Download Geographic distribution and evolution of Sindbis virus in Australia

Journal
of General Virology (1999), 80, 739–748. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Geographic distribution and evolution of Sindbis virus in
Australia
Leanne M. Sammels, Michael D. Lindsay, Michael Poidinger,† Robert J. Coelen‡
and John S. Mackenzie†
Department of Microbiology, The University of Western Australia, Nedlands, Western Australia 6907, Australia
The molecular epidemiology and evolution of Sindbis (SIN) virus in Australia was examined. Several
SIN virus strains isolated from other countries were also included in the analysis. Two regions of the
virus genome were sequenced including a 418 bp region of the E2 gene and a 484 bp region
containing part of the junction region and the 5h end of the C gene. Analysis of the nucleotide and
deduced amino acid sequence data from 40 SIN virus isolates clearly separated the Paleoarctic/
Ethiopian and Oriental/Australian genetic types of SIN virus. Examination of the Australian strains
showed a temporal rather than geographic relationship. This is consistent with the virus having
migratory birds as the major vertebrate host, as it allows for movement of virus over vast areas of
the continent over a relatively short period of time. The results suggest that the virus is being
periodically redistributed over the continent from an enzootic focus of evolving SIN virus. However,
SIN virus strains isolated from mosquitoes collected in the south-west of Australia appear to
represent a new SIN virus lineage, which is distinct from the Paleoarctic/Ethiopian and
Oriental/Australian lineages. Given the widespread geographic dispersal of the Paleoarctic/
Ethiopian and Oriental/Australian lineages, it is surprising that the South-west genetic type is so
restricted in its area of circulation. Nucleotide sequence data from the C gene of the prototype
strain of the alphavirus Whataroa were also determined. This virus was found to be genetically
distinct from the SIN virus isolates included in the present study ; however, it is clearly SIN-like and
appears to have evolved from a SIN-like ancestral virus.
Introduction
Sindbis (SIN) and SIN-like viruses are the most widely
distributed of all known arboviruses, with regular isolations
occurring in four of the six zoogeographic regions of the
world. Human SIN virus infection was considered a minor
medical problem from a public health point of view until
epidemics in South Africa in 1974 (McIntosh et al., 1976) and
Author for correspondence : Leanne Sammels.
Fax j61 8 9346 2912. e-mail lsammels!cyllene.uwa.edu.au
† Present address : Department of Microbiology, The University of
Queensland, Brisbane, Queensland 4072, Australia.
‡ Present address : Department of Biomedical and Tropical Veterinary
Sciences, The James Cook University, Townsville, Queensland 4811,
Australia.
The GenBank accession numbers of the sequences reported in this
paper are AF061205–AF061239 and AF061684–AF071715.
0001-5904 # 1999 SGM
later in areas of Northern Europe in the mid 1980s (Skogh &
Espmark, 1982 ; Lvov et al., 1984), often involving hundreds of
cases, were reported. SIN virus is the most commonly isolated
arbovirus from mosquitoes in Australia (Mackenzie et al.,
1994). Despite this, human disease has only been reported on
two occasions in Australia (Doherty et al., 1969 ; Guard et al.,
1982), although seroepidemiological studies suggest that
frequent subclinical infections do occur (Boughton et al., 1984 ;
Doherty, 1973 ; Kanamitsu et al., 1979 ; Liehne et al., 1976).
Strain variation or the presence of clinically similar diseases or
antigenically related alphaviruses may explain the low incidence of clinical infections in humans in Australia.
In a study of genetic identity among 15 strains of SIN virus
using RNA–RNA hybridizations, Rentier-Delrue & Young
(1980) found that two distinct geographic types of SIN virus
could be distinguished. These authors suggested that divergent
evolution had occurred between European\African and
Indian\Far Eastern\Australian types. A subsequent study of
antigenic variation among SIN virus isolates from Paleoarctic,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31
HDJ
L. M. Sammels and others
Ethiopian, Oriental and Australian areas demonstrated antigenic diversion between viruses from the Paleoarctic\
Ethiopian and Oriental\Australian regions (Olson & Trent,
1985). Genetic studies, by RNase T1 mapping, indicated that
the primary structures of RNA from viruses isolated in the four
zoogeographic regions were distinct from one another (Olson
& Trent, 1985). An RNase T1 mapping study of a limited
number of Australian SIN virus isolates indicated that the virus
may exist throughout Australia as a single topotype (Brand,
1989).
The aim of this study was to examine the geographic
distribution and evolution of SIN virus in Australia by
determining the nucleotide sequence of regions of the E2 and
C genes of a number of SIN virus strains isolated in various
geographic locations in Australia. Molecular epidemiological
studies of another Australian alphavirus, Ross River (RR) virus,
have shown that distinct genetic types of the virus are
associated with geographic origin (Faragher et al., 1985 ;
Lindsay et al., 1993 ; Sammels et al., 1995). Thus, this study was
undertaken to better understand the genetic variation in
alphaviruses and to identify potential mechanisms of spread of
these viruses in Australia. A limited number of SIN virus strains
isolated in other countries, including Egypt, South Africa,
Malaysia and Papua New Guinea (PNG), was also included in
the study to examine the genetic variation of SIN virus in
different zoogeographic regions of the world. An isolate of
Whataroa (WHA) virus, an alphavirus belonging to the
Western equine encephalitis antigenic complex, which is
enzootic in parts of New Zealand (Miles, 1973), was also
included. Neutralization tests have indicated that WHA virus
is antigenically related to SIN and SIN-like viruses (Calisher et
al., 1988 ; Karabatsos, 1975). Recently, nucleotide sequence
analysis has shown that WHA virus is SIN-like, although its
sequence diverges by about 20 % from SIN virus (Weaver et al.,
1997).
nucleotide variability. Therefore, a second, more highly conserved region
of the SIN virus genome was targeted for sequencing (Strauss et al., 1984).
