Journal of Virological Methods, 30 (1990)41-54 Elsevier

41

VIRMET

01065

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood Vincent Deubel’, Manola Laille’, Jean-Philippe Hugnot’, Eliane Chungue3, Jean-Luc Guesdon4, Marie ThCrkse Drouet’, Sylviane Bassot’ and Dani&le Chevriei’ ‘Institut Pasteur, Luboratoire des Arbovirus, Paris, France, 21nstitut Pasteur de Nouvelle Calkdonie, Laboratoire des Arhovirus, New Caledonia, ‘Institut Territorial de Recherches Mkdicales Louis Malardk, Lohoratoire de Virologie, Papeete, Tahiti, French Polynesia and ‘Institut Pasteur, Laboratoire des Sondes Froides, Paris, France (Accepted

5 June

1990)

Summary

Polymerase chain reaction (PCR) was developed,for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the .four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-bIot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNAPAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens. Dengue virus; Polynucleotide

chain reaction: Virus diagnosis

Correspondence to: Vincent

Laboratoire

75724

Paris Cedex

0168-8510/90/$03.500

Deubel, 15, France.

des Arbovirus,

1990 Elsevier Science Publishers

lnstitut

B.V. (Biomedical

Pasteur,

Division)

25 rue du Dr Roux,

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Introduction The flavivirus genus contains approximately 70 related members classified in subgroups including dengue, yellow fever, Japanese encephalitis and tick-borne encephalitis. Their genome is a single-strand positive sense RNA of about 11 000 nucleotides which lacks a poly(A) tract at its 3’-extremity. In terms of morbidity, dengue fever is the most important mosquito-borne viral disease of man, causing in tropical latitudes millions of cases annually (Halstead, 1988). Each of the four dengue serotypes can be genetically (Blok et al., 1989; Chu et al., 1989; Trent et al., 1989) and antigenically (Monath et al., 1986) subdivided into subtypes that may vary in virulence (Rosen, 1987). The extent of dengue virus circulation and the increasing rate of more severe symptoms in children known as dengue haemorrhagic fever/dengue shock syndrome cause a major. problem of public health (Schlesinger, 1977). The rise of epidemics outside the Southeast Asian endemic part of the world results from the introduction of new variants and serotypes, mainly by means of air travel. Therefore, rapid characterization of the causal agent would help institute measures to control outbreaks. Moreover, the identification of the variations and evolution of the viral genome would contribute to our understanding of the epidemiology and the molecular aspects of the disease. The clinical and epidemiological studies on dengue are based upon serological diagnoses that are time-consuming or non specific (Burke et al., 1988) and virus isolation (Kuno et al., 1985). Recovering virus from viremic sera might fail if the specimens contained anti-dengue antibodies or were improperly preserved. The virus serotype can usually be determined by using type-specific monoclonal antibodies to detect the virus envelope protein in infected cultured cells (Henchal et al., 1982). Dengue 2 virus has been identified in specimens by specific hybridization using cDNA probes (Henchal et al., 1987; Khan and Wright, 1987). However, direct detection with nucleic acid probes from blood samples is limited by the low viremia in some samples and the need for specific probes corresponding to each serotype. The recent publication of the sequence of the structural genes (Deubel et al., 1986; Mason et al., 1987; Osatomi et al., 1988; Zhao et al., 1986) allows the preparation of such probes. The development of the polymerase chain reaction (PCR) allowed a selective and rapid amplification of short segments of genome (Saiki et al., 1988). It has proved its utility in the detection of viral pathogens (Persing and Landry, 1989) and its power to acquire direct sequences from the amplified gene molecules (Engelke et al., 1988). In this study, we have developed a rapid and sensitive diagnostic tool for identification of dengue viruses in serum specimens by cDNA amplification coupled to hybridization with type-specific cDNA probes (DNA-priming-amplificationhybridization or DNA-PAH).

