VIROLOGY 172,374-376 (1989)

Sequence Variation of the P Gene among Mumps Virus Strains

AKIO YAMADA,’ KAORU TAKEUCHI, KIYOSHI TANABAYASHI, MICHIKO HISHIYAMA, AND AKIRA SUGIURA

Department of Measles Virus, National Institute of Health, 4-7- 1 Gakuen, Musashimurayama, 190- 12 Tokyo, Japan

Received March 15, 1989; accepted May 3 1, 1989

We have determined nucleotide sequences of a 183-nucleotide long region of the P gene of 10 mumps virus strains after gene amplification mediated by DNA polymerase catalyzed chain reaction (PCR) and have compared them with those of two strains which had been reported earlier (K. Takeuchi et a/., J. Gen. Viral., 69, 2043-2049 (1988)). It was shown that mutation is generally noncumulative, i.e., most nucleotide substitutions in earlier strains do not appear in later strains. Viruses of different lineages appeared to cocirculate, but the comparison of American and Japanese strains suggested that those isolated in one country are more closely related to each other than to those isolated in the other country. 0 1989 Academic Press, Inc.

Mumps virus had been thought to be monotypic (I). Antigenic diversity of mumps virus strains was demon- strated in recent studies, however, using monoclonal antibodies directed to the surface glycoproteins as well as internal proteins such as nucleoprotein (NP) and phosphoprotein (P) (Z-5).

We recently determined the complete nucleotide se- quence of the P gene of the Miyahara and Enders strains of mumps virus showing that most nucleotide differences between the two strains are clustered in two regions of the coding sequence of the gene (6). It was expected, therefore, that the comparison of the nucleotide sequence of the supposedly variable re- gions of the P gene may serve to distinguish individual mumps virus strains, but the relatively poor growth of most mumps virus strains in cell cultures has been a hindrance for obtaining a sufficient amount of virus- specific RNAfor such analysis. The recently developed gene amplification through polymerase catalyzed chain reaction (PCR) using heat-resistant DNA poly- merase (7) made it possible to circumvent the above problem. Comparison of twelve mumps virus strains utilizing the method revealed that these strains could be divided into two groups.

The Vero cell-adapted Enders (EN)/45 strain and at- tenuated Jeryl Lynn (JL)/63 vaccine strain were from the United States. All other strains were isolated in Japan. Wild-type Urabe (WU)/67, Torii (T0)/67, and Miyahara (Ml)/70 strains were the progenitors of attenuated live vaccines. Hoshino (HO)/72 is a vaccine strain, but its progenitorwas not available. TH/77, SA/77, SU/77, MY 85, 1 l/85, and 21/85 were independent field isolates. The egg-adapted line of the Enders strain was also in- cluded in this study and was propagated in 7-day-old embryonated chicken eggs. All other virus strains were

’ To whom requests for reprints should be addressed.

propagated in Vero cells as described previously (8). Oligonucleotide primers, P9 (5’-CTCATTGGCAATC- CAGAGCA-3’, in the mRNA sense) and PlO (5’-AT- GAACCTGTTGGTTGGATA-3’, in the vRNA sense), were prepared using an automated DNA synthesizer (Applied Biosystems, Foster City, CA) and purified by polyacrylamide gel electrophoresis (9). These se- quences were chosen because it was shown that the P9 and PlO sequences were identical between the En- ders and Miyahara strains, whereas there were 15 nu- cleotide difference in the 183-nucleotide intervening segment. Total cellular RNA (2 pg) extracted from virus- infected cells by the guanidine thiocyanate method (9) was transcribed into cDNA using the P9 and P10 prim- ers (0.5 PM each) and 200 units of reverse transcrip- tase (BRL, Gaithersburg, MD). In the case of the egg- adapted Enders strain, viral RNA (vRNA) extracted from purified virions was used as template. After hydrolysis of RNA by alkali treatment, the DNA was purified using Spin column G-50 (Boehringer-Mannheim, West Ger- many). The cDNA transcripts were amplified by PCR as described before (7). In brief, 100 ~1 reaction mixture containing cDNA transcripts in 50 m&I KCI, 10 ml\/l Tris-HCI (pH 8.4) 6 ml\/l MgCI,, 1 @I each of P9 and PlO primers, 200 PLM each of dATP, dCTP, dlTP, and dGTP, 170 pg/ml bovine serum albumin (Pharmacia, Uppsala, Sweden), and 2 units of DNA polymerase from Therms aquaticus (New England Biolabs, Bev- erly, MA) was incubated sequentially at 95” for 1 min (denaturation), 40” for 2 min (annealing), and 70” for 1.5 min (extension). The cycle of denaturation, annealing, and extension was repeated 20 to 25 times. The PCR products were electrophoretically purified using 6% polyacrylamide gel and cloned into the Smal site of M 13 mpl0 phage (9). Nucleotide sequences were de- termined by the dideoxy-termination method described by Sanger et al. (70). In order to avoid being misled by

