Molecular evolution and distribution of dengue viruses type 1 and 2 in nature

VIROLOGY

174,479-493

(1990)

Molecular

Evolution and Distribution

of Dengue Viruses Type 1 and 2 in Nature

REBECA RICO-HESSE’ Department

of Epidemiology

and Public Health, Yale University School of Medicine, ReceivedAugust

11, 1989; acceptedoctober

60 College St., New Haven, Connecticut

065 10

11, 1989

During the past several decades, dengue viruses have progressively extended their geographic distribution, and are currently some of the most important mosquito-borne viruses associated with human illness. Determining the genetic variability and transmission patterns of these RNA viruses is crucial in developing effective control strategies for the disease. Primer-extension sequencing of <39/o of the dengue genome (across the E/NSl gene junction) provided sufficient information for estimating genetic relationships among 40 dengue type 1 and 40 type 2 virus isolates from diverse geographic areas and hosts. A quantitative comparison of these 240-nucleotide-long sequences revealed previously unrecognized evolutionary relationships between disease outbreaks. Five distinct virus genotypic groups were detected for each of the two serotypes. The evolutionary rates of epidemic dengue viruses of types 1 and 2 were similar, although the transmission pathways of these viruses around the world are different. For dengue type 2, one genotypic group represents an isolated, forest virus cycle which seems to have evolved independently in West Africa. This is the first genetic evidence of the existence of a sylvatic cycle of dengue virus, which is clearly distinct from outbreak 0 1990 Academic Press, Inc. viruses.

ing the genetic variability and worldwide distribution of these RNA viruses. Viruses have been traditionally classified into serotypes, on the basis of their antigenic characteristics. A more sensitive means of classifying viruses is the comparison of nucleotide or gene sequences that share a common ancestry; from such data, phylogenetic trees can be inferred quantitatively (Zuckerkandl and Pauling, 1965). Because of the high degree of fixation of mutation in the viral RNA genome, often only a small portion of a gene or genes needs to be compared to infer evolutionary relationships. Primer-extension sequencing of a short region of the viral RNA genome has been used previously in the study of poliovirus epidemiology (Rico-Hesse et al., 1987). Primer extension has the major advantage of directly determining the nucleotide sequences and thereby identifying also the location of the differences among strains, something that is not available by the currently used T, oligonucleotide fingerprinting methods. Furthermore, fingerprinting and monoclonal antibodies that recognize serotype-, group-, or family-specific epitopes do not identify or detect differences that are phenotypically silent. Thus, the evolutionary and epidemiologic relationships between many isolates of the same virus become apparent only by comparing nucleotide sequences. The research described here demonstrates the use of this highly productive approach on a RNA virus with a relatively low mutation rate (Repik et al,, 1983; Trent et a/., 1983; Blok et al., 1989; Chu et a/., 1989).

INTRODUCTION Dengue virus, a mosquito-borne Flavivirus, is responsible for a growing health problem in much of the tropical world. Dengue viruses produce a spectrum of illness in humans, varying from a flu-like disease (dengue fever) to a fulminating illness, dengue hemorrhagic fever (DHF), which can progress to dengue shock syndrome (DSS) and death. The known geographic range of dengue includes the Americas, Africa, Asia, and the South Pacific, following the distribution of its mosquito vectors. In terms of human morbidity, dengue fever is currently one of the most important mosquito-transmitted viral diseases; millions of persons are infected annually (Institute of Medicine, 1986; Halstead, 1988). Not only is the incidence of dengue fever on the rise, but the more severe forms of the disease (DHWDSS) have recently appeared in the Americas (Kouri et a/., 1986). Introduction of virus strains from dengue endemic regions has resulted in epidemics with the subsequent establishment of the virus in ecosystems where the disease had not occurred before (Gubler et a/., 1978; Pan American Health Organization, 1979; Centers for Disease Control, 1988). It is presently unknown whether specific virus genotypes are responsible for the more severe forms of the disease or for particular widespread epidemics. Therefore, important information for the design of vaccines and strategies for control of the disease should come from understand’ To whom requests for reprints should be addressed.

479

0042-6822/90

$3.00

CopyrIght Q 1990 by Academic Press, Inc. All rtghts of reproduction in any form reserved.

REBECA RICO-HESSE

480

The analysis of short genomic sequences was adapted to dengue viruses so as to extend the epidemiologic range of molecular methods for their identification. Dengue viruses of serotypes 1 and 2 were chosen because they are associated with the highest amount of morbidity and mortality. Eighty dengue type 1 and 2 (DEN-l and DEN-2) viruses, isolated over a 45-year span from humans and mosquitoes in 36 countries, were studied by primer-extension sequencing, using the viral RNA as template. A quantitative comparison of 240 nucleotides from the E/NSl junction (2.2% of the total genome) revealed previously unknown evolutionary relationships between disease outbreaks and the first genetic evidence of a distinct jungle cycle of dengue virus. The expansion of this data base will allow us to rapidly determine the geographic origin of new outbreak viruses and to monitor the effectiveness of control measures. MATERIALS

AND METHODS

Viruses

Dendrogram

The 80 dengue virus isolates analyzed by primer-extension sequencing are listed in Tables 1 and 2. Most viruses were obtained from the Yale Arbovirus Research Unit reference collection and were donated previously by numerous laboratories. All isolates were propagated once in C6/36 (Aedes albopictus) cell monolayers, to prepare high-titered stocks. Virus stocks were identified according to serotype by indirect fluorescent antibody tests (Tesh, 1979) with type-specific monoclonal antibodies obtained from Dr. N. Karabatsos, Centers for Disease Control (Fort Collins, CO). Virus growth, purification,