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Materials and Methods Cells and viruses Dengue virus strains used in this study (dengue 1: 331-98, Tahiti 1988; dengue 2: 1409, Jamaica 1983; dengue 3: PaH 881, Thailand 1988; dengue 4: PaH 813, Martinique 198 1) were isolated from human sera and propagated on C6/36 (Aedes albopictus) cell monolayers. The serotype was confirmed by indirect fluorescent antibody tests with type-specific monoclonal antibodies (Henchal et al., 1982). Human sera Sera used in the comparative diagnosis were collected from patients who had a dengue illness confirmed by virus detection or serological investigation. Serological confirmation of dengue was achieved by demonstration of IgM (Chungue et al., 1989) in an early sample or, if negative, in a convalescent sample collected 2 weeks later. The acute sera were collected within 5 days after the onset of the fever. Except for one case of dengue 2, samples were from humans infected with dengue 1, 3 or 4 during dengue epidemics in 1988-1989 in the South Pacific. Information to determine whether the patients sustained primary dengue or dengue superinfection was not available. Virus growth, RNA extruaction and cDNA synthesis Dengue viruses were grown in C6/36 cells (m.o.i.=l-5 PFU/cell) for 3 days in Eagle’s minimum essential medium containing 2% fetal calf serum. Cells were washed in 1 x TNE (T~s~aCl~~TA) buffer and lysed in 0.1 x TNE containing 0.5% NP40. Cell nuclei were pelleted by low speed centrifugation and the cytoplasmic extract was deproteinized with 3 treatments of phenol in presence of 1% SDS. To recover RNA from serum, 200 ~1 of sample were incubated for 1 h at S6OC in presence of 0.5% SDS, 10 pg proteinase K (Boehringer) and 80 units RNAse inhibitor (Promega). Sample was diluted with 300 ~1 TNE buffer and subjected to 2 phenol trea~ents. RNA was precipitated with 2.5 volumes of pure ethanol/O.3 M ammonium acetate. Five pg of intracellular RNA or RNA corresponding to 50 ~1 of serum sample were primed with oligonucleotide primers for first strand cDNA synthesis using reverse transcriptase (Deubel et al., 1986). Four oligonucleotide primers were synthesized complementary to E gene sequences of dengue 1 (strain CV 1636/77). dengue 2 (strain 1409). dengue 3 (strain H87) and dengue 4 (strain 814669) viruses (Chu et al., 1989; Deubel et al., 1986; Osatomi et al., 1988; Zhao et al., 1986). respectively (Table 1). Dried RNA was dissolved in 10 ~1 of water with 100 ng of oligonucleotide. The mixture was heated at 90°C for 2 min, then cooled on ice. cDNA synthesis was performed in 20 ~1 of Tris.HCl 50 mM, pH 8.3, KC1 50 mM, MgCl2 8 mM, dithiothreitol 10 mM, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dCTP, 0.5 mM dTTP, 40 units RNAse inhibitor and 2 units

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TABLE 1 Sequences of synthetic oligonucleotide

primers

Prime?

Numberh

Sequence’

Length of the PCR product (bp)

Dl(+) Df(-)

52 288

GCAACGTGGGTTGACGTGGTATTGG AAACGTTCGTC~ACACACAAAGTTCG

231

D2(+) D2( -)

39 305

GGGGTTTCAGGAGGAAGCTGGGTTGAC CCCCATCCTCTGTCTACCATG

266

D3(+) D3(-)

47 303

CGGGAGCTACGTGGGTTGACGTGG CCAGCCTCTGTCTACGTATGTATGC -

257

D4(+) D4(-)

40 306

GGAGTCTCAGGTGGAGCATGGGTCGAC GCCCCACCCTCTGTCTACCACATC

267

“D1.2.3.4: primer of dengue serotype number 1.2,3,4of genomic (+) or anti-genomic (-) sense. bNumber indicates the map site at which the 5’-end of the oligonucleotide hybridizes on the strain gene E. ‘Mismatch in the nucleotide sequence for site-directed mutagenesis is underlined. bp: base pairs.

reverse transcriptase (Boehringer). The reaction was incubated for 1 h at 42’C and stopped in 0.2 M EDTA. DNA was phenol extracted and ethanol precipitated. (DNA amplification For standard PCR (Saiki et al., 19SS), one fifth of the cDNA .product was added to a final volume of 50 ~1 containing 50 mM KCl, 10 mM Tris.HCl pH 8.4, 2 mM MgCl?, 10 ,ug gelatin, 250 ,QM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP. dTTP), and 0.3 pg of each oligonucleotide primer. The sequences of the primers corresponding to the viral genomic sense were chosen at about 250 nucleotides upstream from those of the primers used for cDNA synthesis (Table 1). First, a denaturation of the RNA-cDNA hybrids was done at 95°C for 10 min followed by primer annealing for 2 min at 55OC. 2.5 units of Thermus aquaticus (Taq) polymerase (Perkin-Elmer Cetus) were added and the reaction was maintained at 72°C for 90 s. Amplification was achieved by 36 cycles with the following step cycle: denaturation at 94’C for 15 s, annealing at 55’C for 1 mitt, and extension at 72’C for 45 s. After the.last cycle, samples were maintained at 72’C for 12 min. Five ~1 of each reaction mixture were electrophoresed through 1.5% agarose gel in TBE (Tris/Borate/EDTA) stained with ethidium bromide for DNA visualization. Cloning

of dengue

(DNA

One half of the amplified DNA mixture was incubated with restriction enzymes HirzcII and AccI (Table l), then loaded on a 1% low melting point agarose gel

