Previous Article | Next Article 
J Virol, January 1998, p. 73-83, Vol. 72, No. 1
0022-538X/98/$00.00+0
Phylogeny of the Genus Flavivirus
Goro
Kuno,*
Gwong-Jen
J.
Chang,
K. Richard
Tsuchiya,
Nick
Karabatsos, and
C. Bruce
Cropp
Arbovirus Diseases Branch, Division of
Vector-Borne Infectious Diseases, National Center for Infectious
Diseases, Centers for Disease Control and Prevention, Fort Collins,
Colorado 80522-2087
Received 15 July 1997/Accepted 19 September 1997
 |
ABSTRACT |
We undertook a comprehensive phylogenetic study to establish the
genetic relationship among the viruses of the genus
Flavivirus and to compare the classification based on
molecular phylogeny with the existing serologic method. By using a
combination of quantitative definitions (bootstrap support level and
the pairwise nucleotide sequence identity), the viruses could be
classified into clusters, clades, and species. Our phylogenetic study
revealed for the first time that from the putative ancestor two
branches, non-vector and vector-borne virus clusters, evolved and from
the latter cluster emerged tick-borne and mosquito-borne virus
clusters. Provided that the theory of arthropod association being an
acquired trait was correct, pairwise nucleotide sequence identity among these three clusters provided supporting data for a possibility that
the non-vector cluster evolved first, followed by the separation of
tick-borne and mosquito-borne virus clusters in that order. Clades
established in our study correlated significantly with existing
antigenic complexes. We also resolved many of the past taxonomic
problems by establishing phylogenetic relationships of the
antigenically unclassified viruses with the well-established viruses
and by identifying synonymous viruses.
| |
INTRODUCTION |
The genus Flavivirus of
the family Flaviviridae comprises over 70 viruses, many of
which, such as the dengue (DEN) viruses, Japanese encephalitis (JE)
virus, St. Louis encephalitis (SLE) virus, and yellow fever (YF) virus
are important human pathogens (22, 31). Dengue and its
severe and sometimes fatal forms, dengue hemorrhagic fever and dengue
shock syndrome, alone affect nearly 80 million people a year
(30). As demonstrated in recent outbreaks of meningitis by
West Nile (WN) virus in Algeria and Romania, viruses of this group
sometimes cause serious public health concern in unexpected locations
(27).
Most of these viruses were serologically classified into eight
antigenic complexes, but many viruses, including the prototype of this
group, YF virus, could not be affiliated with any complexes (6). Furthermore, many new viruses have been documented
since the establishment of the serological classification, but their overall relationship with the other viruses has not been determined. The difficulty encountered with flavivirus classification partly derives from the extensive geographic distribution and the diversity of
the arthropod vectors or vertebrates hosts associated with biological
transmission of these viruses. Also, it derives from a confusion in
virus nomenclature. For example, tick-borne encephalitis virus strains
isolated primarily in western parts of Eurasia have been called TBE
viruses, but, as clearly pointed out by Calisher (5), no
such virus as tick-borne encephalitis virus (or TBE) has ever been
registered to an international body dedicated to virus taxonomy. To
compound the problem further, an increasing number of viruses have been
added as new members of so-called TBE complex without a virus
definition provided (16, 18, 29, 42, 52). This practice
clearly demonstrates a need for establishing objective criteria for a
better classification of those viruses.
Molecular genetic classification of these viruses has been attempted
before. In all previous studies, fewer than one-third of the members,
primarily mosquito-borne and tick-borne viruses, were used to create
phylogenetic trees, which showed evolution of mosquito-borne and
tick-borne viruses from the presumed ancestor (3, 11). Since
few sequence data were available from other viruses, in particular the
viruses without known vectors (hereafter called the non-vector group),
those phylogenetic trees provided only partial information.
To establish a comprehensive phylogeny of the genus
Flavivirus, we attempted to obtain the genomic sequence of a
1.0-kb segment at the 3' terminus of the NS5 gene from all viruses
whose sequences were not available. We analyzed, together with the
other sequence data already published, the genetic relationships among
the members of this group. Quantitative criteria based on a combination
of the bootstrap support level and the pairwise nucleotide sequence identity were established to define subgeneric taxa. These included cluster, clade, and species. With our new taxonomic definitions, we
then compared our genetic classification with the traditional system
based on serological data.
| |
MATERIALS AND METHODS |
Viruses.
The 58 viruses and one geographic strain of YF
virus sequenced in this study and 13 viruses (including cell fusing
agent [CFA]) whose NS5 gene sequences had been already available in
GenBank are listed in Table 1. The
majority of the viruses sequenced were obtained from
the World Health Organization (WHO)
Collaborating Center for Reference and Research in our institution. For
three tick-borne viruses (Kyasanur Forest Disease [KFD]), Russian
spring summer encephalitis [RSSE], and Omsk hemorrhagic fever (Omsk
HF]), extracted viral RNAs were obtained from the Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease
Control and Prevention. Iguape and Kedougou viruses were obtained from
the WHO International Reference Center, University of Texas Health
Center, Galveston. A total of nine viruses were not used in this study.
These included louping ill, Wesselsbron, Spanish sheep tick-borne
encephalitis, Greek goat encephalitis, and Turkish sheep tick-borne
encephalitis viruses, whose importation and usage is restricted by the
U.S. Department of Agriculture. In addition, TBE complex viruses
(Absettarov, Kumlinge, Hanzalova, and Hypr), which require higher
biosafety facilities, were unavailable in our laboratory.
Reverse transcription (RT)-PCR.
Viral RNA was extracted from
126 µl of 10% suckling mouse brain suspension or cell culture
supernatant fluid by using a Qia-HCV kit (Qiagen, Santa Clarita,
Calif.) or from 50 µl of bulk mouse brain tissue by using RNeasy
(Qiagen). RNA adsorbed on silica membrane was eluted in 50 µl of
water.
For cDNA synthesis, 20 µl of viral RNA was mixed with 1 µl each of
a forward and a reverse primer (50 µM) as well as 8 µl
of water,
and the mixture was heated at 92°C for 1 min and then
cooled to
45°C. Thirty microliters of enzyme mix (10 µl of 5×
reverse
transcription buffer [Boehringer Mannheim, Indianapolis,
Ind.], 5 µl of deoxynucleoside triphosphate [dNTP] mixture [10
mM each
dNTP], 9 U of Rous sarcoma virus reverse transcriptase
[RAV-2;
Amersham, Cleveland, Ohio], and 4.5 µl of water) was added
per tube,
and tubes were incubated at 45°C for 45 min.
PCR was performed with a commercial kit (Expand Long Template PCR
system; Boehringer Mannheim). Five microliters of cDNA was
mixed with
5.0 µl of dNTPs (10 mM each), 1 µl each of forward
and reverse
primers (50 µM), and 33 µl of water. The reaction
mixture was
heated to 94°C, and then 50 µl of the enzyme mixture
(5 µl of
10× PCR buffer, 0.75 to 1.5 µl of enzymes, 44 µl of water)
was
added. After heat denaturation at 94°C for 4 min, temperature
was
shifted to 45°C for 1 min and then to 68°C for 1 min. The
thermocycle program was as follows: 3 cycles (94°C for 20 s,
45°C
for 1 min, 68°C for 1 min), 10 cycles (94°C for 20 s,
50°C for
30 s, 68°C for 1 min), 16 cycles (94°C for 20 s, 50°C for 30 s,
68°C for 1 min in the first cycle, with an
increment of 20 s per
cycle thereafter). A final extension was at
68°C for 5 min.
Primers for DNA template amplification.
Primers were
selected to sequence the genomic regions (nearly 1 kb long) at the 3'
terminus of the NS5 gene delineated between FU1 and cFD3 in Fig.
1. All primers used for DNA template
amplification are listed in Table 2, and
their relative genomic locations are shown in Fig. 1. For most viruses,
a pair of primers (FU1 and cFD3), which had been previously determined
(7) produced the desired amplicons. However, for those
viruses which did not produce the expected amplicons, templates of
various sizes were produced by using the other primers shown in Table
2.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 1.
Relative genomic positions of primers used for
amplification of DNA templates from the NS5 gene of flaviviruses and
for sequencing. *, From reference 14. **, The
name in parentheses indicates a degenerate primer at the same location
corresponding to the primer immediately above. ***, From
reference 33.
|
|
Nucleotide sequencing.
