Generic RT-nested-PCR for detection of flaviviruses using degenerated primers and internal control followed by sequencing for specific identification

Journal of Virological Methods 126 (2005) 101–109

Generic RT-nested-PCR for detection of flaviviruses using degenerated primers and internal control followed by sequencing for specific identification M.P. S´anchez-Seco a,b,∗ , D. Rosario c , C. Domingo a , L. Hern´andez a , K. Vald´es c , M.G. Guzm´an c , A. Tenorio a a

Laboratory of Arboviruses and Imported Viral Diseases, Diagnostic Microbiology Service, National Center for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain b Alert and Emergency Unit, National Center for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain c Virology Department, PAHO, WHO Collaborating Center for Viral Diseases, Instituto de Medicina Tropical “Pedro Kour´ı”, Ciudad Habana, Cuba Received 15 September 2003; received in revised form 17 January 2005; accepted 18 January 2005 Available online 2 March 2005

Abstract Flaviviruses are a widespread and numerous group of arboviruses that can cause serious illness in humans. The continuous and slow spread of certain flaviviruses, such as Dengue viruses, and the recent entry and spread of West Nile virus to the American continent, point to the need to control these infections. This control requires the use of suitable techniques for diagnostic and surveillance programmes. A generic RT-nested-PCR that is, theoretically, able to detect each member of the group has been designed. The identification of the detected virus is carried out by sequencing. The introduction of an internal control would reduce the number of false negative results and could be used to quantify the viral load in clinical samples where the method works well. © 2005 Elsevier B.V. All rights reserved. Keywords: Flavivirus; Generic PCR; Detection; Degenerated primers

1. Introduction The genus Flavivirus, spread worldwide, belonging to the Flaviviridae family, is composed of a wide group of pathogenic viruses transmitted by arthropods. These are enveloped viruses, the genome of which is a single-stranded, positive polarity RNA molecule, of approximately 11 kb in length (Murphy et al., 1995). At least 30 flaviviruses cause disease in humans. Most of these infections are asymptomatic, and disease may vary from a febrile illness with or without rash, to life-threatening conditions, such as hemorrhagic fever or encephalitis. Moreover, flaviviruses cause severe and economically significant diseases in domestic animals. Tick-borne encephali∗

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tis virus (TBEV), Japanese encephalitis virus (JEV), Saint Louis encephalitis virus (SLEV), Yellow fever virus (YFV), Dengue viruses, serotypes 1–4 (DENV1–4), or West Nile virus (WNV) are key human pathogens (Burke and Monath, 2001). DENV causes more than 50 million cases of human infection worldwide each year, resulting in around 24,000 deaths (WHO, 1999; Gibbons and Vaughn, 2002). The disease ranges from a mild, febrile illness to severe hemorrhagic Dengue fever. DENV is endemic in the Americas, South-East Asia, the Western Pacific, Africa and the eastern Mediterranean region (WHO, 2000; Gubler, 2001). Yellow fever has gained considerable notoriety in the last few years, due to resurgence in epidemic activity in South America and Africa (Monath, 1997, 2001). Disease in travellers returning from endemic regions, caused by both viruses, has also been reported (Drosten et al., 2003; Stephan et al., 2002). WNV was

