The mumps virus (MuV) causes a systemic illness spread by virus-containing droplets from the upper respiratory tract. Infection with mumps is asympto-matic in one-third of cases. The disease is generally self-limiting and usually characterized by parotitis and mild nonspecific symptoms, but it has the capacity to invade the visceral organs and central nervous system (CNS).
The mumps virus is a member of the Rubulavirus genus of the family Paramyxoviridae. The virus contains a negative-sense RNA genome of 15 384 nucleotides that consists of seven transcription units. The gene order is as follows: nucleoprotein (N), phosphoprotein (P), and matrix (M), fusion (F), small hydrophobic (SH), hemagglutinin-neuraminidase (HN), and large (L) proteins. The SH gene (57 amino acids) is the most variable segment of the MuV genome, with a missense to silent mutation ratio of 2.0 (cf. a ratio of <0.5 for other MuV genes). Phylogenetic analyses are, therefore, mainly based on the SH gene sequence. Sequence comparison based on the SH gene has led to the definition of distinct MuV genotypes (6, 11). As of 2007, 13 genotypes known as A-M had been described (12).
In China, mumps outbreaks due to the genotype F virus still occur (8, 17). Infection with genotype F mainly results in parotitis and rarely in meningitis. No isolates belonging to genotype F were found in cere-brospinal fluid (CSF) samples. Even though a definitive study cannot be performed due to the lack of neuro-pathogenic isolates in China, we speculate that geno-type F (including SP strain) is likely to cause a milder illness than meningitis. However, this genotype may exhibit neurovirulence in some cases. In this study, we determined the entire nucleotide sequence of a genotype F wild-type mumps virus isolated from a patient in ShiPing, China and compared it with those of previously reported strains.
Five throat swab samples were collected from patients with parotitis in ShiPing, China in March, 2006. Vero cells were cultured in five tissue culture flasks in 25cm2 of minimum essential medium (MEM) supplemented with 5% fetal calf serum (FCS). A 1 mL throat swab sample was inoculated into Vero cells. After contact for 1 hr at 37 ℃, samples were removed, and MEM supplemented with 2% FCS was added. Within three passages, samples that induced mumps virus-specific cytopathic effects (CPEs) were consi-dered to be positive for virus isolation. Samples that did not induce CPEs through three passages in Vero cells were considered to be negative. Supernatants from the inoculated wells showing CPEs were harvested. The supernatant was identified by a culture-neutralization test using the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medi-cal College（IMB）mumps virus-antisera. This proce-dure is the standard diagnostic currently used in China.
The 36 primers used in RT-PCR are shown in Table 1. Primers were newly designed based on the publi- shed sequence of the UrabeAM9 strain (GenBank ACC# AB000388, genotype B). Many primers were described in the previous reports (4, 6, 7).
Table 1. Primers used for RT-PCR and sequencing of mumps virus genome
RT-PCR, cloning, and sequencing Viral RNA was extracted from culture fluids using a Viral RNA Kit (Qiagen Inc., USA) according to the manufacturer's instructions. The RNA was reverse transcribed into first strand cDNA with the following primer pairs: 1F, 7F, 13F, 19F, 23F, and 29F. These primers covered the entire genome of the mumps virus (Revertaid TM first strand cDNA synthesis Kit). PCR reactions were carried out using Taq polymerase (TakaRa Dalian Co., Japan) and other essential reagents. The cycling condi-tions consisted of an initial denaturation at 94 ℃ for 5 min, 35 cycles of 94 ℃ for 30 s, 54 ℃ for 30 s, and 72 ℃ for 2 min, and a final extension step for 10 min at 72 ℃ in a thermal cycler. These amplified products were gel-purified and then cloned into a PMD18-T vector (TakaRa Dalian Co., Japan) according to standard procedures. Recombinant plasmids confirmed by PCR were sequenced by Sangon Corporation.
Sequencing results analyzed and compared using the MEGA4.1 (Rainbow Technologies, INC) software package to define the genetic variations and relation-ships with other strains were obtained from GenBank. The complete sequence of the SP strain was submitted to the GenBank under accession number GenBank ACC#DQ-649478, genotype F.
The strain/isolate names and GenBank accession numbers for sequences used in this work are as follows: Complete genome sequences: 87-1004 (AF-314560), 87-1005 (AF314562), 88-1961 (AF467767), Biken (AF314561), Dg1062/Korea/98 (Korea98) (AY 309060), Drag94 (AY669145), Glouc1/UK96 (UK96) (AF280799), JERYL LYNN-2 (vaccine strain, JL2) (A F345290), JERYL LYNN-5 (vaccine strain, JL5) (AF-338106), L-Zagreb vaccine (vaccine strain) (AY685920), Miyahara (AB040874), PetroNov (AY681495); SP (DQ649478); Smith-Kline Beecham (Smith, vaccine strain) (AF314559), SIPAR 02 (AF314558), L3/Russia/ Vector, (AY508995).
