A cDNA clone representing the VP4-encoding gene of human rotavirus strain 69M(VP7 serotype 8) was constructed and inserted into a baculovirus expression vector. Baculovirus recombinants that expressed the 69 M VP4 protein in Spodoptera frugiperda (Sf9) cells were screened by immunofluorescence with hyperimmune antiserum to the 69M strain and purified by terminal dilution. The expressed VP4 was detected by Coomassie blue staining of PAGE-separated proteins. The antigenic relationships between the VP4 of the 69M strain and those of various human and other animal rotavirus strains representing ten established VP4 serotypes were examined by plaque reduction neutralization. Hyperimmune antiserum produced in guinea pigs following immunization with a lysate of Sf9 cells infected with a 69M gene 4-baculovirus recombinant neutralized the infectivity of the homologous human rotavirus 69M strain as well as heterologous equine rotavirus H-2 strain to a high titer. The anti-69M VP4 hyperimmune antiserum did not neutralize significantly other rotavirus strains of human, simian, porcine, bovine, or murine origin. It thus appears that the human rotavirus 69M strain has a distinct VP4 serotype (designated as P serotype 4) which is closely related antigenically to equine rotavirus H-2 VP4.

Most strains of animal rotaviruses are able to agglutinate erythrocytes, and the surface protein VP4 is the virus hemagglutinin. To map the hemagglutination domain on VP4 while preserving the conformation of the protein, we constructed full-length chimeras between the VP4 genes of hemagglutinating (YM) and nonhemagglutinating (KU) rotavirus strains. The parental and chimeric genes were expressed in insect cells, and the recombinant VP4 proteins were evaluated for their capacity to agglutinate human type O erythrocytes. Three chimeric genes, encoding amino acids 1 to 208 (QKU), 93 to 208 (QC), and 93 to 776 (QYM) of the YM VP4 protein in a KU VP4 background, were constructed. YM VP4 and chimeras QKU and QC were shown to specifically hemagglutinate, indicating that the region between amino acids 93 and 208 of YM VP4 is sufficient to determine the hemagglutination activity of the protein.

Two neutralizing monoclonal antibodies (N-mAbs) were utilized to locate amino acid (aa) residues involved in the formation of serotype-cross-reactive epitopes on the VP7 of selected group A rotaviruses. N-mAb 954/159/13 neutralized G serotype 3 as well as porcine G serotype 4 rotaviruses, whereas N-mAb 57/8 neutralized G serotype 3, 4, 6, 9, and 10 strains. Neutralization-resistant variants of each serotype were selected in the presence of these two monoclonal antibodies. Sequence analysis of the gene encoding VP7 of such variants revealed: (i) variable regions VR-5 (aa 88-100), VR-8 (aa 209-223), and VR-9 (aa 235-242) are involved in cross-reactive neutralization; (ii) an aa substitution can occur at the same position on the VP7 of different serotypes selected by a given N-mAb; (iii) the location of an aa substitution on the variant VP7 selected by a given N-mAb can vary depending on the rotavirus serotype; (iv) a substituted single aa species at a specific position on the variant VP7 selected by a single N-mAb can vary, resulting in variants which exhibit antigenic differences; and (v) the VP7 of a porcine rotavirus Gottfried strain has a unique antigenic mosaic of serotype 3 and serotype 4.

A template-dependent, in vitro rotavirus RNA replication system was established. The system initiated and synthesized full-length double-stranded RNAs on rotavirus positive-sense template RNAs. Native rotavirus mRNAs or in vitro transcripts, with bona fide 3' and 5' termini, derived from rotavirus cDNAs functioned as templates. Replicase activity was associated with a subviral particle containing VP1, VP2, and VP3 and was derived from native virions or baculovirus coexpression of rotavirus genes. A cis-acting signal involved in replication was localized within the 26 3'-terminal nucleotides of a reporter template RNA. Various biochemical and biophysical parameters affecting the efficiency of replication were examined to optimize the replication system. A replication system capable of in vitro initiation has not been previously described for Reoviridae.

