Journal of Medical Virology 33:260-267 (1991)

Detection of Flaviviruses by Reverse-Transcriptase Polymerase Chain Reaction Zayd A. Eldadah, David M. Asher, Mark S. Godec, Kitty L. Pomeroy, Lev G. Goldfarb, Stephen M. Feinstone, Herbert Levitan, C. J. Gibbs, Jr., and D. Carleton Gajdusek Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda (Z.A.E., D.M.A., M.S.G., K.L.P., L.G.G., C.J.G., D.C.G.); Food and Drug Administration, Bethesda, (S.M.F.); University of Maryland, College Park, Maryland (H.L.)

RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE),Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR)successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffinembedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PCR) was 1,000-fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.

advantages for the diagnosis of viral infections. It is rapid and highly specific. PCR is a general technique that can be applied to many viruses, and, because extracted viral nucleic acid is used, the technique is comparatively safe. We modified the PCR to amplify selected regions of the genomes of St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. We also compared the sensitivity of PCR in detecting SLE virus to the sensitivity of two conventional assays of viral infectivity.

MATERIALS AND METHODS Viruses Suckling National Institutes of Health (NIH) Swiss albino mice were inoculated intracerebrally with 30 p1 of DEN-2, DEN-4, JBE, SLE (two strains), YF, Langat (LGT), and Powassan (POW) viral suspensions. Viral strains employed and passage data are summarized in Table I. The mice became ill 4 or 5 days postinoculation. They were anesthetized deeply, and their brains were removed and stored at -70°C. C6136 Aedes albopictus mosquito cells [Igarashi, 19781 (kindly provided by Dr. Peter Summers, Walter Reed Army Institute of Research, Washington, D.C.) were inoculated with suspensions of SLE virus in Eagle’s medium, and RNA was extracted 7 days later. KEY WORDS: St. Louis encephalitis, Japanese Nucleic Acid Extraction encephalitis, dengue, yellow feRNA was extracted from brain tissues or cell cultures ver, reverse transcriptase, nestby modification of a method previously described [Chiring primers, RNA gwin et al., 1979; Davis et al., 1986; Godec et al., 19901; 1.3g of brain tissue or lo7 cultured cells were placed in a cylindrical plastic tube (Spex Industries) containing INTRODUCTION 5.0 ml of 4 M guanidine isothiocyanate (GIT) in 3 M The family Flaviviridae (group B arboviruses) in- sodium acetate (pH 6) and 0.1 M P-mercaptoethanol, cludes 66 antigenically related viruses with positive- with four glass beads 4 mm in diameter. Caps of each sense, single-stranded RNA genomes. Twenty-two flaviviruses are known to cause human disease [Monath, 19901. Laboratory diagnosis of flavivirus infections Accepted for publication November 15, 1990. may require the use of suckling rodents, mosquitoes, Address reprint requests to Dr. David M. Asher, Laboratory of and a variety of cell cultures; it also entails some risk Central Nervous System Studies, National Institute of Neurolog[Hammon, 1968; Shope and Sather, 19791. The poly- ical Disorders and Stroke, Building 36, Room 5B21, National merase chain reaction (PCR) offers several obvious Institutes of Health, Bethesda, MD 20892. 0 1991 WILEY-LISS. INC.

Detection of Flaviviruses by PCR

261

TABLE I. Strains and Passage Histories of Flaviviruses Studied Flavivirus Dengue 2 Dengue 4 Japanese encephalitis Langat Powassan St. Louis encephalitis Yellow fever

Abbreviation" DEN-2 DEN-4 JBE LGT POW SLE YF

Strain New Guinea H-241 M543 TP21 MacLean Parton Hubbard 17D vaccine

Passageb 24 42 2

NSc 7

4

5 1

"Abbreviationssuggested by the American Committee on Arthropod-borne Viruses. bNumberof mouse brain passages in Laboratory of CNS Studies, NIH. eNot specified.

