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Bacillus subtilis vaccine strains engineered to express either group A bovine or murine rotavirus VP6 were tested in adult mice for their ability to induce immune responses and provide protection against rotavirus challenge. Mice were inoculated intranasally with spores or vegetative cells of the recombinant strains of B. subtilis. To enhance mucosal immunity, whole cholera toxin (CT) or a mutant form (R192G) of Escherichia coli heat-labile toxin (mLT) were included as adjuvants. To evaluate vaccine efficacy, the immunized mice were challenged orally with EDIM EW murine rotavirus and monitored daily for 7 days for virus shedding in feces. Mice immunized with either VP6 spore or VP6 vegetative cell vaccines raised serum anti-VP6 IgG enzyme-linked immunosorbent assay (ELISA) titers, whereas only the VP6 spore vaccines generated fecal anti-VP6 IgA ELISA titers. Mice in groups that were immunized with VP6 spore vaccines plus CT or mLT showed significant reductions in virus shedding, whereas the groups of mice immunized with VP6 vegetative cell vaccines showed no difference in virus shedding compared with mice immunized with control spores or cells. These results demonstrate that intranasal inoculation with B. subtilis spore-based rotavirus vaccines is effective in generating protective immunity against rotavirus challenge in mice.

Sera obtained from adult volunteers inoculated with genogroup II Norwalk-like viruses (NLVs), Hawaii virus, and Snow Mountain virus and from patients involved in outbreaks of gastroenteritis were tested for genogroup II NLV Mexico virus-specific immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant Mexico virus antigen (rMXV)-based IgM capture enzyme-linked immunosorbent assay (ELISA). Sera from genogroup I Norwalk virus (NV)-inoculated volunteers and from patients involved in a genogroup I NLV outbreak were also tested. In sera from those infected with genogroup I NV or NLVs in volunteer and outbreak studies, only 3 of 25 were rMXV IgM positive; in contrast, 24 of 25 were IgM positive for recombinant NV (rNV). In sera from those infected with genogroup II NLVs in volunteer and outbreak studies, 28 of 47 were rMXV IgM positive and none were IgM positive for rNV, showing the specificity of each IgM test for its respective genogroup. In an outbreak of gastroenteritis not characterized as being of viral etiology but suspected to be due to NV, 7 of 13 persons had IgM responses to rMXV, whereas none had IgM responses to rNV, thus establishing the diagnosis as genogroup II NLV infection. The rMXV-based IgM capture ELISA developed is specific for the diagnosis of genogroup II NLV infections.

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.

Sera obtained from two groups of adult volunteers infected with Norwalk virus (NV) and two groups of patients involved in two natural outbreaks were tested for NV-reactive immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant-antigen-based IgM capture enzyme immunoassay (EIA). No NV-reactive IgM was detected in the preinoculation sera of 15 volunteers, and 14 of 15 showed NV-reactive antibodies postinfection with NV. All of the volunteers showed IgG seroconversion to NV. In the outbreak studies, all 9 persons in one outbreak and 19 of 24 in another outbreak had NV-reactive IgM. In the first outbreak, only three of nine seroconverted to NV, which was likely due to late collection of acute-phase sera. In the second outbreak, 21 of 24 showed IgG seroconversion to NV. Sequencing of viruses isolated from five stool samples selected from those in the second outbreak showed that they were human calicivirus (HuCV) genogroup 1 viruses related, but not identical, to NV. In the volunteer studies, NV-reactive IgM was first detected 8 days postinoculation. The time of development of NV-reactive IgM antibodies in natural outbreaks was estimated to be similar to that found in the volunteer studies. Sera from three Hawaii virus-infected volunteers, four Snow Mountain virus patients, and 80 healthy individuals were negative for NV-reactive IgM, indicating test specificity for HuCV genogroup I infections. This capture IgM EIA is suitable for diagnosis of NV and other HuCV genogroup I infections and is especially useful when sera and fecal samples have not been collected early in the course of an outbreak.