DNA synthesizer (Applied Biosystems, Foster City, CA) and were used without further purification. Aliquots were tested to confirm oligonucleotide length. A total of 12 primers, hybridizing to 6 different genomic intervals, were used in sequencing reactions: D2/262, a 22mer, 5’-CTGTTGGTGGGATTGTTAGGAA-3’; D2/2452, a 17-mer, 5’-CCACAllTCAGTTCllT-3’; D2/2578, a 25-mer, 5’-TTACTGAGCGGATTCCACAGATGCC-3’; D2/2773, an 18-mer, 5’-CAlllXGCllTACCCCA-3’; D2/3015, a 15-mer, 5’-TCTATCCAGTACCCC-3’; and D2/3349, a 23-mer, 5’-GTGCAAGATCGGCAGCACCATTC-3’. Numbers indicate the map site at which the 5’ end of the oligonucleotide hybridizes on the DEN-2 vaccine strain, PR159Sl (Hahn et a/., 1988). The remainderwere DEN-l primers of the same length, made to hybridize to the corresponding site on the Nauru strain, 17672 (Mason et a/., 1987). The 240-nucleotide-long sequences compared among many strains were obtained with two primers, D2/2452 and D2/ 2578 (DEN-l version: D1/2423 and D1/2549).

and sequencing

Dengue viruses were grown in C6/36 cells (m.o.i.:
of sequence similarity

A dendrogram of virus relationships, in effect a phylogenetic tree without a time coordinate, was generated with the NUCLDIFF program and is available from Dr. Mark A. Pallansch (Division of Viral Diseases, Centers for Disease Control, Atlanta, GA). It is based on the algorithm described by Fitch and Margoliash (1967). This program calculated a difference table for the strains by performing all pairwise sequence comparisons. Since all sequences were colinear, each nucleotide difference between strains was assigned an equivalent statistical weight. From the difference table, through a binary agglomerative procedure, a tree was constructed that graphically presents the degree of nucleotide sequence divergence. RESULTS Selection

of the genomic interval for comparisons

To select the region showing the highest rate of mutation, nucleotide sequence variation among 5 DEN-l strains and 5 DEN-2 strains was determined for several dengue virus gene intervals. The dengue virus genome is single-stranded RNA of messenger sense, of approximately 11 -kb in length, and encodes 10 distinct proteins. The gene order is 5’-C-prM(M)-E-NSl -NS2ANS2B-NS3-NS4A-NS4B-NS5-3’ (Speight and Westaway, 1989; Wright et al., 1989). The first three proteins are structural components of the virion while the remainder are found on the infected cell surface (NSl) or as intracellular proteins involved in virus replication.

EVOLUTION

OF DENGUE VIRUSES

Portions of three functional domains of the dengue virus genome were explored by primer-extension sequencing: the capsid, C, the envelope, E, and the nonstructural protein, NSl. A total of approximately 1500 nucleotides of sequence information were obtained for each of the 10 dengue strains analyzed initially (results not shown). The E/NSl gene junction of the genome was chosen as the best source of nucleotide sequences for comparison among strains of the same serotype and across serotypes 1 and 2, to derive evolutionary information which could be interpreted epidemiologically. This choice was based on the following: (a) This area of the genome showed a uniform rate of random mutation, with no hypervariable regions that might be affected by immune selection of epitopes. An attempt was made to avoid regions evolving rapidly (e.g., with reversions) because of immune selection which might be applied by individual hosts along the chain of transmission, thus obscuring long-term evolutionary trends. (b) The large majority of the mutations in this region occurred in the third position of the codon and are mostly silent, probably random mutations which provide no selective advantages. This junction at functional domains shows evolutionary characteristics similar to those of the poliovirus junction VP1/2A, the target region chosen in previous sequencing studies (Rico-Hesse et a/., 1987) although the mutation rate was much lower for dengue viruses (9-16 vs 229/o). Previous evolutionary studies have used only third position changes for the genetic comparison of strains (Zuckerkandl and Pauling, 1965). (c) Complete E gene nucleotide sequences of type 1 and 2 viruses obtained by other workers (Blok et al., 1989; Chu et a/., 1989) have shown this region to vary (maximum divergence of 109/o)in a nonuniform manner, with genetic relationships among strains difficult to interpret. “Hot spots” or intervals of high nucleotide variation seemed to occur in areas encoding epitopes and these regions probably mask the random mutation events that may give clues to evolutionary relationships. Limited sequencing studies performed here confirmed these results; many changes occurred in the first and second positions of the codon. Only the Cterminus sequences of this gene showed few amino acid changes. (d) Sequences from various-areas of the NSl gene (four primer-extension reactions) showed characteristics similar to those of the E gene. Nucleotide differences across the center of the gene coded for changes at the amino acid level and were interspersed with si-

