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Rapid diagnosis of encephalomyocarditis virus infections in pigs using a reverse transcription-polymerase chain reaction
Journal of Virological Methods ELSEVIER
Journal of Virological Methods 66 (1997) 83-89
Rapid diagnosis of encephalomyocarditis virus infections in pigs using a reverse transcription-polymerase chain reaction H. Vanderhallen,
F. Koenen
*
Accepted 20 February 1997
Abstract
Encephalomyocarditis virus (EMCV) is widespread and the economic losses caused by an EMCV outbreak in pig holdings and the similarity between a foot-and-mouth disease virus (FMDV) and an EMCV infection in young piglets stress the need for a rapid, specific and broad diagnostic assay. An alternative to the time-consuming seroneutralisation assay, currently used for the chardcterisation of EMCV, is described. An EMCV specific reverse transcriptionpolymerase chain reaction (RT-PCR), using primers located in a conserved region of the 3D gene of the viral genome. was developed and tested on 114 different EMCV isolates. The identity of the respective amplicons was confirmed by sequencing. The potential of this assay for future diagnostic purposes was demonstrated by applying the RT-PCR on tissue samples collected from an experimentally infected piglet. 0 1997 Elsevier Science B.V. K~JJWYWL/.S~~: RT-PCR:
Encephalomyocarditis
virus; Rapid
1. Introduction Encephalomyocarditis to the genus Cardiovirus naviridae (Minor et al., natural hosts of EMCV, without causing disease rodents to a wide variety
virus (EMCV) belongs of the family Picor1995). In rodents, the the virus usually persists and is transmitted from of animal species and is
* Corresponding author. Tel: + 32 2 3754455; fax: + 32 7 3750979; e-mail: [email protected] 0166-0934:97.$17.00 PII
SO 166.0934(97)022
0
1997
diagnosis
not limited to a defined geographical location (Acland, 1989). EMCV has been recognised as a pathogen of pigs for many years (Murnane et al., 1960). However, the clinical signs of disease vary. Sudden death of pigs (Gainer, 1967; Koenen et al., 1996) and reproductive failure in sows (Christianson et al., 1990; Koenen et al., 1991) have both been attributed to an EMCV infection. This virus is widespread and the economic losses caused by an EMCV outbreak in pig hold-
Elsevier Science B.V. All rights reserved. 14-3
84
H. Vanderhalien, F. Koenen /Journal
ings (Christianson et al., 1990) stress the need for a simple and broad diagnostic assay for EMCV. Furthermore, the marked similarity of the clinical picture caused by EMCV and footand-mouth disease virus (FMDV) in very young piglets (Acland, 1989) requires a diagnostic assay to distinguish unequivocally between EMCV and FMDV infection to limit trade restrictions. Currently, EMCV diagnosis is achieved by virus isolation from pig tissues followed by neutralisation against a specific anti-EMCV serum (Acland, 1989). This characterisation is timeconsuming because seroneutralisation requires two further days of propagation of the virus in-vitro. transcription-polymerase Combined reverse chain reaction (RT-PCR) methods have been developed for many other RNA viruses (Belak and Ballagi-Pordany, 1993). This approach allows the detection of viral RNA by primer-directed enzymatic amplification of specific target RNA sequences. The present study evaluates a RT-PCR, using primers located in the 3D gene of the EMCV genome, for characterisation of a wide range of EMCV isolates. The applicability of this assay for diagnosis of EMCV infection on tissue samples is also discussed.
of Virological Methods 66 (1997) 83-89
isolates, listed in Table various sources.
Geographical origin
Year
Species
Australia
1976 1987
Belgium
1990 1991 1992 1995 1995 1960 1984 1964
Pig Pig Rat Pig Pig Pig Pig Rat Spiny rat Pig Red squirrel Grey squirrel Mouse Pig Pig Pig Pig Pig Pig Pig Pig Pig Rodent Pig Rodent Pig Dormouse Squirrel Pig Pig Pig Pig Pig Elephant Chimpanzee Rodent Mosquito Elephant Lemur Rodent Pig Baboon Warthog Tapir Giraffe
Brazil England Germany Greece
Italy
and methods
2.1. Cells New Zealand
Theiler’s murine encephalomyelitis virus strain GDVII (TMEV-GDVII) and all EMC viruses, except the reference strain (ATCC 129B), were propagated in baby hamster kidney 21 (BHK21) cells (ATCC CCLlO). ATCC 129B was propagated in Vero cells (ATCC CCLSl). Three different types of FMDV (A, Leffinge ‘59, 0, Brugge ‘63, C, Loupoigne ‘53) were propagated in SK-6 cells (Kasza et al., 1972).
