstrated by IEM, as was evidence for two additional strains, UK3 and UK4. Although HCV strains and Norwalk virus were distinct by IEM, 12 of 20 patients with gastroenteritis due to HCV UK4 and two of eight with gastroenteritis due to UK2 showed seroconversions to Norwalk virus by RIA. These naturally occurring antibody responses in humans, as detected by RIA, support the concept that Norwalk virus belongs to the family Caliciviridae. An RIA for HCV Japan antigen also detected HCV UK1, UK2, and UK4 and thus appears to identify a group-specific antigen for these viruses. An RIA for antibody to HCV Japan failed to identify seroconversions in 45 of 47 patients with gastroenteritis due to HCV UK. These results may reflect different reactivities of various immunologic tests for the identification of infections due to small gastroenteritis viruses.