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RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease Molecular Epidemiology Group RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals* N.J. Knowles and A.R. Samuel Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom 6 December 1998 *Originally entitled: “RT-PCR and Sequencing Protocols for Molecular Epidemiology of Virus Diseases”, 12th September 1996 PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 1 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel CONTENTS Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Protocol 1: Extraction of Virus RNA using RNeasy Spin-Columns . . . . . . . . . . . . . . . . . . . 5 Protocol 2: Reverse Transcription of Virus RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Protocol 3: PCR Amplification of Reverse Transcribed RNA . . . . . . . . . . . . . . . . . . . . . . . . 7 Protocol 4: Agarose Gel Electrophoresis of PCR Products . . . . . . . . . . . . . . . . . . . . . . . . . 8 Protocol 5: Purification of PCR Products using Promega Wizard Preps™ . . . . . . . . . . . . . . 9 Protocol 6: End-Labelling of Oligonucleotide Primers using T4 Polynucleotide Kinase . . . 10 Protocol 7: Cycle Sequencing using the Promega f-mol® Kit . . . . . . . . . . . . . . . . . . . . . . . 11 Protocol 8: Polyacrylamide Gel Electrophoresis of Cycle Sequencing Products . . . . . . . . . 13 Sequence Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Specific Applications 1: Molecular Epidemiology of Foot-and-Mouth Disease types O, A, C and Asia 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Specific Applications 2: Molecular Epidemiology of Foot-and-Mouth Disease types SAT 1, SAT 2 and SAT 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Specific Applications 3: Molecular Epidemiology of Swine Vesicular Disease . . . . . . . . . . 22 Specific Applications 4: Molecular Epidemiology of Encephalomyocarditis Virus Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Appendix I: Reagents for Acrylamide Gel Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Appendix II: Polymerase Chain Reaction Amplification and Cycle Sequencing of the 1D (VP1) Gene of Foot-and-Mouth Disease Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Appendix III: Recent Developments in the Epidemiology of Foot-and-Mouth Disease: Genetic Relationships Determined by Nucleotide Sequence Analysis . . . . . . . . . . . . . . . . . . 35 PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 2 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel ACKNOWLEDGEMENTS We would like to thank the following staff members and visiting workers who have assisted in the development and application of the techniques described in this manual: David Ansell, Gang Zhang, Lin Fengsheng, Vladimir Drygin and Alexei Sherbakov. The technical assistance of Nicola Dickinson and Ginette Wilsden is also gratefully acknowledged. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 3 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel INTRODUCTION Figure 1 shows the strategy employed in the OIE/FAO World Reference Laboratory for Footand-Mouth Disease (WRLFMD) for the generation of sequence data for molecular epidemiological studies. Preferred route Sample arrives in WRLFMD Typing by ELISA Alternative route Preparation of 10% epithelium susp. Inoculation of 10% susp. onto cell cultures Typing by ELISA Molecular Epidemiology Group vRNA extraction Reverse transcription of vRNA PCR Analysis by agarose gel electorphoresis Wizard Prep purification of PCR amplicon Automated cycle sequencing Manual (radioactive) cycle sequencing Sequence reading and entry onto WRLFMD database Comparison with other sequences on database Production of phylogenetic tree using selected sequences Fig. 1. . Flow chart showing the RT-PCR/sequence analysis strategy employed by the WRLFMD Molecular Epidemiology Group. Subsequent chapters present the protocols used for RNA extraction, reverse transcription, polymerase chain reaction amplification and nucleotide sequencing. Additionally references (and abstracts, where available) are given in which the application of these techniques has been applied to the study of foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV) and encephalomyocarditis virus (EMCV). At the moment the chapter on sequence analysis only contains references to software which is of use for this work; this subject will be expanded upon in a later edition of this manual. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 4 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Protocol 1: Extraction of Virus RNA using RNeasy Spin-Columns 1. Put 460 µl of your sample into a 1.5 ml tube. Add an equal volume of Lysis buffer RLT (containing 1% 2-mercaptoethanol) to your sample and mix by vortexing. 2. Add 460 µl 70% ethanol and mix by vortexing. 3. Apply to RNeasy spin column (700 µl maximum loading volume). Spin in a microfuge for 15 sec at 10,000 rpm. Discard flowthrough and reuse collection tube. Repeat with remaining volume. 4. Wash with 700 µl wash buffer RW1. Centrifuge as before. 5. Wash with 500 µl wash buffer RPE. Centrifuge as above. 6. Repeat wash with 500 µl wash buffer RPE. Centrifuge at max speed for 2 min to dry membrane. 7. Elute RNA with 50 µl DEPC-H2O into a new clean collection tube. Spin in a microfuge for 60 sec at 10,000 rpm. 8. Store in clean tube at -20°C. Label the tube clearly with “RNA”, the virus name, today’s date and your name. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 5 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Protocol 2: Reverse Transcription of Virus RNA 1. In a sterile 0.75 ml eppendorf tube prepare the following mixture (depending on the number of RNAs to be reverse transcribed). Reagent No. of RNA preps 1 2 3 4 5 10 mM dNTPs 2.5* 5 7.5 10 12.5 5× RT Buffer (with DTT) 5 10 20 25 MMLV (200 U/µl) 0.5 1 1.5 2 2.5 RNasin (3.3 U/µl) 1 2 3 4 5 DEPC-H2O 1 2 3 4 5 Primer (25 pmol/µl) 1 2 3 4 5 Total 11 22 33 44 55 15 * amount in µl 2. Add 11 µl of the mixture to 14 µl of each RNA template giving at total volume of 25 µl. 3. Spin briefly in microfuge. 