Antibody response to serotype-specific and cross-reactive neutralization epitopes on VP4 and VP7 after rotavirus infection or vaccination.

By using a competitive solid-phase immunoassay with serotype-specific and cross-reactive neutralizing monoclonal antibodies directed at VP4 and VP7, we tested the antibody responses to some neutralization epitopes on VP4 and VP7 in individuals infected or vaccinated with rotavirus. Antibody responses to VP7 epitopes of the infecting serotype of virus were found at a high frequency in both infants and children. In contrast, antibody responses to VP4 and heterotypic VP7 were observed only when the individuals possessed antibodies to any serotype of rotavirus in their acute-phase or prevaccination sera.     

Rotavirus VP7 neutralization epitopes of serotype 3 strains

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on 27 human and animal rotavirus strains of serotype 3 in order to examine genetic variation within strains of identical serotype. Comparisons of the deduced amino acid sequences of the VP7s showed overall sequence identities of 85% or higher. A higher degree of overall VP7 sequence similarity was observed among strains from the same animal species when compared to strains from different animal species, suggesting that there are species-specific sequences in the VP7 protein. Alignment of the amino acid sequences demonstrated that amino acid sequence divergence among serotype 3 strains from different species was located primarily in previously established VP7 serotype-specific regions where genetic variation was identified among strains of different serotype. These regions were highly conserved among serotype 3 strains derived from the same species. The varying reactivities of three anti-VP7 monoclonal antibodies with the 27 strains was consistent with the occurrence of antigenic variation among serotype 3 strains. Moreover the reactivity of monoclonal antibodies correlated with the amino acid sequence found in two serotype-specific regions (VR5 and VR8). A computer-derived predicted phylogenetic tree suggests that rotavirus strains from different animal species belonging to serotype 3 are more closely related to each other than to rotavirus strains of different serotypes.     

Human rotavirus VP4 contains strain-specific,serotype-specific and cross-reactive neutralization sites

Summary The neutralization epitopes of human rotavirus VP4 were studied by using a panel of neutralizing monoclonal antibodies previously shown to be strain-specific (RV-3:3), serotype-specific (RV-5:2, ST-3:3) or cross-reactive (F45:4). Antigenic variants of human rotaviruses RV-3, ST-3, RV-5 and F45 resistant to neutralization by the appropriate of VP4 specific monoclonal antibodies (RV-3:3, ST-3:3, RV-5:2 and F45:4 respectively) were selected. By nucleotide sequence analysis and single strand conformational polymorphism analysis of these variants, three sites of neutralization on VP5* and one site on VP8* were identified. At or near to the putative fusion region on VP5*, a strain-specific site (aa383), a serotype P1A-P2 cross-reactive site (aa392) and a serotype P2-specific site (aa397) were found. On VP8*, a serotype P1B-specific site at aa148 was detected. These results confirmed the importance of the putative fusion region in neutralization and have identified a new neutralization site in the hypervariable region of VP8* which is specific for serotype P1B human rotaviruses.     

Cross-reactive,serotype- and monotype-specific neutralization epitopes on VP7 of serotype G3 and G5 porcine rotavirus strains

Summary VP7 specific monoclonal antibodies raised against serotype G5 porcine rotavirus strains isolated in Venezuela showed either a serotype G5- or monotype-specific pattern of reactivity by neutralization against a panel of 53 group A rotavirus isolates representative of all established G serotypes. Monoclonal antibodies raised against two G3 porcine strains were either specific for a subset of porcine G3 strains or reactive with another subset of porcine G3 strains and with most G5 strains. Neither were reactive with G3 strains from other species. Analysis of neutralization resistant mutants selected with these monoclonal antibodies indicated that epitopes defined by cross-reactive, serotype-and monotype-specific monoclonal antibodies overlap functionally and that binding and neutralization by these antibodies depended on specific amino acid residues in the region A or C of VP7. Results indicate that a high degree of monotypic variation occurs among G5 and G3 porcine rotavirus strains and the existence of at least one common epitope shared by G5 and G3 porcine strains, in the major neutralization domain of these VP7s.     

Heterogeneity of VP4 neutralization epitopes among serotype P1A human rotavirus strains.

