Journal of Virological Methods, 31 (1992) 275-288 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/%05.00

275

VIRMET 01317

Direct sequence analysis of amplified dengue virus genomic RNA from cultured cells, mosquitoes and

mouse brain

Eva Lee, Ann Nestorowicz, Ian D. Marshall, Ronald. C. Weir and Lynn Dalgarno

Division of Biochemistry and Molecular Biology, School of Life Sciences, Faculty of Science, Australian National University, Canberra, ACT, Australia

(Accepted 4 December 1991)

Summary

A method is described for direct sequence analysis of selected regions of dengue virus genomic RNA in infected tissues. Using specific primers, total high-molecular-weight infected-cell RNA is reverse transcribed to single- stranded (ss) complementary DNA, amplified using the polymerase chain reaction (PCR) and sequenced using ssDNA obtained after lambda exonuclease digestion of one strand of the PCR product (R.G. Higuchi and H. Ochman, Nucleic Acids Research, 17, 5865, 1989). Sequence data for the envelope protein gene of two dengue-3 virus isolates were obtained using RNA from small numbers (10’) of cultured mosquito or monkey kidney cells, from one mg of infected mouse brain and from 1/300th of an infected Toxorhynchites amboinensis mosquito. Independent determinations showed that errors occurring during reverse transcription or PCR were not represented to a significant degree in the sequence of the amplified DNA. The method does not depend on extensive passaging of virus or large-scale growth to generate material for sequencing and therefore provides a means of obtaining sequence data for unadapted dengue virus isolates.

Dengue virus; Polymerase chain reaction; Sequence analysis

Correspondence to: E. Lee, Division of Biochemistry and Molecular Biology, School of Life Sciences, Faculty of Science, Australian National University, GPO Box 4, Canberra, ACT, Australia 2601.

276

Introduction

The dengue viruses are small, enveloped, positive-strand RNA viruses which form an antigenic complex within the family Flaviviridae (Westaway et al., 1985: Calisher et al., 1989). They are transmitted to humans principally by Aedes aegypti and Ae. albopictus mosquitoes and circulate over extensive regions of the tropics and sub-tropics. The dengue viruses are an increasingly important public health problem worldwide (Gubler, 1991) and are associated with a range of symptoms including dengue fever and the severe hemorrhagic fever/dengue shock syndrome (see reviews by Monath, 1986; Halstead, 1988).

The factors which determine the severity of dengue virus disease manifestations have been the subject of debate. It has been argued that severe disease is largely the result of immune enhancement on secondary infection by a different serotype (Halstead, 1979, 1982; Halstead et al., 1980; Halstead, 1982; Kliks et al., 1989). However, there is epidemiological evidence that dengue viruses differ in pathogenic potential, both between and within serotypes, and that differences in disease severity result from intrinsic differences in viral virulence (Rosen, 1977; Gubler et al., 1978, 1979, 1981; Eram et al., 1979). To resolve these issues extensive data on dengue virus strain differentiation and evolution are needed. Nucleotide sequence data will be required for unadapted virus strains which are close, phenotypically and genetically, to the agent as isolated from the patient or mosquito. Current procedures for studying genetic differences between dengue viruses (Vezza et al., 1980; Trent et al., 1983; Walker et al., 1988; Blok et al., 1989; Rico-Hesse, 1990) require large-scale growth of virus (which may have been extensively passaged in the laboratory) with the consequent likelihood of variant selection and alterations in virus phenotype.

In this communication we describe the direct sequencing of dengue virus genomes present in small quantities of cellular material, including infected mosquitoes. The procedure involves reverse transcription of dengue RNA present in the tissue, amplification of the cDNA to double-stranded DNA using the polymerase chain reaction (PCR) and the digestion of one strand of the double-stranded DNA to yield single-stranded DNA for sequencing. Importantly, the method does not require large scale virus propagation or extensive passaging to generate template RNA and the virus is therefore retained in a relatively unadapted state. As test viruses we have used two human dengue-3 (DEN-3) isolates obtained in the course of dengue fever and dengue hemorrhagic fever/dengue shock syndrome outbreaks in Java, Indonesia in the 1970s (Gubler et al., 1979, 1981; Sumarmo et al., 1983).

Materials and Methods

Cells and virus

Ae. albopictus (C6/36) cells (Igarashi, 1978) were grown at 28°C in Eagle’s basal medium plus non-essential amino acids ‘and 8% foetal calf serum (FCS); African Green monkey kidney (Vero) cells (ATCC, CCL 81) were grown at 35°C in medium 199 supplemented with lactalbumin hydrolysate and 5% new born calf serum (NBS) in 5% C02/95% air. Vero cells were used between passage levels 122 and 137.

