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Genomic divergence among sindbis virus strains
VIROLOGY
106, 59-70 (1980)
Genomic FRANCOISE
Divergence
among
RENTIER-DELRUE’
Sindbis AND
Virus Strains
NATHANIEL
A. YOUNG2
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 Accepted May 17, 1980
Antigenic variants of the alphavirus Sindbis have been isolated from the Palearctic, Ethiopian, Oriental, and Australian zoogeographic regions. The genomes of these variants were analyzed for homology by the hybridization of virion RNAs to double-stranded RNAs isolated from infected cells. Under nonstringent conditions (T, - 55”) the RNAs of OrientalAustralian strains showed only 35 to 51% nucleotide sequence homology with the RNAs of the Palearctic-Ethiopian strains although homology was essentially complete among isolates within the Oriental and Australian regions and among isolates within the Palearctic and Ethiopian regions. Under more stringent conditions (T, - 26”), nucleotide sequence differences of 2 to 45% were detected among the RNAs of virus strains from geographically distant localities within each of these two major zoogeographic subdivisions. Year of isolation, passage history, and vertebrate or invertebrate host of origin were not major determinants of sequence heterology. The hypothesis that ancestral Sindbis virus became separated by geographic barriers and evolved into two distinct types is presented. Further divergent evolution has obviously occurred within each of these types.
Malaysia (Traub et al., 1957; Simpson et al., Previous studies have shown that anti- 1970; Lim et al., 1972), India (Shah et al., genie similarities among strains of several 1960), the Philippines (Rudnick et al., 1962), togaviruses follow geographic lines. Among Australia (Doherty et al., 1963), West Africa (Institut Pasteur de Dakar, 1972), alphaviruses, Casals (1961, 1964) reported Israel (Nir et al., 1967, 1972), Mozambique antigenic variants of Chikungunya (CHIK), (S. Afr. Inst. Med. Res., 1973), CzechosloSindbis (SV), and Eastern equine encephvakia (Ernek et al., 1973; Gresikova et al., alitis virus (EEE), using a modification of 1973; Kozuch et al., 1978), USSR (Gaidamothe hemagglutination inhibition test. Karavitch et al., 1978), and Sicily (Gresikova batsos et al. (1963) found antigenically et al., 1978). By antigenic analysis, three distinct isolates of Western equine encephalitis (WEE) virus in the eastern and Indian strains, an Australian strain, and western United States. Young and Johnson two African strains were shown to form (1969) examined antigenic variation among three distinct subgroups correlating to their 124 strains of Venezuelian equine enceph- geographic distribution (Clarke, 1963). The genome of Sindbis virus is a singlealitis (VEE) virus and found a correlation stranded RNA with a molecular weight of between antigenie variation and the geo4.3 x lo6 and with a sedimentation cographic location of the original virus efficient of about 42 S (Dobos and Faulkner, isolation. Sindbis virus has a wide geographic dis- 1970; Simmons and Strauss, 1972a). In intribution. It was first isolated in Egypt by fected cells, in addition to the 42 S RNA, a Taylor (1955) and subsequently isolated in 26 S virus-specified, single-stranded RNA South Africa (Weinbren et al., 1956; Mal- of the same polarity is synthesized (Levin and Friedman, 1971) which serves as the herbe et al., 1963), Uganda (Eavri, 1962>, specific messenger for the structural proteins of the virus particles (Cancedda 1 To whom reprint requests should be addressed. et al., 1974; Simmons and Strauss, 1974). e This work was performed in the laboratory of Dr. In addition, double-stranded viral RNAs Nathaniel A. Young, who died on February 4, 1979. INTRODUCTION
59
0042-6822/80/130059-12$02.00/O Copyright All rights
Q 1980 by Academic Rem, Inc. of reproduction in any form reserved
60
RENTIER-DELRUE
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TABLE SUMMARY Geographic country
OF THE
PEDIGREE
1
OF FIFTEEN
SINDBIS
origin Locality
Strain
Y&V isolated
Origin
Sindbis
AR-339
1952
C&z
Entebbe
MP-634
1953
spring
AR-36
upington
of isolation
univittatus
STRAINS Laboratory p.%Wge hosts
STUDIED Number of passage
S0UKe
Mice
13
Yale Arbovirus Research Unit
Mamoniafwcopennata
Mice
7
Yale Arbovirus Research Unit
1954
Culer
spp.
Mice
3
Yale Arbovirus Research Unit
AR-18132
1976
Cules
univittatw
Mice
1
National Institute for Virology, Transvaal
AR-6671
1964
Mice
3
National Institute for Virology, Transvaal
Johannesburg
Giidwood
196.3
Isl%si
HadsiX
M-1355
1967
USSR
Aserbsiisn
AZ-16
1971
iVycticmm (bird)
Czeeboslowkia
Western
R-33
1971
Amcephalw scirpacewr
India
Mysm
A-1036
1953
Bdellcmysaus
B-33%3/34
1953
Wagtail alba)
Malaya
MM-2215
1955
Philippines
P-336
Uganda
South
Africa
Australia
state
Cdl-Jlh
Mice
3-l
National Institute for Virology, Transvaal
LOW
Israel Institute for Biological Research
Mice
5
Institute for Virology, Moscow, USSR
Mice
I
Institute of Virology, Slovak Academy SCiWXe
Mice
13
Yale Arbovirus Research Unit
Mice
10
National Institute of Virology, Poona, India
Culer tritaeniorhynchus
Mice
2
Yale Arbovinis Research Unit
1956
C&z
Mice
1
Yale Arbovinis Research Unit
C-377
1960
Mansonia septempunctata
Mice
1
Yale Arbovirus Research Unit
Ch-19470
1976
Pool of mosquitoes
Mice
4
Queensland Institute of Medical Research, Brisbane
_ KIhims) Chsrleville
MEUI
nycticomz
bursa
(Motucillu
bitaeniorhynchus
are formed (Levin and Friedman, 1971; streptomycin, and 0.5% of a combination of Simmons and Strauss, 1972b; Segal and three organic buffers: 10 mM BES (NJ, Streevalsan, 1974). In this report, we bis-(2-hydroxyethyl-2-aminoethanesulfonic compared the genome of 15 strains of acid); 15 mM HEPES (N-&hydroxyethylpiperazine-N-2-ethanesulfonic acid), and 10 Sindbis virus by saturation hybridization experiments using unlabeled virion RNA an- mM HEPPS (N-2-hydroxyethylpiperazinenealed to 3H-labeled viral double-stranded N-2-propanesulfonic acid) (BHE) (Eagle, 1971). RNA. Viruses. Table 1 summarizes the information on the 15 Sindbis isolates used in this MATERIALS AND METHODS study. Each isolate had a low passage history Cells. Primary chick embryo fibroblasts and was subjected to three successive (CEF) were cultured in 199 medium sup- plaque purifications. Virus stocks were plemented with 2.5% fetal calf serum established from these plaque isolates with (Gibco), 2.5% chicken serum, penicillin and no further passage. Other alphavirus strains
GENOMIC
DIVERGENCE
AMONG
received from other laboratories included: WEE Cal., EEE PE-6, and VEE TC-83 kindly provided by Dr. G. Eddy (Fort Detrick, Md.), and Whataroa (WHA) virus M-78 given by Dr. R. E. Shope (Yale Arbovirus Research Unit). Virus production and purijkation. Confluent monolayers of CEF were washed with Hanks’ balanced salt solution and infected with 0.01 PFU per cell in 199 medium containing 0.01 pg/ml actinomycin D (Calbiochem) and 0.5% BHE. After adsorption for 1 hr at 33”, the inoculum was replaced by the same medium supplemented with 1% fetal calf serum. After incubation at 33” for 18 to 20 hr, the extracellular fluid was harvested and usually contained 5.0 x lo7 to 5.0 x lOlo PFU/ml depending on the virus strain used. The clarified supernatant was adjusted to 0.5 M NaCI and 10% polyethylene glycol 6000 (BDH Chemicals). The suspension was stirred overnight at 4” and the precipitate collected by centrifugation at 12,000 g for 30 min. The pellet was resuspended in l/lOth of the original volume of 2x TNE (TNE: 0.05 M Tris; 0.1 M NaCl, 10m3M EDTA, pH 7.6) and sonicated on ice for 2 min at a setting of 4 in a Branson W-350 sonifier. The suspension was layered on a 15-30% sucrose gradient (w/w) in TNE buffer and centrifuged at 85,000 g for 2.5 hr at 4” in a SW 27 rotor (Beckman). The opalescent bands containing virus were diluted fourfold in TNE buffer and the virus was pelleted at 170,000 g for 1 hr at 4” in a R60 Ti rotor (Beckman). The pellets were resuspended in 2~ TNE and used within a few hours. Viral RNA pur$.cation. Sodium dodecyl sulfate (SDS) was added to the viral suspension at a final concentration of 0.5%. RNA extraction was carried out at room temperature with equal volumes of phenolcresol (7:l) equilibrated with 2X TNE, pH 7.6, containing 0.0125% (w/v) 8-hydroxyquinoline, and chloroform-isoamyl alcohol (1OO:l). A second extraction with chloroform-isoamyl alcohol (1OO:l) was then performed, followed by five ether extractions. Ether was removed by bubbling nitrogen through the tinal aqueous phase and the viral RNA was adjusted to 0.4 M NaCl (final
SINDBIS
VIRUS
STRAINS
61
concentration). Viral RNA was ethanol precipitated, resuspended in 2~ TNE, pH 7.6, and stored at -70”. The ss RNA concentration was determined from the optical density at 260 nm. Preparation stranded viral
of radiolabeled doubleRNA. Confluent primary
CEF monolayers were washed with Hanks’ balanced salt solution and infected with virus at a multiplicity of 50 PFU per cell. After 1 hr at 34”, virus inoculum was replaced by 199 medium containing 1% fetal calf serum and 2 pg/ml actinomycin D. One hour later, 100 PCiiml [3H]uridine (45-47 Ci/mmol) (New England Nuclear) was added. Seven hours after infection, the fluid was discarded and the cells were washed with phosphate buffered saline (PBS) and resuspended in SDS-acetate buffer (0.35% SDS, 0.01 M sodium acetate pH 5.1, 0.1 M NaCl). Total RNA was extracted at 60” with phenol equilibrated with acetate buffer (0.01 M sodium acetate pH 5.1, 0.1 M NaCl) and chloroform-isoamyl alcohol (100: 1) (Penman, 1966). RNA was precipitated with 2 vol of ethanol at -2o”, overnight and resuspended in TNE buffer. Differential salt precipitation of single-stranded RNA was carried out according to the method of Bishop and Koch (1967). Doublestranded RNA in the supernatant was ethanol precipitated, redissolved in TNE buffer at a maximum concentration of 0.5 mg RNA/ml, and purified by two cycles of chromatography on cellulose columns (Whatman CF,,). Successive elutions with 15% ethanol in TNE buffer and with buffer alone were carried out as described by Franklin (1966). RNA-RNA hybridization. Viral doublestranded RNA was denatured in SSC (0.15 M NaCl, 0.015 M Na-citrate) at 120” for 10 min, in the presence of highly polymerized yeast RNA (15 pg/ml) (Calbiochem) and rapidly cooled. A maximum of 0.002 pg of 3H-labeled double-stranded RNA (specific activity: 8.5 X lo”-1.0 X 10” cpmipg) was allowed to anneal with increasing amounts of homologous or heterologous unlabeled virion RNA. Reaction mixture (l.O-ml volumes) were incubated for 4 hr, in 5 x SSC at 57” for “nonstringent” reactions and in 2x SSC and 33% formamide (Eastman
