VIROLOGY

189, 363-367

(1992)

Primer Design for Specific Diagnosis by PCR of Highly Variable Typing of Foot-and-Mouth Disease Virus

RNA Viruses:

ANA RODRIGUEZ,”ENCARNACI~N MARTiNEz-%.AS,t JOAQU~NDOPAZO,* MERCEDESDAvIM,t JUAN CARLOSSAz,*a’ AND FRANCISCOSOBRINO*~’ *Departamento de Sanidad Animal, CIT-INIA. CIEmbajadores 68, Madrid 280 12, Spa/n; and tCentro de Biologia Molecular, UAM, Canto Blanco, 28049, Madrid, Spain Received January

13, 1992; accepted

March 27, 1992

A PCRassay for the specific detection and identification of viral sequences that correlate with established serotypes of foot-and-mouth disease virus (FMDV) has been developed. A new analysis based on homology profiles among reported sequences was used for primer design. RNA replicase (3D) gene regions that showed high homology among FMDVs, and low homology to other picornaviruses, were used for PCR amplification. Specific and highly sensitive detection was achieved for RNA of FMDV types C, A, and 0, either purified or extracted from vesicular fluids of infected animals, under reaction conditions permissive for the detection of variants present in the virus population. Similarly, serotype-specific primers were designed to amplify the carboxy-terminal end of VP1 gene of FMDVtypes either C, A, or 0. The results of PCR amplification of 15 different FMDV RNAs using type-specific primers are in agreement with the serological typing of the corresponding viruses and show that the primer-selection procedure developed for FMDV constitutes a reliable method of viral diagnosis.

o 1992 Academic

Polymerase chain reaction (PCR) is an extremely sensitive method (I) that has become widely used for identification of viruses, including the picornaviruses (2-4). Specific detection by PCR amplification has to take into account the problem introduced by the extensive heterogeneity exhibited by viral RNA genomes (5). Since tolerance for priming events is required to enhance the detection of possible variants, the design of primers is crucial in attaining specificity for a virus group and, at the same time, in differentiating among variants within the group. In an attempt to investigate efficient strategies for the specific identification of conserved regions within highly variable genomes, we carried out a study of nucleotide sequence homology profiles corresponding to either “conserved” (30) or “variable” (VPl) genomic regions of foot-andmouth disease virus (FMDV), to design appropriate primers for virus detection and genetic characterization by PCR. FMDV is a member of the Picornaviridae family that causes an economically damaging disease of domestic animals (6). Capsid protein VP1 contains major antigenie determinants and is highly variable in sequence, not only among different serotypes but also within isolates of the same serotype (reviewed in (7)). Compari-

PESS. IK.

son of RNA polymerase (3D) coding sequences indicates that the degree of intertypic homology is higher for this region of the genome than for VP1 coding sequences (8). Based on this evidence, we have explored the ability of PCR amplification of the polymerase gene to enable detection of small amounts of RNA from any FMDV isolate and of amplification of the VP1 gene to provide type-specific identification. The comparison of the 30 gene sequences was aimed at finding conserved regions among different FMDVs that were, however, divergent in other picornaviruses. A consensus sequence, derived from the alignment of FMDV sequences, was used to obtain the homology profiles either among FMDVs or other picornaviruses as described in the legend of Fig. 1‘. Several regions highly conserved among FMDVs sequences, but not with other picornaviruses, werefound. The suitability of these sequences as primers for PCR amplification was confirmed by direct sequence comparison. The sequence corresponding to 3D-1 was selected as (+) primer, while sequences corresponding to 3D-2 and 3D-3 were chosen as (-) primers (Fig. 1). Two combinations of these primers (3D-2/3D-1 and 3D-3/ 30-l) were used for PCR amplification of RNA from 12 different FMDVs of serotypes C, A, and 0. In all cases, a DNA band of the expected size was observed after analysis- of the PCR products by gel electrophoresis (Fig. 2). In contrast, and confirming the specificity of the assay, no products were amplified when using

’ Present address: Centro de Blologia Molecular, IJAM, Canto Blanco, Madrid, Spain. ’ To whom requests for reprints should be addressed.

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0042.6822/92

$5.00

Copyright 0 1992 by Academic Press, Inc. All rtghts of reproduction in any form resewed

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364

301 5 ._______

3D-2 -~~~~~---~-~~~.~~~--~~.--

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FIG. 1. Profiles of nucleotide homology among 3D coding sequences of picornaviruses. The average nucleotide substitutions obtained, accumulated every five nucleotides (among FMDVs and their consensus (-), and between the FMDV consensus and other picornaviruses ( +. . . . )) are plotted versus the nucleotide position. A computer program (PROFILES), available upon request, was used for primer selection. Sequences (8, 74-29) aligned by means of the CLUSTAL program (30) were split into two groups: a group for which

