Characterization of two decades of temporal co-circulation of four mumps virus genotypes in Denmark: identification of a new genotype

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1 Journal of General Virology (2001), 82, Printed in Great Britain... SHORT COMMUNICATION Characterization of two decades of temporal co-circulation of four mumps virus genotypes in Denmark: identification of a new genotype Tesfaldet Tecle, 1 Blenda Bo ttiger, 2 Claes O rvell 1 and Bo Johansson 1 1 Huddinge University Hospital, Department of Clinical Virology, Karolinska Institut, SE Stockholm, Sweden 2 Statens Serum Institut, Department of Virology, Copenhagen, Denmark Twenty-nine Danish virus isolates and 14 serum samples from patients with mumps were genotyped by nucleotide sequencing of the small hydrophobic (SH) protein gene and the deduced 57 amino acid sequences were aligned with sequences of mumps virus strains published previously. Four neurovirulent genotypes of the SH protein gene, genotypes C, D, H and a new genotype, designated J, were found. There was a dynamic fluctuation of the different genotypes over the two decade period of time. Genotype J was found from 1981 to 1988; genotypes C and H exhibited a similar distribution in time. Genotype D was found between 1979 and 1982, it then disappeared and reappeared again in From 1996 onwards, genotype D was found to be the predominant genotype, which is in contrast to the situation seen in the neighbouring country of Sweden, where, since 1985, only genotype A has been found. The human pathogen mumps virus is classified as a member of the Paramyxoviridae family, Rubulavirus genus (Rima et al., 1995). The virus genome is a single-stranded negative-sense RNA, which contains seven genes in the following order on the genome map: the nucleocapsid (N), phospho (P), membrane (M), fusion (F), small hydrophobic (SH), haemagglutinin neuraminidase (HN) and large (L) protein genes (Elango et al., 1988; Elliott et al., 1989). The SH gene is the most variable part of the mumps virus genome. Its mrna is 310 nucleotides in length and encodes a viral membrane protein of 57 amino acids (Elango et al., 1989; Elliott et al., 1989; Takeuchi et al., 1996). The SH protein has been identified immunologically but its function is unknown. It is believed to represent a non-structural protein (Takeuchiet al., 1996). Phylogenetic comparison of the Author for correspondence: Claes O rvell. Fax clor labd01.hs.sll.se SH gene from different virus isolates from around the world have shown the existence of nine genotypes, designated A I (Wu et al., 1998; Jin et al., 1999; Kim et al., 2000). Most of the virus strains belonging to the recently discovered genotypes G and H were isolated in the UK from 1995 to 1998, whereas genotype I was isolated during a mumps outbreak in Korea in 1998 (Jin et al., 1999; Kim et al., 2000). Different virus genotypes have been shown to co-circulate in the same country (Afzal et al., 1997a, b; Tecle et al., 1998; Takahashi et al., 2000) and the distribution of genotypes may vary even between closely located regions within the same country (Takahashi et al., 2000). Different mumps virus strains exhibit varying degrees of neurovirulence (Merz & Wolinsky, 1981; Saito et al., 1996; Tecle et al., 1998; Rubin et al., 1998, 2000). In Sweden, the relatively non-neurovirulent SBL-1 strain is the dominating strain (Tecle et al., 1998). In the present study, the circulation of mumps virus genotypes in Denmark was studied to see if there existed a different epidemiological situation compared to Sweden. A total of 29 mumps virus strains isolated from the cerebrospinal fluid (CSF) from patients with mumps collected between 1979 and 1989 from different parts of Denmark was studied (Table 1). After 1989, no mumps virus strains have been recovered at the State Serum Institute in Copenhagen. A total of 14 serum samples collected between 1996 and 1999 from patients with mumps was used directly for genotyping (Table 1). All serum samples were demonstrated by serology to contain IgM antibodies against mumps virus. Background clinical information was collected. Age of patients and symptoms were recorded where this information was available. Isolation of mumps virus RNA from the different samples was performed using the QIAamp RNA mini kit (Qiagen), as described by the manufacturer. The procedures for PCR amplification and DNA sequencing of the SH gene have been described previously (O rvell et al., 1997a, b). Eight of the 29 mumps virus strains isolated from the CSF of patients with mumps were found to contain a previously undetected genotype of mumps virus. Genotype J, the new genotype, was found in strains collected between 1981 and SGM CGHF