62
RENTIER-DELRUE
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since the G-C content of SV RNA (50%) (Pfefferkorn and Hunter, 1963) is very close to that of SFV RNA (51%) (Kaariainen and Gomatos, 1969), these same T,,, values were employed in calculating the effective temperatures of our hybridization reactions. Purified ds RNA preparations had specific activities ranging from 8.5 x lo5 to 1 x 10” cpmlpg and were 97 to 100% resistant to pancreatic ribonuclease A (40 pg/ 1.0 2.0 3.0 ml, in 2 x SSC). After denaturation, 3 to 5% tq of RNA of the RNA remained resistant to panFIG. 1. Annealing of 3H-radiolabeled Sindbis Egypt creatic ribonuclease A and since this mateAR-339 ds RNA with unlabeled virion ss RNA. rial could participate in annealing reactions, Samples containing 3H-labeled ds RNA (1.5 ng, 1000 it was considered as background. Homolocpm) and various amounts (0 to 3 pg) virion ss RNA in 1 gous annealing of several Sindbis strains ml of 2 x SSC, 33% formamide, were annealed at 57” for were first performed in 2x SSC, 33% 2 hr. Each RNA sample was digested with pancreatic formamide at 57” for 2 hr as a control of the ribonuclease A (40 &ml) for 30 min at 37”. The RNA was precipitated with trichloroacetic acid and counted purity of the reagents. Formamide was for radioactivity. Results were expressed as the used in order to lower the T,,, of the RNA percentage of residual input radioactivity that de- duplex and permit hybridization experiments to be carried out at lower temperaveloped RNAse resistance. tures. Since formamide lowers the T,,, of a DNA duplex by 0.72” for every 1% increase in formamide concentration (ConKodak) at 5’7” for “stringent” reactions. aughy et al., 1969), the “stringent condiThe samples were further incubated in the presence of 40 pglml pancreatic ribonuclease tions” of hybridization used in these studies correspond to an effective temperature of A (Worthington Biochemical Corporation) about 26” below the T,,,. A representative for 30 min at 37”. Ribonuclease-resistant RNA was precipitated with cold 5% tri- saturation curve obtained with prototype chloroacetic acid (TCA) in the presence of Sindbis Egypt AR-339 is shown in Fig. 1. 50 pg/ml yeast RNA (Schwarz/Mann) as Since the virion RNA is complementary to carrier, and collected on type HA filters only one of the radiolabeled strands, the (Millipore). Filters were dried and counted maximum hybridization reached in the absence of the probe reannealing with itusing scintillation fluid (Econofluor, NEN) in a Beckman LS 250 scintillation spec- self would be 50%. In the experiment presented in Fig. 1, this plateau level was trometer. achieved with 0.7 pg of virion RNA. RESULTS
The melting temperature (T,) of SV ds RNA used in our calculations was estimated by comparison to T,,, values for Semliki Forest virus (SFV) ds RNA, another alphavirus. SFV ds RNA is reported to be 89” in 0.1~ SSC (Wengler et al., 1976). In preliminary experiments (unpublished results) we found the T,,, of SFV ds RNA to be 102” in SSC and 106” in 2x SSC. By extrapolation from the data of Billeter et al. (1966), the T, in 5x SSC was estimated to be 112- 114”. Since the T, is a function of the G-C content of RNAs (Billeter et al., 1966), and
Analysis of RNA Homology between Sindbis Virus Strains under Nonstringent Hybridization Conditions The initial analysis of homology among Sindbis virus isolates from remote geographic area was performed under nonstringent hybridization conditions in order that regions containing significant base mismatch would be thermally stable and detected as duplex in the enzymatic assay (Howley et al., 1979). The hybridizations were performed in 5 x SSC at 57” for 4 hr, an effective temperature of about 55” below the
GENOMIC DIVERGENCE
AMONG SINDBIS VIRUS STRAINS
63
,&I OF RNA
FIG. 2. Annealing of four strains of Sindbis virus ss RNA with Sindbis Egypt AR-339ds RNA isolated from infected cells. Denatured 3H-radiolabeled SV Egypt AR-339 ds RNA, 1.5 ng per tube, 1000cpm, was annealed with increasing quantities of ss RNA from either homologous or heterologous strains of Sindbis virus. Reactions were carried out in 5 x SSC at 57” for 4 hr. Acid-precipitable radioactivity was determined after treatment with 40 pg/ml pancreatic ribonuclease A. The homologous reaction was normalized to a value of 1.0 and the results were expressed as a decimal fraction of the homologous reaction. (m) SV Egypt AR-339 ss RNA: (Cl) SV South Africa AR-36 ss RNA: (0) SV India A-1036 ss RNA; (0) SV Australia C-377 ss RNA; and (A) poliovirus ss RNA.
T,,, for SV ds RNA. The initial analysis involved SV Egypt AR-339, SV India A1036, SV Australia C-377, and SV South Africa AR-86. Each radiolabeled ds RNA was denatured and reacted in the presence of increasing amounts of virion ss RNA. Under these conditions, plateau levels were easily reached with from 1 to 3 pg ss RNA. In each case, the homologous reaction which achieved a plateau level of approximately 50% was normalized to a value of 1.0 and the results were expressed as a decimal fraction of the homologous reaction. By this analysis, a radiolabeled SV Egypt AR-339
probe was found to be almost completely homologous with SV South Africa AR-86 virion RNA (0.99). SV India A-1036 and SV Australia C-377 respectively, annealed to 51 and 47% of the complementary strand with the probe (Fig. 2). The specificity of the hybrids formed was demonstrated by the absence of detectable annealing with poliovirus ss RNA. Reciprocal experiments were performed utilizing SV India A-1036 ds [3H]RNA and SV Australia C-377 ds [3H]RNA as probes (Figs. 3 and 4), respectively. SV India A1036 and SV Australia C-377 were found
pg OF RNA
FIG. 3. Annealing of Sindbis India A-1036 denatured 3H-radiolabeled ds RNA with (0) SV India A1036ss RNA; (0) SV Australia C-377ss RNA; (m SV Egypt AR-339 as RNA; (A) SV South Africa AR-86 ss RNA; and (A) poliovirus ss RNA. Hybridization conditions were the same as in Fig. 2.
64
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FIG. 4. Annealing of Sindbis Australia C-377denatured 3H-radiolabeled ds RNA with (0) homologous ss RNA; (0) SV India A-1036 ss RNA; (W) SV Egypt AR-339 ss RNA; (A) SV South Africa AR-86 ss RNA; and (A) poliovirus ss RNA. Annealing conditions are described in Fig. 2.
to be 90-91% homologous under these conditions in that each virion ss RNA was capable of protecting that percentage of the complementary heterologous strand of the radiolabeled ds RNA probe from RNase digestion. Hybridization values obtained between the SV India A-1036 probe and the SV Egypt AR-339 and SV South Africa AR-86 virion RNAs were 0.44 and 0.47, respectively (Fig. 3). Similarly, the fraction hybridized between the SV Australia C-377 probe and the unlabeled SV Egypt AR-339 and SV South Africa AR-86 virion RNAs were 0.41 and 0.38 (Fig. 4). Thus, on the basis of the nonstringent hybridization results, SV India A-1036 can be grouped with SV Australia C-377, and SV Egypt AR-339 can be shown to be closely related to Africa AR-86. Similar hybridizations were performed using 3H-labeled probes for SV Egypt AR-339, SV India A-1036, or SV Australia C-377 and virion RNAs of seven additional SV strains, WEE, EEE, VEE, WHA, and poliovirus (Table 2). The Sindbis virus strains could be separated into two discrete groups on the basis of homology, those which were homologous with SV Egypt AR-339 ds RNA and those which were essentially homologous with both SV India A-1036 and SV Australia C-377 ds RNAs. The strains which hybridized extensively with the Egypt AR-339 probe were all isolated from Europe and Africa and those which hybridized extensively with SV India A-1036 and SV Australia C-377 were all
isolated from India, the Far East, and Australia. Each member of these two geographically defined groups demonstrated between 35 and 50% homology with intergroup ds RNA probes under these nonstringent conditions (Table 2). Also summarized in Table 2 are the results of the nonstringent hybridizations between the 3H-labeled SV probes and ss RNA from other selected alphaviruses. From 6-9% homology was found between each of the SV RNAs and WEE virion RNA and from 19-23% homology was found between each of these RNAs and WHA virion RNA. No homology was detected between SV RNAs and these virion RNAs of VEE, EEE or poliovirus. Analysis of RNA Homology betweenSindbis Virus Strains under Stringent Condition Hybridization To more closely examine the extent of RNA homology among the strains of the European-African group of SV and the Indian-Far Eastern-Australian group of SV, hybridizations were performed under the stringent conditions of 57”, in 2~ SSC and 33% formamide (an effective temperature of 26” below the T,,, for SV ds RNA). These conditions are optimal for the rate of reassociation for nucleic acid (Wetmur and Davidson, 1968) but require at least 82% base matching for a homologous hybridized molecule to be thermally stable (Howley et al., 1979).