We have used serotype-specific VP4 and VP7 neutralizing monoclonal antibodies (Nt-MAbs), as well as subgroup (SG)-specific MAbs, to characterize by enzyme immunoassay rotavirus strains isolated from diarrheic infants in the city of Monterrey, Mexico, from July 1993 to March 1994. Of a total of 465 children studied, 140 were rotavirus positive, including 3 patients infected with non-group A rotaviruses. The SG and VP7 (G) serotype specificities could be determined for 118 (84%) of the 140 rotavirus-positive stool specimens; 4 rotavirus strains were serotype G1 and SGII; 1 strain was serotype G2 and SGI+II; 112 strains were serotype G3 and SGII; 1 strain was serotype G3 and SGI; and none of the strains was serotype G4. Fifty-eight specimens, representing the 13 different group A rotavirus electropherotypes detected, were chosen for VP4 (P) serotyping. Of these, 48 (83%) strains reacted with the P1A serotype-specific Nt-MAb 1A10. None of the strains reacted with the serotype P2-specific Nt-MAbs tested. Not all viruses that reacted with Nt-MAb 1A10 were recognized by Nt-MAbs 2A3 and 2G1, which also recognize P1A strains, indicating heterogeneity of neutralization epitopes among serotype P1A human rotaviruses. This heterogeneity could be relevant for the specificity of the VP4-mediated neutralizing antibody immune response and indicates the need for antigenic characterization, in addition to genomic typing, of the VP4 proteins of circulating human rotavirus field strains.     

人轮状病毒G2和G3型主要中和抗原VP7基因在重组腺病毒中的表达

目的 研究我国G2和G3型轮状病毒主要中和抗原VP7基因在重组腺病毒中的表达。方法 在前期成功表达Gl型VP7的基础上，选用我国G2和G3型主要流行株97S43和97S48 VP7基因，用非复制型腺病毒载体对上述基因进行表达。结果 获得了表达我国G2型和G3型人轮状病毒流行株VP7基因的非复制型重组腺病毒rvAdG2VP7和rvAdG3VP7，应用PCR及Southem blot技术证实在重组腺病毒中整合有轮状病毒G2型VP7和G3型VP7基因，RT-PCR证明重组腺病毒在感染的293细胞内均能有效地转录插入基因，Western blot检测到轮状病毒VP7基因的表达。结论 这一工作为进一步进行动物实验，发展多价轮状病毒疫苗打下了基础。     

Single gene substitution rotavirus reassortants containing the major neutralization protein (VP7) of human rotavirus serotype 4.

A series of reassortants was isolated from coinfection of cell cultures with wild-type bovine rotavirus (UK strain [serotype 6]) or rhesus rotavirus (strain MMU18006 [serotype 3]) and a tissue culture-adapted human rotavirus strain, ST3 (serotype 4). Monospecific antiserum or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7, of the animal rotavirus parent was used to select for reassortants with human rotavirus serotype 4 neutralization specificity. The majority of reassortants contained only gene 9 of the human rotavirus parent, ST3, whereas the remaining genes were derived from the animal rotavirus parent. These single human rotavirus gene substitution reassortants were neutralized to high titer by hyperimmune serum directed at ST3, thus demonstrating that gene 9 of ST3 codes for the major neutralization protein of this strain. Moreover, these single gene substitution, reassortants were also neutralized to low titer by antiserum directed at their animal rotavirus parent, probably because they derived gene 4, which codes for another outer capsid protein, VP3, from their animal rotavirus parent. None of the reassortants derived gene 4, which had previously been shown to be responsible for host range restriction of human rotaviruses in tissue culture, from ST3, despite the fact that the ST3 strain used for gene reassortment had been tissue culture adapted.     

Determination of human rotavirus VP4 using serotype-specific cDNA probes

Summary The VP4 genetic groups of 151 field strains of human rotaviruses obtained from infants and young children with diarrhea from four locations in Malaysia were analyzed. The strains were adapted to growth in tissue culture and studied further by molecular hybridization of northern blotted RNA to PCR-generated cDNA probes representing amino acids 84–180 of the KU strain VP4, 83–181 of the DS-1 strain VP4, and 83–180 of either the 1076 or K8 strain VP4, representing VP4 genetic groups 1–4 (P1A, P1B, P2, and P3), respectively. The majority (79% of the field strains hybridized with the KU VP4 genetic group 1 probe and were associated with G1, G3, G4, untypable, or mixed G serotypes. VP4 genetic group 1 (P1A) strains were the most common in all locations in Malaysia between 1978–1988. Three strains which exhibited G3 and subgroup I specificity hybridized with the K8 VP4 genetic group 4 probe. These three VP4 genetic group 4 (P3) strains were detected in two different years and locations, extending the initial detection of this VP4 genetic group (the K8 strain) in Japan to a larger geographical area of Asia.     