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introduced for the first time into New York, USA, during the summer of 1999 (Briese et al., 1999). Its vector has spread quickly, and during the following years the virus has invaded 46 states in the USA, causing more than 9000 human cases, which were fatal in 264 (Anonimous, 2003). These viruses are a real concern for public health in developed countries, which are setting up surveillance programs for their rapid and reliable detection and identification. However, developing countries also require rapid, reliable and simple techniques for diagnosis. Flaviviruses share common antigens, which make serology studies hard to interpret, and diagnosis based on this methodology difficult. Many of these viruses also share their transmission vectors, leading to frequent co-circulation of different viruses, and thus hampering precise identification (WHO, 1999). Specific molecular diagnosis has been designed for some flaviviruses, and a number of attempts have also been made at generic detection (Chow et al., 1993; Fulop et al., 1993; Tanaka, 1993; Chang et al., 1994; Puri et al., 1994; Pierre et al., 1994; Meiyu et al., 1997; Kuno, 1998), although only one generic RT-heminested-PCR reaction with a high degree of sensitivity has been developed (Scaramozzino et al., 2001; Lanciotti, 2003). The aim of this study was to develop a sensitive and reliable technique for flavivirus detection and identification, to be used as a suitable diagnostic and surveillance tool. The introduction of an exogenous target provided us with an internal control (IC) for the extraction and amplification processes. The amplification of this internal control can also be measured and used to quantify the viral load. 2. Materials and methods 2.1. Viruses and their propagation YFV, strain 17D, was obtained from Stamaril (Aventis Pasteur, Strasbourg, France). Vero cell monolayers, maintained in MEM, supplemented with FBS 2% and with a mixture of penicillin and streptomycin 0.01% (Gibco BRL), were infected with YFV and cultured for 5 days, until a cytopathic effect was evident. Infected cultures were scraped subsequently and the infected cells harvested in MEM. Titration was undertaken following the protocol described by Morens et al. (1985). 2.2. Obtaining of RNAs Viral RNA was extracted from the infected cell cultures and from clinical samples, following a procedure described previously (Casas et al., 1995). In brief, infected cells were disrupted and 50 ␮l of the supernatant fluids or clinical samples were incubated with 200 ␮l of a guanidinium thiocyanate lysis buffer, nucleic acid was precipitated with an equal volume of isopropanol and the pellet was dried out after washing with ethanol 70% and dissolved in 10 ␮l of ribonuclease-free

water. Alternatively, QIAamp Viral RNA Mini Kit (Quiagen) was used following manufactures indications. RNAs from DENV1, Hawaii strain, DENV2, New Guinea C strain, DENV3, H87 strain, DENV4, H241 strain, JEV, Nakagana strain, TBEV, vaccine strain, SLEV, 52 strain, WNV, B956 strain, and Murray Valley Encephalitis Virus (MVEV), MV/1/1951strain, were acquired commercially from the National Collection of Pathogenic Viruses (UK). 2.3. Generic oligonucleotides Alignments were undertaken using nucleotide sequences of RNA of different flaviviruses, obtained from GenBank (National Institute of Health, Bethesda, MD, USA), using the MACAW 2.0.5 programme (Schuler et al., 1991). Table 1 shows the abbreviations and accession numbers for the viruses whose sequences have been used. Degenerated primers were designed based on conserved motifs in a region of gene NS5, which encodes for the polymerase, to perfectly align with known flaviviruses sequences. Primers selected were: Flavi1+: 5 7871 GAYYTIGGITGYGGIIGIGGIRGITGG7897 3 , Flavi1−: 5 9255 TCCCAICCIGCIRTRTCRTCIGC9233 3 , Flavi2+: 5 8987 YGYRTIYAYAWCAYSATGGG9006 3 , Flavi2−: 5 9130 CCARTGITCYKYRTTIAIRAAICC9107 3 . The primers designated as Flavi1 were used in the RTPCR amplification and Flavi2 in the nested amplification. The symbols (+) and (−) respond to sense and antisense sequences, respectively. Indicated positions correspond to those of YFV strain 17DD (accession number: U17066). 2.4. Generic RT-PCR Reverse transcription of RNA to cDNA and subsequent amplification were carried out using the Access RT-PCR System (Promega, Madison, WI, USA) in a PCT-200, Peltier Thermal Cycler (MJ Research, Watertown, MA, USA) utilising thin-walled reaction tubes (REAL, Durviz, Valencia, Spain) with no mineral oil overlay. Briefly, 5 ␮l of nucleic acid preparation was added to 45 ␮l of a RT-PCR Mix containing 2 mM MgSO4 , 0.2 mM of dNTP, 40 pmols of each primer (1+ and 1−), 5 U of avian myeloblastosis virus reverse transcriptase and 5 U of the thermostable Tfl DNA polymerase from Thermus flavus. Samples underwent an initial cycle at 38 ◦ C for 45 min and 94 ◦ C for 2 min, followed by further 40 PCR cycles at 94 ◦ C for 30 s (denaturation), 47 ◦ C for 1 min (annealing) and 68 ◦ C for 1 min and 15 s (elongation). A final extension step was carried out at 68 ◦ C for 5 min. 2.5. Generic nested PCR amplification The nested PCR reaction mixture was carried out in a final volume of 50 ␮l and contained 2.5 mM MgCl2 (Perkin-

