Download Identification of several different lineages of measles virus

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unpublished observations), vary widely in this region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
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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
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Cattaneo et
This work
Cattaneo et
Cattaneo et
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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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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
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Edm
Hal
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4
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4
5
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8
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1
13
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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
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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
. . . . . . . . .
. . . . . . . . .
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]
.
"*°°°°°°°°'°°°°°,°o,-,,,,°°°°°°,,*,°o,.,.oo,°°°°,°°°,,°.G~°°°°°~°.°°°°°°°,,,
,*°°°°°°G°°°-°°°,°°°°.,°°°°°°°.,°,o°°°°°°°,,°°°°°°,,°°°°G°°°.°°°,.,°.°,°°°°o]
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--°°°°°°°°°°,,°,°-,,,,,°°°°°°°°~,~°°.,°.°°°,~,°,°.°,.°,°G.°°°°°,~,°,,,,°°°°°
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• °°°°°°°°°°°°°°°,,,,°,°°,°°°°°°,°°,°,..°°°,,°°.°°°°°°,°,..°°°°°°,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
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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.
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