Circulation of Mumps Virus Genotypes in Spain from 1996 to

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2010, p. 1245–1254 Vol. 48, No. 40095-1137/10/$12.00 doi:10.1128/JCM.02386-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Circulation of Mumps Virus Genotypes in Spain from 1996 to 2007�

J. E. Echevarría,1,2* A. Castellanos,1,2 J. C. Sanz,2,3 C. Perez,4 G. Palacios,5M. V. Martínez de Aragon,2,6 I. Pena Rey,2,6 M. Mosquera,1,2

F. de Ory,1,2 and E. Royuela1,2

Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain1; CIBER en Epidemiología ySalud Publica, CIBERESP, Madrid, Spain2; Laboratorio Regional de Salud Publica, Madrid, Comunidad de Madrid,

Spain3; Hospital General de Gran Canaria Dr. Negrín, Las Palmas de Gran Canaria, Canary Islands, Spain4;Center for Infection and Immunity, Columbia University, New York, New York5; and Centro Nacional de

Epidemiología, Instituto de Salud Carlos III, Madrid, Madrid, Spain6

Received 7 December 2009/Returned for modification 14 December 2009/Accepted 19 January 2010

Although the WHO recommends the use of genotyping as a tool for epidemiological surveillance for mumps,limited data on mumps virus (MV) genotype circulation that may be used to trace the patterns of virus spread areavailable. We describe the first complete series of data from Spain. The small hydrophobic region was sequencedfrom 237 MV-positive samples from several regions of Spain collected between 1996 and 2007. Six differentgenotypes were identified: A, C, D (D1), G (G1, G2), H (H1, H2), and J. Genotype H1 was predominant during theepidemic that occurred from 1999 to 2003 but was replaced by genotype G1 as the dominant genotype in theepidemic that occurred from 2005 to 2007. The same genotype G1 strain caused concomitant outbreaks in differentparts of the world (the United States, Canada, and the United Kingdom). The remaining genotypes (genotypes A,C, D, and J) appeared in sporadic cases or small limited outbreaks. This pattern of circulation seems to reflectcontinuous viral circulation at the national level, despite the high rates of vaccine coverage.

Mumps is a highly transmissible but usually benign diseaseconsisting of bilateral swelling of the salivary glands. In someinstances, though, clinical complications can arise. Bilateralorchitis and clinical self-limited meningitis or more seriouscomplications, such as encephalitis, deafness, male sexual ste-rility, and pancreatitis, may occur in rare cases (9).

Mumps virus (MV), a virus belonging to the genus Rubula-virus of the subfamily Paramyxovirinae, is considered mono-typic regarding its antigenicity. Thus, mumps vaccination ispart of the regular immunization schedule of many countries,usually along with the measles and rubella vaccination (i.e., theMMR vaccine) in a single formulation. However, in contrast torubella and measles, secondary vaccine failure frequently al-lows MV circulation within highly immunized populations (1,5, 8, 24, 36). The use of a poorly immunogenic genotype Astrain called Rubini has been proposed as a cause of thesemajor failures, although the occurrence of mumps in patientsimmunized with other vaccine strains has also been described(26).

Genetic variation in the small hydrophobic (SH) gene hasled to the characterization of 12 genotypes, which are recog-nized by the WHO (13, 14, 23). Differential efficiency on crossneutralization among different genotypes has been suggested(20, 21), as have the differential capacities of certain strains toinvade the neural system (25, 32). Finally, previous experiencewith the elimination programs for other preventable viral dis-eases, such as measles, rubella, and polio, suggest that geno-

typing would facilitate mumps surveillance (38), since the pat-tern of viral circulation can be traced. Consequently, molecularepidemiology studies have been performed around the world(2, 7, 10, 11, 12, 15, 17, 18, 23, 31, 32, 33, 34, 35, 41).