481

lent, third position mutations. Only the 5’ end of NSl showed the desired characteristics. (e) Nucleotide sequences encoding a portion of the capsid gene showed very little variation (maximum of 6%) across strains within a serotype, confirming the need for structural conservation. Apparently this region does not tolerate third position changes; this suggests that RNA secondary structure may also be constrained within this area. Sequences of the E/NSl junction dengue viruses

of type 1 and 2

The molecular evolution of the E/NSl region of dengue viruses in nature and under laboratory conditions became evident from the comparison of nucleotide sequences from 80 different viruses. Sequence information from the E/NSl region of all viruses listed in Tables 1 and 2 are shown in Figs. 1 and 2, respectively. When an interval of 240 nucleotides (1 1 1 from the 3’ end of E and 129 from the 5’ end of NSl) from numerous dengue isolates was compared to a prototype strain (Nauru Island for DEN-l and Puerto Rico vaccine strain for DEN-2) substantial divergence was found within serotypes. Maximum divergence over this region was approximately 9% for type 1 viruses, while for type 2 strains it was 16%. Sequence divergence across the two serotypes was approximately 3 1%; thus, isolates also fell into two distinct serotype groups when comparing nucleotide sequences from this region of the genome. The size of the interval used for comparison among strains was chosen based on the rate of variation. It was necessary to detect a significant number of nucleotide differences among strains to be able to distinguish them quantitatively and to improve the resolution of the dendrogram (see below). The comparison of the PR159Sl vaccine strain and its parent, PR159, demonstrated that no mutations were fixed in this area of the genome after 17 passages in vitro. Other viruses which had been grown numerous times in mice also maintained the characteristics or consensus sequence of viruses from the same geographic area (e.g., Mochizuki, TR1751, and HD10674 strains). Their respective parent viruses are unavailable for comparison, so the actual effect of passage in viva cannot be measured. The rate of fixation of mutations during natural passage could not be calculated from these data because of the lack of sequential isolates from the same individual or geographic location, but the prevalence of mutants and their geographic distribution was evident. The deduced amino acids (80) encoded by these nucleotide sequences differed at few sites when com-

REBECA RICO-HESSE

482

TABLE 1 DENGUETYPE 1 ISOLATESCOMPAREDBYGENOMIC SEQUENCEANALYSIS Strain Mochizuki Hawaii 691475 IBH28326 094 106 16299 228682 222683 228686 777849 228690 1186 1236 IBH13689 DAK29 177 1298 PUO-359 D81-135 816879 Tl4 CSl 1310/82 1344 1351 1413 1412 1378 347869 ArA15120” CEAt47 766602 765101 351094 1916 027 28973 36589 779172 2000

Passage history’ MOUSE 176, SM.4 MONK.1, MOSQ.6, C 3 SM.2, MOSQ.2, MK2 1 SM.21, C6/36 2 MOSQ. 1, C6/36 2 MOSQ. 1, C6/36 2 MOSQ.?. C6/36 1 MOSQ.?, C6/36 1 MOSQ.4, C6/36 1 MOSQ.?, C6/36 1 AP61 l,MK21,C3 MOSQ.?, C6/36 1 MOSQ.2, C6/36 1 MOSQ.2, C6/36 1 SM.8 MOSQ. 1, C6/36 1 MOSQ.2, C6/36 1 C6/36 1 MK2 3, C6/36 4 NONE C6/36 1 C6/36 3 MK2 2, C6/36 2 MOSQ.2, C6/36 1 MOSQ.2, C6/36 1 MOSQ.2, C6/36 1 MOSQ.2, C6/36 1 MOSQ.2. C6/36 1 C6/36 3 C6/36 1 ? C6/36 2. AP61 1 C6/36 3 C6/36 3 MOSQ. 1 AP61 2 C6/36 8 C6/36 2 C6/36 3 MOSQ. 1

Location

Year

Donorb

Nagasaki, Japan Hawaii Sri Lanka Nigeria Bangkok, Thailand Bangkok, Thailand Nauru Manila, Philippines Fiji Burma Bahamas Jamaica Jakarta, Indonesia Jakarta, Indonesia Nigeria Bandia, Senegal Mexico Bangkok, Thailand Bangkok, Thailand Surinam Thursday Island. Australia Cairns, Australia Kuala Lumpur, Malaysia Mexico Colombia Haiti Mexico Mexico Caqueta. Colombia Ivory Coast Ceara, Brazil Kaohsiung, Taiwan Tungkang, Taiwan Guaviare, Colombia El Salvador Manila, Philippines Brazil Angola Kaohsiung, Taiwan Paraguay

1943 1945 1969 1968 1973 1974 1974 1974 1975 1976 1977 1977 1977 1978 1978 1979 1980 1980 1981 1981 1981 1982 1982 1982 1982 1983 1983 1983 1985 1985 1986 1987 1987 1987 1987 1988 1988 1988 1988 1988

YARU LR PR PR SCK SCK YARU YARU YARU YARU PR YARU YARU YARU YARU JPD YARU YARU PR BH BHK BHK PR YARU YARU YARU YARU YARU JB JPD ATR YCW YCW JB DG CH HS HS YCW DG

“Abbreviations used: AP61, Aedes pseudoscutellaris cell line: C or C6/36. A. albopicfus cell line; MK2, (LLC-MK2) monkey kidney cell line; MONK., rhesus monkey; MOSQ., whole mosquito; NONE, acute serum of patient; SM., suckling mouse brain. b Donors of specimens or isolates are J. Boshell, J. P. Digoutte. D. Gubler. C. Hayes, B. Hull, B. H. Kay, S. C. Kliks, P. Repik, L. Rosen, H. Schatzmayr, A. Travassos daRosa, Yale Arbovirus Research Unit, Y. C. Wu. c Mosquito isolate, from A. aegypti. All others are from human serum.

pared to their prototype viruses (Figs. 3 and 4). The large majority (>70%) of nucleotide differences between strains were therefore silent. The 37 amino acids from the E gene which were compared here form internalized, mainly hydrophobic domains, while the 43 amino acids from the NSl gene form both hydrophobic and hydrophilic domains of their respective proteins (Smith and Wright, 1985; Putnak et a/., 1988; Winkler

era/., 1989). None of the amino acid differences in this region caused significant changes in the overall hydrophobicity of these protein domains (results not shown), suggesting that the primary amino acid sequence is not as important as maintaining the tertiary structure of the protein. For DEN-l viruses, an isolate from Sri Lanka showed the maximum number of amino acid differences, 8/80 (1 O%), while the remaining strains

