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The Aino virus is an arthropod-borne virus belonging to the Simbu group of the family Bunyaviridae, genus Bunyavirus. The virus is widely distributed in Southeast Asia, Australia, and East Asia. Neutralizing antibodies against the Aino virus have been detected in the precolostral sera of Japanese (5, 8, 11, 16) and Australian (3) calves exhibiting arthrogryposis, hydranencephaly, and cerebellar hypoplasia. In addition, the antigen of the Aino virus has been immunohistochemically demonstrated in the brain of an aborted bovine fetus (18), and the Aino virus has also been isolated from an aborted fetus (22). These findings strongly suggest that the Aino virus may be involved in the etiology of hydranencephaly, arthrogryposis, and cerebellar hypoplasia in cattle. In fact, studies have shown that chick embryos infected with the Aino virus went on to develop arthrogryposis, hydranencephaly, and cerebellar hypoplasia (12, 13, 14). However, in order to develop a vaccine or other preventive measures against this virus, further information on its antigenic diversity within the epidemic area will be needed.

In this study, we prepared neutralizing monoclonal antibodies (MAbs) against the Aino virus and defined the neutralizing epitopes of the virus by competitive binding assays. We then compared the antigenicities of 22 field isolates using MAbs against each distinct epitope.

The JaNAr28 strain that is the prototype strain of the Aino virus, isolated from Culex tritaeniorhynchus (19), was used for preparation of MAbs. The HmLu-1 cells were infected with the JaNAr28 strain of virus at a multiplicity of infection of 0.01 to 0.1 and were incubated at 37°C until they showed a complete cytopathic effect. Purification of virus was performed by the method by Ide et al. (7). The methods of production of MAbs, neutralization tests, and determination of antibody subtype were as described previously by Yoshida and Tsuda (23).

Viral antigens were separated as described by Laemmli (15) by 10% polyacrylamide gel electrophoresis and were then electrically transferred to a membrane (polyvinylidene difluoride; pore size, 0.45 µm; Immobilon; Millipore Corp., Bedford, Mass.) (Western blotting). Immunoenzymatic staining with the 400-fold dilutions of MAbs from mouse ascitic fluid was performed as essentially described by Towbin et al. (21), except that 4% skim milk was used for blocking and 0.027% 3,3'-diaminobenzidine tetrahydrochloride was used for visualization.

MAbs were purified from ascitic fluid by using a MAb G II affinity chromatography kit (Pharmacia, Uppsala, Sweden). Conjugation of peroxidase to the MAbs was performed essentially as described by Tijssen and Kurstak (20). The competitive binding assay was performed essentially as described by Kimura-Kuroda and Yasui (9). Briefly, enzyme-linked immunosorbent assay (ELISA) plates (Immulon 2; Dynatech Laboratories Inc., Chantilly, Va.) were coated for 48 h at 4°C with purified JaNAr28 antigen diluted to the appropriate concentration with carbonate-bicarbonate buffer (0.05 M [pH 9.6]) and were washed three times with washing solution (0.15 M NaCl, 0.02% Tween 20). Each of the competing antibodies was diluted serially with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 4% skim milk, and then the diluted antibodies were added to the antigen-coated wells of the ELISA plates. The plates were incubated for 2 h at 37°C and washed once. Peroxidase-conjugated MAb, at a predetermined dilution that gave an absorbance of between 0.5 and 0.8 with PBS containing 0.1% Tween 20 and 8% skim milk, was added; and the plates were incubated for 1 h at 37°C. The plates were washed six times, and substrate solution (0.1 M citric acid, 0.2 M Na₂HPO₄, 0.04% o-phenylenediamine dihydrochloride, 0.007% H₂O₂) was added. After incubation for 40 min at room temperature, the reaction was stopped with H₂SO₄ and the absorbance was measured with an ELISA reader (S Jeia II; Sanko Junyaku Co., Tokyo, Japan) with a 492-nm filter. The percent competition was determined at the 800-fold dilution of competitive MAb by the following formula (6): 100× (optical density [OD] without competitor  OD with competitor)/(OD without competitor  OD with homologous MAb).

Twelve MAbs possessed strong neutralizing activity against the Aino virus JaNAr28 strain, and their neutralizing titers of the ascitic fluid were more than 1,024. Of these MAbs, six reacted with the G1 glycoprotein of the virus. These MAbs were used to determine their topological relationships to the neutralizing epitopes. The subtype, the neutralizing titer, and a summary of the competitive binding assays are shown in Table 1.