Panama Puerto Rico South Africa USA
1949 1986 1987 I988 1989 1990 1991 1992 1994 1995
different
EMCV
1988 1990 1992 1995 1976 1978 1958 1990 1994 I944 1958 1963 1976 1985 1988 1990 NA NA NA
2.2. Samples 2.2.1. Cell culture supernatants One hundred and fourteen
obtained
Table 1 Overview of the virus isolates which are positively ised as EMCV by RT-PCR and seroneutralisation
1996
2. Materials
1, were
NA, not available.
from
character-
No. of isolates
6 17 31 4
2
4 2
Characterisation of the EMCV isolates was carried out by neutralisation of 100 median tissue culture infective doses (TCID,,) of the isolated virus with a specific EMCV antiserum directed against the reference strain (Koenen et al., 1991). 2.22. Clinicd samples One piglet of about 20 kg was infected with a recent Belgian EMCV isolate (Koenen et al.. 1996). At post-mortem, heart and spleen tissues were collected and processed for RNA extraction and subsequent RT-PCR analysis. The amount of EMCV in 100 mg tissue was IO4 TCID,,. An EMCV-antigen and -antibody negative piglet was slaughtered to collect negative samples.
RNA-extraction was carried out based on the single-step method described by Chomczynski and Sacchi (1987). The precautions recommended by Sambrook et al. (1989) were observed to avoid contamination with ribonucleases. 2.3.1. RNA extrcrction jkm infected cell cultures A cell monolayer, covered with minimal essential medium (MEM) supplemented with lO”/o fetal calf serum (FCS), was infected with 300 ~1 of a virus suspension containing approximately 10’ TCID,,,;SO ,ll of the isolate. This culture was incubated at 37°C and inspected twice a day. When cytopathic effect (CPE) was apparent the infected cell culture fluid was frozen. For extraction, the fluid was thawed and centrifuged at 5000 rpm for 5 min. The virus was pelleted from 15 ml of the resulting supernatant by ultracentrifugation at 16 000 rpm for 16 h at 4°C. Subsequently, the virus pellet was dissolved in 1 ml TRIzolTM reagent (Gibco BRL). The mixture of virus pellet and the monophasic solution of phenol and guanidine isothiocyanate was incubated at room temperature. After 5 min, 200 111 chloroform was added. The solution was shaken by hand and incubated at room temperature for 2-~ 3 min. After centrifugation at 12 000 x s for 15 min at 4”C, the aqueous phase was transferred to a fresh tube, mixed with 500 111 of isopropanol and placed at room temperature foi
10 min. Sedimentation at 12 000 x g for 10 min at 4°C was performed and the resulting RNA pellet was mixed with 1 ml of 75% ethanol. After vortexing this mixture was centrifuged at 12 000 x g for 5 min at 4°C. The pellet was air-dried and RNA was dissolved in 40 ~1 diethyl pyrocarbonate (DEPC) treated H,O by incubating 10 min at 55°C. 2.3.2. RNA extraction jkm clinicul sumpIes Approximately 100 mg of tissue were homogenised in I ml TRIzol reagent using an UltraTurrax (24000 rpm). After 5 min, 200 ~11 chloroform was added and the solution was vortexed and incubated at room temperature for 2-3 min. Following centrifugation ( 12 000 x g, 20 min. 4°C) the upper aqueous phase was transferred to a fresh tube and 500 111 ice-cold isopropanol was added. This mixture was stored at - 20°C for 1 h. Finally, the RNA was precipitated completely by centrifugation at 12000 g for 30 min at 4°C. The resulting pellet was processed as described before (see Section 2.3.1). 2.4. Reverse
transcription
(RT)
For each RT reaction, 3.8 jr1 of RNA extraction product was added to 6.2 111of a RT premix to obtain a final concentration of 1 x first strand buffer (Gibco BRL), 0.5 mM dNTP mix (Eurogentec), 10 mM DTT (Gibco BRL), 100 U moloney murine leukaem.ia virus reverse transcriptase (Gibco BRL), and 3.5 pmolipl of random hexamers (Pharmacia Biotech). Subsequently reverse transcription was carried out at 37°C for 15 min and followed by heating for 5 min at 95°C. 2.5. Oligonucleotide
primers
The primers were based on published sequences of the EMCV reference strain (Duke et al., 1992). strains B and D (Bae et al., 1989) and PV2 1 (Zimmermann et al., 1994). These sequences were aligned and the computer program OLIGOTM4 (Rychlik and Rhoads, 1989) was used to determine primer sequences best suited for the proposed application. The priming capabilities on the 0, Kaufbeuren FMDV genome (Beck and
7360
7370
7380
7380
763”
7640
I
I
I
I
I
I
7650
I
7660
I
ATCC 1298 GGATTTTGATARDDTDA~~~~~~~-~~~~~~~...................,,...................GCTCTTTOCCCCATTCCOTCACCTACGOGTTGTCG TA..........A...G..........G................................,,..................................~.....*........~..~. EMCV-B TA..........R...G..........6................................,,..................................~.....~........~..~. EMCV-D ..________............................................ //...................................................... PV21 PZ
P1
Fig. I. Computer alignment of selected sequences within the 3D gene sequences of published EMCV genomes. The selected prime1 set, PI-P2, is indicated. Nucleotide numbering is that used for publication of the EMCV reference strain by Duke et al. (1992).
2.8. Agarose
gel electrophoresis
Strohmaier, 1987) and on the genomes of TMEVand DA (Ohara et al., 1988), TMEV-Bean TMEV-GDVII (Law and Brown, 1990) were analysed with 0LIGOTM4 to evaluate the specificity of the designed primers. The oligonucleotides were synthesised chemically by Pharmacia Biotech.