Amplicons were purified with a Qiagen
PCR purification kit, and aliquots of approximately 60 to 160 ng of the
purified DNA templates were used for direct cycle sequencing using an
ABI (Foster City, Calif.) Prism DNA sequencing kit for dye terminator
cycle sequencing with AmpliTaq-FS enzyme. The sequencing primers
including the primers used for DNA template preparation and their
corresponding degenerate primers are listed in Tables 2 and
3. Thirty cycles of a thermocycle program
(96°C for 15 s, 50°C for 15 s, and 60°C for 4 min) were
performed with Gene Amp PCR System 9600 thermocycler (Perkin-Elmer,
Norwalk, Conn.). The products were purified in Centri-Sep spin columns
(Princeton Separations, Adelphi, N.J.) and directly sequenced with ABI
model 377 sequencer. Nucleotide sequences were edited and compilied by
using a computer program, DNASIS for Window (version 1.1; Hitachi
Software Engineering America, Ltd., South San Francisco, Calif.).
Phylogenetic analysis.
The multiple sequence alignment
program Clustal W (version 1.6) (43) was used to obtain an
optimal nucleotide or amino acid sequence alignment file. Phylograms
for the entire sequence (about 1 kb between primers FU1 and cFD3 [Fig.
1]) were obtained either by MEGA (version 1.01) (25) or
PHYLIP (version 3.57c) (12, 13) based on aligned nucleotide
or amino acid sequences. MEGA was also applied to analyze a subset of
the nucleotide sequence (about 220 bp between primers FU1 and cFD2
[Fig. 1]).
In constructing phylograms with distance methods of MEGA, we determined
genetic distance by the proportional distance method
(
37),
Kimura's two-parameter method (
24), and the Tajima-Nei
method (
40), applying pairwise deletion of gaps and equally
weighting both transition and transversion for all three codon
positions. A proportional distance matrix was transformed to calculate
the pairwise nucleotide sequence identity between all virus pairs.
For
tree building, various genetic distance matrices were used
for the
neighbor-joining method (
37) which calculated bootstrap
confidence intervals of 500 heuristic search replicates and confidence
probability of the genetic distance by a standard error test.
We also
tested a character state tree-building algorithm which
consisted of a
sequential programs in the PHYLIP package. A strict
consensus bootstrap
tree was obtained by using the following programs:
(i) SEQBOOT to
generate 100 reiterated replicas; (ii) DNAPARS
or PROTPARS to acquire
the most parsimony tree of each reiterated
data, (iii) CONSENSE to
build a strict consensus bootstrap tree,
and (iv) DRAWGRAM to draw the
phylogenetic tree.
Virus identification.
Numerous attempts with various primer
combinations (Tables 2 and 3) failed to obtain amplicon from Tamana bat
virus RNA by RT-PCR. We tried to reidentify the virus by the
immunofluorescence technique using 13 polyvalent hyperimmune mouse
ascitic fluids prepared against 9 antigenic groups in the family
Bunyaviridae and the Tacaribe group of the
Arenaviridae, monovalent antiserum against vesicular
stomatitis virus, rabies virus, flaviviruses (DEN, Murray Valley
encephalitis, YF, WN, Powassan [POW], and Tamana bat viruses), and
flavivirus group-reactive monoclonal antibodies 4G2 and 6B6C-1 on
virus-infected Vero cells. The infected cells were also embedded in
LX-112 Araldite mixture and examined with a model 410 Life Science
Phillips electron microscope (Phillips, Eindhoven, The Netherlands)
operating at 80 kV as described earlier (9).
| |
RESULTS |
PCR primers.
The pair of flavivirus cross-reactive primers
(FU1 and cFD3) proved to be highly efficient for generating about
1-kb-long DNA templates near the 3' terminus of the NS5 gene for most
of the viruses by RT-PCR. A small number of viruses that were not amplified or poorly amplified with this pair (Apoi, Karshi, Kokobera, Rio Bravo, Sal Vieja, and Sokuluk viruses) could be sequenced with
overlapping DNA templates of various sizes generated with other pairs
of primers shown in Table 2.
Molecular classification.
For convenience, throughout the
text, the hierarchal levels for molecular systematics of this genus are
organized in descending order as follows: cluster, clade, and species.
A cluster was designated based on the bootstrap support exceeding 95%
and host-vector association. A clade was defined as a group of viruses
that share the 69% or higher pairwise nucleotide sequence identity
among the members. This 69% quantitative criterion was chosen from the
pairwise identity minus 2 standard deviations among four serotypes of
DEN virus, because DEN complex viruses are easily separated from other
flaviviruses not only by a serologic test but also by analysis of
nucleotide sequence data (6, 11). A species was defined as a
class of viruses with higher than 84% nucleotide sequence identity
among them. The cutoff criterion was derived from two strains of YF viruses, the prototype Asibi and the TN96 isolate, which were separated
by 69 years and belonged to two distinct genotypes (7). An
extensive envelope gene sequence study on many strains of four DEN, JE,
SLE, and YF viruses from all known areas of the world where there
viruses are endemic had earlier indicated that YF virus was the most
diversified species of all, with a maximum of 14% nucleotide sequence
difference among strains. Among 71 viruses of this genus, non-vector,
tick-borne, and mosquito-borne clusters contained 14, 15, and 42 viruses, respectively (Table 4).
Sixty-eight viruses were further separated into 14 clades, and three
viruses, CFA, Apoi, and Kedougou viruses, were not associated with any
clade. The following virus pairs had pairwise nucleotide sequence
identity of 91, 88, 92, 90, and 95%, respectively, and were determined
to be genetic variants of the same virus: Phnom Penh bat and Batu Cave;
TBE-central European subtype (TBE-CE) and Negishi; Potiskum and Saboya;
THCAr (21) and Tembusu; and Israel turkey
meningoencephalitis and Bagaza.
The genome of Tamana bat virus, originally isolated in Trinidad
(
35), could not be amplified satisfactorily with any
combination
of the primers used in this study. In immunofluorescence
tests,
only a weak reactivity was observed with a WN virus monospecific
antiserum, and the virus did not react with 20 other polyclonal
and 2 flavivirus group-reactive monoclonal antibodies, despite
a positive
reaction to the homologous polyclonal ascitic fluid
against Tamana bat
virus (data not shown). However, electron microscopy
revealed
conclusively that Tamana bat virus-infected Vero cells
exhibited
numerous virion-like particles that had a morphological
characteristics
of a typical flavivirus (Fig.
2).
Phylograms.
The unrooted neighbor-joining tree based on a
proportional distance of 1-kb nucleotide sequence is shown in Fig.
3. The phylogram demonstrates clearly
that although the exact host association of CFA virus in nature remains
unknown, it is the most distally related flavivirus sequenced so far.
CFA virus has pairwise nucleotide sequence identities with viruses of
the designated clades (Table 4) as follows: with Apoi virus, 57%; with
clade I, 56%; with clade II, 53 to 56%; with clade III, 54 to 57%;
with clade IV, 56 to 57%; with clade V, 55 to 57%; with clade VI, 53 to 56%; with clade VII, 55 to 56%; with clade VIII, 54 to 55%; with
clade IX, 55%; with clade X, 54 to 55%; with clade XI, 53 to 57%;
with clade XII, 54 to 56%; and with clade XIV, 53 to 56%.
Furthermore, the phylogram reveals that non-vector and vector-borne
clusters emerged first from the putative origin of the genus
Flavivirus. The latter further branched off to form
tick-borne and mosquito-borne virus clusters. These three clusters are
well supported by 99% of bootstrap replicates and 99% confidence
probabilities (CPs) of a standard error test.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 3.
Phylogenetic tree of the genus Flavivirus,
using nucleotide sequence. The tree was constructed by the
neighbor-joining method of MEGA. Each number at nodes is the percentage
of 500 bootstrap replicate support; * indicates confidence
probability higher than 90%. Vertical length is arbitrary. Scale is
percentage of genetic distance.
|
|
The non-vector cluster further branched into three clades with Apoi
virus by itself outside any of the three (Fig.
3 and Table
4). San
Perlita and Jutiapa viruses in clade I and four viruses
in clade II
with the exception of Montana myotis leukoencephalitis
virus are
rodent-associated viruses. The six viruses in clade
III are all bat
associated. The tick-borne cluster consists of
two clades, represented
by Gadgets Gully virus and a collection
of 10 viruses (clade IV), most
of which belong to the so-called
TBE complex (
10), and
Kadam, Tyuleniy, Meaban, and Saumarez
Reef viruses (clade V) (Fig.
3).
In the aforementioned two clusters,
CPs generally parallel bootstrap
supports; and even when bootstrap
supports are weak, the corresponding
CPs are very high, as demonstrated
in the clade III (52 versus 99% in
Fig.
3).