M.P. S´anchez-Seco et al. / Journal of Virological Methods 126 (2005) 101–109 Table 1 Strains of flaviviruses used indicating their accesion number Virus identification

Strain

Accession number

Japanese encephalitis

K94P05 RP-2MS strain RP-9 strain SA-14-2-8 strain P3 strain SA-14 virus

AF045551 AF014160 AF014161 U15763 U47032 M55506

Kunjin

MRM61C strain

D00246

Yellow fever

Vaccine strain 17DD Vaccine strain 17D-213 French neurotropic strain French viscerotropic strain 85-82H Ivory Coast

U17066 U17067 U21055 U21056 U54798

Louping ill

367/T2 strain

Y07863

West Nile

33/G8;34/F6 virus clone Isolate Italy 1998-Equine “NY 2000-crow 3356 “NY-grouse 3286 Eg101

M12294 AF404757 AF404756 AF404755 AF260968

Dengue virus type 1

Clone WestPac Singapore strain S275/90 259 par00 Den1BR/90

U88535 M87512 AF514883 AF226685

Dengue virus type 2

New Guinea C strain S1 vaccine strain ThNH-7/93 strain ThNH-p16/93 New Guinea C/PUO-218 Jamaica/N.1409 16681 strain PDK53 strain

M29095 M19197 AF022434 AF022440 AF038402 M20558 U87411 U87412

H87 strain CH53489 (D73-1) 80-2

M93130 AF017733 Af317645

China Guangzhou B5 Clone 2A 814669

M14931 AF289029 AF375822 AF326573

Neudoerfl virus Hypr virus Sofjin-HO

U27495 U39292 AB662064

Dengue virus type 3

Dengue virus type 4

Tick-borne encephalitis

Murray Valley virus Langat

Powassan virus

Cell fusing agent virus

NC 003690 Attenuated strain E5 TP21 strain TP21 TP21

AF253420 AF253419 NC 003690 M86650

LB strain 791A-52 Isolate CO52 64-7062 Isolate NY64A

L06436 AF310950 AF310944 NC 001564

Elmer-Cetus, Norwalk, CT), 0.1 mM of each dNTP (Amersham Pharmacia Biotech, Sweden), 40 pmols of each primer (2+ and 2−), 2.5 U of AmpliTaq DNA Polymerase (PerkinElmer-Cetus, Norwalk, CT) and 1 ␮l of the product of the first amplification. The mix was then subjected to an initial