Gel electrophoresis was carried out on a 2% agarose gel in 1 x TAE buffer using 10 ,ul of each amplified product. The amplicons were visualised by staining the gel with ethidium bromide and using UV light.
2.6. Polymeruse
2.9. Spec!jicitJ,
chuin reuction
(PCR)
The general guidelines for PCR manipulations, as described by Kwok and Higuchi (1989) were observed. The RT-product was diluted with 90 ~1 DEPC treated HzO. Each PCR reaction mixture consisted of the following reagents in a total volume of 10 ~1:2 ~11 of the diluted RT-product; 1 ~11 10 x PCR buffer (Eurogentec); 1 mM MgCl, (Eurogentec); 0.2 mM dNTP mix (Eurogentec); 0.15 U of GoldStur Polymerase (Eurogentec); DEPC treated H,O and 15 pmol Pl and P2. Using RNA extracted from a cell culture infected with the EMCV ATCC129B, various PCR parameters (temperature, time and number of cycles) were examined individually to design a rapid and efficient PCR protocol.
The specificity of the assay was determined by applying the optimised RT-PCR protocol on RNA extracted from the supernatant of an uninfected cell culture and on RNA extracted from the supernatant of cell cultures infected with three different types of FMDV (C, Loupoigne ‘53, A, Leffinge ‘59, 0, Brugge ‘63) and TMEV-GDVII. 2.10. Sensitivity Tenfold dilutions of the supernatant of a BHK21 cell culture infected with the Belgian EMCV isolate described by Koenen et al. (1991) were made in MEM. MEM was used as a negative control. Each dilution was handled to determine the TCID,,, and was treated as described to carry out RT-PCR.
2.7. Sequencing The amplicons were purified using Microcon100 microconcentrators (Amicon). The identity of the amplified fragments was determined by sequencing using the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase (FS) and an ABI PRISM 310 Genetic Analyzer according to the manufacturer’s protocol (Perkin Elmer). Reverse transcription, DNA amplification and the sequencing reaction were undertaken using a GeneAmp PCR System 2400 (Perkin Elmer).
3. Results After aligning the published 3D gene sequences, two regions of about 30 nucleotides at the 3’ end of the gene, exhibiting minimal variation, were selected. 0LIGOTM4 analysis of these regions resulted in two primer sequences; the downstream, Pl, and the upstream primer, P2 (Fig. 1). Computer simulation of the proposed RT-PCR, on the EMCV sequence as published by Duke et al. (1992) and on the examined genome sequences of
H.
Vanderhallen,
F. Kornen
/ Journtrl
related Picornaviridae, predicted an EMCV specific 286 bp amplicon. The melting temperature (r,,,) of Pl and P2 was calculated to be 66.0 and 65.1”C. respectively. No 3’-terminal dimer or hairpin formation was evident after 0LIGOTM4 analysis. An initial denaturation at 95°C for 10 s, 25 cycles of ramping up and down from 94 to 6O”C, followed by a final extension at 60°C for 10 s proved to be the most appropriate amplification method. RT-PCR revealed an amplification product of the expected length, 286 bp, on the agarose gels for all the tested EMCV isolates (Table 1) and for heart and spleen tissues of the infected piglet. EMCV specific nucleic acids were not demonstrated by RT-PCR in uninfected cells, in cell cultures infected with three different types of FMDV (C, Loupoigne ‘53, A, Leffinge ‘59, 0, Brugge ‘63) and TMEV-GDVII and in heart and spleen tissues collected from an EMCV negative piglet The assay allowed demonstration of EMCV specific amplicons in virus dilutions containing at least IO4 TCID50 of EMCV. Sequencing of the amplified region of all the tested EMCV isolates (data not shown) revealed a homology of 81.7% at the nucleotide and 96.7% at the amino acid level. Primer P2 proved to be the most effective sequencing primer.
4. Discussion The nucleotide sequence amplified in this study corresponds to a 286 bp fragment in the 3D gene of the EMCV genome. This gene codes for the RNA polymerase (Duke et al., 1992) and contains nucleotide regions of about 30 nucleotides exhibiting minimal variation among the published EMCV sequences as shown by aligning these sequences (Fig. 1). Aphtoviruses. a genus of the family of Picornaviridae closely related to the Cardioviruses (Minor et al., 1995), also show little variation in the nucleotide sequences of the 3D gene for all reported genomes (Martinez-Salas et al., 1985). Several RT-PCR assays targeting this region on the FMDV genome have been described for the diagnosis and characterisation of all seven FMD viral serotypes (Meyer et al., 1991: Laor et al., 1992). The present
of’ Virok@al
A4ethoci.s 66 (1997)
8-C 89
87
study confirms that the target sequence described lies within an area conserved among EMCV genomes, since 114 isolates collected from different geographical locations, different species and at different times are positively characterised by the RT-PCR assay. The intrinsic characteristics of the two selected primers, equivalent T,,, and absence of possible dimer and hairpin formation, allowed the use of a very fast PCR program. PCR was completed in less than 30 min. The specificity of the characterisation assay was demonstrated by the absence of amplification in all reactions with RNA extracted from cell cultures infected with TMEV or FMDV (C, Loupoigne ‘53, A, Leffinge ‘59, 0, Brugge ‘63). In particular, the ability to distinguish from FMDV, which causes a clinical picture similar to EMCV (Acland, 1989) is critical because a false FMDV diagnosis can result in considerable economic losses (Sellers and Daggupaty, 1990). Although the primer sequences are conserved, the region they enclose is variable. Since neutralisation of all 114 isolates confirmed their identity as EMCV, the obtained variance of 18.3% obtained at the nucleotide level can be considered as the primissible diversity in this region of the viral genome. In addition, this variability justifies consideration of the amplified region as a molecular marker of the virus. Linking these molecular data with epidemiological and pathological observations might lead to a better insight of this infection (Koenen et al.. 1996). Similar to the strategy applied for the development of a RT-PCR assay for the diagnosis of a FMDV infection (Laor et al., 1992), the RT-PCR described above was applied for diagnostic purposes, resulting in a more rapid and accurate diagnosis of EMCV outbreak. The results on heart and spleen tissues, containing lo4 TCID,,, EMCV/ 100 mg organ tissue, illustrate this application. Taking into account these findings and the data collected by Meyer et al. (1991), indicating that the amount of FMDV present in tissue samples is at least lo4 TCID,,, the majority of EMCV positive clinical samples will presumably be detected. Until validation of the procedure by testing more field samples, a negative diagnosis by RT-PCR necessitates confirmation by VI. It is expected that the
88
RT-PCR current with the allowing relation
H. VundrrhaNen,
F.