Neutralization of rotavirus and recognition of immunologically important epitopes on VP4 and VP7 by human IgA

Summary.  Rotavirus is an important cause of gastroenteritis in young children. Locally produced antibodies in the intestinal mucosa are proposed to play an important role in the defence against rotavirus infection, but it is not established whether IgA alone can neutralize rotavirus, nor if IgA antibodies recognize epitopes involved in protective immunity. To evaluate whether human IgA plays a role in virus neutralization, serum IgA was purified from nineteen rotavirus seropositive individuals and examined for its neutralizing capacity by a peroxidase focus reduction test. In all nineteen sera IgA neutralizing antibodies against serotype 3 (rhesus rotavirus) were demonstrated. Purified IgA was further investigated and shown not only to neutralize rotavirus in solution but also to neutralize rotavirus already pre-bound to epithelial cells (MA-104). IgA epitope blocking assays with monoclonal antibodies directed against heterotypic epitopes on VP4 and VP7, revealed that IgA antibodies from 4/16 sera recognized epitopes on VP4, while 5/16 sera recognized a VP7 epitope. When whole sera were investigated for comparison 7 and 9/16 sera recognized epitopes on VP4 and VP7 respectively. Accepted January 22, 1997; Received October26, 1996     

Characterization of cross-reactive and serotype-specific epitopes on the nucleocapsid proteins of hantaviruses

The hantavirus nucleocapsid (N) protein fulfills several key roles in virus replication and assembly and is the major antigen in humoral immune responses in humans and mice. Here we report on epitopes involved in serotype-specific and cross-reactive recognition of the N proteins of hantaviruses using monoclonal antibodies (mAbs) against the N proteins of Andes virus (ANDV) and Sin Nombre virus (SNV). The mAbs define at least twelve different epitopic patterns which span eight sequences, including amino acids 17-59, 66-78, 79-91, 157-169, 222-234, 244-263, 274-286 and 326-338 on the SNV and ANDV N proteins. Studies on the cross-reactivity of these mAbs with different hantavirus N proteins indicated that epitopes located within amino acids 244-286 are related to serotype specificity. We analyzed further the location of epitopes with available three-dimensional structure information including the N-terminal coiled-coil and derived exposed and hidden residues of these epitopes. The generated recombinant N proteins and the characterized mAbs are functional tools being now available for hantavirus diagnostics and replication studies.     

A porcine rotavirus strain with dual VP7 serotype specificity

Porcine rotavirus MDR-13, which on original isolation showed a two-way antigenic relationship with human rotavirus RV-3, shows VP7 relationships with serotype G5 as well as G3 viruses upon gene reassortment. Analysis of porcine MDR-13 and the MD-UK reassortant revealed marked nucleotide and amino acid similarity of VP7 genes of these viruses with those of both serotype G3 and G5 viruses. Evolution of such a strain, possibly by sequential mutations in the VP7 gene, is discussed.     

Characterization of homotypic and heterotypic VP7 neutralization sites of rhesus rotavirus

The gene 9 nucleotide sequence was determined for rhesus rotavirus and each of 14 viral variants selected for their resistance to neutralizing monoclonal antibodies. Each variant contains a single gene 9, VP7, mutation which permits viral growth in the presence of the antibody. Variant mutations were identified in two distinct neutralization regions. Region A was identified by monoclonal antibodies that are involved in both serotype-specific and serotype cross-reactive neutralization. Region C was identified by serotype-specific neutralizing monoclonal antibodies. Heterotypic neutralizing monoclonal antibody 57-8 selected variants with a mutation at amino acid 94 in the A region, the same amino acid location selected by serotype-specific monoclonal antibodies. Monoclonal antibody 3 selected a VP7 mutation at amino acid 99 resulting in additional N-linked glycosylation of the VP7 protein. Despite the added VP7 glycosylation, variant v3 was not broadly resistant to additional VP7-specific neutralizing monoclonal antibodies.     

Genetic relatedness among human rotavirus genes coding for VP7, a major neutralization protein, and its application to serotype identification.

