J. gen. Virol. (1985), 66, 1323-1325.
Printed in Great Britain
1323
Key words: dengue viruses/eDNA-RNA hybridization/serotypes
Genetic Relationships of the Dengue Virus Serotypes
By J. B L O K
Queensland Institute of Medical Research, Brarnston Terrace, Herston, Brisbane, Queensland
4006, Australia
(Accepted 4 March 1985)
SUMMARY
Previous studies have compared RNA genomes from the different dengue virus
serotypes by c D N A - R N A hybridization using dengue-1 virus- and dengue-2 virusspecific cDNA probes. These probes revealed that there is a close genetic relationship
between dengue virus serotypes 1 and 4. In this communication, the c D N A - R N A
hybridization results using dengue-3- and dengue-4-specific cDNA probes to
determine the genetic relatedness of all four dengue virus serotypes are reported. The
results indicate that serotypes 1 and 4 are genetically very closely related (sharing about
70% of their genomes as detected by both the dengue-1 and dengue-4 CDNA probes), as
are serotypes 3 and 4 (sharing about 50~o of their genomes as detected by both the
dengue-3 and dengue-4 cDNA probes). Serotype 2 does not seem to be very closely
related to the other dengue virus serotypes by c D N A - R N A hybridization analysis.
Flaviviruses are a group of related viruses within the family Togaviridae, which replicate in
arthropod and vertebrate cells. Some of these viruses cause major diseases such as Japanese
encephalitis, yellow fever and dengue haemorrhagic fever in humans. Flaviviruses are
subdivided by their reactions in serological tests. Each virus contains antigenic determinants
common to the group, others which specify a complex of viruses and those which determine
serotype specificity (Trent, t977). There are four dengue virus serotypes which form a discrete
complex of flaviviruses (Porterfield, 1980), and each of these serotypes is associated with a wide
range of clinical manifestations.
Dengue viruses consist of a single-stranded RNA genome (about 12000 nucleotides long), a
capsid protein (C), a low mol. wt. membrane protein (M) and an envelope glycoprotein (E) (for
review, see Schlesinger, 1977). To determine the relatedness of dengue virus RNA genomes
from each of the four serotypes, near full-length serotype-specific cDNA probes were
synthesized and used in c D N A - R N A hybridization experiments. Studies with dengue-l- and
dengue-2-specific cDNA probes were reported previously (Blok et al., 1984) and results with
dengue-3- and dengue-4-specific probes are described below.
Prototype viruses of the four dengue serotypes (dengue-l, Haw; dengue-2, NGC; dengue-3,
H87; dengue-4, H241) were grown in the C6/36 clone of Aedes albopictus cells, and virus particles
extruded into the medium were purified by polyethylene glycol precipitation. Viral RNA was
extracted from the virus pellets and used either as a template for cDNA synthesis in vitro using
an oligo(dT) primer and the enzyme reverse transcriptase, or in c D N A - R N A hybridization
experiments as described earlier (Blok et al., 1984).
In order to synthesize the amount of radioactively labelled cDNA needed for c D N A - R N A
hybridization analyses, the concentrations of dengue-3- and dengue-4-specific RNA, of oligo
(dT)12_18 primer, [32p]dCTP and avian myeloblastosis virus reverse transcriptase were
increased tenfold over that used in previous studies with RNA from dengue-1 and dengue-2
serotypes (Blok et al., 1984). One possible explanation for the necessity to increase the
concentrations of reactants is that the poly(A) or adenosine-rich region at or near the 3' end of
the RNA genomes of dengue-3 and dengue-4 prototype viruses is shorter than that found in the
other two serotypes. The 3' terminal sequences of dengue virus RNA genomes have not yet been
determined but published sequence data from the West Nile and Japanese encephalitis
0000-6488 © 1985 SGM
Short communication
1324
Table 1. Estimated sequence homology among dengue viruses*
R N A used for
c D N A synthesis
Dengue-4
Dengue-3
Dengue-2
Dengue-1
(H241)
(H87)
(NGC)'J"
(Haw)t
c
Dengue-4
100
52
23
73
R N A used in hybridization reaction
~
Dengue-3
Dengue-2
Dengue-1
C6/36
55:~
100
24
38
34
34
100
42
73
30
36
100
9
1l
-
Yeast
11
10
10
17
* c D N A R N A hybrids were formed and assayed with $1 nuclease as described in the text and the estimated
percentage sequence homology was calculated as described (Gould & Symons, 1977; Gonda & Symons, 1978).
