Huddinge University Hospital, Department of Clinical Virology, Karolinska Institutet, SE-141 86 Stockholm, Sweden1
Department of Microbiology, Akita Prefectural Institute of Public Health, Akita 010-0874, Japan2
Author for correspondence: Claes Örvell. Fax +46 8 58 58 13 05. e-mail clor{at}labd01.hs.sll.se
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Abstract |
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Introduction |
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Reinfections occur with mumps virus and vaccination with monovalent vaccines may show a lack of efficient protection against all mumps virus genotypes (Künkel et al., 1994 ; Gut et al., 1995
; Afzal et al., 1997b
; Kim et al., 2000
; Nöjd et al., 2001
). This phenomenon may be due to pronounced antigenic differences that exist between the HN proteins of different mumps virus strains (Server et al., 1982
; Örvell, 1984
; Yates et al., 1996
; Örvell et al., 1997b
; Nöjd et al., 2001
). Genotype A has been shown to be antigenically distinct from genotypes B, C and D, but knowledge about genotypes EJ is lacking (Yates et al., 1996
; Örvell et al., 1997b
; Nöjd et al., 2001
). For surveillance of immunity against mumps virus, it is important to follow the distribution of genotypes in different countries and also to measure genotype-specific immunity in the population (Nöjd et al., 2001
). In Sweden, mass vaccination against mumps virus was started with the Jeryl Lynn (JL) strain of genotype A in 1982. This vaccine has been reported to contain two different viruses, JL isolates 2 and 5 (JL2 and JL5) (Afzal et al., 1993
). There was a temporal relationship between the start of vaccination and the disappearance of the neuropathogenic genotypes C and D from the community from 1986 onwards. In contrast, the less neuropathogenic SBL-1 strain of genotype A is still endemic in Sweden (Tecle et al., 1998
; Nöjd et al., 2001
).
The aim of the present study was to investigate the antigenic relationships between mumps virus strains by including a larger number of virus strains of different genotypes into the comparison. An attempt was made to define both different serotypes of mumps virus and explain the seeming paradox of the persistence of the SBL-1 strain of mumps virus in Sweden, in spite of the continued mass vaccination with the JL virus strain of the same genotype (Tecle et al., 1998 ).
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Methods |
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Antibodies.
Monoclonal antibodies (mAbs) directed against the HN protein of the SBL-1 strain of mumps virus were used. The 11 mAbs used have been characterized by serological analysis and competitive ELISA in a previous study (Örvell, 1984 ). One group of three antibodies (group III), mAbs 2041, 5500 and 2072, could inhibit haemagglutinating (HA) activity and exhibited the highest neutralizing titres compared to other antibodies. Their binding locations on the HN protein has been defined previously (Örvell et al., 1997b
). Other antibodies (group IV), mAbs 2034, 2082 and 5342, inhibited NA activity but did not block HA activity. Five rabbits were hyperimmunized with purified virions of the SBL-1, Kilham (genotype A) and RW (genotype D) strains of mumps virus. Ten serum samples obtained from Swedish children aged between 2 and 4 years old who had been vaccinated at about 18 months of age with the JL mumps virus component included in the MMR vaccine were also used; these children were vaccinated between 1998 and 2000, at a time when the MMR vaccine coverage of 2-year-old children was approximately 95%.
Immunofluorescence (IF) analysis.
The procedure for IF analysis was similar to that described previously (Rydbeck et al., 1986 ). GMK cells infected with the different strains of mumps virus were transferred to glass slides. The cells were then air dried and fixed in cold (-20 °C) acetone. mAbs were used at a 1:50 dilution of the original ascites fluid. After incubation with the antibodies at 37 °C for 30 min., the slides were washed with PBS, after which goat anti-mouse fluorescein-labelled antibodies were added and the incubation was repeated. After washing, Evans blue at a final concentration of 0·03% was added and the preparations were examined under a fluorescence microscope.
Neutralization assays.
The procedure for end-point neutralization has been described previously (Örvell, 1976 ). Serial twofold dilutions of rabbit hyperimmune sera or human sera in a volume of 0·15 ml were mixed with an equal volume of virus (100200 TCID50/0·1 ml). The mixtures were shaken and incubated at room temperature for 1 h. After that time-period, 0·1 ml of the antigenantibody mixtures were inoculated onto GMK cells in tissue culture tubes; two tubes were inoculated per antibody dilution. The inoculated tubes were incubated at 37 °C and inspected for cytopathic effect and final readings after 7 days of incubation.
Nucleotide sequencing.
A selected coding area of the HN gene covering aa 341380 of the HN protein was sequenced for 16 virus strains belonging to genotypes C, D, G, H, I and J. The SH gene of ten clonal isolates of the JL virus strain were also sequenced. The procedures for PCR, nucleotide sequencing and construction of phylogenetic trees have been described previously (Örvell et al., 1997a , b
; Tecle et al., 2000
).