Koemw Jourrud qf Virologid
described will be a powerful alternative to laboratory assays for EMCV diagnosis advantage of generating molecular data, immediate classification of EMCV in to other EMCV isolates.
Acknowledgements The authors wish to thank Miss R. Debaugnies for excellent technical assistance. We are grateful to Dr K. De Clercq (Nationaal Instituut voor Diergeneeskundig Onderzoek, Brussels), M. Phil. N.J. Knowles (World Reference Laboratory for FMD at the Institute for Animal Health, Pirbright), Prof Dr 0. Papadopoulos (Faculty of Veterinary Medicine at the Aristotle University, Thessaloniki), Dr E. Paschaleri (Institute of Infectious and Parasitic Diseases, Thessaloniki), Dr F. Desimone and Dr E. Brocchi (Instituto Zooprofilattice Sperimentale della Lombardia e dell’Emilia, Bresciaj for providing isolates. This work was supported by the European Union (Contract no. AIR3-CT93-1465).
Appendix A This gel shows the amplification of RNA extracted from cell cultures infected with eight of the 31 EMCVs isolated from pigs in 1995 in Belgium using the described method. The expected bands of 286 bp are evident and now parasite bands can be observed. Lane 1, 100 bp ladder; lanes 2--9, EMCV isolates; lanes lo- 11, negative controls. 12
345678910
11
Method.~ 66 (1997) 83-89
References Acland, H.M. (1989) Encephalomyocarditis virus. In M.B. Pensaert (Ed.), Virus infections of porcines. Vol. II, pp. 25% 263. Bae, Y.-S., Eun. H.-M. and Yoon, J.-W. (1989) Genomic differences between the diabetogenic and nondiabetogenic variants of the encephalomyocarditis virus. Virology 170. 282 287. Beck. E. and Strohmaier. K. (1987) Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. J. Virol. 61. 1621 1629. Belak, S. and BaUagi-Pordany, A. (1993) Application of the polymerase chain reaction (PCR) in veterinary diagnostic virology. Vet. Res. Corn. 17, 55 72. Chomczynski. P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162, I56 159. Christianson, W.T.. Kim. H.S.. Joo, H.S. and Barnes, D.M. (1990) Reproductive and neonatal losses associated with possible encephalomyocarditis virus infection in pigs. Vet. Rec. 126. 54 57. Duke. G.M.. Hoffman. M.A. and Palmenberg, A.C. (1992) Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. J. Virol. 66 (3). 1602 -1609. Gainer, J.H. (1967) Encephalomyocarditis virus infections in Florida, 1960 1966. JAVMA I5 I (4), 421~ 425. Kasza, L., Shadduck, J.A. and Christofinis. G.J. (1972) Establishment. viral susceptibility and biological characteristics of a Swine Kidney cell line SK-6. Res. Vet. Sci. 13, 46 51. Koenen, F., Declercq. K. and Strobbe, R. (1991) Isolation of encephalomyocarditis virus in swine with reproductive failure in Belgium. VI. Diergeneesk. Tijdschr. 60, I I3 I IS. Koenen. F.. Vanderhallen. H., Castryck. F., Miry, C. and Fockedey. M. (1996) Epidemiologic, pathogenetic and molecular analysis of the first encephalomyocarditis outbreak in fattening pigs in Belgium. Proceedings of the 14th IPVS Congress, Bologna, Italy, 7 IO July 1996, pp. 96. Kwok. S. and Higuchi, R. (1989) Avoiding False positives with PCR. Nature 339. 237 238. Laor, O., Torgersen, H., Yadin, H. and Becker. Y. (1992) Detection of FMDV RNA amplified by the polymerase chain reaction (PCR). J. Viral. Meth. 36. 197-208. Law. K.M. and Brown. T.D.K. (1990) The complete nuclcotide sequence of the GDVII strain of Theiler’s murine encephalomyelitis virus (TMEV). Nucleic Acids Res. 18(22), 6707 6708. Martinez-Salas, E.. Ortin, J. and Domingo, E. (1985) Sequence of the viral replicasc gene from foot-and-mouth disease virus Cl Santa Pau ((X8). Gene 35. 55 61. Meyer, R.F.. Brown, C.C., House, C.. House, J.A. and Molitor. T.W. (1991) Rapid and sensitive detection of foot-andmouth disease virus in tissues by enzymatic RNA amplification of the polymerase gene. J. Virol. Meth. 34, 161 172.
