Identification of serotype 9 human rotavirus by enzyme-linked immunosorbent assay with monoclonal antibodies.

Hybridomas producing monoclonal antibodies to VP7, a major neutralizing protein of serotype 9 rotavirus (strain W161), were prepared. One monoclonal antibody, W161-6A1, was shown to neutralize only serotype 9 rotavirus strains and reacted specifically with serotype 9 rotaviruses in an enzyme-linked immunosorbent assay. The development of an immunoassay for detection of serotype 9 rotaviruses should facilitate epidemiologic studies.     

Reactivity of VP4-specific monoclonal antibodies to a serotype 4 porcine rotavirus with distinct serotypes of human (symptomatic and asymptomatic) and animal rotaviruses.

Thirteen hybridomas secreting VP4-specific monoclonal antibodies against the Gottfried strain of porcine rotavirus (serotype 4) were produced and characterized. Nine of the hybridomas secreted neutralizing monoclonal antibodies (N-MAbs) against Gottfried rotavirus. These N-MAbs were divided into five distinct groups (groups I to V) according to their patterns of reactivity with different serotypes of human and animal rotaviruses. Group I N-MAbs (n = 3) were cross-reactive with five different serotypes of human rotavirus examined by a plaque reduction virus neutralization test. Group II N-MAbs (n = 3) neutralized all symptomatic human rotavirus serotypes tested and asymptomatic human rotavirus serotype 4 to a low titer. The single group III N-MAb neutralized mainly symptomatic human rotavirus serotypes 2 and 9 and none of the asymptomatic human rotavirus serotypes. The one N-MAb in group IV reacted at low titers with only asymptomatic human rotavirus serotypes 1 through 4. A group V N-MAb recognized serotype 4 porcine rotaviruses (Gottfried and SB-2) but no other human or animal rotaviruses examined. None of the N-MAbs recognized any animal rotaviruses tested (SA-11, RRV, OSU, NCDV, and B223), except for the Gottfried and SB-2 rotaviruses. The failure of N-MAbs (groups I to IV) to react with any animal rotaviruses tested but their ability to react variably with all human rotaviruses tested suggest that neutralizing epitopes on the VP4 protein are highly conserved between the Gottfried porcine and human rotaviruses. The Gottfried rotavirus may possibly represent a naturally occurring reassortant between pig and human rotaviruses or a rotavirus which is human in origin but pathogenic for swine.     

Direct serotyping of porcine rotaviruses using VP7-specific monoclonal antibodies by an enzyme immunoassay.

Employing a serotyping EIA test using MAbs both cell culture adapted and faecal porcine rotaviruses were classified into serotypes G3, G3/5, G4, and G5. The MAbs have confirmed and extended the serotyping results obtained using polyclonal antisera. These MAbs are therefore potential reagents for serotyping of porcine rotaviruses. Using subgroup specific MAbs serotypes G3, G3/5, and G5 were found to contain subgroup I antigens while G4 rotaviruses contained either subgroup II or subgroup I antigens.     

Bovine rotavirus type detection by neutralizing monoclonal antibodies

Summary A series of monoclonal antibodies were developed against bovine rotavirus Q₁₇. Among the five high affinity antibodies characterized, two (RQ 31 and RQ 4) were able to neutralize type G 6 viruses and may be specific for PB 1 type virus. Seventy seven feces from diarrheic calves were tested by double sandwich ELISA using four monoclonal and one polyclonal anti-rotavirus antibodies. The combination of mono- and polyclonal antibodies thus appears to be a more efficient strategy for detection and typing of bovine rotaviruses.     

Identification and characterization of serotype 4-specific antigens of Ureaplasma urealyticum by use of monoclonal antibodies.

Monoclonal antibodies against Ureaplasma urealyticum serotype 4 were produced by immunizing BALB/c mice with whole-cell antigens of the U. urealyticum serotype 4 reference strain. Ten monoclonal antibodies differentiated into two groups were found: one group included five monoclonal antibodies recognizing a band in immunoblotting that had a molecular mass of 81 kDa, and a second group included another five monoclonal antibodies recognizing three bands in immunoblotting that had molecular masses of 81, 75, and 71 kDa. Fifteen clinical U. urealyticum isolates were selected for serotyping with serotype 4-specific monoclonal antibodies and polyclonal antisera 1 to 14. The results obtained with polyclonal and monoclonal antibodies suggest the existence of heterogeneity of the serotype antigens among clinical isolates of U. urealyticum serotype 4.     

