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Journal of General Virology(1991), 72, 83-88. Printedin Great Britain 83 Identification of several different lineages of measles virus M . J. Taylor, 1 E. Godfrey, 1 K. Baczko, 2 V. ter Meulen, 2 T. F. Wild 3 and B. K. Rima 1. 1Division of Genetic Engineering, The Queen's University of Belfast, Belfast BT9 7BL, U.K., 2Institut fftr Virologic und Immunbiologie, University of Wftrzburg, Versbacherstrasse 7, D-8700 Wiirzburg, Germany and 3CNRS, Faeultd de M~deeine Alexis Carrel, Rue Guillaume Paradin, 69008 Lyon, France The sequences o f a region of the nucleocapsid protein gene, between nucleotides 1231 and 1686, encoding the C-terminal 151 amino acid residues of the nucleocapsid protein have been determined for 16 strains of measles virus. Analysis of this region showed that it is highly divergent (up to 7 - 2 % divergence in the nucleotide sequence and 10.6% divergence in the amino acid sequence between most distant strains) and that several lineages of measles virus can be found to co-circulate at a given time. Some o f the lineages show geographical restriction. The results for measles virus are similar to those reported for other h u m a n paramyxoviruses such as m u m p s virus, parainfluenza type 3 virus and the avian Newcastle disease virus. Introduction low number of passages in tissue culture. We have concentrated our sequence analysis on a variable region of the N gene of MV because it has been shown (Rozenblatt et al., 1985) that the sequence of canine distemper virus and MV, as well as that of phocine distemper virus (M. D. Curran & B. K. Rima, unpublished observations), vary widely in this region showing the lowest level of similarity of any region of the genome except that of the 5' untranslated part of the F m R N A of morbilliviruses. Furthermore, Buckland et al. (1989) have identified important B cell epitopes in this region and it is not unreasonable to suspect that T cell epitopes are present in this region as well. We report here that the analysis of these sequences shows that at least four different lineages of strains can be distinguished. Measles virus (MV) is a monotypic virus which has been considered extraordinarily stable in terms of its serology and immune responses to it. However, analysis of the ability of different strains of the virus to bind monoclonal antibodies has revealed a low level of variability in the major antigens (Sheshberadaran et al., 1983) and, more recently, strain variations have been studied directly by analysis of the nucleotide sequences of MV strains (data compiled in Cattaneo et al., 1989). Most data are available for the matrix protein (M) gene of MV where a number of lytic growing strains (Bellini et al., 1986, Wong et al., 1987; Curran & Rima, 1988) and subacute sclerosing parencephalitis (SSPE)-derived strains (compiled in Cattaneo et al., 1989; Enami et al., 1989) have been analysed. This gene has been studied in detail because it has been assumed that it, in particular, is defectively expressed in SSPE. Further analyses of the nucleocapsid protein (N), phosphoprotein (P), fusion protein (F) and haemagglutinin (H) genes have been published in the last 2 years, almost always comparing the only sequence known in its totality (i.e. that of the Edmonston strain, as passaged in various laboratories) with those of SSPE-derived strains or MV derived from measles inclusion body encephalitis (MIBE) (compiled in Cattaneo et al., 1989). There have been many indications that the Edmonston strain is rather unrepresentative and that this comparison may give a false impression of the true rates of change between lytic MV and those sequences from SSPE-derived strains. Therefore, we have started sequencing studies on other, more recently isolated MV strains and wild-type viruses with a 0000-9826 © 1991 SGM Methods Viruses. The origin and nucleotide sequence of some of the virus strains analysed here have been reported earlier (See Table 1). The origins of strains EdP9, Hu2, MVO and MVP have been described before (Rima et al., 