Reassortant rotaviruses containing structural proteins vp3 and vp7 from different parents induce antibodies protective against each parental serotype.

Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SA11 and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SA11 or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SA11. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SA11 X NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections.     

Genetic mapping indicates that VP4 is the rotavirus cell attachment protein in vitro and in vivo.

To identify the rotavirus protein which mediates attachment to cells in culture, viral reassortants between the simian rotavirus strain RRV and the murine strains EHP and EW or between the simian strain SA-11 and the human strain DS-1 were isolated. These parental strains differ in the requirement for sialic acid to bind and infect cells in culture. Infectivity and binding assays with the parental and reassortant rotaviruses indicate that gene 4 encodes the rotavirus protein which mediates attachment to cells in culture for both sialic acid-dependent and -independent strains. Using ligated intestinal segments of newborn mice and reassortants obtained between the murine strain EW and RRV, we developed an in vivo infectivity assay. In this system, the infectivity of EW was not affected by prior treatment of the enterocytes with neuraminidase, while neuraminidase treatment reduced the infectivity of a reassortant carrying gene 4 from RRV on an EW background more than 80% relative to the controls. Thus, VP4 appears to function as the cell attachment protein in vivo as well as in vitro.     

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.     

NS35 and not vp7 is the soluble rotavirus protein which binds to target cells.

Recent studies using radiolabeled rotavirus lysates have demonstrated a 35-kilodalton viral protein that binds specifically to the surface of MA104 cells (N. Fukuhara, O. Yoshie, S. Kitakoa, and T. Konno, J. Virol. 62:2209-2218, 1988; M. Sabara, J. Gilchrist, G.R. Hudson, and L.A. Babiuk, J. Virol. 53:58-66, 1985). The binding protein was identified as vp7, an outer capsid glycoprotein and the product of rotavirus gene 9. These studies concluded that vp7 mediated viral attachment to MA104 cells and that the binding of a soluble viral protein to a cell monolayer mirrored the attachment of infectious rotavirus to permissive tissue culture cells. In the process of determining which viral protein adheres to the in vivo target cell in rotavirus infection, the mammalian enterocyte, we found that a similar 35-kilodalton rhesus rotavirus (RRV) protein bound to both MA104 cells and murine enterocytes. However, further analysis of this protein by immunoprecipitation, inhibition of glycosylation, and partial proteolysis showed that it was not the RRV gene 9 product, vp7, but the gene 8 product, NS35. Similar results were obtained by using porcine rotavirus (OSU) and bovine rotavirus (NCDV) strains. Binding studies using the in vitro-expressed products of RRV genes 8 and 9 confirmed these results. Since double-shelled virions inhibited the binding of NS35 to cells, we looked for the presence of this protein in preparations of purified virus. Examination of density gradient-purified virus preparations revealed biochemical and immunological evidence that NS35 copurifies in small amounts with double-shelled virions. Thus, these studies clearly demonstrated that when rotavirus proteins are prepared in a soluble form from infected cells, NS35, and not vp7, binds to the surfaces of MA104 cells and murine enterocytes. The observations do not confirm previous experimental results which supported the hypothesis that vp7 was the viral attachment protein. They are consistent with but do not prove the hypothesis that NS35 functions as the rotavirus attachment protein.     

RNA of rotavirus: comparison of RNAs of human and animal rotaviruses.

Single-stranded RNA (ssRNA) was transcribed in vitro from inner-shell particles of human rotavirus strain Wa (HRV-Wa) and a bovine rotavirus (neonatal calf diarrhea virus [NCDV]) by virion-associated RNA polymerase activity. The ssRNA product consisted of 11 RNA segments which were separated by polyacrylamide gel electrophoresis. In vitro-transcribed 32P-labeled ssRNA was used to study the genetic relatedness between rotaviruses by annealing with genomic double-stranded RNA (dsRNA) of homologous or heterologous rotavirus. All segments of HRV-Wa ssRNA were hybridized with dsRNA of HRV TK80, collected from the feces of a gastroenteritis patient, at the level of 88 to 100% of the homologous reaction. On the other hand, no segments of ssRNA from HRV-Wa hybridized with dsRNA of NCDV or simian rotavirus (simian agent 11). Similarly, ssRNA from NCDV did not hybridize with dsRNA of HRV-Wa, but hybridized with dsRNA of simian agent 11 at the level of 30% of the homologous value.     

Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins vp3 and vp7.