tube were secured with tape. Pairs of tubes were taped together, placed in a Spex mixer mill (Spex Industries) and agitated for 60 sec (Emmons RW, Ascher MS, Dondero D, 1989, personal communication). Each tissue suspension was then layered on 4.0 ml of cesium chloride buffer (5.7 M CsC1, 3 M sodium acetate, pH 6) in a 10-ml conical polypropylene ultracentrifuge tube (Beckman 358120) and centrifuged at 20°C for 21 hr at 32,000 rpm (174,OOOg)in a Beckman SW41 rotor. After centrifugation, the supernatant solution, containing lipids, proteins, and DNA, was aspirated. The RNA pellet was then suspended by repeated pipetting in 200 pl of 0.3 M sodium acetate (pH 6) and transferred to a 1.5-ml microfuge tube. The ultracentrifuge tube was rinsed with 100 p1 of sodium acetate, which was then added to the microfuge tube. The RNA was precipitated by adding 750 pl of absolute ethanol, freezing at -70°C for 20 min, and centrifuging at 12,OOOgfor 10 min. The supernatant was discarded and the pellet resuspended in 300 pl of 80% ethanol. The microfuge tube was centrifuged again at 12,OOOgfor 2 min. The RNA pellet was then dried at room temperature for 18 hr and finally resuspended in 100 p1 of distilled water. The concentration and purity of the RNA samples were assessed spectrophotometrically,measuring absorptions at 260 and 280 nm.

Selection of Oligonucleotide Primers The flaviviral genomic sequences to be amplified and their respective oligonucleotide primer pairs were selected using the GenBank nucleic acid database and the Sequence Analysis Software (SAS) package of the Genetics Computer Group, University of Wisconsin [Devereux et al., 19841. We sought viral genomic sequences that appeared to be well conserved among several flaviviruses, reasoning that sequences well conserved among related viruses within a genus are likely to be at least as well conserved among strains of a single virus. Comparisons were made using the SAS Bestfit and Gap programs, which employ the algorithms of Smith and Waterman [1981] and Needleman and Wunsch [19701, respectively. A second criterion was selection of sequences that contain an internal endonuclease cleavage site to produce an easily recognizable pattern after endonuclease digestion and gel

electrophoresis for confirmation of the identity of the product. Table I1 lists the sequences of the primer pairs selected for DEN-2, DEN-4, JBE, SLE, and YF viruses as well as the length of the genomic sequence amplified by each pair, the lengths of the fragments produced by endonuclease digestion of the PCR product, the location of the target sequence in the viral genome, and the strain and source of the reference viral sequence. Two sets of primers are listed for SLE virus in the table: the outer primers anneal to locations flanking the positions of two inner (nested) primers in the template viral genome. The product of amplification with the outer primers can serve as a template for amplification with the nested primers.

PCR For each specimen to be tested by RT-PCR, a mixture of the following reagents was made to a final total volume of 80 ~ 155.7 : pl of distilled water, 10 p1 of x 10 reaction buffer (500 mM KC1, 100 mM Tris-HC1, pH 8.3, Perkin-Elmer-Cetus), 4.0 pl of MgC12 solution (25 mM, Perkin-Elmer-Cetus), 0.5 p1 of Amplitaq DNA polymerase (5 U/p1, Perkin-Elmer-Cetus), 0.8 pl of avian myeloblastosis virus reverse transcriptase (9 UI pl, Promega), 1.0 p1 of RNase inhibitor (RNasin, 30 U/p1, Promega), and 2.0 p1 each of dATP, dCTP, dGTP, dTTP (10 mM each, Perkin-Elmer-Cetus). A volume of this reagent mixture sufficient to conduct all anticipated RT-PCR tests was prepared in advance, using only "dedicated" pipetting devices that were never used for templates or primers, in order to reduce the possibility of contamination [Kwok and Higuchi, 19891. Next, 80 pl of this mixture was pipetted into a 0.5-ml conical microcentrifuge tube (Perkin-Elmer-Cetus); 25 p1 of mineral oil was added, followed by 5 p1 of each oligonucleotide primer (20 pM in distilled water) and 0.1-5 pg of the template RNA in 10 p1 of distilled water; only dedicated positive-displacement pipettes were used for primers and templates. Each tube was then briefly centrifuged and placed in a Perkin-ElmerCetus thermal cycler set to incubate at 42°C for 1hr (for reverse transcription), followed by 40 cycles of the following three steps: 94°C for 1 min, 55°C for 1 min, and 72°C for 3 min. For PCR with nested primers

Eldadah et al.