Sequence data of the 3h end of the highly conserved junction region and
the 5h end of the C gene were obtained for all the selected SIN virus
isolates.
RT–PCR of cDNA. Details of the methods used to amplify a 482 bp
cDNA fragment of the SIN virus E2 gene and a 510 bp cDNA fragment
containing part of the junction region and the 5h end of the C gene have
been described previously (Sellner et al., 1992). Briefly, a 0n5 µl aliquot of
tissue culture supernatant was digested with 5 µg proteinase K in a total
volume of 12n5 µl for 30 min at 42 mC. Proteinase K was inactivated by
heating to 94 mC for 5 min. The proteinase K reaction mix was then
combined with the reagents for a one-step RT–PCR reaction in a total
volume of 25 µl. Each reaction contained 20 pmoles each primer
(SIN9170* and SIN9652 for the E2 gene or SIN7575* and SIN8086 for
the C gene), 2 U Taq DNA polymerase, 0n5 U AMV reverse transcriptase,
100 µM dNTPs, 2 mM MgCl , 2 µg yeast tRNA and 5 µl 5i reaction
#
buffer resulting in final concentrations of 67 mM Tris–HCl (pH 8n8),
50 mM KCl, 6 µM EDTA and 0n1 % Triton X-100, 0n5 mM DTT. The
tubes were overlaid with 60 µl paraffin oil and incubated in a thermal
cycler for 30 min at 42 mC followed by 94 mC for 5 min (to activate and
then inactivate the AMV reverse transcriptase) and then 35 cycles of
94 mC for 30 s, 60 mC for 30 s and 72 mC for 60 s, with a final extension
at 72 mC for 7 min.
Nucleic acid sequencing and analysis. Nucleic acid sequence
analysis was performed on an automated Applied Biosystems 373A
DNA sequencer. Both strands of the amplified cDNA fragments were
sequenced using primers that were identical to those used in the RT–PCR
reaction. The purified dsDNA was sequenced on an automated sequencer
using the Prism DyeDeoxy Terminator Cycle Sequencing kit (Applied
Biosystems) and the cycle sequencing parameters used were as described
in the manufacturer’s protocol.
Multiple nucleotide and amino acid sequence alignments were
performed with the CLUSTAL V program (Higgins & Sharp, 1989).
Aligned sequences underwent phylogenetic analysis using programs
from the PHYLIP package (Felsenstein, 1989). Briefly, distances were
calculated using the Kimura two-parameter method and trees were
calculated using the neighbour-joining method. Bootstrap resampling of
1000 replicates was used to place confidence values on groupings within
trees.
Methods
Virus stocks. All SIN virus strains used in this study are listed in
Table 1. Virus stocks were prepared on Vero cell (Monkey kidney ; ATCC
CCL 81) monolayers at 37 mC at an m.o.i. of 0n1 p.f.u. per cell. At 48 h
post-infection, cell culture supernatants were harvested, clarified by
centrifugation at 4000 g for 30 min and stored at k70 mC.
Design and synthesis of oligonucleotide primers. All oligonucleotide primers used in this study were synthesized on an automated
DNA synthesizer (380B ; Applied Biosystems) to hybridize to specific
locations of the SIN virus genome, according to the reported sequence
(Strauss et al., 1984). The primers are identified according to the
nucleotide site at which the 3h end of the primer was designed to anneal.
All primers have complementary-sense orientation except those marked
with an asterisk which have plus-sense orientation.
Initially, the E2 gene was chosen for sequencing because the structural
genes are known to be more variable than the non-structural genes in
alphaviruses and also because the E2 gene carries the major antigenic
determinants. However, numerous attempts to produce cDNA of the
region for several SIN virus isolates failed, suggesting considerable
HEA
Results
The 41 SIN virus strains and the WHA isolate (M78)
included in the analysis, with their location and host of origin,
are listed in Table 1. The SIN virus strains were isolated from
four different zoogeographic regions of the world over a 40
year period. Thirty-five of these strains were isolated from
mosquitoes trapped in various geographic locations in
Australia over a 33 year period. The published sequence of the
prototype strain of Ockelbo virus, Edsbyn82 (Shirako et al.,
1991), was obtained from GenBank and included in the
analysis.
Nucleotide sequencing studies in the E2 gene
The nucleotide sequences of a 418 bp region of the E2 gene
were determined for 32 SIN virus isolates. Five strains of virus,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31
Table 1. Details of Sindbis and SIN-like virus isolates
Abbreviations used : PNG, Papua New Guinea ; QLD, Queensland, Australia ; NSW, New South Wales, Australia ; Vic, Victoria, Australia ; WA, Western Australia, Australia ; NT, Northern
Territory, Australia.