GENOMIC DIVERGENCE
65
AMONG SINDBIS VIRUS STRAINS TABLE 2
SUMMARYOFPOLYNUCLEOTIDESEQUENCERELATIONSHIP AMONGRNAs OFSINDBISVIRUS STRAINS, OTHER ALPHAVIRUSES, AND POLIOVIRUSUNDER NONSTRINGENTCONDITIONSOF HYBRIDIZATION ds RNA Virion RNA
SV Egypt AR-339
SV Egypt AR-339 Uganda MP-684 South Africa AR-86 Israel M-1855 Czechoslovakia R-33 USSR AZ-16
0.99 t 0.01 0.99 k 0.01 0.98 f 0.01 1.00 1.00
India A-1036 India B-322/23124 Malaya MM-2215 Australia C-377 Australia Ch 19470
0.51 ” 0.02 ND 0.42 5 0.02 0.47 r 0.02 0.49
VEE EEE WEE WHA Poliovirus
1.00
SV India A-1036
SV Australia C-377
0.44 t 0.04 0.46 0.47 0.39 0.40 0.41
0.41 2 0.01 0.40 0.38 0.41 0.36 0.35
1.00
1.00 0.90 k 0.02 0.90 2 0.01 0.89
0.91 ? 0.01 0.90 0.97 ‘-c 0.01 1.00 1.00
ND ND 0.06 0.19 co.01
Note. Details of the reactions are given under Materials and Methods and in Fig. 2. Results represent the maximal homology obtained at plateau level of the saturation curve and are expressed as decimal fractions of the value for homologous annealing. Some of the results are the mean of three experiments and are indicated by standard deviation values.
In the European-African group, 3Hlabeled ds RNA of SV Egypt AR-339 was annealed with virion RNA of isolates belonging to the same group (Table 3). Values indicating a close relationship of 0.92, 0.99, and 0.95 were obtained for SV Egypt AR339, SV Israel M-1855, Czechoslovakia R-33, and USSR AZ-16, respectively. The hybridized fraction obtained with SV Uganda MP-684 was 0.83 and with SV South Africa AR-86,0.72. When SV South Africa AR-86 ds RNA was used as a source of complementary strand, a fraction of 0.94 was found with SV Uganda MP-684 ss RNA, 0.83 with SV Egypt AR-339 and SV Israel M-1855 ss RNA, and 0.76 with SV Czechoslovakia R-33 ss RNA. Under these stringent conditions, a very low degree of homology was detectable with the ss RNAs of the Indian-Far Eastern-Australian group (0.06 with SV India A-1036 and 0.05 with SV Australia C-377). Representative SV strains of the Indian-
Far Eastern-Australian group demonstrated a somewhat greater degree of heterogeneity in their nuclei acids (Table 4). Using SV India A-1036 ds RNA, relatedness values of 0.98 and 0.87 were obtained with SV India B-322/23/24 virion RNA and SV Philippines P-886 virion RNA whereas 0.66, 0.66, and 0.63 were found with SV Malaya AM-2215, SV Australia C-377, and SV Australia Ch-19470 ss RNAs, respectively. The RNA of the two Indian strains and the Philippine strain appear to share from 0.55 to 0.61 of their nucleotide sequence with SV Australia C-377 RNA. The homologous fraction of the same Australian strain reached 0.90 with SV Malaya AM2215 and 0.98 with SV Australia Ch-19470. InJuence
of Host and Year of Isolation
Sindbis virus strains used in these studies were originally isolated from humans, birds,
66
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TABLE 3
DISCUSSION
POLYNUCLE~TIDE SEQUENCE RELATIONSHIP AMONG RNAs OF SINDBIS VIRUS STRAINS BELONGING TO THE EUROPEAN-AFRICAN GROUP
The extent of homology among viral genomes of a family of viruses, related by serological and morphological criteria, has very often been underestimated (Howley et al., 1979). Most likely, this is due to the stringency of the hybridization techniques employed. Under standard hybridization conditions, only those regions of DNA with as much as 80-85% base matching will be thermally stable, and significant regions of homology with greater than 15% base mismatch will be scored as nonhomologous. Due to the degeneracy of the genetic code, identical proteins may be encoded by nucleic acids with as much as 33% base mismatch. In the experiments presented, we have examined the nucleic acid homology of Sindbis virus strains under both stringent and nonstringent conditions. In standard salt concentration (2 x SSC) pancreatic ribonuclease is able to digest short doublestranded RNAs dispersed among nonhomologous sequences. Under higher salt concentration, short double-stranded segments adjacent to single-stranded RNA segments
ds RNA
Virion RNA SV Egypt Israel M-1855 Czechoslovakia R-33 USSR AZ-16 Uganda MP-684 South Africa AR-86 India A-1036 Australia C-377
SV Egypt AR-339 1.00
0.92 0.99 0.95 0.83
f k 2 k
sv South Africa AR-86 0.83 + 0.02
0.02 0.01 0.01 0.01
0.72 f 0.04
0.83 f 0.03 0.76 f 0.01 0.94 f 0.01 1.00
0.06
-
0.05
-
Note. Hybridizations were carried out under stringent conditions as described under Materials and Methods. Results are expressed as decimal fractions of the value for homologous annealing. Standard deviations are based on the mean ofthree experiments.
mites, and mosquitoes (Table 1). The lapse of time between the earliest Sindbis isolation and the most recent isolation available was 24 years. In order to know whether year of isolation or animal host played any role in the heterogeneity of the virus nucleic acids, cross annealings were performed under stringent conditions with strains isolated in the same country from different hosts over a long period of time. Hybridizations utilizing SV South Africa AR-86 ds 13H]RNA as probe and virion RNAs of SV South Africa AR-6071, AR18132, both isolated from Culex univittatus, 10 and 22 years after the 1954 isolation of SV South Africa AR-86 as well as vii-ion RNA of SV South Africa Girdwood, isolated from man 11 years after the SV AR-86 strain showed no significant differences (Table 5A). Hybridizations of 2 strains isolated in India from two different hosts in the same year (Table 5B) showed no significant sequence divergence detectable by this method as well as hybridizations performed with two SV strains isolated in Australia from mosquitoes, 16 years apart (Table 5C).
TABLE 4 POLYNUCLE~TIDE SEQUENCE RELATIONSHIP AMONG RNAs OF SINDBIS STRAINS BELONGING TO THE INDIAN-FAR EASTERN-AUSTRALIAN GROUP ds RNA sv
Virion RNA SV India A-1036 India B-322/23/24 Philippines P-886 Malaya MM-2215 Australia C-377 Australia Ch-19470 Egypt AR-339 USSR AZ-16 Israel M-1855
SV India A-1036
Australia
1.00
0.61 ” 0.04
0.98 + 0.01 0.87 ” 0.02 0.66 f 0.03 0.66 k 0.02
0.58 0.55 + 0.02 0.90 * 0.02 1.00
0.63
0.98
0.08 0.05 0.03
0.05 0.04 0.05
c-377
Note. Hybridizations were carried out under stringent conditions as described under Materials and Methods. Results are expressed as a decimal fraction of the homologous reaction.
GENGMIC DIVERGENCE
67
AMONG SINDBIS VIRUS STRAINS TABLE 5
POLYNUCLEOTIDESEQUENCERELATIONSHIP AMONGRNAs OF SINDBIS VIRUS STRAINS ISOLATED IN SAME AREA FROMVARIOUS HOSTSAND/ORAT VARIOUS PERIODSOF TIME
THE
ds RNA
Virion RNA SV South Africa AR-86 (A) SV South Africa AR-86 South Africa AR-6071 South Africa AR-18132 South Africa Girdwood
1954 1964 1976 1963
Spring Johannesburg Upington Johannesburg
Culex Culex Culex Man
1.00
1.00 0.97 + 0.01 1.00
SV India A-1036 (B) SV India A-1036 India B-322/23/24
1953 1953
Mysore State Bombay State
Mite Wagtail (bird)
0.98
Mosquito Mosquito
1.00 0.98
1.00
SV Australia C-377 (C) SV Australia C-377 Australia Ch-19470
1960 1976
Cairns (Queensland) Charleville
Note. Hybridizations were carried out under stringent conditions. Results are expressed as a decimal fraction of the homologous reaction. (A) South African strains; (B) Indian strains; (C) Australian strains.