Monoclonal antibodies against the prototype 8FIIa strain of Norwalk virus were prepared and applied to an enzyme immunoassay (EIA) for detecting Norwalk virus in stool specimens. The monoclonal antibodies immunoprecipitated a 58-kDa protein which had been produced by in vitro transcription-translation of Norwalk virus cloned cDNA, and they reacted by EIA with recombinant Norwalk virus capsid protein at a sensitivity level of 1 ng/ml. The EIA detected virus in all tested samples from 15 different Norwalk virus-infected volunteers. No cross-reactions were seen in stools containing other caliciviruses or in stools containing rotaviruses, astroviruses, or enteric adenoviruses.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.

We report the cloning of antigenic, protein-coding regions of human astrovirus serotype 1 that appear to be common to most, if not all, serotypes of human astrovirus. Screening of lambda gt11 libraries identified three different but overlapping clones (A43, A35, and A1) and one independent clone (A14) that reacted with serum from a rabbit repeatedly immunized with purified astrovirus particles but not with its preimmunization serum. These clones were shown to be astrovirus specific. Of note, a radiolabeled probe representing the immunoreactive clones A43-A35-A1 hybridized exclusively to the 7.2-kb astrovirus genomic RNA, while a clone A14-specific probe hybridized with both the genomic and the 2.8-kb astrovirus subgenomic RNAs. This suggests that the immunoreactive epitopes, selected by antiserum to purified astrovirus particles, are encoded by the subgenomic RNA as well as other regions of the genomic RNA.

A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.

Fecal excretion of astroviruses was monitored in 321 children, 0 to 3 years old, living in the rural highlands of Guatemala. During the longitudinal study, from February 1987 to February 1989, we examined 5,000 stool specimens, including 1,805 collected during 1,369 episodes of diarrhea, 830 collected during the convalescent week, and 216 and 244 collected 2 weeks and 1 week, respectively, before the onset of diarrhea. Routine specimens were taken once a month from every child who had been free from diarrhea for at least three consecutive weeks. Of the children, 124 (38.6%) excreted astroviruses during the study. In total, we identified 184 infections by astroviruses. Of the samples collected 2 weeks and 1 week before the initiation of symptoms, 0.9 and 4.9%, respectively, were positive, while 7.3% of the diarrhea episodes were associated with astroviruses. Of the convalescent specimens, 3.4% were shown to be positive; 2.4% of the 1,905 specimens taken in diarrhea-free periods contained astroviruses. Infections by other potential enteropathogens were documented in 54 and 65% of the asymptomatic and symptomatic astrovirus infections, respectively. Diarrhea associated with astroviruses alone had a median duration of 5 days and was associated with vomiting in 8.6%, with fever in 17.1%, with dehydration in 5.7%, and with loss of appetite in 34.3% of the episodes. Diarrhea due to astroviruses was accompanied by negative changes in weight gain. Astrovirus diarrhea contributes to the high morbidity observed in young children living under poor conditions and has a deleterious effect on their nutritional status.

Two new astrovirus assays, a rapid biotin-avidin enzyme immunoassay (EIA) and RNA probe hybridization, were developed and compared with an established astrovirus assay, an indirect EIA, and immune electron microscopy. Sensitivity and specificity were evaluated by using a screening panel of 22 astrovirus-positive and 305 astrovirus-negative fecal specimens. The biotin-avidin assay was equivalent in performance to the reference indirect assay, and both could detect about 10 ng of viral protein. Although the probe was more sensitive than either EIA and could detect higher dilutions of virus in tissue culture and stool specimens, it did not detect more astrovirus-positive fecal specimens. Of the 22 astrovirus-positive specimens detected by the EIAs, 20 were confirmed by immune electron microscopy with hyperimmune rabbit antiserum. To determine the usefulness of EIAs for large epidemiologic studies, EIAs were used to screen 1,289 stool specimens from three studies of children with and without diarrhea. Astrovirus was detected in 3.5% of specimens from children with diarrhea and 1.9% of specimens from those without diarrhea. Our results indicate that the biotin-avidin EIA is an efficient, sensitive, and specific method for routinely screening large numbers of fecal samples and that its application in epidemiologic studies may yield higher rates of astrovirus infection than have been found previously by other methods.