DEN-3 isolates 1153 and 1239 were provided by Dr. D.J. Gubler (Division of Vector-Borne Infectious Diseases, Centers for Disease Control, Fort Collins, CO, USA). Strain 1153 was isolated in 1977 from a case of fatal dengue shock syndrome in Djakarta, Indonesia; strain 1239 was isolated in 1978 from a case of dengue fever in Sleman, Indonesia (D.J. Gubler, personal communication). The original isolation was by intrathoracic inoculation of female Ae. aegypti mosquitoes with acute serum from patients (Gubler et al., 1981; Sumarmo et al., 1983). This material was inoculated into Toxorhynchites amboinensis mosquitoes which were later despatched to our laboratories. Mosquitoes were homogenised individually in Hanks’ balanced salt solution (HBSS) containing 3% FCS plus penicillin and streptomycin (see below). After light centrifuga- tion, the supernatant solution was inoculated on to C6/36 cell monolayers; during the re-isolation of DEN-3 from TX. amboinensis mosquitoes, a- ketoconisole (2.5 pg/ml final concentration) was added to the C6/36 cell growth medium to inhibit fungal growth. Growth medium was harvested at 2-5 days post-infection to give virus seed stock. Working stocks of isolates 1153 and 1239 had received a total of 2 and 3 passages, respectively in C6/36 cells.

Plaque titration

Virus diluted in HBSS (pH 8) was inoculated on to Vero cell monolayers in 6-well plastic trays (TC grade; Linbro Scientific Inc., USA). After 1.5 h, medium 199 overlay (3.5 ml) containing 1% Bacto agar (Difco Laboratories, Detroit, MI, USA), 2% FCS, 2% NBS and 0.2% (w/v) diethylaminoethyl dextran was added; incubation was at 35°C for 5 days. Plaques were stained by overlaying with 0.7% agar containing 1% neutral red and counted after overnight incubation. DEN-3 1153 and 1239 plaques (x0.5 mm diameter at 5 days) were readily obtained from TX. amboinensis extracts and from the subsequent C6/36 passage material.

RNA extraction

(1) C6/36 cells or Vero cells in 35-mm dishes (Nunc Inc., Roskilde, Denmark) were infected (m.o.i. w 0.01 PFU/cell) and incubated at 28°C or 35°C respectively. Monolayers were washed twice with phosphate-buffered

218

saline (PBS); 250~1 each of sodium p-aminosalicylate (6% w/v; BDH Chemicals Ltd., Poole, UK) and phenol-cresol (79%, v/v phenol; 11% v/v cresol; 10% deionized water; 0.08% w/v 8hydroxyquinoline) was then added (Shine and Dalgarno, 1973). The mixture was transferred to microfuge tubes, vortexed for 1 min and centrifuged for 5 min. NaCl(50 ,ul; 15% w/v) and phenol-cresol(l25 ~1) were added to the aqueous phase and extracted as above. Nucleic acids were precipitated with 2 vols of ethanol, held on ice for 30 min, centrifuged (5 min) and pellets resuspended in 100 ~1 NET buffer (0.12 M NaCl, 1 mM EDTA, 12 mM Tris-HCl, pH 7.5). An equal volume of 4 M LiCl (Merck, Darmstadt) was added and the mixture held on ice for 1 h. High-molecular-weight, single- stranded (ss) RNA was pelleted by centrifugation in a microfuge for 5 min. After a second lithium chloride precipitation the RNA pellet was resuspended in NET buffer (300 ~1) and precipitated with 2.5 vols of ethanol. The ethanol precipitation step was repeated and the pellets washed with 70% ethanol; after drying, pellets were resuspended in 10 ~1 TE buffer (5 mM Tris-HCl, 0.5 mM EDTA, pH 7.4). Quantitation of RNA was by UV-spectrophotometry; yields were approximately 10 ,ug per 35-mm dish of C6/36 or Vero cells (approximately lo6 cells).

(2) TX. amboinensis mosquitoes infected by intra-thoracic inoculation with DEN-3 (1239) (D.J. Gubler, personal communication), were stored at -60°C to -70°C. HBSS homogenates from which virus was propagated (see above) were used for RNA extraction. Homogenate (100 ~1 from 3 ml total) was mixed with 25 ~1 TES solution (20 mM Tris-HCl, pH 8; 2 mM EDTA, 0.12 M NaCl, 2% sodium dodecyl sulphate) and extracted twice with an equal volume of phenol/cresol. Nucleic acids were precipitated with 2 vols of ethanol and lithium chloride extracted as above.

(3) Day-old outbred Swiss white mice were inoculated intracerebrally with lo4 PFU of DEN-3 1153 or 1239 (30 ,ul; in C6/36 cell growth medium) using a 26-gauge needle. At 8 days post-inoculation, brains (approximately 200 mg wet weight) were removed and homogenized in 2 ml ice-cold PBS (pH 7.4); the homogenate was stored at -70°C. For RNA extraction, 50 ~1 TES solution was added to 200 ~1 of homogenate (equivalent to 20 mg tissue) and extracted as above. RNA yield was approximately 1 mg/g of brain tissue.