or unpaired bases between double-stranded regions appear to be more protected from ribonuclease digestion. Under stringent conditions (2x SSC, 33% formamide, 57”), the nucleic acid homology of some of the Sindbis viruses isolated from different countries was very low, but under nonstringent conditions (5x SSC, 57”), the degree of homology of various strains was maximized, and well-defined groups were recognized (Fig. 5). While this technique of using ribonuclease is useful in comparing the relative degree of homology of the Sindbis virus genomes, the data do not allow us to quantitate the number of homologous nucleotides. The relevance of hybridization data to serological relatedness is well illustrated in Table 2. Alphaviruses that show some antigenic cross-reactivity with Sindbis virus, such as WEE and WHA, are measurably related also by nonstringent hybridization. Similar findings were described by Wengler et al. (1977) in an analysis of RNA homology among four alphaviruses. Under the hybridization conditions utilized, significant RNA homology was found only between serologically closely related viruses such as Chikungunya (CHIK) virus and O’nyong-nyong (ONN) virus
(Porterfield, 1961). In order to minimize the effect of defective viral RNA in our studies, each virus strain was plaque purified three times and viral stocks were established with no further passage. The 15 strains of Sindbis virus studied were obtained from 10 different countries and 12 different hosts. Their isolations spanned a period of 25 years. Using RNARNA hybridization, under nonstringent conditions, these 15 isolates could be divided into two groups which corresponded to two geographic areas. The first group includes the isolates from Europe and Africa and the second group includes the isolates from India, the Far East, and Australia. Using stringent conditions of hybridization, further genetic subdivisions were observed within each main group. The host and the year of isolation were considered as potential factors leading to this genetic divergence. However, no variation was observed among South African strains isolated from different hosts and only a 2% difference was observed with the Indian strains isolated the same year from different hosts. Two South African strains isolated from the same host, 22 years apart, differed by only 3% and two Aus-
68
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PALE ARCTIC REGION
FIG. 5. Sindbis virus isolates can be divided into major groups based on their genomic similarities as well as geographic distribution.
tralian strains isolated 16 years apart shared 98% of homology. Strikingly, Egyptian strain AR-339 isolated in 1952 from a pool of Culex mosquitoes and Czechoslovakian strain R-33 recovered in 1971 from a bird were identical under stringent conditions. Thus the variations detected with regard to year of isolation or host were within the standard error (1 to 4%) of the hybridization technique. These observations would indicate that the subdivision of the various Sindbis strains was primarily a function of geographic region of isolation. A display of the results of hybridizations on the map (Fig. 5) shows that the subdivision of SV variants into subgroups follows the Wallace line (Darlington, 1957; Simpson, 1968) which divides the world into six zoogeographic regions. Sindbis virus is found in four of these regions, the Palearctic, Ethiopian, Oriental, and Australian regions. The degree of relationship between the RNAs of Sindbis isolates from Egypt, Israel, USSR, and Czechoslovakia varied between 92 and 100%. All these countries belong to the Palearctic region. The genetic
relationship of the isolates from Uganda and South Africa, located in the Ethiopian region, was very high (92 to 100%) and formed another subgroup. The Sindbis strains isolated from the Oriental region (India, Philippines) formed a homogeneous group distinct from the Australian isolates. An exception is the Malayan strain MM-2215 which appeared to be closer to the Australian group. These zoogeographic regions are based on mammalian distribution and are separated by natural migration barriers both topographic and climatic. The frequency of Sindbis virus isolation from birds suggests that they are maintenance hosts and participants in the virus transmission cycle (McIntosh et al., 1969). Since migratory birds are not necessarily subject to the barriers separating the zoogeographic regions, the distribution of Sindbis subgroups within these barriers seems to minimize the importance of birds in disseminating the virus. Sindbis virus has been isolated from a variety of hosts including the frog, several species of birds and mammals, and a variety
GENOMIC DIVERGENCE
AMONG SINDBIS VIRUS STRAINS
of vectors including several species of Culex, Aedes, Mansonia mosquitoes. Other alphaviruses are not found in so wide a variety of hosts, and so this observation may explain the wide geographic distribution of Sindbis virus, which is, in fact, the most widely distributed of all arboviruses (Theiler and Downs, 19’73). The variation in the genome of the Sindbis strains, and the consequent antigenic differences, appears to be a result of a divergent evolution from a common ancestor. Our data cannot determine whether the divergence occurred primarily through genetic drift, or through adaptation to unique, ecological conditions within each zoogeographical zone. The data obtained from hybridizations performed with RNA of other alphaviruses (Table 2) under nonstringent conditions show virtually no homology between Sindbis and either VEE or EEE genomes. Homology to SV of about 9 and 22% was observed with WEE and WHA RNAs, respectively, indicating a genetic relationship between these viruses. For both viruses, the same fractional hybridization was observed regardless of the Sindbis strain used as source of the complementary strand. These results suggest that Sindbis virus, Western equine encephalitis virus, and Whataroa virus share common nucleotide sequences and could have evolved from a common ancestor. Divergence of these three viruses most likely would have begun before the divergence of the various Sindbis geographic groups described in this paper. In summary, we have shown that, by relaxing the usual stringent conditions of RNA homology testing, incomplete homologies can be recognized in the genome of certain alphaviruses. In the case of Sindbis virus, the data indicate that a given subgroup is restricted to a single zoogeographic region. ACKNOWLEDGMENTS I wish to thank Dr. P. M. Howley for his support and detailed review of the manuscript, Dr. J. M. Dalrymple for his encouragement during the completion of the work and his valuable suggestions, and Dr. B. W. Burge for his helpful comments. I also wish to thank Ms. Susan Hostler for her skillful typing.
69
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CASALS,J. (1964). Antigenic variants of eastern equine encephalitis virus. J. Exp. Med. 119, 547- 565. CLARKE, D. H. (1963). Antigenie variation and geographic distribution of arboviruses. Ann. Microbial. 11, 143-148. DARLINGTON,P. J. (1957).Zoogeography. In “The Geographical Distribution of Animals.” p. 2, Fig. 1. Wiley, New York. DOBOS, P., and FAULKNER, P. (1970). Molecular weight of Sindbis virus ribonucleic acid, as measured by polyacrylamide gel1 electrophoresis. J. Viral. 6, 145- 147. DOHERTY, R. L., CARLEY, J. G., MACKERRAS,M. J., and MARKS, E. N. (1963). Studieson arthropodborne virus infections in Queensland. III. Isolation and characterization of virus strains from wildcaught mosquitoes in North Queensland. Aust. J. Erp. Biol. Med. Sci. 41, 17-40. EAGLE, H. (1971). Buffer combinations for mammalian cell culture. Science 174, 500-503. EAVRI (196-1962). Annual Report. ERNEK, E., KOZUCH,E., GRESIKOVA,M., NOSEK,J., and SEKEYOVA,M. (1973). Isolation of Sindbis virus from the reed warbler ( Acrocephalus scirpaceus ) in Slovakia. Acta Viral. 17, 359-361. ERNEK, E., KOZUCHO., NOSEK, J., and LABUDA, M. (1973-1974). Evidence for circulation of Sindbis virus and other arboviruses by using sentinel animals in Western Slovakia. Intervirology 2, 186192. FRANKLIN, R. M. (1966). Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc. Nut. Acad. Sci. USA 55,15041511. GAIDAMOVICH,S. YA., ISMAILOV, A. SH., KLISENKO, G. A., and MIRZOEVA, N. M. (1978). Detection of Sindbis and West Nile viruses in the blood of living birds by indirect haemagglutination. Acta Viral. 22, 430.
GRESIKOVA,M., SEKEYOVA, M., BATIKOVA, M., and BIELIKOVA, V. (1973). Isolation of Sindbis virus from
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the organs of a hamster in East Slovakia. In “Internatiomdes arbeitzkolloquium uber Naturkerde von Infektionskrankheiten in Zentraleuropa,” April 1973, 17-19, Illmitz 1971/l pp. 59-63. GRESIKOVA, M., SEKEYOVA, M., TEMPERA, G., GUGLIELMINO, S., and CASTRO, A. (1978). Identi-
fication of a Sindbis virus strain isolated from marginatum ticks in Sicily. Acta Viral. 22, 231-232. HOWLEY, P. M., ISRAEL, M. A., LAW, M.-F., and MARTIN, M. A. (1979). A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes. J. Biol. Chum. 254, 4876-4883. Institut Pasteur de Dakar (1972). Rapport Annuel. KAARIAINEN, L., and GOMATOS,P. J. (1969). A kinetic analysis of the synthesis in BHK 21 cells of RNAs specific for Semliki Forest virus. J. Gen. Virol. 5, 251-265. KARABATSOS,N., BOURKE,A. T. C., and HENDERSON, J. R. (1963). Antigenic variation among strains of western equine encephalomyelitis virus. Amer. J. Trop. Med. 12, 408-412. KOZUCH, O., LABUDA, M., and NOSEK, J. (1978). Isolation of Sindbis virus from the frog Rana Hyalomu
ridibunda.
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LEVIN, J. G., and FREIDMAN, R. M. (1971). Analysis of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis. J. Viral 7, 504-514. LIM, T. W., BURHAINUDDIN, M., and ABBAS, A. (1972). A case of Sindbis virus infection in Kuala Lumpur. Med. J. Malaysia 27, 147-149. MALHERBE, H., STRICKLAND-CHOLMLEY, M., and JACKSON, A. L. (1963). Sindbis virus infection in man. S. Afr. Med. J. 37, 547-552. MCCONAUGHY,B. L., LAIRD, C. D., and MCCARTHY, B. J. (1969). Nucleic acid reassociation in formamide. Biochemistry
8, 3289-3295.