Mumps vaccination was introduced in the Spanish nationalvaccination repertoire in 1981 as part of the triple viral vaccine(the MMR vaccine). As a result, the number of mumps casesfell from 286,887 in 1984 to 1,527 in 2004. However, the gen-eral descendant trend was interrupted by some peaks: in 1989(from 48,393 in 1987 to 83,527 in 1989), 1996 (from 7,002 in1995 to 14,411 in 1996), 2000 (from 2,857 in 1998 to 9,391 in2000), and 2007 (from 1,527 in 2004 to 10,219 in 2007). Sur-prisingly, these peaks occurred despite a national vaccine cov-erage rate of over 95% of the population by 1999. As has beenpreviously reported (29, 30), the occurrence of vaccinated in-dividuals with MV RNA present in their saliva and/or urinewith viral IgG but not IgM in acute-phase serum was frequent,suggesting secondary vaccine failure. Although most secondaryvaccine failures were associated with the use of at least onedose of the Rubini strain, cases of mumps in patients vacci-nated with two doses of the Jerryl Lynn strain were also ob-served (29, 30).

In this report, we describe the MV genotypes circulating inSpain over the past 8 years. This represents the first series ofdata obtained for the national level. Interestingly, this seriescomprises three different epidemic peaks with two interepi-demic periods with scarce viral circulation, allowing compari-son of the pattern of genotype circulation under different ep-idemiological conditions.

MATERIALS AND METHODS

Samples. Two hundred thirty-seven samples positive for MV RNA by reversetranscription-PCR (RT-PCR) (see below) were included in the study and com-prised the following three sets of samples. Set 1 consisted of 224 samples (202

* Corresponding author. Mailing address: Unidad de Aislamiento yDeteccion de Virus, Servicio de Microbiología Diagnostica, CentroNacional de Microbiología, Instituto de Salud Carlos III, Ctra. Maja-dahonda-Pozuelo s/n, Majadahonda 28220, Madrid, Spain. Phone: 3491 822 3676. Fax: 34 91 509 7919. E-mail: [email protected].

� Published ahead of print on 27 January 2010.

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saliva, 5 urine, 4 spinal fluid, and 13 oropharyngeal swab specimens in viraltransport medium) that were received at the National Center for Microbiology(CNM) for the diagnosis of MV infection from 2000 to 2007 (Fig. 1). Themajority of the samples collected between 2000 and 2003 originated from theautonomous community of Madrid (51 of 74 cases) in central Spain. After 2004,sampling was extended to other autonomous communities. Two aliquots of 100�l each were made in the Sample Reception Unit upon arrival at CNM and werestored to �80°C until they were processed in the laboratory. Set 2 consisted of10 virus isolates selected from a collection of 68 MV strains obtained at the GranCanaria General Hospital Dr. Negrín, Gran Canaria Island (Canary Islands),Spain, from 1998 to 2000. These strains were obtained from the spinal fluid ofpatients with MV-associated meningitis. Set 3 consisted of 3 samples (2 spinalfluid samples and 1 serum sample) obtained from a retrospective panel (1988 to1999) of 243 MV IgM-positive serum specimens and 21 spinal fluid specimensfrom patients with MV-associated meningitis. These specimens had been storedat �20°C for serological studies and had been thawed and frozen an unknownnumber of times.

Nucleic acid extraction. Total nucleic acids were extracted from 100 �l of theoriginal specimen with a Magna Pure LC total nucleic acid isolation kit in aMagna Pure LC automatic extractor (Roche Diagnostics, Mannheim, Germany).Nucleic acids were resuspended in a final elution volume of 50 �l. Ten thousandmolecules of synthetic RNA were included in the lysis solution as an internalcontrol (6).

Amplification and sequencing methods. Initial screening of sets 1 and 2 wasperformed by a previously described RT-PCR method, but we used a slightmodification (22). The modification consisted of performance of the reversetranscription and the first amplification in a single step by means of the AccessRT-PCR system kit (Promega Co., Madison, WI). Genotyping of the MV-positive samples was done by partial sequencing of the SH gene (23). Set 3 wasstudied directly by the genotyping RT-PCR described previously (23).