Comparisons of rotavirus VP7-typing monoclonal antibodies by competition binding assay.

Three sets of neutralizing monoclonal antibodies (MAbs) used to type the outer capsid protein VP7 of four group A rotavirus serotypes (1 through 4) were compared in competition immunoassays. Reciprocal competition was observed for each of the VP7 type 2-, 3-, and 4-specific MAbs. The VP7 type 1 MAbs exhibited variable competition patterns with other VP7 type 1 MAbs. MAb RV4:3, which has been used to recognize antigenic variants within VP7 type 1 strains, showed reciprocal competition with the four VP7 type 3 MAbs (RV3:1, YO-1E2, 4F8, and 159) using a VP7 type 3 virus (SA11) as antigen. MAb 2C9, also prepared against VP7 type 1, reacted with VP7 type 3 strains and competed with a VP7 type 3 MAb, 159, using RRV as antigen. Use of the different sets of VP7 type-specific MAbs in the enzyme-linked immunosorbent assay permitted the recognition of six antigenic variants within VP7 types 1, 2, and 3 among specimens whose VP7 type could not be determined previously with only one set of typing MAbs. These results demonstrate differences of typing ability among these VP7-specific MAbs and emphasize the need to improve the sensitivity of typing systems by incorporating panels of MAbs reacting with several neutralizing epitopes.     

Antigenic analysis of avian rotavirus VP6 using monoclonal antibodies

Summary Monoclonal antibodies (MAbs) were prepared to analyze antigens on the major inner capsid protein, VP 6 of avian group A rotavirus. Based on the results of a competitive binding assay using 15 MAbs directed against VP 6 of the PO-13 rotavirus strain, isolated from a pigeon in Japan, it was found that VP 6 of avian rotavirus possesses at least four spatially distinct antigenic sites. Two antigenic sites (I and II) were topologically distinct from the other two (III and IV), which were in close proximity. From the reaction of MAbs in indirect immunofluorescent antibody tests to a series of known rotaviruses, epitopes representing common antigens of all group A rotavirus including avian rotavirus were localized in sites II and III. One epitope in site IV appeared to have a subgroup antigenic specificity that reacted only with rotaviruses belonging to subgroup I. Interestingly, avian rotaviruses isolated from turkeys and chickens in Northern Ireland also reacted only with these subgroup I specific MAbs, but not with subgroup II specific MAb. This indicates that avian rotavirus has subgroup I specific antigen, which is antigenically similar to that of other mammalian rotavirus strains.     

Identification of four distinct neutralizing epitopes on bluetongue virus serotype 10 using neutralizing monoclonal antibodies and neutralization-escape variants.

Neutralizing sites on bluetongue virus serotype 10 (BTV-10) were investigated with a panel of seven neutralizing monoclonal antibodies (MAbs). Each MAb was coupled to agarose beads and tested against the other MAbs and a nonneutralizing control MAb in a competitive immueprecipitation assay. In addition, neutralization-escape viral variants of BTV-10 were identified and cloned by selecting individual plaques that formed in the presence of neutralizing MAbs. Four antigenic variants were isolated, each under the selective pressure of a different MAb. Parental BTV-10 and the four antigenic variants were compared by microneutralization assay using the MAb panel. The panel of neutralizing MAbs was subdivided into four groups on the basis of these assays, indicating that at least four distinct neutralizing epitopes exist on the BTV-10 virion. These epitopes are individually defined by representative MAbs 034, 039, 041, and 045, and the results indicate that the four epitopes are interacting sites within a single antigenic domain on BTV-10 outer capsid protein VP2. This conclusion is further supported by the fact that a double-site neutralization-escape variant designated DE34/39 (sequentially produced against MAbs 034 and 039) was not neutralized by any MAb of the panel.     