MCINTOSH, B. M., DICKINSON, D. B., and MCGILLIVRAY, G. M. (1969). Ecological studies on Sindbis and West Nile virus in South Africa. V. The response of birds to inoculation of virus. S. Afr. J. Med. Sci. 34, 77-82. NIR, Y., AVIVI, A., LASOVSKI,Y., MARGALIT, J., and GOLDWASSER,R. (1972). Arbovirus activity in Israel. Zsr. J. Med. Sci. 8, 1695-1701. NIR, Y., GOLDWASSER,R., LASOVSKI, Y., andAv1v1, A. (1967). Isolation of arboviruses from wild birds in Israel. Amer. J. Epidemiol. 86, 372-378. PENMAN, S. (1966). RNA metabolism in the Hela cell nucleus. J. Mol. Biol. 17, 117-130. PFEFFERKORN, E. R., and HUNTER, H. S. (1963). Purification and partial chemical analysis of Sindbis virus. Virology 20,433-445. PORTERFIELD,J. S. (1961). Cross-neutralization studies with group A arthropod-borne viruses. Bull. WHO 24, 735-741. RUDNICK, A., HAMMON,W.McD., and SATHER, G. E. (1962). A strain of Sindbis virus isolated from Culex bitaeniorhynchus mosquitoes in the Philippines. Amer. J. Trop. Med. Hyg. 11, 546-549.
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SEGAL, S., and STREEVALSAN,T. (1974). Sindbis virus replicative intermediates: Purification and characterization. Virology 59, 428-442. SHAH, K. V., JOHNSON,H. N., RAO, T. R., RAJAGOPALAN,P. K., and LAMBA, B. S. (1960). Isolation of five strains of Sindbis virus in India. Indian J. Med. Res. 48.300-308. SIMMONS, D. T., and STRAUSS,J. H. (1972a). Replica-
tion of Sindbis virus. I. Relative size and genetic content of 26s and 49s RNA. J. Mol. Biol. 71, 599-613. SIMMONS,D. T., and STRAUSS,J. H. (1972b). Replication of Sindbis virus. II. Multiple forms of doublestranded RNA isolated from infected cells. J. Mol. Biol. 71, 615-631. SIMMONS,D. T., and STRAUSS,J. H. (1974). Translation of Sindbis virus 26s RNA and 49s RNA in lysates of rabbit reticulocytes. J. Mol. Biol. 86, 397-409.
SIMPSON,D. I. H., GORDONSMITH, C. E., BOWEN, E. T. W., PLATT, G. S., WAY, H., MCMAHON, D., BRIGHT, W. F., HILL, M. N., MAHADEVAN, S., and MCDONALD, W. W. (1970). Arbovirus infection in Sarawak; virus isolations from mosquitoes. Ann. Trop. Med. Parasitol. 64, 137-151. S. Afr. Inst. Med. Res. (1973). Annu. Rep., p. 2. SIMPSON,G. C. (1968). “Evolution and Geography.” Oregon State System of Higher Education, Eugene. TAYLOR, R. M., HURLBUT, H. S., WORK, T. H., KINGSTON,J. R., and FROTHINGHAM,T. E. (1955). Sindbis virus: A newly recognized arthropod-transmitted virus. Amer. J. Trop. Med. Hyg. 4,844-862. THEILER, M., and DOWNS,W. G. (1973). “The Arthropod-Borne Viruses of Vertebrates,” p.115. Yale University Press, New Haven/London. TRAUB, R., ELISBERG, B. L., WEBB, P. A., SHOPE, R., WEBB, H., VANREENEN, R. M., POND, W. L., and WAID, S. T. (1958). “Annual Report of the Institute for Medical Research for 1957, Federation of Malaya,” pp. lOO- 106. Government Press, KuaIa Lumpur. WEINBREN, M. P., KOKERNOT, R. H., and SMITHBURN, K. C. (1956). Strains of Sindbis-like virus isolated from culicine mosquitoes in the Union of South Africa. 8. Afr. Med. J. 30,631-636. WENGLER, G., WENGLER, G., and FILIPE, A. R. (1977). A study of nucleotide sequence homology between the nucleic acids of different alphaviruses. Virology 78, 124-134. WENGLER, G., WENGLER, G., and WAHN, K. (1976). Isolation and characterization of double-stranded RNA containing infectious viral genome RNA from cells infected with Semliki Forest virus. Arch. Viral. 50, 45-53. WETMUR, J. G., and DAVIDSON,N. (1968). Kinetics of renaturation of DNA. J. Mol. Biol. 31, 349-370. YOUNG, N. A., and JOHNSON,K. M. (1969). Antigenic variants of Venezuelan equine encephalitis virus: Their geographic distribution and epidemiologic significance. Amer. J. Epklemiol. 89, 286-307.
106, 59-70 (1980)
Genomic FRANCOISE
Divergence
among
RENTIER-DELRUE’
Sindbis AND
Virus Strains
NATHANIEL
A. YOUNG2
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 Accepted May 17, 1980
Antigenic variants of the alphavirus Sindbis have been isolated from the Palearctic, Ethiopian, Oriental, and Australian zoogeographic regions. The genomes of these variants were analyzed for homology by the hybridization of virion RNAs to double-stranded RNAs isolated from infected cells. Under nonstringent conditions (T, - 55”) the RNAs of OrientalAustralian strains showed only 35 to 51% nucleotide sequence homology with the RNAs of the Palearctic-Ethiopian strains although homology was essentially complete among isolates within the Oriental and Australian regions and among isolates within the Palearctic and Ethiopian regions. Under more stringent conditions (T, - 26”), nucleotide sequence differences of 2 to 45% were detected among the RNAs of virus strains from geographically distant localities within each of these two major zoogeographic subdivisions. Year of isolation, passage history, and vertebrate or invertebrate host of origin were not major determinants of sequence heterology. The hypothesis that ancestral Sindbis virus became separated by geographic barriers and evolved into two distinct types is presented. Further divergent evolution has obviously occurred within each of these types.
Malaysia (Traub et al., 1957; Simpson et al., Previous studies have shown that anti- 1970; Lim et al., 1972), India (Shah et al., genie similarities among strains of several 1960), the Philippines (Rudnick et al., 1962), togaviruses follow geographic lines. Among Australia (Doherty et al., 1963), West Africa (Institut Pasteur de Dakar, 1972), alphaviruses, Casals (1961, 1964) reported Israel (Nir et al., 1967, 1972), Mozambique antigenic variants of Chikungunya (CHIK), (S. Afr. Inst. Med. Res., 1973), CzechosloSindbis (SV), and Eastern equine encephvakia (Ernek et al., 1973; Gresikova et al., alitis virus (EEE), using a modification of 1973; Kozuch et al., 1978), USSR (Gaidamothe hemagglutination inhibition test. Karavitch et al., 1978), and Sicily (Gresikova batsos et al. (1963) found antigenically et al., 1978). By antigenic analysis, three distinct isolates of Western equine encephalitis (WEE) virus in the eastern and Indian strains, an Australian strain, and western United States. Young and Johnson two African strains were shown to form (1969) examined antigenic variation among three distinct subgroups correlating to their 124 strains of Venezuelian equine enceph- geographic distribution (Clarke, 1963). The genome of Sindbis virus is a singlealitis (VEE) virus and found a correlation stranded RNA with a molecular weight of between antigenie variation and the geo4.3 x lo6 and with a sedimentation cographic location of the original virus efficient of about 42 S (Dobos and Faulkner, isolation. Sindbis virus has a wide geographic dis- 1970; Simmons and Strauss, 1972a). In intribution. It was first isolated in Egypt by fected cells, in addition to the 42 S RNA, a Taylor (1955) and subsequently isolated in 26 S virus-specified, single-stranded RNA South Africa (Weinbren et al., 1956; Mal- of the same polarity is synthesized (Levin and Friedman, 1971) which serves as the herbe et al., 1963), Uganda (Eavri, 1962>, specific messenger for the structural proteins of the virus particles (Cancedda 1 To whom reprint requests should be addressed. et al., 1974; Simmons and Strauss, 1974). e This work was performed in the laboratory of Dr. In addition, double-stranded viral RNAs Nathaniel A. Young, who died on February 4, 1979. INTRODUCTION
59
0042-6822/80/130059-12$02.00/O Copyright All rights
Q 1980 by Academic Rem, Inc. of reproduction in any form reserved
60
RENTIER-DELRUE
AND YOUNG
TABLE SUMMARY Geographic country
OF THE
PEDIGREE
1
OF FIFTEEN
SINDBIS
origin Locality
Strain
Y&V isolated
Origin
Sindbis
AR-339
1952
C&z
Entebbe
MP-634
1953
spring
AR-36
upington
of isolation
univittatus
STRAINS Laboratory p.%Wge hosts
STUDIED Number of passage
S0UKe
Mice
13
Yale Arbovirus Research Unit
Mamoniafwcopennata
Mice
7
Yale Arbovirus Research Unit
1954
Culer
spp.
