Identification of group A rotavirus genes associated with virulence of a porcine rotavirus and host range restriction of a human rotavirus in the gnotobiotic piglet model.

Rotaviruses are the leading cause of severe diarrhea in infants and young children worldwide. Thus, the development of an effective rotavirus vaccine is a major public health goal. This study was performed to identify the gene or genes responsible for rotavirus virulence or host range restriction and attenuation in a natural host. Such knowledge could have an important bearing on the selection of candidate live vaccine strains. We addressed this issue by analyzing the response of gnotobiotic piglets to orally administered porcine x human rotavirus reassortants. It was possible to determine (i) which porcine rotavirus genes were required for the induction of diarrhea, and (ii) which human rotavirus genes are associated with the host range restriction because the parental porcine rotavirus (SB-1A strain) caused diarrhea in piglets, whereas the parental human rotavirus (DS-1 strain) was attenuated in piglets. Substitution of the 3rd (VP3) or 4th (VP4) or 9th (VP7) or 10th (NS28 (NSP4)) gene of the avirulent human strain for the corresponding gene of porcine rotavirus that was virulent for gnotobiotic piglets yielded viral reassortants that failed to induce diarrhea. Further analysis indicated that reassortants which possessed only one, two, or three of these porcine rotavirus genes on a background of human rotavirus genes also failed to induce diarrhea. However, diarrhea was induced when all four of these porcine rotavirus genes were included in a reassortant in which the remaining seven genes were derived from the human rotavirus. These observations suggest that it may be possible to attenuate wild-type human rotavirus strains that are virulent for humans by selective genetic reassortment with an animal rotavirus strain that is attenuated for humans.

The outer capsid protein VP4 of murine rotavirus strain Eb represents a tentative new P type.

The nucleotide and deduced amino acid sequence of the gene 4 of murine rotavirus strain Eb were determined. The gene is 2359 nucleotides in length and encodes for a protein of 775 amino acids. Comparison of the VP4 amino acid sequence of the Eb strain with several human and animal rotavirus strains which represent all of the currently recognized distinct VP4 genotypes revealed amino acid identities of from 55.7-75.1% for VP4, 37.1-63.3% for VP8, and 23.9-52.1% for the B region (amino acids 84-180). In addition, antisera to recombinant VP4s of five distinct rotavirus serotypes and two subtypes failed to react significantly by neutralization assay with the Eb strain. Thus, it appears that the Eb strain should be considered a new VP4 genotype and/or serotype.

Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen.

A single-gene substitution reassortant 11-1 was generated from two porcine rotaviruses, OSU (serotype 5) and Gottfried (serotype 4). This reassortant derived 10 genes, including gene 4 encoding VP3, from the OSU strain and only gene 9, encoding a major neutralization glycoprotein (VP7), from the Gottfried strain and was thus designated VP3:5; VP7:4. Oral administration of this reassortant to colostrum-deprived gnotobiotic newborn pigs induced a high level of neutralizing antibodies not only to Gottfried VP7 but also to OSU VP3, thus demonstrating that VP3 is as potent an immunogen as VP7 in inducing neutralizing antibodies during experimental oral infection. Gnotobiotic piglets infected previously with the reassortant were completely resistant to oral challenge with the virulent Gottfried strain (VP3:4; VP7:4), as indicated by failure of symptoms to develop and lack of virus shedding. Similarly, prior infection with the reassortant induced almost complete protection against diarrhea and significant restriction of virus replication after oral challenge with the virulent OSU strain (VP3:5; VP7:5). Thus, it appears that (i) the immune system of the piglet responds equally well to two rotavirus outer capsid proteins, VP3 and VP7, during primary enteric rotavirus infection; (ii) antibody to VP3 and antibody to VP7 are each associated with resistance to diarrhea; and (iii) infection with a reassortant rotavirus bearing VP3 and VP7 neutralization antigens derived from two viruses of different serotype induces immunity to both parental viruses. The relevance of these findings to the development of effective reassortant rotavirus vaccines is discussed.

Analysis by plaque reduction neutralization assay of intertypic rotaviruses suggests that gene reassortment occurs in vivo.

The SB-1A rotavirus recovered from a diarrheic piglet in the United States is a naturally occurring intertypic rotavirus. When studied by reciprocal neutralization tests, the SB-1A virus was similar, if not identical, to the porcine Gottfried virus (serotype 4) and the porcine OSU virus (serotype 5). Analysis of reassortant viruses prepared from the SB-1A virus and the serotype 2 human DS-1 virus revealed that the antigenic specificity of the outer capsid protein VP3 of SB-1A was shared with the OSU virus, while the antigenic specificity of another outer capsid protein, VP7, of SB-1A appeared to be shared with the Gottfried virus. This suggests that SB-1A is a naturally occurring reassortant rotavirus between OSU-like and Gottfried-like porcine rotaviruses. In addition, using a genetic approach, we found evidence that the fourth gene was responsible for the predominantly one-way cross-neutralizing reactivity between canine rotavirus strain CU-1 (serotype 3) and porcine rotavirus strains SB-1A (serotypes 4 and 5) and OSU (serotype 5). Assignment of hemagglutination function to the fourth genome segment of porcine rotaviruses SB-1A and OSU and canine rotavirus CU-1 confirmed a similar previous gene assignment established for certain rotaviruses. Analysis of single gene 4 substitution reassortants confirmed our previous finding that VP3 was as potent in stimulating neutralizing antibodies as VP7. The observations confirm the need for a binary system of rotavirus classification and nomenclature similar to that used for the influenza A viruses; in such a system the neutralization specificity of both VP3 and VP7 would be indicated.