Nine clades (VI to XIV) comprise the mosquito-borne cluster. Kedougou
virus is not associated with any clade and has a range
of 60 to 65%
nucleotide sequence identities with other mosquito-borne
viruses (data
not shown). Sepik and YF viruses comprise clade
VII; Sokuluk, Entebbe
bat, and Yokose viruses comprise clade VIII;
Zika and Spondweni viruses
comprise clade X; and Naranjal, Busssuquara,
Aroa, and Iguape viruses
comprise clade XII. None of those viruses
have been placed in any
antigenic complexes before. Clade VI consists
mostly of Uganda S
complex viruses and two previously unclassified
viruses, Jugra and
Saboya viruses. Potiskum virus in this clade
is considered a subtype of
Uganda S virus by neutralization test
(
23). The DEN complex,
consisting of four serotypes alone, is
assigned a separate clade (IX).
Former JE complex viruses were
separated into clades XI, XIII, and XIV.
Clade XI includes four
viruses of the Ntaya antigenic complex as well
as members of the
JE complex, such as SLE, Rocio, and Ilheus viruses.
The segregation
of the other JE complex viruses, Stratford and Kokobera
viruses,
into one clade (clade XIII) by themselves agrees well with the
previous conclusion that those viruses are distinct from the other
JE
complex viruses (
34). Clade XIV includes Cacipacore virus,
Yaounde virus, and the remaining seven JE complex viruses. Bootstrap
supports of the clade XI and XII viruses were 53 and 70%,
respectively.
Nevertheless, the corresponding CPs are both 99%,
providing a
strong support to our classification.
Among other phylograms created by the combination of distance and
tree-constructing methods examined, the tree based on Kimura's
two-parameter method produced a phylogram very similar to those
in Fig.
3 and
4. The Tajima-Nei distance method
produced a phylogram
quite different from those in Fig.
3 and
4 and was
judged inappropriate
because at the optimal cutoff level for clade
(using 65%, rather
than 69%, nucleotide sequence identity), Tyuleniy
group (clade
V) was split to two groups and clade (IV) was further
divided,
creating a phylogram considerably different from the
phylograms
obtained by us, as mentioned above, and by other
investigators.
The phylogram based on nucleotide sequences between FU1
and cFD2
(220 bp) demonstrated a similar tree topology as with 1 kb for
most clades (data not shown), despite the low bootstrap supports
at
some nodes and shift in affiliation of some viruses at the
terminal
branches.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 4.
Phylogenetic tree of the genus Flavivirus,
using amino acid sequence. The tree was constructed by the
neighbor-joining method using MEGA. Each number at nodes is the
percentage of 500 bootstrap replicates. Vertical length is arbitrary.
Scale is percentage of amino acid distance.
|
|
The strict consensus tree, obtained by character-state of the most
parsimony algorithm, showed a similar tree topology as
by the distance
method (data not shown). By this method clade
XIII, comprising
Stratford and Kokobera viruses, was weakly associated
with clade XII.
Kedougou virus also was weakly linked to DEN complex
viruses (clade
IX).
The most notable differences between the amino acid sequence-based tree
(Fig.
4) and the nucleotide sequence-based tree are
the more distant
relation of clade XIII from and the closer relation
of clade X to clade
XIV in the former tree. A shift of affiliation
of Sepik and Montana
myotis leukoencephalitis viruses to a different
subset of viruses is
also observed. Nevertheless, topologies of
the trees by both nucleotide
and amino acid sequences are essentially
identical.
Amino acids and motifs.
In addition to the GDD motif, other
highly conserved motifs include YADDTAGWDT, QRGSGQV, DDCVV, TACL,
YFHRRDLR, and SAVP (Fig. 5). In other,
less conserved motifs, amino acids unique to a particular cluster (or
clusters) are found. For a non-vector cluster, they are SF (amino acids
[aa] 28 to 29) and G (aa 190) (Fig. 5). For vector-borne clusters,
they are A (aa 13) and P (aa 255); for the tick-borne cluster, they are
W (aa 57) and C (aa 292).
View larger version (91K):
[in this window]
[in a new window]
|
FIG. 5.
Multiple amino acid sequence alignment of the CFA, Apoi,
and Kedougou viruses and one member representing each clade of the
genus Flavivirus. Amino acid 1 corresponds to nucleotides
9018 to 9020 of YF virus. A dash indicates missing one amino acid. A
dot indicates that the amino acid (or absence of it) in a given amino
acid sequence is the same as in the corresponding sequence of CFA virus
above of the aligned sequences.
|
|
Codon usage.
For analyzing the host association among two
clusters (non-vector and vector borne) of viruses, we examined the
frequencies of the dinucleotide CpG. When the viruses were classified
into two categories, those with 
9 CG-containing amino acids and those with 
10 such amino acids, 9 of 14 non-vector group but only 5 of 58 vector-borne viruses belonged to the former category (chi-square test:
2 = 55.8; P = 0.0000322). When the
cutoff number of CG-containing amino acids was changed to 13, the
numbers of the viruses in non-vector and vector-borne clusters with
13 such amino acids were 11 and 29, respectively (chi-square test:
P = 0.053).
| |
DISCUSSION |
The phylograms of flaviviruses created in the past were based on
the sequences of only about one-third or fewer of the members and thus
provided only partial information (3, 11, 15, 28, 48).
Nevertheless, the dichotomy between mosquito-borne and tick-borne
viruses has been clearly recognized by those investigators and was
again confirmed in our study. As shown in our phylograms, the genus
Flavivirus presents as a monophyletic tree. Unlike previous studies, however, our study reveals further that from the putative ancestor of the genus Flavivirus, two major branches
emerged, non-vector and vector-borne clusters, and that from the latter cluster emerged tick-borne and mosquito-borne clusters. The above topology as well as subsequent branching patterns leading to clades in
each cluster were found to be basically identical between the trees
based on nucleotide as well as amino acid sequences.
In our study, we constructed trees without selecting CFA virus as an
outgroup taxon, as was done before (28). Such an unrooted tree is expected to provide the least biased phylogenetic tree. Irrespective of the difference in requirement of an outgroup in the
software used, CFA virus was placed at the root of the tree by MEGA
(Fig. 3) and by PHYLIP (data not shown).
The phylogenetic segregation of the viruses into three major clusters
was not surprising because of a clear distinction in the size of the
sequences between amplimers FU1 and cFD3: members of non-vector cluster
all had 1,011 bases, tick-borne viruses had a median length of 1,026 bases, and mosquito-borne viruses had a median length of 1,035 bases.
Thus, when all sequences were aligned optimally by introducing gaps to
make all lengths equal, 13 aa were missing from all viruses in the
non-vector cluster and 8 aa were missing from all viruses of the
tick-borne cluster, with all missing amino acids being located at the
same sites as the non-vector viruses. On the other hand, the pattern of
missing amino acids was more variable in the mosquito-borne cluster.
While the majority of the mosquito-borne viruses had 5 aa missing,
DEN-1, -2, -3, and -4, Kedougou, Kokobera, and Stratford viruses had an
additional missing amino acid. All of these, as well as Zika and
Spondweni viruses, had 2 aa missing at the same locations where
non-vector and tick-borne viruses similarly were missing amino acids.
Although the flavivirus phylograms produced in the past were primarily
based on envelope gene sequences, it has been reported that the
topologies based on envelope and NS5 genes showed perfect agreement
(28). The envelope gene of flaviviruses is less conserved than the NS5 gene, and this difference is reflected in greater differences in the amino acid sequence. Thus, while the ranges of
pairwise amino acid sequence identities in the envelope gene were 72.3 to 80.4% in DEN complex viruses, 81 to 94.6% in JE complex viruses,
and 66.3% between Banzi and YF viruses (15), the
corresponding ranges for NS5 gene in our study were of 75 to 86%, 83 to 97%, and 72%, respectively, confirming conservative nature of the
latter gene.
Regarding the evolution of three clusters of viruses, theoretically any
cluster could have been ancestral, since unrooted methods were chosen
for our phylograms. However, Calisher (5) speculated that
"evolutionary pressure may have created a divergence of the
virus-vector relationships, perhaps from a common original one." Blok
and Gibbs (3) are of the opinion that the arthropod-mediated transmission of the flaviviruses is an acquired trait, although they
also recognized the opposite possibility. One prevailing theory is that
tick-borne and mosquito-borne clusters independently evolved from the
common ancestor. Marin et al. (28) previously concluded that
the Tyuleniy group (Table 4), which branched off early after the
vector-borne group split to mosquito-borne and tick-borne clusters, had
the traits typical of mosquito-borne viruses, such as the absence of
hexapeptide insertion, possession of the common glycosylation site in
the envelope gene, and ability to replicate in mosquito cell cultures.