262

Viral primers

TABLE 11. Flaviviral Oligonucleotide Primer Sequences and PCR Products Predicted Predicted sizes of Position of size of PCR A h I digest target sequence product fragments in viral RNA Primer sequence (5'-3') (base pairs) (base pairs) (gene)

1 CGC GCT GCC 2 GCT TTG TCT 1 TTG TGG TTG DEN-4 2 GTT GGT CGT 1 ATG ACT AAA JBE 2 CTT GCG AGC 1 GGG AAT TAC SLE (outer) 2 ACT GCA ATG 1 GAA ACC GGG SLE (nested) 2 GAG CAA CGA YF 1 TAC CCT GGA 2 GCT TTT CCA

DEN-2

CAA TTC ACA GAA AAA CAC CCA AAC TTG TCT GCA TAC

CAC ATT ACG CAT CCA ATG ATG TCG TCA GGT AGA CCA

AAG TGC TGC GCC GGA ATT TCT CCA ATA CCC CAA ATG

GG AG AC AA GG GA AA GT TG TC GT AA

Strain of reference viral sequence

400

217,183"

Envelope

S1 vaccineb

440

232, 208a

Nucleocapsid

Caribbeanc

350

243,107

Envelope

JaOArS982d

382

198, 184

Nucleocapsid

MSI.7"

302

158, 144

Nucleocapsid

MSI.7"

465

287,178

Envelope

17D vaccinef

"Sizes of EcoN I (DEN-2) and Hinc 11(DEN-4) digest fragments. (No AZu I sites in products.) bData from Hahn et al. [1988]. "Data from Zhao et al. [1986]. dData from Sumiyoshi et al. [1986]. "Data from Trent et al. [1987]. fData from Rice et al. [1985].

TABLE 111. Attempts to Detect RNA Sequences in Seven Related Flaviviruses by Polymerase Chain Reaction Viralprimers SLE JBE YF DEN-2 DEN-4

SLE

JBE

YF

+a

-

-

-

+

Viral cDNA template DEN-2 DEN-4

-

-

-

-

-

+-

-

-

-

-

-

-

+ -

-

-

+

LGT -

NDb ND ND ND

POW -

ND ND ND ND

"Product of predicted size (Table 11) amplified by RT-PCR. bNot done.

(N-PCR), using the cDNA products of RT-PCR as templates, reverse transcriptase and RNase inhibitor were omitted from the master reaction mixture and replaced with distilled water, and there was no preliminary incubation at 42°C.

PCR Product Detection PCR products were handled with dedicated pipetting devices in a room separate from that in which the templates, primers, and master reagent mixtures were prepared. PCR products were visualized by electrophoresis in 1.2%or 4.0% agarose-ethidium bromide gel in TBE buffer. A 10 pl aliquot of the fluid from each PCR tube was mixed with 1.5 pl of a standard gel loading solution (US Bioproducts); 10 pl of this mixture was then loaded into a 4-mm x 1-mm slot in an agarose gel under TBE buffer (100 mM Tris at pH 7.8, 100 mM boric acid, 2 mM EDTA). DNA size standards (1 kb DNA Ladder, Bethesda Research Laboratories) were included in each gel. To confirm the specificity of the reactions by enzymatic cleavage, 20 U of Alu I (New England BioLabs) in 20 ~1 of buffer was added to 20 p1 of PCR product and incubated for 1hr at 37°C. These fragments were then

TABLE IV. Comparison of PCR and Infectivity Assays for Detection of St. Louis Encephalitis Virus Highest dilution of infected brain tissue in which virus was detected (Tissue sample number) Assav 1 2 3 SMBa 7.0b 4.3 5.3 C6/36-SMBC NDd 5e ND RT-PCR~ 5 5 7 8 8 N-PCRg ND aSuckling mouse intracerebral inoculation, 0.03 ml per inoculum. bReciprocal of log,, estimated 50% lethal end-point dilution [Reed and Muench, 19381. CAmplificationof virus in C6/36 cells, then inoculation of cells and supernatant fluid into suckling mice. dNot done. eReciprocal of loglo highest positive dilution. Qeverse-transcriptase PCR. gNested-primer PCR.

visualized by gel electrophoresis as above. Products that failed to cleave were digested with a second restriction endonuclease (Eco NI or Hinc 11, NE BioLabs). A partial nucleic acid sequence of one product

