Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.     

Nucleotide sequence of VP4 and VP7 genes of human rotaviruses with subgroup I specificity and long RNA pattern: implication for new G serotype specificity.

We sequenced the genes coding for the two neutralization proteins, VP4 and VP7, of human rotavirus strains L26 and L27 with subgroup I specificity but the long RNA pattern. The deduced VP7 amino acid sequence of strains L26 and L27 showed a low homology (73.6 to 81.9%) to those of rotavirus strains of the established serotypes. This finding, together with the previous serological characterizations, suggests that the VP7 (G) serotype of the L26 and L27 strains is distinct from those of strains of the previously established serotypes. In contrast, the VP4 sequences of the L26 and L27 strains were quite similar to those of virulent serotype 2 strains (DS-1, S2, and RV-5).     

Human rotavirus K8 strain represents a new VP4 serotype.

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.     

Serotype-specific epitope(s) present on the VP8 subunit of rotavirus VP4 protein.

cDNA clones representing the VP8 and VP5 subunits of VP4 of symptomatic human rotavirus strain KU (VP7 serotype 1 and VP4 serotype 1A) or DS-1 (VP7 serotype 2 and VP4 serotype 1B) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2 and VP4 serotype 2) were constructed and inserted into the pGEMEX-1 plasmid and expressed in Escherichia coli. Immunization of guinea pigs with the VP8 or VP5 protein of each strain induced antibodies that neutralized the rotavirus from which the VP4 subunits were derived. In a previous study (M. Gorziglia, G. Larralde, A.Z. Kapikian, and R. M. Chanock, Proc. Natl. Acad. Sci. USA 87:7155-7159, 1990), three distinct serotypes and one subtype of VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to five distinct VP7 serotypes. The results obtained by cross-immunoprecipitation and by neutralization assay with antisera to the VP8- and VP5-expressed proteins suggest that the VP8 subunit of VP4 contains the major antigenic site(s) responsible for serotype-specific neutralization of rotavirus via VP4, whereas the VP5 subunit of VP4 is responsible for much of the cross-reactivity observed among strains that belong to different VP4 serotypes.     

Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus.

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.     

Comparative amino acid sequence analysis of VP4 for VP7 serotype 6 bovine rotavirus strains NCDV, B641, and UK.

In a previous study (S. Zheng, G. N. Woode, D. R. Melendy, and R. F. Ramig, J. Clin. Microbiol. 27:1939-1945, 1989), it was predicted that the VP7 serotype 6 bovine rotavirus strains NCDV and B641 do not share antigenically similar VP4s. In this study, gene 4 and the VP7 gene of B641 were sequenced, and the amino acid sequences were deduced and compared with those of NCDV and bovine rotavirus strain UK. Amino acid sequence homology in VP7 between the three strains was greater than 94%, confirming their relationship as VP7 serotype 6 viruses. VP4 of B641 showed amino acid homology to UK of 94% but only 73% homology to NCDV. Sequence comparison of a variable region of VP8 demonstrated amino acid homology of 53% between B641 and NCDV, whereas B641 and UK were 89% homologous in this region. These results confirm the earlier prediction that although the same serotype by VP7 reactivity, B641 and NCDV represent different VP4 serotypes. This difference in VP4 may have contributed to the lack of homotypic protection observed in calves, implicating VP4 as an important antigen in the active immune response to rotavirus infection in bovines.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

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.     

Sequence of the fourth gene of human rotaviruses recovered from asymptomatic or symptomatic infections.