We isolated five stable SA11 clones (TN-S1, TN-S2, TN-L1, TN-L2, and BN-S4) with different plaque sizes from two SA11 stocks. In polyacrylamide gel electrophoresis, the mobilities of the fourth, fifth, and seventh RNA segments of SA11 clones with large plaque size (TN-L1 and TN-L2) were faster than those of clones with small plaque size (TN-S1, TN-S2, and BN-S4). Nucleotide sequence determination of the fourth RNA segment identified a five-amino-acid difference in VP4s between the clones with large and small plaque sizes. The VP4 sequences of two clones with small plaque size (TN-S1 and BN-S4) were the same as the sequence of SA11-SEM reported by K. Nishikawa et al. (1988, J. Virol. 62, 4022-4026), while the VP4 sequences of the clones with large plaque size (TN-L1 and TN-L2) were similar, but not identical, to that reported by D. B. Mitchell and G. W. Both (1989, Nucleic Acids Res., 17, 2122). A single-gene reassortant, K8-L2.4, in which RNA segment 4 was derived from clone TN-L2 of SA11 virus and the other RNA segments were from strain K8, produced large plaques like TN-L2, suggesting that the five-amino-acid difference in VP4 between the clones with large and small plaque sizes might be associated with the difference in plaque size.

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.

Rotavirus strain A253, isolated from the faeces of a diarrhoeic piglet in Venezuela, was classified as serotype G11 by cross-neutralization studies and by comparison of the deduced amino acid sequence of the VP7 surface protein. The epitopes involved in neutralization of the two G11 porcine rotavirus strains A253 and YM were analysed using neutralization-resistant mutants selected with seven neutralizing monoclonal antibodies (MAbs), monotype-specific (M-) MAbs and serotype-specific (S-) MAbs, produced against VP7 of strain A253. Cross-neutralization tests and sequence analysis of the escape mutants selected from strains A253 and YM indicated the presence of two antigenic sites, one common to both M-MAbs and S-MAbs in region A (positions 87, 91 and 96) and the other defined by one S-MAb in region C (position 223). All A253 variants selected with M-MAbs and two S-MAbs, although having different amino acid substitutions, had a change at amino acid position 87, whereas YM variants involved residues 91 and 96, part of the same antigenic site. Compared to strain A253, the YM stain presents an amino acid substitution at position 87 and was not recognized by M-MAbs. These results suggest that in the VP7 of G11 serotype specificity, the amino acid at position 87 is an important component of a neutralization site associated with region A and the intraserotypic variation between strains A253 and YM may account for the selection of mutations at different positions by a single MAb.

The complete VP4 gene of the human rotavirus (HRV) K8 strain (G1 serotype) was cloned and inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. A K8VP4 recombinant baculovirus was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells with transfer vector DNA containing the K8VP4 gene and wild-type baculovirus DNA. Infection of Sf9 cells with this VP4 recombinant baculovirus resulted in the production of a protein that is similar in size and antigenic activity to the authentic VP4 of the K8 strain. Guinea pigs immunized with the expressed VP4 developed antibodies that neutralized the infectivity of the K8 strain. This antiserum neutralized HRV strains belonging to VP4 serotypes 1A, 1B, and 2 with efficiency eightfold or lower than that of the homologous virus, indicating that the human rotavirus K8 strain represents a distinct VP4 serotype (P3). In addition, low levels of cross-immunoprecipitation of the K8VP4 and its VP5 and VP8 subunits with hyperimmune antisera to HRV strains representing different VP4 serotype specificities also suggested that the K8 strain possesses a unique VP4 with few epitopes in common with other P-serotype strains.

