Genetic variation among dengue 2 viruses of different geographic origin

VIROLOGY

Genetic

128, ml-284

Variation

(1983)

among

Dengue

2 Viruses

J. A. GRANT,*

D. W. TRENT,*r’

of Different

Geographic

Origin

L. ROSEN,? AND T. P. MONATH*

*Division of Vector-Borne Viral Diseases, Center for I&c&us Disease, Centers for Disease Control, Public Health Service, U S Lkpartment of Health and Human sentices, P.O. Box 9087, Fort CoUin~, Colorads 805.2%N87, and fArbovirus Program, Pacijic Biomedicul Research Center, University of Hawaii, Honolulu, Hawaii 96806 Received November

8,

accepted April

1982;

12, 1989

Genetic variation in dengue 2 isolates from various geographic areas was examined by oligonucleotide fingerprinting of the 40 S genome RNA. Oligonucleotide maps of geographically isolated and epidemiologically unrelated viruses were very distinct. Direct comparison of the oligonucleotide map of the dengue 2 prototype New Guinea 2 virus, isolated in 1944, with the fingerprints of more recent isolates from the South Pacific indicated that the genome of dengue 2 virus had undergone extensive change although the viruses are serologically indistinguishable. The oligonucleotide map of an isolate from a recent case in Jamaica and a mosquito isolate from Upper Volta, Africa, were reeognixed to be almost identical, suggesting that virus may have been introduced into the Caribbean from West Africa. Likewise, the fingerprints of isolates from Puerto Rico and the South Pacific shared 39 to 95% of their large oligonucleotides, suggesting that the virus involved in these epidemics may have spread throughout Tahiti, American Samoa, Fiji, and to Puerto Rico in the Caribbean or vice versa. On the basis of these studies, five genetic variants or topotypes of dengue 2 virus have been established: (1) Puerto Rico-South Pacific, (2) Burma-Thailand, (3) the Seychelles, (4) the Philippines, and (5) Jamaica-West Africa. Oligonucleotide fingerprinting offers a highly sensitive and reproducible technical approach to the investigation of dengue 2 virus intratypic variation and possibly to the understanding of the biological variation associated with dengue fever and hemorrhagic disease. INTRODUCTION

In 1945 Sabin and Schlesinger used dengue (DEN) virus isolates from Hawaii and New Guinea in human cross-challenge experiments which resulted in the designation of DEN virus serotypes 1 and 2 (Sabin, 1950). Later, during epidemics in the Philippines, Hammon et al. (1960) isolated DEN viruses which were antigenically related to types 1 and 2 but clearly distinct by complement fixation and neutralization. These new virus strains were designated serotypes 3 and 4. Dengue viruses of the four serotypes can readily be distinguished and identified by neutralization (Russell and Nisalak, 1967); however, the criteria for serologic classification do not provide information about the epidemiologic ori’ Author addressed.

to whom requests

for reprint

should be

gins or phenotypic characteristics of new isolates, nor do they provide clear evidence of intratypic genetic relatedness among strains of a single serotype which have been isolated over time. Accurate characterization of the strain variation within a serotype is critical to our understanding of the patterns of DEN epidemics and the involvement of specific strains of virus in DEN hemorrhagic fever. Intratypic variation among strains has been studied by serologic techniques using crude antigens (Hammon and Sather, 1964) and purified nonstructural soluble complement-fixing (SCF) antigens (McCloud et d, 1971). Although SCF antigens of selected DEN 1 strains could be differentiated by their relative mobility, the SCF antigens of DEN 2 strains could not be distinguished. Antigenic and biological variation related to the geographic distribution of Aa271 004%6822183

$3.00

2.72

TRENT ET AL.