Detection of Flaviviruses by PCR

Fig. 1. Agarose gels (4.’0%) of PCR products. a: Titration of SLE virus-infected mouse brain (Table 111, sample 2) assa ed by RT-PCR using outer rimer pair Lanes 1 and 14, 1-kb DNd7 ladder: lanes lane 12, control SLE 2-11, mousegrain dilutions through RNA from infected mouse brain; lane 13, normal mouse brain RNA. Amplification of the redicted 382 base-pair product was detected through b: FurtRer amplification of PCR products from Figure

263

l a by N-PCR. Lanes 1 and 14, l-kb DNA ladder; lanes 2-11, N-PCR using the cDNA products from initial round of RT-PCR SLE-infected mouse brain dilutions 10-3-10-13 as templates; lane 12, N-PCR am lification of control RT-PCR SLE virus cDNA product; lane 13, N - h R amplification of control RT-PCR normal mouse brain cDNA product. The predicted 302-base product was detected through

Titration experiments were then undertaken with SLE virus to determine the sensitivity of PCR. In the first titration, RNA from SLE-infected mouse brain of low infectivity was extracted and suspended in distilled water. It was found by spectrophotometry to contain 0.5 Specificity and Sensitivity of PCR pg of nucleic acid per pl. Six 10-fold dilutions of this RNA from each of five flaviviruses was tested with sample were then made in distilled water; 10 p1 of each oligonucleotide primer pairs selected for the same five dilution was used as the template RNA for separate viruses to determine cross-reactivity within the group. amplifications. For three titrations, 20% suspensions of

was determined by P. Shields (Lofstrand Laboratories) with the method of Sanger et al. [19771 using singlestranded DNA prepared from cDNA by asymmetric PCR [Gyllensten and Erlich, 19881.

Eldadah et al.

264

Fig. 2. Agarose gel (1.2%)of PCR roducts (left) and Alu I digests of those products (right) for five flaviviruses. First and last lanes, l - i b DNA ladder.

normal mouse brain were first prepared in PBS. Next, a 20% suspension of SLE-infected mouse brain was made in PBS; equal volumes of the two suspensions were mixed. The resulting suspension was a 1:lO dilution of SLE-infected tissue in a 20% mouse brain suspension; 10-fold dilutions of those suspensions were made in 20% normal mouse brain, keeping the amount of normal mouse nucleic acid constant. Aliquots of 1.7 ml from each dilution were added to 5.0 ml of GIT, and RNA was extracted as described above. The remaining unextracted suspensions at each dilution were frozen at -70°C. RNA samples from the serial dilutions were subjected to RT-PCR with the outer pair of primers that amplified the longer SLE sequence, and products were visualized on electrophoretic gels as above; 10-pl aliquots of the cDNA products of those reactions served as the templates for N-PCR. The frozen unextracted suspensions of SLE virus in 20% mouse brain were thawed and assayed by two conventional methods for detecting viral infectivity: (1)30-pl aliquots of each dilution were inoculated intracerebrally into eight suckling mice; mortality at 3-14 days was considered t o be specific, and (2) cultures of C6/36 Aedes albopictus mosquito cells were raised in 15-ml cell culture tubes (Falcon). A 0.25-ml aliquot of each dilution of SLE virus-infected mouse brain suspension was added to 1.0 ml of Eagle's minimal essential medium (MEM) with 10% fetal bovine serum (FBS) and glutamine. Gentamicin sulfate was added to a final concentration of 100 pg/ml and amphotericin-B to 2.5 kg/ml. The suspension was centrifuged for 30 min at 3,OOOg and held at room temperature for 1 hr. Cell culture tubes were drained of medium and then inoculated with 0.6 ml of each dilution of virus. The culture tubes were centrifuged at 200g for 30 min, incubated at 28°C for 30 min; 0.4 ml of

Eagle's MEM was then added to each. After incubating for 7 days at 28"C, the cultures were harvested by freezing and thawing; 30-p1 aliquots from each tube were inoculated intracerebrally into eight suckling mice, and mortality was recorded. An attempt was also made to detect SLE genome in RNA from a sample of SLE-infected mouse brain fixed in formalin, embedded in paraffin, sectioned on a microtome with a disposable blade, and extracted by a modification of the procedure described above [Godec et al., 1990; Shibata et al., 19883.