Independent segregation of two antigenic specificities (VP3 and VP7) involved in neutralization of rotavirus infectivity.

Antiserum prepared against the M37 strain of rotavirus, recovered from an asymptomatic newborn infant in Venezuela, neutralized two prototype human rotaviruses that define two separate serotypes: serotype 1 (Wa) and serotype 4 (ST3). Thus, the M37 strain is a naturally occurring intertypic rotavirus. Analysis of reassortant viruses produced during coinfection in vitro indicated that the observed dual serotype specificity of M37 resulted from sharing a related outer capsid protein, VP3, with the ST3 virus and another related outer capsid protein, VP7, with the Wa virus. Analysis of single (VP3)-gene-substitution reassortants indicated that VP3 was as potent an immunogen as VP7. In addition, direct evidence was obtained that the serotype specificity of neutralizing antibody elicited by VP3 can differ from the serotype specificity of neutralizing antibody elicited by VP7, indicating the need for a dual system of rotavirus classification in which the neutralization specificity of both VP3 and VP7 outer capsid proteins are identified.

Monoclonal antibodies to subgroup 1 rotavirus.

Subgroup 1-specific monoclones were analyzed and used successfully in an enzyme-linked immunosorbent assay to recognize certain subgroup 1 rotaviruses.

Enhancement of antigen incorporation and infectivity of cell cultures by human rotavirus.

Infection of cell cultures with human rotavirus preparations was attempted and the effects of trypsin and low-speed centrifugation on antigen incorporation, as demonstrated by immunofluorescence and radioimmunoassay, were determined. In addition, the effect of viral aggregation on antigen incorporation was investigated by filtering viral preparations. Four strains of human rotavirus were employed, and the results were compared to those obtained with two tissue culture-adapted animal rotaviruses. Centrifugation and trypsin appeared to have little or no effect on infectivity of the tissue culture-adapted (simian rotavirus) or -adaptable (Nebraska calf diarrhea virus) strains, whereas centrifugation and viral aggregation appeared to be essential for the human viruses. In addition, trypsin enhanced antigen incorporation of the human strains to some extent. Infectivity for cell cultures and in vitro human rotavirus protein formation was demonstrated by [35S]methionine incorporation, and the specificity of this human viral protein was established by radio-immunoprecipitation.

A microtiter solid phase radioimmunoassay for detection of the human reovirus-like agent in stools.

The development of a microtiter solid-phase radioimmunoassay for detection of the human reovirus-like (RVL) agent is described. The test is simple to perform and uses small amounts of reagents; a large number of specimens can rapidly be tested in a single test. Both filtered and unfiltered stool suspensions can be employed. The test was as sensitive as immune electron microscopy, and with an appropriate blocking test, was specific for the human RVL agent.

Diarrhea in gnotobiotic calves caused by the reovirus-like agent of human infantile gastroenteritis.

Gnotobiotic newborn calves were found to be susceptible to infection with the reovirus-like agent of human infantile gastroenteritis (HRVL). Infection was based on (i) seroresponse using immunofluorescence and (ii) fecal shedding of virus particles using electron microscopy. Virus was detected in fecal samples for at least 2 to as long as 7 days after inoculation, although peak virus concentrations were observed on days 1 to 4. Diarrheal illness was observed in seven calves on second to fourth serial passage of HRVL in calves but in none of four animals studied on first passage. Diarrhea began 15 to 30.5 h (mean = 22.3 h) post-inoculation and lasted less than 24 h; three of the seven animals that developed diarrhea were also depressed or anorectic.

Detection and localization of viruses in human fetal intestinal organ cultures by immunofluorescence.

Viral antigens from viruses belonging to four different viral groups were detected directly in human fetal intestinal organ cultures by the application of immunofluorescent techniques. The time of appearance and the cellular localization of fluorescent-stainable antigen varied with the type of virus under investigation. After infection with adenovirus or with adeno-associated virus, fluorescent-stainable antigen was seen in the epithelial cells of the explants, though no light microscopic changes could be observed. In infection with herpes simplex virus and echovirus, fluorescence was noted in both the epithelium and the lamina propria, along with histological changes throughout the organ culture. These techniques offer promise for the investigation of possible viral agents implicated in gastrointestinal disease.