viviruses has been demonstrated with strains of Japanese encephalitis (Okuno et cc&1968), American and African strains of yellow fever (Clarke, 1960), St. Louis encephalitis (Monath et al, 1980), and DEN (Russell and M&own, 1972). Analysis of the genome of many RNA-containing viruses by fingerprinting of the RNase Tlresistant oligonucleotides has facilitated specific identification by the genetic variation observed among virus isolates of a single serotype (Robson et ok, 1979; Nottay et a& 1981; Trent and Grant, 1980; El Said et CQ!,1979; Clewley et al, 1977). Analysis of St. Louis encephalitis virus strains from different geographic regions has revealed that strains isolated at the same time in different geographic areas are genetically distinct (Trent et d, 1981). Vezza et al (1980) reported that the oligonucleotide fingerprints of the 40 S RNA of each of the DEN virus serotypes were distinct. These studies provided a basis for the molecular analysis of DEN viruses of a single serotype which have been isolated in different geographic areas and from outbreaks of disease with varying clinical manifestations. We have analyzed the oligonucleotide fingerprints of 42 strains of DEN 2 virus isolated from various geographic areas throughout the world. Isolates from each major geographic region could be readily distinguished from viruses isolated from other areas at approximately the same time and from the prototype DEN 2 virus isolated in New Guinea in 1944. Five geographically and genetically distinct variants (topotypes) of DEN 2 virus have been established by oligonucleotide fingerprinting the genome RNA: Thailand, the Seychelles, Puerto Rico-South Pacific, Philippines, and Jamaica-Africa. MATERIALS

AND METHODS

virmses. DEN viruses analyzed were from the collection of one of us (L.R.) and were sent with coded identifying numbers to the Division of Vector-Borne Viral Diseases, Ft. Collins, where they were tested and analyzed without knowledge of their source. The sources, geographic location,

and year of isolation, revealed after the fingerprint analyses were finished, are listed in Table 1. All virus strains had been previously serologically confirmed to be DEN 2 by virus neutralization (Russell and Nisalak, 1967) or complement fixation (Kuberski and Rosen, 1977). Passage history of the prototype New Guinea C strain of dengue 2 virus used in this study is presented in detail by Vezza et al (1980). Most of the other strains used in the study were isolated from human blood by the intraa,lbqvictm or thorasic inoculation of A& Tawrhynchites amboinensis mosquitoes and were passed only in mosquitoes prior to the preparation of seed stocks in monolayer cultures of C6/36 Aedes cdbopictus cells (Igarashi, 1978). Working virus stocks were titrated on monolayers of LLC-MKz cells (Sukhavachana et d, 1966) in 60-mm, 6-well plates. Irlfecttm of celk and vim pur@catim Monolayer cultures of C6/C36 Ae. aZbp ictus cells were grown at 28” in Dulbecco’s modified minimum essential medium (DMEM) (Dulbecco and Freeman, 1959) containing 10% heat-inactivated fetal calf serum (FCS). Monolayer cultures in 175~cm2plastic flasks (Falcon Labware) were infected at a multiplicity of 0.01 to 0.1 plaque-forming unit per cell. Virus was allowed to adsorb for 1.5 hr at room temperature, and DMEM containing 5% FCS was added. The cultures were incubated at 28” for 5 days and virus in the medium was concentrated by polyethylene glycol precipitation and purified by rate-zonal and isopycnic centrifugation in potassium tartrate-glycerol gradients (Trent and Grant, 1980). RNase Tl oligonudeotide jingerp-int ana&& Virion RNA was extracted from purified virus with sodium dodecyl sulfate (SDS) and phenol-chloroform as previously described (Trent and Grant, 1980). RNase Tl-resistant oligonucleotides were end labeled by a modification of the method of Pedersen and Haseltine (1980). One to five micrograms of viral RNA was hydrated in 40 ~1 of water, heated to 90” for 5 min, immediately frozen in dry ice, and lyophilized. The pellet was hydrated in 10 ~1 of 20 mMTris-HCl, pH 8.0,2 mMED’l?A, and

DENGUE

2 GENETIC

273

VARIATION

TABLE 1 DENGUE2 VIRUS STRAWSUSED IN COMPARATIVEOLIGONUCLEOTIDE FINGERPRINTSTUDIES Strain

Source

Geographic location

Year

New Guinea C New Guinea C New Guinea C New Guinea C New Guinea C New Guinea C New Guinea C Tr 1751 IBH 10126 s-10099 S-10098 pa-1407 PR 152 PR 156 PR 159 s-33421 S-7848 s-33417 S-7850 S-5312 s-9155 VL63A VL 68A S-16803 S-16782 S-31695 s-19949 s-35179 S-40548 S-40916 s-40659 s-40921 s-40979 S-44552 s-44554 S-44556 S-44558 uv 2039 Dak ArA 578 ARAC-8110827 1666/81