Antigenic characterization of human rotaviruses by plaque reduction neutralization assay has revealed four distinct serotypes. The outer capsid protein VP7, coded for by gene 8 or 9, is a major neutralization protein; however, studies of rotaviruses derived from genetic reassortment between two strains have confirmed that another outer capsid protein, VP3, is in some cases equally important in neutralization. In this study, the genetic relatedness of the genes coding for VP7 of human rotaviruses belonging to serotypes 1 through 4 was examined by hybridization of their denatured double-stranded genomic RNAs to labeled single-stranded mRNA probes derived from human-animal rotavirus reassortants containing only the VP7 gene of their human rotavirus parent. A high degree of homology was demonstrated between the VP7 genes of strain D and other serotype 1 human rotaviruses, strain DS-1 and other serotype 2 human rotaviruses, strain P and other serotype 3 human rotaviruses, and strain ST3 and other serotype 4 human rotaviruses. Hybrid bands could not be demonstrated between the VP7 gene of D, DS-1, P, or ST3 and the corresponding gene of human rotaviruses belonging to a different serotype. RNA specimens extracted from the stools of 15 Venezuelan children hospitalized with rotavirus diarrhea were hybridized to each of the reassortant probes representing the four human serotypes. All five viruses with short RNA patterns showed homology with the DS-1 strain VP7 gene; two of these were previously adapted to tissue culture and shown to be serotype 2 strains by tissue culture neutralization. Of the remaining 10 viruses with long RNA patterns, 2 hybridized only to the D strain VP7 gene, 6 hybridized only to the P strain VP7 gene, and 2 hybridized only to the ST3 strain VP7 gene. Hybridization using single human rotavirus gene substitution reassortants as probes may provide an alternative method for identifying the VP7 serotype of field isolates that would circumvent the need for tissue culture adaptation.     

A porcine G9 rotavirus strain shares neutralization and VP7 phylogenetic sequence lineage 3 characteristics with contemporary human G9 rotavirus strains

Of five globally important VP7 (G) serotypes (G1-4 and 9) of group A rotaviruses (the single most important etiologic agents of infantile diarrhea worldwide), G9 continues to attract considerable attention because of its unique natural history. Serotype G9 rotavirus was isolated from a child with diarrhea first in the United States in 1983 and subsequently in Japan in 1985. Curiously, soon after their detection, G9 rotaviruses were not detected for about a decade in both countries and then reemerged in both countries in the mid-1990s. Unexpectedly, however, such reemerged G9 strains were distinct genetically and molecularly from those isolated in the 1980s. Thus, the origin of the reemerged G9 viruses remains an enigma. Sequence analysis has demonstrated that the G9 rotavirus VP7 gene belongs to one of at least three phylogenetic lineages: lineage 1 (strains isolated in the 1980s in the United States and Japan), lineage 2 (strains first isolated in 1986 and exclusively in India thus far), and lineage 3 (strains that emerged/reemerged in the mid-1990s). Currently, lineage 3 G9 viruses are the most frequently detected G9 strains globally. We characterized a porcine rotavirus (A2 strain) isolated in the United States that was known to belong to the P[7] genotype but had not been serotyped by neutralization. The A2 strain was found to bear serotype G9 and P9 specificities as well as NSP4 [B] and subgroup I characteristics. By VP7-specific neutralization, the porcine G9 strain was more closely related to lineage 3 viruses than to lineage 1 or 2 viruses. Furthermore, by sequence analysis, the A2 VP7 was shown to belong to lineage 3 G9. These findings raise intriguing questions regarding possible explanations for the emergence of variations among the G9 strains.     

Sequence analysis of the gene encoding the serotype-specific glycoprotein (VP7) of two new human rotavirus serotypes

Human rotavirus strains 69M and WI61 are distinct from human rotavirus serotypes 1, 2, 3, and 4 and from each other by plaque reduction neutralization and have been proposed as new human rotavirus serotypes (serotype 8 and serotype 9, respectively). The nucleotide sequence of the gene encoding the serotype-specific capsid glycoprotein, VP7, of strains 69M and WI61 was determined. In addition, the sequence of the VP7 gene of strain F45 (serotypically indistinguishable from WI61) was determined. Comparative analyses of the nucleotide and deduced amino acid sequences with those of reference strains from serotypes 1,2,3,4,5, and 6 demonstrated that WI61 and F45 share a high degree of sequence similarity with each other and that strains 69M, WI61, and F45 are distinct from established serotypes 1,2,3,4,5, and 6 in nine defined regions of the VP7 which are variable across rotavirus serotypes.     