The neutralization epitopes of the outer capsid protein VP7 of a porcine group A rotavirus were studied by using neutralizing monoclonal antibodies (N-MAbs). Six N-MAbs which were specific for the VP7 protein of the Gottfried strain of porcine rotavirus (serotype G4) were used for analyzing the antigenic sites of VP7. Three different approaches were used for this analysis: testing the serological reactivity of each N-MAb against different G serotypes of human and animal rotaviruses, analyzing N-MAb-resistant viral antigenic variants, and performing a nucleotide sequence analysis of the VP7 gene of each of the viral antigenic variants generated. From the serological analyses, three different reactivity patterns were recognized by plaque reduction virus neutralization and cell culture immunofluorescence tests. A single MAb (RG36H9) reacted with animal rotavirus serotypes G3 and G4 but not with human serotypes G3 and G4. The MAb 57/8 (D. A. Benfield, E. A. Nelson, and Y. Hoshino, p. 111, in Abstr. VIIth Internat. Congr. Virol., 1987, and E. R. Mackow, R. D. Shaw, S. M. Matsui, P. T. Vo, D. A. Benfield, and H. B. Greenberg, Virology 165:511-517, 1988) reacted with animal and human rotavirus serotypes G3 and G4 and also with human serotype G9 and bovine serotype G6. The other four MAbs reacted only with the porcine rotavirus serotype G4. The epitope defined by MAb 57/8 and the epitope defined by the other five MAbs appeared to be partially overlapping or close to each other, as identified by viral antigenic variant analysis. However, data from nucleotide and deduced amino acid sequence analyses of the VP7 of each of the viral antigenic variants showed that these two epitopes constituted a large, single neutralization domain.

Recombinant OSU VP4 protein, an outer capsid antigen of porcine rotavirus, was purified to a high level from the spent broth of baculovirus-infected Spodoptera frugiperda insect cells. Initial clarification of the broth with a 0-60% ammonium sulfate cut retained 93% of the total VP4. Q-sepharose ion exchange chromatography performed at pH 6.5 yielded 67% of the initial amount of VP4 in the pooled fractions, with more than four times the purity of the original sample. Gel filtration chromatography followed ion exchange. VP4 eluted from this column at a volume corresponding to a protein of a molecular weight of approximately 85 kDa, the single chain molecular weight of VP4. This step retained 34% of the initial VP4, with a 28-fold purification. Affinity chromatography, using heparin and glycophorin as ligands, was chosen as a final polishing step. The selective binding of VP4 to the two ligands suggested that VP4 may play a role during in vivo rota-viral infection. These specific interactions based on the biological properties of rotaviruses achieved a VP4 purity level of 85-95%. The overall purification scheme recovered about 1.5 mg per liter of VP4 spent broth from about 50 mg/liter (5% of total protein) present initially in the broth.

Three human rotavirus (HRV) VP4 serotypes and one subtype have been described on the basis of a fourfold or an eightfold-or-greater difference in neutralization titer when tested with hyperimmune antisera to recombinant VP4 or VP8* (serotypes P1A, P1B, P2, and P3). To start to analyze the antigenic basis underlying serotype specificity, we produced a library of 13 VP4-specific neutralizing monoclonal antibodies (NMAbs) to two HRVs, the serotype P1A strain Wa and the serotype P2 strain ST3, and characterized the reactivity of these NMAbs with a panel of serotypically diverse HRV strains by neutralization assay and enzyme-linked immunosorbent assay (ELISA). We characterized the serotypic specificity of the NMAbs by using a fourfold or an eightfold-or-greater difference in titer against the homologous (i.e., immunogen) and heterologous strains as a criterion for serotype. Some ST3-derived NMAbs reacted specifically with serotype P2 HRVs by ELISA and/or neutralization assay, while some Wa-derived NMAbs reacted specifically by ELISA and/or neutralization assay with some or all serotype P1A HRVs. Other Wa- and ST3-derived NMAbs reacted with some or all serotype P1A and P2 HRV strains by neutralization assay and ELISA. Most NMAbs did not react with serotype P1B or P3 strains. In previous studies, three distinct operationally defined epitopes have been identified on VP4 by examining the reactivity patterns of selected antigenic variants of HRV strain KU. At least one of the NMAbs described here recognizes an epitope unrelated to these previously identified epitopes, since it neutralized both KU and its variants.