Mosquito passage Cell culture passage Mouse passage Lab infection, Day 1 Lab infection, Day 3 Lab infection, Day 5 Lab infection, Day 6 Human Human Human Human Sentinel monkey Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human

New Guinea New Guinea New Guinea New Guinea New Guinea New Guinea New Guinea Trinidad Nigeria, Africa Philippines Malaysia Malaysia Puerto Rico Puerto Rico Puerto Rico Fiji Tahiti Fiji Tahiti American Samoa New Caledonia Puerto Rico Puerto Rico Thailand Thailand Vietnam Tahiti Manila, Philippines Burma Burma Burma Burma Burma Seychelles Seychelles Seychelles Seychelles Soumousso, Upper Volta, Africa Dabakala, Ivory Coast, Africa Jamaica Jamaica

1944 1944 1944 1944 1944 1944 1944 1954 1966 1966 1967 1968 1969 1969 1969 1971 1971 1971 1971 1972 1972 1973 1973 1974 1974 1975 1975 1975 1976 1976 1976 1976 1976 1977 1977 1977 1977 1980 1980 1981 1981

A& luteocephalus Aedes ta$n-i group

Human Human

digested with 5.0 U of RNase Tl (Calbiochem) at 37” for 60 min. Fifty microliters of polynucleotide kinase reaction mixture [40 mM Tris-HCl, pH 9.0, 10 miI4 Nlg (OAC)*, 5 m&f DTT, 100 rCi @V-labeled ATP, and 10 U of polynucleotide kinase (New England Nuclear)] was added to the

digested RNA, and the incubation was continued for 3 hr at 37”. The reaction was terminated by the addition of 50 ~1 of 0.6 MNH,(OAC). Yeast carrier RNA (100 kg) was added and the mixture was precipitated with 2.5 vol of ethanol at -70”. Twodimensional polyacrylamide gel electro-

274

TRENT ET AL.

phoresis of the 32P-labeled RNase Tlresistant oligonucleotides was performed according to DeWachter and Fiers (1972) as modified by Lee et al. (1979). Fingerprints were visually compared and analyzed as previously described by Trent et al. (1981).