Isolate name
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
EgAR339
EgAR338
SAAR86
M78‡
MRE16
MK6962
M78
MRM39
16700
RRD764
RRD800
AS19016
BB19153
NL26287
RB27266
BH2907
MM840
BH40503
OR6
OR339
OR374
OR405
OR437
OR499
OR922
WK465
K2673
CX184
K2588
K1961
K2924
K3141
K3028
K3057
K10390
K12710
SW6562
SW20055
SW25713
SW33029
V620
V634
Location
Cairo, Egypt
Cairo, Egypt
South Africa
New Zealand
Sarawak, Malaysia
Joinjakaka, northern PNG
New Zealand
Kowanyama, Cape York Peninsula, far north QLD
Charleville, south-central QLD
Ross River Dam, north coast QLD
Ross River Dam, north coast QLD
Lachlan River, south-central NSW
Griffith, south-central NSW
Narran Lake, north-central NSW
Redbank Weir, south-west NSW
Barmah Forest, north-west Vic
Lake Moodemere, Vic
Barmah Forest, north-west Vic
Ord River, NE Kimberley, WA
Ord River, NE Kimberley, WA
Ord River, NE Kimberley, WA
Ord River, NE Kimberley, WA
Ord River, NE Kimberley, WA
Ord River, NE Kimberley, WA
Ord River, NE Kimberley, WA
Camballin, W Kimberley, WA
Lake Argyle, NE Kimberley, WA
Ord River, NE Kimberley, WA
Billiluna, SE Kimberley, WA
Billiluna, SE Kimberley, WA
Parrys Ck, NE Kimberley, WA
Parrys Ck, NE Kimberley, WA
Nullagine, Pilbara, WA
Nullagine, Pilbara, WA
Derby, SW Kimberley, WA
Billiluna, SE Kimberley, WA
Southern suburbs, Perth, south-west WA
Peel region, south-west WA
Leschenault region, south-west WA
Peel region, south-west WA
Darwin, north-central coast NT
Maratanka, north-central NT
Date collected
Source (vector or host)
1953
1953
1954
1962
1975
Feb. 1966
1962
Apr. 1960
1974
Feb. 1991
Feb. 1991
Feb. 1979
Feb. 1979
Apr. 1981
Nov. 1981
Feb. 1974
Feb. 1974
Jan. 1984
6\6\72
30\3\74
2\4\74
6\4\74
Mar.\Apr. 1974
Mar.\Apr. 1974
June\July 1976
3\8\79
22\4\82
13\6\82
21\4\89
24\4\89
25\4\89
25\4\89
17\2\90
19\2\90
6\4\93
20\4\93
6\2\90
20\1\92
13\10\92
5\10\93
6\3\84
15\3\84
Culex univittatus
Unavailable
Culex species
Culex pervigilans
Unavailable
Ficalbia flavens
Culex pervigilans
Culex annulirostris
Unavailable
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Anopheles amictus
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Aedes normanensis
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Aedes eidsvoldensis
Culex annulirostris
Culex annulirostris
Aedes pseudonormanensis
Aedes pseudonormanensis
Culex annulirostris
Culex annulirostris
Culex annulirostris
Culex annulirostris
Aedes camptorhynchus
Aedes camptorhynchus
Culex annulirostris
Aedes normanensis
Passage history*
Supplier†
14ismb, 1iVero
12ismb, 1iVero
4ismb, 1iVero
Unavailable
6imosq, 1ismb, 1iVero
3ismb, 1iVero
3ismb, 1iBHK
1ismb, 1iVero
3ismb, 1iVero
1iC6\36, 1iBHK
1iC6\36, 1iBHK
1ismb, 1iVero
1ismb, 1iVero
1ismb, 1iVero
1ismb, 1iVero
1ismb, 1iVero
1ismb, 1iVero
1ismb, 1iVero
2ismb, 1iVero
2ismb, 1iVero
2ismb, 1iVero
2ismb, 1iVero
2ismb, 1iVero
2ismb, 1iVero
2ismb, 1iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
2iC6\36, 2iVero
1iC6\36, 3iVero
1iC6\36, 3iVero
1iC6\36, 3iVero
1iC6\36, 3iBHK
1iC6\36, 3iBHK
1iC6\36, 3iBHK
IM
IM
IM
DP
IM
IM
DP
DP
DP
BK
BK
IM
IM
IM
IM
IM
IM
IM
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
UWA
RW
RW
HEB
* Passage history, history of each isolate upon arrival in this laboratory. C6\36, C6\36 clone of Aedes albopictus cells ; smb, suckling mouse brain ; Vero, Vero (African green monkey kidney) cells ; and BHK, baby hamster kidney cells.
† UWA, Arbovirus Laboratory, Department of Microbiology, University of Western Australia, Nedlands, Australia ; RW, Richard Weir, Berrimah Agricultural Centre, NT, Australia ; IM, Ian Marshall, Department of Biochemistry,
Australian National University, Canberra, ACT, Australia ; DP, Debbie Phillips, Laboratory of Microbiology and Pathology, State Health Laboratory, Department of Health, QLD, Australia ; and BK, Brian Kay, Queensland Institute of
Medical Research, QLD, Australia.
‡ M78 is a strain of WHA virus.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31
Evolution of Sindbis virus
No.