2 T. Tecle and others Table 1. Mumps virus samples Samples are arranged by year of collection and sequential number of the sample collected in the same year (for example, DK 79 01). NK, not known; E, encephalitis; M, meningitis; P, parotitis; CSF, cerebrospinal fluid. Sample no. Genotype Source Age of patient (years) Clinical symptoms DK D CSF NK NK DK D CSF NK NK DK D CSF NK NK DK D CSF NK NK DK D CSF 5 P, M DK D CSF 6 M DK D CSF 60 M DK C CSF 5 M DK J CSF NK NK DK C CSF 20 M DK D CSF NK NK DK H CSF NK NK DK D CSF 6 P, M DK C CSF NK NK DK J CSF 5 M DK C CSF NK NK DK D CSF NK NK DK C CSF 13 M DK H CSF 13 M DK H CSF 6 M DK J CSF 7 M DK J CSF 4 M DK J CSF 6 M DK J CSF 1 M DK C CSF 27 P, M DK J CSF NK NK DK H CSF NK NK DK J CSF NK NK DK C CSF NK NK DK D Serum 13 NK DK D Serum 37 NK DK D Serum 27 P DK C Serum 59 NK DK D Serum 68 P DK D Serum 35 NK DK D Serum 29 NK DK D Serum 40 NK DK D Serum 49 NK DK D Serum 26 NK DK D Serum 25 NK DK D Serum 31 NK DK D Serum 22 NK DK D Serum 49 P 1988 (Table 1). By nucleotide distance analysis, virus isolates of the new genotype were separated from the genotypes of mumps virus recognized previously. (Fig. 1). By analogy with the established nomenclature for mumps virus genotypes, the new genotype was designated genotype J. From the 43 samples that were genotyped, none was found to belong to genotypes A, B, E, F, G or I. Eight samples were found to belong to genotype C, 23 to genotype D, four to genotype H and eight to genotype J. The distribution of the different virus genotypes are listed in Table 1. A relative homogeneity with the dominating genotype D was found in the years 1979 and 1980 (seven genotype D strains and one genotype C strain) and 1996 to 1999 (13 genotype D strains and one genotype C strain). In the time interval between 1981 and 1989, a pronounced heterogeneity was found. In 1982, all four virus genotypes were identified and, in 1983, three of the four genotypes were found. In other years (1984, 1985, 1988 and 1989), the number of samples was too small to allow the detection of more than two different genotypes in the same year. Clinical symptoms were recorded from 15 of 29 patients from whom mumps virus had been isolated. All four genotypes were associated with meningitis. Five patients with the new genotype J (DK and DK ) suffered from meningitis, which was their only symptom. Four, four and two patients with genotypes C, D and H, respectively, also suffered from meningitis and in three of them, DK (genotype D), DK (genotype D) and DK (genotype C), parotitis was also recorded. The nucleotide sequences of the SH gene of the 43 samples were reported to GenBank (accession nos AF AF365925). A nucleotide tree was constructed and the sequences were compared with those of mumps virus strains reported previously (Fig. 1). The different nucleotide sequences clustered to virus strains genotyped previously. The samples belonging to the new genotype J formed a distinct lineage of high homogeneity and the cluster was supported by 100% bootstrap values (Fig. 1). Three of the four genotype H strains grouped together, but one strain, DK 88 01, was divergent and was shown to be closely related to the Dev1 strain isolated in the UK in 1998 (Jin et al., 1999). When the ten different genotypes were compared, genotype A was relatively more distant from the other nine genotypes. The amino acid sequences deduced from a large number of SH genes from different parts of the world were compared (Fig. 2). Some genotypes, such as genotypes C, E, G, and J, showed a high degree of conservation, whereas a somewhat lower degree of conservation was found in others, such as genotypes A, B, D, F, H and I. The new genotype J contains the amino acid triplet TIS at positions and the amino acid L at position 6, characteristic of the genotype. A high degree of correlation between an amino acid triplet at positions and genotype has been reported (O rvell et al., 1997a; Tecle et al., 1998). The amino acid triplets TIL, IIS, SLS, IML, TIS at these positions were characteristic of genotypes A, B, E, F and J, respectively. Most strains of genotypes C and D were found to contain the amino acid triplets VVS and IIL, respectively. The amino acid triplet IIL was also found in genotypes G and I. The present study is the second one that deals with the topic of the circulation of different mumps virus genotypes in a country over a prolonged period of time. In Sweden, the CGHG

3 Identification of a new mumps virus genotype Fig. 1. Phylogenetic tree of prototype and different Danish mumps virus SH genotype sequences. Analysis was performed using the TREECON program (Van de Peer & DeWachter, 1994). Sequence distances were calculated from alignments using the Jukes Cantor method and clustering was performed by the neighbour-joining method. Evaluation of the robustness of the tree was performed by bootstrap analysis (100 trees). CGHH