We calculated a pairwise nucleotide sequence identity of 63 to 65%
between the members of different clusters. A higher percentage of
proportional pairwise nucleotide sequence identity could reflect the
close genetic and evolutionary relationship between the members of the
two clusters. As shown in Fig. 6, the
proportion of pairwise nucleotide sequence identities falling in this
range was 20.9% between non-vector and tick-borne clusters, whereas it
was only 1.2% when the non-vector cluster was compared with the
mosquito-borne cluster. On the other hand, when tick-borne and
mosquito-borne clusters were compared, as much as 55.7% of the virus
pairs had a nucleotide sequence identity in this range. Furthermore,
with respect to vector association, while some viruses in the
mosquito-borne cluster, such as SLE, WN, and YF viruses, have been
sometimes isolated from ticks, the reverse observation has been
recorded only in the case of POW virus of the tick-borne cluster. It is
noteworthy that none of the members of non-vector cluster replicated in
mosquito cell culture (46). Thus, the casual association of
mosquito-borne viruses with ticks may be considered a vestigial trait
of the past association with ticks before adaptation to mosquitoes.
Taken together, the observations provide evidence in support of second possibility that the viruses of this genus evolved from non-vector group to tick-borne and then to mosquito-borne group. Exceptions to the
above speculation are Aroa, Entebbe bat, Saboya, and Sokuluk viruses,
which are placed in the mosquito-borne cluster in our phylogram despite
the absence of arthropod vectors. This may be partly due to the lack of
in-depth field investigations to search for arthropod vectors of these
viruses. In fact, all of them are known to replicate in a mosquito cell
culture (46).
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 6.
Pairwise nucleotide sequence identity relationship among
three clusters of the genus Flavivirus. *, Number of pairs
with 63 to 65% nucleotide sequence identity/total number of pair
sequence compared.
|
|
It has been recognized that the genomes of higher vertebrates and birds
are deficient in the frequency of the dinucleotides CpG, which, in
turn, reflect on the biased codon usage containing this dinucleotide. A
review on this subject concluded that "there is a partial but not a
complete correlation between CG content and evolutionary history of
life cycle of different viruses" (39). Among the family
Togaviridae, such a deficit was found in many alphaviruses
(50). Our analysis similarly confirms a strong relationship
between CG deficit and exclusive association with mammalian hosts in
the natural transmission cycle when viruses were evaluated in the
studied NS5 region.
A cline theory has been proposed to describe the correlation of genetic
distance and geographic locations for the so-called TBE complex viruses
(10). Assuming that POW virus was more ancestral, the data
suggested westward movement of TBE complex viruses to Europe across
northern Eurasia. The distribution of the viruses that did not satisfy
the geographic movement, such as Negishi and Kyasanur Forest disease
viruses, was explained by accidental virus transportation by
tick-infested migratory birds. Our results do not support the above
cline theory because of geographic distribution of two additional
members of the TBE complex (clade IV), Gadgets Gully of Australia and
Royal Farm of Afghanistan. Information on geographic distribution of
those viruses used for the basis of the above theory was incomplete.
For example, indigenous transmission of RSSE virus, which had been
previously thought to be confined to eastern parts of Russia, has been
confirmed in Japan (41). Furthermore, both Negishi and
Langat viruses were reported to have been isolated in the former Soviet
Union (36). Regarding the speculations on the origin of
Negishi virus, while the role of migratory birds transporting louping
ill virus to Far East Asia (47) remains a possibility, it is
noted that neutralizing antibody to Negishi virus was detected in
mammals there (44). The other speculation that it was
actually a reference virus used during identification tests as a result
of laboratory contamination or mislabeling (17) is ruled out
for the following reason. The published documents reveal that neither
TBE-CE nor louping ill virus, most closely related to Negishi virus,
was used during virus isolation, passage, and identification phases
(1, 19, 32); rather, RSSE and POW viruses were used for
identification tests.
Division of a genus to subgeneric levels based on molecular sequence
depends on the definition of species. Currently, virus species is
defined as "a polythetic class of viruses constituting a replicating
lineage and occupying a particular ecological niches" (45), a definition which was adopted by the International
Committee on the Taxonomy of Viruses (31). While all
classification systems, including serologic technique and nucleotide
sequence-based classification, are not without problems
(45), combination of those two methods with a minimum amount
of discrepancy between them will improve virus classification based on
polythetic concept of species definition. As far as quantitative
species definition based on nucleotide sequence data is concerned,
criteria used for RNA viruses have been variable. For example,
bluetongue viruses comprising 24 serotypes are considered one species,
while the DEN virus serotypes are considered four distinct species
(31). Furthermore, while species are distinguished at a
64.6% nucleotide sequence identity for members of the arenaviruses
(4), 67 to 77% identity of the NS5 gene has been adopted
for the definition of genotypes within hepatitis C virus
(38). The variation of quantitative criteria for various
levels of taxa reflects partly the difference in the rate of evolution
among different virus groups and partly philosophical difference on the
concept of virus species among virologists (45). In our
study, the classification into clades using 69% pairwise nucleotide
sequence identity between viruses as a criterion agreed well with
grouping of viruses in the phylogram. Similarly, our definition of
>84% pairwise sequence identity as a criterion for species of the
members of the genus Flavivirus agreed with the results
obtained by neutralization testing. For example, Batu Cave virus, which
was shown to be identical to Phnom Penh bat virus according to our
definition, had been withdrawn from registration because it was found
to be identical to the latter virus by a neutralization test
(46). Likewise, THCAr was found to be a subtype of Tembusu
virus both by neutralization (23) and sequence analyses.
The application of our criteria should help resolve the confusing
taxonomic status of the tick-borne encephalitis viruses primarily
isolated in western Eurasia. Although sometimes the Neudoerfl strain is
described as the prototype of central European subtype of "TBE
virus" (49), neither it nor any virus by the name of
tick-borne encephalitis (or TBE) virus has ever been registered (5). In the meanwhile, the number of tick-borne viruses
bearing the name TBE virus proliferated. The recently completely
sequenced louping ill virus as well as Negishi virus, Spanish sheep
tick-borne encephalitis, Turkish sheep tick-borne encephalitis, and
Greek goat encephalitis viruses show more than 83% nucleotide sequence identity in the envelope gene region with either the Neudoerfl or the
Kumlinge strain (16, 18, 29, 52), and they are serologically
indistinguishable (17). The distinction of so-called TBE
viruses into two subtypes (far eastern and central or western European)
did not help to correct the taxonomic problem because of overlapping
geographic distributions (36). Four viruses (Absettarov, Kumlinge, Hanzalova, and Hypr) are registered but considered variants of the same virus by serological classification (5). Thus, it is highly conceivable that when nucleotide sequence data of the
unsequenced viruses are made available for comparison, most (if not
all) of those tick-borne viruses are determined to be variants of one
virus species with its geographic distribution stretching from Far East
Asia to the British Isles and from Scandinavia to the countries along
the Mediterranean, leaving RSSE as a virus distinct from them. The
recently described deer tick virus has a high (>84%) pairwise
nucleotide sequence identity in the NS5-3' untranslated region compared
with POW virus (42). For another virus recently described as
a new tick-borne virus, Vasilchenko strain, similarly no justification
or criteria for classification into a new virus were described
(18). Thus, the species status of each of these viruses
needs to be carefully reexamined. Then, an appropriate strain must be
designated and registered, if not yet done; consequently, all
registered synonyms need to be withdrawn from registration. Whatever
the outcome of reexamination, when nucleotide sequence identity is high
(>80%) compared with a known virus, it is prudent to perform a
neutralization test in two directions rather than relying solely on
sequence data before one attempts to establish a new virus.
Recently, it was reported that Kunjin virus was a member of WN virus
based on short sequence in envelope gene (2). Since those
viruses are distinct species according to our classification, we offer
our thoughts to identify the possible sources of discrepancy. In the WN
virus study above, nucleotide sequence of only one strain of Kunjin
virus, which is well known for its close relation to WN virus, was
compared with sequences of many strains of WN virus for a phylogenetic
study. When only two viruses are compared in a phylogenetic study, it
is not surprising that the sole sequence of one virus (Kunjin) is
automatically grouped in one of the branches (called lineages in the
above study) of the other species, simply because of shared sequence
identity. For a more conclusive study, inclusion of more Kunjin virus
strains, Asian strains of WN virus, and at least one less related
flavivirus is essential, particularly because both viruses are found in
Asia. Second, phylograms generated based on very short sequences (<300
bases) are sometimes different from those generated on much longer
sequences. For example, while only one genotype was identified for
DEN-4 viruses worldwide, using short sequences (8), two
genotypes were identified by using the identical criterion (6%
divergence), virus strains from the same geographic regions, and a much
longer (1.5 kb) sequence of the same viral gene (26). In
this study, phylograms based on short sequences were similar to those
based on 1-kb sequences, but the bootstrap supports at some nodes were
much lower, rendering phylograms unreliable. Thus, a caution was voiced
against the use of such short sequences for phylogenetic studies of
flaviviruses (51). Regardless, more Asian strains of both
viruses are needed to resolve the species status of Kunjin virus,
particularly because a Kunjin virus with intermediate characteristics
with WN virus was reported (34).