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denaturation stage (94 ◦ C for 2 min) followed by 40 PCR cycles. Conditions used were similar to those described in the previous paragraph. Minor changes were introduced in elongation and final extension. The first was carried out at 72 ◦ C for 15 s, and the final step also at this temperature for 5 min. Upon completion of the amplifications, 10 ␮l of each reaction mixture was analysed by electrophoresis with a 2% agarose gel (MS8, Hispanlab, Spain) containing 0.5 ␮g/ml of ethidium bromide in TBE buffer gels. Products were visualised under UV light. A 1 Kb DNA ladder (Boehringer Manheim) was included on each gel. 2.6. Construction of the internal control The MIMIC approach (Siebert and Larrick, 1993) was modified to obtain an internal standard as previously described by Fedele et al. (2000). Briefly, two tailed primers were designed to amplify the heterologous gene coding for the Pseudorabies virus (PrV) polymerase. At its 3 end the primer named MimFlavSen contains a sequence, which aligns with PrV, and at its 5 end a sequence matching that of Flavi1+ follows that of Flavi2+. The other tailed primer, MimFlavAnt, also contains a sequence of the heterologous gene at its 3 end and sequence corresponding to Flavi1−, which follows that of Flavi2− at the opposite end. Tailed primers were as follows: MimFlavSen: 5 -gacctaggttgtggatgcggtagctggtgtattcacatcacgatgggatccgcttctcgggcatcgc-3 MimFlavAnt: 5 -tcccatccggctatatcgtcagcccaatgttcctcattgagaaatccccggaaggtcttctcgcactc-3 PrV sequences are written in lower case, Flavi2+ and − sequences are underlined, and Flavi1+ and − sequences are represented in bold type. The product resulting from PrV amplification using these tailed primers contains the four recognition sites for the flavivirus generic primers (Fig. 1). The fragment amplified by these tailed primers was purified in agarose gels and cloned in the pCR 4-TOPO vector, as described in the following section. 2.7. Cloning The DNA band, resulting from first generic amplification of YFV RNA, was excised from the agarose gel using a Geneclean kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions and ligated with the plasmid pGEM-T (Invitrogen, The Netherlands) following the T/A cloning strategy. The resulting plasmid was named pYFL. Epicurean Coli XL1-Blue electroporation-competent cells (Stratagene) were transformed with the ligation mix and plated onto l-agar containing ampicillin, IPTG and X-Gal (Sambrook et al., 1989). Colonies containing recombinant plasmids were identified by PCR using the flavivirus generic

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gen, Germany). Sequencing reactions on both strands were performed with the ABI Prism BigDye Terminator Cycle Sequencing v2.0 Ready Reaction (Applied Biosystems, Foster City, CA), and analysed using an ABI model 377 automated sequencer (Applied Biosystems, USA). 2.10. Handling of sequences The sequences obtained were compared with those recorded in databases, in order to identify the detected agent and to study the level of homology. The multiple sequence alignment programme Clustal W (1.6 Version) was used to obtain an optimal sequence alignment file. A tree was built using the neighbour-joining method, which calculated bootstrap confidence values of 1000 bootstrapping trials using the MEGA2 program (Kumar et al., 2001). 2.11. Clinical samples Fig. 1. Diagram showing the preparation and cloning of the internal control molecule. The primer MimFlavSen contains the sequences of Flavi1+ (dark box), Flavi2+ (grey box) and a fragment of PrV (white box). MimFlavAnt contains the sequences of Flavi1− (dark box), Flavi2− (grey box) and another fragment of PrV (white box). The fragment amplified by these primers, which contains the recognition sites for the primers used in the flaviviruses generic PCR, was cloned and used later as internal control.

primers, so that the correct band was obtained only when the insert was present. Plasmid DNA was extracted from bacterial cells using the Wizard Plus SV Minipreps DNA Purification System (Promega). Purified plasmid was quantified spectrophotometrically and digested with Pst I (Boehringer Manheim) to linearise it. To clone the IC, the band resulting from amplification of PrV by the tailed primers was excised from the agarose gels using the QIAquick Gel Extraction Kit (QIAGEN) and ligated with the HTP TOPO TA Cloning Kit (Invitrogen) on to the pCR 4-TOPO vector, following manufacturers’ instructions. One Shot TOP10 Competent Cells (Invitrogen) were used to transform the resulting plasmid. Colonies containing recombinant plasmids were identified as described in the previous paragraph. Purified plasmid was also digested with Pst I (Boehringer Manheim). 2.8. Amplification with the internal control Amplification was carried out under the same conditions previously described for the generic reaction. IC and pYFL were amplified in the same tube. After electrophoresis through a 3% agarose gel, the intensities of the two bands were determined by densitometry using 1D Manager Software (TDI, Spain). 2.9. Direct sequencing Bands from the second round of generic amplification were purified using QIAquick PCR Purification Kit (Quia-