Sequence analysis. Sequences were assigned to a given genotype by compar-ison with the sequences of reference strains (13). Subgrouping within genotypesH, G, and D was performed as described by Palacios et al. (23). The sequenceswere aligned by the use of Clustal X software and were analyzed by the MEGA(version 2.1) program. Phylogenetic trees were obtained by using the neighbor-joining and the Kimura two-parameter model of substitution with 1,000 boot-strap replications. Additional analysis by Bayesian inference with MrBayes (ver-sion 3.1) software improved the strength of the genotype assignation, whennecessary (see below). When discrepancies with published data arose, the wholeSH gene was sequenced to apply the reference criteria for genotype definition onthe basis of simple homology (13).

Nucleotide sequence accession numbers. The GenBank accession numbers ofthe nucleotide sequences obtained in this study are found in Table 1.

RESULTS

Genotype assignation of sequences obtained from samples.Six different MV genotypes (A, C, D, G, H, and J) wereidentified to have been circulating in Spain. Assignment togenotypes C, G, and H was based on low significant bootstrapvalues in the initial neighbor-joining phylogenetic analysis.Subsequent analysis by Bayesian inference clustered every se-quence to a known genotype with significant bootstrappingvalues (Fig. 2).

Of the 237 sequences obtained, 134 were identical sequencesobtained from specimens collected between 2005 and 2007(and represented in the tree as strain 804O05) and were clas-sified as genotype G. These sequences were also identical tosequences obtained from mumps outbreaks in the UnitedStates and Canada in 2006 (37) and to the previously publishedsequence Ast/SP07. While the North American sequences hadbeen characterized as genotype G, the sample collected inAsturias, Spain, in 2007 was proposed to be part of a newgenotype whose reference strain would be strain UNK02-19(4). To resolve this discrepancy, the entire SH gene (GenBankaccession no. AM766002) was sequenced. Strain 804O05 wasgreater than 97.5% homologous with both genotype G refer-ence strains but less than 92.7% homologous with the refer-ence strains of the other genotypes as well as 92.4% homolo-gous with strain UNK02-19. According to these data andfollowing the criterion of the need for 95% homology in the

TABLE 1. Sequences described in this work

Sequencename

GenBankaccession

no.

No. ofsequencesa Source location Yr of

recovery Genotype

2485NE FJ919343 1 Pontevedra 2005 G11501O FJ919344 2 Almerıa 2005 G12006O FJ919345 2 Logrono 2006 G1804O FJ919346 134 Almerıa 2005 G11028O FJ919347 2 Almerıa 2005 G1772O FJ919348 1 Caceres 2007 G12393O FJ919349 1 Segovia 2007 G13736O FJ919350 2 Logrono 2006 G11459O FJ919351 2 Madrid 2005 G12384O FJ919352 1 Madrid 2007 G2591O FJ919353 13 Madrid 2005 H21337O FJ919354 1 Madrid 2005 H22104O FJ919355 31 Madrid 2000 H18Canarias FJ919356 1 Las Palmas de

Gran Canaria2000 H1

1295O FJ919357 1 Palencia 2002 H110Canarias FJ919358 1 Las Palmas de

Gran Canaria2000 H1

556O FJ919359 4 Madrid 2001 H1646O FJ919360 1 Madrid 2001 H11269O FJ919361 10 Madrid 2000 H1857NE FJ919362 2 Ferrol 1996 H11290O FJ919363 1 Madrid 2005 H1985O FJ919364 8 Almerıa 2000 D19820 FJ919365 1 Madrid 2001 D11818O FJ919366 2 Ceuta 2007 D11913O FJ919367 2 Ceuta 2007 D1878O FJ919368 3 Murcia 2006 C1610NE FJ919369 1 Alicante 1999 C2780O FJ919370 3 Madrid 2003 J167O FJ919371 2 Madrid 2001 A

a The number of identical sequences represented by this accession number.

FIG. 1. Circulation of different mumps virus genotypes in Spain inrelation to the clinical declaration of mumps to the mandatory infor-mation system. Both the sequences obtained in this study and thesequences published previously were included: 4 sequences from ref-erence 23, 10 sequences from reference 18, 1 sequences from reference4, and 8 sequences from reference 27.

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FIG. 2. Phylogenetic relationships among mumps virus genotypes. The phylogenetic tree was obtained by Bayesian inference.