H. Vandrrhallen,
F. Koenen /Journal
Minor, P.D.. Brown, F.. Domingo, E., Hoey, E., King, A., KnowJes. N., Lemon, S., Palmenberg, A., Rueckert. R.. Stanway, G. Wimmer. E. and Yin-Murphy, M. (1995) Picornaviridae. In F.A. Murphy, C.M. Fauquet, D.H.L. Bishop, S.A. Ghabrial and A.W. Jarvis (Eds.), Virus Taxonomy. Classification and Nomenclature of Viruses. Sixth Report of the International Committee on Taxonomy of Viruses. Arch. Virol., Springer-Verlag, Vienna, (SuppI. 10) pp. 329-336. Murnane. T.G., Graighead. J.E., Mondragon, H. and Shelokov, A. (1960) Fatal disease of swine due to encephalomyocarditis virus. Science 131, 498-499. Ohara. Y., Stein. S.. Fu, J.. Stillman, L.. Klaman, L. and Roos. R.P. (1988) Molecular cloning and sequence determination of DA strain of Theiler’s murine encephalomyelitis viruses. Virology 164, 145-255.
of Virological Methods 66 11997) S-?-S9
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Rychlik. W. and Rhoads, R.E. (1989) A computer program for choosing optimal oligonucleotides for filter hybridization,
sequencing
cleic Acids Sambrook.
and in vitro
J., Fritsch,
E.F. and Maniatis.
lar Cloning:
A Laboratory
Laboratory.
New York,
Sellers,
R.F.
and
1952. Can. Eggers, and highly 366
of DNA.
Nu-
disease
T. (1989) MolecuCold
Spring
Harbot-
USA. SM.
(1990)
The epidemic
in Saskatchewan.
Canada.
ol
I95
I
J. Vet. Res. 54, 457-464. A., Nielsen-Salz,
H.J.
(1994)
construction virulent 372.
Manual.
Daggupdty,
foot-and-mouth Zimmermann,
amplification
Res. 17, 8543-8551.
of
B., Kruppenbacher.
The
complete
an
infectious
encephalomyocarditis
nucleotide
J.P. and sequence
cDNA
clone
virus.
Virology
of
a
203.
Journal of Virological Methods 66 (1997) 83-89
Rapid diagnosis of encephalomyocarditis virus infections in pigs using a reverse transcription-polymerase chain reaction H. Vanderhallen,
F. Koenen
*
Accepted 20 February 1997
Abstract
Encephalomyocarditis virus (EMCV) is widespread and the economic losses caused by an EMCV outbreak in pig holdings and the similarity between a foot-and-mouth disease virus (FMDV) and an EMCV infection in young piglets stress the need for a rapid, specific and broad diagnostic assay. An alternative to the time-consuming seroneutralisation assay, currently used for the chardcterisation of EMCV, is described. An EMCV specific reverse transcriptionpolymerase chain reaction (RT-PCR), using primers located in a conserved region of the 3D gene of the viral genome. was developed and tested on 114 different EMCV isolates. The identity of the respective amplicons was confirmed by sequencing. The potential of this assay for future diagnostic purposes was demonstrated by applying the RT-PCR on tissue samples collected from an experimentally infected piglet. 0 1997 Elsevier Science B.V. K~JJWYWL/.S~~: RT-PCR:
Encephalomyocarditis
virus; Rapid
1. Introduction Encephalomyocarditis to the genus Cardiovirus naviridae (Minor et al., natural hosts of EMCV, without causing disease rodents to a wide variety
virus (EMCV) belongs of the family Picor1995). In rodents, the the virus usually persists and is transmitted from of animal species and is
* Corresponding author. Tel: + 32 2 3754455; fax: + 32 7 3750979; e-mail: [email protected] 0166-0934:97.$17.00 PII
SO 166.0934(97)022
0
1997
diagnosis
not limited to a defined geographical location (Acland, 1989). EMCV has been recognised as a pathogen of pigs for many years (Murnane et al., 1960). However, the clinical signs of disease vary. Sudden death of pigs (Gainer, 1967; Koenen et al., 1996) and reproductive failure in sows (Christianson et al., 1990; Koenen et al., 1991) have both been attributed to an EMCV infection. This virus is widespread and the economic losses caused by an EMCV outbreak in pig hold-
Elsevier Science B.V. All rights reserved. 14-3
84
H. Vanderhalien, F. Koenen /Journal
ings (Christianson et al., 1990) stress the need for a simple and broad diagnostic assay for EMCV. Furthermore, the marked similarity of the clinical picture caused by EMCV and footand-mouth disease virus (FMDV) in very young piglets (Acland, 1989) requires a diagnostic assay to distinguish unequivocally between EMCV and FMDV infection to limit trade restrictions. Currently, EMCV diagnosis is achieved by virus isolation from pig tissues followed by neutralisation against a specific anti-EMCV serum (Acland, 1989). This characterisation is timeconsuming because seroneutralisation requires two further days of propagation of the virus in-vitro. transcription-polymerase Combined reverse chain reaction (RT-PCR) methods have been developed for many other RNA viruses (Belak and Ballagi-Pordany, 1993). This approach allows the detection of viral RNA by primer-directed enzymatic amplification of specific target RNA sequences. The present study evaluates a RT-PCR, using primers located in the 3D gene of the EMCV genome, for characterisation of a wide range of EMCV isolates. The applicability of this assay for diagnosis of EMCV infection on tissue samples is also discussed.