L. M. Sammels and others
Fig. 1. Sequences of the 137 amino acid residues deduced from the nucleotide sequences of a 418 nucleotide region of the
E2 gene of 33 SIN virus isolates described in Table 1. The dots indicate identity with the prototype strain EgAR339, while
letters indicate amino acid substitutions. The N-glycosylation sites are underlined.
including the WHA isolate (M78) and the four strains of SIN
virus isolated in the south-west region of Western Australia
(WA), failed to produce cDNA in an RT–PCR reaction. Several
attempts to produce cDNA from these strains using different
reaction conditions were performed without success. The
sequences obtained for the prototype strain of SIN virus,
EgAR339, and the South African strain, SAAR86, were
identical to the published E2 gene sequences of these two
isolates (Davis et al., 1986 ; Russell et al., 1989). The nucleotide
sequences of each strain were aligned and compared with the
SIN prototype EgAR339 sequence. When all of the strains
were included in pairwise comparisons, the nucleotide sequence variability of the 418 bp segment of E2 was high, with
a maximum divergence of 26n1 %. The majority of the
nucleotide substitutions occurred at the third position of the
codon and 72 % were silent. The maximum nucleotide
divergence was much lower (5n1 %) when only Australian
strains were included. Interestingly, in pairwise comparisons of
the Malaysian (MRE16) and PNG (MK6962) strains with the
Australian strains, the maximum nucleotide divergence was
only 3n9 and 4n1 %, respectively, suggesting a close genetic
relationship between SIN virus isolates from these different
geographic areas. Malaysia lies within the Oriental zoogeographic region while PNG is part of the Australian zoogeographic region (Wallace, 1962) ; therefore this group of
isolates was designated the Oriental\Australian genotype.
The isolates from Egypt, South Africa and Sweden also shared
high nucleotide conservation in pairwise comparisons with a
maximum nucleotide divergence of 6n5 % ; the group was
designated the Paleoarctic\Ethiopian genotype.
Within the Australian SIN strains, there were several
nucleotide fixations associated with time of isolation. No
HEC
nucleotide fixations observed in the Australian SIN virus
isolates correlated with geographic location, host or passage
history.
The divergence in nucleic acid sequence observed between
the Paleoarctic\Ethiopian and Oriental\Australian strains was
also reflected in the encoded amino acid sequence alignments
(Fig. 1). There were 37 different amino acid substitutions, of
which 17 were conservative and 20 were non-conservative
changes. The amino acid sequence divergence between the
two groups of isolates was 13–16 %. The amino acid sequences
within the Paleoarctic\Ethiopian and Oriental\Australian
genotypes were highly conserved with maximum variations of
4n4 and 3n6 %, respectively.
The phylogenetic tree constructed from the E2 nucleotide
sequence data (Fig. 2) defined two clusters of SIN virus isolates
corresponding to the Paleoarctic\Ethiopian and Oriental\
Australian genotypes identified from the nucleotide and
deduced amino acid alignments. Within the Paleoarctic\
Ethiopian genotype, the South African and Ockelbo strains
were more closely related to each other than they were to the
two Egyptian isolates. All of the isolates within the Oriental\
Australian genotype, including the Malaysian and PNG
isolates, were closely related, sharing greater than 95 %
nucleotide sequence.
Due to the high nucleotide sequence divergence between
the Paleoarctic\Ethiopian strains and the Oriental\Australian
strains, the branch lengths within the Oriental\Australian
cluster of isolates were short, making it difficult to examine the
relationships between the strains within this genotype. Therefore, the analysis was repeated without the data from the
Paleoarctic\Ethiopian strains in order to produce a tree which
would demonstrate the phylogeny of the Oriental\Australian
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31
Evolution of Sindbis virus
Fig. 2. Phylogenetic tree showing the genetic relationships based on
partial sequence of the E2 gene of the SIN virus isolates included in this
study. Numbers above branches show bootstrap values. Vertical distances
are arbitrary ; horizontal branch lengths are proportional to mutation
distance as indicated (bar, 0n1 substitutions per nucleotide).
strains more clearly (Fig. 3). In this phylogenetic tree, the
original 1960 Australian SIN virus isolate was located on a
separate branch (designated group A) and the remaining
isolates could be divided into three clusters, designated groups
B, C and D. The clustering of the isolates did not correlate with
geographic origin, mosquito species or passage history.
However, there was a very strong temporal relationship
between the strains included in each group. Group B contained
eight strains, including the PNG strain isolated in 1966 and all
of the strains isolated in Australia in the 1970s (except strain
OR922). The PNG isolate was somewhat distinct from the
other isolates although the maximum nucleotide divergence
between the strains in group B was only 1n7 %. The 16 isolates
within group C were also highly conserved with a maximum
divergence of 1n2 %. The group included strains isolated in the
Pilbara and Kimberley regions of WA, north- and south-central
New South Wales (NSW), north-west Victoria (Vic), central
and north-coastal Northern Territory (NT). All of the strains
were isolated between 1976 and 1990. Group D contained four
Australian SIN virus strains, which shared at least 99n5 %
nucleotide sequence identity, and the somewhat distinct
Malaysian strain. Each of these groups contained isolates
Fig. 3. Phylogenetic tree showing the genetic relationships based on
partial sequence of the E2 gene of the SIN virus isolates from the
Oriental/Australian genotype. The location (MAL, Malaysia ; see legend to
Table 1 for other abbreviations) and year of isolation of each strain are
listed. Numbers above branches show bootstrap values and genetic
groupings (A–D) are indicated on the right hand side. Vertical distances
are arbitrary ; horizontal branch lengths are proportional to mutation
distance as indicated (bar, 0n01 substitutions per nucleotide).
which shared identical nucleotide sequences in the E2 gene
fragment despite being isolated in widely separated geographic
regions of Australia.
Nucleotide sequence studies of the C gene
The sequences obtained for the majority of the strains
included 48 nucleotides of the junction region, which are
untranslated, and 436 nucleotides of the 3h end of the capsid
gene which encode the first 145 amino acids of the capsid
protein. The RT–PCR reactions on the SIN virus strains
isolated in the south-west region of WA consistently produced
a cDNA product approximately 200 bp shorter than that
produced from all other isolates. Several attempts to produce
cDNA fragments of the correct size from these strains using
different reaction conditions were performed without success.