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(Gibco Bethesda Research Laboratory) in TAE buffer (Tris/acetate/EDTA). The DNA was excised from the gel, phenol extracted, and ethanol precipitated in 2.5 volumes of pure ethanol/O.3 M NaCl. The DNA was cloned into the EcoRVand HincII-cut, calf intestine phosphatase-dephosphorylated plasmid bluescript pKS(-) (Alting-Mees and Short, 1989; Maniatis et al., 1982). Dengue doublestranded recombinant DNA was sequenced using the M 13 universal primer and T7 polymerase (Sequenase, US Bio~hemicals). Southern and dot-blot hybridization

DNAs from agarose gel were transferred to cellulose nitrate membrane by the Southern method (Maniatis et al., 1982). For dot hybridization, samples of DNA corresponding to one tenth of the PCR product were denatured in 50 mM Tris.HCl, pH 7.4, 0.2 N NaOH, 6 x SSC for 10 min at 80°C. After neut~lization with 0.2 M TrisHCl, pH 7.4, the samples were spotted onto nitro~ellulo~ filters by using a manifold (Minifold I, Schleicher & Schuell). The membranes were heated for 2 h at 80°C and prehybridized for 30 min at 42°C in a buffer containing 4 x SSC, 5% powdered milk, 50% formamide, 0.1% SDS, then hybridized overnight at 37OCin the same solution containing the labelled denguespecific probe. The recombinant plasmids containing each a fraction of E gene corresponding to a dengue serotype were labelled with [“ZP]dCTP (Amersham) using the Nick-tr~slation procedure (Boeh~nger) and were used as radioactive probes for hybridization tests. Membranes were washed (Maniatis et al., 1982), dried and exposed at -7OOC to X-ray film (Hypertilm-MP, Amersham) using an intensifier screen, Nonradioactive probes were prepared by labelling with a N-acetoxy-N-2-acetylaminofluorene (AAAF) and used for DNA-DNA hybrid detection with anti-AAF monoclonal antibody as previously described (Chevrier et al., 1989). Briefly, the filter was hybridized in the same condition as above with 200 ng of heat-denatured AAF-labeled DNA probe per ml. After washing, the filter was incubated for 1 h with anti-AAF monoclonal antibody, washed and incubated for 1 h with alkaline phosphatase-la~lled sheep anti-mouse IgG antibody. The enzyme on positive hybridization was revealed for 30 min with a mixture of 15 ml of 0.33 mg/ml of Nitro Blue Tetrazolium and 15 ml of 0.16 mg/ml of 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris.HCl, pH 9.5, 100 mM NaCl, 50 ml MgCl2. Results Selection, amplification specific hybridization

and evaluation of the dengue E gene fragments for

The secondary structure of the envelope protein E of the ffaviviruses is highly conserved and contains 3 major antigenic domains, A, B and C (Mandl et al., 1989). Domain A located at the N-terminus of the protein E is stabilized by 3

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Fig. I. Amplification products. RNA from cells infected with dengue I (I), dengue 3 (3) or dengue 4 (4) virus was amplified by PCR using specific primer sets (Table I). Plasmid p30-VD2 (Deubel et al., 1986) was digested with Awl and SCUI (MW) or with Hincll and Au.1 (2). Shown is the ethidium bromide-stained 1.5%agarosc gel. Arrowhead indicates the amplified DNA fragment.

disulfide bridges and the majority of its antigenicity is destroyed by detergent and low pH (Guirakhoo et al., 1989). In this domain, a hydrophilic peptide ranging from amino acid 22 to 97 contains a linear epitope (Deubel, unpublished results), one neutralizing epitope (Mandl et al., 1989; Lobigs et al., 1987) and a glycosylation site. The corresponding nucleotide sequence shows great changes between variants belonging to the same serotype (Blok et al., 1989; Chu et al., 1989). Moreover, this gene fragment is flanked by sequences relatively conserved between geographical variants that have been used as primer for gene amplification. Two restriction sites, Hi~11 and AccI, natural or introduced by PCR (Table l), were used to clone the amplified fragments (Fig. 1). Clearcut PCR products of about 250 base pairs (bp) were obtained with each set of primers used for dengue 1, 3 and 4 gene amplification, respectively, and were compared to the product of HincII-A

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood.

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the en...
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