in experimental hosts and during a single infection in man, DEN virus was subject to very little selective pressure and that the fingerprints of virus strains maintained by laboratory passage were indeed representative of that of the original isolate. Comparison of oligonucleotide jingerprints of DEN 2 isolates from di&mnt gee RESULTS graphic ureas The homology of the genome F6ngerprinting of RNase Tl 5’-end-labeled RNAs from virus isolates from widely sepoligonucleotides. The oligonucleotide fin- arated geographic areas was initially degerprint of the New Guinea C (NGC) pro- termined by a comparison of the fingertotype DEN 2 strain, which was isolated prints of virus isolates from Puerto Rico from human serum and had been passed (Fig. 2A), Jamaica (Fig. 2C), the Seychelles only in mosquitoes (first virus listed in Ta- (Fig. 2E), Burma (Fig. 3A), and the Philippines (Fig. 3C) with that of the 1944 ble 1) prior to being grown in mosquito cell culture for fingerprinting, was com- isolate from New Guinea (Fig. 1A). Visual pared to genome maps of this strain fol- comparison of these fingerprints revealed lowing different laboratory passage his- that the patterns of the isolates were very tories (remaining NGC strains in Table 1). different from that of the prototype DEN The fingerprint of this NGC strain (mos- 2 NGC and that they were very different from each other. The fingerprints of the quito passage only) is shown in Fig. lA, different type 2 DEN virus RNAs from diftogether with a key to the large oligonucleotides present in the lower half of the ferent areas differed from each other by at least 75% in their large oligonucleotides autoradiograph (Fig. 1B). The oligonucleotides numbered 1 to 41 were used to com- and therefore, based on computer analysis pare this map with the fingerprints of the of the effect of sequence changes on RNase other NGC DEN 2 strains with various Tl maps, would be approximately 90% passage histories. The oligonucleotide fin- identical in their overall sequence (Aarongerprint obtained by in vitro labeling of son et aL, 1982). The fingerprints for the viruses isolated from each of the geothe 5’ terminus of the RNase Tl-resistant DEN 2 oligonucleotides of this NGC virus graphic regions are so unique that they strain is essentially identical to that of the could not be directly compared with the previously published in viva labeled oli- fingerprints of viruses from other areas. gonucleotide map of this same virus (Vezza Therefore, a prototype or geographic variet a& 1980). This observation clearly in- ant, designated a topotype, was selected dicated that it was technically possible to from the viruses from each region and used as a standard to determine the extent of study, by the oligonucleotide fingerprint technique, DEN virus isolates which do not genetic variation among DEN 2 viruses grow to high titer and for which in vivo- isolated within a limited period of time labeled nucleic acid is not available in suf- from each of the ecological niches (Trent et al, 1981). ficient quantity for study. Virus isolates from Puerto Rico in the A comparison of the fingerprints of the New Guinea C isolate after mouse passage, Caribbean and tilands in the South Pac$c. cell culture passage, and after reisolation When the oligonucleotide fingerprints were in mosquitoes from human blood after ac- analyzed and compared to each other and cidental laboratory infection (Table 1) were with representative.isolates from the other very similar if not identical (Table 2). The geographic areas, five isolates of DEN 2 viruses from Puerto Rico, seven from the variations observed in the oligonucleotide map are within the technical limitations South Pacific, and one from Burma were of the test and are similar to those reported found to have similar patterns (Table 3). by Vezza et al. (1980). This indicated that The oligonucleotide fingerprints of these under the conditions of laboratory passage 12 viruses, when compared with PR 152, a

DENGUE

0

2 GENETIC

0

VARIATION

275

Dengue 2 Prototype ( New Guinea C 1

FIG. 1. RNase Tl fingerprint of 40 S DEN 2 New Guinea 2 virus (A). A 500-ng amount of RNA was digested by RNase Tl in a volume of 10 ~1,followed by 5’ labeling in 60 ~1of the polynucleotide kinase reaction mixture for 60 min. The resulting B-end-labeled oligonucleotides were resolved by two-dimensional polyacrylamide gel electrophoresis and autoradiographed. In this, and in all subsequent autoradiograms, migration in the first dimension is from left to right and the second dimension from bottom to top. The position of the two dye markers as indicated: bromophenol blue (X, top center), xylene cyan01 FF (X, bottom left). A schematic drawing of the fingerprint is shown in (B); 41 large oligonucleotides have been assigned arbitrary numbers for reference purposes.

1969 isolate from Puerto Rico, varied from 95 to 80% in the number of large oligonucleotides which they shared (Table 3). The fingerprints of the DEN 2 viruses from Puerto Rico and the South Pacific, isolated over a T-year period, from a closely related

genomic set; recently isolated virus strains from the South Pacific have oligonucleotide fingerprints missing many of the same oligonucleotides when compared to the selected topotype PR 152 (oligonucleotides No. 8,29,30, and 39). A comparison of the

2’76

TRENT ET AL. TABLE 2 A C~MPARIXJN OF THE LARGE OLICONIJCLEOTIDES OF DENGUE 2 VIRUS Saps OF NEW GUINEA C WITH VARIOUS PASSAGE HISTORIES

New Guinea C virus strains with varying passage histories New New New New New New

Guinea Guinea Guinea Guinea Guinea Guinea

New Guinea C mosquito passage strain oligonucleotides shared

C, mouse passage C, lab infection Day 1 C, lab infection Day 3 C, lab infection Day 5 C, lab infection Day 6 C, cell culture passage

(39/41) (39/41) (41/41) (38/41) (39/41) (38/41)

95* 95 196 93 95 93

No. of the oligonucleotide in the New Guinea C fingerprint missing’ 21,31 40, 41 0 28,40,41 28,40 1, 2, 22

’ Figures 1A and B. * Percentage.