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Results |
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Comparison of a partial protein sequence of the HN protein from 21 virus strains
The results described showed that viruses of genotypes C, D, G, H and I were antigenically different from the SBL-1 strain of genotype A. It was also found that there existed antigenic differences between the three virus strains of genotype A. It was considered of interest to compare the amino acid sequence in the region where antibodies 2041 and 5500 bind. Of 16 virus strains collected at different time-points, a portion of the HN gene of each strain was sequenced and the amino acid sequence deduced (Fig. 2). In comparison to the SBL-1 strain, all virus strains of genotypes CJ investigated contained glutamine instead of proline at position 354 and aspartic acid instead of glutamic acid at position 356. Genotype H differed by containing the amino acids serine, tyrosine and proline at positions 357359. These results could explain why the two antibodies could not bind to viruses of genotypes C, D, G, H or I. The lack of reactivity of mAb 2041 with the JL virus could not be explained, as the virus material should contain both the JL2 and the JL5 virus clones (Afzal et al., 1993
); the sequence of the JL2 virus is identical to the SBL-1 strain in this region (Yates et al., 1996
) and thus a reaction with mAb 2041 would have been expected if the virus material had contained the JL2 virus.
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Discussion |
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The results from this and previous studies show that there exists a significant antigenic difference between the SBL-1 strain of genotype A and genotypes B, C, D, G, H and I (Örvell, 1984 ; Yates et al., 1996
; Örvell et al., 1997b
). By using mAbs directed against the Urabe strain of genotype B, Yates et al. (1996)
could show that their two mAbs, mAbs 1970 and 1711, with the highest HA-inhibiting and neutralizing titres did not react with the Enders, Rubini, SBL-1 or JL2 strains of genotype A but reacted with the group C virus strains. Antibody 1970 bound to JL5 but not to JL2, whereas antibody 1711 bound neither JL5 nor JL2. Yates et al. (1996)
also showed an antigenic difference between the SBL-1 and the JL2 and JL5 virus strains. A similar antigenic difference was demonstrated in the present study between the JL5 and the SBL-1 strain. The JL virus preparation in the present study was concluded to contain the JL5 virus but not the JL2 virus. The results from cross-neutralization experiments showed that the SBL-1, JL5 and Kilham virus strains form three distinct serotypes within genotype A. An unexpected finding was that the JL5 isolate was efficiently neutralized by antibodies directed against the RW strain of genotype D. The difference between JL5 and RW viruses in the antigenic area of antibody 2041 is the presence of aspartic acid at position 356 in RW and glutamic acid in JL5. These two amino acids show structural similarities. It is therefore possible that the pronounced neutralization of JL5 by the rabbit hyperimmune sera directed against strain RW is due to the presence of glutamine at position 354 in both virus strains. A new neutralizing epitope at positions 329340 has been described recently (Cusi et al., 2001
). A unique structural similarity between RW and JL5 with the amino acid leucine instead of serine at position 336 (Yates et al., 1996
) may also be of importance for the pronounced reactivity between JL5 and RW.
The JL virus has been used from 1982 for vaccination of Swedish children. The significantly lower titres against the SBL-1 virus strain in the sera from vaccinated children may indicate that the JL5 virus is subobtimal for protection against the SBL-1 strain in Sweden. The low neutralization titres against the SBL-1 virus may be one of more possible explanations for the continued occurrence of the SBL-1 strain in Sweden (Tecle et al., 1998 ; Nöjd et al., 2001
). The JL virus strain has been reported to contain the JL2 and JL5 strains in the same vaccine preparation in proportions of 1:5 (Afzal et al., 1993
); however, a large amount of phylogenetic data on the dominating sequence after growing the virus in tissue culture has shown that this proportion between JL2 and JL5 is not a static phenomenon. For example, the JL variant of mumps vaccine grown on Vero cells and investigated by Yeo et al. (1993)
was found to contain the JL2 virus (X63707 in Fig. 3
). The JL2 virus (D90232 in Fig. 3
) described by Takeuchi et al. (1991)
was also found in the total virus material of infected cells but, in the present study, the vaccine preparation was found to contain JL5 after growing the virus in tissue culture. The JL2 virus has an amino acid sequence that is similar to the SBL-1, Enders and Rubini strains of genotype A in the epitope(s) of mAbs 2041 and 5500, whereas the JL5 virus shows an amino acid sequence that is partly similar to both genotypes A and non-A (glutamic acid at position 356 like genotype A and glutamine in position 354 like non-A genotypes). It is possible that the JL2 virus will give better protection than JL5 against the SBL-1 strain. However, the antigenic match between JL2 and SBL-1 is not ideal. The amino acid sequence of JL2 does not conform with the SBL-1 strain in the neutralizing epitope of antibody 2072 around position 269 (Yates et al., 1996
; Örvell et al., 1997b
). The epitope of antibody 2072 is unique for SBL-1, as all other known mumps virus strains have two different amino acids, positions 265 and 266, compared to SBL-1 in this region (Yates et al., 1996
; Örvell et al., 1997b
; Tecle et al., 1998
). Also, Yates et al. (1996)
have found an antigenic difference between SBL-1 and JL2 by using mAbs directed against the Urabe strain. Three of their neutralizing mAbs reacted with the SBL-1 virus but did not react with the JL2 strain.
In recent years, the neuropathogenicity of different mumps virus strains has been investigated. A number of genotypes (genotypes C, D, G, H, I and J) has been found to exhibit a pronounced neuropathogenic capacity (Saito et al., 1996 , 1998
; Rubin et al., 1998
, 2000
; Tecle et al., 1998
, 2001
, 2002
; Saika et al., 2002
). The present study could not find any clear-cut antigenic difference between the non-A genotype neuropathogenic virus strains. In the case that future research will confirm these findings, it may be important to direct vaccine protection against this group of neuropathogenic viruses.
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Acknowledgments |
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References |
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Received 28 March 2002;
accepted 11 June 2002.