Clades established in our study are not exactly comparable to antigenic
complexes in terms of membership (6). For example, Carey
Island virus, formerly a member of TBE complex, is now classified as a
member of the non-vector cluster, while Saboya virus, formerly a member
of the Rio Bravo antigenic complex, now belongs to the mosquito-borne
cluster. The discrepancy between molecular and serologic
classifications partly reflects the difficulty of achieving a 100%
agreement between the two systems based on different principles, given
diversity of the viruses involved. Nevertheless, we believe that our
molecular classification produces the smallest amount of discrepancy
compared with serologic classification and together, the two methods
would greatly improve our understanding of the relationship among the
members of the genus Flavivirus.
| |
ACKNOWLEDGMENTS |
We thank Robert E. Shope, University of Texas, Galveston, for the
gift of Iguape and Kedougou viruses; Thomas G. Ksiazek, Special
Pathogens Branch, National Center for Infectious Diseases, Centers for
Disease Control for Prevention, Atlanta, Ga., for the gift of extracted
RNA of KFD, Omsk HF, and RSSE viruses; and Yuki Eshita, Kurume
University School of Medicine, Kurume, Japan, for providing documents
on Negishi virus.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Arbovirus
Diseases Branch, Division of Vector-Borne Infectious Diseases, National
Center for Infectious Diseases, Centers for Disease Control and
Prevention, P.O. Box 2087, Fort Collins, CO 80522-2087. Phone: (970)
221-6431. Fax: (970) 221-6476. E-mail: GOK1{at}CDC.GOV.
| |
REFERENCES |
| 1.
|
Ando, K.,
K. Kuratsuka,
S. Arima,
N. Hironaka,
Y. Honda, and K. Ishii.
1952.
Studies on the viruses isolated during epidemic of Japanese B encephalitis in 1948 in Tokyo area.
Kitasato Arch. Exp. Med.
24:49-61.
|
| 2.
| Berthet, F.-X., H. G. Zeller,
M.-T. Drouet, J. Rauzier, J.-P. Digoutte, and V. Deubel. Extensive nucleotide changes and deletions within the
envelope glycoprotein gene of Euro-African West Nile viruses. J. Gen. Virol. 78:2293-2297.
|
| 3.
|
Blok, J., and A. J. Gibbs.
1995.
Molecular systematics of the flaviviruses and their relatives, p. 270-289. In
A. Gibbs, C. H. Calisher, and F. Garcia-Arenal (ed.), Molecular basis of virus evolution.
Cambridge University Press, Cambridge, England.
|
| 4.
|
Bowen, M. D.,
C. J. Peters,
J. N. Mills, and S. T. Nichol.
1996.
Oliveiros virus: a novel arenavirus from Argentina.
Virology
217:362-366[Medline].
|
| 5.
|
Calisher, C. H.
1988.
Antigenic classification and taxonomy of flaviviruses (family Flaviviridae) emphasizing a universal system for the taxonomy of viruses causing tick-borne encephalitis.
Acta Virol.
32:469-478[Medline].
|
| 6.
|
Calisher, C. H.,
K. Karabatsos,
J. M. Dalrymple,
R. E. Shope,
J. S. Porterfield,
E. G. Westaway, and W. E. Brandt.
1989.
Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera.
J. Gen. Virol.
70:37-43[Abstract/Free Full Text].
|
| 7.
| Chang, G.-J. J. Unpublished data.
|
| 8.
|
Chungue, E.,
O. Cassar,
M. T. Drouet,
M. G. Guzman,
M. Laille,
L. Rosen, and V. Deubel.
1995.
Molecular epidemiology of dengue-1 and dengue-4 viruses.
J. Gen. Virol.
76:1877-1884[Abstract/Free Full Text].
|
| 9.
|
Cropp, C. B.,
W. C. Prange, and T. P. Monath.
1985.
Le Dantec virus: identification as a rhabodovirus associated with human infection and formation of a new serogroup.
J. Gen. Virol.
66:2749-2754[Abstract/Free Full Text].
|
| 10.
|
de Zanotto, P. M.,
G. F. Gao,
T. Gritsun,
M. S. Marin,
W. R. Jiang,
K. Venugopal,
H. W. Reid, and E. A. Gould.
1995.
An arbovirus cline across the northern hemisphere.
Virology
210:152-159[Medline].
|
| 11.
|
de Zanotto, P. M.,
E. A. Gould,
G. F. Gao,
P. H. Harvey, and E. C. Holmes.
1996.
Population dynamics of flaviviruses revealed by molecular phylogenies.
Proc. Natl. Acad. Sci. USA
93:548-553[Abstract/Free Full Text].
|
| 12.
|
Felsenstein, J.
1993.
.
PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author.
Department of Genetics, University of Washington, Seattle.
|
| 13.
|
Felsenstein, J.
1989.
PHYLIP Phylogeny Inference Package (version 3.2).
Cladistics
5:164-166.
|
| 14.
|
Fulop, L.,
A. D. T. Barrett,
R. Phillpotts,
K. Martin,
D. Leslie, and R. W. Titball.
1993.
Rapid identification of flaviviruses based on conserved NS5 gene sequences.
J. Virol. Methods
44:179-188[Medline].
|
| 15.
|
Fulop, L. D., and A. D. T. Barrett.
1995.
Nucleotide sequence of NS5 gene of Banzi virus: comparison with other flaviviruses.
J. Gen. Virol.
76:2317-2321[Abstract/Free Full Text].
|
| 16.
|
Gao, G. F.,
M. H. Hussain,
H. W. Reid, and E. A. Gould.
1993.
Classification of a new member of the TBE flavivirus subgroup by its immunological, pathogenetic and molecular characteristics: identification of subgroup-specific pentapeptides.
Virus Res.
30:129-144[Medline].
|
| 17.
|
Gould, E.,
P. M. A. Zanotto, and E. C. Holmes.
1997.
The genetic evolution of flaviviruses, p. 51-63. In
J. F. Saluzzo, and B. Dodet (ed.), Factors in the emergence of arbovirus diseases.
Elsevier, Paris, France.
|
| 18.
|
Gritsun, T. S.,
K. Venugopal,
P. M. de Zanotto,
M. V. Mikhailov,
A. A. Sall,
I. Polkinghorne,
T. V. Frolova, and V. V. Pogodina.
1997.
Complete sequence of two tick-borne flaviviruses isolated from Siberia (Vasilchenko strain) and United Kingdom (louping ill): analysis and significance of the 5' and 3' UTRs.
Virus Res.
49:27-39[Medline].
|
| 19.
|
Honda, Y.
1951.
Comparison of the immunological characters of a new virus "Negishi strain" and Russian Spring Summer encephalitis virus (in Japanese).
Kitasato Jikken Igaku
24:91-93.
|
| 20.
| Igarashi, A. Unpublished data.
|
| 21.
| Igarashi, A. Personal communication.
|
| 22.
|
Karabatsos, N.
1985.
.
International catalogue of arboviruses, 3rd ed.
American Society of Tropical Medicine and Hygiene, San Antonio, Tex.
|
| 23.
| Karabatsos, N. Unpublished data.
|
| 24.
|
Kimura, M.
1980.
A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences.
J. Mol. Evol.
16:111-120[Medline].
|
| 25.
|
Kumar, S.,
K. Tamura, and M. Nei.
1993.
.
Molecular evolutionary genetics analysis version 1.01.
The Pennsylvania State University, University Park, Pa.
|
| 26.
|
Lanciotti, R. S.,
D. J. Gubler, and D. W. Trent.
1997.
Molecular evolution and phylogeny of dengue-4 viruses.
J. Gen. Virol.
78:2279-2286[Abstract].
|
| 27.
|
Le Guenno, B.,
A. Bougermouh,
T. Azzam, and R. Bouakaz.
1996.
West Nile: a deadly virus?
Lancet
348:1315[Medline].
|
| 28.
|
Marin, M. S.,
C. W. Mandl,
C. Kunz, and F. X. Heinz.
1995.
The flavivirus 3'-noncoding region: extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virus.
Virology
213:169-178[Medline].
|
| 29.
|
Marin, M. S.,
J. McKenzie,
G. F. Gao,
A. Antoniadis, and E. A. Gould.
1995.
The virus causing encephalomyelitis in sheep in Spain: a new member of the tick-borne encephalitis group.
Res. Vet. Sci.
58:11-13[Medline].
|
| 30.
|
Monath, T. P.
1994.
Yellow fever and dengue-the interactions of virus, vector and host in the re-emergence of epidemic disease.
Semin. Virol.