Samples of whole blood, taken from four Spanish travellers returning from Dengue endemic regions, were used to validate the method. DENV infection has been previously diagnosed in these samples using a RT-nested PCR protocol developed for the detection of the four serotypes of dengue virus in clinical samples (Domingo et al., 2004). Sample was mixed with the lysis buffer to which IC had been added to obtain a final concentration of 500 copies per 5 ␮l. Nucleic acids were obtained as previously described. Blood was also obtained from a YFV vaccinated subject 0, 4, 6, 8, and 15 days post-vaccination in order to control viremia levels.

3. Results 3.1. Generic amplification of vRNAs Primer annealing temperatures and concentrations, thermocycling parameters, and each reaction component were standardised by experimentation. The conditions described in Section 2 are the ones selected; however, the reaction also works in a wide range of salt conditions and annealing temperatures (data not shown). A band of the correct size (1385 bp) was obtained following RT-PCR amplification and also a correct one after the nested reaction (143 bp) with all flaviviruses tested: JEV, MVV, SLEV, TBEV, WNV, DENV1–4 and YFV (Fig. 2). The amplification is also specific, since no bands were obtained when RNA from a non-infected cell culture was used as target. The sensitivity of the reaction was determined using the extracted RNA of serial dilutions of a YFV stock with 2 × 105 TCID50 ml−1 . We were able to detect about 10 TCID50 per tube using the nested reaction (Fig. 3A). The limit of detection, in terms of number of copies of cDNA, was also calculated. With this aim, the product of the first

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Fig. 2. Amplification of RNAs from different flaviviruses using the generic RT-nested-PCR. A fragment of 143 bp was obtained when the RT-nested-PCR was carried out with RNAs from JEV, MVV, SLEV, TBEV, WNV, DENV1–4 and YFV. (MW) DNA marker; (Neg) RNA obtained from Vero cells.

amplification of YFV was cloned and serial dilutions of the linearised plasmid (pYF) were assayed. The limit of detection was about 1000 copies in the first reaction and about 10 copies in the second (Fig. 3B).

is added, however this could be improved with minor modifications in the reaction, by doubling the concentration of primers and changing the annealing temperature to 40 ◦ C for 4 min (Fig. 4B).

3.2. Internal control

3.3. Clinical samples

Serial dilutions of target pYFL DNA were co-amplified in the presence of 100 copies of IC per tube (Fig. 4A), which affords a broad range of linearity, as could be shown when the standard curve was constructed by plotting the target/IC signal ratio against the logarithm of the initial amount of the target in the reaction tube (not shown). The sensitivity of the reaction is slightly hampered when the competitor molecule

Some clinical samples from Spanish patients were used to validate the methodology and confirm the suitability of the technique for use as a diagnostic tool (Fig. 5). Viremia was detected 6 days after vaccination with YFV vaccine. Positive results were also obtained in three patients with DENV infection (samples 2, 3, and 4). The inhibition of sample 1 is demonstrated by the lack of amplification of IC. 3.4. Identification and grouping of the sequences Double-stranded DNA products amplified in the generic nested PCR were directly sequenced as described in Section 2. The sequences of RNAs from the controls used, those obtained from clinical samples and equivalent ones, selected from the viruses listed in Table 1, were aligned with the Clustal programme and analysed using MEGA software to construct a tree based on their relationships (Fig. 6). Two main groups were formed: the mosquito-borne and tick-borne viruses with Cell Fusing Agent virus (CFAV) as the outgroup sequence. Sequences of viruses that belong to the same species group together with very high bootstrapping values.