4. Heat at 42°C for 60 min and then at 94°C for 10 min (either in a waterbath or in the thermocycler). 5. Store at -20°C. Label the tube with “RT product”, the virus name, today’s date and your name. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 6 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Protocol 3: PCR Amplification of Reverse Transcribed RNA 1. Into a sterile 0.75 ml tube add the following. Take care to use sterile tips and avoid cross contamination.Use new tips for each reagent and template. Reagent Concentration Amount (µl) 25 mM MgCl2 1.5 mM 3 10 mM dNTPs 200 µM 1 1x 5 primer 1 (10-25 pmol/µl) 02.-0.5 pmol/µl 1 primer 2 (10-25 pmol/µl) 02.-0.5 pmol/µl 1 10× buffer Taq DNA polymerase (5 U/µl) 2.5 U RT product (template) 0.5 5 DEPC-H2O 33.5 Total volume: 50 2. Add 20 µl of mineral oil to the top of mixture, spin. 3. Run on Thermocycler as follows: Temperature Time No. of cycles 94°C 4 min 1 94°C 60 sec 55°C 60 sec 72°C 90 sec 72°C 5 min 30 1 4. Take 5 µl of PCR product and check it on an agarose gel. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 7 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Protocol 4: Agarose Gel Electrophoresis of PCR Products Note: Take care ethidium bromide is harmful, gloves should be worn at all times. 1. Prepare 60 ml of 2% agarose in 1x TBE buffer. 2. Either heat in microwave for ~2 min on full power or place in beaker of boiling water until melted. 3. Allow to cool to about 45°C and add 1 µl ethidium bromide (stock=5 mg/µl) per 10 ml, giving a final concentration of 0.5 µg/ml. This can be increased to 1 µg/ml if no ethidium bromide is added to the buffer (see below). 4. Pour gel and insert well former (comb). Allow to set on a flat surface for about 15 min. 5. Pour buffer 1x TBE (containing 0.5 µg/ml ethidium bromide, i.e. 1 µl of 5 mg/ml stock to every 10 ml of buffer) into tank and remove comb from gel. 6. Prepare samples in tubes, a multiwell plate or on parafilm. 1 µl loading buffer 5 µl PCR product 7. Prepare molecular weight marker. 0.5 µl molecular weight marker VI (Boehringer) 1 µl loading buffer 4.5 µl H2O 8. Load samples into the wells formed in the gel. It is often useful to load the molecular weight markers in both the first and last lanes. 9. Electrophorese at 100 volts for 20 min (minimum) or 10 volts overnight. 10. View and photograph the gel on an UV-transilluminator. Use UV-safety spectacles. 6x Loading Buffer PROTOCOL.WPD Reagent Final conc. Amount (µl) Glycerol 30% 300 10% bromophenol blue 0.25% 25 10% xylene cyanol 0.25% 25 H2O 650 Total 1000 WRL-FMD: Molecular Epidemiology Group Page 8 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Protocol 5: Purification of PCR Products using Promega Wizard Preps™ Note:- It is advisable to prepare all tubes/columns and syringes before use. 1. Transfer PCR reaction (after checking on a agarose gel) to a 1.5 ml tube (take care not to take up any mineral oil) . 2. a) Add 100 µl Direct Purification Buffer. b) Mix. 3. a) Add 1 ml of Resin. b) Mix 3 times over 1 min. 4. Prepare one Wizard Prep mini-column for each PCR product. a) Remove the plunger from a 2 ml disposable syringe. b) Attach syringe to mini-column (lock together). c) Rest on the top of a 1.5 ml tube which has had its lid removed. 5. a) Pipette the Resin/PCR mix into the syringe barrel (from step 3). b) Insert the syringe plunger slowly and push the mix into the mini-column, letting liquid go to waste. 6. a) Remove syringe from mini-column and remove plunger (this is to stop you pulling the mix back out of the mini-column). b) Reattach syringe to the mini-column. c) Pipette 2 ml of 80% isopropanol into the syringe. d) Insert the syringe plunger and push the isopropanol through the mini-column to waste. 7. Remove the syringe and centrifuge for 20 sec at 12,000 rpm to dry resin. 8. a) b) c) d) Transfer mini-column to new 1.5 ml tube. Add 50 µl DEPC-H2O. Leave for 1+ min (30 min maximum). Microfuge for 20 sec at 12,000 rpm. 9. Store -20oC in 0.75 ml tube. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 9 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Protocol 6: End-Labelling of Oligonucleotide Primers using T4 Polynucleotide Kinase 1. Prepare the following mixture in an eppendorf tube: Reagent Amount (µl) Primer (24 pmol/µl) 1 32 5 P -ATP (1.85 MBq) 10x PNK Buffer 3 T4 polynucleotide kinase (10 U/µl) 2 DEPC-H2O 19 Total 30 2. Spin briefly in a microfuge and incubate as indicated below: Temperature Time 37°C 30-60 min 90°C 5 min 3. Use 1.2 pmol of primer (1.5 µl) per sample (i.e. set of four reactions) in the cycle sequencing protocol. The above is enough for about 20 samples. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 10 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Protocol 7: Cycle Sequencing using the Promega f-mol® Kit 1. a) Prepare one set of 4 tubes per isolate to be sequenced, label tubes T, C, G and A. A thin walled 96 well plate may also be used. b) Add 1 µl of the appropriate dd/dNTPs into each tube/well (in the order of T, C, G, A) c) Spin briefly in microfuge. d) Store at 4°C until needed (see part 4). 2. For each set of reactions add in a separate tube the following:Reagent Amount (µl) Template DNA (Wizard Prep™ purified)* 3 5 10 5x sequencing buffer 4 4 4 32 1.5 1.5 1.5 Sequencing grade Taq polymerase (5 U/µl) 1 1 1 P -ATP labelled primer DEPC-H2O 10.5 8.5 3.5 Total 20 20 20 * the amount of template used will depend on band intensity/conc, vary volume of water accordingly. 3. Mix and spin 4. a) Add 4 µl of mixture to each dd/dNTP tube/well previously prepared. b) Spin down (use a flat bed centrifuge if using a 96 well plate, 1000 rpm maximum) 5. Add a drop of mineral oil on top of reaction mixture. 6. Put onto pre-heated thermal cycler at 94°C. Running conditions: 94°C 2 min 92°C 1 min 55°C 1 min 72°C 1 min 30 sec 20°C. Hold 1 cycle 30 cycles 1 cycle 7. a) Add 4 µl of stop solution (see table below) to each tube/well. b) Spin down. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 11 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel 8. Heat at 80-90°C for 2-5 min before running on a gel. 9. Load 2.5-3.5 µl into each well. Fmol® Sequencing Stop Solution Reagent Final conc. Amount (µl) Formamide 95% 950 2 M NaOH 10 mM 5 10% Bromophenol Blue 0.05% 5 10% Xylene Cyanol FF 0.05% 5 DEPC-H2O Total PROTOCOL.WPD 35 1000 WRL-FMD: Molecular Epidemiology Group Page 12 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Protocol 8: Polyacrylamide Gel Electrophoresis of Cycle Sequencing Products 1. a. Carefully clean a set of gel plates with a detergent that does not leave a residue. b. Wash with distilled water and then wipe clean with ethanol. 2. Coat one side of the SMALL plate with repelcote. If possible, mark the plate the first time it is used with a diamond-tipped pen and always repelcote the same side. 3. Put the plates together with the repelcoted side on the inside and the spacers at either side. 4. Check that the sharkstooth combs fit tightly between the plates and then tape the plates on either side and at the bottom. 5. Mix gel solution: (note: wear gloves - acrylamide is dangerous) - 125 ml 0.5x TBE gel mix - 250 µl TEMED - 250 µl 25% ammonium persulphate (AMPS) Note: the gel will start setting within about 5 min of the addition of the AMPS. If using Sequagel use 100 ml and 800 µl 10% AMPS. 6. Pour the gel solution between the glass plates using a 50 ml syringe. 7. Insert combs upside down (i.e. flat side next to gel and nearly as far in as the holes in the comb). 8. Clamp tightly using clips. 9. Leave to set for at least 1 hour. The gel can be stored at 4°C overnight if the top is kept moist with 1x TBE and covered with cling film. 10. Pre-running the gel: - remove the combs carefully - the teeth break easily. - remove the tape. - rinse the top of the gel with distilled water or 1x TBE. - Insert combs with sharks tooths forming wells (with points just touching top of gel). - Put into gel tank and clamp tightly. 11. Prepare 1000 ml 1x TBE and pour into top and bottom tanks. 12. Wash urea out of wells with syringe and bent needle and pre-run the gel at a constant 70 watts for 1 hour. 2 µl of formamide dyes may be loaded in every other well prior to prerunning to see if any leakage from well-to-well is occurring. 13. Heat the samples at 95-100°C for 2 min before just before loading. 12. Wash urea out of wells with a 50 ml syringe and bent needle IMMEDIATELY prior to loading samples. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 13 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel 14. Add 3 µl of each sample into the wells formed by the combs following the order T, C, G, A. 15. Run at a constant 70 watts for 2 hours (the bromophenol blue marker should just run of the end of the gel into the buffer). The samples can also be run of 4 hours. 16. Remove the plates from the gel tank, carefully remove the combs and split the glass plates apart. The gel should remain attached to the larger glass plate! 17. Optionally fix in at tray of 10% acetic acid, 10% ethanol, 80% water for 20-30 min. 18. Remove from fixative and lay a piece of 3MM filter paper over the gel. Carefully peel off with the gel sticking to the 3MM paper. 19. Dry down for 45 min to 1.5 hours on slab gel dryer set to heat at 80-85oC. 20. Mark one corner of the dried gel (e.g. by cutting off a corner). Expose gel to X-ray film in a light tight cassette at room temperature (cut off the corresponding corner of the X-ray film). If using an intensifying screen store at -70°C. Exposure times usually range from 18-48 h with an intensifying screen and 18-60 h without, depending on the activity of the radioisotope. 21. Develop autoradiograph and immediately label with the date, order of loading (i.e. TCGA) and the name of each sample. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 14 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Sequence Analysis At the moment the chapter on sequence analysis only contains references to software which is of use for this work; this subject will be expanded upon in a later edition of this manual. Software packages for the PC which are useful for sequence analysis EpiSeq for DOS EpiSeq for Windows PHYLIP ClustalW ClustalX GeneDoc TreeView Neighbor-joining algorithm UPGMA algorithm Bootstrap re-sampling PROTOCOL.WPD Knowles, N.J. (1991-98). EpiSeq for DOS: a suite of programs for epidemiological sequencing. Unpublished. [Availability limited] Knowles, N.J. (1994-98). EpiSeq for Windows: a suite of programs for epidemiological sequencing. Unpublished. [Availability limited] Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, 1993. http://evolution.genetics.washington.edu/phylip.html Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680. http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/clustalw.html Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876-4882. http://www-igbmc.u-strasbg.fr/BioInfo/ClustalX/ Nicholas, K.B. and Nicholas, H.B. Jr. (1996). GeneDoc: a tool for editing and annotating multiple sequence alignments. Distributed by the author. http://www.cris.com/~ketchup/genedoc.shtml Page, R.D.M. (1996). TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: 357-358. http://taxonomy.zoology.gla.ac.uk/rod/treeview.html Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406425. Prager, E.M. and Wilson, A.C. (1978). Construction of phylogenetic trees for proteins and nucleic acids: empirical evaluation of alternative matrix methods. Journal of Molecular Evolution 11: 129-142. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791. Efron, B., Halloran, E. and Holmes, S. (1996). Bootstrap confidence levels for phylogenetic trees. Proceedings of the National Academy of Sciences USA 93: 13429-13434. WRL-FMD: Molecular Epidemiology Group Page 15 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel SPECIFIC APPLICATIONS 1: MOLECULAR EPIDEMIOLOGY OF FOOT-ANDMOUTH DISEASE TYPES O, A, C AND ASIA 1 Please refer to the following papers (and references therein) for examples of work on this subject: Beck, E. and Strohmaier, K. (1987). Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. Journal of Virology 61: 1621-1629. Abstract: The VP1-coding regions of foot-and-mouth disease virus strains from 18 recent European outbreaks and of 9 strains isolated more than 20 years ago and used in part as vaccines were determined by direct cDNA sequencing. Comparison of the sequences revealed that most of the isolated outbreak viruses are closely related to the vaccine strains used. Isolates from the Italian epizootic of 1984 to 1985 correspond, for example, to the vaccine strain A5 Parma 62; the outbreak in 1984 in Bernbeuren, Federal Republic of Germany, was induced by A5 Allier 60; outbreaks in 1982 in Funen, Denmark, and in Murchin, German Democratic Republic, were caused by O1 Lausanne 65. Viruses isolated during the 1983 Iberian epizootic show a close relationship to the vaccine strain A5 Allier 60 but were probably derived from another not yet identified vaccine strain from Spain. Only two minor outbreaks in the Federal Republic of Germany, A Aachen in 1976 and O Wuppertal in 1982, did not correspond to the classical European strains but were obviously introduced from outside. We suggest that nucleotide sequence analysis should be used as a standard method of diagnosis, because when compared with other techniques it more clearly reveals the origin and course of epizootics and offers the possibility of preventing further outbreaks. Samuel, A.R., Knowles, N.J. and Kitching, R.P. (1988). Serological and biochemical analysis of some recent type A foot-and-mouth disease virus isolates from the Middle East. Epidemiology and Infection 101: 577-590. Abstract: In 1986 and 1987 foot-and-mouth disease virus (FMDV) serotype A was isolated from outbreaks of disease in Saudi Arabia and Iran. Selected virus isolates were antigenically distinct from the prototype A22 virus strain (A22/Iraq/64), but were serologically related to each other. However, polyacrylamide gel electrophoresis showed that whilst the respective Saudi Arabian structural polypeptides were homogeneous, those from an Iran isolate were distinct. Direct sequencing of part of the P-1D (VP1) gene demonstrated considerable difference in nucleotide homology between the two groups of viruses; the Saudi Arabian viruses were closely related to each other but only distantly related to both the A22 prototype virus strain and the Iranian virus isolate. The latter viruses were only slightly more closely related to each other. Thus there appeared to be at least two distinct FMDV type A variants co-circulating in the Middle East, both of which differed considerably from the classical A22 subtype. Martínez, M.A., Dopazo, J., Hernández, J., Mateu, M.G., Sobrino, F., Domingo, E. and Knowles, N.J. (1992). Evolution of the capsid protein genes of foot-and-mouth disease virus. Antigenic variation without accumulation of amino acid substitutions over six decades. Journal of Virology 66: 3557-3565. Abstract: The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 16 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time. Samuel, A.R., Ansell, D.M., Rendle, R.T., Armstrong, R.M., Davidson, F.L., Knowles, N.J. and Kitching, R.P. (1993). Field and laboratory analysis of an outbreak of foot-and-mouth disease in Bulgaria in 1991. Revue scientifique et technique de l’Office International des Epizooties 12: 839-848. Abstract: In July 1991, an outbreak of foot and mouth disease (FMD) occurred near Stefan Karadjovo village in Boliarovo (south-east Bulgaria, close to the Turkish border). The virus isolated was identified in Bulgaria as serotype O and this was subsequently confirmed by the World Reference Laboratory for Foot and Mouth Disease in Pirbright (United Kingdom). Serological studies using bovine sera and monoclonal antibody analysis were made. In addition, the sequence of approximately 170 nucleotides at the 3’ end of the 1D gene was determined for the field isolate and for vaccine strains used in Bulgaria. These were compared with other sequences of type O FMD viruses from outbreaks in the Middle East. Serum samples were taken from domestic animals in the region close to the outbreak and examined for anti-FMD virus antibodies to assess the extent (if any) of spread of the virus before or after the outbreak. No evidence of infection was found in these animals. The virus involved in the Bulgarian outbreak was antigenically similar to the O1 vaccine strains but probably did not originate from these strains. The virus was closely related genetically to a group of viruses isolated in the Middle East since 1987, suggesting that it may have been introduced into Bulgaria from an area in the Middle East by unidentified means. Ansell, D.M., Samuel, A.R., Carpenter, W.C. and Knowles, N.J. (1994). Genetic relationships between foot-and-mouth disease type Asia 1 viruses. Epidemiology and Infection 112: 213224. Abstract: The sequence of 165 nucleotides at the 3’ end of the 1D (VP1) gene of foot-and-mouth disease (FMD) virus was determined for 44 type Asia 1 strains isolated from throughout Asia between 1954-92. Analysis of the relationships between the virus genomes showed epidemiological links not previously evident. The possible origin of the only outbreak of FMD Asia 1 to have occurred in Europe, in Greece in 1984, was identified because the nucleotide sequence of this virus was closely-related to the sequences of those present in the Middle East between 1983-5. Variation in the region sequenced was not as great as that seen in the other FMDV serotypes and all viruses shared greater than 85% nucleotide identity. Thus all the virus isolates examined were considered to belong to a single genotype. A database of Asia 1 virus sequences has been established which will facilitate the rapid analysis of new outbreaks strains. Armstrong, R.M., Samuel, A.R., Carpenter, W.C., Rama Kant and Knowles, N.J. (1994). A comparative study of serological and biochemical methods for strain differentiation of foot-and-mouth disease type A viruses. Veterinary Microbiology 39: 285-298. Abstract: Three serological and three biochemical methods were used to compare five field isolates