5:133-145.
|
| 31.
|
Murphy, F. A.,
C. M. Fauquet,
D. H. L. Bishop,
S. A. Ghabrial,
A. W. Jarvis,
G. P. Martelli,
M. A. Mayo, and M. D. Summers (ed.).
1995.
, p. 415-421.
Virus taxonomy: classification and nomenclature of virus
Spring-Verlag, New York, N.Y.
|
| 32.
|
Okuno, T.,
A. Oya, and T. Ito.
1961.
The identification of Negishi virus: a presumably new member of Russian Spring Summer encephalitis virus family isolated in Japan.
Jpn. J. Med. Sci. Biol.
14:51-59[Medline].
|
| 33.
|
Pierre, V.,
M.-T. Drouet, and V. Deubel.
1994.
Identification of mosquito-borne flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction.
Res. Virol.
145:179-188.
|
| 34.
|
Poidinger, M.,
R. A. Hall, and J. S. MacKenzie.
1996.
Molecular characterization of the Japanese encephalitis serocomplex of the Flavivirus genus.
Virology
218:417-421[Medline].
|
| 35.
|
Price, J. L.
1978.
Isolation of Rio Bravo and a hitherto undescribed agent Tamana Bat virus, from insectivorous bats in Trinidad with serological evidence of infection in bats and man.
Am. J. Trop. Med. Hyg.
27:153-161.
|
| 36.
|
Rubin, S. G., and M. P. Chumakov.
1980.
New data on the antigenic types of tick-borne encephalitis (TBE) virus.
Zentralbl. Bakteriol. Suppl.
9:231-236.
|
| 37.
|
Saitou, N., and M. Nei.
1987.
The neighbor-joining method: a new method for reconstructing phylogenetic trees.
Mol. Biol. Evol.
4:406-425[Abstract].
|
| 38.
|
Simmonds, P.,
D. B. Smith,
F. McOmish,
P. L. Yap,
J. Kolberg,
M. S. Urdea, and E. C. Holmes.
1994.
Identification of genotypes of hepatitis C virus by sequence comparison in the core, E1, and NS-5 regions.
J. Gen. Virol.
75:1075-1061.
|
| 39.
|
Strauss, E. G.,
J. M. Strauss, and A. J. Levine.
1990.
Virus evolution, p. 167-190. In
B. N. Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsch, J. L. Melnick, T. P. Monath, and B. Roizman (ed.), Virology, 2nd ed.
Raven Press, New York, N.Y.
|
| 40.
|
Tajima, F., and M. Nei.
1984.
Estimation of evolutionary distance between nucleotide sequences.
Mol. Biol. Evol.
1:269-285[Abstract].
|
| 41.
|
Takashima, I.,
K. Morita,
M. Chiba,
D. Hayasaka,
T. Sato,
C. Takezawa,
A. Igarashi,
H. Kariwa,
K. Yoshimatsu,
J. Arikawa, and N. Hashimoto.
1997.
A case of tick-borne encephalitis in Japan and isolation of the virus.
J. Clin. Microbiol.
35:1943-1947[Abstract].
|
| 42.
|
Telford, S.,
P. M. Armstrong,
P. Katavolos,
I. Foppa,
A. S. Olmeda Garcia,
M. L. Wilson, and A. Spielman.
1997.
A new tick-borne encephalitis-like virus infecting New England deer ticks, Ixodes dammini.
Emerging Infect. Dis.
3:165-170.
[Medline] |
| 43.
|
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680[Abstract/Free Full Text].
|
| 44.
|
Ueda, M.
1980.
A serological survey of cattle and small wild mammals for evidence of tick-borne encephalitis virus infection in Hokkaido, Japan.
Jpn. J. Vet. Res.
28:51-52.
|
| 45.
|
Van Regenmortel, M. H. V.
1990.
Virus species, a much overlooked but essential concept in virus classification.
Intervirology
31:241-254[Medline].
|
| 46.
|
Varelas-Wesley, I., and C. H. Calisher.
1982.
Antigenic relationships of flaviviruses with undetermined arthropod-borne status.
Am. J. Trop. Med. Hyg.
31:1273-1284.
|
| 47.
|
Venugopal, K.,
A. Buckley,
H. W. Reid, and E. A. Gould.
1992.
Nucleotide sequence of the envelope glycoprotein of Negishi virus shows a close homology to Louping ill virus.
Virology
190:515-521[Medline].
|
| 48.
|
Venugopal, K.,
T. Gritsun,
V. A. Lashkevich, and E. A. Gould.
1994.
Analysis of the structural protein gene sequence shows Kyasanur Forest disease virus as a distinct member in the tick-borne encephalitis virus serocomplex.
J. Gen. Virol.
75:227-232[Abstract/Free Full Text].
|
| 49.
|
Wallner, G.,
C. W. Mandl,
M. Ecker,
H. Holzmann,
K. Stiasny,
C. K. Kunz, and F. X. Heinz.
1996.
Characterization and complete genome sequences of high- and low-virulence variants of tick-borne encephalitis virus.
J. Gen. Virol.
77:1035-1042[Abstract/Free Full Text].
|
| 50.
|
Weaver, S. C.,
A. Hagenbaugh,
L. A. Bellew,
S. V. Netesov,
V. E. Volchkov,
G.-J. J. Chang,
D. K. Clarke,
L. Gousset,
T. W. Scott,
D. W. Trent, and J. J. Holland.
1993.
A comparison of the nucleotide sequences of eastern and western equine encephalitis viruses with those of other alphaviruses and related RNA viruses.
Virology
197:375-390[Medline].
|
| 51.
|
Westaway, E. G., and J. Blok.
1997.
Taxonomy and evolutionary relationships of flaviviruses, p. 147-173. In
D. J. Gubler, and G. Kuno (ed.), Dengue and dengue hemorrhagic fever.
CAB International, Wallingford, England.
|
| 52.
|
Whitby, J. E.,
S. N. Whitby,
A. D. Jennings,
J. R. Stephenson, and A. D. T. Barrett.
1993.
Nucleotide sequence of the envelope protein of a Turkish isolate of tick-borne encephalitis (TBE) virus is distinct from other viruses of the TBE virus complex.
J. Gen. Virol.
74:921-924[Abstract/Free Full Text].
|
J Virol, January 1998, p. 73-83, Vol. 72, No. 1
0022-538X/98/$00.00+0
This article has been cited by other articles:
-
Bondre, V. P., Sapkal, G. N., Yergolkar, P. N., Fulmali, P. V., Sankararaman, V., Ayachit, V. M., Mishra, A. C., Gore, M. M.
(2009). Genetic characterization of Bagaza virus (BAGV) isolated in India and evidence of anti-BAGV antibodies in sera collected from encephalitis patients. J. Gen. Virol.
90: 2644-2649
[Abstract]
[Full Text]
-
Cook, S., Moureau, G., Harbach, R. E., Mukwaya, L., Goodger, K., Ssenfuka, F., Gould, E., Holmes, E. C., de Lamballerie, X.
(2009). Isolation of a novel species of flavivirus and a new strain of Culex flavivirus (Flaviviridae) from a natural mosquito population in Uganda. J. Gen. Virol.
90: 2669-2678
[Abstract]
[Full Text]
-
Blitvich, B. J., Juarez, L. I., Tucker, B. J., Rowley, W. A., Platt, K. B.
(2009). Antibodies to West Nile Virus in Raccoons and Other Wild Peridomestic Mammals in Iowa. J Wildl Dis
45: 1163-1168
[Abstract]
[Full Text]
-
Huhtamo, E., Putkuri, N., Kurkela, S., Manni, T., Vaheri, A., Vapalahti, O., Uzcategui, N. Y.
(2009). Characterization of a Novel Flavivirus from Mosquitoes in Northern Europe That Is Related to Mosquito-Borne Flaviviruses of the Tropics. J. Virol.
83: 9532-9540
[Abstract]
[Full Text]
-
Johnson, B. W., Kosoy, O., Hunsperger, E., Beltran, M., Delorey, M., Guirakhoo, F., Monath, T.
(2009). Evaluation of Chimeric Japanese Encephalitis and Dengue Viruses for Use in Diagnostic Plaque Reduction Neutralization Tests. CVI
16: 1052-1059
[Abstract]
[Full Text]
-
Duffy, M. R., Chen, T.-H., Hancock, W. T., Powers, A. M., Kool, J. L., Lanciotti, R. S., Pretrick, M., Marfel, M., Holzbauer, S., Dubray, C., Guillaumot, L., Griggs, A., Bel, M., Lambert, A. J., Laven, J., Kosoy, O., Panella, A., Biggerstaff, B. J., Fischer, M., Hayes, E. B.