Primers and primer phosphorylation

Oligonucleotide primers (HPLC-purified) were from the Biomolecular Resource Facility, John Curtin School of Medical Research, Australian National University. Envelope protein (E) gene primers (Fig. 1) were based on the DEN-3 H87 prototype sequence (Osatomi and Sumiyoshi, 1990). Primers Pl and P2 were of plus polarity; P3 and P4 were of minus polarity. Primer concentration was determined by UV-spectrophotometry. Primers were phosphorylated in a final volume of 10 ,ul containing 2 pg of primer, 1 ~1 of x 10 reaction buffer (700 mM Tris-HCl, pH 7.4; 100 mM MgC12, 50 mM

dithiothreitol, 5 mg/ml bovine serum albumin), 0.5 ~1 of 20 mM ATP and 2 U

279

1000 1500 2000 2500 NT

I.. . . I . I. * 1.. . . 1

1 DEN-3 E GENE I

-B Pl

+ P3

978bp

+ P2

+

P4

759bp

Fig. 1. Primers used in RT-PCR and sequencing of E gene fragments of DEN-3 isolates. The E gene is represented by an open box and the positions of primers, derived from the genome sequence of DEN-3 H87 (Osatomi and Sumiyoshi, 1990), by arrows. Primers Pl and P2 are of plus polarity; P3 and P4 are of minus polarity; sequences are as follows: Pl, GCCCATTACATAGGCACT (18mer); P2, CATACAG- CACTGACAGGAGC (20mer); P3, GCACATTGCATAGCTC (16mer) and P4, TCCACTTCCA- CATTTGAG (18mer). P3 has a single mismatch (A-C, underlined) with the sequence for isolate 1153 (E. Lee, unpublished results); no evidence was obtained that this adversely affected yields of cDNA or sequencing results. Predicted cDNA fragments generated by RT-PCR (see text) are represented by solid

boxes.

of T4 polynucleotide kinase (Bethesda Research Laboratories, Gaithersburg, MD, USA); incubation was at 37°C for 30 min and the reaction stopped by incubation at 70°C for 10 min. Phosphorylated primers were used for cDNA synthesis and amplification directly from the reaction mix,

cDNA synthesis and amplification

cDNA synthesis was performed using the Perkin-Elmer Cetus (Norwalk, CT, USA) GeneAmp RNA PCR kit. Infected-cell RNA (approximately 1 pg) was incubated in a reaction mix (20 ~1) containing 1 ~1 of 50 PM (0.3 pug/l) downstream primer (P3 or P4), 2 ~1 each of dGTP, dATP, dTTP and dCTP (10 mM each), 2 ~1 of x 10 PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3), 4~1 of 25 mM MgC12, 1 ~1 of RNase inhibitor (20 U/p& Perkin-Elmer Cetus) and 1 ~1 of Moloney Murine Leukemia Virus Reverse Transcriptase (50 U/pi, Perkin-Elmer Cetus). The mix was incubated at 37°C for 1 h followed by heating at 95°C for 5 min. RNA from infected mosquitoes was not quantitated; nucleic acid from the equivalent of 1/300th of a mosquito was used for reverse transcription. In our hands random hexamers (Perkin-Elmer Cetus) were not suitable for reverse transcription as they were not readily removed during ethanol precipitation of DNA. For the generation of ssDNA for sequencing (see below) one of the primers used in reverse transcription (RT) and PCR was phosphorylated (Higuchi and Ochman, 1989). The use of phosphorylated primers Pl, P2, P3 or P4 in RT-PCR gave similar yields of cDNA fragments as were obtained using non-phosphorylated primers. For PCR, the reverse

280

transcription mix (20 ~1) was made up to a final volume of 100 ~1 with the following: 1 ~1 of 50 PM upstream primer (Pl or P2), 4 ~1 of 25 mM KCl, 8 ~1 of x 10 PCR buffer and 0.5 ~1 of AmpliTaq DNA polymerase (5 II/,& Perkin- Elmer Cetus). The mixture was overlaid with 50 ~1 heavy-white oil (Sigma) and placed in a Hybaid Thermal Reactor set to incubate at 95°C for 2 min followed by 35 cycles at 95°C (1 min), 39942°C (1 min) and 70°C (1.5 min). After the final cycle, incubation was continued at 70°C for 7 min. A negative control (no infected cell RNA) was routinely included. After PCR, an aliquot (5 ~1) of the mixture was removed for agarose gel electrophoresis.