of foot-and-mouth disease virus (FMDV) from Western India with nine reference vaccine strains and five field isolates from other countries. The serological tests (liquid-phase ELISA and virus neutralization) were able to distinguish between the three reference vaccine strains examined, but the five Indian field isolates reacted poorly with antisera produced against these vaccine strains. Analysis of monoclonal antibody (mAb) data was difficult to interpret although clearly the field isolate A/IND/5/87 reacted to a lesser extent with one of the mAb panels (A 10/Holland/42) than the other four Indian isolates. The A22/Iraq/24/64 mAbs did not react with any of the Indian field isolates and only significantly with one of the reference vaccine strains, A/IND/57/79. Polyacrylamide gel electrophoresis distinguished the reference vaccine strains from each other and from the field isolates. Additionally, one of the Indian isolates (A/IND/5/87) could be differentiated from the other four. Electrofocusing showed similarities between the reference vaccine strain A22/Iraq/24/64 and three of the Indian field isolates (A/IND/1/87, A/IND/2/87 and A/IND/3/87), however, A/IND/4/87 and A/IND/5/87 were distinct. Nucleotide sequencing showed that the isolates A/IND/1/87, A/IND/2/87 and A/IND/3/87 were very closely related to each other and related to A/IND/4/87, however, A/IND/5/87 was different. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 17 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Samuel, A.R., Knowles, N.J., Kitching, R.P. and Hafez, S.M. (1997). Molecular analysis of type O foot-and-mouth disease viruses isolated in Saudi Arabia between 1983 and 1995. Epidemiology and Infection 119: 381-389. Abstract: Partial nucleotide sequence of the capsid polypeptide coding gene ID (VP1) was determined for 68 serotype O foot-and-mouth disease viruses isolated between 1983 and 1995 from outbreaks occurring in Saudi Arabia. The sequences were compared with previously published sequences: 14 viruses of Middle Eastern origin (isolated between 1987 and 1991); and with four vaccine virus strain sequences, three originating from the Middle East (O1/Turkey/Manisa/69, O1/Sharquia/Egypt/72 and O1/Israel/2/85) and one from Europe (O1/BFS 1860/UK/67). The virus isolates from Saudi Arabia and the Middle East vaccine virus strains formed a related genetic group distinct from the European O1 virus. Within this large group 12 distinct genetic sublineages were observed. Pattnaik, B., Venkataramanan, R., Tosh, C., Sanyal, A., Hemadri, D., Samuel, A.R., Knowles, N.J. and Kitching, R.P. (1998). Genetic heterogeneity of Indian field isolates of foot-andmouth disease virus serotype O as revealed by partial sequencing of 1D gene. Virus Research 55: 115-127. Abstract: The sequence of 165 nucleotides at the 3’ end of the ID gene, determined from RT-PCR amplified cDNA fragments, of 25 type O strains isolated from different parts/regions of India during 1987-1995 and the vaccine strain (R2/75) currently in use in India were subjected to phylogenetic analysis. One isolate from the neighbouring country Nepal was also included in the study. The virus/field strains showed high degree of genetic heterogeneity among themselves with % divergence in nucleotide sequence ranging from 1.2 to 19.4%. The Indian strains were much away (13.3-20.6%) from the exotic type O strains of O1 BFS, O1 K, and O1 Campos. The type O strains-analyzed were classified into three genotypes basing on level of divergence observed in nucleotide sequence. The type O vaccine virus (R2/75) was > 7% divergent (7.3- 15.2%) from the field strains which revealed significant (> 5%) genetic heterogeneity between the two. The phylogenetic analysis identified three distinct lineages, viz., (i) lineage 1 represented by the exotic strains, (ii) lineage 2 represented by 25 of the field strains which clustered into seven subgroups/sublines (2a-2g), and (iii) lineage 3 represented by a unique field isolate which shared the branching/origin with the vaccine strain. The lineage 2 which comprised of 25 of the 26 type O field strains analyzed, was placed almost at equidistance from the lineages 1 and 3 in the phylogenetic tree. The vaccine strain was closer to the viruses in lineage 2. Though there was no specific distribution pattern of sequences in different geographical regions of Indla, the viruses/sequences in subgroup 2f appeared to be restricted to the southern states. Comparison of deduced amino acid sequence in the immunodominant regions 133-160 and 200-208 of the 1D gene product (VP1) showed that the two viruses in lineage 3 had unique amino acid residues at the positions 138 (D), 139 (G), 144 (I), and 158 (A) compared to rest of the strains including the exotic ones. Comparison of amino acid residues at critical positions 144, 148, 149, 151, 153, 154, and 208 revealed similarity between the type O strains analyzed. The virus strains showed variation (V/L/I) at position 144. One field strain showed replacement from Q149 - E and another from P208 - L. Thus, the study revealed that the type O FMD virus populations circulating in India and causing disease outbreaks are genetically much heterogenous but related at the immunodominant region of VP1 polypeptide, and there are more than one genetically distinct virus populations in almost every region of the country which is possible due to unrestricted animal movement in the country. The involvement of vaccine virus in disease outbreaks was ruled out as the field strains (excluding the one in lineage 3) were phylogenetically distinct from it. Knowles, N.J. and Samuel, A.R. (1998). Molecular techniques in foot-and-mouth disease epidemiology. Towards livestock disease diagnosis and control in the 21st century: proceedings of an international symposium on diagnosis and control of livestock diseases jointly organized by the International Atomic Energy Agency and the Food and Agriculture Organization of the United Nations, held in Vienna, 7-11 April 1997. Vienna: International Atomic Energy Agency, p. 185-201. Abstract: The