1983). Sequencesof the $33 and $81 strains were obtained from brain autopsy material from two cases of SSPE diagnosed in Northern Ireland. Case $33 concerns a male who died at age 16 and who had shown symptomsfor 7 years and 10 months, and case $81 a male who died at age 21 who had shown symptoms for 2 years and 10 months. cDNA cloningand polymerase chain reaction(PCR). The N genes of strains EdP9, JM and CM were cloned from mRNA extracted from infected Vero cells using Bluescript plasmids as vectors, essentially described by Schmid et al. (1987). cDNAs of strains Hu2, Y22, Rl18, MVO, MVP, $33 and $81 were obtained by reverse transcription of total RNA extracted from infected Vero cells, or from the brains of SSPE cases with a specific primer representing the antisense strand Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 23:25:30 84 M . J. T a y l o r a n d others Table 1. Origin o f measles virus strains Name Description Sequence reference Edm Hal EdP9 Hu2 EdmonstonB vaccine strain Hall6 SSPE strain Salt-dependenthaemagglutinating variant of Edmonston strain Schwarzvaccine-related case derived from a child with dysgammaglobulinaemia Moraten vaccine-related case of measles Wild-type isolate from Cameroun (Giraudon et al., 1988) Wild-type isolate from Gabon (Giraudon et al., 1988) SSPE case B from Austria Wild-type isolate; Bethesda, Md., U.S.A. (obtained from J. Milstein) SSPE case IP3 SSPE case from Germany Wild-type isolate from Bristol, U.K. Wild-type isolate from Bristol, U.K SSPE case from Northern Ireland; 16 year old male (1983) SSPE case from Northern Ireland; 21 year old male (1986) MIBE case; U.S.A. Wild-type isolate; Bethesda, Md., U.S.A. (obtained from B. Fields) Rozenblatt et al. (1985) Buckland et al. (1988) This work Mor Y22 R118 S(B) JM SIP3a S(A) MVO MVP $33 $81 IE(C) CM complementaryto the 3' end of the mRNA (Schmidet al., 1987). PCR amplification of the resulting cDNA with an mRNA sense primer (5' TTAGGCAAGAGATGGTAAGG3", representing nucleotides 1198 to 1218 of the N gene of MV) and the forward primer for reverse transcription was carried out by following the instructions of the supplier (Cetus). PCR products were cut with appropriate restriction enzymes and cloned in M13mp8 or Bluescript plasmid vectors. Nucleotide sequencing was carried out using the dideoxynucleotide chain termination technique. Results Nucleotide sequencing was carried out with both vectorspecific primers as well as with internal primers complementary to the sequence of the last 456 nucleotides of the coding sequence of the N gene. Fig. 1 shows a sequence alignment of the 16 sequences with that of a consensus sequence derived from them. 'Mutations' away from the consensus have been indicated. The sequence of a seventeenth MV strain derived from a Moraten vaccine-associated case, obtained from Dr A. Osterhaus (Bilthoven, The Netherlands), was found to be identical to that of EdP9 given here. Fig. 2 shows an analysis of nucleotide changes away from the consensus sequence which are shared by the various strains. A number of these 'mutations' are not mutations in the true sense of the word because we cannot know at present whether the consensus sequence is biased towards a subset of all circulating MV strains. However, this type of table makes relationships between strains clearer than one which gives the numbers of nucleotides that are divergent between strains or percentage divergences between strains, particularly when the consensus sequence is made up of a high This work This work This work This work Cattaneo et This work Cattaneo et Cattaneo et This work This work This work This work Cattaneo et This work al. (1989) al. (1989) al. (1989) aL (1989) number of divergent sequences. The data show that the MV strains analysed so far fall into a number of more or less closely related groups. Group A consists of the laboratory-adapted Edmonston vaccine strains, including the Moraten and Schwarz vaccine-associated cases and the Hall6 strain of SSPE virus; group B consists of the very distantly related African strains; group C consists of a number of