Mice
3
Yale Arbovirus Research Unit
AR-18132
1976
Cules
univittatw
Mice
1
National Institute for Virology, Transvaal
AR-6671
1964
Mice
3
National Institute for Virology, Transvaal
Johannesburg
Giidwood
196.3
Isl%si
HadsiX
M-1355
1967
USSR
Aserbsiisn
AZ-16
1971
iVycticmm (bird)
Czeeboslowkia
Western
R-33
1971
Amcephalw scirpacewr
India
Mysm
A-1036
1953
Bdellcmysaus
B-33%3/34
1953
Wagtail alba)
Malaya
MM-2215
1955
Philippines
P-336
Uganda
South
Africa
Australia
state
Cdl-Jlh
Mice
3-l
National Institute for Virology, Transvaal
LOW
Israel Institute for Biological Research
Mice
5
Institute for Virology, Moscow, USSR
Mice
I
Institute of Virology, Slovak Academy SCiWXe
Mice
13
Yale Arbovirus Research Unit
Mice
10
National Institute of Virology, Poona, India
Culer tritaeniorhynchus
Mice
2
Yale Arbovinis Research Unit
1956
C&z
Mice
1
Yale Arbovinis Research Unit
C-377
1960
Mansonia septempunctata
Mice
1
Yale Arbovirus Research Unit
Ch-19470
1976
Pool of mosquitoes
Mice
4
Queensland Institute of Medical Research, Brisbane
_ KIhims) Chsrleville
MEUI
nycticomz
bursa
(Motucillu
bitaeniorhynchus
are formed (Levin and Friedman, 1971; streptomycin, and 0.5% of a combination of Simmons and Strauss, 1972b; Segal and three organic buffers: 10 mM BES (NJ, Streevalsan, 1974). In this report, we bis-(2-hydroxyethyl-2-aminoethanesulfonic compared the genome of 15 strains of acid); 15 mM HEPES (N-&hydroxyethylpiperazine-N-2-ethanesulfonic acid), and 10 Sindbis virus by saturation hybridization experiments using unlabeled virion RNA an- mM HEPPS (N-2-hydroxyethylpiperazinenealed to 3H-labeled viral double-stranded N-2-propanesulfonic acid) (BHE) (Eagle, 1971). RNA. Viruses. Table 1 summarizes the information on the 15 Sindbis isolates used in this MATERIALS AND METHODS study. Each isolate had a low passage history Cells. Primary chick embryo fibroblasts and was subjected to three successive (CEF) were cultured in 199 medium sup- plaque purifications. Virus stocks were plemented with 2.5% fetal calf serum established from these plaque isolates with (Gibco), 2.5% chicken serum, penicillin and no further passage. Other alphavirus strains
GENOMIC
DIVERGENCE
AMONG
received from other laboratories included: WEE Cal., EEE PE-6, and VEE TC-83 kindly provided by Dr. G. Eddy (Fort Detrick, Md.), and Whataroa (WHA) virus M-78 given by Dr. R. E. Shope (Yale Arbovirus Research Unit). Virus production and purijkation. Confluent monolayers of CEF were washed with Hanks’ balanced salt solution and infected with 0.01 PFU per cell in 199 medium containing 0.01 pg/ml actinomycin D (Calbiochem) and 0.5% BHE. After adsorption for 1 hr at 33”, the inoculum was replaced by the same medium supplemented with 1% fetal calf serum. After incubation at 33” for 18 to 20 hr, the extracellular fluid was harvested and usually contained 5.0 x lo7 to 5.0 x lOlo PFU/ml depending on the virus strain used. The clarified supernatant was adjusted to 0.5 M NaCI and 10% polyethylene glycol 6000 (BDH Chemicals). The suspension was stirred overnight at 4” and the precipitate collected by centrifugation at 12,000 g for 30 min. The pellet was resuspended in l/lOth of the original volume of 2x TNE (TNE: 0.05 M Tris; 0.1 M NaCl, 10m3M EDTA, pH 7.6) and sonicated on ice for 2 min at a setting of 4 in a Branson W-350 sonifier. The suspension was layered on a 15-30% sucrose gradient (w/w) in TNE buffer and centrifuged at 85,000 g for 2.5 hr at 4” in a SW 27 rotor (Beckman). The opalescent bands containing virus were diluted fourfold in TNE buffer and the virus was pelleted at 170,000 g for 1 hr at 4” in a R60 Ti rotor (Beckman). The pellets were resuspended in 2~ TNE and used within a few hours. Viral RNA pur$.cation. Sodium dodecyl sulfate (SDS) was added to the viral suspension at a final concentration of 0.5%. RNA extraction was carried out at room temperature with equal volumes of phenolcresol (7:l) equilibrated with 2X TNE, pH 7.6, containing 0.0125% (w/v) 8-hydroxyquinoline, and chloroform-isoamyl alcohol (1OO:l). A second extraction with chloroform-isoamyl alcohol (1OO:l) was then performed, followed by five ether extractions. Ether was removed by bubbling nitrogen through the tinal aqueous phase and the viral RNA was adjusted to 0.4 M NaCl (final
SINDBIS
VIRUS
STRAINS
61
concentration). Viral RNA was ethanol precipitated, resuspended in 2~ TNE, pH 7.6, and stored at -70”. The ss RNA concentration was determined from the optical density at 260 nm. Preparation stranded viral
of radiolabeled doubleRNA. Confluent primary
CEF monolayers were washed with Hanks’ balanced salt solution and infected with virus at a multiplicity of 50 PFU per cell. After 1 hr at 34”, virus inoculum was replaced by 199 medium containing 1% fetal calf serum and 2 pg/ml actinomycin D. One hour later, 100 PCiiml [3H]uridine (45-47 Ci/mmol) (New England Nuclear) was added. Seven hours after infection, the fluid was discarded and the cells were washed with phosphate buffered saline (PBS) and resuspended in SDS-acetate buffer (0.35% SDS, 0.01 M sodium acetate pH 5.1, 0.1 M NaCl). Total RNA was extracted at 60” with phenol equilibrated with acetate buffer (0.01 M sodium acetate pH 5.1, 0.1 M NaCl) and chloroform-isoamyl alcohol (100: 1) (Penman, 1966). RNA was precipitated with 2 vol of ethanol at -2o”, overnight and resuspended in TNE buffer. Differential salt precipitation of single-stranded RNA was carried out according to the method of Bishop and Koch (1967). Doublestranded RNA in the supernatant was ethanol precipitated, redissolved in TNE buffer at a maximum concentration of 0.5 mg RNA/ml, and purified by two cycles of chromatography on cellulose columns (Whatman CF,,). Successive elutions with 15% ethanol in TNE buffer and with buffer alone were carried out as described by Franklin (1966). RNA-RNA hybridization. Viral doublestranded RNA was denatured in SSC (0.15 M NaCl, 0.015 M Na-citrate) at 120” for 10 min, in the presence of highly polymerized yeast RNA (15 pg/ml) (Calbiochem) and rapidly cooled. A maximum of 0.002 pg of 3H-labeled double-stranded RNA (specific activity: 8.5 X lo”-1.0 X 10” cpmipg) was allowed to anneal with increasing amounts of homologous or heterologous unlabeled virion RNA. Reaction mixture (l.O-ml volumes) were incubated for 4 hr, in 5 x SSC at 57” for “nonstringent” reactions and in 2x SSC and 33% formamide (Eastman
62
RENTIER-DELRUE
AND YOUNG
since the G-C content of SV RNA (50%) (Pfefferkorn and Hunter, 1963) is very close to that of SFV RNA (51%) (Kaariainen and Gomatos, 1969), these same T,,, values were employed in calculating the effective temperatures of our hybridization reactions. Purified ds RNA preparations had specific activities ranging from 8.5 x lo5 to 1 x 10” cpmlpg and were 97 to 100% resistant to pancreatic ribonuclease A (40 pg/ 1.0 2.0 3.0 ml, in 2 x SSC). After denaturation, 3 to 5% tq of RNA of the RNA remained resistant to panFIG. 1. Annealing of 3H-radiolabeled Sindbis Egypt creatic ribonuclease A and since this mateAR-339 ds RNA with unlabeled virion ss RNA. rial could participate in annealing reactions, Samples containing 3H-labeled ds RNA (1.5 ng, 1000 it was considered as background. Homolocpm) and various amounts (0 to 3 pg) virion ss RNA in 1 gous annealing of several Sindbis strains ml of 2 x SSC, 33% formamide, were annealed at 57” for were first performed in 2x SSC, 33% 2 hr. Each RNA sample was digested with pancreatic formamide at 57” for 2 hr as a control of the ribonuclease A (40 &ml) for 30 min at 37”. The RNA was precipitated with trichloroacetic acid and counted purity of the reagents. Formamide was for radioactivity. Results were expressed as the used in order to lower the T,,, of the RNA percentage of residual input radioactivity that de- duplex and permit hybridization experiments to be carried out at lower temperaveloped RNAse resistance. tures. Since formamide lowers the T,,, of a DNA duplex by 0.72” for every 1% increase in formamide concentration (ConKodak) at 5’7” for “stringent” reactions. aughy et al., 1969), the “stringent condiThe samples were further incubated in the presence of 40 pglml pancreatic ribonuclease tions” of hybridization used in these studies correspond to an effective temperature of A (Worthington Biochemical Corporation) about 26” below the T,,,. A representative for 30 min at 37”. Ribonuclease-resistant RNA was precipitated with cold 5% tri- saturation curve obtained with prototype chloroacetic acid (TCA) in the presence of Sindbis Egypt AR-339 is shown in Fig. 1. 50 pg/ml yeast RNA (Schwarz/Mann) as Since the virion RNA is complementary to carrier, and collected on type HA filters only one of the radiolabeled strands, the (Millipore). Filters were dried and counted maximum hybridization reached in the absence of the probe reannealing with itusing scintillation fluid (Econofluor, NEN) in a Beckman LS 250 scintillation spec- self would be 50%. In the experiment presented in Fig. 1, this plateau level was trometer. achieved with 0.7 pg of virion RNA. RESULTS
The melting temperature (T,) of SV ds RNA used in our calculations was estimated by comparison to T,,, values for Semliki Forest virus (SFV) ds RNA, another alphavirus. SFV ds RNA is reported to be 89” in 0.1~ SSC (Wengler et al., 1976). In preliminary experiments (unpublished results) we found the T,,, of SFV ds RNA to be 102” in SSC and 106” in 2x SSC. By extrapolation from the data of Billeter et al. (1966), the T, in 5x SSC was estimated to be 112- 114”. Since the T, is a function of the G-C content of RNAs (Billeter et al., 1966), and
Analysis of RNA Homology between Sindbis Virus Strains under Nonstringent Hybridization Conditions The initial analysis of homology among Sindbis virus isolates from remote geographic area was performed under nonstringent hybridization conditions in order that regions containing significant base mismatch would be thermally stable and detected as duplex in the enzymatic assay (Howley et al., 1979). The hybridizations were performed in 5 x SSC at 57” for 4 hr, an effective temperature of about 55” below the
GENOMIC DIVERGENCE
AMONG SINDBIS VIRUS STRAINS
63
,&I OF RNA
FIG. 2. Annealing of four strains of Sindbis virus ss RNA with Sindbis Egypt AR-339ds RNA isolated from infected cells. Denatured 3H-radiolabeled SV Egypt AR-339 ds RNA, 1.5 ng per tube, 1000cpm, was annealed with increasing quantities of ss RNA from either homologous or heterologous strains of Sindbis virus. Reactions were carried out in 5 x SSC at 57” for 4 hr. Acid-precipitable radioactivity was determined after treatment with 40 pg/ml pancreatic ribonuclease A. The homologous reaction was normalized to a value of 1.0 and the results were expressed as a decimal fraction of the homologous reaction. (m) SV Egypt AR-339 ss RNA: (Cl) SV South Africa AR-36 ss RNA: (0) SV India A-1036 ss RNA; (0) SV Australia C-377 ss RNA; and (A) poliovirus ss RNA.