The nucleotide and deduced amino acid sequence of G serotype 3 equine rotavirus strain H-2 was determined. A predicted 776-amino-acid H-2 VP4 shows less than or equal to 85.3% identity to other rotavirus VP4 types sequenced to date and thus represents a new P serotype. A PCR-generated probe derived from a cDNA clone of H-2 gene 4 hybridized to gene 4 of several tissue-culture-adapted equine rotavirus isolates, demonstrating that the gene 4 allele present in the H-2 strain is present in the equine rotavirus population.

The antigenic relationships of the VP4 serotype of porcine rotavirus Gottfried strain with other rotaviruses were determined by using antiserum to Gottfried VP4-baculovirus recombinant. This antiserum failed to react significantly with virus of serotypes P1A, P1B, P3, and OSU; however, it reacted with P2 strains. In the reciprocal assay, antiserum to VP4 of an asymptomatic strain (P2) failed to neutralize the Gottfried strain virus to a significant level. It thus appears that the Gottfried strain should be considered a subtype of P2.

Annals of the New York Academy of SciencesVolume 665, Issue 1 p. 210-218 Genetically Engineered Viral Antigens from Insect Cell Culturea MICHAEL J. BETENBAUGH, MICHAEL J. BETENBAUGH Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorDAVID A. LINDSAY, DAVID A. LINDSAY Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorLUIS G. JUARBE-OSORIO, LUIS G. JUARBE-OSORIO Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorMARIO GORZIGLIA, MARIO GORZIGLIA Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892Search for more papers by this authorSTEVEN VONDERFECHT, STEVEN VONDERFECHT Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205Search for more papers by this authorJOSEPH J. EIDEN, JOSEPH J. EIDEN Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205Search for more papers by this author MICHAEL J. BETENBAUGH, MICHAEL J. BETENBAUGH Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorDAVID A. LINDSAY, DAVID A. LINDSAY Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorLUIS G. JUARBE-OSORIO, LUIS G. JUARBE-OSORIO Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218Search for more papers by this authorMARIO GORZIGLIA, MARIO GORZIGLIA Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892Search for more papers by this authorSTEVEN VONDERFECHT, STEVEN VONDERFECHT Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205Search for more papers by this authorJOSEPH J. EIDEN, JOSEPH J. EIDEN Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205Search for more papers by this author First published: October 1992 https://doi.org/10.1111/j.1749-6632.1992.tb42585.xCitations: 1 a Partial support of this research was provided by the Engineering Foundation (Grant No. RI-A-90-6) and the National Science Foundation (Grant No. BCS-9007762). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1 Ratafia, M. 1987. Bio/Technology 5: 1154– 1158. 2 Thayer, A. M. 1991. Chem. Eng. News 69(8: 27– 48. 3 Allison, A. C. 1987. 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A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agricultural Experiment Station Bulletin No. 1555. College Station, Texas . 22 Shuler, M. L., T. Cho, T. Wickham, O. Ogonah, M. Kool, D. A. Hammer, R. R. Granados & H. A. Wood. 1990. Ann. N.Y. Acad. Sci. 589: 399– 422. 23 Hink, W. F., D. R. Thomsen, D. J. Davidson, A. L. Meyer & F. J. Castellino. 1991. Biotechnol. Prog. 7: 9– 14. 24 Betenbaugh, M. J., L. E. Balog & P-S. Lee. 1991. Biotechnol. Prog. 7: 462– 467. 25 Lynn, D. E., E. M. Dougherty, J. T. McClintock & M. Loeb. 1988. In Invertebrate and Fish Tissue Culture. Y. Kuroda, E. Kurstak & K. Maramorosch, Eds.: 239– 242. Japan Scientific Societies Press/Springer-Verlag. Tokyo / Berlin . 26 Juarbe-Osorio, L. G., M. Gorziglia & M. J. Betenbaugh. 1991. Protein expression and purification. Submitted. 27 Mackow, E. R., J. W. Barnett, H. Chan & H. B. Greenberg. 1989. J. Virol. 63: 1661– 1668. 28 Zhang, Y-M., E. P. Hayes, T. C. McCarty, D. R. Dubois, P. L. Summers, K. H. Eckels, R. M. Chanock & C-H. Lai. 1988. J. Virol. 62: 3027– 3031. 29 Juarbe-Osorio, L. G., M. Gorziglia & M. J. Betenbaugh. 1991. In Bioseparation Technologies. AIChE Symposium Series. M. M. Ataai & S. K. Sikdar, Eds. In press. Citing Literature Volume665, Issue1Biochemical Engineering VII: Cellular and Reactor EngineeringOctober 1992Pages 210-218 ReferencesRelatedInformation