EVOLUTION

OF DENGUE VIRUSES

483

TABLE 2

Strain NewGuineaC TR1751 16681 PR159 PR159Sl HD10674 28741 NC9163 8720 1251 ArD2076 1 c 8730 1232 DakA578” ArA5iOC ArA2022” UV2039” PM33974C 1318 8110827 PL-046 PL-001 1334 1353 1329 JAH 1349 PhH2172 516 044 975 ArA6894” 348600 350447 24H 57s VEN2 766635 028 351863

Passage histo@

Location

Year

Donor*

MONK.11 MOSQ.4, r SM.57 MK2 1, C6/36 5 PGMK 6 PGMK 19, FRhL2 4 SM.25, C6/36 1 MOSQ. 1 C6/36 1 MOSQ.?, C6/36 1 MOSQ.2 SM.8, C6/36 1 MOSQ.?, C6/36 1 MOSQ.2, C6/36 2 SM.8, C6/36 1 SM.4, C6/36 1 SM.6, C6/36 1 SM.6, C6/36 1 MOSQ.1, C6/36 1 MOSQ.2, C6/36 1 NONE C6/36 3 C6/36 3 MOSQ. 1, C6/36 2 MOSQ. 1, C6/36 2 MOSQ. 1, C6/36 2 C6/36 2 MOSQ. 1, C6/36 2 AP61 2, C6/36 1 MOSQ. 1, C6/36 2 C6/36 2 C6/36 3 ?, C6/36 1 C6/36 3 C6/36 3 C6/36 1 C6/36 1 AP61 2 C6/36 4 AP61 2 C6/36 3

New Guinea Trinidad Thailand Puerto Rico Puerto Rico Bandia, Senegal Tahiti New Caledonia Java, Indonesia Tonga Kedougou. Senegal Seychelles Jakarta, Indonesia Ivory Coast Ivory Coast Burkina Faso Burkina Faso Republic of Guinea Puerto Rico Jamaica Tungkang, Taiwan Liouchyou, Taiwan Colombo. Sri Lanka Colombo, Sri Lanka Jamaica Jamaica Burkina Faso Manila, Philippines Thailand Mexico Sri Lanka Burkina Faso Tumaco, Colombia Colombia Hanoi, Vietnam Saigon, Vietnam Venezuela Kaohsiung, Taiwan Manila, Philippines Tolima. Colombia

1944 1954 1964 1969 1969 1970 1971 1972 1973 1974 1974 1977 1978 1980 1980 1980 1980 1981 1981 1981 1981 1981 1981 1982 1982 1982 1982 1983 1983 1983 1985 1986 1986 1987 1987 1987 1987 1987 1988 1988

YARU YARU YARU KE KE JPD LR YARU YARU DG JPD YARU YARU JPD JPD JPD JPD JPD YARU BH YCW YCW YARU YARU YARU RBT YARU YARU SCK SCK SCK JPD JB JB HTN DQH FB YCW CH JB

“Abbreviations used: AP61, Aedes pseudoscutellaris cell line; C or C6/36, A. albopicfus cell line; FrhL2, fetal rhesus monkey lung cell line; MK2, (LLC-MK2) monkey kidney cell line; MONK., rhesus monkey; MOSQ., whole mosquito; NONE, acute serum of patient; PGMK, primary green monkey kidney cells; SM., suckling mouse brain. b Donors of specimens or isolates are F. Belle, J. Boshell, J. P. Digoutte, K. H. Eckels. D. Gubler, D. Q. Ha, C. Hayes, B. Hull, B. H. Kay, S. C. Kliks, H. T. Nguyen, P. Repik, L. Rosen, H. Schatzmayr, R. B. Tesh, A. Travassos daRosa, Yale Arbovirus Research Unit, Y. C. Wu. ’ Mosquito isolates, beginning at top of list, from A. luteocephalus, A. taylori, A. taylori, A. africanus, A. luteocephalus, A. africenus. A. aegypti. All others are from human serum.

showed few changes. For DEN-2 viruses, amino acid differences were limited to 6180 (7.5%) but this maximum occurred in many more strains (see West Africa group, Indonesia, and Philippines). Few evolutionary relationships could be established by comparing amino acid sequences, mainly because they were conserved within a serotype.

Genetic relationships dengue viruses

among type 1 and 2

Genetic relationships were quantitated by a computer program that performed all pairwise comparisons of the selected genomic interval. In this manner, any bias introduced by comparison to a single reference

REBECA RICO-HESSE

484 A 16299 222683 228682 027 1186 CSl 114 1298 1236 HAYAl 1 094

ET

22a2 NAUR 74

2401

AUAGCCAWCUGWGACAUGGCUAGGAW~CUCCCUCGGGA

,c,J, 75 _.---_____-.--__.-.-------~~~---------.-~~._.___._______.____-_____________________.----~~~~-----~---.--.~*~.-----.-~--. PHIL PHIL I ND0 AUST AUST UEXI I ND0

1344 1378 347869 777849 228690 1351 1412 816879

HEX1 UEXI

82 83 COLO 85 BANA 77 77 JAM COLO 82 HEX1 83 SURI 81 COLO 07 351094 28973 BRA2 88 1413 HAIT 63 CEA147 BRA2 66 36589 ANGO 88 1916 SAL” a7 2000 PARA a8 IS,,13689 NIGE 78 DAK29177 SENE 79 lRH28326 NIGE 68 ARA15120 IVOR 85 1310/82 MALI 82 081-135 THAI 81 228686 EURM 76 106 THAI 74 691475