RNA from poliovirus type 1 (Mahoney), two isolates of swine vesicular disease virus (SVDV), the causative agent of a disease for which a differential diagnosis from FMD is required, or BHK-21 cells. Amplification of nucleic acids extracted directly from vesicular fluid of FMDV-infected pigs confirmed the ability of this procedure to detect virus in lesions of infected animals. In all cases, the expected specific PCR products were obtained, as further confirmed by Southern-blotting hybridization with an internal probe (data not shown). The sensitivity of the assay was determined using serial dilutions of known amounts of FMDV RNA as template for cDNA synthesis and PCR amplification of the VP1 gene. Twenty thousand RNA molecules were detected under reaction conditions described in the legend of Pig. 2. In addition, positive amplification was achieved tihen using viral suspensions from animal lesions containing about 3 PFU as starting material for the nucleic acid extraction (data not shown). The procedure used constitutes a fast, sensitive, and reliable system for FMDV detection that avoids the time-consuming procedures of cell culture infections. The potential of this approach can be further explored since several other regions, mainly on the 3’ end of 3D gene (see Fig. l), remain to be tested. Application of the same analysis to 20 VP1 gene sequences, representative of viruses (of serotypes C, A, and 0) that have circulated during the last decades (9) has allowed the specific typing of FMDV RNA by PCR amplification. Sequences were aligned and the intratypic/intertypic homology profiles were obtained for each of the serotypes compared (Fig. 3). Oligonucleotides corresponding to conserved sequences within serotypeeitherc (VPlC-1 and VPlC-2) A(VPlA-1 and VPl A-2) or 0 (VPlO-1, VP1 O-2, and VPlO-3) were used for PCR amplification in combination with (-) primer 2A-1, whose sequence is widely conserved among FMDVs of serotypes A, 0, and C (Fig. 3 and the determination of average conserved sequences was intended (that of FMDV isolates) and a second group that included those sequences to be differentiated from the previous ones. A consensus sequence was constructed from the first group of sequences. In both groups, and for each position, the number of sequences showing a nucleotide difference from the consensus was recorded. The values estimated for each position were divided by the number of sequences in each group The values calculated were used to obtain the nucleotide homology profiles. Primers for efficient detection of FMDV were chosen, after direct comparison of sequence alignments, among the 3D regions conserved in FMDV and not conserved among other picornaviruses, especially at the region corresponding to the 3’end of each of the primers. The bars included on the top indicate the sequences spanned by the primers used that corresponded to positions 18-37 (3D-l), 207-225 (3D-2) and 520539 (3D-3), according to (8).

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t A

663 :g = 394 298 234/220154-

FIG. 2. Agarose gel electrophoresis of the amplified 3D gene products from FMDV types C, A, and 0. (A) Amplification obtained usrng primers 3D-2 and 3D-1. (B) Amplification obtained using primers 3D3 and 3D-1. The nomenclature of the viruses is based on that proposed (31). Vrral RNA was prepared either from supernatants of BHK-21 -infected cells or from lesions of infected pigs by addition of 1 pg of yeast tRNA, treatment with 1 ~1 of HPRI, phenol-chloroform extraction, and ethanol precipitation. Viral nucleic acid was mixed with 200 ng of the (-) primer, complementary to the genomic sense RNA, in a total volume of 10 pl of sterile water. After 2 min of heatrng at 92” hybridization was allowed to proceed for 5 min at 45”. Sample was adjusted to 1 X PCR buffer (10 mMTris. pH 8.3; 50 mM KCI; 1.5 mM MgCI,; 0.01% gelatine), 10 mM dithiothreitol. each dNTP at 100 pM, 5 units HPRI, and 10 units of AMRT In a total volume of 100 ~1. The cDNA reaction mixture was incubated at 42” for 30 min and boiled. An alrquot of 33 ~1 was supplemented with 66 ng of the correspondrng (+) primer, complementary to the antigenomic sense cDNA, and 2.5 units of Taq polymerase (Perkin-Elmer, Cetus). The mixture was amplified by 30 successive cycles of heating at 93” for 1 min. renaturing at 37” for 1 mm, and polymerization at 72” for 1.5 min, except when indicated. From 1 to 8 ~1 of the final reaction was analyzed in a 19/oagarose gel contarning 0.5 pg/ml of ethidium bromide. Asterisks denote those samples prepared from lesions of infected animals.