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FIG. 3. Phylogenetic relationships among genotype H strains. Œ, reference strains; F, strains detected during this study.

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whole SH gene (13, 38) to be able to assign a sequence to apreexisting genotype, both Ast/SP07 and our sequences of thesame cluster belonged to genotype G.

Genotype circulation. The different MV genotypes identifiedin this study are listed in Fig. 1. The results obtained before theyear 1999 are based entirely on the results for 6 samples(0.024% of the 24,263 declared samples). A greater than 10-fold increase in the percentage of genotyped cases occurredduring the epidemic between 1999 and 2004 (0.3%, 74/24,767samples) due to enforced sampling in Madrid and the CanaryIslands and even more in the last epidemic wave from 2005 to2007, when most of the Spanish territory was covered, reaching0.94% of mumps cases identified at the national level (181 of19,237 cases; Fig. 1).

Genotype H1 was clearly dominant during the epidemicwave of 1999 to 2004. This genotype was also found in the fewsamples available from 1996 to 1998. The cocirculation ofgenotype D1 (9/43, 20.9%) together with the major genotype,genotype H1 (32/43, 74.4%), was regularly observed in Madridduring 2000 and 2001, as had been previously reported in SanSebastian (Basque Country; northern Spain) (18). However,genotype D1 was not found among the 10 strains identifiedfrom the Canary Islands. No associations between differentepidemiological factors (age, gender, geographical area, vac-cine status) and the presence of genotype H1 or D1 were foundamong the cases from Madrid (data not shown). The circula-tion of genotype H1 as the major genotype seemed to subsidein 2004; coincidentally, the lowest number of cases in the serieswas recorded in that year (Fig. 1). Spanish strains clusteredwithin the genotype H1 branch (Fig. 3) with other strains fromSwitzerland, the United Kingdom, and Argentina recovered inthe same years, as well as with a group of sequences fromBelarus (3). Genotype D1 strains were the most similar tothose from an outbreak in Portugal in 1996 within a biggergenotype D1 group containing contemporary strains fromother countries in Europe (Fig. 4). A single sequence of geno-type C detected in 1999 was similar to the sequences of a groupof strains circulating in Switzerland at that time (Fig. 5). Twocases with the same genotype A sequence were also detected inMadrid in January and February of 2001, together with geno-types H1 and D1. The comparison of this sequence with othergenotype A sequences showed striking differences from thesequences of both vaccine and wild-type strains (Table 2). Thepositive controls used in the laboratory were also included torule out laboratory contamination. According to these data,these two sequences seem to be those of new wild-type strainsof genotype A. Both patients had received a single dose ofvaccine 5 and 8 years before the mumps episode, respectively,and their vaccination records did not show that they had re-ceived further vaccinations. Further investigation is needed forthe definitive classification of this strain, since the circulationof wild-type strains of genotype A has not been reported inrecent years.

The number of notifications started to increase again in 2005due to simultaneous outbreaks caused by genotype H2 strainsin Segovia (Castilla-Leon, central Spain) and genotype G1strains in Almería (Andalusia, southern Spain) from May toJuly, causing concomitant individual cases in Madrid togetherwith others caused by genotype J (Fig. 1). These genotype H2strains clustered with a group of sequences from Israel and

Palestine collected in 2004 (Fig. 3). A single genotype G1strain detected in an encephalitis patient from Pontevedra(Galicia, northwestern Spain) in October 2005 was the onlyavailable representative of that genotype from the hundreds ofcases during that outbreak. In 2006, genotype G1 spreadthroughout the Spanish territory. At the time of this work’ssubmission, genotype G1 was still the dominant circulatinggenotype, as demonstrated by reports from Asturias (4) andValencia (27). Since 2006, only three cases of MV genotype Cinfections in Murcia (southeastern Spain), a single case of anMV genotype D1 infection in the North African city of Ceuta,Morocco, and an isolated case of a genotype G2 infection fromMadrid have been detected. The dominant genotype G1 strainwas very similar to those causing large outbreaks in the UnitedStates and Canada, as well as to a single strain from Croatia(28, 37) (Fig. 6).