study of the epidemiology of FMD has been revolutionized by the introduction of molecular biological techniques which can establish genetic relationships between the causative viruses. Early biochemical techniques such as SDS-polyacylamide gel electrophoresis (PAGE), electrofocusing and ribonuclease T1 PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 18 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel oligonucleotide mapping were used to augment traditional antigenic comparisons to relate different FMDV isolates and strains. We have been studying FMD epidemiology using nucleotide sequencing since 1987 and have accumulated a database of nearly 1500 partial VP1 sequences representing all seven serotypes of the virus. This has put us in a unique position to study the global epidemiology of the disease. Our studies have shown that FMD viruses may be grouped into genetic types which correlate with geographical location. We have proposed that these geographically distinct genotypes be termed ‘topotypes’. For FMD type O at least six topotypes have been defined, one of which is probably now extinct; for type A four topotypes have so far been identified; for type C about six genotypes and for Asia 1 only one genotype. Studies on the SAT 1 and SAT 3 serotypes in southern Africa have shown the presence of three distinct topotypes for each. These have probably arisen through the geographic isolation of wild buffalo herds and multiple introductions into domesticated cattle. The situation with the SAT 2 serotype was, however, different; only two genotypes were found, which did not correlate with geographical origin. Samuel, A.R., Knowles, N.J. and Mackay, D.K.J. (1998). Genetic analysis of type O viruses responsible for epidemics of foot-and-mouth disease in North Africa. Epidemiology and Infection, in press. Abstract: The nucleotide sequences of the 3' end of the capsid-coding region were determined for 30 serotype O foot-and-mouth disease (FMD) viruses isolated between 1987 and 1994 from outbreaks in North Africa and the Middle East. These sequences were compared with the previously published sequences of nine field virus isolates from the Middle East and five vaccine virus strains, three of which originated from the Middle East (O 1/Turkey/Manisa/69, O1/Sharquia/Egypt/72 and O1/Israel/2/85) and two from Europe (O1/Lausanne/Switzerland/65 and O2/Brescia/Italy/47). Cluster analysis of these sequences using the unweighted pair group mean average (UPGMA) method showed i) that the FMD viruses isolated from North Africa and the Middle East were very different from the classical European vaccine strains; ii) that all the viruses isolated during the 1989-92 North African epidemic formed a cluster differing by no more than 6% from each other; iii) a virus isolated in Libya in 1988 was unrelated to the aforementioned epidemic; and iv) viruses from a second, less extensive epidemic, occurring in 1994, fell into yet another cluster. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 19 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel SPECIFIC APPLICATIONS 2: MOLECULAR EPIDEMIOLOGY OF FOOT-ANDMOUTH DISEASE TYPES SAT 1, SAT 2 AND SAT 3 Note: The primers set used for the RT-PCR amplification of the South African Territories (SAT) serotypes of FMDV is as follows (N.J. Knowles, unpublished data): Name Primer sequence (5’ to 3’) SAT-1D209F CCACATACTACTTTTGTGACCTGGA FMD-2B208R ACAGCGGCCATGCACGACAG NK72 GAAGGGCCCAGGGTTGGACTC Location Use Gene Nt. VP1 209-234 Forward primer 2B 208-227 Reverse primer 2A/2B 34-48/1-6 Sequencing primer This primer set works for all three SAT serotypes and results in a PCR products of approximately 730 bp, 715 bp and 718 bp for SAT 1, SAT 2 and SAT 3, respectively. Please refer to the following papers (and references therein) for examples of work on this subject: Vosloo, W., Knowles, N.J. and Thomson, G.R. (1992). Genetic relationships between southern African SAT-2 isolates of foot-and-mouth disease virus. Epidemiology and Infection 109: 547-558. Abstract: Sequencing of part of the 1D gene of foot-and-mouth disease virus was used to determine the relationships between SAT-2 viruses isolated from outbreaks which occurred in cattle in Zimbabwe and Namibia and in impala in South Africa between 1979 and 1989. The results demonstrated that the outbreaks in different countries were unrelated. Surprisingly close relationships were shown between all SAT-2 viruses isolated from cattle in Zimbabwe since 1983 but the two major epizootics which occurred in 1989 were caused by viruses which were clearly different. Conversely, two apparently unrelated outbreaks in impala in South Africa were caused by viruses which could not be distinguished. Dawe, P.S., Flanagan, F.O, Madekurozwa, R.L., Sorensen, K.J., Anderson, E.C., Foggin, C.M., Ferris, N.P. and Knowles, N.J. (1994). Natural transmission of foot-and-mouth disease virus from African buffalo (Syncerus caffer) to cattle in a wildlife area of Zimbabwe. Veterinary Record 134: 230-232. Abstract: An outbreak of foot-and-mouth disease (FMD) occurred during April 1991 in a trypanosomiasis sentinel cattle herd by the Rifa River to the east of Lake Kariba, Zimbabwe. Despite the cattle having been vaccinated biannually for the previous five years the disease was severe. The viruses isolated from the affected animals were typed as FMD virus type SAT 1. Free-living African buffalo (Syncerus caffer) which had been using the same watering place as the affected cattle were sampled and FMD type SAT 1 virus was isolated. Partial nucleotide sequencing of the gene coding for the capsid protein 1D (VP1) of one of the viruses isolated from cattle and two of the viruses isolated from buffalo demonstrated a close relationship between the three viruses. Since no other cattle were present in the area and no outbreaks of SAT 1 had occurred in Zimbabwe since 1989, it was concluded that the disease had been transmitted from buffalo to cattle. Vosloo, W., Kirkbride, E., Bengis, R.G., Keet, D.F. and Thompson, G.R. (1995). Genome variation in the SAT types of foot-and-mouth disease viruses prevalent in buffalo (Syncerus caffer) in the Kruger National Park and other regions of southern Africa, 1986-93. Epidemiology and Infection 114: 203-218. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 20 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Abstract: Dideoxy nucleotide sequencing of a portion of the 1D gene of SAT-type foot-and-mouth disease viruses (FMDV) was used to derive phylogenetic relationships between viruses recovered from the oesophageo-pharyngeal secretions of buffalo in the Kruger National Park as well as several other wildlife areas in southern Africa. The three serotypes differed from one another by more than 40% while intratypic variation did not exceed 29%. Within each type, isolates from particular countries were more closely related to one another than to isolates from other countries lending credence to previous observations that FMDV evolve independently in different regions of the subcontinent. Vosloo, W., Bastos, A.D., Kirkbride, E., Esterhuysen, J.J., Rensburg, D.J. Van, Bengis, R.G., Keet, D.W and Thomson, G.R. (1996). Persistent infection of African buffalo (Syncerus caffer) with SAT-type foot-and-mouth disease viruses: rate of fixation of mutations, antigenic change and interspecies transmission. Journal of General Virology 77: 1457-1467. Abstract: Transmission of a plaque-purified SAT-2 foot-and-mouth disease virus (FMDV) occurred erratically from artificially infected African buffaloes in captivity to susceptible buffaloes and cattle in the same enclosure; in some instances transmission occurred only after contact between persistently infected carriers and susceptible animals lasting a number of months. Because the rate at which FMDV mutations accumulated in persistently infected buffaloes was approximately linear (1.64% nucleotide substitutions per year over the region of the 1D gene sequenced), both buffaloes and cattle that became infected some months after the start of the experiment were infected with viruses that differed from the original clone. The nucleotide differences were reflected in significant antigenic change. A SAT-1 FMDV from a separate experiment inadvertently infected some of the buffalo in the SAT-2 experiment. The SAT-1 FMDV also accumulated mutations at a constant rate in individual buffaloes (1.54% nucleotide changes per year) but the resultant antigenic variation was less than for SAT-2. It is concluded that persistently infected buffaloes in the wild constantly generate variants of SAT-1 and SAT-2 which explains the wide range of genomic and antigenic variants which occur in SAT-1 and SAT-2 viruses in southern Africa. Keet, D.F., Hunter, P., Bengis, R.G., Bastos, A. and Thomson, G.R. (1996). The 1992 foot-and-mouth disease epizootic in the Kruger National Park. Journal of the South African Veterinary Association 67: 83-87. Abstract: The monitoring of a foot-and-mouth disease epizootic amongst impala (Aepyceros melampus) in the Kruger National Park is described. Infection rates of different sex and age classes of impala within the outbreak focus were determined. Seroprevalence rates in other cloven-hoofed species were also determined. RNA sequencing of a portion of the 1D gene of viruses isolated from SAT-2 viruses obtained from diseased impala showed that they were unrelated to previous SAT-2 isolates made from animals in the Kruger National Park. Bastos, A.D.S. (1998). Detection and characterization of foot-and-mouth disease virus in sub-Saharan Africa. Onderstepoort Journal of Veterinary Research 65: 37-47. Abstract: Genomic amplification of the VP1 gene of SAT-type foot-and-mouth disease virus (FMDV) was performed with published and novel oligonucleotide primers. The primer pair with the highest SAT-type recognition (67%) was identified and selected for optimization. Modifications to primers significantly improved SAT-type detection (100%), broadened the recognition range to European (A, O and C) and Asian (Asia-1) serotypes and improved test sensitivity. In addition to being able to confirm the presence of FMDV in a clinical specimen within 6 h of receipt, the PCR product, which is amenable to nucleotide sequencing, enables genetic characterization of viruses into serotype and topotype within 48 h. VP1 gene sequence analysis of isolates from seven African countries and representative of five of the six serotypes occurring on the continent, revealed that SAT-types have the highest levels of intratypic variation. Intratypic variation for the SAT-types ranged from 34-40,4% on nucleotide level, and from 24,1-27,5% on amino acid level. In addition, the methodology presented here was shown to be useful for determining the origin and tracing the course of epizootics in both wild and domestic cloven-hoofed animals. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 21 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel SPECIFIC APPLICATIONS 3: MOLECULAR EPIDEMIOLOGY OF SWINE VESICULAR DISEASE Please refer to the following papers (and references therein) for examples of work on this subject: Brocchi, E., Zhang, G., Knowles, N.J., Wilsden, G., McCauley, J.W., Marquardt, O., Ohlinger, V.F. and De Simone, F. (1997). Molecular epidemiology of recent outbreaks of swine vesicular disease: two genetically and antigenically distinct variants in Europe, 1987-1994. Epidemiology and Infection 118: 51-61. Abstract: Viruses from the recent epidemic of swine vesicular disease (SVD) in Europe have been isolated and characterized by antigenic and genetic methods to examine the likely epidemiological origins of the disease. Antigenic analysis was performed on 77 SVD viruses (SVDV) isolated in Europe between 1966 and 1994 using two panels of monoclonal antibodies (MAb) in a trapping ELISA. Genetic analysis of 33 of the SVD viruses by reverse transcription-polymerase chain-reaction (RT-PCR) amplification and nucleotide sequencing of the ID (VP1) coding region was also performed. Comparison of the nucleotide sequences with each other and with three other previously published SVDV sequences revealed four