(2009). Zika Virus Outbreak on Yap Island, Federated States of Micronesia. NEJM
360: 2536-2543
[Abstract]
[Full Text]
-
Junglen, S., Kopp, A., Kurth, A., Pauli, G., Ellerbrok, H., Leendertz, F. H.
(2009). A New Flavivirus and a New Vector: Characterization of a Novel Flavivirus Isolated from Uranotaenia Mosquitoes from a Tropical Rain Forest. J. Virol.
83: 4462-4468
[Abstract]
[Full Text]
-
Farfan-Ale, J. A., Lorono-Pino, M. A., Garcia-Rejon, J. E., Hovav, E., Powers, A. M., Lin, M., Dorman, K. S., Platt, K. B., Bartholomay, L. C., Soto, V., Beaty, B. J., Lanciotti, R. S., Blitvich, B. J.
(2009). Detection of RNA from a Novel West Nile-like Virus and High Prevalence of an Insect-specific Flavivirus in Mosquitoes in the Yucatan Peninsula of Mexico. Am J Trop Med Hyg
80: 85-95
[Abstract]
[Full Text]
-
Kim, J.-M., Yun, S.-I., Song, B.-H., Hahn, Y.-S., Lee, C.-H., Oh, H.-W., Lee, Y.-M.
(2008). A Single N-Linked Glycosylation Site in the Japanese Encephalitis Virus prM Protein Is Critical for Cell Type-Specific prM Protein Biogenesis, Virus Particle Release, and Pathogenicity in Mice. J. Virol.
82: 7846-7862
[Abstract]
[Full Text]
-
Yoshii, K., Goto, A., Kawakami, K., Kariwa, H., Takashima, I.
(2008). Construction and application of chimeric virus-like particles of tick-borne encephalitis virus and mosquito-borne Japanese encephalitis virus. J. Gen. Virol.
89: 200-211
[Abstract]
[Full Text]
-
Misra, M., Schein, C. H.
(2007). Flavitrack: an annotated database of flavivirus sequences. Bioinformatics
23: 2645-2647
[Abstract]
[Full Text]
-
Medeiros, D. B. A., Nunes, M. R. T., Vasconcelos, P. F. C., Chang, G.-J. J., Kuno, G.
(2007). Complete genome characterization of Rocio virus (Flavivirus: Flaviviridae), a Brazilian flavivirus isolated from a fatal case of encephalitis during an epidemic in Sao Paulo state. J. Gen. Virol.
88: 2237-2246
[Abstract]
[Full Text]
-
Silva, P. A. G. C., Molenkamp, R., Dalebout, T. J., Charlier, N., Neyts, J. H., Spaan, W. J. M., Bredenbeek, P. J.
(2007). Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3' stem-loop structure is not required for replication. J. Gen. Virol.
88: 1738-1747
[Abstract]
[Full Text]
-
Wang, H. Y., Takasaki, T., Fu, S. H., Sun, X. H., Zhang, H. L., Wang, Z. X., Hao, Z. Y., Zhang, J. K., Tang, Q., Kotaki, A., Tajima, S., Liang, X. F., Yang, W. Z., Kurane, I., Liang, G. D.
(2007). Molecular epidemiological analysis of Japanese encephalitis virus in China. J. Gen. Virol.
88: 885-894
[Abstract]
[Full Text]
-
Chao, D.-Y., Davis, B. S., Chang, G.-J. J.
(2007). Development of Multiplex Real-Time Reverse Transcriptase PCR Assays for Detecting Eight Medically Important Flaviviruses in Mosquitoes. J. Clin. Microbiol.
45: 584-589
[Abstract]
[Full Text]
-
Palmer, D. R., Fernandez, S., Bisbing, J., Peachman, K. K., Rao, M., Barvir, D., Gunther, V., Burgess, T., Kohno, Y., Padmanabhan, R., Sun, W.
(2007). Restricted replication and lysosomal trafficking of yellow fever 17D vaccine virus in human dendritic cells. J. Gen. Virol.
88: 148-156
[Abstract]
[Full Text]
-
BENTLER, K. T., HALL, J. S., ROOT, J. J., KLENK, K., SCHMIT, B., BLACKWELL, B. F., RAMEY, P. C., CLARK, L.
(2007). SEROLOGIC EVIDENCE OF WEST NILE VIRUS EXPOSURE IN NORTH AMERICAN MESOPREDATORS. Am J Trop Med Hyg
76: 173-179
[Abstract]
[Full Text]
-
KUNO, G., CHANG, G.-J. J.
(2006). CHARACTERIZATION OF SEPIK AND ENTEBBE BAT VIRUSES CLOSELY RELATED TO YELLOW FEVER VIRUS. Am J Trop Med Hyg
75: 1165-1170
[Abstract]
[Full Text]
-
Grard, G., Lemasson, J.-J., Sylla, M., Dubot, A., Cook, S., Molez, J.-F., Pourrut, X., Charrel, R., Gonzalez, J.-P., Munderloh, U., Holmes, E. C., de Lamballerie, X.
(2006). Ngoye virus: a novel evolutionary lineage within the genus Flavivirus.. J. Gen. Virol.
87: 3273-3277
[Abstract]
[Full Text]
-
Gritsun, T. S., Gould, E. A.
(2006). Direct repeats in the 3' untranslated regions of mosquito-borne flaviviruses: possible implications for virus transmission.. J. Gen. Virol.
87: 3297-3305
[Abstract]
[Full Text]
-
Casati, S., Gern, L., Piffaretti, J.-C.
(2006). Diversity of the population of Tick-borne encephalitis virus infecting Ixodes ricinus ticks in an endemic area of central Switzerland (Canton Bern).. J. Gen. Virol.
87: 2235-2241
[Abstract]
[Full Text]
-
Chien, L.-J., Liao, T.-L., Shu, P.-Y., Huang, J.-H., Gubler, D. J., Chang, G.-J. J.
(2006). Development of Real-Time Reverse Transcriptase PCR Assays To Detect and Serotype Dengue Viruses. J. Clin. Microbiol.
44: 1295-1304
[Abstract]
[Full Text]
-
May, F. J., Lobigs, M., Lee, E., Gendle, D. J., Mackenzie, J. S., Broom, A. K., Conlan, J. V., Hall, R. A.
(2006). Biological, antigenic and phylogenetic characterization of the flavivirus Alfuy. J. Gen. Virol.
87: 329-337
[Abstract]
[Full Text]
-
Carrington, C. V. F., Foster, J. E., Pybus, O. G., Bennett, S. N., Holmes, E. C.
(2005). Invasion and Maintenance of Dengue Virus Type 2 and Type 4 in the Americas. J. Virol.
79: 14680-14687
[Abstract]
[Full Text]
-
Hanna, S. L., Pierson, T. C., Sanchez, M. D., Ahmed, A. A., Murtadha, M. M., Doms, R. W.
(2005). N-Linked Glycosylation of West Nile Virus Envelope Proteins Influences Particle Assembly and Infectivity. J. Virol.
79: 13262-13274
[Abstract]
[Full Text]
-
Kuno, G., Chang, G.-J. J.
(2005). Biological Transmission of Arboviruses: Reexamination of and New Insights into Components, Mechanisms, and Unique Traits as Well as Their Evolutionary Trends. Clin. Microbiol. Rev.
18: 608-637
[Abstract]
[Full Text]
-
BRYANT, J. E., CRABTREE, M. B., NAM, V. S., YEN, N. T., DUC, H. M., MILLER, B. R.
(2005). ISOLATION OF ARBOVIRUSES FROM MOSQUITOES COLLECTED IN NORTHERN VIETNAM. Am J Trop Med Hyg
73: 470-473
[Abstract]
[Full Text]
-
ROOT, J. J., HALL, J. S., MCLEAN, R. G., MARLENEE, N. L., BEATY, B. J., GANSOWSKI, J., CLARK, L.
(2005). SEROLOGIC EVIDENCE OF EXPOSURE OF WILD MAMMALS TO FLAVIVIRUSES IN THE CENTRAL AND EASTERN UNITED STATES. Am J Trop Med Hyg
72: 622-630
[Abstract]
[Full Text]
-
SBRANA, E., TONRY, J. H., XIAO, S.-Y., DA ROSA, A. P. A. T., HIGGS, S., TESH, R. B.
(2005). ORAL TRANSMISSION OF WEST NILE VIRUS IN A HAMSTER MODEL. Am J Trop Med Hyg
72: 325-329
[Abstract]
[Full Text]
-
Nisbet, D. J., Lee, K. J., van den Hurk, A. F., Johansen, C. A., Kuno, G., Chang, G.-J. J., Mackenzie, J. S., Ritchie, S. A., Hall, R. A.
(2005). Identification of new flaviviruses in the Kokobera virus complex. J. Gen. Virol.
86: 121-124
[Abstract]
[Full Text]
-
Crill, W. D., Chang, G.-J. J.