DISCUSSION

Genotyping is a basic tool used for epidemiological surveil-lance of vaccine-preventable viral diseases. In order to providethese surveillance programs with universal tools enablingworldwide surveillance, the WHO has established standardgenotype nomenclature, reference strains, and target genomicsequences for the viruses included in the triple viral MMRvaccine: measles virus (39), mumps virus (13, 38), and rubellavirus (40). Data from as many countries as possible are re-quired to understand the epidemiological significance of geno-typing data. However, in the case of the mumps virus, theamount of available data is far from optimal. In this work, wecontribute the first series of data covering a period of 10 yearson the MV genotypes in general circulation in Spain.

Previous available data on local outbreaks in San Sebastian(18), Valencia (27), and Asturias (4) concur with our data. Thedominance of a single genotype at the national level was asso-ciated with periods of reporting of high rates of mumps to thenational surveillance system.

In this series, three epidemic waves of mumps cases wereobserved. Interestingly, dominant genotype replacement oc-curred after mumps reporting decreased to minimum levels.Genotype H1 was dominant between 1999 and 2003 but wassuccessfully replaced by G1 after a “silent” period of 2 yearswith scarce mumps circulation. On the other hand, althoughdata from 1996 to 1998 are limited, the genotype H1 strain wasalso detected during that period, suggesting that the end of anepidemic wave was not always followed by a genotype replace-ment. Although a temporal shift in MV genotype circulationwas described previously (10, 11, 12, 16, 26, 33), we believe thatthis is the first report establishing a link between cyclic varia-tions, genotype, and mumps prevalence.

The pattern observed during the epidemic waves (the circu-lation of a dominant genotype) seems to reflect continuousviral circulation, in accordance with the clinical data. Eventhough the rates of vaccine coverage were identically high formeasles and mumps viruses, the measles virus showed a dif-ferent pattern (e.g., a variety of genotypes associated withunrelated local outbreaks) (19). The latter pattern correlateswith the interruption of measles virus circulation at the na-tional level, as reflected by case reports (19). These resultshighlight the relatively low efficiency of the mumps vaccine in

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FIG. 4. Phylogenetic relationships among genotype D strains. Œ, reference strains; F, strains detected during this study.

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FIG. 5. Phylogenetic relationships among genotype C strains. Œ, reference strains; F, strains detected during this study.


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comparison with that of the measles vaccine. The use of theRubini strain in Spain between 1993 and 1999 for mumpsvaccination could be an important factor to explain this lowefficiency.

Interestingly, genotype H2 strains were not able to establishcirculation at the national level, even though they caused alocal outbreak in Segovia that radiated cases to Madrid. How-

ever, genotype G1 succeeded in spreading shortly after that.The same G1 strain was also reported from many other partsof the world (28, 37), suggesting an enhanced ability to spreadwithin vaccinated populations. Similarly, another genotype, ge-notype D1, was able to establish circulation concomitantly withthe dominant genotype, genotype H1, during 2000 and 2001, asreported in Switzerland (35). The selective forces that led tothe extinction of circulating genotypes and caused the furtheronset of particular strains are unknown. The differential abilityof vaccine-induced antibodies to neutralize different MV ge-notypes suggested by some authors (21) might account for partof the explanation for these positive selection events; we be-lieve that our data support this theory and that they thuswarrant additional investigation. The different histories of ge-notype importation, the variations in vaccine coverage rates,and the use of different vaccine strains in each country draw acomplex global picture that could be the cause of the differentgeographical patterns of mumps virus genotype circulation ob-served in the United Kingdom (12), Japan (31), and Switzer-land (35).

ACKNOWLEDGMENTS

We thank the Genomics Unit of the Instituto de Salud Carlos III forcarrying out all the automatic sequencing. We also thank the techni-cians of the Viral Detection and Isolation Unit (Service of DiagnosticMicrobiology) of the National Center of Microbiology for their assis-tance and especially Irene Gonzalez for the nucleic acid extractionsand Francisco Salvador for the preparation of the PCR master mix-tures for diagnosis. We also thank the staff of the Sample ReceptionUnit of the National Center of Microbiology for their work on sampleidentification and preparation. We also thank Nazir Savji from theCenter for Infection and Immunity for his help with editing the manu-script.