SR,L

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FIG. 1. Comparison of nwcleotide sequences at the E/NSl gene junction of 40 type 1 dengue virus isolates, obtained from 1943 to 1988, from 26 different countries. Sequencing was performed by extension of two synthetic DNA primers with reverse transcriptase in the presence of [a35S]dATP and dideoxynucleoside triphosphates as described under Materials and Methods. Strain numbers are listed first, followed by abbreviations for country of isolation (see Fig. 5). and the last two digits of year of isolation. Nucleotide differences from 16299, a virus isolated during an outbreak on Nauru Island, are shown; dashes indicate identities. Nucleotide positions are numbered according to Mason et a/. (1967). The 240 nucleotides shown constitute the total sequence information used for each strain to construct the dendrogram in Fig. 5, and have been arranged into genotypic groups.

strain was eliminated; in addition, genetic relationships were visualized by plotting differences as a dendrogram (Figs. 5 and 6). When genomic intervals with lowervariation rates were compared, only the abscissa of the dendrogram changed, leaving the broad genetic relationships between strains intact (unpublished observations). In other words, the resolution between genotypic groups diminished in proportion to the rate of variation, but the genetic relationships were similar. This agreed with previous studies of poliovirus evolution (Rico-Hesse eta/., 1987). No instances of discontinuities in evolution across the E/NSl junction were de-

tected here, as they were for the VP1/2A junction of several wild type 1 polioviruses. Thus, intramolecular recombination does not seem to occur in this region of the dengue virus genome. Because the rate of variation of dengue viruses in nature seems to be lower than that seen in other RNA viruses (e.g., Buonagurio et al., 1986; Rico-Hesse et al., 1987; Nichol eta/., 1989) a lower arbitrary value for the definition of a genotype was chosen here. In this comparison, a genotype is defined as a group of dengue viruses having no more than 6% sequence divergence within the chosen interval (E/NSl junction). This

EVOLUTION

16299

Wl"R

74

222683

FIJI

75

228682

PHIL

74

027

PHIL

88

1186

I NO0

77

CSl

AUST

82

714

*ius,

81

1298

MEXl

80

1236

1NO0

78

HAUAl I

HAVl

45

094

TM,

73

tlOC”l2.

JWA

43

PUO-359

THAI

80

766602

TAlV

87

765101

T*IY

87

779172

,Al!A

88

1344

WEX I

82

....

..~..........~...-..........-......--”.-...-....-.-.“....-...-.....“.-..-..-.-.-..C..-...-----”..~.~.--~~.~~~~~~~~

1378

MEXl

83

....

..G..........G..................-...-”-......-......“.-............“..-.....---...C--.--.-...-”.--..-.-~-.*-----o~-

347869

COLO

85

........

OF DENGUE VIRUSES

485

.. .

..-....-.~...-..-.......~.-...-..”...-.-..-.-.-.“..-......~....”......--.---.-C---.--.----”~.--...~~--~.~.~~~

777849

BAHA

77

..............

228690

JAM

77

.............

1351

COLO

82

...............

1412

"EXI

83

.........

816879

SUR I

81

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351094

COLO

87

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28973

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823

...............

...

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.

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.

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1413

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83

...............

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.

cm147

BRA2

86

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.

36589

ANGO

88

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1916

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87

...............

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2000

PARI\

88

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... .. ....

18813689

LlltE

78

...............

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...

DAK29177

SENE

79

...............

..~...........~...........“..-...........”..............“..........-...C.-....-....“...-...-..-~..-.-~

...

IBH28326

NI‘E

68

...............

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...

ARAl5120

,"OR

85

...............

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...

..~................-......“.....C..~.....”..............“..........................“........~..~

1310182

MALA

82

...............

Cal-135

THAI

81

...

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228686

8"R"

76

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106

TH.41

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691475

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FIG. 1-Continued

limit was based on the clustering of isolates, for which linkages would be expected on epidemiological grounds. This segregation was especially obvious in the DEN-2 dendrogram (Fig. 6). When 6% difference was taken as a cutoff point for virus relationships, five genotypic groups were discerned for each of the two serotypes. For DEN-l viruses, the first group contained viruses from the Americas, Africa, and Southeast Asia; the second was composed of one isolate from Sri Lanka; the third was also composed of one isolate, Japan 1943; the fourth included strains from Southeast Asia, the South Pacific, Australia, and Mexico; and the fifth group contained viruses from Taiwan and Thailand. For DEN-2 viruses, the first group included viruses from the Caribbean and South Pacific; the second contained isolates from Taiwan, the Philippines, the New Guinea prototype virus, and an older Thai strain; the third included Vietnamese, Jamaican, and

Thai strains; the fourth contained isolates from Indonesia, the Seychelles, Burkina Faso, and Sri Lanka; and the fifth group included isolates from rural Africa. The extent of genetic variability across this region of the virus genome seems to be independent of host origin or laboratory passage level, and genetic relationships were detectable after at least 34 years of natural evolution (e.g., for DEN-2, Trinidad 1954 to Colombia 1988). Distribution

of type 1 and 2 dengue viruses in nature

The worldwide transmission of dengue viruses could be followed by comparing relatively short nucleotide sequences. The dendrogram gave evidence of previously unknown evolutionary relationships among viruses over a vast geographic range and time period. Several examples are discussed in detail here, to demonstrate the applicability of this technique to understanding dengue virus epidemiology.