Table 1). Primers VPlC-1, VPlA-1, VPlA-2, and VPlO-1 permitted amplification of the intended viral RNAs but not of those of other viral serotypes, yielding DNA bands of the expected size (Fig. 4). In four cases, the nucleotide sequence was deter-

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mined, using a modification of the procedure described (3) and was as expected (results not shown). Thus, FMDV characterization by specific VP1 PCR amplification in combination with direct sequencing of the amplified DNA provides a promising method for epidemiological studies. Three primers tested (VPlC-2, VPlO-2, and VPlO3) showed nonserotype specific cDNA amplification (Table 1). The contribution to the instability of DNAprimer hybrids during the Taq DNA polymerase and reverse transcriptase extension is not equivalent for different mismatches; C-C, A-G, G-A, and A-A being the ones that more drastically contribute to the instability; any mismatch involving T seems to have no effect (11, 12). An analysis of the sequences referred in Figs. 1 and 3, in those cases when the PCR was tested (data not shown), allowed us to suggest that amplification of A-type viruses with primer VPlC-2 can be due to the irrelevance of the G-T mismatches produced at the 3’ end of the primer. However, the presence of an additional A-G mismatch in this region probably limited the amplification of 0 serotype viruses. In the case of VPlO-2 the lack of specificity is probably due to the presence of a single A-G mismatch at the 3’end of the primer. The ability of VP1 O-3 to amplify RNA from serotype A can be explained by the absence of mismatches at the 3’end of the primer. In contrast, the specificity of this primer for C serotype appears to be due to the C-C or A-A mismatches. In summary, primers that produce amplification in a serotype-specific manner have 1.8-fold more mismatches/primer than nonspecific ones, and the num-

TABLE 1 PCR AMPLIFICATIONOF FMDV VP1 GENES

Primer” VPlC-1 VPlC2 VPlA-1 VPlA-2 VP1 O-1 VPlO-2 VP1 o-3

Expected serotype specrficity C C Ad A 0 0 0

Non-Specific amplificatrons

Ab up to 57””

C and A” up to 66“ ’ A type up to 66” c

a The nucleotides spanned by each of the primers used are rndicated in the legend of Fig. 3. In all cases olrgonucleotrde 2A-1, which spans nucleotrdes 35-54 of 2A gen (IO), was chosen as (-) primer for VP1 RNA amplification. b No DNA amplificatron was observed for A5 Sp83. ’ Annealing temperatures. d A weak DNA amplificatron was observed for A Arg79. e No DNA amplification was observed for Cl Santa Pau Sp70, Cl Girona Sp79, and A Arg79.

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100

150 200

250 300 350 400 Nuclmtide position

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250 300 350 400 NucMide position

VP1 - o-2 ---.-.-.-.--

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FIG. 4. Agarose gel electrophoresis of the amplified products of VP1 gene from FMDV types C, A, and 0. (A) Amplification obtained using oligonucleotide primers 2A-1 and VPlC-1, (B) 2A-1 and VP1 A2, (C) 2A-1 and VP1 0-l. Asterisks denote those samples prepared from lesions of infected animals.

Nuchtide pition FIG. 3. Profiles of intratypic and intertypic nucleotide homology of VP1 gene among FMDV of serotypes C (A), A(B), and 0 (C). In each case, the average nucleotide substitutions obtained, accumulated every five nucleotides (among FMDV of the corresponding serotype (-) and among the consensus sequence of this serotype with respect to the others ( e.. . - )) are plotted versus the nucleotide position. Sequences (10, 32-42) were aligned as previously described (9) and

ber of mismatches located at the 3’end of the primer is 3.5-fold more, in agreement with previous reports (11, 13). The results obtained indicate that although the primer effectiveness for specific amplification has to be the homology profiles were obtained as in Fig. 1. The bars included on the top indicate the sequences spanned by the primers used that corresponded to VP1 positions 96-l 14 (VPlC-1) and 471-490 (VP1 C-2) of the consensus sequence in Ref. (32); 134-l 51 (VPl A-1) and 516-535 (VPlA-2) of A27 in Ref. (38); and 113-131 (VPlO-I), 293-312 (VP1 O-2) and 4777496 (VP1 O-3) of 01 K in Ref. (33).

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experimentally tested, estimation of the homologies by the procedures developed here can greatly improve the selective PCR amplification. The method constitutes a valuable strategy to design PCR primers, provided that a sufficient number of sequences are available.

ACKNOWLEDGMENTS We are indebted to M. Borca, N.J. Knowles, and F. Abarca for providing us with viral RNAs and to E. Domingo for valuable suggestlons as well as for correction of the manuscript. We thank J. Plana and B. Hass for provldlng us with viral strains, J. HernBndez for advice In direct PCR products sequencing, and M. Gonzelez and J. Taz6n for technlcal assistance. Work at INIA was supported by ClCYT (Grant BIO 88.0452.CO503) and INIA (Grant 05-7537). Work at CBM was supported by CICYT (grants BIO 88.0452.CO5-01 and BIO 89.0668.CO3-02). A.R. and J.C.S. were supported by fellowships from CICYT. J.D was supported by a fellowship from INIA.

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Primer design for specific diagnosis by PCR of highly variable RNA viruses: typing of foot-and-mouth disease virus.

A PCR assay for the specific detection and identification of viral sequences that correlate with established serotypes of foot-and-mouth disease virus...
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