Astroviruses are nonenveloped particles with a distinctive star-shaped surface structure that have been detected by electron microscopy in stool samples from humans and animals with gastroenteritis. We examined the patterns of macromolecular synthesis in astrovirus-infected cells with a goal of establishing a molecular basis for taxonomic classification. Trypsin is required for continuous replication of astrovirus in cultured cells; however, during a single cycle of infection, astrovirus antigen was synthesized earlier and at higher levels when serum, rather than trypsin, was included in the growth medium. This enhanced production of antigen, as measured by enzyme immunoassay, was accompanied by the appearance of aggregates of virus particles in the cytoplasm of infected cells. During astrovirus replication in cells cultured in the presence of serum, we detected a single infection-specific protein (90 kDa) beginning at 12 h postinfection. This protein was recognized by antiastrovirus rabbit serum and was sensitive to trypsin digestion in vitro, with the concomitant appearance of three smaller immunoreactive proteins (31, 29, and 20 kDa). We also detected two dactinomycin-resistant RNAs (7.2 and 2.8 kb), both of which were polyadenylated, in the cytoplasm of astrovirus-infected cells. The larger of these two RNAs is presumably the viral genome, whereas the smaller species may be a subgenomic messenger. Comparison of the proteins and RNAs synthesized in astrovirus-infected cells with those of the recognized families of nonenveloped single-stranded RNA animal viruses suggests that astroviruses should not be classified as members of either Caliciviridae or Picornaviridae.

We compared a direct fluorescent-antibody stain (DFA) and an enzyme immunoassay (EIA) with a standard cell culture technique for the detection of Chlamydia trachomatis infection in women in an urban family practice setting. We also evaluated a DFA sample in a commercial laboratory to determine the interlaboratory reliability of this test. There were 268 women in the study; the EIA provided a higher sensitivity (83 versus 50%) and a higher positive predictive value (83 versus 69%) than the DFA test and comparably high specificity (99 versus 98%). Concordance between the two laboratories on the DFA test was not high when data were adjusted for chance agreement (kappa coefficient = 0.64). DFA validity was optimal with an elementary body cutoff of greater than 5, while EIA validity was optimal at the recommended cutoff of 0.1 optical density unit. None of 11 women with negative cultures after treatment had false-positive antigen tests. False-negative results with both tests were associated with low culture inclusion counts but were not strongly associated with the presence or absence of symptoms, menses, pregnancy, or recent antibiotic use. False-positive results with EIA were seen only for three women who had a chief complaint of vaginal discharge. Although the positive predictive value of DFA could be increased in high-prevalence subpopulations, EIA was still more valid in two such groups: teenagers and prenatal patients. These results indicate that EIA might be preferable for low- or moderate-prevalence populations in primary care settings and that a falloff in DFA sensitivity could be explained by lower infection burdens in low-prevalence groups.

Chlamydiazyme is a 4-h enzyme-linked immunoassay that detects an antigen of Chlamydia trachomatis directly in clinical specimens. This immunoassay was compared with cell culture for the diagnosis of chlamydial infections of the genital tract. The assay was evaluated at five clinics with a total of 1,277 cervical specimens of which 239 were culture positive. At three of these clinics where urethral samples were taken from males, 99 of 363 samples were culture positive. The sensitivity of the assay averaged 89.5% for detecting cervical infections and 78.8% for detecting male urethral infections. Specificity was 97.0% when samples from either males or females were tested. Some patients who were culture negative were infected with chlamydiae according to both Chlamydiazyme and a monoclonal antibody test that detected a chlamydial antigen distinct from the antigen detected by Chlamydiazyme. If the 15 females and 2 males who were positive by both immunoassays but culture negative were considered positive for chlamydial infection, the specificity of the assay was 98.4% in females and 97.7% in males. Chlamydiazyme is a simple and relatively rapid immunoassay that has sufficient sensitivity and specificity to supplant culture in the detection of genital chlamydial infections.

