Use of in vitro gas production models in ruminal kinetics.

Physiological systems models for ruminant animals are used to predict the extent of ruminal carbohydrate digestion, based on rates of intake, digestion, and passage to the lower tract. Digestion of feed carbohydrates is described in these models by a first-order rate constant. Recently, an in vitro gas production technique has been developed to determine the digestion kinetics in batch fermentation, and nonlinear mathematical models have been fitted to the cumulative gas production data from these experiments. In this paper, we present an analysis that converts these gas production models to an effective first-order rate constant that can be used directly in rumen systems models. The analysis considers the digestion of an incremental mass of substrate entering the rumen. The occurrence of passage is represented probabilistically, and integration through time gives the total mass of substrate and total rate of digestion in the rumen. To demonstrate the analysis, several gas production models are fitted to a sample data set for corn silage, and the effective first-order rate constants are calculated. The rate constants for digestion depend on ruminal passage rate, an interaction that arises from the nonlinearity of the gas production models.

Immunogenicity, antigenicity, and protection efficacy of baculovirus expressed VP4 trypsin cleavage products, VP5(1)* and VP8* from rhesus rotavirus.

Rhesus rotavirus (RRV) VP4 trypsin cleavage product VP5(1)*, a truncated form of VP5*, was expressed in baculovirus and found by immunoprecipitation to be antigenically similar to VP5* on the virion. Immunization of mice with VP5(1)* elicited neutralizing antibody that was found to be cross-reactive with viruses representing P genotypes 1, 3, 4, 6, 7, and 8. Baculovirus expressed trypsin cleavage products, VP8* (amino acids 1-246) and VP5(1)* (amino acids 247-474), were tested for their ability to elicit a protective response in a murine model of passive protection. These results were compared to those obtained with baculovirus expressed RRV VP4. Dams immunized with baculovirus expressed RRV VP4 gave birth to pups protected from RRV virus challenge. Neither VP5(1)* nor VP8* was as effective at generating protective immunity as full length VP4. However, antibody to VP5(1)* was more effective than antibody to VP8* at mediating protection even though the neutralizing antibody titers as measured by hemagglutination inhibition and focus reduction neutralization were similar.

Comparison of VP4 and VP7 of five murine rotavirus strains.

The nucleotide and deduced amino acid sequences of outer capsid proteins VP4 and VP7 of five murine rotavirus strains (EW, EB, EC, EL, EHP) were determined. Comparisons of the VP7 amino acid sequences of the five murine rotavirus strains with rotavirus strains representative of G serotypes 1-14 showed that the murine strains were highly homologous to one another and more closely related to strains representing G3 than to any other G type. Analysis of the VP4 amino acid sequences of the murine strains revealed the presence of at least two murine P types. Therefore, sequence analysis would predict that the murine rotaviruses are G3 or G3-like and represent at least two unique P types (tentatively P17 for EW, EB, EC, and EL and P18 for EHP). When we attempted to categorize the murine strains by serum neutralization tests, our results were less clear. Serum to two murine strains, EHP and EW, displayed one-way reactivity by focus-reduction neutralization assays using several prototype G3 strains. The G3 serotyping monoclonal antibody 159 failed to neutralize EHP and EW, while the G3-specific monoclonal YO-1E2 neutralized EHP, but not EW. A reassortant (A15) containing VP7 from EW and VP4 from RRV was neutralized by these two G3-specific monoclonal antibodies to a level 8- to 20-fold more than EW, but was still 64- to 250-fold less than either SA11 and RRV. These results suggest that VP4 can influence the antigenicity of VP7.

Comparison of the rotavirus nonstructural protein NSP1 (NS53) from different species by sequence analysis and northern blot hybridization.

The nucleotide sequence of gene 5 encoding the rotavirus nonstructural protein NSP1 (NS53) of 6 strains (EW, EHP, RRV, I321, OSU, and Gottfried) was determined and compared to 6 previously reported strains (SA11, UK, RF, Hu803, DS-1, and Wa). The 12 rotavirus strains were derived from a total of five separate species (murine, bovine, simian, porcine, and human). Gene sizes ranged from 1564 to 1611 nucleotides in length and the deduced protein sequences were found to be 486 to 495 amino acids in length. Comparisons of NSP1 amino acid sequences showed identities ranging from 36 to 92%. This diversity was most evident between strains from different species. Phylogenetic analysis revealed a clustering of NSP1 sequences according to species origin with the exception that the human and porcine strains were included in a single grouping. Northern blot hybridizations using additional rotavirus strains from the five species confirmed the grouping found by sequence analysis. The species specificity of NSP1 is consistent with the hypothesis that NSP1 plays a role in host range restriction.

Identification of a new neutralization epitope on VP7 of human serotype 2 rotavirus and evidence for electropherotype differences caused by single nucleotide substitutions.

Neutralizing monoclonal antibodies 2F1 and 1C10, which are specific for VP7 of serotype 2 rotaviruses (G2), were used to select neutralization escape variants of the human serotype 2 rotavirus, DS-1. Neutralization survival patterns generated by monoclonal antibodies 2F1, 1C10, and RV5:3 indicated that 2F1 and 1C10 did not recognize identical epitopes. Direct sequencing of PCR products of gene 8, encoding VP7, revealed that each escape variant possessed only a single nucleotide mutation which resulted in a single amino acid substitution. In one variant, a second nucleotide change occurred, but did not result in an amino acid change. Four independently selected 2F1 mutants showed mutations in four separate sites in antigenic regions A, C, and D. Two independently selected 1C10 variants had mutations in either the A region or an unreported site at amino acid 190. Two of the mutations resulted in the creation of new glycosylation sites which were utilized, but did not appear to greatly affect antigenicity. Of note, three of the mutants also demonstrated alterations in the migration patterns of gene 8 on PAGE. Such electrophoretic mobility shifts caused by single base neutralization escape mutations have not previously been reported for rotavirus. Since multiple mutations were selected with the same monoclonal antibody it appears that the antigenic regions of VP7, although widely separated in the linear sequence, are parts of a single, large and complex neutralization domain which includes amino acid 190 as well as the other previously reported epitopes.