This study has been partially supported by project MPY1190/02 ofthe research program of the Carlos III Health Institute.

REFERENCES

1. Afzal, M. A., J. Buchanan, J. A. Dias, M. Cordeiro, M. L. Bentley, C. A.Shorrock, and P. D. Minor. 1997. RT-PCR based diagnosis and molecularcharacterisation of mumps viruses derived from clinical specimens collectedduring the 1996 mumps outbreak in Portugal. J. Med. Virol. 52:349–353.

2. Atrasheuskaya, A. V., M. V. Kulak, S. Rubin, and G. M. Ignatyev. 2007.Mumps vaccine failure investigation in Novosibirsk, Russia, 2002–2004. Clin.Microbiol. Infect. 13:670–676.

3. Atrasheuskaya, A. V., E. M. Blatun, M. V. Kulak, A. Atrasheuskaya, I. A.Karpov, S. Rubin, and G. M. Ignatyev. 2007. Investigation of mumps vaccinefailures in Minsk, Belarus, 2001–2003. Vaccine 25:4651–4658.

4. Boga, J. A., M. de Ona, A. Fernandez-Verdugo, D. Gonzalez, A. Morilla, M.Arias, L. Barreiro, F. Hidalgo, and S. Melon. 2008. Molecular identificationof two genotypes of mumps virus causing two regional outbreaks in Asturias,Spain. J. Clin. Virol. 42:425–428.

5. Briss, P. A., L. J. Fehrs, R. A. Parker, P. F. Wright, E. C. Sannella, R. H.Hutcheson, and W. Schaffner. 1994. Sustained transmission of mumps in ahighly vaccinated population: assessment of primary vaccine failure andwaning vaccine-induced immunity. J. Infect. Dis. 169:77–82.

6. Echevarría, J. E., A. Avellon, J. Juste, M. Vera, and C. Ibanez. 2001. Screen-ing of active lyssavirus infection in wild bat populations by viral RNA onoropharyngeal swabs. J. Clin. Microbiol. 39:3678–3683.

7. Heider, A., G. Ignatyev, E. Gerike, and E. Schreier. 1997. Genotypic char-acterization of mumps virus isolated in Russia (Siberia). Res. Virol. 148:433–435.

8. Hersh, B. S., P. E. M. Fine, W. K. Kent, S. L. Cochi, L. H. Khan, E. R. Zell,et al. 1991. Mumps outbreak in a highly vaccinated population. J. Pediatr.119:187–193.

9. Hviid, A., S. Rubin, and K. Muhlemann. 2008. Mumps. Lancet 371:932–944.10. Inou, Y., T. Nakayama, N. Yoshida, H. Uejima, K. Yuri, M. Kamada, T.

Kumagai, H. Sakiyama, A. Miyata, H. Ochiai, T. Ihara, T. Okafuji, T.Okafuji, T. Nagai, E. Suzuki, K. Shimomura, Y. Ito, and C. Miyazaki. 2004.Molecular epidemiology of mumps virus in Japan and proposal of two newgenotypes. J. Med. Virol. 73:97–104.

11. Jin, L., S. Beard, and D. W. Brown. 1999. Genetic heterogeneity of mumps

FIG. 6. Phylogenetic relationships among genotype G strains. Œ,reference strains; F, strains detected during this study.


at CO

LUM

BIA

UN

IVE

RS

ITY

on May 13, 2010

jcm.asm

.orgD

ownloaded from

http://jcm.asm.org

virus in the United Kingdom: identification of two new genotypes. J. Infect.Dis. 180:829–833.

12. Jin, L., D. W. Brown, P. A. Litton, and J. M. White. 2004. Genetic diversityof mumps virus in oral fluid specimens: application to mumps epidemiolog-ical study. J. Infect. Dis. 189:1001–1008.