SH gene sequences (SA702Ja99, AB056145), Ya-maguchi99 (Ab105482), TokyoM-21 (AB105478), Mu-VS-ISR05-98649-G5 (AM293334), UK(AF280799); New Jersey (US2006), L-Zagreb (AY685920), L3/ Russia (AY508995), Islip1-UK97(AF142766), Kent1-3UK97 (AF142767), Se50647 (U50284), SEVII (U 50 291), DK8307 (AF365919), DK8206 (AF365897), DK8306(AF365918), Fukuoka49-JPN.00 (AB105483), TokyoS-III-10-JPN.01 (AB105480), TokyoM-50-JPN. 00 (DQ136174), 36-1079 (AB116011), Minsk.Be-larus/ 10.02 (DQ136175), Minsk.Belarus 44.01 (DQ 136174), MinskBelarus09.03, (DQ25004), MuVs-PAL-04-86994 (AM293337), 871004 (AF314560), 871005 (AF314-562), 88-1961 (AF467767), Biken (AF314561), Kor98 (AY309060), DRAG94 (AY669145), Glouc1/ UK96 (UK96) (AF280799), JL2 (F345290), JL5 (AF 338106), PetroNov (AY681495), SP (DQ649478), Yeo ju1503 (AY048995), 776274 (AF526410), SA475Ja 97 (AB0 56148), AS97-8 (AF180376), IS98-58 (AF 180384), SIPAR02 (AF314558), MUVs-PAL04-89033 (AM29 3338), Edingburgh2 (X63711), Edingburgh6 (X637 12), Edingburgh4 (X93177), Belfast (X63709), Bri-stoll (X63713), Polos-Portugal-96 (Y08214), Wlz1 (Z77158), Wsh1 (Z77160), Wsh2 (z81005), Wsh3 (U 80435), Wlz2 (Z77161), Wlz3 (Z77159), Zhejiang 06-30-10 (EF102880), UK99-102x13 (AY380065), UK99-190 (AY380068), MuVs-ISR04-94507 (AM 293339), 1700 (AB115996), MP94-H (AB003417), TK087 Ja 97 (AB05614).
One virus strain known as SP was isolated with CPE characteristics in Vero cells. This strain was confi-rmed by a culture-neutralization test and RT-PCR.
Two mumps virus phylogenetic trees based on the 316 nucleotide region of the SH gene and the com-plete genome were generated by the neighbor-joining method. In Fig 1, the SP strain was closely related with Wlz2 and Wsh3 (Fig. 1). The data showed that these strains may belong to the same genotype (geno-type F). In Fig. 2, the no closest genetic relationship to SP strain is compared with the complete genome of JERYL LYNN-2 and JERYLLYNN-5.
Figure 1. Mumps virus phylogenetic tree generated by the neighbor-joining method, based on the complete genome of mumps virus. The phylogenetic tree was constructed by the neighbor-joining method with MEGA version 4.1 software package, and the reliabilities indicated at the branch nodes were evaluated using 500 bootstrap replications. The length the horizontal branches reflects the phylogenetic distance.
Figure 2. Mumps virus phylogenetic tree generated by the neighbor-joining method, based on a 316 nucleotide region of the SH gene. The phylogenetic tree was constructed by the neighbor-joining method with the MEGA version 4.1 software package and the reliabilities indicated at the branch nodes were evaluated using bootstrap 500 replications. The length the horizontal of the branches reflects phylogenetic distance.
The complete sequence of the SP strain was sub-mitted to the GenBank under accession number Gen-Bank ACC#DQ-649478, genotype F. The genome of the SP isolate was 15 384 base pairs in length. Similar to the other 15 strains, it is organized around eight major proteins. The homology with the other 15 mumps virus strains was 96%-93.2%. Amino acid variations were observed.
The homologies of the SP strain to 15 other mumps virus strains in the region of the N and L genes were 94.2% to 95.8% and 94.1% to 96.6%, respectively (1 650 nt); the amino acid sequences were 97.2% to 98.9% and 97.8 to 99.2% homologous to the 15 other strains, respectively. For the comparison of the P protein, two extra guanosine residues were inserted at a G-rich region as shown in other reports (3). Faithful transcription without the insertion of a P gene yields the V protein, which may be important for the viral multiplication of the mumps virus in cells. The P and V genes were 92.3% to 96.2% and 92.7% to 96.7% homologous to the 15 other strains, respectively, at the nucleotide sequence levels; the amino acid sequences were 95.4% to 98.3% and 93.3% to 97.3% homo-logous to the 15 other strains, respectively. These data show that the amino acid sequences were mostly conserved, particularly at the NP and L genes (Table. 2), and they confirm findings from a previous report (2).
Table 2. Nucleotide and amino acid homology comparison between SP and other Mumps virus isolates
The N protein also plays a role during viral assembly through interactions with the matrix protein. Antigenic sites of the MuV N protein have been found to reside within its C-terminus in amino acid regions 412-475 and 475-549 (5). We found that the deduced sequence of the SP strain was quite different when compared with vaccine strains belonging to genotypes A or B. Instead, it was more homologous to the L-Zagreb strain (Fig. 3).
The SP strain was 93.7% to 96.1% and 92.2% to 96.6% homologous to the 15 other mumps virus strains at the regions of the F and HN genes, respec-tively. The amino acid sequences were 95.2% to 97.4% and 97.2 to 99.1% homologous, respectively. The amino acid sequences of the HN gene were particularly similar to the JL5 and JL2 strains (vaccine strain, A genotype), with 95.4% and 96.2% homology, respectively. The M gene was 94.7% to 97.1% homologous to the 15 other strains at the nucleotide sequence levels, whereas the amino acid sequence was 98.9% to 99.7% homologous to the 15 other strains. Our data thus show that the M gene was mostly conserved (Table. 2).
So far, known epitopes neutralizing antibodies against the MuV are located in the HN protein in amino acid regions 265-288, 329-340, and 352-360. The HN protein, which is important for receptor binding, neuraminidase activity, and promotion of fusion protein function is isolated at the virion surface and provides the major target for the humoral immune response against MuV infection (5). In the deduced HN protein sequence of the SP strain, the antigenic epitopes were quite different than those from vaccine strains belonging to genotypes A or B. Instead, they were identical to those from the L-Zagreb strain (Fig. 4).
A genotype-specific motif at positions 28-30 in the SH gene was observed in the form of Ile-Met-Leu.No similar patterns were observed in other genotypes (10, 14, 15).