RESULTS RT-PCR detected the target region in the nucleocapsid gene of two strains of SLE virus propagated in both mouse brain tissue and C6/36 cell culture. The results of experiments assessing specificity of detection are summarized in Table 111. Each pair of flavivirus PCR primers amplified only the template sequence from which the pair was selected. The primer pairs did not amplify sequences from any other flavivirus examined, nor did they amplify sequences from RNA extracts of normal mouse brains or control uninfected C6/36 cell cultures. The end point of a titration of an SLE RNA template was at a dilution in water of 1:10,000, suggesting that 500 pg of total RNA provided sufficient viral RNA to amplify the specific region of the SLE genome to a level detectable by gel electrophoresis. A comparison of PCR with classical infectivity titrations is presented in Table IV. Using the outer SLE primers, even the SLE virus-infected mouse brain of lowest infectivity (104.3 LD5,/0.03-ml inoculum) produced a detectable product at a dilution of after RT-PCR. N-PCR extended the end point of detection to lop8. (Infectivity assay of the same preparation of SLE virus in suckling mice after

Detection of Flaviviruses by PCR

265

TABLE V. Homologies Between Oligonucleotide Primers for Five Flaviviruses and Heterologous Flaviviral Templates

Viral primers DEN-2

No. 1

2 1

2 1 2 1

2 1

DEN-4

JBE

SLE

YF

2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

Viral template DEN-2 DEN-2 DEN-4 DEN-4 JBE JBE SLE SLE YF YF DEN-2 DEN-2 DEN-4 DEN-4 JBE JBE SLE SLE YF YF DEN-2 DEN-2 DEN-4 DEN-4 JBE JBE SLE SLE YF

YF DEN-2 DEN-2 DEN-4 DEN-4 JBE JBE SLE SLE YF YF DEN-2 DEN-2 DEN-4 DEN-4 JBE JBE SLE SLE YF YF

Position of 5’ terminus in genome 1151 1531 1153 1533 a

a

1177 a a

1560 2480 2900 2479 2899 2536 2738 2443 a

2688 2364 a a a

a

96 426 99 a

a a

122 a a a

124 565 127 409 144 495 1176 a a a a a

1182 a

1042 1487

Total/20 nucleotides 0 0 6 5 N A ~ NA 7 NA NA 7 7 5 0 0 7 6 7 NA 7 6 NA NA NA NA 0 0 3 NA NA NA 5 NA NA NA 3 6 0 0 5 6 7 NA NA NA NA NA 7 NA 0 0

Mismatches between primer and template 3’ terminal Number within 5 Number within 10 nucleotide nucleotides from nucleotides from mismatched? 3’ terminus 3’ terminus No 0 0 No 0 0 Yes 1 2 2 Yes 2 NA NA NA NA NA NA Yes 4 2 NA NA NA NA NA NA No 4 2 2 Yes 1 1 No 3 0 No 0 0 No 0 Yes 2 3 2 3 Yes 1 2 Yes NA NA NA No 1 3 0 No 3 NA NA NA NA NA NA NA NA NA NA NA NA 0 0 No 0 No 0 2 2 Yes NA NA NA NA NA NA NA NA NA 0 1 No NA NA NA NA NA NA NA NA NA 1 0 No 1 2 No 0 0 No 0 0 No 1 0 No 4 5 No 3 4 No NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 2 3 Yes NA NA NA 0 0 No No 0 0

*No sequence having fewer than 8 mismatches with 20 bases of the oligonucleotide primer was identified. bIn the absence of any close match, the analysis is not applicable.

266

Eldadah et al.

