Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent.

Molecular characterization of Hawaii virus and other Norwalk-like viruses: evidence for genetic polymorphism among human caliciviruses.

Hawaii virus (HV), from a 1971 family outbreak of gastroenteritis, is serotypically distinct from Norwalk virus (NV), recently identified as a human calicivirus by molecular analysis. About 2600 consecutive nucleotides of the HV genome (including those encoding the viral capsid protein) and part of the polymerase region of three other viruses (MDV1, MDV6 and SV7) were sequenced. Comparison of the amino acid sequence of the capsid protein of HV with NV and other human caliciviruses (Toronto virus [TV24], Desert Shield virus [DSV395], and Southampton virus [SHV]) demonstrated the existence of two major genetic groups (genogroups) typified by HV and NV. HV had 76% identity with TV24 and 48% identity with NV, DSV395, or SHV. In addition, comparison of part of the polymerase protein of HV with other human caliciviruses also showed that there were these two genogroups. The large genetic diversity between the capsid sequence of HV and NV is consistent with their serotypic distinctiveness.

Detection of Norwalk virus or Norwalk-like virus infections in Finnish infants and young children.

Norwalk virus (NV) and Norwalk-like viruses are important causes of epidemic nonbacterial gastroenteritis in older children and adults. Serologic responses to NV of 154 Finnish infants and young children participating in a rotavirus vaccine study were examined by ELISA with a recently available baculovirus-expressed recombinant NV capsid protein. In 4 serially collected sera (at the median ages of 3, 4, 14, and 23 months), 49% of children had at least one NV infection over the approximately 2-year study period. Children with low NV-specific IgG titers (< 1:50) at the median age of 4 or 14 months were significantly more likely to acquire an NV infection by the median age of 14 or 23 months, respectively, than children who had higher NV IgG titers (> 1:50) (P < .05). Thus, NV or Norwalk-like virus infections are more common in infants and young children than previously believed, and antibody to NV may be protective against such infections.

Identification of serotype 9 human rotavirus by enzyme-linked immunosorbent assay with monoclonal antibodies.

Hybridomas producing monoclonal antibodies to VP7, a major neutralizing protein of serotype 9 rotavirus (strain W161), were prepared. One monoclonal antibody, W161-6A1, was shown to neutralize only serotype 9 rotavirus strains and reacted specifically with serotype 9 rotaviruses in an enzyme-linked immunosorbent assay. The development of an immunoassay for detection of serotype 9 rotaviruses should facilitate epidemiologic studies.

An equine rotavirus (FI-14 strain) which bears both subgroup I and subgroup II specificities on its VP6.

An equinine rotavirus FI-14 strain, originally isolated from a diarrheic foal in New York state, was shown to belong to serotype 3 by neutralization assay. In addition, it was found to react with both subgroup I and subgroup II monoclonal antibodies by enzyme-linked immunosorbent assay (ELISA), thus representing the first rotavirus strain to exhibit both subgroup specificities. By using hybridoma technology, we successfully produced monoclonal antibodies directed against the major inner capsid protein VP6 (the sixth gene product) of FI-14 virus. Such monoclonal antibodies reacted specifically with either subgroup I or subgroup II rotaviruses thus demonstrating that the VP6 of FI-14 virus has both subgroup I- and subgroup II-specific epitopes. Four additional monoclones directed to the VP6 of FI-14 demonstrated distinct reactivities by ELISA with a panel of 49 rotavirus strains derived from 11 different animal and avian species. Thus, at least six distinct antigenic sites were shown to exist on VP6 of FI-14 virus. When these 49 rotavirus strains were arranged based on their reactivity patterns with the six representative monoclones, they fell into one of eight reactivity groups. Analysis of the reactivity patterns of rotaviruses derived from various animal species suggested that human rotaviruses may have two ancestral lineages: one (subgroup II, serotype 1, 3, and 4) with pig-human lineage, and the other (subgroup I, serotype 2) with bovine-simian-human lineage. When analyzed by radioimmunoprecipitation, the molecular weight of the FI-14 virus VP6 (subgroups I and II) appeared to be larger (approx 45K) than those (approx 42K) of rhesus monkey MMU18006 virus VP6 (subgroup I) or human Wa virus VP6 (subgroup II). By RNA-RNA hybridization analysis, the FI-14 virus was shown not to share significant homology with viruses belonging to the four known human rotavirus serotypes.