of Virological Methods 66 (1997) 83-89
isolates, listed in Table various sources.
Geographical origin
Year
Species
Australia
1976 1987
Belgium
1990 1991 1992 1995 1995 1960 1984 1964
Pig Pig Rat Pig Pig Pig Pig Rat Spiny rat Pig Red squirrel Grey squirrel Mouse Pig Pig Pig Pig Pig Pig Pig Pig Pig Rodent Pig Rodent Pig Dormouse Squirrel Pig Pig Pig Pig Pig Elephant Chimpanzee Rodent Mosquito Elephant Lemur Rodent Pig Baboon Warthog Tapir Giraffe
Brazil England Germany Greece
Italy
and methods
2.1. Cells New Zealand
Theiler’s murine encephalomyelitis virus strain GDVII (TMEV-GDVII) and all EMC viruses, except the reference strain (ATCC 129B), were propagated in baby hamster kidney 21 (BHK21) cells (ATCC CCLlO). ATCC 129B was propagated in Vero cells (ATCC CCLSl). Three different types of FMDV (A, Leffinge ‘59, 0, Brugge ‘63, C, Loupoigne ‘53) were propagated in SK-6 cells (Kasza et al., 1972).
Panama Puerto Rico South Africa USA
1949 1986 1987 I988 1989 1990 1991 1992 1994 1995
different
EMCV
1988 1990 1992 1995 1976 1978 1958 1990 1994 I944 1958 1963 1976 1985 1988 1990 NA NA NA
2.2. Samples 2.2.1. Cell culture supernatants One hundred and fourteen
obtained
Table 1 Overview of the virus isolates which are positively ised as EMCV by RT-PCR and seroneutralisation
1996
2. Materials
1, were
NA, not available.
from
character-
No. of isolates
6 17 31 4
2
4 2
Characterisation of the EMCV isolates was carried out by neutralisation of 100 median tissue culture infective doses (TCID,,) of the isolated virus with a specific EMCV antiserum directed against the reference strain (Koenen et al., 1991). 2.22. Clinicd samples One piglet of about 20 kg was infected with a recent Belgian EMCV isolate (Koenen et al.. 1996). At post-mortem, heart and spleen tissues were collected and processed for RNA extraction and subsequent RT-PCR analysis. The amount of EMCV in 100 mg tissue was IO4 TCID,,. An EMCV-antigen and -antibody negative piglet was slaughtered to collect negative samples.
RNA-extraction was carried out based on the single-step method described by Chomczynski and Sacchi (1987). The precautions recommended by Sambrook et al. (1989) were observed to avoid contamination with ribonucleases. 2.3.1. RNA extrcrction jkm infected cell cultures A cell monolayer, covered with minimal essential medium (MEM) supplemented with lO”/o fetal calf serum (FCS), was infected with 300 ~1 of a virus suspension containing approximately 10’ TCID,,,;SO ,ll of the isolate. This culture was incubated at 37°C and inspected twice a day. When cytopathic effect (CPE) was apparent the infected cell culture fluid was frozen. For extraction, the fluid was thawed and centrifuged at 5000 rpm for 5 min. The virus was pelleted from 15 ml of the resulting supernatant by ultracentrifugation at 16 000 rpm for 16 h at 4°C. Subsequently, the virus pellet was dissolved in 1 ml TRIzolTM reagent (Gibco BRL). The mixture of virus pellet and the monophasic solution of phenol and guanidine isothiocyanate was incubated at room temperature. After 5 min, 200 111 chloroform was added. The solution was shaken by hand and incubated at room temperature for 2-~ 3 min. After centrifugation at 12 000 x s for 15 min at 4”C, the aqueous phase was transferred to a fresh tube, mixed with 500 111 of isopropanol and placed at room temperature foi
10 min. Sedimentation at 12 000 x g for 10 min at 4°C was performed and the resulting RNA pellet was mixed with 1 ml of 75% ethanol. After vortexing this mixture was centrifuged at 12 000 x g for 5 min at 4°C. The pellet was air-dried and RNA was dissolved in 40 ~1 diethyl pyrocarbonate (DEPC) treated H,O by incubating 10 min at 55°C. 2.3.2. RNA extraction jkm clinicul sumpIes Approximately 100 mg of tissue were homogenised in I ml TRIzol reagent using an UltraTurrax (24000 rpm). After 5 min, 200 ~11 chloroform was added and the solution was vortexed and incubated at room temperature for 2-3 min. Following centrifugation ( 12 000 x g, 20 min. 4°C) the upper aqueous phase was transferred to a fresh tube and 500 111 ice-cold isopropanol was added. This mixture was stored at - 20°C for 1 h. Finally, the RNA was precipitated completely by centrifugation at 12000 g for 30 min at 4°C. The resulting pellet was processed as described before (see Section 2.3.1). 2.4. Reverse
transcription
(RT)