4. Discussion

Fig. 3. Limit of detection of the generic RT-nested-PCR. RNA obtained from serial 10-fold dilutions of a titred stock of YFV (A) or DNA obtained from serial 10-fold dilutions of the pYF plasmid (B) was assayed with the generic RT-nested-PCR. (MW) DNA marker; (Neg) H2 O.

Techniques used for diagnostic and surveillance programmes must be rapid and simple, and should detect and identify a wide range of pathogens to a high level of sensitivity. Generic RT-nested-PCRs, followed by sequencing, match all these requirements. For this reason a generic RT-nestedPCR for flavivirus detection has been developed. The method

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Fig. 4. Co-amplification of YFV and IC sequences. An increasing number of copies of the pYFL was used, with a fixed quantity of target (IC) to assay the range of linearity of the reaction (A). The same assay was carried out increasing the concentration of the primers and the time of annealing in the reaction (80 pmols and 4 min, respectively) (B). (MW) DNA marker.

is able to detect a wide spectrum of viruses, is sensitive and can be used for clinical samples. None of the methods described for generic or specific detection of flaviviruses are able to detect every member of this genus with a suitable degree of sensitivity. Only one RT-heminested-PCR has been described that would detect each one of the flavivirus group members when the viral load is low (Scaramozzino et al., 2001; Lanciotti, 2003). Degenerated oligonucleotides have been designed in homologous regions of the conserved NS5 gene that encodes for the polymerase, one of the best preserved proteins in RNA viruses, since avoiding specific primer sequences is required to amplify a wide spectrum of RNA viruses (Sanchez-Seco et al., 2003). We have tried to avoid mismatches by studying a great

number of sequences of different viruses and using degenerated primers with special care over the third position of the codons. DENV1–4 (dengue subgroup), JEV, MVV, SLEV and WNV (Japanese encephalitis subgroup) and yellow fever subgroup (YFV), which belong to the mosquito-borne group, were assayed and all of them rendered positive amplification (Fig. 2). One virus (TBEV) belonging to the tick-borne group also rendered a correct band when assayed. The wide spectrum of viruses detected; the design of our degenerated oligonucleotides; their high level of sensitivity; and their suitability for use with clinical samples (Figs. 2–5) make this a very useful method for detection of both known and unknown flaviviruses. The amplified flavivirus is identified by

Fig. 5. Clinical samples assayed with the generic reaction. Whole blood was obtained from a vaccinated case before vaccination with YFV vaccine (0), and at 4, 6, 8 and 15 days afterwards, and assayed with our reaction. Whole blood from four patients (samples 1, 2, 3, and 4) in which DENV was diagnosed was also assayed. (MW) DNA marker; (−) RNA obtained from Vero cells; (+) RNA obtained from Vero cells infected by YFV.

Fig. 6. Phylogenetic tree obtained with flavivirus sequences. The tree shows the relationships between some flavivirus strains obtained with the MEGA2 programme, using the NJ method. Each number at the nodes represents the percentage of bootstrap replicates. The sequences of the strains obtained from databases are indicated by their accession number. In lower case letters sequences from RNAs of the viruses used in this work are indicated: yf (YFV, strain 17D); dengue4 (DENV4, H241 strain); cs2 (clinical sample 2); je (JEV, Nakagana strain); mv (MVEV, MV/1/1951strain); sle (SLEV, 52 strain); dengue2 (DENV2, New Guinea C strain); cs3 (clinical sample 3); dengue3 (DENV3, H87 strain); cs4 (clinical sample 4); dengue1 (DENV1, Hawaii strain); wn (WNV, B956 strain); tbe (TBEV, vaccine strain).