oligonucleotide fingerprints of DEN-Z isolate S-33421 from Fiji (Fig. 4A) with that of VL 68A, a 1973 isolate from Puerto Rico (Fig. 4B), illustrates the genetic variation observed within the group. The map of the Fiji isolate has five new oligonucleotides not present in the fingerprint of the topotype virus PR 152 (indicated by arrows in Fig. 4A) and is missing oligonucleotides No. 27, 29, and 30, indicated by asterisks (compare Figs. 2A and B and Table 3). The fingerprint of the Puerto Rico isolate VL 68A is more distinct from the topotype than the Fiji isolate and is missing seven oligonucleotides, No. 7,8,12,39,28,29, and 30, indicated by asterisks (compare Figs. 2A and B) and has four new oligonucleotides indicated by arrows in Fig. 4B (see Table 3). The oligonucleotide fingerprints of the 1976 Burma isolate S-40548 and 1972 American Samoan strain S-5312 were identical. A comparison of j&p-prints of dengue2 virus strains from Jamaica and West A$ rice+ In 1981 DEN 2 virus was isolated from a patient in Jamaica. This isolate was designated as strain ARAC-8110827 by the Caribbean Research Epidemiological Center and strain 1666 by the Walter Reed Army Institute of Research Laboratory. Fingerprints of this virus obtained from both institutions were identical and very different from those of other DEN 2 strains isolated in the Caribbean, including the 1969 to 1973 Puerto Rican isolates (Table

3) and the Tr 1751 strain from Trinidad isolated in 1954 (compare Figs. 2A and C). A visual comparison of the oligonucleotide map of the ARAC-8110827 virus with those of other isolates revealed that the fingerprint of the Jamaican virus was very similar to that of the DEN 2 virus strain UV 2039 isolated by the Institut Pasteur of Ivory Coast in 1980 from Ae. luteocephalus mosquitoes in Soumoussa, Upper Volta, and identified by the Institut of Pasteur, Dakar, Senegal (Table 4). These two viruses shared 38 of 43 (88%) of their long RNase Tl-resistant oligonucleotides. A comparison of fingerprints of the 1981 Jamaican isolate ARAC-8110827 with the 1980 isolate from the Ivory Coast (DAK ArA 578) and a 1966Nigerian isolate (IBH 0126) revealed that these African DEN 2 viruses were not genetically related to each other nor were they of the same genetic topotype as the Jamaican virus. A comparison of jingerprints of DEN 2 virus strains from Asia. The genomes of nine DEN 2 virus isolates from Burma, Thailand, and the Philippines were compared by the oligonucleotide fingerprint technique. The oligonucleotide map of a 1976 isolate from Burma (S-40916,Fig. 3A), together with a diagram of large oligonucleotides, shown in Fig. 3B, is typical of the virus isolates from Burma and Thailand over the period of 1974 to 1976. The comparative analysis of these fingerprints is summarized in Table 5. These virus

DENGUE

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2 GENETIC VARIATION

Puerto Rico (PR 152)

Jamaica (ARAC -8110827)

0

Seychelle

blonds

(S-44554)

FIG. 2. The oligonucleotide fingerprints of RNase Tl digested ~-labeled RNA of DEN 2 virus isolates PR 152 Puerto Rico (A), ARAC-811082’7 Jamaica (C), and S-44554 the Seychelles (E) with schematic drawings of each of the fingerprints (B, D, and F). The large oligonucleotides of each viral RNA have been arbitrarily assigned numbers for reference purposes.

strains shared from 81 to 100% percent of unique and was more ‘like the 1976 Burma their long oIigonucleotides; virus S-16782, topotype isolate S-40979 than were the fina 1974 isolate from Thailand, was the most gerprints of the other isolates examined.

278

TRENT

a

@

ET AL.

Burma (S-40916)

FIG. 3. The oligonucleotide fingerprints of RNase Tl digested q-labeled RNA of DEN 2 virus isolates S-40916 Burma (A) and S-10099 the Philippines (C) with schematic drawings of each of the fingerprints (B and D). The large oligonucleotides of each viral RNA have been arbitrarily assigned numbers for reference purposes.