REBECA RICO-HESSE

486

1232 8720 8730 1349 ArA6894 975 1334 1353

FIG. 2. Comparison of nucleotide sequences at the E/NSl gene junction of 40 type 2 dengue virus isolates, obtained from 1944 to 1988, from 21 different countries. Sequencing was performed by extension of two synthetic DNA primers with reverse transcriptase in the presence of [a%]dATP and dideoxynucleoside triphosphates as described under Materials and Methods. Strain numbers are listed first, followed by abbreviations for country of isolation (see Fig. 6). and the last two digits of year of isolation. Nucleotide differences from PR159S1, an attenuated virus derived by serial passage of isolate PR159 (Eckels et a/., 1976), are shown; dashes indicate identities. Nucleotide positions are numbered according to Hahn et a/. (1988). The 240 nucleotides shown constitute the total sequence information used for each strain to construct the dendrogram in Fig. 6, and have been arranged into genotypic groups.

Although the direction of spread of dengue viruses is not obvious from the genetic relationships shown here, the route of transmission could be inferred by using classical epidemiologic observations. For example, the close relationship between DEN-2 isolates from Taiwan and the Philippines suggested an origin for recent Taiwanese dengue outbreaks (Figs. 2 and 6). Dengue virus is endemic in the Philippines (World Health Organization, 1986), but produces disease only sporadically in Taiwan. Therefore, the direction of spread is probably from the Philippines to Taiwan, and has occurred repeatedly; two different variants of the virus were in-

traduced in 1981 to southern Taiwan and possibly again, in 1987. In contrast, recent outbreaks caused by DEN-l viruses in Taiwan seem to have been imported from Thailand, with a 1980 Thai virus showing the closest relationship to three Taiwanese isolates (Fig. 5). Analysis of an older DEN-l isolate, Japan 1943, provided evidence for the probable disappearance of a genotype from the gene pool. This virus along with an isolate from Sri Lanka (1969) were so distantly related to more recent isolates that they were the only members of their genotypic group. Disease caused by this sero-

EVOLUTION

6 PR159sl PRl59

OF DENGUE VIRUSES

487 2550

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FIG. 2-Continued

type has not been reported from Japan since then, and thus supports this conclusion. Because the selection of isolates for this comparison does not reflect the abundance of specific genotypes in nature, the inclusion of more isolates from other areas of the world will examine whether these genotypes presently exist outside of their site of origin. Comparison of nucleotide sequences may reveal genetic relationships between virus isolates that were distantly separated by time and place, in lieu of virus samples from intervening years and outbreaks. By primer-extension sequencing, three DEN-2 Jamaican isolates (1981, 1982 and 1982) were shown to be closely related to Vietnamese (1987) and Thai (1983) isolates, while two West African isolates (Burkina Faso 1982 and 1986) were shown to be related to a virus from the Seychelles (1977) and these, in turn, to isolates from Sri Lanka (1981, 1982, and 1985) (Fig. 6).

The relationship of the three Jamaican DEN-2 virus isolates to Southeast Asian virus strains was unexpected; the former are quite different from the other American dengue viruses (see top group in Fig. 6). In this case, the sequence data may be helpful in determining the origin of the 1981 Cuban dengue epidemic, the first major outbreak of dengue hemorrhagic fever in the Americas (Kouri et al., 1986). From 1977 to 1980, Vietnam experienced severe dengue epidemics annually (World Health Organization, 1986). During this same period, Cuban military personnel were present in Vietnam. The Cuban dengue epidemic was first detected in May, 1981, raising the possibility that a viremic human may have transported a Southeast Asian strain of dengue virus from Vietnam to Cuba, thereby initiating the epidemic which ultimately affected an estimated 344,000 persons. Since there was very little reported DEN-2 activity in Jamaica in 1981 (World Health Organi-

REBECA RICO-HESSE

488

739 NAUR 74 FIJl

75

JAPA 43

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FIG. 3. Amino acid sequences deduced from the 240 nucleotides Strains were arranged into genotypic groups.

zation, 1981) the appearance of an Asian genotype during 1981-l 982 suggests that it may have been imported from Cuba. However, this hypothesis can only be proven by primer-extension sequencing of dengue isolates from the 1981 Cuban epidemic, which until now have not been rnade available for study. It has been suggested that frequent trade between the islands of the Indian Ocean and East Africa has resulted in spread of DEN-2 virus and in epidemics of dengue in the Seychelles (1976-l 979) and East Africa (1982) (Calisher et al., 1981; Centers for Disease Control, 1982). This is supported by the sequencing data shown here. The route of transmission appears to have

used for determining

genetic relatedness

among dengue type 1 isolates.

been from Sri Lanka (or India), through the Seychelles to East Africa, and then to West Africa. The missing points of reference in this sequence of events are India and East Africa. It is unlikely that the route of transmission was in the opposite direction (from West African sylvatic foci to East Africa), as previously suggested (Centers for Disease Control, 1982). The West African sylvatic virus, circulating in the jungles of Burkina Faso, does not seem to have extended to the urban, eastern areas of the same country, since another virus genotype was isolated during the 1982 and 1986 outbreaks. This latter group of isolates, from African sylvatic mosquitoes and from one human (Senegal 1970)

EVOLUTION 739 PUER 69 PUER 69

MEXl

83

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OF DENGUE VIRUSES

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FIG. 4. Amino acid sequences deduced from the 240 nucleotides Strains were arranged into genotypic groups.