SSPE-derived strains and the wild-type JM strain, group D consists of the United Kingdom isolates; group E consists of the strain derived from two United States strains, i.e one wild-type strain and a strain derived from a case of MIBE. The groupings are also evident from an alignment of the deduced N sequence in this hypervariable region (Fig. 3). Group A carries group-specific residues at positions 405 (K) and 522 (N) of the N protein; group B at position 448 (G); group C at positions 432 (W), 438 (M) and 470 (D); group D at positions 431 (G), 456 (S) and 470 (S); group E at positions 440 (A), 450 (I), 485 (Q) and 489 (Q). These changes can be related to epitopes II and III determined by Buckland et al. (1989). They would predict that only strains of group A bind to the monoclonal antibody that delineates epitope III (residues 519 to 525) and it is known that the two group B strains do not bind this antibody (Giraudon et al., 1988). Furthermore it would be predicted that the monoclonal antibody that delineates epitope II (residues 457 to 476) would probably not bind to the group C and D strains. African strain Y22 does show a number of nonconservative amino acid replacements in this site and does not bind the monoclonal antibody (Giraudon et al., 1988). 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" " ~ > . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , o , , o , . , . , , , , , . ~ ., .. . .I .~ . , . ,. o. . . . . . . . . > . . ~ . o . . . . . . . . . . . . . . . . . . . . . . o . . o ~ : • > o o o o o o o o o o o o o o o . ~ . o . o . . . o . . . . o , . . ~ . . . . . . . . . . . . . . . . . o " o . o o , o . . . . . . . . . o . o . , , , . . . . . . . . ~. ~ ~ ~. ~ > • O0 t~ 86 M. J. Taylor and others con Edm 1650 1686 AAGGCTCAGACACGGACAC C CCTAGAGTGTACAATGACAGAGATCTTCTAGACTAG ........................ C ................ A .............. Hal EdP9 Hu2 MVO MVP $33 ........................ ~ ................ X.............. ........................ T ................ A. . . . . . . . . . . . . . ........................ ~ ................ X.............. ........................ _ ................ _ .............. ....................... c ................................ ...A ..................................................... S81 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rl18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y22 ....................................................... SIP3a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S(A) ................... A T .................................... S(B) ........................................................ JM .......................................... IE(C). ....................................................... C M . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . ............. . . . . . . ° . . . . . . . . . . . . . . . . . Fig. 1. Comparison of the sequences of the hypervariable region of the N gene of different MV strains. Mutations from the consensus (con) sequence are indicated. Those that are underlined are expressed changes. Sequences of the EdP9, Hu2, MVO, MVP, $33, $81, R118, Y22, JM and CM strains were determined in this study. Those for the other strains, i.e. Edm, Hal, SIP3a, S(A), S(B) and IE(C), were described earlier (see Table 1 for references). Edm Hal EdP9 Hu2 Rl18 Y22 S(B) JM SIP3a S(A) MVO MVP $33 $81 IE(C) CM 0 1 0 0 --I Edm Hal 10 4 4 4 5 5 8 EdP9 Hu2 Y22 A 1 2 0 0 1 5 1 2 0 0 0 0 0 0 0 0 0 0 8 3 2 0 0 0 0 0 0 0 0 0 0 10 4 2 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 16 RII8 S(B) B 4 1 13 0 18 JM SIP3a S(A) MVO t 2 I i 1 I 1 0 0 14 t 5 4 0 0 0 0 0 1 18 [ 4 4 0 0 0 0 0 1 10 5 0 0 0 1 1 1 11 0 0 0 0 0 0 9 8 8 1 1 13 9 8 1 1 15 9 1 1 10 1 1 9 MVP $33 $81 IE(C) D 10 9 CM 10 E Fig. 2. Nucleotide sequence variations in the variable region of the N gene of various MV strains. The figures indicate the number of nucleotide changes away from the consensus sequence shared between two strains; for example, the Edm strain sequence has 10 nucleotides different from the consensus sequence of which four are