13. Jin, L., B. Rima, D. Brown, C. Orvell, T. Tecle, M. Afzal, K. Uchida, T.Nakayama, J. W. Song, C. Kang, P. A. Rota, W. Xu, and D. Featherstone.2005. Proposal for genetic characterisation of wild-type mumps strains: pre-liminary standardisation of the nomenclature. Arch. Virol. 150:1903–1909.

14. Johansson, B., T. Tecle, and C. Orvell. 2002. Proposed criteria for classifi-cation of new genotypes of mumps virus. Scand. J. Infect. Dis. 34:355–357.

15. Kim, S. H., K. J. Song, Y. K. Shin, J. H. Kim, S. M. Choi, K. S. Park, L. J.Baek, Y. J. Lee, and J. W. Song. 2000. Phylogenetic analysis of the smallhydrophobic (SH) gene of mumps virus in Korea: identification of a newgenotype. Microbiol. Immunol. 44:173–177.

16. Lee, J. Y., Y. Y. Kim, G. C. Shin, B. K. Na, J. S. Lee, H. D. Lee, J. H. Kim,W. J. Kim, J. Kim, C. Kang, and H. W. Cho. 2003. Molecular characteriza-tion of two genotypes of mumps virus circulated in Korea during 1998–2001.Virus Res. 97:111–116.

17. Lee, J. Y., B. K. Na, J. H. Kim, J. S. Lee, J. W. Park, G. C. Shin, H. W. Cho,H. D. Lee, U. Y. Gou, B. K. Yang, J. Kim, C. Kang, and W. J. Kim. 2004.Regional outbreak of mumps due to genotype H in Korea in 1999. J. Med.Virol. 73:85–90.

18. Montes, M., G. Cilla, J. Artieda, D. Vicente, and M. Basterretxea. 2002.Mumps outbreak in vaccinated children in Gipuzkoa (Basque Country),Spain. Epidemiol. Infect. 129:551–556.

19. Mosquera, M., F. de Ory, J. E. Echevarría, and the Network of Laboratoriesin the Spanish National Measles Elimination Plan. 2005. Measles virusgenotype circulation in Spain after implementation of the National MeaslesElimination Plan 2001–2003. J. Med. Virol. 75:137–146.

20. Nojd, J., T. Tecle, A. Samuelsson, and C. Orvell. 2001. Mumps virus neu-tralizing antibodies do not protect against reinfection with a heterologousmumps virus genotype. Vaccine 19:1727–1731.

21. Orvell, C., T. Tecle, B. Johansson, H. Saito, and A. Samuelson. 2002. Anti-genic relationships between six genotypes of the small hydrophobic proteingene of mumps virus. J. Gen. Virol. 83:2489–2496.

22. Palacios, G., C. Rodriguez, D. Cisterna, M. C. Freire, and J. Cello. 2000.Nested PCR for rapid detection of mumps virus in cerebrospinal fluid frompatients with neurological diseases. J. Clin. Microbiol. 38:274–278.

23. Palacios, G., O. Jabado, D. Cisterna, F. de Ory, N. Renwick, J. E. Echevar-ria, A. Castellanos, M. Mosquera, M. C. Freire, R. H. Campos, and W. I.Lipkin. 2005. Molecular identification of mumps virus genotypes from clin-ical samples: standardized method of analysis. J. Clin. Microbiol. 43:1869–1878.

24. Pons, C., T. Pelayo, I. Pachon, A. Galmes, L. Gonzalez, G. Sanchez, and F.Martinez. 2000. Two outbreaks of mumps in children vaccinated with theRubini strain in Spain indicate low vaccine efficacy. Euro Surveill. 5:80–84.

25. Rafiefard, F., B. Johansson, T. Tecle, and C. Orvell. 2005. Characterizationof mumps virus strains with varying neurovirulence. Scand. J. Infect. Dis.37:330–337.

26. Richard, J. L., M. Zwahlen, M. Feuz, and M. C. Matter. 2003. Comparison

of the effectiveness of two mumps vaccines during an outbreak in Switzer-land in 1999 and 2000: a case-cohort study. Eur. J. Epidemiol. 18:569–577.