Fingerprints of the 1976 Burma isolates S40921 and S-40916 were identical to each other and very similar to that of the S40659 strain. Fingerprints of two virus isolates from the Philippines, S-35179and S-10099 (Table

1) were different from other DEN 2 viruses. These two fingerprints shared 32 of their 37 long oligonucleotides, and although the two Philippine viruses were isolated 9 years apart, the fingerprint of the 1975 isolate was more similar to that of the 1966

DENGUE

2 GENETIC

0 GD

Philippine isolate than it was to any of the other DEN 2 viruses examined. This would seem to indicate that DEN virus in this geographic area has been limited in its spread by biological or physical barriers. from the Sey&e& 1sZunok DEN2 WirzGses Four virus isolates recovered during an ep-

279

VARIATION

Philippines

(S-l0099)

idemic in 1977 in the Seychelles were analyzed by mapping of their T1-resistant oligonucleotides (Table 6). A comparison of the oligonucleotide map of strain S-44554 (Fig. 2E) and the diagram of the large oligonucleotides (Fig. 2F) with the other isol&es from these small islands in the Indian

280

TRENT ET AL. TABLE 3 CMPARISON OFTHE LARGE OLIGONUCLJZOTIDES OFDENCUE 2 VIRUS STRAINSFROM THE SOUTHPACIFICAND CARIBBEANWITH (PR 152) FROMPUERTORICO

Virus isolate/year of isolate, geographic area

Puerto Rico (PR 152) oligonucleotides shared

No. of the (PR 152) oligonucleotides missing’

Caribbean PR 156 Puerto Rico/69 PR 159 Puerto Rico/69 VL 68A Puerto Rico/73 VL 63A Puerto Rico/73

(34/40) 85* (38140) 95 (33/40) 83 (38/40) 95

6, 7, 16, 1’7,27, 34 19, 34 7, 8, 12, 28, 29, 30, 39 a,29

South Pacific S-33421 Fiji/71 S-7848 Tahiti/71 S-33417 Fiji/71 S-7856 Tahiti/71 S-5312 American Samoa/72 S-9155 New Caledonia/W S-19949 Tahiti/75 S-40548 Burma/76

(37140) 93* (34/40) 85 (33140) 83 (32/40) 80 (36/40) 90 (35140) 88 (32/49) 80 (36149) 90

27,29,30 8, 12,25x,29, 30,39 8, 11, 25,29,36,34,39 4, 8, 13, 14, 28, 29, 37, 39 8,29,30,39 8, 12, 28,29,30 5, 8, 12, 17, 21, 22, 28, 29 8,29,30,39

a Figures 2A and B. ‘Percentage.

Ocean revealed that the strains of .virus involved in the 197’7 outbreak are genetically very similar. Strains of DEN-2 with ~dtitinet oligmucleotide$ngeqwak?s. The fingerprints of six DEN 2 viruses were so unique that they could not be classified into one of the existing geographic sets. This group included the virus isolate from New Guinea (NGC), 1944; two isolates from Africa, Dak ArA 578 and IBH 10126 from Nigeria; two Malaysian strains, P8-1407 from a sentinel monkey and S-10098 from a human; a 19’75 virus isolate from Vietnam (S-31695); and virus TR 1751 isolated in Trinidad in 1954. The DEN strains from Malaysia showed definite, but distant relationship to one another (50% homology). The prototype New Guinea C strain and the TR 1751 Trinidad strain are old virus isolates, and undoubtedly because of genetic change, the fingerprints of DEN 2 viruses isolated recently from these areas would be expected to be significantly different from the older isolates (Trent et al, 1981). DISCUSSION