et al., 1989; Rotbart, 19901. Such a sequence was not found. PCR can also be highly sensitive. A sample of 500 pg of total host plus viral RNA provided sufficient template to amplify SLE cDNA with RT-PCR to detectable levels. The sensitivity of PCR compared favorably with those of two conventional infectivity assays. Indeed, after two rounds of PCR amplification (RT-PCR followed by N-PCR), SLE virus was detected at an endpoint dilution of l o p s or nearly 1,000-foldhigher than the corresponding end points of infectivity assays with the same materials. The amplification of SLE viral sequences in RNA extracted from formalin-fixed, paraffin-embedded tissue also shows that PCR can detect flaviviruses in specimens completely unsuitable for classical isolation attempts. However, the very sensitivity of PCR and its ability to amplify target sequences and generate enormous amounts of specific DNA product can result in artifactual carryover of product into starting material, producing false-positive results; that should be prevented by strict adherence to careful laboratory practices [Kwok and Higuchi, 19891. The flaviviruses are important causes of human disease worldwide [Monath, 19901. Even in urbanized areas of the USA small outbreaks of mosquito-borne flaviviral encephalitis have recently caused illnesses and deaths [CDC, 1990a,bl with considerable disrupDISCUSSION tion in normal community activities. Rapid detection The polymerase chain reaction provides a powerful by PCR should simplify and accelerate the diagnosis of technique for detecting a DNA for which at least a infections with flaviviruses and aid in the surveillance partial nucleotide sequence is known [Saiki et al., of their animal reservoirs and arthropod vectors. 19811. It has been adapted to detect RNA, including ACKNOWLEDGMENTS viral RNA, by preliminary reverse transcription into cDNA [Cristiano et al., 1991; Gama et al., 1989; Godec We thank Alfred Bacote and Michael Sulima for their et al., 1990; Jones and Foulkes, 1989; Rotbart, 1990; skilled technical assistance. Tse and Forget, 19901. PCR offers several advantages REFERENCES over classical techniques for detecting viruses. It is rapid. In this study, flaviviruses could be detected by Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-van Dillen PME, van der Noordaa J (1990): Ra id and sim le method for PCR and their identity confirmed by restriction endopurification of nucleic acids. Journa? of Clinica! Microbiology nuclease cleavage in 3 days, while infectivity assays in 28:495-503. animals or cell cultures with confirmation by serum CDC (1990a): Arboviral surveillance-United States 1990. Morbidity neutralization tests may require 3 2 weeks. Simplified and Mortality Weekly Report 39593-598. techniques for preparing nucleic acids [Boom et al., CDC (1990b): Update: Arboviral surveillance. Morbidity and Mortality Weekly Report 39:650-651. 19901 may reduce even further the time needed to Chirgwin JM; Przibyla AE, MacDonald RJ, Rutter WJ 11979): Isoladetect viruses by PCR. tion of biologically active ribonucleic acid from sources enriched in Under stringent annealing conditions, PCR is also ribonuclease. Biochemistry 18:5294-5299. highly specific. Cross-reactions between primers for Cristiano K, De Bisceglie AM, Hoofnagle JH, Feinstone SM (1991): Hepatitis C viral RNA in serum of patients with chronic non-A, SLE, JBE, YF, DEN-2, and DEN-4 viruses and heternon-B hepatitis: Detection by the polymerase chain reaction using ologous templates were not found. Examination of the multiple primer sets. Hepatology, in press. mismatches between primers and heterologous tem- Davis L, Dibner M, Batte K (1986): “Basic Methods in Molecular Biology.” New York: Ecevier, pp 130-133. plates may aid in explaining this specificity (Table V). J , Haeberli P, Smithies 0 (1984): A comprehensive set of Even in the most closely matched heterologous system Devereux sequence analysis programs for the VAX. Nucleic Acids Research (SLE primers with JBE template), there were three 12:387-395. mismatched bases between SLE primer #1 and the JBE Gama RE, Horsnell PR, Hughes PJ, North C, Bruce CB, Al-Nakib W, Stanway G (1989): Amplification of rhinovirus specific nucleic cDNA sequence and six mismatched bases between acids from clinical specimens using the polymerase chain reaction. SLE primer #2 and JBE. We hoped to find a region of Journal of Medical Virology 285’3-77. the flaviviral genome sufficiently well conserved so Godec MS, Asher DM, Swoveland PT, Eldadah ZA, Feinstone SM, Goldfarb LG, Gibbs CJ J r , Gajdusek DC (1990): Detection of that a single pair of oligonucleotide primers could measles virus genomic sequences in SSPE brain tissue by the amplify cDNA of several different viruses within the polymerase chain reaction. Journal of Medical Virology 30:237group, as has been done with picornaviruses [Gama 244. preliminary amplification in C6/36 cells had an end point of only lop5.)With an RNA templat,e extracted from a preparation of SLE virus-infected mouse brain of somewhat higher infectivity (105.3),the end point of detection by RT-PCR was lop7,and the end point by the second N-PCR was lop8. Figure 1 displays the PCR products from one of these titrations (tissue sample 2) separated in a 4% agarose gel. We also successfully amplified by RT-PCR the SLE target sequence with RNA extracted from a single 10-pm section of a formalin-fixed, paraffin-embedded mouse brain infected with SLE virus; titrations and N-PCR were not performed. The PCR products of SLE, JBE, and YF viruses were cleaved by treatment with AZu I to yield smaller fragments of predicted sizes (Fig. 2). The RT-PCR product of DEN-2 virus was not cleaved by AZu I; however, treatment with Eco NI yielded fragments of predicted sizes. A partial sequence of 161 bases of the DEN-2 product demonstrated considerable homology with the published sequence, although there was a 9% mismatch. Unfortunately, neither the AZu I nor the Eco NI endonuclease cleavage sites fell within the region successfully sequenced. The RT-PCR product of DEN-4 virus was not cleaved by AZu I or Hinc 11. Further attempts t o sequence the PCR products of DEN-2 and DEN-4 viruses are in progress.