27. Roig, F. J., M. A. Bracho, R. Caebo, E. Giner, F. Gonzalez, S. Guiral, C.Monedero, and A. Salazar. 2006. Brote epidemico por virus de la parotiditisgenotipo G1. Bol. Epidemiol. Sem. 14:25–36. http://www.isciii.es/htdocs/centros /epidemiologia/boletin_semanal/bes0606.pdf.

28. Santak, M., T. Kosutic-Gulija, G. Tesovic, S. Ljubin-Sternak, I. Gjenero-Margan, L. Betica-Radic, and D. Forcic. 2006. Mumps virus strains isolatedin Croatia in 1998 and 2005: genotyping and putative antigenic relatedness tovaccine strains. J. Med. Virol. 78:638–643.

29. Sanz, J. C., M. Mosquera, J. E. Echevarría, M. Fernandez, N. Herranz, G.Palacios, and F. de Ory. 2006. Sensitivity and specificity of immunoglobulinG titer for diagnostics of mumps virus in infected patients depending onvaccination status. APMIS 114:788–794.

30. Sanz-Moreno, J. C., A. Limia-Sanchez, L. García-Comas, M. M. Mosquera-Gutierrez, J. E. Echevarría-Mayo, A. Castellanos-Nadal, and F. de Ory-Manchon. 2005. Detection of secondary mumps vaccine failure by means ofavidity testing for specific immunoglobulin G. Vaccine 23:4921–4925.

31. Takahashi, M., T. Nakayama, Y. Kashiwagi, T. Takami, S. Sonoda, T. Ya-manaka, H. Ochiai, T. Ihara, and T. Tajima. 2000. Single genotype ofmeasles virus is dominant whereas several genotypes of mumps virus areco-circulating. J. Med. Virol. 62:278–285.

32. Tecle, T., B. Johansson, A. Jejcic, M. Forsgren, and C. Orvell. 1998. Char-acterization of three co-circulating genotypes of the small hydrophobic pro-tein gene of mumps virus. J. Gen. Virol. 79:2929–2937.

33. Tecle, T., B. Bottiger, C. Orvell, and B. Johansson. 2001. Characterization oftwo decades of temporal co-circulation of four mumps virus genotypes inDenmark: identification of a new genotype. J. Gen. Virol. 82:2675–2680.

34. Uchida, K., M. Shinohara, S. Shimada, Y. Segawa, and Y. Hoshino. 2001.Characterization of mumps virus isolated in Saitama Prefecture, Japan, bysequence analysis of the SH gene. Microbiol. Immunol. 45:851–855.

35. Utz, S., J. L. Richard, S. Capaul, H. C. Matter, M. G. Hrisoho, and K.Muhlemann. 2004. Phylogenetic analysis of clinical mumps virus isolatesfrom vaccinated and non-vaccinated patients with mumps during an out-break, Switzerland 1998–2000. J. Med. Virol. 73:91–96.

36. Vandermeulen, C., M. Roelants, M. Vermoere, K. Roseeuw, P. Goubau, andK. Hoppenbrouwers. 2004. Outbreak of mumps in a vaccinated child popu-lation: a question of vaccine failure? Vaccine 22:2713–2716.

37. Watson-Creed, G., A. Saunders, J. Scott, L. Lowe, J. Pettipas, and T. Hat-tchete. 2006. Two successive outbreaks of mumps in Nova Scotia amongvaccinated adolescents and young adults. Can. Med. Assoc. J. 175:484–488.

38. WHO. 2005. Global status of mumps immunization and surveillance. Wkly.Epidemiol. Rec. 48:418–424.

39. WHO. 2005. New genotype of measles virus and update on global distribu-tion of measles genotypes. Wkly. Epidemiol. Rec. 80:347–351.

40. WHO. 2007. Update of standard nomenclature for wild type rubella viruses,2007. Wkly. Epidemiol. Rec. 82:216–222.

41. Wu, L., Z. Bai, Y. Li, B. K. Rima, and M. A. Afzal. 1998. Wild type mumpsviruses circulating in China establish a new genotype. Vaccine 16:281–285.(Erratum, 16:759.)


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Circulation of Mumps Virus Genotypes in Spain from 1996 to