Lambda exonuclease treatment and preparation of SSDNA and dsDNA for sequencing

To generate ssDNA for sequencing, 10 ~1 of exonuclease buffer (0.67 M glycine, pH 9.4 adjusted with KOH; 25 mM MgCl$ and 0.5 ~1 of lambda exonuclease (4 U/pi; BRL) were added directly to the PCR mix (95 ~1). After incubation at 37°C for 30 min, 5 ~1 was removed for agarose gel electrophoresis. EDTA (0.5 ~1, 0.5 M) was added followed by heating at 70°C for 5 min and extraction with phenol, phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol. DNA was precipitated by addition of 50 ~1 of 7.5 M ammonium acetate and 150 ~1 of ethanol followed by holding on ice for 1 h and centrifugation. After another round of precipitation, pellets were washed with 80% ethanol, air-dried and resuspended in 10 ~1 of TE buffer. Ethanol precipitation of DNA was on water-ice; freezing the sample on dry-ice, in liquid nitrogen or at -20°C led to co-precipitation of short oligonucleotides which interfered with sequencing. It was estimated that 100 ~1 of PCR product generated sufficient ssDNA for 4 sets of sequencing reactions.

For dsDNA sequencing, PCR products were extracted directly with phenol, phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol. DNA was precipitated with ammonium acetate and ethanol, washed with 80% ethanol and resuspended in 10 ~1 of TE buffer.

Nucleo tide sequence analysis

The Sequenase Version 2.0 kit (United States Biochemical Corp., Cleveland, OH, USA) was used for sequencing ssDNA corresponding to the 3’ half of the DEN-3 E gene; the manufacturer’s protocol was followed. An annealing mix (10 p-11) was made up with 2.5 ~1 of ssDNA template (see above), 1 ~1 of 2 PM primer and 2 ~1 of x 5 reaction buffer (200 mM, Tris-HCl, pH 7.5; 100 mM Mg&; 250 mM NaCl). The mix was heated at 65°C for 2 min and slow-cooled to room temperature. The labelling reaction contained the annealing mix, 0.5 ~1 of adenosine 5’[a-35S]thiotriphosphate (3000 Ci/mmol, Amersham), 1 ~1 of dithiothreitol (0.1 M) and 2 ~1 dNTP (0.2 or 1 ,uM each of dGTP, dCTP and dTTP depending on the transcript length required). Sequenase 2.0 (13 U/pi) was diluted 1:8 in cold buffer (10 mM Tris-HCl, pH 7.5; 5 mM DTT; 0.5 mg/ml

281

BSA); 2 ~1 of diluted enzyme was added last; incubation was at 20°C for 3 min. Labelling mix (3.5 ~1) was added to ddG, ddA, ddT and ddC termination mixes (2.5 ~1 each). The termination mix contained ddGTP, ddATP, ddTTP or ddCTP (8 PM) and 4 deoxynucleotides (80 PM each). After incubation at 37°C for 5 min, 4 ,ul of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyan01 FF) was added. For sequencing dsDNA, 5 ~1 of template and primer (5 pmol) were heated at 95°C for 10 min as above and chilled in ice-water. Labelling and termination reactions were as above. Sequencing reactions were electrophoresed on 6% acrylamide gels (0.4 mm thick; 38 cm long) in TBE buffer (90 mM Tris-HCl, 90 mM H3B03, 2.5 mM EDTA, pH 8.3) at 40 mA for 1.5 h or 4.5 h. Gels were fixed in 5% acetic acid-5% methanol for 30 min, dried under vacuum and exposed for a day to Fuji RX100 film for autoradiography.

Amphjikation of DEN-3 RNA sequences

Our aim was to amplify, and subsequently to sequence, DEN-3 envelope protein (E) gene sequences in small quantities of infected-cell RNA, using reverse transcription and the polymerase chain reaction (RT-PCR). The DEN- 3 strains used were Indonesian isolates from clinical cases of dengue shock syndrome (isolate 1153) and dengue fever (isolate 1239) and had been isolated by inoculation of human serum into Ae. aegypti mosquitoes; subsequent passage was in TX. amboinensis mosquitoes (once). Prior to experimental use, virus had a further 2 (1153) or 3 (1239) passages in C6/36 cells to generate working stocks; titres were in the range of 105-lo6 PFU/ml.

C6/36 cells were infected with isolates 1153 or 1239 (m.o.i. ~0.01 PFU/cell) and RNA extracted at 5 days post-infection (p.i.) when extracellular virus titres were 5 x lo5 and 4 x lo6 PFU/ml, respectively (see below). Cell RNA was extracted using phenol/cresol/sodium aminosalicylate followed by lithium chloride precipitation to remove low-molecular-weight RNAs and the bulk of the cellular DNA. Two primer pairs were used for RT-PCR of the E gene. Primers Pl and P3 were used for the 5’ end and P2 and P4 for the 3’ end (Fig. 1). Primers were based on the E gene sequence of H87, the DEN-3 prototype strain (Osatomi and Sumiyoshi, 1990).