For each RT reaction, 3.8 jr1 of RNA extraction product was added to 6.2 111of a RT premix to obtain a final concentration of 1 x first strand buffer (Gibco BRL), 0.5 mM dNTP mix (Eurogentec), 10 mM DTT (Gibco BRL), 100 U moloney murine leukaem.ia virus reverse transcriptase (Gibco BRL), and 3.5 pmolipl of random hexamers (Pharmacia Biotech). Subsequently reverse transcription was carried out at 37°C for 15 min and followed by heating for 5 min at 95°C. 2.5. Oligonucleotide
primers
The primers were based on published sequences of the EMCV reference strain (Duke et al., 1992). strains B and D (Bae et al., 1989) and PV2 1 (Zimmermann et al., 1994). These sequences were aligned and the computer program OLIGOTM4 (Rychlik and Rhoads, 1989) was used to determine primer sequences best suited for the proposed application. The priming capabilities on the 0, Kaufbeuren FMDV genome (Beck and
7360
7370
7380
7380
763”
7640
I
I
I
I
I
I
7650
I
7660
I
ATCC 1298 GGATTTTGATARDDTDA~~~~~~~-~~~~~~~...................,,...................GCTCTTTOCCCCATTCCOTCACCTACGOGTTGTCG TA..........A...G..........G................................,,..................................~.....*........~..~. EMCV-B TA..........R...G..........6................................,,..................................~.....~........~..~. EMCV-D ..________............................................ //...................................................... PV21 PZ
P1
Fig. I. Computer alignment of selected sequences within the 3D gene sequences of published EMCV genomes. The selected prime1 set, PI-P2, is indicated. Nucleotide numbering is that used for publication of the EMCV reference strain by Duke et al. (1992).
2.8. Agarose
gel electrophoresis
Strohmaier, 1987) and on the genomes of TMEVand DA (Ohara et al., 1988), TMEV-Bean TMEV-GDVII (Law and Brown, 1990) were analysed with 0LIGOTM4 to evaluate the specificity of the designed primers. The oligonucleotides were synthesised chemically by Pharmacia Biotech.
Gel electrophoresis was carried out on a 2% agarose gel in 1 x TAE buffer using 10 ,ul of each amplified product. The amplicons were visualised by staining the gel with ethidium bromide and using UV light.
2.6. Polymeruse
2.9. Spec!jicitJ,
chuin reuction
(PCR)
The general guidelines for PCR manipulations, as described by Kwok and Higuchi (1989) were observed. The RT-product was diluted with 90 ~1 DEPC treated HzO. Each PCR reaction mixture consisted of the following reagents in a total volume of 10 ~1:2 ~11 of the diluted RT-product; 1 ~11 10 x PCR buffer (Eurogentec); 1 mM MgCl, (Eurogentec); 0.2 mM dNTP mix (Eurogentec); 0.15 U of GoldStur Polymerase (Eurogentec); DEPC treated H,O and 15 pmol Pl and P2. Using RNA extracted from a cell culture infected with the EMCV ATCC129B, various PCR parameters (temperature, time and number of cycles) were examined individually to design a rapid and efficient PCR protocol.
The specificity of the assay was determined by applying the optimised RT-PCR protocol on RNA extracted from the supernatant of an uninfected cell culture and on RNA extracted from the supernatant of cell cultures infected with three different types of FMDV (C, Loupoigne ‘53, A, Leffinge ‘59, 0, Brugge ‘63) and TMEV-GDVII. 2.10. Sensitivity Tenfold dilutions of the supernatant of a BHK21 cell culture infected with the Belgian EMCV isolate described by Koenen et al. (1991) were made in MEM. MEM was used as a negative control. Each dilution was handled to determine the TCID,,, and was treated as described to carry out RT-PCR.
2.7. Sequencing The amplicons were purified using Microcon100 microconcentrators (Amicon). The identity of the amplified fragments was determined by sequencing using the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase (FS) and an ABI PRISM 310 Genetic Analyzer according to the manufacturer’s protocol (Perkin Elmer). Reverse transcription, DNA amplification and the sequencing reaction were undertaken using a GeneAmp PCR System 2400 (Perkin Elmer).
3. Results After aligning the published 3D gene sequences, two regions of about 30 nucleotides at the 3’ end of the gene, exhibiting minimal variation, were selected. 0LIGOTM4 analysis of these regions resulted in two primer sequences; the downstream, Pl, and the upstream primer, P2 (Fig. 1). Computer simulation of the proposed RT-PCR, on the EMCV sequence as published by Duke et al. (1992) and on the examined genome sequences of
H.