Oligonucleotide fingerprinting is a powe&l technique for studying the molecular

epidemiology of RNA viruses (Nakajima et a& 1979; Robson et al, 1979; Nottay et aL, 1981). Genetic variation among strains isolated within a defined geographic area and time identify changes in virus populations which may be related to antigenic character and/or virulence. The rapidly changing evolution of the oligonucleotide fingerprints of RNA viruses (Holland et d, 1982), and specifically flaviviruses, is clearly evident when the maps of the 1944 New Guinea C strain of DEN 2 are compared with those of other isolates from the South Pacific and Southeast Asia. This phenomenon of evolutionary change in the primary nucleotide sequence of the flavivirus genome has been previously documented for St. Louis encephalitis virus (Trent et d, 1981). Nottay et al. (1981) documented the rapid change in the oligonucleotide fingerprints of poliovirus during passage through humans. The highly sensitive technique of fingerprinting the RNase Tl-resistant oligonucleotides can be applied in epidemiologic studies of RNA viruses only when virus specimens are available which have been col-lected during a limited time interval in a specific area. The analyses of genome changes by oligonucleotide mapping may provide an un-

DENGUE

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281

FIG. 4. The oligonucleotide fingerprints of RNase Tl digested and B-end-labeled oligonucleotides of dengue 2 virus isolates S-99421 and Fiji (A) and S-19416 from Puerto Rico (B). Numbered oligonucleotides in the fingerprint of topotype strain S-16491 (Figs. 2A, B) which are missing from these fingerprints are indicated by asterisks. New oligonucleotides are indicated by arrows. The two marker dyes, bromophenol blue and xylene cyanol, are indicated by heavy asterisks in the top center and bottom left, respectively.

derstanding of biological and antigenic variation which needs to be further defined by monoclonal antibody analysis of the envelope glycoproteins, and nucleotide sequence analysis of the genome structure. With the limited number of DEN 2 strains examined in this study, we have not been able to define or detect genetic evolutionary changes in viruses which have been associated with severe outbreaks of DEN disease. An analysis of many viruses from a specific area isolated over an extended period of time would be necessary in order to correlate genetic and phenotypic TABLE 4 COMPARMN OF THE LARGE OLICONUCLZ~TIDES OF DENGUE 2 VIRUS STRAWS FROM JAMAICA AND WEST AFRICA WITH JAMAICA (ARAC-8110827) Jamaica ARAC-8110827 oligonucleotides shared” 1633 Jamaica/81 UV 2039 Africa, Upper Volta&O ’ Figures 2C and D. bPercentage.

(43/43) loo* (W43) 88

changes with virulence and envelope glycoprotein antigenic markers. From an epidemiologic point of view, we have shown that 27 DEN 2 virus strains collected over an 11-year period (1969 to 1980), can be divided into five sets or “topotypes” based on the comparative analysis of their RNase Tl oligonucleotide fingerprints (Table 7). Strains of DEN 2 virus isolated within a specific geographic area within a specific time period have oligonucleotide fingerprints more like each other than do strains of virus from other areas, although genetic variation among strains is frequently observed (Trent et u,L, 1981). The finding that topotype I DEN 2 strains isolated during outbreaks in Puerto Rico and the South Pacific between 1969 and 1973 were closely related genetically suggests that the virus may have been introduced into the South Pacific from the Caribbean (or vice versa). Dengue 2 virus had not been isolated in the Caribbean region between 1978 and 1981, when a large outbreak appeared in Cuba. The Cuban epidemic was unusual because of the appearance of large numbers of cases of

282

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TABLE

5

COMPARMN OF THE LARGE OLIGONUCLEOTIDES OF DENGUE 2 VIRUS STRAINS FROM SOUTHEAST ASIA, BURMA, THAILAND, AND THE PHIJJPPINES

Virus isolate S-16782 S-16803 S-40979 S-40659 S-40921 S-40548

Thailand/74 Thailand/74 Burma/76 Burma/76 Burma/76 Burma/76

Burma (S-40916) oligonucleotides shared

No. of the Burma (S-40916) oligonucleotides missing4

(38/47) 81* (45/47) 96 (41147) 87 (46/47) 98 (47/47) 100 (43147) 91

2, 12, 15, 16, 37, 38, 42, 43, 44 12, 13 12, 37, 38, 43, 43, 44 12 0 7, 10, 12, 33

Philippines (S-10099) oligonucleotides shared S-35179 Manila/75

(32/37)

86*

No. of the S-10099 oligonucleotides missing 15, 17, 22, 27, 34

‘Figures 3A and B. * Percentage. ’ Figures 3C and D.