Both the 5’ and 3’ sections of the E gene of DEN-3 isolates 1153 and 1239 were amplified from high-molecular-weight infected cell RNA. Fig. 2A (lanes 1 and 2) show the results of an experiment in which infected-cell RNA from lo5 C6/36 cells was reverse-transcribed using primer P4 and cDNA subjected to PCR after the addition of P2. The PCR products for both DEN-3 isolates showed a major band of 0.76 kb on agarose gel electrophoresis which corresponded to the expected cDNA fragment size. For amplification of the 5’ half of the E gene, RT-PCR with primers Pl and P3 gave the expected 0.98-kb

282

Ml2 M3 4 Ml 2 3 4

1353 1078 872

603

310

Fig. 2. Agarose gel electrophoresis of RT-PCR products amplified from DEN-3 infected tissues. (A) RNA (1 fig) from C6/36 cells infected with DEN-3 isolates 1153 or 1239 (3 days p.i.) was subjected to RT-PCR and the product (2 ~1 from a total of 100 ~1 reaction mix) electrophoresed on a 1.3% agarose gel followed by ethidium bromide staining. Lane 1: isolate 1153, P2/P4; lane 2: isolate 1239, P2/P4; lane 3: isolate 1153, Pl/P3; lane 4: isolate 1239, Pl/P3; lane M 50 ng HueIII-digested +X174 DNA. (B) RNA from TX. umboinensis mosquitoes, mouse brain or Vero cells infected with DEN-3 (strain 1239) was subjected to RT- PCR as above using P2/P4. Aliquots were electrophoresed and stained as above. Lane 1; no RNA added to RT-PCR mix; lane 2: TX. amboinensis RNA: lane 3: mouse brain RNA; lane 4: Vero cell RNA; lane M: 50

ng HaeIII-digested 4X174 DNA.

fragment for both isolates (Fig. 2A; lanes 3 and 4). The yield in both sets of reactions (for 5’ and 3’ halves of the E gene) was l-2 pmol per 100 ~1 of RT- PCR mix.

Vero cells were infected with DEN-3 (m.o.i.xO.01 PFU/cell) and RNA

10” -

IO3 -

I I I I I

123 4 5 67

Time post-infection (days)

Fig. 3. Timeecourse of DEN-3 (isolate 1239) replication in C6/36 cells. C6/36 cells in 35-mm Nunc dishes were infected with isolate 1239 (m.o.i . ~0.001); after 1.5 h the inoculum was removed and medium added. Incubation was at 28°C. Aliquots were removed at 24-h intervals over a 7-day period; assay of

extracellular virus was by plaque formation on Vero cell monolayers.

283

A B

1153 1239 MOS BRAIN

Fig. 4. Sequence analysis of DEN-3 RNA in cell culture, mosquitoes and mouse brain. RNA from tissue infected with isolates 1153 or 1239 was subjected to RT-PCR using primers P2/P4 to amplify the 3’ half of the E gene; ssDNA was generated from cDNA with lambda exonuclease and sequenced using P4. (A) Sequence ladders for isolates 1153 and 1239: C6/36 cell extracts. (B) Sequence ladders for isolate .1239: TX. amboinen#s mosquitoes (MOS) and mouse brain (BRAIN) extracts. Arrows indicate sequence differences

between isolates 1153 and 1239. Numbers indicate distance from the primer in nucleotides.

extracted at 5 days p.i. RT-PCR amplification of the 3’ end of the E gene gave similar results to those observed for C6/36 cells (Fig. 2B; lane 4). The yield of cDNA was similar to that obtained from C6/36 cells.

284

To determine whether similar procedures could be applied to virus genomes present in infected mosquitoes, RNA was extracted from homogenates of infected TX. amboinensis mosquitoes. These had been stored at -60°C for several years after infection with isolate 1239 and were homogenised individually in 3 ml of HBSS containing FCS; 100 ,ul of homogenate (titre: 2 x lo4 PFU/ml) was mixed with a quarter volume of TES buffer (containing

Tris, EDTA, NaCl and 2% sodium dodecyl sulphate; see Materials and Methods) and extracted with phenol/cresol and lithium chloride as for cultured cells. Following RT-PCR with primers P2/P4, using RNA equivalent to l/ 300th of a mosquito, the expected cDNA fragment of 0.76 kb was obtained (Fig. 2B; lane 2). Successful amplification was also obtained with primers Pl/P3 in RT-PCR. However, yields of cDNA from TX. amboinensis extracts were less than those obtained when RNA from tissue culture cells was amplified.