4 T. Tecle and others Fig. 2. Alignment of deduced amino acid sequences of different genotypes of the mumps virus SH protein gene. Strains highlighted in bold (genotypes C, D, H and J) were obtained from the present work, whereas all other strains were obtained from GenBank. Proposed genotype amino acids at positions seem to provide a useful signature for rough genotyping, except for genotypes C and H. Genotypes D, G and I have the same central motif, but a distinction of genotype I from the others is possible by comparing the amino acid at position 20, which is M in genotype I, but L in genotypes D or G. distribution of mumps virus genotypes was studied from 1971 to 1997 (O rvell et al., 1997a; Tecle et al., 1998). Similarities and differences were found when the circulation of genotypes in Sweden and Denmark were compared. In the present study, four different genotypes, C, D, H and J, were co-circulating at certain time periods, whereas in the Swedish studies, genotypes A, C and D were found. During the years 1981 to 1988, four genotypes were found in Denmark and in one and the same CGHI

5 Identification of a new mumps virus genotype year, 1982, all four genotypes were identified. In contrast to this finding, in the time periods from 1979 to 1980 and 1996 to 1999, genotype D was dominating in Denmark, whereas genotypes A, B, E, F, G and I were not found. Genotype A has been identified in Sweden from 1971 to 1999 (Tecle et al., 1998). Genotype A has also been found in Germany in the years 1987 and Genotype B has been found among naturally circulating strains in Japan, but not in Europe (Afzal et al., 1997a; O rvell et al., 1997a; Takahashi et al., 2000). The authenticity of genotype E has been questioned (Jin et al., 1999; M. A. Afzal, personal communication). Genotype G has been found in the UK, Europe and Nepal (Jin et al., 1999) and genotype F has been described from China and Korea (Wu et al., 1998). Genotypes C and D were found in Denmark and Sweden. Genotype D was dominating in Sweden in the 1970s and in Denmark in 1979 and 1980; genotype D was found up to 1982 in the present study. In Danish virus isolates from 1983 to 1984, genotype D has been found (Afzal et al., 1997 a). It is possible, therefore, that genotype D disappeared from Denmark at about the same time as that in Sweden. A surprising difference was that genotype D is now present in Denmark, but not in Sweden. After 1989, no virus isolates from the CSF of patients with meningitis have been recovered in Denmark. This is similar to the situation in Stockholm, where mumps virus isolates were not recovered after 1985 (Tecle et al., 1998). Different strains of mumps virus have been shown to exhibit a variable degree of neurovirulence when tested in a neonatal rat model (Rubin et al., 1998, 2000). The Urabe strain of genotype B and the Lo1 strain of genotype D were found to be more neurovirulent than the Jeryl Lynn strain of genotype A. Also, in clinical situations, some virus strains have been reported to be more neuropathogenic than others (Saito et al., 1996; Tecle et al., 1998). In the study by Tecle et al. (1998), genotypes C and D were found to be more neuropathogenic than the SBL-1 strain of genotype A. In the present study, all four genotypes, C, D, H and J, were found to be neurovirulent, as they were isolated from the CSF of patients with meningitis. However, the relative degree of neurovirulence between them could not be determined. An interesting finding was that five patients with genotype J showed meningitis as their only symptom. Mumps virus outbreaks have been reported to occur in populations with high vaccine coverage (Wharton et al., 1988; Hersh et al., 1991; Briss et al., 1994; Ku nkel et al., 1994, 1995; Cheek et al., 1995; Germann et al., 1996; Afzal et al., 1997 b; Kim et al., 2000). An immunological difference between genotype A and genotype D has been demonstrated (O rvell, 1984; Yates et al., 1996; O rvell et al., 1997b;No jd et al., 2001). Neutralizing antibodies formed after infection with genotype D could not protect against reinfection with genotype A in a patient (No jd et al., 2001) and vaccination with genotype A did not protect against epidemics with either genotype C or genotype D (Ku nkel et al., 1994; Stro hle et al., 1996; Afzal et al., 1997b). It is intriguing that genotype A is dominating in Sweden, while genotype D is dominating in the neighbouring country of Denmark. One may speculate about the possibility that the immunity against mumps virus in the two countries may not be sufficient to protect against the invasion of a heterologous genotype. In 1996, an accumulation of mumps cases was observed in the south of Sweden, Ska ne, the area most closely located to Denmark. The virus causing this local epidemic has not yet been identified. 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