We compared the diagnostic value of an enzyme immunoassay method for detection of gonococcal antigen in genital secretions with culture results and direct Gram stain for Neisseria gonorrhoeae in 1,171 men and 723 women attending a sexually transmitted disease clinic. When compared with culture results in men, the immunoassay provided a sensitivity of 94% and a specificity of 98% and was essentially equivalent to the urethral Gram stain. The predictive value of a positive immunoassay was 97% in men with a urethral discharge in whom the prevalence of gonorrhea was 36%, and 30% in men without urethral discharge, who had a 2% prevalence of gonorrhea (P less than 0.001). The sensitivity of the immunoassay was 95% in men with and 67% in men without urethral discharge (P less than 0.01). In women, the immunoassay resulted in a sensitivity of 78% and a specificity of 98% compared with cervical culture and had a significantly better sensitivity than the cervical Gram stain (78 versus 48%, P less than 0.001). Analysis of patients with discrepant culture and immunoassay results suggested that most culture-negative, immunoassay-positive patients probably did not have gonorrhea. After treatment, all but 1 of 59 originally culture- and immunoassay-positive patients became negative in both tests by 3 days. Results of the immunoassay were not affected by transport or by refrigeration for up to 30 days.

A total of 176 human fecal specimens were examined for the presence of rotavirus by four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, North Chicago, Ill. (Rotazyme I); a modification of this assay which is now commercially available (Rotazyme II); and a latex agglutination test (Rotalex) recently introduced by Medical Technology Corp., Somerset, N.J. In addition, selected specimens were examined for the presence of rotavirus by electron microscopy, immune electron microscopy, and RNA gel electrophoresis. A total of 40 specimens were positive in the monoclonal antibody enzyme immunoassay, and 136 were negative. Using the results obtained with this procedure as the reference standard, we found the sensitivities of the Rotazyme I, Rotazyme II, and Rotalex tests to be 97.4, 100, and 81.6%, respectively. The specificities of these three procedures were 88.8, 83.9, and 100%, respectively.

Cotton swabs were used to collect two specimens each from 416 patients (206 males, 210 females) attending a sexually transmitted disease clinic. The first swab was transported in Specimen Storage Reagent and extracted in Specimen Dilution Buffer for enzyme immunoassay by Chlamydiazyme (Abbott Laboratories); the second swab was extracted into 2SP and inoculated into McCoy cell cultures. In the first phase of the study (215 patients: 111 males, 114 females) enzyme immunoassay results were positive (optical density greater than or equal to 0.1) in 30 of 35 instances in which Chlamydia trachomatis was isolated (sensitivity, 86%). Of 18 false-positive enzyme immunoassay results, 15 (83%) were cervical swabs (specificity, 90%). In a phase II study, using a modified Chlamydiazyme kit, 201 patients were tested (95 males, 106 females). Of 41 chlamydial isolates, 8 were not detected by the Chlamydiazyme test (sensitivity, 81%). Only three positive Chlamydiazyme test results could not be confirmed by culture (specificity, 98%). Overall, Chlamydiazyme assay provided a rapid (4 h), sensitive, and specific assay for the detection of chlamydial antigens.

In countries with temperate climates, enteric adenoviruses have been shown to be a substantial cause of pediatric gastroenteritis. To determine the incidence of adenovirus infection in a tropical climate, stools were collected from children under age 7 during a 1-year period at an outpatient clinic in Bangkok, Thailand. Stools from 1,114 children with gastroenteritis and from 947 children without gastroenteritis were tested. Each stool was tested for adenovirus group antigen and for specific enteric adenovirus types (Ad40 and Ad41) by monoclonal antibody enzyme immunoassays. We found that 4.4% (49 of 1,114) of children with gastroenteritis and 1.8% (17 of 947) of children without gastroenteritis were positive for adenovirus group antigen. In tests for specific enteric adenovirus types, 2.0% (22 of 1,114) of the tests were positive in children with gastroenteritis and 0.6% (6 of 947) were positive in children without gastroenteritis. There was a significant correlation (P less than 0.02) of gastroenteritis with nonenteric adenovirus types (27 of 1,114) as well as with specific enteric adenovirus types (P less than 0.01). By comparison, 19.7% of children with gastroenteritis and 0.7% of those without gastroenteritis were positive for rotavirus infection. In the adenovirus-infected children with gastroenteritis, there were coinfections with rotavirus only in those with nonenteric adenovirus infection (7 of 27 children). There were no significant differences in the association of bacterial or parasitic infections with either enteric or nonenteric adenovirus infections in either group of children studied. These data demonstrate that Ad40 and Ad41 are causes of gastroenteritis in this population, but among the spectrum of diarrheal etiologies, they may be proportionately less important than they are in countries with temperate climates.