These shorter products were subsequently sequenced and the
data included 48 untranslated nucleotides of the junction
region and 232 nucleotides of the C gene, encoding 77 amino
acid residues.
Pairwise comparisons of the nucleotide sequences revealed
a high degree of genetic divergence with a maximum of 38 %
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31
HED
L. M. Sammels and others
Fig. 4. Sequences of the 145 amino acid residues deduced from the nucleotide sequences of a 436 nucleotide region of the C
gene from 37 SIN virus isolates and the WHA virus isolate (M78) described in Table 1. The dots indicate identity with the
prototype strain EgAR339, while letters indicate amino acid substitutions.
between the Australian 1960 strain MRM39 and the WHA
isolate (M78). The sequences in the untranslated junction
region were highly variable, although the three in-phase
termination codons were maintained in all strains except the
four south-west WA isolates, which have only two in-phase
stop codons.
Pairwise comparisons clearly distinguished the
Paleoarctic\Ethiopian and Oriental\Australian isolates. In
addition, a third genotype of strains comprising the four
isolates from the south-west of WA (designated the Southwest genotype) could be distinguished in the pairwise
comparisons. The nucleotide sequence of the WHA (M78)
virus isolate was substantially different to the sequence of all
the other strains. The level of nucleotide divergence between
the Paleoarctic\Ethiopian and the Oriental\Australian isolates
was 24–26n5 %. The ranges of divergence between the
nucleotide sequences of strains in the South-west genotype
and isolates of the Paleoarctic\Ethiopian and Oriental\
Australian genotypes were 16n8–19n4 and 25n4–28n9 %, respectively, suggesting that the South-west genotype is more
closely related to the Paleoarctic\Ethiopian isolates than to the
Oriental\Australian isolates. The WHA virus isolate (M78)
appeared to be equally divergent from the Paleoarctic\
Ethiopian, Oriental\Australian and South-west isolates with
sequence divergence values of 33–34, 33n5–34n9 and 35–
35n4 %, respectively.
The level of nucleotide divergence observed between
isolates within each of the three main genotypes was very low
indicating high nucleotide conservation in this region of the
capsid gene. The maximum divergence between the four
HEE
strains included in the Paleoarctic\Ethiopian genotype was
4 %. The Oriental\Australian isolates showed a maximum
divergence of 5n7 %. The South-west isolates, which were all
obtained from mosquitoes trapped in the south-west region of
WA between 1990 and 1993, shared greater than 99 %
nucleotide sequence identity.
The deduced amino acid sequence alignments (Fig. 4)
included 143 residues of the capsid protein of all the SIN virus
strains except those belonging to the South-west genotype,
from which only 77 amino acids were identified. There was a
total of 92 amino acid substitutions, of which 28 were
conservative and 64 were non-conservative changes. The
alignment indicated a highly variable region between residues
57 and 96 with numerous substitutions, although there were
substitutions scattered throughout the entire length of the
capsid fragment. The amino acid divergence values between
M78 and the Paleoarctic\Ethiopian, Oriental\Australian and
South-west genotypes were 31n2–32n6, 28–29n4 and 36 %,
respectively. The maximum amino acid variation between the
isolates of the Oriental\Australian genotype was 6n9 %. The
variations between the Oriental\Australian and Paleoarctic\
Ethiopian genotypes and between the Oriental\Australian and
South-west genotypes were 16–21n7 and 20n8–26 %, respectively. The amino acid variation between the Paleoarctic\
Ethiopian and South-west genotypes was somewhat lower at
9–11n7 %.
The phylogenetic tree constructed from the nucleotide
sequence data (Fig. 5) defined three clusters of SIN virus
isolates corresponding to the Paleoarctic\Ethiopian, Oriental\
Australian and South-west genotypes. The WHA virus isolate
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31
Evolution of Sindbis virus
Fig. 5. Phylogenetic tree showing the genetic relationships based on
partial sequence of the C gene of the SIN virus isolates and the WHA virus
prototype strain included in this study. Numbers above branches show
bootstrap values. Vertical distances are arbitrary ; horizontal branch lengths
are proportional to mutation distance (bar, 0n1 substitutions per
nucleotide).
(M78), with its unique nucleotide and amino acid sequences,
was located on a separate branch of the tree. The tree also
indicated that isolates belonging to the South-west genotype
were more closely related to strains from the Paleoarctic\
Ethiopian genotype than they were to those of the Oriental\
Australian genotype. All of the remaining Australian SIN virus
isolates were grouped within the Oriental\Australian genotype. The analysis was repeated without data from the
Paleoarctic\Ethiopian and South-west genotypes to produce a
tree which would demonstrate the phylogeny of the Oriental\
Australian strains more clearly (Fig. 6). The analysis placed the
1960 Australian SIN virus isolate MRM39 on a separate
branch of the tree (designated group A) and the remaining
isolates were divided into three clusters, groups B, C and D.
Group B contained the 1966 PNG isolate and nine strains
isolated from various geographic locations in Australia during
the 1970s. The nucleotide sequences of the Australian isolates
within this group were highly conserved with a maximum
nucleotide divergence of less than 0n5 %. The PNG strain was
approximately 1n5 % divergent from the Australian isolates.