(Robin et a/., 1980; Roche et al., 1983) showed the highest degree of divergence among DEN-2 viruses (16%~).This suggests that a geographically isolated and genetically distinct forest cycle of dengue virus exists in West Africa, and that “epidemic” viruses from other areas have evolved independently for some time. Since these sylvatic viruses share only a distant progenitor with the remainder of the DEN-2 viruses, this former group is probably not a natural maintenance cycle of epidemic viruses, as others have suggested (reviewed by Gubler, 1987). In other words, the West African forest viruses have not recently contributed to the epidemic dengue virus gene pool represented here.

used for determining

genetic relatedness

among dengue type 2 isolates.

Other, clinically silent, jungle cycles of dengue virus have also been described in Malaysia (Rudnick, 1965) and Vietnam (World Health Organization, 1976) but representative strains have not yet been studied genetically. The wide geographic distribution of one DEN-l genotype was evident from the comparison of sequences. The largest genotypic group consisted of DEN-l viruses from three continents: the Americas, Africa, and Southeast Asia (Fig. 5). This suggests that in the recent past, possibly during the early 60s with the advent of jet travel (Gubler, 1987) a progenitor to these viruses was widely disseminated and established itself

REBECA RICO-HESSE

490 MBXICO MEXICO COLOMBIA BAHAMAS JAMAICA COLOMBIA MEXICO SURINAM COLOMBIA BRAZIL HAITI BRAZIL ANGOLA EL SALVADOR PARAOUAY NIGERIA SENEGAL NIGERIA IVORY COAST MALAYSIA THAILAND BURMA THAILAND SRI LANKA JAPAN PHILIPPINES PHILIPPINES

82 83 65 77 77 82 63 61 67 66 63 66 88 67 66 76 79 66 65 82 61 76 74 69 43 66 74

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% DIFFERENCE

FIG.5. Dendrogram of genetic relationships of 40 dengue type 1 viruses from different geographic areas. The percentage nucleotide sequence divergence between any two strains is twice the distance along the x axis to the node that connects them. The dendrogram was constructed by pairwise comparison of all nucleotide sequences (map positions 2282 through 252 1) (Mason et a/., 1987), by a computer program that assigned each base substitution an equivalent statistical weight

in numerous niches. After this initial event, multiple lineages were formed by divergent evolution; this is implied by the random, nonlinear distribution of amino acid differences among these isolates. The source and direction of this pandemic-like virus transmission is unknown and cannot be derived from these data. Classical epidemiologic observations must therefore supplement the sequence data for an accurate interpretation of these evolutionary relationships. DISCUSSION The efficient worldwide control of dengue virus requires the definition of sources of epidemic viruses and, thus, the precise identification of virus genotypes. Limited genomic sequencing of uncloned isolates provided a rapid way of discriminating dengue viruses according to serotype, subtype, and variant (or genotype). This technique allowed for a much more precise measurement of the relationship between isolates than the serological and chemical methods used before. Thus,

this approach appears to be generally applicable to determining the epidemiology of RNA viruses, despite lower observable rates of genetic variation. The genotypic groups distinguished here by primerextension sequencing generally correspond to the topotypes segregated by RNase T, oligonucleotide fingerprinting (Repik et al., 1983; Trent eta/., 1983; Monath et a/., 1986; Kershner et al., 1986; Walker et al,, 1988; Chu et al., 1989). Broader genetic relationships were revealed by comparing nucleotide sequences; the discrepancy is probably caused by limitations on the interpretation of oligonucleotide fingerprints. The evolutionary range of fingerprinting is quite short, because it is highly sensitive to point mutations; other evidence is needed to support relationships between viruses that are more than 5% divergent (Kew et al., 1984). In contrast, the limits of interpretation of virus genetic relationships in the dengue virus dendrograms are reached at the lower end, at approximately 1% divergence, caused by error implicit in the RNA sequenc-

EVOLUTION VENEZUELA MEXICO COLOMBIA TAHITI PUERTO RICO PUERTO RICO TONGA

491

OF DENGUE VIRUSES

87 63 1 86 71 60 69 74

TAIWAN 81 PHILIPPINES 88

NEW GUINEA 44 THAILAND 64

INDONESIA 78 INDONESlA 73 SEYCHELLES 77 BURKINA FASO 82 BURKINA FASO 86 SRI LANKA 85 SRI LANKA 81 SRI LANKA 82 GUINEA81 -

I 9 0

I

I 0 ei

I

I 9 P

I

I 9 (0

I

I 9 m

% DIFFERENCE FIG. 6. Dendrogram of genetic relationships of 40 dengue type 2 viruses from different geographic areas. The percentage nucleotide sequence divergence between any two strains is twice the distance along the x axis to the connecting node. The dendrogram was constructed by pair-wise comparison of all nucleotide sequences (map positions 231 1 through 2550) (Hahn et al., 1988), by a computer program that assigned each base substitution an equivalent statistical weight

ing technique. The limited sequencing approach provides data which are easier to interpret and can be analyzed quantitatively; in addition, it is amenable to comparing isolates across serotypes. Once a genomic interval for comparison is selected, the study of numerous isolates is straightforward and produces rapid results. Although the rate of fixation of mutation cannot be calculated from these data, maximum divergence among epidemic viruses of both serotypes was the same (9%) while only the sylvatic DEN-2 genotype was more divergent (16%). This implies that this rate is similar only within highly transmissible viruses and that this genomic region is under the influence of similar selective pressures or molecular constraints at the nucleotide level, across serotypes. The inclusion of the 6 DEN-2 isolates from canopy-dwelling mosquitoes and one human from Western Africa illustrates the plasticity of the denguevirus genome, while undergoing possibly unique evolutionary processes in isolation. Whether these and other genomic differences have any effect