shared with the Hal strain and two with strain R118. Discussion This sequence analysis indicates that several lineages exist for MV. One group of viruses (A) consists of the Edmonston-related sequences including, not surprisingly, the EdP9 salt-dependent haemagglutinating variant and the identical Moraten vaccine strain, but also strain Hu2, derived from a Schwarz vaccine-related death, and the Hall6 strain of virus isolated from an SSPE case. The M gene of the Hu2 strain shows only a few differences to the Edmonston strain (Curran & Rima, 1988) as does the F gene (Hull et al., 1987). Particularly, some of the differences in the M gene are probably significant in indicating re-adaptation of the vaccine strain to human Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 23:25:30 Measles virus lineages 380 con Edm Hal EdP9 Hu2 Rl18 Y22 . 400 . 420 . 440 W T ASE GITAEDAR VSEI HTTEDRISRAVGP Q VSrLHGDNSENELP LGG EORRW SRGEARE °--°-°°.,°°°°°°°,°°..,°.°..°..m°°,°°o.°°°°°°° ..... °°,°°°,o°o°o,.....,°°°°°.° ,,-°°°°..°°°°°°°,°,o°°o..°.°°°K° ,,.-°-.-,-°°°,°°,°°°°°,..,°°°,°,°t,°°°°°°°.,...°°°°°°o°°,,°,.,.,,°°,°,,,,G° A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S(B) . . . . . . . . . . . JM SIP3a S(A) MVO MVP $33 S81 IE(C) CM v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s . . . . . . . . . . . . . . . . . . w N . . . . . . M ..... . . . . . ~..']B 7.. 1 . . . . . . . . . . . . . . . . . . . . . | c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .w. .M .. .. .. .. .. ... .. .. .. .. .. ] . "*°°°°°°°°'°°°°°,°o,-,,,,°°°°°°,,*,°o,.,.oo,°°°°,°°°,,°.G~°°°°°~°.°°°°°°°,,, ,*°°°°°°G°°°-°°°,°°°°.,°°°°°°°.,°,o°°°°°°°,,°°°°°°,,°°°°G°°°.°°°,.,°.°,°°°°o] ~.°°°.,...o..°°°o.,op,~.°°.°...~°,°...,..,o,°...°°.°....~oo.oo..,~...°°oo°,° --°°°°°°°°°°,,°,°-,,,,,°°°°°°°°~,~°°.,°.°°°,~,°,°.°,.°,°G.°°°°°,~,°,,,,°°°°° "'°°°'°°°°°°-°--°°o°--°°°°°°-,--°,°°°~o.....°°o°°°°°°°°°~°°°..°o.Ao°°°°°°°°~ • °°°°°°°°°°°°°°°,,,,°,°°,°°°°°°,°°,°,..°°°,,°°.°°°°°°,°,..°°°°°°,A°°°.°°°.,I 460 con Edm Hal EdP9 Hu2 87 480 m m 500 520 YRETGPSRASDARAAHLPTGTPLDIDTASESSQDPQDSRRSADALLRLQAMAGISEEQGSDTDTPRVYNDRDLLD* °°°''°°°°'''°°--°°°°°°°°°°°°°°~,,°-...°°°°°°.,~,,,..°...°°°..,.°,I°°°°°N ............. R .............. T .................................... ............. R .............. M .................................... Rll8 Y22 . . . . . . . . . . . . . . . ......... ~ .... • ............ R?.P . . . . . . . . . . v?._.~ . . . ~ ..... N y ..... N ........................................... F ..... L . . . . ~. . . . . . . P..D .......... 7 ........... E ................................. .... R ? . . . . . . . . T . P . . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S(B) JM SIP3a .... ? ......... S(A) MVO MVP $33 $81 ................... V.?..5 ...................... ~ ................... ............. .... ........ R....................... . 'siii!!iiiiiiii!!iiiiiiiiiiiiiiii i!i!! iiiii!iii!iiiiiii :l p ................. ~..Q ]B ] l E ................................. K..E ......... . . . ? . S .... m . . . . . . . . S. IE(C) ................ CM A .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................................... c ] ° ]E Fig. 3. Alignment of the deduced amino acid sequences of the C-terminal variable region of the N protein of various MV strains. Changes away from the consensus sequence are indicated. Those that are underlined concern non-conservative replacements (see legend of Fig. 1, and Table 1 for descriptions of the strains). passage. In the F gene of the Hu2 strain the major difference occurs in the cytoplasmic tail of F, just after the m e m b r a n e anchor. Similarly, SSPE-derived viruses often appear to be altered in this area (Cattaneo et al., 1989). The Hall6 strain also shows remarkable identity to the Edmonston strain in the more conserved F gene (Buckland et ok, 1987). The five sequences in this group form a very tight cluster and thus most of the vaccine viruses available at present are in this group. The cocirculating wild-type viruses appear to have very different sequences in this part of the N gene. The African isolates (group B) show low levels of similarity with the strains in the first group and high levels of divergence between themselves and the other strains analysed. They appear to have evolved away from the other sequences and it will be of interest to sequence further isolates from Africa to investigate whether this is a general pattern due to the r a m p a n t nature of the virus in that continent. Strains S(B) and J M (group C) have a high degree of identity and are clearly related despite the geographical separation of the areas of isolation ( G e r m a n y and N e w England, respectively). The results obtained for the M gene (K. Baczko et al., unpublished results) confirm the very close relationship between these two strains. The S I P 3 a and S(A) SSPE-derived isolates are also related to the former two strains but are more divergent than strains S(B) and JM. The next group (D) consisting of four U.K. isolates from the early 1970s onwards, is clearly distinct and comprises two wild viruses from Bristol and two SSPEderived sequences from brains of autopsy cases from Northern Ireland. The level of identity in this group is very high. A low level of identity exists (one shared nucleotide change only) between these strains and the final group (E) of two strains, comprising the IE(C) isolate from a case of M I B E in the U.S.A. and a wildtype strain from the N e w England area (CM). Again, the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 23:25:30 88 M. J. Taylor and others relationships between the IE(C) and CM strain sequences are further confirmed by the M gene sequences of these strains (K. Baczko et al., unpublished results). Evolutionary trees based on these data have been calculated but not included because the relationships between the various lineages are not clear. It is not known how long ago they have diverged from each other and have been maintained in various geographical areas. This poses problems in establishing the roots of the trees. In conclusion this study shows that several lineages of MV appear to be co-circulating at any particular time and that some level of geographical restriction occurs. These results are similar to those obtained for the human parainfluenza virus type 3 (van Wyke Coelingh et al., 1988) and for mumps virus (Yamada et al., 1989; R. P. Yen, M. A. Afzal & B. K. Rima, unpublished observations). In mumps virus no apparent evolution of mutations at silent sites was detected in strains isolated over a time span of several years in Japan (Yamada et al., 1989); the same applied to human parainfluenza type 3 viruses. Another negative strand virus, vesicular stomatitis virus, also appears to be evolving within a number of co-circulating lineages (Bilsel et al., 1990). Thus most of the non-segmented negative-strand viruses analysed so far appear to evolve like the influenza C viruses rather than the influenza A viruses (Yamashita et al., 1988). We thank Professor I. V. Allen for brain material from SSPE patients, Dr P. Sharp for advice on the analysis of sequence comparisons, Steven Flanagan for providing a cDNA clone of the N gene of EdP9 and the EEC, the Wellcome Trust and Multiple Sclerosis Society of Great Britain and Northern Ireland and the Deutsche Forschungsgemeinschaft for financial support for these studies. References BELLINI, W. J., ENGLUND,G., RICHARDSON,C. D., ROZENBLA'F'f,S. & LAZZARINI,R. A. (1986). Matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences and deduced amino acid sequences. Journal of Virology 58, 408-416. BILSEL, P. A., ROWE, J. E., FITCH, W. M. & NICHOLL, S. T. (1990). Phosphoprotein and nucleocapsid protein evolution of vesicular stomatitis virus New Jersey. 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Virology 172, 374-376. YAMASH1TA,M., KRYSTAL, M., FITCH, W.M. & PALESE, P. (1988), Influenza B virus evolution: co-circulating lineages and comparison of evolutionary pattern with those of influenza A and C viruses. Virology 163, 112-122. (Received 26 July 1990: Accepted 12 October 1990) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 23:25:30