Monoclonal antibodies directed against two rotavirus surface proteins (vp3 and vp7) as well as a rotavirus inner capsid protein (vp6) were tested for their ability to protect suckling mice against virulent rotavirus challenge. Monoclonal antibodies to two distinct epitopes of vp7 of simian rotavirus strain RRV neutralized RRV in vitro and passively protected suckling mice against RRV challenge. A monoclonal antibody directed against vp3 of porcine rotavirus strain OSU neutralized three distinct serotypes in vitro (OSU, RRV, and UK) and passively protected suckling mice against OSU, RRV, and UK virus-induced diarrhea. The role of vp3 in eliciting protection against heterotypic rotavirus challenge should be considered when developing a vaccine with cloned rotavirus genes. Alternatively, immunization with a reassortant rotavirus containing vp3 and vp7 from two antigenically distinct rotavirus parents might protect against diarrhea induced by two or more rotavirus serotypes.     

Comparative analysis of the VP3 gene of divergent strains of the rotaviruses simian SA11 and bovine Nebraska calf diarrhea virus.

The gene encoding outer capsid protein VP3 of subpopulations of two animal rotaviruses, simian SA11 and Nebraska calf diarrhea virus (NCDV), was analyzed. Two laboratory strains of simian SA11 rotavirus (SA11-SEM and SA11-FEM) differed with respect to VP3. This dimorphism was indicated by a difference in electrophoretic mobility and a difference in reactivity with anti-VP3 monoclonal antibodies. The overall VP3 amino acid homology between the two SA11 VP3 proteins was 82.7%, whereas the VP3 protein of SA11-FEM was 98.5% homologous in amino acid sequence to NCDV VP3, suggesting that SA11-FEM VP3 was derived by gene reassortment in the laboratory during contamination with a bovine rotavirus. A comparison of the deduced amino acid sequence of the VP3 of two virulent NCDV strains and an attenuated NCDV strain (RIT 4237), revealed only five amino acid differences which were scattered throughout the protein but did not involve the trypsin cleavage sites. Of interest, the VP3 of the standard strain of NCDV which is virulent for cows differed in only one amino acid (position 23, Gln to Lys) from the VP3 of an NCDV mutant which was attenuated both for cows and for children.     

Reassortant rotaviruses as potential live rotavirus vaccine candidates.

A series of reassortants was isolated from coinfection of cell cultures with a wild-type animal rotavirus and a "noncultivatable" human rotavirus. Wild-type bovine rotavirus (UK strain) was reassorted with human rotavirus strains D, DS-1, and P; wild-type rhesus rotavirus was reassorted with human rotavirus strains D and DS-1. The D, DS-1, and P strains represent human rotavirus serotypes 1, 2, and 3, respectively. Monospecific antiserum (to bovine rotavirus, NCDV strain) or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7 (of the rhesus rotavirus), was used to select for reassortants with human rotavirus neutralization specificity. This selection technique yielded many reassortants which received only the gene segment coding for the major neutralization protein from the human rotavirus parent, whereas the remaining genes were derived from the animal rotavirus parent. Single human rotavirus gene substitution reassortants of this sort represent potential live vaccine strains.     

Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcalpha3-Galbeta) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcalpha3Galbeta is preferred to NeuAcalpha3Galbeta. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.     

Analysis of reassortment of genome segments in mice mixedly infected with rotaviruses SA11 and RRV.

Seven-day-old CD-1 mice born to seronegative dams were orally inoculated with a mixture of wild-type simian rotavirus SA11 and wild-type rhesus rotavirus RRV. At various times postinfection, progeny clones were randomly isolated from intestinal homogenates by limiting dilution. Analysis of genome RNAs by polyacrylamide gel electrophoresis was used to identify and genotype reassortant progeny. Reassortment of genome segments was observed in 252 of 662 (38%) clones analyzed from in vivo mixed infections. Kinetic studies indicated that reassortment was an early event in the in vivo infectious cycle; more than 25% of the progeny clones were reassortant by 12 h postinfection. The frequency of reassortant progeny increased to 80 to 100% by 72 to 96 h postinfection. A few reassortants with specific constellations of SA11 and RRV genome segments were repeatedly isolated from different litters or different animals within single litters, suggesting that these genotypes were independently and specifically selected in vivo. Analysis of segregation of individual genome segments among the 252 reassortant progeny revealed that, although most segments segregated randomly, segments 3 and 5 nonrandomly segregated from the SA11 parent. The possible selective pressures active during in vivo reassortment of rotavirus genome segments are discussed.     

Molecular basis of bluetongue virus neutralization.