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374

SHORT COMMUNICATIONS 375

EN/45 JL/63 WU/67 TO/67 MI/70 HO/72 TH/77 SRI/77 su/77 MJ/85 11/85 21/85

EN JL wu TO MI HO TH SA su MJ 11 21

EN JL wu TO MI HO TH SA su MJ 11 21

EN JL wu TO MI HO TH SA su MJ 11 21

150 160 170 180 190 ~TCATT GGCAATCCAG AGCPJPAAGAA CAT-~CAGCAC CCTACCGCAT cACATCAGGG

T C A T C A T C A T C TA

A T CC A T C A T c A T C A T C A T C A

200 210 220 230 240 250 ATCCAAGTCA AAGGGCAGAG GCTCAGGGGT CAGGTCCATC ATAGTCCCAC CCTCCGAAGC

C G C G C C T T G

C C AC T T G C C T T G C C T T c

C C C T T G C C T T G

A C C T T G A C C T T G

C C T T G G C C T T G

260 270 280 290 300 310 AGGCAATGGA GGGACTCAGA TTCCTGAGCC CCTTTTTGCA CAAACAGGAC AGGGTGGTAT

C C C C C G C C C C C C C C C C C C C C

320 330 340 AGTCACCACA GTTTATCAGG ATCCAACT-

cc c T C T GC T C T C T C T C T C T C T C T C

A C A C A C A c

A C A C A C A C A C

FIG. 1. Nucleotide sequences of the region of the P genes which lres between the P9 and PlO primer sequences of varrous mumps virus strains. Only nucleotides different from those of the EN/45 strain are shown. The P9 and PlO primer sequences are boxed and underlined, respectively. Numbers indicate nucleotide positions starting from the first ATG codon (6).

errors that might arise during PCRs and subsequent cloning, single-stranded DNAfrom several clones were combined for sequence analysis, and the opposite strands were also sequenced. For analysis of nucleo- tide sequences, a computer software GENETYX (Soft ware Development, Japan) was used.

The nucleotide sequences of the region of the P gene lying between the P9 and PlO primer sequences from eleven mumps virus strains were compared with that of the prototype Enders (EN/45) strain (Fig. 1). The egg-adapted line of the Enders strain was identical to

the Vero cell-adapted one (EN/45) in the nucleotide se- quence of the above region (data not shown). In addi- tion to those virus strains, we have determined nucleo- tide sequences of three vaccine strains derived from WU/67, T0/67, and Ml/70 and found that there was no difference in the nucleotide sequence between pro- genitor strains and their respective vaccine strains (data not shown). Overall, nucleotide differences were found at 32 positions with the 12 strains examined. Differences in 12 out of 32 nucleotides were common to all 10 strains isolated in Japan. Up to 8 nucleotide

376 SHORT COMMUNICATIONS

TABLE 1

A PAIRWISE COMPARISON OF NIJCLEOTIDE DIFFERENCE AMONG MUMPS VIRUS STRAINS

JL wu TO MI HO TH SA su MJ 11 21

EN/45 7 15 17 15 15 15 14 15 15 14 15 JU63 20 22 20 19 20 19 20 20 19 20 WUl67 4 2 4 6 1 2 2 1 2 TO/67 4 2 8 3 4 4 3 4 Ml/70 2 6 1 2 2 1 1 HOI72 6 1 2 2 1 1 THl77 5 6 6 5 6 sAf77 1 1 0 1 SUl77 2 1 2 MJ/85 1 2 1 l/85 1

differences were found between pairs of Japanese strains isolated between 1967 and 1985 (Table 1). In addition, each of 18 out of 32 nucleotide differences occurred in only one strain, and no nucleotide changes which occurred at a specific time were retained in sub- sequent isolates. It was, therefore, suggested that pro- gressive accumulation of nucleotide changes with time did not occur. This view was strengthened by a pair- wise comparison of the sequences as shown in Table 1. The nucleotide sequence of the SA/77 strain iso- lated in 1977 was identical to that of the 1 l/85 strain isolated 8 years later (in 1985), whereas there were five nucleotide changes between the former and the TH/77 strain isolated in the same year (1977).

There were seven nucleotide differences between EN/45 and JU63 isolated in the United States 18 years apart and subjected to numerous laboratory passages thereafter. By contrast, variation of nucleotide se- quences was much more prominent when virus strains isolated in the United States and Japan were com- pared. It appears that mumps virus strains examined could be delineated by geographic origin.

These observations strongly suggest that the evolu- tion of mumps virus is similar to that of influenza C virus and type 3 human parainfluenza virus (I I- 13) in that epidemiologically dominant variants do not emerge se- quentially, and viruses of different lineages are cocircu- lating at a given time. This view differs from that re- ported by Rydbeck et al. (2) who suggested that only one mumps virus is circulating in the population at a given time.

The observation that three vaccine strains licensed in Japan showed identical nucleotide sequences to those of the respective progenitor strains indicates that the attenuation process was not associated with alter- ations of nucleotides in the region lying between the P9 and PlO sequences. Moreover, no difference was observed between egg-adapted and Vero cell-adapted

Enders strains. The nucleotide sequence of this region of the P gene, although variable from strain to strain, is relatively stable upon laboratory propagation of the vi- rus and may be used as the marker of individual mumps virus strains.

Finally, the PCR method offers a great advantage to molecular epidemiological studies of RNA viruses es- pecially for viruses growing poorly in cell cultures.

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