Detection of Flaviviruses by PCR Gyllensten UB, Erlich HA (1988):Generation of sinyle-stranded DNA by the polymerase chain reaction and its app ication to direct sequencing of the HLA-DQA locus. Proceedings of the National Academy of Sciences, USA 857652-7656. Hahn YS, Galler R, Hunkapiller T, Dalrym le JM, Strauss JH, Strauss EG (1988): Nucleotide sequence ofden e 2 RNA and comparisonof the encoded proteins with those of o x r flaviviruses. Virology 162:167-180. Hammon WM (1968): Human infection acquired in the laboratory. Journal of the American Medical Association 203:647-648. Igarashi A (1978): Isolation of a Singhs Aedes albopictus cell clone sensitive to dengue and Chikungunya viruses. Journal of General Virology 40531-544. Jones MD-Foulkes NS (1989): Reverse transcription of mRNA b Thermus aquaticus DNA polymerase. Nucleic Acids Researcg 1723387-8388. Kwok S, Higuchi R (1989):Avoiding false positives with PCR. Nature (London)339:237-238. Monath T (1990):Flaviviridae. In Mandell GL, Douglas RG, Bennett JE (eds): “Princi les and Practice of Infectious Diseases.” New York: Churchill fivingstone, pp 1248-1251. Needleman SB, Wunsch CD (1970): A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of Molecular Biology 48:443-453. Reed LJ, Muench H (1938): A simple method of estimating fifty percent endpoints. American Journal of Hygiene 27:493497. Rice CM, Lenches EM, Eddy SR, Shin SJ, Sheets RL, Strauss J H (1985):Nucleotide sequence of yellow fever virus: Implications for flavivirus gene expression and evolution. Science 229:726-733. Rotbart HA (1990): Enzymatic RNA amplification of the enteroviruses. Journal of Clinical Microbiology 28:438-442.

267 Saiki RK, Gelfand DH, Stoffel S, Scharf DJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988): Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. Sanger F, Nicklen S, Coulson AR (1977):DNA sequencing with chain terminatin inhibitors. Proceedings of the National Academy of Sciences, &A 745463-5467. Shibata DK, Arnheim N, Martin WJ (1988): Detection of human papilloma virus in parafin-embedded tissue using the polymerase chain reaction. Journal of Experimental Medicine 167:225-230. Shope RE, Sather GE (1979):Arboviruses. In Lennette EH, Schmidt NJ (eds): “Viral, Rickettsia1 and Chlamydia1 Infections.” Washington, DC: American Public Health Association, pp 776-778. Smith TF, Waterman MS (1981): Comparison of biosequences. Advances in Applied Mathematics 2:482-489. Sumiyoshi H, Morita K, Mori C, Fuke I, Shiba T, Sakaki Y, Igarashi A (1986): Sequence of 3000 nucleotides at the 5’ end of Japanese encephalitis virus RNA. Gene 48:195-201. Trent DW, Kinney RM, Johnson BJB, Vorndam AV, Grant JA, Deubel V, Rice CM, Hahn C (1987): Partial nucleotide se uence of St. ns2a, and Louis ence halitis virus RNA: Structural proteins, ns2b. Virorogy 156:293-304. Tse WT, Forget BG (1990): Reverse transcription and direct am lifi cation of cellular RNA transcripts by Taq polymerase. 8en; 88:293-296. Zhao B, Mackow E, Buckler-White A, Markoff L, Chanock RM, Lai C J , Makino Y (1986): Cloning full-length dengue 4 viral DNA sequences: Analysis of genes coding for structural proteins. Virology 155:77-88.

Nil,