A similar approach was applied to brain tissue of DEN-3 infected mice. Outbred Swiss White mice (day-old) were inoculated intracerebrally with lo5 PFU of DEN-3 1239 (passage 3 C6/36 cell supernatant). Brains were harvested at 8 days p.i. and homogenised in PBS (titre: 4 x lo4 PFU/ml). A quarter volume of TES buffer was added and RNA extracted as before; RNA from 1 mg of tissue was used for RT-PCR with primers P2/P4. A cDNA fragment of 0.76 kb was obtained in similar yields to that observed with RNA from cultured cells (Fig. 2B; lane 3). It is possible that genome RNA from the original inoculum was transcribed and amplified in this procedure. However, other studies (E. Lee, unpublished results) have demonstrated the replication of DEN-3 in mouse brain under ‘the experimental conditions used; replicated RNA presumably represents the majority sequences which are, reverse- transcribed and amplified.

To determine the relationship between virus growth and the ability to amplify DEN-3 sequences in the corresponding infected cells, C6/36 cells were infected with strain 1239 (m.0.i.x 0.001 PFU/cell) and assayed for virus production over a 7-day period. The first increase in extracellular virus titres was seen between 2 and 3 days p.i. (Fig. 3); at 1 day p.i. titres had not increased above background and RNA extracted at this time gave no detectable cDNA products; RNA extracted at 2, 3 and 5 days gave similar yields of PCR- amplified cDNA (data not shown). There was no increase in PCR yield as virus titres increased by 2 log units between 2 and 5 days p.i.

Nucleotide sequence analysis of ampliJied DNA

To generate ssDNA for sequencing, the method of Higuchi and Ochman (1989) was used. This involves the use of a phosphorylated primer in RT-PCR. Double-stranded DNA with a phosphorylated 5’ end is degraded to ssDNA using lambda exonuclease which preferentially digests DNA with phosphory- lated 5’ termini. To generate ssDNA corresponding to the 5’ end of the E gene, either Pl or P3 was phosphorylated (depending on whether plus or minus strand DNA was required for sequencing) and used for RT-PCR. Similarly

285

either P2 or P4 was phosphorylated to generate ssDNA corresponding to the 3’ end. Lambda exonuclease and the appropriate buffer was added directly to the PCR mix (see Materials and Methods). Following incubation (37°C 30 min) the mix was phenol-extracted and DNA precipitated twice with ammonium acetate/ethanol. Sequencing ssDNA was by standard procedures.

Fig. 4A shows sequence ladders obtained with ssDNA amplified from cultured C6/36 cells infected with isolates 1153 and 1239. The ladders represent the 3’ end of the E gene using P4 as a sequencing primer for the 0.76-kb fragment. No sequencing ambiguities were observed and 35&400 nucleotides were readable from a single reaction mix; similar results were obtained with P2. The 0.9%kb fragment was sequenced in the same way using the PI/P3 primer pair plus two additional internal primers. Two independent RT-PCR amplifications were performed, followed by ssDNA sequencing; we found no differences in sequence within the E gene for isolate 1153 and the same result was obtained for 1239. Sequence data were also obtained with single-stranded cDNA from infected mosquitoes and from infected mouse brain (Fig. 4B). Owing to the lower yield of cDNA from RT-PCR of mosquito RNA, half of the ssDNA produced from a lOO+l PCR mixture, rather than a quarter, was used in sequencing. Over the region sequenced (the 3’ half of the E gene) there were no differences observed between the nucleotide sequence of isolate 1239 in Toxorhynchites mosquitoes, C6/36 cells or mouse brain. The data indicate the existence of sequence differences between isolates 1239 and 1153 (arrows, Fig. 4A). We are at present using the procedure to determine the complete E gene sequence of 4 Indonesian DEN-3 isolates (including 1239 and 1153).

Double-stranded DNA sequencing with RT-PCR products was examined. Satisfactory sequence ladders were obtained from the 0.76-kb dsDNA fragment (with primers P2/P4) by denaturing at 95°C for 10 min and snap cooling at 4°C (data not shown). However, dsDNA templates did not give uniformly good results with all primers; relatively poor results were observed with Pl/P3 (see Discussion).

Discussion

This report describes a method for obtaining sequence data for dengue virus genomic RNA present in small quantities of infected tissue (cultured vertebrate and mosquito cells, mouse brain and intact mosquitoes). The method involves the isolation of high-molecular-weight infected-cell RNA which is reverse transcribed and amplified using PCR. To generate ssDNA for sequencing, one of the amplification primers was phosphorylated and the PCR products digested with lambda exonuclease to remove the 5’-phosphorylated strand (Higuchi and Ochman, 1989). Unambiguous sequence data were obtained with ssDNA from the 3 sources tested using amplification primers for sequencing; internal primers were not required except when sequencing cDNA longer than 0.76 kb. Single-stranded DNA derived from PCR products consistently gave

286

clean sequence data; with dsDNA as template, the clarity of the sequence ladders depended on the primer used. The procedure could be conveniently carried out in two days. RNA extraction and RT-PCR were on day 1 and ssDNA production and sequencing reactions on day 2.