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sequencing the resulting fragment. The sequence obtained is compared with those of known flaviviruses, in order to precisely identify the detected virus, as shown in Fig. 6. Different strains group together with those belonging to the same species, with high bootstrapping values, although strains of WNV form two groups according to previously published data (Briese et al., 1999; Scherret et al., 2001; Scaramozzino et al., 2001). It is also shown that tick-borne and mosquitoborne viruses form two different groups as previously described (Kuno et al., 1998; Gaunt et al., 2001), and that CFAV must be considered as an outgroup. The introduction of a DNA fragment that functions as an internal control in each reaction enabled us to detect the inhibited samples, when the band corresponding to the amplification of this control was absent, thus reducing the risk of reporting false negative results. Inhibition of samples is thus detected by lack of amplification of IC, whereas negative samples will render only the band corresponding to IC, and in positive samples both the target and the IC will be amplified. This is particularly critical for clinical samples, in which the presence of inhibitors might affect the amplification process, as can be seen in Fig. 5. Since flaviviruses are RNA viruses, it would be desirable to have an RNA molecule to use as internal control. Some methods use cellular RNAs as controls but this approach is not useful when cell-free samples are used. An exogenous RNA could also be used but its high lability would make the process tedious, and it would not be suitable for diagnostic purposes. Other approaches using chemically modified RNAs should be researched, however the production of the molecule makes the process difficult, and modification of RNAs should be evaluated to prove whether or not they really behave like an RNA molecule. The amplification process is indeed verified by our internal control. Quantitative reactions for some flaviviruses have been described (Wang et al., 2000; Drosten et al., 2002; Schwaiger and Cassinotti, 2003) but none of these can be used for all flavivirus genus members. Our internal control has been designed to compete in the co-amplification process with any molecule of nucleic acid from any flavivirus, since both sets of molecules contain the same primer binding sites, making it possible to measure the amount of amplification in the target and competitor molecule. This method will enable the study of the relative amount of virus in a sample, making it easier to establish the relationship between viral load during the course of infection and the severity of the resulting disease. This could be particularly important as regards the appearance of hemorrhagic symptoms after DENV infections, and in the case of flaviviral encephalitis, for which three patterns of pathogenesis have been described (Burke and Monath, 2001): (a) fatal encephalitis, usually preceded by early viremia and extensive extraneural replication; (b) subclinical encephalitis, usually preceded by low viremia, late onset of brain infection, and clearance with minimal destructive pathology; and (c) unapparent infection, characterised by trace viremia, limited extraneural replication, and no neuroinvasion.

Flaviviruses are distributed in both developed and developing countries, so the ability of different laboratories to access the reaction components must be taken into consideration. Consequently, the methodology used for their study must be simple and easily performable. Our RT-nested-PCR is suitable for use with a wide variety of buffers and temperatures, and the volume of the reaction and the primers concentrations are low, in order to minimise costs. Nested amplification requires no more equipment or expertise than the first RT-PCR. Care, however, must be taken to avoid contamination, using isolated locations if possible. The procedure described could, therefore, be used for surveillance purposes in regions in which sophisticated technologies have not yet been implemented. Its design will permit development of reactions for specific amplification of certain flaviviruses to make their diagnosis easy. With the aim of specific detection of the members of this genus that are able to co-circulate and cause hemorrhagic fever symptoms, we have developed a reaction for the specific detection of DENV1–4 and YFV. In order to do this, the RT-PCR described in this work has been used, and the nested one has been transformed in a multiplex nested-PCR (Sanchez-Seco et al., submitted for publication).

Acknowledgements We would like to thank the Biopolymers Unit for their technical assistance, Fionna Westbury for her assistance in the preparation of this manuscript and Dr. L.E. Mart´ın Otero and his team from La F´abrica Nacional “La Mara˜nosa” (Spanish Ministry of Defence). This work has been partially backed by grants from the Fondo de Investigaciones Sanitarias (FIS) (Spanish Ministry of Health) (FIS 98/0229) and the Spanish Ministry of Defence. Drs. Sanchez-Seco, Tenorio and Domingo are members of the Red EVITAR (Enfermedades V´ıricas Transmitidas por Artr´opodos y Roedores) and Red RICET (Red de Investigaci´on Cooperativa en Enfermedades Tropicales), groups founded by the FIS (G03/059 and C03/04, respectively).

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