Vanderhallen,
F. Kornen
/ Journtrl
related Picornaviridae, predicted an EMCV specific 286 bp amplicon. The melting temperature (r,,,) of Pl and P2 was calculated to be 66.0 and 65.1”C. respectively. No 3’-terminal dimer or hairpin formation was evident after 0LIGOTM4 analysis. An initial denaturation at 95°C for 10 s, 25 cycles of ramping up and down from 94 to 6O”C, followed by a final extension at 60°C for 10 s proved to be the most appropriate amplification method. RT-PCR revealed an amplification product of the expected length, 286 bp, on the agarose gels for all the tested EMCV isolates (Table 1) and for heart and spleen tissues of the infected piglet. EMCV specific nucleic acids were not demonstrated by RT-PCR in uninfected cells, in cell cultures infected with three different types of FMDV (C, Loupoigne ‘53, A, Leffinge ‘59, 0, Brugge ‘63) and TMEV-GDVII and in heart and spleen tissues collected from an EMCV negative piglet The assay allowed demonstration of EMCV specific amplicons in virus dilutions containing at least IO4 TCID50 of EMCV. Sequencing of the amplified region of all the tested EMCV isolates (data not shown) revealed a homology of 81.7% at the nucleotide and 96.7% at the amino acid level. Primer P2 proved to be the most effective sequencing primer.
4. Discussion The nucleotide sequence amplified in this study corresponds to a 286 bp fragment in the 3D gene of the EMCV genome. This gene codes for the RNA polymerase (Duke et al., 1992) and contains nucleotide regions of about 30 nucleotides exhibiting minimal variation among the published EMCV sequences as shown by aligning these sequences (Fig. 1). Aphtoviruses. a genus of the family of Picornaviridae closely related to the Cardioviruses (Minor et al., 1995), also show little variation in the nucleotide sequences of the 3D gene for all reported genomes (Martinez-Salas et al., 1985). Several RT-PCR assays targeting this region on the FMDV genome have been described for the diagnosis and characterisation of all seven FMD viral serotypes (Meyer et al., 1991: Laor et al., 1992). The present
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87
study confirms that the target sequence described lies within an area conserved among EMCV genomes, since 114 isolates collected from different geographical locations, different species and at different times are positively characterised by the RT-PCR assay. The intrinsic characteristics of the two selected primers, equivalent T,,, and absence of possible dimer and hairpin formation, allowed the use of a very fast PCR program. PCR was completed in less than 30 min. The specificity of the characterisation assay was demonstrated by the absence of amplification in all reactions with RNA extracted from cell cultures infected with TMEV or FMDV (C, Loupoigne ‘53, A, Leffinge ‘59, 0, Brugge ‘63). In particular, the ability to distinguish from FMDV, which causes a clinical picture similar to EMCV (Acland, 1989) is critical because a false FMDV diagnosis can result in considerable economic losses (Sellers and Daggupaty, 1990). Although the primer sequences are conserved, the region they enclose is variable. Since neutralisation of all 114 isolates confirmed their identity as EMCV, the obtained variance of 18.3% obtained at the nucleotide level can be considered as the primissible diversity in this region of the viral genome. In addition, this variability justifies consideration of the amplified region as a molecular marker of the virus. Linking these molecular data with epidemiological and pathological observations might lead to a better insight of this infection (Koenen et al.. 1996). Similar to the strategy applied for the development of a RT-PCR assay for the diagnosis of a FMDV infection (Laor et al., 1992), the RT-PCR described above was applied for diagnostic purposes, resulting in a more rapid and accurate diagnosis of EMCV outbreak. The results on heart and spleen tissues, containing lo4 TCID,,, EMCV/ 100 mg organ tissue, illustrate this application. Taking into account these findings and the data collected by Meyer et al. (1991), indicating that the amount of FMDV present in tissue samples is at least lo4 TCID,,, the majority of EMCV positive clinical samples will presumably be detected. Until validation of the procedure by testing more field samples, a negative diagnosis by RT-PCR necessitates confirmation by VI. It is expected that the
88
RT-PCR current with the allowing relation
H. VundrrhaNen,
F.
Koemw Jourrud qf Virologid
described will be a powerful alternative to laboratory assays for EMCV diagnosis advantage of generating molecular data, immediate classification of EMCV in to other EMCV isolates.
Acknowledgements The authors wish to thank Miss R. Debaugnies for excellent technical assistance. We are grateful to Dr K. De Clercq (Nationaal Instituut voor Diergeneeskundig Onderzoek, Brussels), M. Phil. N.J. Knowles (World Reference Laboratory for FMD at the Institute for Animal Health, Pirbright), Prof Dr 0. Papadopoulos (Faculty of Veterinary Medicine at the Aristotle University, Thessaloniki), Dr E. Paschaleri (Institute of Infectious and Parasitic Diseases, Thessaloniki), Dr F. Desimone and Dr E. Brocchi (Instituto Zooprofilattice Sperimentale della Lombardia e dell’Emilia, Bresciaj for providing isolates. This work was supported by the European Union (Contract no. AIR3-CT93-1465).
Appendix A This gel shows the amplification of RNA extracted from cell cultures infected with eight of the 31 EMCVs isolated from pigs in 1995 in Belgium using the described method. The expected bands of 286 bp are evident and now parasite bands can be observed. Lane 1, 100 bp ladder; lanes 2--9, EMCV isolates; lanes lo- 11, negative controls. 12
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Method.~ 66 (1997) 83-89
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