(2004). Localization and Characterization of Flavivirus Envelope Glycoprotein Cross-Reactive Epitopes. J. Virol.
78: 13975-13986
[Abstract]
[Full Text]
-
EBEL, G. D., CARRICABURU, J., YOUNG, D., BERNARD, K. A., KRAMER, L. D.
(2004). GENETIC AND PHENOTYPIC VARIATION OF WEST NILE VIRUS IN NEW YORK, 2000-2003. Am J Trop Med Hyg
71: 493-500
[Abstract]
[Full Text]
-
Mutebi, J.-P., Rijnbrand, R. C. A., Wang, H., Ryman, K. D., Wang, E., Fulop, L. D., Titball, R., Barrett, A. D. T.
(2004). Genetic Relationships and Evolution of Genotypes of Yellow Fever Virus and Other Members of the Yellow Fever Virus Group within the Flavivirus Genus Based on the 3' Noncoding Region. J. Virol.
78: 9652-9665
[Abstract]
[Full Text]
-
Lee, E., Hall, R. A., Lobigs, M.
(2004). Common E Protein Determinants for Attenuation of Glycosaminoglycan-Binding Variants of Japanese Encephalitis and West Nile Viruses. J. Virol.
78: 8271-8280
[Abstract]
[Full Text]
-
Keelapang, P., Sriburi, R., Supasa, S., Panyadee, N., Songjaeng, A., Jairungsri, A., Puttikhunt, C., Kasinrerk, W., Malasit, P., Sittisombut, N.
(2004). Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses. J. Virol.
78: 2367-2381
[Abstract]
[Full Text]
-
Saha, S., Rangarajan, P. N.
(2003). Common host genes are activated in mouse brain by Japanese encephalitis and rabies viruses. J. Gen. Virol.
84: 1729-1735
[Abstract]
[Full Text]
-
Holmes, E. C.
(2003). Molecular Clocks and the Puzzle of RNA Virus Origins. J. Virol.
77: 3893-3897
[Full Text]
-
Chambers, T. J., Liang, Y., Droll, D. A., Schlesinger, J. J., Davidson, A. D., Wright, P. J., Jiang, X.
(2003). Yellow Fever Virus/Dengue-2 Virus and Yellow Fever Virus/Dengue-4 Virus Chimeras: Biological Characterization, Immunogenicity, and Protection against Dengue Encephalitis in the Mouse Model. J. Virol.
77: 3655-3668
[Abstract]
[Full Text]
-
Solomon, T., Ni, H., Beasley, D. W. C., Ekkelenkamp, M., Cardosa, M. J., Barrett, A. D. T.
(2003). Origin and Evolution of Japanese Encephalitis Virus in Southeast Asia. J. Virol.
77: 3091-3098
[Abstract]
[Full Text]
-
Wang, W.-K., Sung, T.-L., Tsai, Y.-C., Kao, C.-L., Chang, S.-M., King, C.-C.
(2002). Detection of Dengue Virus Replication in Peripheral Blood Mononuclear Cells from Dengue Virus Type 2-Infected Patients by a Reverse Transcription-Real-Time PCR Assay. J. Clin. Microbiol.
40: 4472-4478
[Abstract]
[Full Text]
-
de Lamballerie, X., Crochu, S., Billoir, F., Neyts, J., de Micco, P., Holmes, E. C., Gould, E. A.
(2002). Genome sequence analysis of Tamana bat virus and its relationship with the genus Flavivirus. J. Gen. Virol.
83: 2443-2454
[Abstract]
[Full Text]
-
Charlier, N., Leyssen, P., Pleij, C. W. A., Lemey, P., Billoir, F., Van Laethem, K., Vandamme, A.-M., De Clercq, E., de Lamballerie, X., Neyts, J.
(2002). Complete genome sequence of Montana Myotis leukoencephalitis virus, phylogenetic analysis and comparative study of the 3' untranslated region of flaviviruses with no known vector. J. Gen. Virol.
83: 1875-1885
[Abstract]
[Full Text]
-
Charlier, N., Leyssen, P., Paeshuyse, J., Drosten, C., Schmitz, H., Van Lommel, A., De Clercq, E., Neyts, J.
(2002). Infection of SCID mice with Montana Myotis leukoencephalitis virus as a model for flavivirus encephalitis. J. Gen. Virol.
83: 1887-1896
[Abstract]
[Full Text]
-
Huelsenbeck, J. P.
(2002). Testing a Covariotide Model of DNA Substitution. Mol Biol Evol
19: 698-707
[Abstract]
[Full Text]
-
Callahan, J. D., Wu, S.-J. L., Dion-Schultz, A., Mangold, B. E., Peruski, L. F., Watts, D. M., Porter, K. R., Murphy, G. R., Suharyono, W., King, C.-C., Hayes, C. G., Temenak, J. J.
(2001). Development and Evaluation of Serotype- and Group-Specific Fluorogenic Reverse Transcriptase PCR (TaqMan) Assays for Dengue Virus. J. Clin. Microbiol.
39: 4119-4124
[Abstract]
[Full Text]
-
Gaunt, M. W., Sall, A. A., Lamballerie, X. d., Falconar, A. K. I., Dzhivanian, T. I., Gould, E. A.
(2001). Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography. J. Gen. Virol.
82: 1867-1876
[Abstract]
[Full Text]
-
Ebel, G. D., Spielman, A., Telford, S. R. III
(2001). Phylogeny of North American Powassan virus. J. Gen. Virol.
82: 1657-1665
[Abstract]
[Full Text]
-
Scaramozzino, N., Crance, J.-M., Jouan, A., DeBriel, D. A., Stoll, F., Garin, D.
(2001). Comparison of Flavivirus Universal Primer Pairs and Development of a Rapid, Highly Sensitive Heminested Reverse Transcription-PCR Assay for Detection of Flaviviruses Targeted to a Conserved Region of the NS5 Gene Sequences. J. Clin. Microbiol.
39: 1922-1927
[Abstract]
[Full Text]
-
Bhardwaj, S., Holbrook, M., Shope, R. E., Barrett, A. D. T., Watowich, S. J.
(2001). Biophysical Characterization and Vector-Specific Antagonist Activity of Domain III of the Tick-Borne Flavivirus Envelope Protein. J. Virol.
75: 4002-4007
[Abstract]
[Full Text]
-
Regner, M., Lobigs, M., Blanden, R. V., Milburn, P., Mullbacher, A.
(2001). Antiviral Cytotoxic T Cells Cross-Reactively Recognize Disparate Peptide Determinants from Related Viruses but Ignore More Similar Self- and Foreign Determinants. J. Immunol.
166: 3820-3828
[Abstract]
[Full Text]
-
Williams, D. T., Wang, L.-F., Daniels, P. W., Mackenzie, J. S.
(2000). Molecular characterization of the first Australian isolate of Japanese encephalitis virus, the FU strain. J. Gen. Virol.
81: 2471-2480
[Abstract]
[Full Text]
-
Wang, W.-K., Lee, C.-N., Kao, C.-L., Lin, Y.-L., King, C.-C.
(2000). Quantitative Competitive Reverse Transcription-PCR for Quantification of Dengue Virus RNA. J. Clin. Microbiol.
38: 3306-3310
[Abstract]
[Full Text]
-
Chang, G.-J. J., Hunt, A. R., Davis, B.
(2000). A Single Intramuscular Injection of Recombinant Plasmid DNA Induces Protective Immunity and Prevents Japanese Encephalitis in Mice. J. Virol.
74: 4244-4252
[Abstract]
[Full Text]
-
Yang, Z., Nielsen, R., Goldman, N., Pedersen, A.-M. K.
(2000). Codon-Substitution Models for Heterogeneous Selection Pressure at Amino Acid Sites. Genetics
155: 431-449
[Abstract]
[Full Text]
-
Wang, E., Ni, H., Xu, R., Barrett, A. D. T., Watowich, S. J., Gubler, D. J., Weaver, S. C.
(2000). Evolutionary Relationships of Endemic/Epidemic and Sylvatic Dengue Viruses. J. Virol.
74: 3227-3234
[Abstract]
[Full Text]
-
Billoir, F., de Chesse, R., Tolou, H., de Micco, P., Gould, E. A., de Lamballerie, X.
(2000). Phylogeny of the genus Flavivirus using complete coding sequences of arthropod-borne viruses and viruses with no known vector. J. Gen. Virol.
81: 781-790
[Abstract]
[Full Text]
-
Leyssen, P., De Clercq, E., Neyts, J.
(2000). Perspectives for the Treatment of Infections with Flaviviridae. Clin. Microbiol. Rev.
13: 67-82
[Abstract]
[Full Text]
Top
Abstract
Introduction