T,,, for SV ds RNA. The initial analysis involved SV Egypt AR-339, SV India A1036, SV Australia C-377, and SV South Africa AR-86. Each radiolabeled ds RNA was denatured and reacted in the presence of increasing amounts of virion ss RNA. Under these conditions, plateau levels were easily reached with from 1 to 3 pg ss RNA. In each case, the homologous reaction which achieved a plateau level of approximately 50% was normalized to a value of 1.0 and the results were expressed as a decimal fraction of the homologous reaction. By this analysis, a radiolabeled SV Egypt AR-339
probe was found to be almost completely homologous with SV South Africa AR-86 virion RNA (0.99). SV India A-1036 and SV Australia C-377 respectively, annealed to 51 and 47% of the complementary strand with the probe (Fig. 2). The specificity of the hybrids formed was demonstrated by the absence of detectable annealing with poliovirus ss RNA. Reciprocal experiments were performed utilizing SV India A-1036 ds [3H]RNA and SV Australia C-377 ds [3H]RNA as probes (Figs. 3 and 4), respectively. SV India A1036 and SV Australia C-377 were found
pg OF RNA
FIG. 3. Annealing of Sindbis India A-1036 denatured 3H-radiolabeled ds RNA with (0) SV India A1036ss RNA; (0) SV Australia C-377ss RNA; (m SV Egypt AR-339 as RNA; (A) SV South Africa AR-86 ss RNA; and (A) poliovirus ss RNA. Hybridization conditions were the same as in Fig. 2.
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FIG. 4. Annealing of Sindbis Australia C-377denatured 3H-radiolabeled ds RNA with (0) homologous ss RNA; (0) SV India A-1036 ss RNA; (W) SV Egypt AR-339 ss RNA; (A) SV South Africa AR-86 ss RNA; and (A) poliovirus ss RNA. Annealing conditions are described in Fig. 2.
to be 90-91% homologous under these conditions in that each virion ss RNA was capable of protecting that percentage of the complementary heterologous strand of the radiolabeled ds RNA probe from RNase digestion. Hybridization values obtained between the SV India A-1036 probe and the SV Egypt AR-339 and SV South Africa AR-86 virion RNAs were 0.44 and 0.47, respectively (Fig. 3). Similarly, the fraction hybridized between the SV Australia C-377 probe and the unlabeled SV Egypt AR-339 and SV South Africa AR-86 virion RNAs were 0.41 and 0.38 (Fig. 4). Thus, on the basis of the nonstringent hybridization results, SV India A-1036 can be grouped with SV Australia C-377, and SV Egypt AR-339 can be shown to be closely related to Africa AR-86. Similar hybridizations were performed using 3H-labeled probes for SV Egypt AR-339, SV India A-1036, or SV Australia C-377 and virion RNAs of seven additional SV strains, WEE, EEE, VEE, WHA, and poliovirus (Table 2). The Sindbis virus strains could be separated into two discrete groups on the basis of homology, those which were homologous with SV Egypt AR-339 ds RNA and those which were essentially homologous with both SV India A-1036 and SV Australia C-377 ds RNAs. The strains which hybridized extensively with the Egypt AR-339 probe were all isolated from Europe and Africa and those which hybridized extensively with SV India A-1036 and SV Australia C-377 were all
isolated from India, the Far East, and Australia. Each member of these two geographically defined groups demonstrated between 35 and 50% homology with intergroup ds RNA probes under these nonstringent conditions (Table 2). Also summarized in Table 2 are the results of the nonstringent hybridizations between the 3H-labeled SV probes and ss RNA from other selected alphaviruses. From 6-9% homology was found between each of the SV RNAs and WEE virion RNA and from 19-23% homology was found between each of these RNAs and WHA virion RNA. No homology was detected between SV RNAs and these virion RNAs of VEE, EEE or poliovirus. Analysis of RNA Homology betweenSindbis Virus Strains under Stringent Condition Hybridization To more closely examine the extent of RNA homology among the strains of the European-African group of SV and the Indian-Far Eastern-Australian group of SV, hybridizations were performed under the stringent conditions of 57”, in 2~ SSC and 33% formamide (an effective temperature of 26” below the T,,, for SV ds RNA). These conditions are optimal for the rate of reassociation for nucleic acid (Wetmur and Davidson, 1968) but require at least 82% base matching for a homologous hybridized molecule to be thermally stable (Howley et al., 1979).
GENOMIC DIVERGENCE
65
AMONG SINDBIS VIRUS STRAINS TABLE 2
SUMMARYOFPOLYNUCLEOTIDESEQUENCERELATIONSHIP AMONGRNAs OFSINDBISVIRUS STRAINS, OTHER ALPHAVIRUSES, AND POLIOVIRUSUNDER NONSTRINGENTCONDITIONSOF HYBRIDIZATION ds RNA Virion RNA
SV Egypt AR-339
SV Egypt AR-339 Uganda MP-684 South Africa AR-86 Israel M-1855 Czechoslovakia R-33 USSR AZ-16
0.99 t 0.01 0.99 k 0.01 0.98 f 0.01 1.00 1.00
India A-1036 India B-322/23124 Malaya MM-2215 Australia C-377 Australia Ch 19470
0.51 ” 0.02 ND 0.42 5 0.02 0.47 r 0.02 0.49
VEE EEE WEE WHA Poliovirus
1.00
SV India A-1036
SV Australia C-377
0.44 t 0.04 0.46 0.47 0.39 0.40 0.41
0.41 2 0.01 0.40 0.38 0.41 0.36 0.35
1.00
1.00 0.90 k 0.02 0.90 2 0.01 0.89
0.91 ? 0.01 0.90 0.97 ‘-c 0.01 1.00 1.00
ND ND 0.06 0.19 co.01
Note. Details of the reactions are given under Materials and Methods and in Fig. 2. Results represent the maximal homology obtained at plateau level of the saturation curve and are expressed as decimal fractions of the value for homologous annealing. Some of the results are the mean of three experiments and are indicated by standard deviation values.
In the European-African group, 3Hlabeled ds RNA of SV Egypt AR-339 was annealed with virion RNA of isolates belonging to the same group (Table 3). Values indicating a close relationship of 0.92, 0.99, and 0.95 were obtained for SV Egypt AR339, SV Israel M-1855, Czechoslovakia R-33, and USSR AZ-16, respectively. The hybridized fraction obtained with SV Uganda MP-684 was 0.83 and with SV South Africa AR-86,0.72. When SV South Africa AR-86 ds RNA was used as a source of complementary strand, a fraction of 0.94 was found with SV Uganda MP-684 ss RNA, 0.83 with SV Egypt AR-339 and SV Israel M-1855 ss RNA, and 0.76 with SV Czechoslovakia R-33 ss RNA. Under these stringent conditions, a very low degree of homology was detectable with the ss RNAs of the Indian-Far Eastern-Australian group (0.06 with SV India A-1036 and 0.05 with SV Australia C-377). Representative SV strains of the Indian-
Far Eastern-Australian group demonstrated a somewhat greater degree of heterogeneity in their nuclei acids (Table 4). Using SV India A-1036 ds RNA, relatedness values of 0.98 and 0.87 were obtained with SV India B-322/23/24 virion RNA and SV Philippines P-886 virion RNA whereas 0.66, 0.66, and 0.63 were found with SV Malaya AM-2215, SV Australia C-377, and SV Australia Ch-19470 ss RNAs, respectively. The RNA of the two Indian strains and the Philippine strain appear to share from 0.55 to 0.61 of their nucleotide sequence with SV Australia C-377 RNA. The homologous fraction of the same Australian strain reached 0.90 with SV Malaya AM2215 and 0.98 with SV Australia Ch-19470. InJuence
of Host and Year of Isolation
Sindbis virus strains used in these studies were originally isolated from humans, birds,
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TABLE 3
DISCUSSION
POLYNUCLE~TIDE SEQUENCE RELATIONSHIP AMONG RNAs OF SINDBIS VIRUS STRAINS BELONGING TO THE EUROPEAN-AFRICAN GROUP
The extent of homology among viral genomes of a family of viruses, related by serological and morphological criteria, has very often been underestimated (Howley et al., 1979). Most likely, this is due to the stringency of the hybridization techniques employed. Under standard hybridization conditions, only those regions of DNA with as much as 80-85% base matching will be thermally stable, and significant regions of homology with greater than 15% base mismatch will be scored as nonhomologous. Due to the degeneracy of the genetic code, identical proteins may be encoded by nucleic acids with as much as 33% base mismatch. In the experiments presented, we have examined the nucleic acid homology of Sindbis virus strains under both stringent and nonstringent conditions. In standard salt concentration (2 x SSC) pancreatic ribonuclease is able to digest short doublestranded RNAs dispersed among nonhomologous sequences. Under higher salt concentration, short double-stranded segments adjacent to single-stranded RNA segments
ds RNA
Virion RNA SV Egypt Israel M-1855 Czechoslovakia R-33 USSR AZ-16 Uganda MP-684 South Africa AR-86 India A-1036 Australia C-377
SV Egypt AR-339 1.00
0.92 0.99 0.95 0.83
f k 2 k
sv South Africa AR-86 0.83 + 0.02
0.02 0.01 0.01 0.01
0.72 f 0.04
0.83 f 0.03 0.76 f 0.01 0.94 f 0.01 1.00
0.06
-
0.05
-
Note. Hybridizations were carried out under stringent conditions as described under Materials and Methods. Results are expressed as decimal fractions of the value for homologous annealing. Standard deviations are based on the mean ofthree experiments.