Group C contained 15 isolates, most of which were isolated in
the late 1970s and the 1980s from widely separated geographic
Fig. 6. Phylogenetic tree showing the genetic relationships based on
partial sequence of the C gene of the SIN virus isolates from the
Oriental/Australian genotype. The location (MAL, Malaysia ; see legend to
Table 1 for other abbreviations) and year of isolation of each strain are
listed. Numbers above branches show bootstrap values and genetic
groupings (A–D) are indicated on the right hand side. Vertical distances
are arbitrary ; horizontal branch lengths are proportional to mutation
distance (bar, 0n01 substitutions per nucleotide).
locations within Australia. The Malaysian strain (MRE16) was
most closely related to these isolates although it was located
on a separate branch of the tree. The maximum divergence
between the isolates in group C was 1n9 %. The four strains
included in group D, two from northern Queensland (QLD) in
1991 (RRD764 and RRD800) and two isolated in Kimberley
(WA) in 1993, shared 99 % nucleotide sequence identity. The
phylogeny inferred from the C gene sequences (Fig. 6) and E2
nucleotide sequences (Fig. 3) of the Oriental\Australian SIN
virus isolates was very similar.
Discussion
Our results show that at least two distinct genotypes of
SIN virus are circulating in Australia. One of these, the
Oriental\Australian genotype, circulates throughout most of
Australia and in PNG and Sarawak, Malaysia, whereas a
second, previously unrecognized genotype appears to be
endemic in a distinct geographic region located in the
temperate south-west of Australia.
The results confirm earlier reports that significant genetic
diversity exists between isolates of SIN virus from the
Paleoarctic\Ethiopian and Oriental\Australian zoogeographic
regions (Norder et al., 1996 ; Olson & Trent, 1985 ; Rentier-
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31
HEF
L. M. Sammels and others
Delrue & Young, 1980). The isolates contained within each of
the two genotypes were closely related genetically. One
genotype included most of the Australian isolates and the
single strains from PNG and Malaysia. These were isolated
over a 33 year period and were obtained from widely
distributed geographic locations including many areas of the
Australian continent. Although all of the isolates within this
genotype were genetically similar, four genetic subtypes
(groups A–D) were identified within the panel of isolates.
There is an obvious temporal relationship between isolates in
the different groups which suggests that SIN virus strains
circulating across most of the Australian continent have been
replaced at various points through time. The first strain of SIN
virus isolated in Australia, MRM39, was obtained from
mosquitoes trapped in far northern Queensland in 1960. The
genotype of MRM39 was not observed in any other SIN virus
isolates examined, although no Australian strains isolated
between 1960 and 1973 were available for inclusion in this
study. The results indicated that the group B genetic type
predominated across most of Australia during the 1970s. The
close genetic relationship observed between these strains and
the 1966 PNG isolate suggests that this genetic type could
have been introduced into Australia from PNG. Virus strains
isolated in the late 1970s to 1990, from widely separated
geographic locations in Australia, were identified as members
of group C. Similarly, the most recent isolates included in the
study, collected in 1991 and 1993, were separated into group
D. The strain isolated in Malaysia in 1975, MRE16, was closely
related to the Australian strains in groups C and D. These
results suggest that the genetic type represented by group C
may have been introduced into Australia from the Oriental
region in the 1970s, when group B was still the predominant
genetic type, and subsequently replaced group B across the
Australian continent. The emergence of group D as the
dominant genetic type may have occurred as a result of genetic
drift leading to a significant growth advantage or by the
reintroduction of a new strain from the Oriental region. The
temporal clustering of the isolates in the different genetic
types, represented by groups A–D, indicates that the virus
may be maintained in an enzootic focus from where it is rapidly
spread to other regions of Australia. This study could not
determine whether the enzootic focus is located within
Australia or another geographic location, such as PNG or
Malaysia. However, it appears that the virus is disseminated
over vast geographic areas of Australia with isolates from
widely separated locations sharing identical nucleotide
sequences. Virus isolations and antibody studies have established that birds are the major vertebrate host in Australia
(Doherty et al., 1966). Many species of birds migrate annually
within the Australian continent between the north-west and
south-east regions and north-east and south-east regions of
Australia (Williams & Williams, 1990). Thus, it seems likely
that such species may play an important role in widely
disseminating SIN virus in Australia.
HEG
The similarity of the Malaysian SIN virus isolate to the
Australian isolates in this study is of interest, as Malaysia is in
the Oriental zoogeographic region. Rentier-Delrue & Young
(1980) and Olson & Trent (1985), using a different Malaysian
isolate of SIN virus, have also demonstrated that the Malaysian
isolate was more closely related to Australian isolates than to
other isolates from the Oriental zoogeographic region. The
Oriental and Australasian zoogeographic regions are separated
by the Wallace Line in the Indo–Australian archipelago, and
seroepidemiological studies have indicated that the alphavirus
Chikungunya virus and the flavivirus Japanese encephalitis (JE)
virus may be restricted to the Oriental zoogeographic region,
whereas the alphavirus RR virus and flavivirus Murray Valley
encephalitis (MVE) virus are restricted to the Australian
zoogeographic region (Mackenzie et al., 1994). Thus, the
finding that SIN virus isolates from Malaysia and Australia are
genetically similar may indicate that the apparent restricted
distribution of these arboviruses (Chikungunya, RR, JE and
MVE viruses) may be less rigid than previously believed.
Indeed, KUN virus, a flavivirus which is widely distributed in
Australia, has also been isolated from mosquitoes collected in
Malaysia (Bowen et al., 1970) and JE virus has recently been
isolated in the Torres Strait of north-eastern Australia (Hanna
et al., 1995). Many species of birds migrate annually between
south-east Asia and northern Australia (Williams & Williams,
1990) providing an opportunity for virus spread between these
geographic regions by movement of viraemic birds.