Since the complete nucleotide sequence of all 4 DEN serotypes has been established (see Chambers et al., 1990) the method is applicable to all DEN serotypes and to virtually all regions of the genome. We have used the procedure to determine the E gene sequences of 4 DEN-3 isolates from Indonesia (Gubler et al., 1981; Sumarmo et al., 1983) including 1153 and 1239 (E. Lee, unpublished data). In testing the reproducibility of the method, two separate RT-PCR reactions were carried out giving rise to two independent sets of sequence data for the E gene. The sequences for 1153 and 1239 were non- identical but the replicate determinations for each isolate gave identical data (E. Lee, unpublished results). The error frequency of Taq polymerase is approximately 2 x 10e4 per base duplication (Keohavong and Thilly, 1989); it appears that under the conditions used here, any errors which occurred during either reverse transcription of RNA or PCR were randomised sufficiently to ensure that they did not represent a significant proportion of the sequence in the amplified DNA.

An advantage of the method is its small scale. RT-PCR reactions were performed with RNA from lo5 C6/36 cells, 1 mg of infected mouse brain and l/ 300th of a single TX. amboinensis mosquito. The method can therefore give sequence data for relatively unadapted field material which has not been extensively amplified in cultured cells. Many current methods for studying genetic differences between dengue virus isolates involve Tl-oligonucleotide mapping (Vezza et al., 1980; Trent et al., 1983) or RNA sequencing (Blok et al., 1981; Walker et al., 1988; Rico-Hesse, 1990) for which relatively large-scale virus growth is required in order to generate the necessary quantities of viral RNA. Dengue and other flaviviruses can change significantly at the antigenic, biologic and genetic levels on laboratory adaptation (e.g. during growth in mice or in cultured cells) (Hardy, 1963; Hearn et al., 1966; Halstead and Simasthien, 1970; Halstead et al., 1984; Dunster et al., 1990; Lobigs et al., 1990). Passaging could therefore complicate studies on the relationship between genotype and phenotype for dengue virus. The method described reduces this possibility and should assist in studies on dengue virus adaptation, evolution and virulence determinants. Direct sequencing of dengue RNA present in human sera may also be possible using methods based on antigen-capture ELISA (Monath and Nystrom, 1984) for the initial isolation of virus.

Acknowledgements

This study was supported by grants from the Australian Research Council and the National Health and Medical Research Council of Australia.

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References

Blok, J., Samuel, S., Gibbs, A.J. and Vitarana, U.T. (1989) Variation of the nucleotide and encoded amino acid sequences of the envelope gene from eight dengue-2 viruses. Arch. Viral. 105, 39-53.

Calisher, C.H., Karabatsos, N., Dalrymple, J.H., Shope, R.E., Porterfield, J.S., Westaway, E.G. and Brandt, W.E. (1989) Antigenic relationships between flaviviruses as determined by cross- neutralization tests with polyclonal antisera J. Gen. Viral. 70, 3743.

Chambers, T.J., Hahn, C.S., Galler, R. and Rice, CM. (1990) Flavivirus genome organization, expression, and replication. Annu. Rev. Microbial. 44, 6499688.

Dunster, L.M., Gibson, C.A., Stephenson, J.R., Minor, P.D. and Barrett, A.D.T. (1990) Attenuation of virulence of flaviviruses following passage in HeLa cells. J. Gen. Virol. 71, 601607.

Eram, S., Setyabudi, Y., Sadono, T.I., Sutrisno, D.S., Gubler, D.J. and Sulianti Saroso, J. (1979) Epidemic dengue hemorrhagic fever in rural Indonesia, II. Clinical studies. Am. J. Trop. Med. Hyg. 28, 711-716.

Gubler, D.J. (1991) Dengue haemorrhagic fever: a global update. Virus Information Exchange Newsletter 8, 2-3.

Gubler, D.J., Reed, D., Rosen, L. and Hitchcock, J.C. (1978) Epidemiologic, clinical, and virologic observations on dengue in the Kingdom of Tonga. Am. J. Trop. Med. Hyg. 27, 581-589.

Gubler, D.J., Suharyono, W., Sumarmo, Wulur, H., Jahja, E. and Suhanti Saroso, J. (1979) Virological surveillance for dengue haemorrhagic fever in Indonesia using the mosquito inoculation technique. Bull. WHO 57, 931-936.