A quantitative enzyme-linked immunosorbent assay was used for identification of selected enteroviruses: poliovirus type 1, echovirus type 6, coxsackievirus A type 9, and coxsackievirus B types 1 through 6. Partially purified viral antigens or virus-specific antibodies were adsorbed to polystyrene spectrophotometer cuvettes, which permitted the assays to be reported and compared in terms of enzyme units specifically reacting. Both the adsorbed antigen and the adsorbed antibody methods were approximately equal in terms of sensitivity and specificity of reaction. By use [14C]leucine-labeled enteroviruses, the amount of virus that bound to the plastics used was shown to be dependent on the purity of the virus preparation used, but it was higher than the amount that was bound by plastics coated with viral antibody. Diluents which contained 0.15% (vol/vol) Tween 20 and 2.0% (wt/vol) bovine serum albumin in phosphate-buffered saline, pH 7.2, were found to be the most effective in inhibiting nonspecific adsorption of immunoreagents. However, the presence of these inhibitors in phosphate-buffered saline solutions also caused desorption of virus or viral antibody during immunoassays; the amount of virus desorption varied with the type of preparation used, and antibody desorption was dependent on the concentration of antibody initially used for adsorption. For specific identification of a given enterovirus type by the enzyme-linked immunosorbent assay method used, approximately 10(5) plaque-forming units of virus per assay tube were required.

Two enteroviruses were inactivated more rapidly in a lake than in sterile lake water; then their coat proteins were degraded and, perhaps, used by microorganisms.

Eighty-two stool samples from children with gastroenteritis in Canada, England, and Thailand which had been shown to contain adenovirus antigen (by a group-specific enzyme-linked immunosorbent assay) or adenovirus particles (by electron microscopy) or both, were tested for primary isolation of enteric adenoviruses in HEp-2 and Graham 293 cells. Graham 293 cells are known to support the replication of enteric adenovirus types (Ad40 and Ad41) on primary isolation, whereas HEp-2 cells reportedly do not. Of the 82 adenovirus isolates, 73 could be typed as Ad40 or Ad41 by type-specific monoclonal antibody enzyme-linked immunosorbent assay and by analysis of SmaI endonuclease digests. Of these 73, 30 (41%) could be isolated in HEp-2 cells, which included 43% (9/21) of those typed as Ad40 and 40% (21/52) of those typed as Ad41. On the basis of these results, the growth characteristics of adenoviruses in HEp-2 cell cultures, commonly used to distinguish enteric from nonenteric adenovirus types, are not valid for either diagnosis or epidemiological studies. For the samples studied here, use of these nondefinitive criteria would result in underestimation of the incidence of enteric adenoviruses in viral gastroenteritis.

Peroxidase-conjugated antibodies were found to be as sensitive as those conjugated to fluorescein-isothiocyanate (FITC) for identification of selected enterovirus types by the indirect technique. Peroxidase conjugates, however, were found to give fewer nonspecific reactions. The reasons for the higher specificity of the immunoperoxidase technique appear to be related to the relative size, charge, and uniformity of the preparations. Monomers of peroxidase-conjugated globulin are larger than those of FITC conjugates, but the latter readily form aggregates. This was shown by chromatography on Sepharose 6B columns: various FITC-conjugated globulins eluted before those conjugated to peroxidase and before (125)I-labeled immunoglobulin G. The net charge of the conjugates was determined by adsorption to ion-exchange columns. FITC-labeled globulins had a negative net charge, eluting at pH 5.1 from diethylamino-ethyl-Sephadex A-50 columns. Peroxidase conjugates were not retained by either cationic or anionic exchangers at pH values ranging from 4.0 to 10.5. Further, the fluorescein/protein ratios of FITC conjugates from different commercial sources and of those prepared in the laboratory were found to be variable; higher fluorescein/protein ratios (>2:1) give a higher degree of nonspecific reactions, whereas the peroxidase/protein ratio does not appear to affect specificity. These characteristics of peroxidase conjugates make the immunoperoxidase technique easier to standardize and more reliable for enterovirus identification than the immunofluorescence technique.