hemorrhagic fever-shock syndrome. Our finding that virus ARAC-811082’7 isolated in Jamaica in 1981 and virus UV 2039 from West Africa in 1980 were readily recognized as being very closely related suggests the possibility that DEN 2 virus may have been introduced into the Caribbean from Africa in 1981. The origin of the DEN 2 virus involved in the 1977 epidemic in the Seychelles, where the virus is probably not endemic, is not known. The oligonucleotide fingerprints of these viruses are very distinct and, therefore, the virus involved in this outbreak may have been introduced from some area not represented in the collection of strains studied, i.e., east Africa or the Indian subcontinent. Such intro-

duction of a new serotype or topotype into a receptive area by infected humans or mosquitoes needs only a suitable mosquito population (Bres, 1979). In our study, we have not examined the oligonucleotide fingerprints of DEN strains from East Africa, Australia, the Indian subcontinent, China, South America, and most notably the recent epidemic in Cuba. Our geographic classification of the strains on the basis of fingerprinting into five groups is undoubtedly incomplete, yet it represents a beginning for subsequent comprehensive, molecular epidemiologic analysis of DEN fever. We have demonstrated that strains of the virus isolated from human blood by mosquito inoculation

TABLE

6

COMPARISON OF THE LARGE OLIGONU~LEOTIDES OF DENGUE 2 VIRUS STRAINS FROM THE SEYCHELLES WITH (S-44554) SEYCHELLES

Virus shared

Seychelles (S-44554) oligonucleotides shared

S-44552177 S-44556177 s-44568/77

(32/34) 94* (34/34) 100 (34/34) 100

DFigures 2E and F. * Percentage.

No. of the (S-44554) oligonucleotides missinga 10, 12 0 0

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2 GENETIC

TABLE

283

VARIATION

7

CLUSTERING OF DENCUE 2 VIRUS ISOLATES BY OLIGONUCLE~TIDE FINGERPRINT SIMILARITY

Towtype Geographic

group I II III IV V

No. of strains analyzed

area

13 3 7 2 4

South Pacific/Puerto Rico Jamaica/West Africa Burma/Thailand Philippines Seychelle Islands

a Oligonucleotide

average similarity

coefficient

Variation similarity

in percentage of within cluster’ 95-80 loo-88 100-81 loo-86 loo-94

of 0.75 or greater.

can be grown in mosquito cell cultures and that the virus can be purified and the RNase Tl oligonucleotides labeled in vitro for fingerprint analysis. Clearly, additional studies of the genome variation among strains from areas of the world yet unstudied are needed, and continued analysis and correlation of genome changes associated with antigenic variation needs to be investigated on a continuing basis. ACKNOWLEDGMENTS This research was partially supported by National Institutes of Health Grants AI-17953 and AI-17995 to L.R. We thank D. J. Gubler, J.-P. Digoutte, W. E. Brandt, R. E. Shope, F. P. Pinheiro, A. Rudnick, P. K. Russell, B. L. Cline, and W. Bancroft for dengue 2 virus strains used in this study. Strains from Upper Volta and Ivory Coast were collected by Dr. R. Cordellier and Dr. J.-P. Hervy and isolated by Dr. J.-C. Roche. REFERENCES AARONSON, R. P., YOUNG, J. F., and PALESE, P. (1982). Oligonucleotide mapping: Evaluation of its sensitivity by computer-simulation. NULL Acids Res 10, 237-246. BRES, P. (1979). Historical review of dengue 1. In “Dengue in the Caribbean, 1977,” 375, pp. 4-10. PAHO Sci. Publ. CLARKE, D. H. (1960). Antigenic analysis of certain Group B arthropodborne viruses by antibody absorption. J. Ezp. Med. 111, 21-32. CLEWLEY, J. P., BISHOP, D. H. L., KANG, C., COFFIN, J., SCHNITZLEIN, W. M., REICHMAN, M. E., and SHOPE, R. E. (1977). Oligonucleotide fingerprints of RNA obtained from rhadboviruses belonging to the vesicular stomatitis virus subgroup. J. Vird 23,152166. DULBECCO, R., and FREEMAN, G. (1959). Plaque formation by polyoma virus. l%-w 8. 396-397.

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