VP4 protein of porcine rotavirus strain OSU expressed by a baculovirus recombinant induces neutralizing antibodies

The complete VP4 gene of porcine rotavirus strain OSU has been inserted into a baculovirus expression vector under the control of the polyhedrin promoter. The VP4 outer capsid protein, which is a major neutralization antigen in rotavirus, was expressed in high yield in Spodoptera frugiperda cells. Reactivity with polyclonal and monoclonal antibodies suggested that neutralizing epitopes were functionally unaltered on the expressed VP4. The VP4 produced in this system also induced antibodies in guinea pigs which inhibited hemagglutination of OSU and neutralized its infectivity to high titer. The available evidence suggests that the VP4 expressed in insect cells maintained its antigenic configuration and may prove useful in elucidation of (1) the extent of VP4 polymorphism among human and animal rotaviruses and (2) the distribution of VP4 among these viruses.     

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.     

Antigenic variability among two subtypes of human adenovirus serotype 7

Human adenovirus type 7 (HAdV-7) is one of the major serotypes responsible for acute respiratory infection. It is important to investigate the antigenic variabilities of different HAdV-7 genomic subtypes for vaccine development. Phylogenetic analysis of global HAdV-7 strains and major antigen proteins showed that HAdV-7 could be classified into two subtypes. There were three highly variable regions (HVR1, HVR4, and HVR7) in the hexon protein that varied between subtypes. Within each of the subtypes, these regions were conserved. Two subtype HAdV-7 strains isolated in China were used to immunize mice for antigenic characterization. Mice immunized with one subtype strain showed 4–8-fold lower neutralizing antibody titers against another subtype strain. ELISA results showed that the variation in HVR1, 4, and 7 regions contributed to antigenic change, and it may be concluded that the three regions contain subtype-specific epitopes. In summary, strains of HAdV-7 could be divided into two subtypes using genome sequence and antigenic analysis; our results could be important for HAdV-7 vaccine development.     

Genetic variation in rotavirus serotype 4 subtypes.

Serotype 4 rotavirus strains have been classified into two antigenic "subtypes" by a solid phase immune electron microscopy technique in which cross-absorbed animal polyclonal immune sera are used as the source of antibodies. The sequences of the gene encoding the outer capsid glycoprotein VP7 from a single serotype 4 rotavirus field strain identified as subtype A ("ST3-like") and from three field strains identified as subtype B ("VA70-like") were determined. A comparison of the deduced amino acid sequences indicated that 15 amino acid residues were divergent between subtypes but were conserved within a subtype. Three of these 15 amino acid residues (at positions 96, 212, and 217) were located in regions of the VP7 that have been defined as serotype-specific antigenic sites. These data suggest that VP7 subtype differences may result from critical amino acid substitutions within an immunodominant serotype 4-specific antigenic site. Whether these differences are an important mechanism in the epidemiology of rotaviruses requires further investigation.     

Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain

Rotavirus genome segment 4, encoding the spike outer capsid VP4 protein, of a porcine rotavirus (PoRV) strain, 134/04-15, identified in Italy was sequenced, and the predicted amino acid (aa) sequence was compared to those of all known VP4 (P) genotypes. The aa sequence of the full-length VP4 protein of the PoRV strain 134/04-15 showed aa identity values ranging from 59.7% (bovine strain KK3, P8[11]) to 86.09% (porcine strain A46, P[13]) with those of the remaining 25 P genotypes. Moreover, aa sequence analysis of the corresponding VP8* trypsin cleavage fragment revealed that the PoRV strain 134/04-15 shared low identity, ranging from 37.52% (bovine strain 993/83, P[17]) to 73.6% (porcine strain MDR-13, P[13]), with those of the remaining 25 P genotypes. Phylogenetic relationships showed that the VP4 of the PoRV strain 134/04-15 shares a common evolutionary origin with porcine P[13] and lapine P[22] rotavirus strains. Additional sequence analyses of the VP7, VP6, and NSP4 genes of the PoRV strain 134/04-15 revealed the highest VP7 aa identity (95.9%) to G5 porcine strains, a porcine-like VP6 within VP6 genogroup I, and a Wa-like (genotype B) NSP4, respectively. Altogether, these results indicate that the PoRV strain 134/04-15 should be considered as prototype of a new VP4 genotype, P[26], and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.     

Identification of serotype 1-, 3-, and 6-specific antigens of Ureaplasma urealyticum by using monoclonal antibodies.