Analysis by RNA-RNA hybridization assay of intertypic rotaviruses suggests that gene reassortment occurs in vivo.

Antigenic characterization of human and animal rotaviruses by the plaque reduction neutralization assay has shown the existence of naturally occurring intertypes. Antiserum to M37, a rotavirus strain isolated from an asymptomatic neonate, neutralizes both Wa and ST3 strains, which are classified as serotype 1 and serotype 4 human rotaviruses, respectively. Likewise, antiserum to SB-1A, a porcine rotavirus, neutralizes rotavirus strains belonging to serotype 4 or 5. Plaque reduction neutralization assay of reassortant rotaviruses produced in vitro from these intertypes indicates that these viruses share one antigenically related outer capsid protein, VP3, with one serotype and another antigenically related outer capsid protein, VP7, with the other serotype. Thus, M37 is related to ST3 on the basis of its fourth-gene product, VP3, and to Wa on the basis of its ninth-gene product, VP7, whereas SB-1A is related to Gottfried (serotype 4 porcine rotavirus) via VP7 and to OSU (serotype 5 porcine rotavirus) via VP3. RNA-RNA hybridization studies revealed a high degree of homology between the VP3 or VP7 gene segments responsible for shared serotype specificity. Thus, the fourth gene segments of M37 and ST3 were highly homologous, while M37 and Wa had homology between their ninth gene segments. SB-1A and Gottfried were homologous not only with respect to the ninth gene but had complete homology in all other genes except the fourth gene. The fourth gene of SB-1A was highly homologous with the fourth gene of OSU. These observations suggested that SB-1A was a naturally occurring reassortant between Gottfried-like and OSU-like porcine rotavirus strains. Our observations also suggested that intertypes may result from genetic reassortment in nature.

Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus.

A series of monoclonal antibodies was isolated which reacted with one of two major surface proteins of rhesus rotavirus. Thirty-six monoclonal antibodies immunoprecipitated the 82-kilodalton outer capsid protein, the product of the fourth gene, the viral hemagglutinin. These monoclonal antibodies exhibited hemagglutination inhibition activity and neutralized rhesus rotavirus to moderate or high titer. Three monoclonal antibodies immunoprecipitated the 38-kilodalton outer capsid glycoprotein, the eighth or ninth gene product. These three monoclonal antibodies neutralized rhesus rotavirus to high titer and also inhibited viral hemagglutination.

Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies.

Ten monoclones directed to the 42,000-dalton inner structural protein of rotavirus were analyzed. Eight monoclones reacted broadly with antigenic domains common to virtually all mammalian rotaviruses. Two monoclones had specificities similar or identical to previously characterized subgroup specificities. These subgroup monoclones were more efficient in detecting subgroup antigen than either hyperimmune or postinfection antisera. Using the subgroup monoclones, we determined that some animal as well as human rotavirus strains carry subgroup 2 specificity and that epizootic diarrhea of infant mice virus and turkey rotavirus are antigenically distinct from other mammalian rotavirus strains.

Proteins of Norwalk virus.

The proteins of the Norwalk virus were studied by polyacrylamide gel electrophoresis. Highly purified specifically immunoprecipitated virions appeared to contain a single primary structural protein with a molecular weight of 59,000. In addition, a soluble Norwalk viral protein with a molecular weight of 30,000 was identified in fecal specimens containing Norwalk virus. The protein structure of the virion is similar to that of the Calciviridae family.

Prevalence of antibody to the Norwalk virus in various countries.

Serum samples from children and adults from several countries were tested by radioimmunoassay for antibody to the Norwalk virus. Antibody was commonly found in adults from all the countries tested. Antibody appears to be acquired more rapidly in children from underdeveloped countries than in children from the United States.

Role of Norwalk virus in outbreaks of nonbacterial gastroenteritis.

Twenty-five separate outbreaks of nonbacterial gastrointestinal illnesses were studied serologically for evidence of infection with the Norwalk virus and the rotaviruses that affect humans. Eight of 25 outbreaks appeared to be related to the Norwalk virus. In one of the 25 outbreaks, there was evidence of rotavirus infection. These observations suggest that the Norwalk virus or serologically related agents play an important role in epidemic nonbacterial gastroenteritis in adults and older children.