mites, and mosquitoes (Table 1). The lapse of time between the earliest Sindbis isolation and the most recent isolation available was 24 years. In order to know whether year of isolation or animal host played any role in the heterogeneity of the virus nucleic acids, cross annealings were performed under stringent conditions with strains isolated in the same country from different hosts over a long period of time. Hybridizations utilizing SV South Africa AR-86 ds 13H]RNA as probe and virion RNAs of SV South Africa AR-6071, AR18132, both isolated from Culex univittatus, 10 and 22 years after the 1954 isolation of SV South Africa AR-86 as well as vii-ion RNA of SV South Africa Girdwood, isolated from man 11 years after the SV AR-86 strain showed no significant differences (Table 5A). Hybridizations of 2 strains isolated in India from two different hosts in the same year (Table 5B) showed no significant sequence divergence detectable by this method as well as hybridizations performed with two SV strains isolated in Australia from mosquitoes, 16 years apart (Table 5C).
TABLE 4 POLYNUCLE~TIDE SEQUENCE RELATIONSHIP AMONG RNAs OF SINDBIS STRAINS BELONGING TO THE INDIAN-FAR EASTERN-AUSTRALIAN GROUP ds RNA sv
Virion RNA SV India A-1036 India B-322/23/24 Philippines P-886 Malaya MM-2215 Australia C-377 Australia Ch-19470 Egypt AR-339 USSR AZ-16 Israel M-1855
SV India A-1036
Australia
1.00
0.61 ” 0.04
0.98 + 0.01 0.87 ” 0.02 0.66 f 0.03 0.66 k 0.02
0.58 0.55 + 0.02 0.90 * 0.02 1.00
0.63
0.98
0.08 0.05 0.03
0.05 0.04 0.05
c-377
Note. Hybridizations were carried out under stringent conditions as described under Materials and Methods. Results are expressed as a decimal fraction of the homologous reaction.
GENGMIC DIVERGENCE
67
AMONG SINDBIS VIRUS STRAINS TABLE 5
POLYNUCLEOTIDESEQUENCERELATIONSHIP AMONGRNAs OF SINDBIS VIRUS STRAINS ISOLATED IN SAME AREA FROMVARIOUS HOSTSAND/ORAT VARIOUS PERIODSOF TIME
THE
ds RNA
Virion RNA SV South Africa AR-86 (A) SV South Africa AR-86 South Africa AR-6071 South Africa AR-18132 South Africa Girdwood
1954 1964 1976 1963
Spring Johannesburg Upington Johannesburg
Culex Culex Culex Man
1.00
1.00 0.97 + 0.01 1.00
SV India A-1036 (B) SV India A-1036 India B-322/23/24
1953 1953
Mysore State Bombay State
Mite Wagtail (bird)
0.98
Mosquito Mosquito
1.00 0.98
1.00
SV Australia C-377 (C) SV Australia C-377 Australia Ch-19470
1960 1976
Cairns (Queensland) Charleville
Note. Hybridizations were carried out under stringent conditions. Results are expressed as a decimal fraction of the homologous reaction. (A) South African strains; (B) Indian strains; (C) Australian strains.
or unpaired bases between double-stranded regions appear to be more protected from ribonuclease digestion. Under stringent conditions (2x SSC, 33% formamide, 57”), the nucleic acid homology of some of the Sindbis viruses isolated from different countries was very low, but under nonstringent conditions (5x SSC, 57”), the degree of homology of various strains was maximized, and well-defined groups were recognized (Fig. 5). While this technique of using ribonuclease is useful in comparing the relative degree of homology of the Sindbis virus genomes, the data do not allow us to quantitate the number of homologous nucleotides. The relevance of hybridization data to serological relatedness is well illustrated in Table 2. Alphaviruses that show some antigenic cross-reactivity with Sindbis virus, such as WEE and WHA, are measurably related also by nonstringent hybridization. Similar findings were described by Wengler et al. (1977) in an analysis of RNA homology among four alphaviruses. Under the hybridization conditions utilized, significant RNA homology was found only between serologically closely related viruses such as Chikungunya (CHIK) virus and O’nyong-nyong (ONN) virus
(Porterfield, 1961). In order to minimize the effect of defective viral RNA in our studies, each virus strain was plaque purified three times and viral stocks were established with no further passage. The 15 strains of Sindbis virus studied were obtained from 10 different countries and 12 different hosts. Their isolations spanned a period of 25 years. Using RNARNA hybridization, under nonstringent conditions, these 15 isolates could be divided into two groups which corresponded to two geographic areas. The first group includes the isolates from Europe and Africa and the second group includes the isolates from India, the Far East, and Australia. Using stringent conditions of hybridization, further genetic subdivisions were observed within each main group. The host and the year of isolation were considered as potential factors leading to this genetic divergence. However, no variation was observed among South African strains isolated from different hosts and only a 2% difference was observed with the Indian strains isolated the same year from different hosts. Two South African strains isolated from the same host, 22 years apart, differed by only 3% and two Aus-
68
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PALE ARCTIC REGION
FIG. 5. Sindbis virus isolates can be divided into major groups based on their genomic similarities as well as geographic distribution.
tralian strains isolated 16 years apart shared 98% of homology. Strikingly, Egyptian strain AR-339 isolated in 1952 from a pool of Culex mosquitoes and Czechoslovakian strain R-33 recovered in 1971 from a bird were identical under stringent conditions. Thus the variations detected with regard to year of isolation or host were within the standard error (1 to 4%) of the hybridization technique. These observations would indicate that the subdivision of the various Sindbis strains was primarily a function of geographic region of isolation. A display of the results of hybridizations on the map (Fig. 5) shows that the subdivision of SV variants into subgroups follows the Wallace line (Darlington, 1957; Simpson, 1968) which divides the world into six zoogeographic regions. Sindbis virus is found in four of these regions, the Palearctic, Ethiopian, Oriental, and Australian regions. The degree of relationship between the RNAs of Sindbis isolates from Egypt, Israel, USSR, and Czechoslovakia varied between 92 and 100%. All these countries belong to the Palearctic region. The genetic
relationship of the isolates from Uganda and South Africa, located in the Ethiopian region, was very high (92 to 100%) and formed another subgroup. The Sindbis strains isolated from the Oriental region (India, Philippines) formed a homogeneous group distinct from the Australian isolates. An exception is the Malayan strain MM-2215 which appeared to be closer to the Australian group. These zoogeographic regions are based on mammalian distribution and are separated by natural migration barriers both topographic and climatic. The frequency of Sindbis virus isolation from birds suggests that they are maintenance hosts and participants in the virus transmission cycle (McIntosh et al., 1969). Since migratory birds are not necessarily subject to the barriers separating the zoogeographic regions, the distribution of Sindbis subgroups within these barriers seems to minimize the importance of birds in disseminating the virus. Sindbis virus has been isolated from a variety of hosts including the frog, several species of birds and mammals, and a variety
GENOMIC DIVERGENCE
AMONG SINDBIS VIRUS STRAINS
of vectors including several species of Culex, Aedes, Mansonia mosquitoes. Other alphaviruses are not found in so wide a variety of hosts, and so this observation may explain the wide geographic distribution of Sindbis virus, which is, in fact, the most widely distributed of all arboviruses (Theiler and Downs, 19’73). The variation in the genome of the Sindbis strains, and the consequent antigenic differences, appears to be a result of a divergent evolution from a common ancestor. Our data cannot determine whether the divergence occurred primarily through genetic drift, or through adaptation to unique, ecological conditions within each zoogeographical zone. The data obtained from hybridizations performed with RNA of other alphaviruses (Table 2) under nonstringent conditions show virtually no homology between Sindbis and either VEE or EEE genomes. Homology to SV of about 9 and 22% was observed with WEE and WHA RNAs, respectively, indicating a genetic relationship between these viruses. For both viruses, the same fractional hybridization was observed regardless of the Sindbis strain used as source of the complementary strand. These results suggest that Sindbis virus, Western equine encephalitis virus, and Whataroa virus share common nucleotide sequences and could have evolved from a common ancestor. Divergence of these three viruses most likely would have begun before the divergence of the various Sindbis geographic groups described in this paper. In summary, we have shown that, by relaxing the usual stringent conditions of RNA homology testing, incomplete homologies can be recognized in the genome of certain alphaviruses. In the case of Sindbis virus, the data indicate that a given subgroup is restricted to a single zoogeographic region. ACKNOWLEDGMENTS I wish to thank Dr. P. M. Howley for his support and detailed review of the manuscript, Dr. J. M. Dalrymple for his encouragement during the completion of the work and his valuable suggestions, and Dr. B. W. Burge for his helpful comments. I also wish to thank Ms. Susan Hostler for her skillful typing.
69
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