The purpose of this study was to develop methods for detection and quantitation of food-borne virus. Samples (25 g) of cottage cheese, contaminated with various quantities of coxsackievirus type A9, comprised the model system. Two of the methods presented have at least a 50% probability of detecting virus at levels below 5 plaque-forming units/25-g sample. Noteworthy aspects of these methods include use of a glycine-NaOH buffer (pH 8.8) containing approximately 1 m MgCl(2) as the diluent in which the sample is slurried, treatment of the slurry with Freon TF and bentonite to facilitate centrifuge clarification, and concentration of the clarified sample extract by a two-stage process employing polyethylene glycol followed by ultracentrifugation. Virus in the final 0.5-ml sample concentrate was detected and quantitated by the plaque technique in rhesus monkey kidney cell cultures. Processing of the sample requires approximately 2 days, and the inoculated cultures may have to be observed for as long as 7 days thereafter. If these levels of sensitivity are desired, and if 12 samples per day are tested on a routine basis, the cost savings achieved by employing these methods rather than testing sample extracts without concentration may range from 75 to 90%.

A method previously reported for detecting virus in a model system composed of cottage cheese contaminated with coxsackievirus type A9 has been adapted to detecting selected strains of enteroviruses in a variety of foods. Bentonite is omitted and serum is added for extracting virus from low-protein foods. Samples of foods, usually 25 g, must contain at least 3 to 4 plaque-forming units for a 50% probability of detecting virus. Sensitivity in detecting echovirus type 6 was lower than that for the other viruses used. After extraction from potato salad, poliovirus type 2 was completely reactivated if it had been neutralized with coproantibody, but it was only partially reactivated if neutralized with hyperimmune rabbit serum.

Antibody to poliovirus type 1 (Po-1) was coupled to peroxidase by use of glutaraldehyde or 4, 4' -difluoro,3, 3' -dinitro diphenyl sulfone. Glutaraldehyde was found to be the superior coupling agent, yielding conjugates that had up to 2.8 x 10(4) enzyme units/ml (75% of total enzyme input). Conjugates migrated as a single band when centrifuged in sucrose density gradients, demonstrating that the purification procedure used was effective in removing both noncoupled enzyme and heterogeneous antibody components. Conjugates were specific for Po-1 and did not adsorb to cells infected with unrelated enterovirus types. Adsorption of conjugates to Po-1-infected cells was demonstrable within 6 h postinfection.

The specificity of labeling enteroviruses with (32)P-labeled NaH(2)PO(4) or (14)C-leucine can be determined by comparing the percent adsorption of infective particles and radioactivity to membrane (Millipore) filters.

The means by which coxsackievirus type A9 (CA9) is inactivated by proteolytic enzymes was investigated. After reaction of (14)C-leucine-labeled CA9 with Pronase, free leucine was liberated as measured by radiochromatography. Treatment of (14)C-leucine-labeled CA9 with trypsin or proteolytic filtrates of Pseudomonas aeruginosa caused the release of a variety of labeled substances. The extent of viral ribonucleic acid (RNA) release after exposure of CA9 to Pronase was determined by RNA infectivity tests or trichloroacetic acid solubility tests. Infective viral RNA was found not to be consistently released by reaction of CA9 with Pronase, but further treatment with 1% sodium dodecyl sulfate at pH 7.0 promoted viral RNA release. Sodium dodecyl sulfate treatment of CA9 that had not been reacted with Pronase did not inactivate virus or cause viral RNA release. Reaction of Pronase with (32)P-labeled CA9 resulted in the liberation of virus components soluble in cold trichloroacetic acid, whereas untreated CA9 or CA9 reacted with ribonuclease were precipitated by cold trichloroacetic acid. These results demonstrate that the primary means by which protease-sensitive enteroviruses are inactivated is by degradation of the virus capsid, with subsequent release of viral RNA.