Little is known about the antigens responsible for serotype specificity in Ureaplasma urealyticum. We produced monoclonal antibodies to U. urealyticum serotypes 1, 3, and 6, the serotypes most commonly found in pregnant women, and analyzed serotype-specific antigens for the three serotypes. Clinical isolates belonging to serotype 1, 3, or 6 were tested in immunoblots with these monoclonal antibodies. The immunoblot patterns of these isolates were, in most cases, different from each other as well as from those of the reference strains, indicating a high rate of antigenic variation among U. urealyticum strains.     

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.     

Production of reassortant viruses containing human rotavirus VP4 and SA11 VP7 for measuring neutralizing antibody following natural infection.

The outer capsid proteins VP4 and VP7 of group A rotaviruses are both targets of neutralizing antibody produced following natural infection in humans. Of interest is the relative importance and immunodominance of each protein in the generation of a protective immune response. In order to measure neutralizing antibody responses to VP4 and VP7 separately, reassortants bearing VP4 of each of the major human rotavirus P types with VP7 of SA11 origin were successfully produced by neutralizing monoclonal antibody selection. The resulting reassortants, together with reassortants representing each of the major VP7 types, were antigenically characterized with serotype-specific neutralizing monoclonal antibodies and hyperimmune sera. The neutralization proteins of human rotavirus origin were found to be unaffected antigenically by reassortment. The abilities of these reassortants to discriminate between VP4 and VP7 immune responses were evaluated with postinfection sera collected from three patients infected with either a P1A[8],G1, a P1B[4],G2, or a P1A[8],G4 rotavirus strain. The reassortants were shown to be capable of separating the neutralizing antibody responses to VP4 and VP7, with each patient showing a different immune response with respect to VP4 or VP7 immunodominance. These reassortants can now be applied to analyses of individual immune responses to VP4 and VP7 proteins after primary rotavirus infections and reinfections in humans.     

Bovine rotavirus strain 678 possesses VP4 of serotype P7[5] specificity

Summary.  Bovine rotavirus strain 678 is the first G8 strain of bovine origin but the literature is confusing as to its P type. In this study, two-way cross neutralization between 678 and 0510, a prototype G6P7[5] virus, was shown by plaque-reduction neutralization assays, establishing the P type of 678 as being P7[5]. The P7[5] specificity of 678 VP4 was reinforced by the finding that the VP8^* portion of 678 VP4 had the highest amino acid identity with those of P7[5] bovine rotaviruses. Apparent contradiction with previous serological studies relates to intricacy of antigenicity and immunogenicity of UK VP4 in reassortants. Received October 28, 1996 Accepted January 6, 1997     

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.     

Effect of the selection pressure with anti-VP7 and anti-VP4 neutralizing monoclonal antibodies on reassortant formation between two human rotaviruses

Summary In order to study the effect of selection pressure of anti-VP4 and anti-VP7 neutralizing monoclonal antibodies(N-MAbs) on reassortant formation, 424 reassortant clones were produced from mixed cultures of human rotavirus strains Wa and HN126 and their genotypes were analysed. Reassortant selection was done with four types of N-MAb: anti-VP4 MAb to Wa and anti-VP7 MAb to HN126(selection A); anti-VP4 MAb to HN126 and anti-VP7 MAb to Wa(selection B); anti-VP7 MAb to Wa(selection C); and anti-VP4 MAb to Wa(selection D). In each selection experiment, more than 100 clones were isolated, and the parental origin of RNA segments was identified by polyacrylamide gel electrophoresis. All clones isolated by selections A and B were found to be antigenic mosaic reassortants with the VP4 gene of HN126 and the VP7 gene of Wa and antigenic mosaic reassortants with the VP4 gene of Wa and the VP7 gene of HN126, respectively. Although in reassortants of both selections, RNA segments 2, 3, 5 and 6 were selected from strain Wa at considerably high rates, selection rates of RNA segments 1, 7, 8, and 11 were significantly different between selection A and B. In reassortants from selection C and D, selection rates of RNA segments 1, 3, 6, 7, 8, and 11 from Wa were significantly lower than those in selection A and B, whereas RNA segments 2 and 5 were almost exclusively selected from Wa as observed in selection A and B. These results indicated the presence of two types of nonrandom gene selection in reassortant formation, one strongly dependent on, and another irrespective of, the selection pressure with N-MAbs.