Molecular and serological analyses of bluetongue virus serotypes 10 and 11 and their intertype reassortants indicate that the viral RNA segment L2 codes for the serotype-specific antigen. Individual RNA segments of parental and reassortant viruses were characterized by oligonucleotide fingerprint analyses. Analyses of their virion polypeptides by Cleveland peptide mapping (Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977) demonstrated that the L2 gene segregated colinearly with the viral P2 protein, implicating it as the antigen that is responsible for the viral serotype specificity.     

Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus.

Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypically distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], less than 20). A reassortant (E19) that contained EDIM VP7 and WC3 VP4 was also very poorly neutralized by these antisera (GMT = 20). In contrast, antibody titers to a reassortant (R20) that contained WC3 VP7 and rhesus rotavirus VP4 were higher than those against WC3 (GMTs of 458 and 313, respectively). Thus, VP7 appeared to be the dominant immunogen for production of neutralizing antibody after intestinal infection of previously uninfected infants vaccinated with WC3 bovine rotavirus.     

A synthetic peptide corresponding to the cleavage region of VP3 from rotavirus SA11 induces neutralizing antibodies.

Antibodies were elicited in rabbits by immunization with the synthetic tetradecapeptide Gln-Asn-Thr-Arg-Asn-Ile-Val-Pro-Val-Ser-Ile-Val-Ser-Arg, corresponding to amino acids 228 to 241 of SA11-VP3. Protein specificity of the antipeptide serum is demonstrated. The antipeptide serum revealed neutralizing activity directed against SA11 in a neutralization assay. Human rotavirus strains Wa, S2, and Hochi and bovine strains NCDV and UK were not neutralized, demonstrating the strain-specific neutralizing activity of the raised antipeptide serum. Upon immune electron microscopy, aggregation of SA11 particles was observed.     

Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge.

A baculovirus-expressed VP4 protein derived from the simian rhesus rotavirus (RRV) was used to parenterally immunize murine dams. VP4-immunized dams developed high levels of neutralizing antibodies against RRV and low levels of cross-reactive neutralizing antibodies against human strains Wa, ST3, and S2 and animal strains SA-11, NCDV, and Eb. Newborn mice suckled on VP4-immunized dams were protected against a virulent challenge dose of the simian strain RRV and against murine rotavirus Eb. The cross-reactive nature of the serum-neutralizing response generated by VP4 immunization and the protective efficacy of the immunization suggest that recombinant-expressed VP4 proteins should be considered as viable vaccine candidates.     

Polypeptide specificity of antiviral serum antibodies in children naturally infected with human rotavirus.

Reassortants between serotype 3 SA11 and serotype 6 NCDV rotaviruses were used to determine the relative amounts of serum-neutralizing antibody to VP4 and VP7 of serotype 3 SA11 rotavirus in children after natural rotavirus exposure. Sera from Ecuadorian children of a population-based study and sera from children of a hospital-based study in Germany (excluding diarrhea patients) demonstrated high titers of VP7-specific but only low titers of VP4-specific antibodies. In contrast, paired sera from German children hospitalized with a symptomatic primary rotavirus gastroenteritis demonstrated a titer increase to VP4 more frequently than to VP7 protein by neutralization test and immunoblotting. For these rotavirus patients, we provided, previously, direct evidence for the development of cross-neutralizing antibodies.     

Molecular and antigenic characterization of porcine rotavirus YM, a possible new rotavirus serotype.

In 1983, we isolated a porcine rotavirus (strain YM) that was prevalent in several regions of Mexico, as judged by the frequency of its characteristic electropherotype. By a focus reduction neutralization test, rotavirus YM was clearly distinguished from prototype rotavirus strains belonging to serotypes 1 (Wa), 2 (S2), 3 (SA11), 4 (ST3), 5 (OSU), and 6 (NCDV). Minor, one-way cross-neutralization (1 to 5%) was observed when antisera to the various rotavirus strains were incubated with rotavirus YM. In addition, the YM virus was not neutralized by neutralizing monoclonal antibodies with specificity to serotypes 1, 2, 3, and 5. The subgroup of the virus was determined to be I by enzyme-linked immunosorbent assay. To characterize the serotype-specific glycoprotein of the virus at the molecular level, we cloned and sequenced the gene coding for VP7. Comparison of the deduced amino acid sequence with reported homologous sequences from human and animal rotavirus strains belonging to six different serotypes further supported the distinct immunological identity of the YM VP7 protein.     

NP, PB1, and PB2 viral genes contribute to altered replication of H5N1 avian influenza viruses in chickens

The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.