The results of the competitive binding assays revealed that the neutralizing epitopes of the Aino virus consisted of two different antigenic domains: domains I and II. Domain I comprised a single site recognized by MAb 3B8. However, domain I might have been slightly dependent on the site recognized by MAb 3A1. Domain II included at least four epitopes: IIA, IIB, IIC, and IID. Epitope IIA comprised a site recognized by MAb 3C6. This epitope broadly overlapped with the epitopes IIB, IIC, and IID. Epitope IIB comprised two sites recognized by MAbs 2F1 and 3F9. Reciprocal competition was found between MAbs 2F1 and 3F9, although the competition levels for these two competitors were slightly different from those for conjugated MAbs 6C7 and 6G10. It was suggested that these sites generally overlapped. Epitope IIC comprised a site recognized by MAb 3A1. Epitope IID could be subdivided into three epitopes: IID1, IID2, and IID3. Epitope IID1 comprised two sites recognized by MAbs 3H11 and 1B3. These two sites were almost the same or overlapped. Epitope IID1 might have been slightly dependent on epitope IIC. Epitope IID2 comprised three sites recognized by MAbs 6C7, 6G10, and 3C8. Although the levels of blocking of these conjugated MAbs were slightly dispersed, these competitor MAbs almost competed with the same conjugated MAbs. It was suggested that these sites generally overlapped. Moreover, these three sites might have been slightly dependent on epitope IIB. Epitope IID3 comprised two sites recognized by MAbs 3E5 and 5G1. Although the levels of blocking of conjugated MAb 6C7 were different, the levels of blocking of conjugated MAbs 3E5 and 5G1 were very similar. It was suggested that these two sites might have generally overlapped. Consequently, it was presumed that the neutralizing epitopes of the Aino virus were narrow, continuous, and overlapped. The proposed interrelationship among these neutralizing epitopes of the Aino virus is shown in Fig. 1. The results of the competitive binding assay with the neutralizing MAbs of the Akabane virus belonging to the Simbu group showed the presence of five independent antigenic domains (23). The neutralizing epitopes of the Aino virus may be more complex than those of the Akabane virus.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Proposed interrelationship of neutralizing epitopes of the Aino virus. The epitopes recognized by the MAbs (in parentheses) are shown.

In order to define the antigenic variation among Aino virus isolates, the antigenicities of 22 isolates were examined by dot immunobinding assays (DIAs) with seven MAbs, MAbs 3B8, 3C6, 2F1, 3A1, 1B3, 6C7, and 3E5, each of which was shown to recognize different neutralizing epitopes: epitopes I, IIA, IIB, IIC, IID1, IID2, and IID3, respectively. The 22 isolates used for this experiment are listed in Table 2, along with their passage levels, origins, collection locations, and years of isolation. DIAs were performed by the method of Yoshida and Tsuda (23). The absorbance of each isolate was divided by the absorbance of JaNAr28 to determine the MAb reaction for each isolate relative to that for the JaNAr28 strain. Furthermore, to correct for variations in viral concentration among the supernatants of the Aino virus isolates, the degree of reaction against each MAb was expressed as a percentage of the total MAb reactions for each isolate. Finally, the data were graphed and the response patterns were identified.

The DIA response patterns of the 22 isolates of Aino virus were classified into four groups. The four groups are shown in Fig. 2, and the isolates included in each group are listed in Table 2. Pattern 1 is represented by isolate JaNAr28, which showed the same level of reaction with all the MAbs. In addition to JaNAr28, this pattern was found for three isolates collected in 1989 and one isolate collected in 1988. Pattern 2 is represented by isolate KSB-10/P/86, whose reactivities with six MAbs were the same as the reactivities of isolates of pattern 1, with the exception of a low level of reactivity with MAb 3B8. All isolates collected in 1986, two isolates collected in Hyogo (central Japan), and one isolate collected in Kagoshima (south western Japan) in 1995 showed this pattern. Pattern 3 is represented by isolate KSB-2/C/88, which demonstrated only slight reactivity with MAbs 2F1 and 1B3. This pattern was found among three isolates obtained in 1988 and one isolate obtained in 1989. Pattern 4 is represented by isolate B7974, which had little reactivity with MAbs 1B3, 6C7, and 3E5, and its reactivity with MAb 3B8 was low, as shown in Fig. 2.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   DIA response patterns of the Aino virus isolates. The abscissa shows the degree of reaction against each MAb (corresponding epitope) as a percentage of the total MAb reactions. Representative strains of the Aino virus with the pattern are indicated in parentheses.

The reactivities to epitopes IIA and IIC were similar for all isolates, and those to epitopes I and IIB were slightly different. However, the reactivities to epitopes IID1, IID2, and IID3 were different. These results suggest that epitopes IIA and IIC are conserved and that epitopes IID1, IID2, and IID3 are mutable. The isolates were divided primarily into four groups represented by strains JaNAr28 (pattern 1), KSB-10/P/86 (pattern 2), KSB-2/C/88 (pattern 3), and B7974 (pattern 4), respectively. Comparison of the patterns for isolates collected in the same years in Japan revealed roughly the same patterns. Furthermore, strains HG-1/B/95 and HG-2/B/95, isolated in 1995 in Hyogo, and strain KSB-3/P/95, isolated in 1995 in Kagoshima, had pattern 2. This result suggests that Aino viruses that possess the same antigenicities are distributed in a wide area in the same season. However, all isolates collected in Japan had pattern 1, 2, or 3, and there was not much difference. This result suggested that the antigenic diversity of the Aino virus might be small even if the host-vector relationship leads to mutation (1, 2, 10). A similar phenomenon has been observed with the Akabane virus (23). On the other hand, the antigenicity of strain B7974 isolated from Culicoides brevitarsis in Australia in 1968 (4) was highly different from those of isolates in Japan. Strains JaNAr28 and B7974 were indistinguishable by cross neutralization, cross hemagglutination-inhibition, and cross complement fixation tests (17). However, DIAs with these MAbs which defined epitopes to the Aino virus clearly revealed the differences in antigenicities of strains JaNAr28 and B7974. Since these MAbs recognize very narrow epitopes, comparison of the reactivities with each MAb is able to reveal more delicate differences in the antigenicities of Aino virus isolates. An inactivated Aino virus vaccine derivative of strain JaNAr28 has been developed and is being used in Japan. However, if there are field isolates with remarkably different patterns by DIA, it may be necessary to examine the vaccine potency in detail. Then, DIA could be used to investigate many field isolates. Therefore, DIAs with these neutralizing MAbs should be a useful tool for investigation of the antigenicities of field isolates of the Aino virus for the evaluation of vaccine potency.