on pathogenesis and transmission of these viruses is unknown. It remains to be determined whether there is a direct association between specific genotypes and severity of disease. The cause(s) of the more severe disease (DHF/DSS) is not fully understood presently. It has been hypothesized that DHF/DSS results from immune enhancement caused by infection by a second dengue virus serotype (Halstead, 1970). It has also been suggested that dengue viruses may vary in virulence and that more severe disease may be associated with specific strains (Rosen, 1977). The evidence obtained here suggests that dengue viruses may have differences in transmission which may prevent them from circulating widely and, thus, from causing outbreaks. The presence of a genetically distinct, sylvatic cycle of DEN-2 which does not seem to spread and cause disease in nearby rural and urban areas populated by susceptibles (Robin et al., 1980; Roche et a/., 1983) implies a decrease in transmission and/or virulence. The analysis of viruses from other, clinically silent, sylvatic cycles

REBECA RICO-HESSE

492

will help determine if this is a general phenomenon. Whether more severe, hemorrhagic disease is caused by specific viruses, immune enhancement, or a combination of factors is another question, not resolved by the data presented here. The analysis of DEN-l isolates from Thailand tells us that the circulation of dengue virus in Southeast Asia may be very complex, with at least three distinct genotypes probably present simultaneously. Because only one strain analyzed here was associated with more severe disease (DEN-2, 16681, from a DSS patient), and it was genetically distinct from another DE.N-2 isolate, obtained from a dengue fever patient, no conclusions could be drawn about the virulence of these genotypes. Thus, before any association can be made between a specific genotype and severe disease, a detailed map of the natural distribution of virus strains must be obtained. When dengue viruses were grouped based on their nucleotide sequence homology, they fell into distinct clusters, according to geographic origin. This grouping would be expected if they evolved from a common progenitor. Thus, each of the many distinct genotypes has a defined focus of endemicity, where it may persist for many years. In some instances (e.g., Southeast Asia to Caribbean), genotypes were transported to another area of the world where they became established. The displacement and subsequent establishment of dengue virus genotypes is not only dependent on susceptible mosquito populations but also on the availability of sizable susceptible human populations. Therefore, the dynamics of dengue virus displacement become complex, and only the successful genotype movements were seen here. The information presented here is valuable to those involved in disease control. Scientists may now systematically design vaccines that will protect against the genotypes which are most often associated with disease. Public health officials can also use these methods to evaluate control strategies and to make informed decisions on where to allocate the limited resources available for the control of mosquito vectors. The expansion of this data base should permit a clearer view of dengue virus epidemiology.

ACKNOWLEDGMENTS I thank Dr. Mark A. Pallansch forthe use of the NUCLDIFF program and Dr. Robert Tesh and the many scientists who contributed dengue virus strains for this study. This work was funded by the Rockefeller Foundation (GA HS8757), the U.S. Army Medical Research and Development Command (DAMD-17-87-G-7005), and the National Institutes of Health (Al 10984).

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EVOLUTION

OF DENGUE VIRUSES

nucleotide sequence of dengue type 4 virus: Analysis of genes coding for nonstructural proteins. virology 159, 2 17-228. MASON, P. W., MCADA, P. C.. MASON, T. L., and FOURNIER, M. J. (1987). Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NSl virology 161, 262-267. MONATH, T. P., WANDS, J. R., HILL, L. J., BROWN, N. V., MARCINIAK. R. A., WONG, M. A., GENTRY, M. A., BURKE, D. S., GRANT, J. A., and TRENT, D. W. (1986). Geographic classification of dengue-2 virus strains by antigen signature analysis. Virology 154, 3 13-324. NICHOL, S. T., ROWE,J. E., and FITCH, W. M. (1989). Glycoprotein evolution of vesicular stomatitis virus New Jersey. Virology 168, 281291. Pan American Health Organization (1979). “Dengue in the Caribbean, 1977,” PAHO Scientific Pub. No. 375, Washington, D.C. PUTNAK,J. R., CHARLES, P. C., PADMANABHAN, R., IRIE, K., HOKE. C. H., and BURKE, D. S. (1988). Functional and antigenic domains of the dengue-2 virus nonstructural glycoprotein NS-1. Virology 163,93103. REPIK, P. M., DALRYMPLE,1. M., BRANDT, W. E., MCCOWN, 1. M., and RUSSELL,P. K. (1983). RNA fingerprinting as a method for distinguishing dengue 1 virus strains. Amer. 1. Trop. Med. Hyg. 32,577589. RICO-HESSE, R., PALLANSCH, M. A., NOTTAY, B. K., and KEW, 0. M. (1987). Geographic distribution of wild poliovirus type 1 genotypes. Virology 160, 31 l-322. ROBIN, Y., CORNET, M., HEME, G., and LE GONIDEC. G. (1980). Isolement du virus de la dengue au Senegal. Ann. Viral. (Inst. Pasteur) 131 E, 149-154. ROCHE, J. C., CORDELLIER,R., HERVY,1. P., DIGOUT~E,1. P., and MONTENY, N. (1983). lsolement de 96 souches de virus dengue 2 d partir de moustiques captures en Cote-D’lvoire et Haute-Volta. Ann. Viral. (Inst. Pasteur) 134 E, 233-244. ROSEN, L. (1977). The Emperor’s New Clothes revisited, or reflections on the pathogenesis of dengue hemorrhagic fever. Amer. J. Trop. Med. Hyg. 26, 337-343.

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