The complete nucleotide sequence of the fourth gene of symptomatic (Wa, DS-1, P, and VA70) and asymptomatic (M37, 1076, McN13, and ST3) rotaviruses of serotype 1, 2, 3, or 4 was determined by the dideoxy chain termination method. In each strain, the fourth gene, which encodes the outer capsid protein VP3, is 2,359 base pairs in length and has 5'- and 3'-noncoding regions of 9 and 25 nucleotides, respectively. The gene has a single long open reading frame of 2,325 base pairs that is capable of coding for a protein of 775 amino acids. A total of 14 N-terminal and 12 C-terminal amino acids are completely conserved or almost completely conserved, respectively, among nine human rotavirus VP3 genes that have been sequenced. In addition, there is conservation of arginine at the two trypsin cleavage sites as well as conservation of clusters of amino acids in different regions of the two VP3 cleavage products, VP8 and VP5. Three distinct forms of VP3 were identified among the nine human rotavirus strains analyzed. Three symptomatic rotaviruses (serotypes 1, 3, and 4) possess highly related VP3 genes (92.2 to 97% nucleotide identity). Two symptomatic serotype 2 rotaviruses possess VP3 genes which are even more closely related to each other (98.6% nucleotide identity) and only moderately related to the aforementioned VP3 genes of serotypes 1, 3, and 4 (87.4 to 88.2% nucleotide identity). The four asymptomatic rotaviruses, which constitute the third group, possess highly related VP3 genes (95.5 to 97.5% nucleotide identity) which are distinct from those of the virulent rotaviruses (73 to 74.8% nucleotide identity). At 91 positions in the protein sequence of VP3, an amino acid is conserved among the asymptomatic rotaviruses, while a different amino acid is conserved among the symptomatic rotaviruses. Notably, five regions are conserved among the symptomatic rotaviruses, while a different set of sequences are conserved among the asymptomatic rotaviruses. It is possible that some or all of these regions of sequence dimorphism may be responsible for the difference in virulence of these two groups of human rotaviruses. There are 13 regions in the VP3 protein sequence which exhibit the greatest variability; the majority of these variable regions are observed between amino acids 106 to 192. These regions may represent potential antigenic sites related to heterotypic rotavirus neutralization.     

Identification of VP7 epitopes associated with protection against human rotavirus illness or shedding in volunteers.

Sera from 17 of 18 adult volunteers challenged with a virulent serotype 1 rotavirus strain (D) were examined for prechallenge antibody levels against several well-defined rotavirus VP7 and VP4 neutralization epitopes by a competitive epitope-blocking immunoassay (EBA) in order to determine whether correlates of resistance to diarrheal illness could be identified. The presence of prechallenge serum antibody at a titer of greater than or equal to 1:20 that blocked the binding of a serotype 1 VP7-specific monoclonal antibody (designated 2C9) that maps to amino acid residue 94 in antigenic site A on the serotype 1 VP7 was significantly associated with resistance to illness or shedding (P less than 0.001) or illness and shedding (P less than 0.01) following challenge with the serotype 1 virus. In addition, an EBA antibody titer of greater than or equal to 1:20 in prechallenge serum against a serotype 3 VP7-specific epitope (defined by monoclonal antibody 954/159) that maps to amino acid 94 on the serotype 3 VP7 was also significantly associated with resistance to illness or shedding (P = 0.02), with a trend for protection against illness and shedding. A trend was also noted between the presence of EBA antibody against a cross-reactive VP4 epitope common to many human rotavirus strains, including the challenge virus, or a rhesus monkey rotavirus strain-specific VP4 antigenic site, and resistance to illness or shedding. These data confirm that the presence of serum antibody correlates with resistance to rotavirus illness or shedding but, in addition, demonstrate the association of antibody to a specific epitope with resistance to illness or shedding. These data also suggest that antigenic site A on the rotavirus VP7, composed of amino acids 87 to 96, may be involved in the formation of a major protective epitope. Further study of the role of this epitope in the development of homotypic and heterotypic immunity to rotaviruses following natural or vaccine-induced infection may be important in the development of strategies for control of rotavirus diarrheal disease.     

Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate‐binding domain (VP8*)

The outer capsid spike protein VP4 of rotaviruses is a major determinant of infectivity and serotype specificity. Proteolytic cleavage of VP4 into 2 domains, VP8* and VP5*, enhances rotaviral infectivity. Interactions between the VP4 carbohydrate‐binding domain (VP8*) and cell surface glycoconjugates facilitate initial virus‐cell attachment and subsequent cell entry. Our saturation transfer difference nuclear magnetic resonance (STD NMR) and isothermal titration calorimetry (ITC) studies demonstrated that VP8*_64‐224 of canine rotavirus strain K9 interacts with N‐acetylneuraminic and N‐glycolylneuraminic acid derivatives, exhibiting comparable binding epitopes to VP8* from other neuraminidase‐sensitive animal rotaviruses from pigs (CRW‐8), cattle (bovine Nebraska calf diarrhoea virus, NCDV), and Rhesus monkeys (Simian rhesus rotavirus, RRV). Importantly, evidence was obtained for a preference by K9 rotavirus for the N‐glycolyl‐ over the N‐acetylneuraminic acid derivative. This indicates that a VP4 serotype 5A rotavirus (such as K9) can exhibit a neuraminic acid receptor preference that differs from that of a serotype 5B rotavirus (such as RRV) and the receptor preference of rotaviruses can vary within a particular VP4 genotype.     