The identification of a new distinct genetic type of SIN
virus circulating in the south-west region of WA was an
unexpected result. At present, we have determined the
nucleotide sequence of 48 bases of the junction region and 232
bases of the C gene from these strains. Nevertheless, enough
data were obtained to indicate that these isolates represented
an independent enzootic focus in south-west Australia.
Analysis of the nucleotide sequences indicated that strains
from the South-west genotype are more closely related to
Paleoarctic\Ethiopian strains of SIN virus isolates than to
those from the Oriental\Australian region. This finding
suggests that SIN virus circulating in the south-west region of
Australia may have been introduced, perhaps by a viraemic
migratory bird or travelling human, and evolved under the
selective pressures of the ecological conditions in this region.
Many of the endemic species of birds in the south-west of WA
are territorial and do not move over very large distances
(Slater, 1974 a, b). It is possible that SIN virus circulating in the
south-west of Australia has adapted to and is being maintained
in the region by these more sedentary bird species.
Alternatively, the virus may have adapted to a new transmission cycle utilizing vertebrate hosts with limited mobility,
such as small mammals including marsupials. A survey of avian
and mammalian species common to the south-west region for
antibody to SIN virus may help to identify potential major
hosts of the virus in this region. Surveillance of arbovirus
activity in the south-west region of WA has been undertaken
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31
Evolution of Sindbis virus
since May 1987 and has involved regular collections of
mosquitoes at various locations. In that time, there have been
over 200 isolations of RR virus but only twelve isolations of
SIN virus. All SIN virus isolates were obtained from mosquitoes trapped between February 1990 and December 1996
(M. D. Lindsay & C. A. Johansen, unpublished results). The
focus of this program is the surveillance of saltmarsh-breeding
mosquito species located in coastal environments where large
outbreaks of RR virus disease occur. Consequently, saltmarsh
mosquitoes in the genus Aedes comprise over 80 % of the adult
mosquito population in the region in which surveillance is
carried out. The low number of isolates of SIN virus compared
to RR virus may indicate that these species are not major
vectors of SIN virus in the region. More extensive genetic and
antigenic characterization of these unique SIN strains and other
SIN strains isolated from mosquitoes collected in the southwest region of WA is currently in progress.
The genetic information obtained from the WHA virus
prototype strain (M78) confirms previous antigenic and genetic
studies which indicated that the virus was distantly related to
SIN and SIN-like viruses (Calisher et al., 1988 ; Weaver et al.,
1997). The results of phylogenetic analysis suggest that WHA
may have evolved in isolation from an ancestral SIN-like virus
which may have originally been introduced into New Zealand
by the arrival of viraemic birds.
We thank those researchers who provided many of the virus isolates
used in the study (listed in Table 1). Thanks also to Ms Cheryl Johansen
and Dr Annette Broom for their assistance with isolation and identification of the Western Australian SIN virus strains. This work was
funded by the National Health and Medical Research Council of Australia
and the Health Department of Western Australia.
body to arboviruses in Aboriginal and other population groups in
northern and eastern Australia, 1968–1971. Transactions of the Royal
Society of Tropical Medicine and Hygiene 67, 197–205.
Doherty, R. L., Gorman, B. M., Whitehead, R. H. & Carley, J. G. (1966).
Studies of arthropod-borne virus infections in Queensland V. Survey of
antibodies to group A arboviruses in man and other animals. Australian
Journal of Experimental Biology and Medical Science 44, 365–378.
Doherty, R. L., Bodey, A. S. & Carew, J. W. (1969). Sindbis virus
infection in Australia. Medical Journal of Australia 2, 1016–1017.
Faragher, S. G., Marshall, I. D. & Dalgarno, L. (1985). Ross River virus
genetic variants in Australia and the Pacific Islands. Australian Journal of
Experimental Biology and Medical Science 63, 473–488.
Felsenstein, J. (1989). PHYLIP : phylogeny inference package (version
3.2). Cladistics 5, 164–166.
Guard, R. W., McAuliffe, M. J., Stallman, N. D. & Bramston, B. A.
(1982). Haemorrhagic manifestations with Sindbis infection. Case
Report. Pathology 14, 89–90.
Hanna, J. N., Ritchie, S. A., Phillips, D. A., Shield, J., Bailey, M. C.,
Mackenzie, J. S., Poidinger, M., McCall, B. J. & Mills, P. J. (1995). An
outbreak of Japanese encephalitis in the Torres Strait, Australia. Medical
Journal of Australia 165, 256–60.
Higgins, D. G. & Sharp, P. M. (1989). Fast and sensitive multiple
sequence alignments on a microcomputer. Computer Applications in the
Biosciences 5, 151–153.
Kanamitsu, M., Taniguchi, K., Urasawa, S., Ogata, T., Wada, Y., Wada,
Y. & Saroso, J. S. (1979). Geographic distribution of arbovirus
antibodies in indigenous human populations in the Indo–Australian
archipelago. American Journal of Tropical Medicine and Hygiene 28,
351–363.
Karabatsos, N. (1975). Antigenic relationships of group A arboviruses
by plaque reduction neutralization testing. American Journal of Tropical
Medicine and Hygiene 24, 527–532.
Liehne, C. G., Stanley, N. F., Alpers, M. P., Paul, S., Liehne, P. F. S. &
Chan, K. H. (1976). Ord River arboviruses – serological epidemiology.
Australian Journal of Experimental Biology and Medical Science 54, 505–512.
Lindsay, M. D. A., Coelen, R. J. & Mackenzie, J. S. (1993). Genetic
References
Boughton, C. R., Hawkes, R. A., Naim, H. M., Wild, J. & Chapman, B.