Solid-phase microtiter radioimmunoassay blocking test for detection of antibodies to Escherichia coli heat-labile enterotoxin.

The development of a solid-phase microtiter radioimmunoassay blocking test to detect serum antibody to Escherichia coli heat-labile enterotoxin is described. The assay is easy to perform and quantitate, and it is sensitive and specific.

Solid-phase microtiter radioimmunoassay for detection of the Norwalk strain of acute nonbacterial, epidemic gastroenteritis virus and its antibodies.

The development of microtiter solid-phase radioimmunoassays for the detection of Norwalk antigen and its antibody is described. The tests are simple to perform and are sensitive and specific. The test for antigen can be used on crude stool filtrates and suspensions. Both tests are at least as sensitive as immune electron microscopy and more sensitive than immune adherence assay.

Purification of hepatitis A antigen from feces and detection of antigen and antibody by immune adherence hemagglutination.

Hepatitis A antigen (HA Ag) was purified from feces collected during acute illness from patients with naturally occurring viral hepatitis, type A. Positive fecal specimens were identified by immune electron microscopy, but for detection of HA Agduring purification immune adherence hemagglutination (IAHA) and microtiter solid-phase radioimmunoassay were used. Isopycnic banding in cesium chloride, rate-zonal separation in sucrose, and preparative zonal electrophoresis were used in various combinations for successive purification, and the purified antigen was successfully used in a test for antibody by IAHA. Seronconversions to HA Ag were demonstrated by IAHA in 20 instances of hepatitis A virus infection, but in none of six cases of type B hepatitis or three cases of post-transfusion hepatitis unrelated to heaptitis A or B viruses, nor in two individuals without hepatitis. In addition, the temporal pattern of antibody development during type A hepatitis was studied in serial sera from an experimentally infected chimpanzee. Antibody titers by IAHA correlated well with antibody ratings determined by immune electron microscopy.

Production and Properties of Antisera to Membrane Glycolipids of Mycoplasma pneumoniae.

The glycolipid haptens of Mycoplasma pneumoniae became immunogenic when bound to membrane proteins of Acholeplasma laidlawii by reaggregation. This process consisted of the solubilization of lipid-depleted A. laidlawii membranes and M. pneumoniae glycolipids in 20 mm sodium dodecyl sulfate and dialysis of the mixed solutions against 20 mm Mg(2+). The antibodies produced in rabbits to the reaggregated glycolipids inhibited the metabolism of M. pneumoniae, fixed complement with M. pneumoniae glycolipids or whole cells, precipitated M. pneumoniae glycolipids, and agglutinated M. pneumoniae cells. All these antibody activities could be blocked or absorbed by the purified glycolipids but not by a series of carbohydrates containing glucose and galactose. It was concluded that the antiserum to the reaggregated glycolipids may be regarded as a specific serum to membrane glycolipids of M. pneumoniae, since the antibodies to A. laidlawii membrane proteins, present in this serum, did not react with the glycolipids or with any other cell component of M. pneumoniae.

Cultivation of Mycoplasmas on glass.

Eight Mycoplasma species of human origin were successfully cultivated on glass. Complement-fixing (CF) antigens prepared from glass-adherent mycoplasmas were potent, specific, and free from anticomplementary activity. PPLO broth medium supplemented with 1 to 5% PPLO serum fraction (bovine), 2.5% fresh yeast extract, and 1% glucose (glycolytic species) or 1% arginine (arginine-utilizing species) supported moderate to luxuriant growth of mycoplasmas on glass. The potency of CF antigens prepared from glass-adherent mycoplasmas varied with the species of Mycoplasma tested and the duration of incubation. When the potency of CF antigens prepared from glass-adherent mycoplasmas was compared with that material sedimented from the broth phase of the same culture, three patterns of growth were observed: M. hominis and M. orale type 2 grew preferentially in the broth phase; M. salivarium, M. orale types 1 and 3, M. pneumoniae, and M. lipophilum preferentially adhered to the glass; and M. fermentans was biphasic. The growth of mycoplasmas on glass provides a simple means of concentrating and purifying such organisms for immunological and biochemical studies.

Electrophoretic analysis of cell proteins of T-strain mycoplasmas isolated from man.

Electrophoretic patterns of the cell proteins of 12 T-strain mycoplasmas isolated from man showed a remarkable similarity. This finding suggests that these strains are genetically closely related and supports their classification in a single species.