Gubler, D.J., Suharyono, W., Lubis, I., Eram, S. and Gunarso, S. (1981) Epidemic dengue 3 in Central Java, associated with low viremia in man. Am. J. Trop. Med. Hyg. 30, 10941099.

Halstead, S.B. (1979) In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody. J. Infect. Dis. 140, 5277533.

Halstead, S.B. (1982) Immune enhancement of viral infection. In: Progress in Allergy, Vol. 31. Karger, Base], pp. 301-364.

Halstead, S.B. (1988) Pathogenesis of dengue: challenges to molecular biology. Science 239, 476 481.

Halstead, S.B. and Simasthien, P. (1970) Observations related to the pathogenesis of dengue hemorrhagic fever, II. Antigenic and biologic properties of dengue viruses and their association with disease response in the host. Yale J. Biol. Med. 42, 276292.

Halstead, S.B., Porterfield, J.S. and O’Rourke, E.J. (1980) Enhancement of dengue virus infection in monocytes by flavivirus antisera. Am. J. Trop. Med. Hyg. 29, 638-642.

Halstead, S.B., Diwan, A.R., Marchette, N.J., Palumbo, N.E. and Srisukonth, L. (1984) Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells, 1. Attributes of uncloned virus at different passage levels. Am. J. Trop. Med. Hyg. 33, 654665.

Hardy, F.M. (1963) The growth of Asibi strain yellow fever virus in tissue cultures, II. Modification of virus and cells. J. Infect. Dis. 113, 9914.

Hearn, H.J., Chappell, W.A., Demchak, P. and Dominik, J.W. (1966) Attenuation of aerosolized yellow fever virus after passage in cell culture. Bacterial. Rev. 30, 6156221.

Higuchi, R.G. and Ochman, H. (1989) Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucleic Acids Res. 17, 5865.

Igarashi, A. (1978) Isolation of a Singh’s Aeaks olbopictus cell clone sensitive to dengue and Chikungunya viruses. J. Gen. Virol. 40, 531-544.

Keohavong, P. and Thilly, W.G. (1989) Fidelity of DNA polymerases in DNA amplification. Proc. Natl. Acad. Sci. USA 86, 9253-9257.

Kliks, S.C., Nisalak, A., Brandt, W.E., Wahl, L. and Burke, D.S. (1989) Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am. J. Trop. Med. Hyg. 40, 444451.

Lobigs, M., Usha, R., Nestorowicz, A., Marshall, I.D., Weir, R.C. and Dalgarno, L. (1990) Host cell selection of Murray Valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse virulence. Virology 176, 587-595.

288

Monath, T.P. (1986) Pathobiology of the flaviviruses. In: S. Schlesinger and M.J. Schlesinger (Eds), The Togaviridae and Flaviviridae. Plenum, New York, Chapter 12, pp. 37540.

Monath, T.P. and Nystrom, R.R. (1984) Detection of yellow fever virus in serum by enzyme immunoassay. Am. J. Trop. Med. Hyg. 33, 151-157.

Osatomi, K. and Sumiyoshi, H. (1990) Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology 176, 643647.

Rico-Hesse, R. (1990) Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology 174, 479493.

Rosen, L. (1977) The Emperor’s New Clothes revisited, or reflections on the pathogenesis of dengue hemorrhagic fever. Am. J. Trop. Hyg. Med. 26, 337-343.

Shine, J. and Dalgarno, L: (1973) Occurrence of heat-dissociable ribosomal RNA in insects: the presence of three polynucleotide chains in 26s RNA from cultured Aedes aegypti cells. J. Mol. Biol. 75, 57-72.

Sumarmo, Wulur, H., Jahja, E., Gubler, D.J., Suharyono, W. and Sorensen, K. (1983) Clinical observations on virologically confirmed fatal dengue infections in Jakarta, Indonesia. Bull. WHO 61, 693-701.

Trent, D.W., Grant, J.A., Rosen, L. and Monath, T.P. (1983) Genetic variation among dengue-2 viruses of different geographic origin. Virology 128, 271-284.

Vezza, AC., Rosen, L., Repik, P., Dalrymple, J. and Bishop, D.H.L. (1980) Characterization of the viral RNA species of prototype dengue viruses. Am. J. Trop. Med. Hyg. 29, 6433652.

Walker, P.J., Henchal, E.A., Blok, J., Repik, P.M., Henchal, L.S., Burke, D.S., Robbins, S.J. and Gorman, B.M. (1988) Variation in dengue type 2 viruses isolated in Bangkok during 1980. J. Gen. Virol. 69, 591-602.

Westaway, E.G., Brinton, M.A., Gaidamovich, S.Ya., Horzinek, MC., Igarashi, A., Kaariainen, L., Lvov, D.K., Porterfield, J.S., Russell, P.K. and Trent, D.W. (1985) Flaviviridae. Intervirology 24, 183-192.