(1984). Arbovirus infections in humans in New South Wales. Sero-
epidemiology of the alphavirus group of togaviruses. Medical Journal of
Australia 141, 700–704.
Bowen, T. T. W., Simpson, D. I. H., Platt, G. S., Way, H. J., Smith,
C. E. G., Ching, C. Y. & Casals, J. (1970). Arbovirus infections in
Sarawak : the isolation of Kunjin virus from mosquitoes of the Culex
pseudovishnui group. Annals of Tropical Medicine and Parasitology 64,
263–268.
Brand, T. N. (1989). Molecular epidemiology of Sindbis virus. Honours
thesis, University of Western Australia.
Calisher, C. H., Karabatsos, N., Lazuick, J. S., Monath, T. P. & Wolff,
K. L. (1988). Re-evaluation of the western equine encephalitis antigenic
complex of alphaviruses (family Togaviridae) as determined by
neutralization tests. American Journal of Tropical Medicine and Hygiene 38,
447–452.
Davis, N. L., Fuller, F. J., Dougherty, W. G., Olmsted, R. A. & Johnston,
R. E. (1986). A single nucleotide change in the E2 glycoprotein gene of
Sindbis virus affects penetration rate in cell culture and virulence in
neonatal mice. Proceedings of the National Academy of Sciences, USA 83,
6771–6775.
Doherty, R. L. (1973). Surveys of haemagglutination-inhibiting anti-
heterogeneity among isolates of Ross River virus from different
geographical regions. Journal of Virology 67, 3576–3585.
Lvov, D. K., Berezina, L. K., Yakovlev, B. I., Aristova, V. A., Gushchina,
E. L., Lvov, S. D., Myasnikova, I. A., Skvortsova, T. M., Gromashevsky,
V. L., Gushchin, B. V., Sidorova, G. A., Klimenko, S. M., Khutoretskaya,
N. V. & Khizhnyakova, T. M. (1984). Isolation of Karelian fever agent
from Aedes communis mosquitoes. Lancet 2, 399.
McIntosh, B. M., Jupp, P. G., Dos Santos, I. & Meenehan, G. M. (1976).
Epidemics of West Nile and Sindbis viruses in South Africa with Culex
univittatus Theobold as vector. South African Journal of Science 72, 295.
Mackenzie, J. S., Smith, D. W., Ellis, T. M., Lindsay, M. D., Broom,
A. K., Coelen, R. J. & Hall, R. A. (1994). Human and animal arboviral
diseases in Australia. Recent Advances in Microbiology 2, 1–91.
Miles, J. A. R. (1973). The ecology of Whataroa virus, an alphavirus, in
South Westland, New Zealand. Journal of Hygiene 71, 701–713.
Norder, H., Lundstrom, J. O., Kozuch, O. & Magnius, L. O. (1996).
Genetic relatedness of Sindbis virus strains from Europe, Middle East, and
Africa. Virology 222, 440–445.
Olson, K. & Trent, D. W. (1985). Genetic and antigenic variations
among geographical isolates of Sindbis virus. Journal of General Virology
66, 797–810.
Rentier-Delrue, F. & Young, N. A. (1980). Genomic divergence among
Sindbis virus strains. Virology 106, 59–70.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31
HEH
L. M. Sammels and others
Russell, D. L., Dalrymple, J. M. & Johnston, R. E. (1989). Sindbis virus
Slater, P. (1974 b). A Field Guide to Australian Birds. Volume II. Passerines.
mutations which coordinately affect glycoprotein processing,
penetration, and virulence in mice. Journal of Virology 63, 1619–1629.
Strauss, E. G., Rice, C. M. & Strauss, J. H. (1984). Complete nucleotide
Sammels, L. M., Coelen, R. J., Lindsay, M. D. & Mackenzie, J. S.
(1995). Geographic distribution and evolution of Ross River virus in
Australia and the Pacific Islands. Virology 212, 20–29.
Sellner, L. N., Coelen, R. J. & Mackenzie, J. S. (1992). A one-tube, one
manipulation RT–PCR reaction for detection of Ross River virus. Journal
of Virological Methods 40, 255–264.
Shirako, Y., Niklasson, B., Dalrymple, J. M., Strauss, E. G. & Strauss,
J. H. (1991). Structure of the Ockelbo genome and its relationship to
other Sindbis viruses. Virology 182, 753–764.
Skogh, M. & Espmark, A. (1982). Ockelbo disease : epidemic arthritis-
exanthema syndrome in Sweden caused by Sindbis-virus like agent.
Lancet 1, 795.
Slater, P. (1974 a). A Field Guide to Australian Birds. Volume I. Nonpasserines. Australia : Rigby.
HEI
Australia : Rigby.
sequence of the genomic RNA of Sindbis virus. Virology 133, 92–110.
Wallace, A. R. (1962). On zoological regions. In The Geographical
Distribution of Animals, vol. 1, pp. 50–82. New York : Hafner Publishing.
Weaver, S. C., Kang, W., Shirako, Y., Rumenapf, T., Strauss, E. G. &
Strauss, J. H. (1997). Recombinational history and molecular evolution
of Western equine encephalomyelitis complex alphaviruses. Journal of
Virology 71, 613–623.
Williams, T. C. & Williams, M. C. (1990). The orientation of transoceanic
migrants. In Bird Migration : Physiology and Ecophysiology, pp. 7–21. Edited
by E. Gwinner. Berlin : Springer-Verlag.
Received 14 August 1998 ; Accepted 4 November 1998
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 07 May 2017 01:06:31