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.     

人A组轮状病毒北京地方株VP4血清型特异片段编码基因的序列分析

轮状病毒是引起婴幼儿严重腹泻的重要病原,其第四基因编码主要中和抗原VP4,而VP4可裂解为VP8和VP5两个片段。VP8为抗原型特异性片段。克隆并测定了具有代表性的三个轮状病毒北京株VP4编码基因5′端（VPS＋VPS一部分）887个核苷酸序列并据此推导出其氨基酸序列。结果表明,相同血清型的地方株和标准株之间具有高度同源性（92％～966％）,不同血清型间则变异较大（70．5％～71％）。氨基酸最大变异处位于aa84～172,并对胰酶作用位点在致病性中的可能性进行了讨论。     

Molecular Characterization of Human Rotavirus VP4 Genes by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assay

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS-1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU-1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV-1 and Cat2) were reverse-transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU-1 genogroup and the other corresponding to the VP4 genes carried by naturally-occurring reassortants between members of the AU-1 and other genogroups.     

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.     

Relation of VP7 amino acid sequence to monoclonal antibody neutralization of rotavirus and rotavirus monotype.

The neutralization epitopes of the VP7 of human rotavirus RV-4 were studied by using five neutralizing mouse monoclonal antibodies to select virus variants resistant to neutralization by each of the antibodies. Antibody resistance patterns and sequence analysis of the RV-4 variants revealed that at least four sites on VP7, located at amino acids 94 (region A), 147 to 148 (region B), 213 (region C), and 291, are involved in neutralization of the human G1 rotavirus RV-4. The A-region site elicited antibody cross-reactive between G types and showed species-restricted immunodominance not related to carbohydrate attachment. The monotype 1b rotavirus M37 lacked this site. The B region contained strain-specific and cross-reactive sites, absent in monotype 1c rotaviruses. The C-region site was present in all G1 rotaviruses tested. Monotype 1a rotaviruses contained all these sites of neutralization. Virus monotype and sensitivity to monoclonal antibody neutralization usually related to the presence of a particular amino acid(s) at or next to the positions at which the mutations were selected in the virus variants.     

Single point mutations may affect the serotype reactivity of serotype G11 porcine rotavirus strains: a widening spectrum?

A panel of single and double neutralization-resistant escape mutants of serotype G11 porcine rotavirus strains A253 and YM, selected with G11 monotype- and serotype-specific neutralizing monoclonal antibodies (MAbs) to VP7, was tested in neutralization assays with hyperimmune sera raised against rotavirus strains of different serotypes. Escape mutants with an amino acid substitution in antigenic region A (amino acids [aa] 87 to 101) resulting in a residue identical or chemically similar to those present at the same positions in serotype G3 strains, at positions 87 for strain A253 and 96 for strain YM, were significantly more sensitive than the parental strains to neutralization with sera against some serotype G3 strains. Also, one YM antigenic variant (YM-5E6.1) acquired reactivity by enzyme-linked immunosorbent assay with MAbs 159, 57/8, and YO-1E2, which react with G3 strains, but not with the serotype G11 parental strain YM. Cross-adsorption studies suggested that the observed cross-neutralization by the G3-specific sera was due to the sera containing antibodies reactive with the parental strain plus antibodies reactive with the epitope(s) on the antigenic variant that mimick the serotype G3 specific one(s). Moreover, antibodies reactive with antigenic region F (aa 235 to 242) of VP7 might also be involved since cross-reactivity to serotype G3 was decreased in double mutants carrying an additional mutation, which creates a potential glycosylation site at position 238. Thus, single point mutations can affect the serotype reactivity of G11 porcine rotavirus strains with both monoclonal and polyclonal antibodies and may explain the origin of rotavirus strains with dual serotype specificity based on sequence divergence of VP7.