New porcine rotavirus serotype antigenically related to human rotavirus serotype 3.

Serotyping of porcine rotaviruses isolated in MA104 cells from Australian piglets with diarrhea showed that two strains belonged to serotype 3 and one strain was antigenically similar to the OSU strain of porcine rotavirus (serotype 5). In addition, neutralizing antibodies to human rotavirus serotype 4 (ST-3 strain) were detected in serum samples from sows in one area, and so it seems probable that porcine rotaviruses of at least three serotypes occur in Australia.     

Identification of two subtypes of serotype 4 human rotavirus by using VP7-specific neutralizing monoclonal antibodies

Two distinct subtypes of human rotavirus serotype 4 were identified by using neutralizing monoclonal antibodies directed to the major outer capsid glycoprotein, VP7, of strains ST3 (subtype 4A) and VA70 (subtype 4B). Specimens containing serotype 4 rotavirus, obtained from different countries, were examined for subtyping by using solid-phase immune electron microscopy, enzyme-linked immunosorbent assay, and, for cell culture-adapted strains, neutralization assay. All 59 human rotavirus strains identified as serotype 4 by using animal antisera were classified into either subtype by monoclonal antibodies. This suggests that the antigenic difference between the two subtypes is a consequence of critical variations within the immunodominant serotype 4-specific neutralization site of rotavirus VP7. Subtype 4A (ST3-like) strains were predominant and were detected in stools from patients with gastroenteritis, as well as from healthy infants and young children.     

轮状病毒结构蛋白VP7 HLA-A2.1限制性CTL表位的初步鉴定

目的 预测并初步鉴定轮状病毒Wa株结构蛋白VP7 HLA—A2．1限制性CTL表位。方法 用BIMAS软件预测VP7 HLA—A2．1限制性CTL表位;分子模拟技术对分值最高的4条肽进行分子模建;最后测定候选肽与HLA-A2．1分子的亲和力及结合稳定性。结果 结合BIMAS及分子模拟的预测结果,选择4条肽QLYCDYNLV（132—140）、LLNYILKSV（18—26）、VLMKYDQSL（140—148）及VNWKKWWQV（287—295）作为候选表位肽;4条肽与HLA—A2．1结合的荧光系数（FI）值分别为2．58、3．83、3．19及0．82,肽-HLA-A2．1复合物半数解离时间（DC50）分别为2-4、6-8、8及2h。结论 QLYCDYNLV（132—140）、LLNYILKSV（18—26）及VLMKYDQSL（140—148）为潜在的HLA-A2．1限制性CTL表位。     

Neutralizing epitopes on herpes simplex virus-1-expressed rotavirus VP7 are dependent on coexpression of other rotavirus proteins.

We constructed a recombinant thymidine kinase-negative herpes simplex virus type 1 (HSV-1) that expressed the rotavirus major outer capsid glycoprotein, VP7. In the recombinant HSV-1, a promoter from the 5' noncoding region of the HSV-1 glycoprotein B locus regulated the expression of VP7 as a HSV-1 gamma 1 gene product. HSV-1-expressed VP7 resembled rotavirus-expressed VP7 in its SDS-PAGE mobility, high mannose-type glycosylation, disulfide bonding, perinuclear to cytoplasmic localization, intracellular retention, and reactivity with polyclonal antisera and nonneutralizing antibodies. Unlike rotavirus-expressed VP7, HSV-1-expressed VP7 lacked several neutralizing epitopes by immuno-histochemical staining and by ELISA. One neutralizing epitope identified on HSV-1-expressed VP7 by ELISA was masked by paraformaldehyde fixation of recombinant HSV-1- but not rotavirus-infected cells. Neutralizing epitopes were restored to HSV-1-expressed VP7 by coinfection of cells with the HSV-1 recombinant and a heterologous rotavirus that lack the neutralizing epitopes. The recovered neutralizing epitopes were detected on double-shelled rotavirus particles produced in the coinfected cells. This study indicates that the formation of several neutralizing epitopes on rotavirus VP7 requires interaction of VP7 with other rotavirus proteins. In addition, HSV-1 was a useful vector for studying the localization, processing, and antigenicity of an RNA virus glycoprotein.