t From Blok et al. (1984).
:~ Each percentage represents an average of at least five experiments.
flaviviruses suggest that they do not contain a poly(A) tail (Wengler & Wengler, 1981 ; Takegami
et al., 1984). However, there is a purine-rich region near the 3' end of the yellow fever virus
genome (C. M. Rice, personal communication) and if this occurs in dengue viruses, it may be
long enough to bind an oligo(dT) primer with variable efficiency.
By optimizing the in vitro c D N A synthesis conditions, a sufficient amount of dengue-3- and
dengue-4-specific c D N A probes was produced to study the genetic relationship of all dengue
virus serotypes by c D N A - R N A hybridization. These cDNA hybridization probes were similar
in electrophoretic mobilities to viral RNA on polyacrylamide gels, and therefore represented
near full-length genome copies. The probes were purified by electroeluting the cDNA from the
region of the gel corresponding to the viral RNA.
The c D N A - R N A hybrids were formed under stringent conditions in buffer containing 0.18
M-NaC1 at 65 °C for 20 h, essentially as described by Gould & Symons (1977). The hybrids
formed were treated with 100 units/ml $1 nuclease at 45 °C for 30 min in buffer containing 0.08
M-NaCI. Rot curves were determined for dengue-3 and dengue-4 c D N A probes and a log10 Rot
value of 1 was used in all hybridization experiments. The results of these hybridizations have
been combined with those obtained with the dengue-l- and dengue-2-specific cDNA probes
(Table 1) and reveal that (i) the background of $1 nuclease-resistant hybrids formed with nonhomologous RNA (either yeast or C6/36 cellular nucleic acid) is about 10~, (ii) there is a very
close genetic relationship between dengue-1 and dengue-4 since a 7 3 ~ sequence is detected by
both dengue-l- and dengue-4-specific cDNA probes and (iii) there is an average sequence
homology of 5 4 ~ between dengue-3 and dengue-4.
There is some evidence of a serological subcomplex of dengue virus serotypes 1 and 3 (Russell
& N isalak, 1967; Henchal et al., 1982), as well as a subcomplex of serotypes 2 and 4 as detected
by monoclonal antibodies (E. A. Henchal, personal communication). However, there does not
need to be a correlation between serology and nucleic acid hybridization since the antigenic
determinants are located on the major virus glycoprotein (E), which is encoded by about 15~ of
the viral RNA genome. The close genetic relationship between serotypes 1 and 4, and 3 and 4,
and the lack of a close sequence homology of serotype 2 with any other dengue virus serotype
suggest that the serotypes may differ in their evolutionary origins.
! would like to thank Fiona Crone for expert technical assistance and Drs B. M. G o r m a n and J. H. Pope for
commenting on this communication. This work was supported by a grant from the National Health and Medical
Research Council, Canberra.
REFERENCES
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cDNA-RNA hybridization analysis and detection of a close relationship between dengue virus serotype 2
and Edge Hill virus. Journal of General Viro/ogy 65, 2173-2181.
OONDA, T. J. & SVMO~S, R. ft. (1978). The use of hybridization analysis with complementary D N A to determine the
RNA sequence homology between strains of plant viruses: its application to several strains of cucumoviruses.
Virology 88, 361 370.
Short communication
1325
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(Received 10 December 1984)