The nucleotide and deduced amino acid sequence of the gene 4 of bovine rotavirus strain B223 is described. The open reading frame is predicted to encode a VP4 of 772 amino acids, shorter than described for any other rotavirus strain sequenced to date. B223 VP4 shows 70 to 73% similarity to other rotavirus VP4 proteins, demonstrating the presence of a unique VP4 type, and confirming a third VP4 allele in the bovine rotavirus population. Multiple sequence alignment with several other rotavirus strains created gaps in the sequence to account for a shorter VP4. The alignment shows a two contiguous amino acid deletions within the trypsin cleavage region of B223 VP4. Comparisons of two regions flanking the trypsin cleavage site, (aa 224 to 235, and aa 257 to 271) which show high homologies between strains, demonstrate that the region 5' to the trypsin cut site has a low homology (66%) to other rotavirus strains, although the region 3' to the trypsin cleavage site shows high homologies (86 to 93%) with other rotavirus strains. The lack of a conserved proline residue within the 5' flanking region suggests a possible altered local conformation of this site in B223 VP4. A second gap inserted into the VP4 of B223 on multiple sequence alignment is a three contiguous amino acid deletion at position 613-615 in the VP5* subunit. Previously defined biologic properties of this strain in relation to the determination of the amino acid composition of VP4 are discussed.

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Cracker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture-propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.

To determine the VP4 (P type) specificity of porcine rotaviruses, full- and partial-length gene 4 probes were produced from cloned Gottfried and OSU porcine rotavirus genomic segment 4 cDNAs. The gene 4 segments from the prototype Gottfried (VP7 serotype 4) and OSU (VP7 serotype 5) porcine rotavirus strains were selected for study because of their distinct P types and the occurrence of rotaviruses with similar serotypes among swine. Partial-length gene 4 cDNAs were produced and amplified by the polymerase chain reaction (PCR) and encompassed portions of the variable region (nucleotides 211 to 612) of VP8 encoded by genomic segment 4. The hybridization stringency conditions necessary for optimal probe specificity and sensitivity were determined by dot or Northern (RNA) blot hybridizations against a diverse group of human and animal rotaviruses of heterologous group A serotypes and against representative group B and C porcine rotaviruses. The PCR-derived gene 4 probes were more specific than the full-length gene 4 probes but demonstrated equivalent sensitivity. The Gottfried PCR-derived probe hybridized with Gottfried, SB2, SB3, and SB5 G serotype 4 porcine rotaviruses. The OSU PCR-derived probe hybridized with OSU, EE, A580, and SB-1A porcine rotaviruses and equine H1 rotavirus. Results of the hybridization reactions of the PCR-derived gene 4 probes with selected porcine rotavirus strains agreed with previous serological or genetic analyses, indicating their suitability as diagnostic reagents.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.