distinct groups which correlated exactly with the results of the pattern of reactivity with MAbs. The first group consisted solely of the earliest SVD virus isolated (ITL/1/66) while the second group comprised viruses present in Europe and Japan between 1972 and 1981. The third group consisted of viruses isolated from outbreaks of SVD in Italy between December 1988 and June 1992. Viruses isolated between 1987 and 1994 from Romania, the Netherlands, Italy and Spain formed a fourth group. The genetic and antigenic similarity of the most recent virus isolates from Western Europe to a virus isolated in Romania 5 years previously suggests that the possible origin of the recent epidemic of swine vesicular disease in Western Europe was in Eastern Europe. Zhang, G., Haydon, D.T., Knowles, N.J., McCauley, J.W. (1999). Molecular evolution of swine vesicular disease virus. Journal of General Virology, in press. Abstract: Phylogenetic analysis was used to examine the evolutionary relationships within a group of coxsackie B viruses that contained representatives of the major serotypes of this group and 45 isolates of swine vesicular disease virus (SVDV) from Asia and Europe. Separate analyses of sequence data from two regions of the viral genomes encoding the VP1 and 3BC genes both revealed the SVDVs to belong to a single monophyletic group, clearly distinguished from all other sampled coxsackie viruses. Regression analysis revealed that within the SVDV clade at least 80% of synonymous variation in evolutionary divergence between isolates was explained by time, indicating the existence of an approximate molecular clock. Calibration of this clock according to synonymous substitutions per year indicated the date of occurrence of a common ancestor for the SVDV clade to be between 1945 and 1965. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 22 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel SPECIFIC APPLICATIONS 4: MOLECULAR EPIDEMIOLOGY OF ENCEPHALOMYOCARDITIS VIRUS INFECTIONS Please refer to the following papers (and references therein) for examples of work on this subject: Koenen, F., Vanderhallen, H., Papadopoulos, O., Billinis, C., Paschaleri-Papadopoulou, E., Brocchi, E., De Simone, F., Carra, E. and Knowles, N.J. (1997). Comparison of the pathogenic, antigenic and molecular characteristics of two encephalomyocarditis virus (EMCV) isolates from Belgium and Greece. Research in Veterinary Science 62: 239-244. Abstract: The pathogenicity of two porcine encephalomyocarditis virus (EMCV) isolates for sows in gestation and young piglets was studied. One virus originated from a case of reproductive failure in pigs in Belgium and the other from a case of acute myocarditis in pigs in Greece. Sows in the mid-gestation period and one- to two-month old piglets were inoculated with each isolate. The molecular relationship between both isolates was studied by determining the nucleotide sequence located across the junction of the 1C and 1D capsid-coding genes. Antigenic analysis was performed using a panel of 35 monoclonal antibodies raised against an Italian field isolate of EMCV. All three approaches revealed differences between both isolates and also confirmed that there was no link between the two outbreaks of disease. Knowles, N.J., Dickinson, N.D., Wilsden, G., Carra, E., Brocchi, E. and De Simone, F. (1998). Molecular analysis of encephalomyocarditis viruses isolated from pigs and rodents in Italy. Virus Research 57: 53-62. Abstract: Partial nucleotide sequences of encephalomyocarditis (EMC) viruses isolated from five, apparently independent, outbreaks of fatal myocarditis in pigs in Italy were compared with three EMC viruses isolated from wild rodents from a different geographic region in the same country. These viruses were also compared with EMC viruses isolated from pigs in other European countries and three historical strains. All the Italian EMC viruses were closely related (> 94.6% nucleotide identity), but were distinct from viruses occurring in Belgium in 1991 (< 80.5% nucleotide identity), Greece in 1990 (< 83.3% nucleotide identity) and the three older viruses (<82.9% nucleotide identity). An EMC virus isolated from pigs in the Netherlands in 1988, was closely related to the Italian viruses (95.3-99.3% nucleotide identity). It is suggested that pigs may play a role in the movement of EMC viruses between different geographic regions. Koenen, F., Vanderhallen, H., Dickinson, N.D. and Knowles, N.J. (1999). Phylogenetic analysis of European encephalomyocarditis viruses: comparison of two genomic regions. Archives of Virology, in press. Abstract: The phylogenetic relationships of encephalomyocarditis (EMC) viruses isolated from pigs and rodents in Europe were determined by comparison of nucleotide sequences from two different regions of the virus genome, the VP3/VP1 gene junction (part of the capsid-coding region) and part of the 3D polymerase-coding region. Thirty-five European EMC viruses could be divided into two genetic groups, one which contained viruses from Greece isolated between 1986 and 1997 and from Belgium in 1991 and the other which contained viruses from Italy (1986-1996), Cyprus (1994-1995), France (1995) and Belgium (1995-1996). PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 23 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Appendix I: Reagents for Acrylamide Gel Fractionation 10x TBE (Tris-borate-EDTA) buffer 432 g Tris base 220 g Boric acid 37.2 g EDTA make up to 4 litres with water titrate to pH 8.3 with conc. HCl Store at room temperature. 40% stock acrylamide solution 380 g Acrylamide 20 g Bis-acrylamide make up to 1 litre with water. Stir with 20 g Amberlite MB-1, filter off, store at 4°C. 0.5x TBE gel mix 150 ml 40% stock acrylamide solution 50 ml 10x TBE 460 g urea make up to 1 litre with water, store at 4°C. Final preparation of acrylamide gel solution For a 0.4 mm gel prepare: 50 ml 0.5x TBE gel solution 100 µl TEMED 100 µl 25% ammonium persulphate (AMPS; after adding AMPS gel sets in ~5-10 min). Mix well. For a 0.4-1.2mm wedge gel prepare: 125 ml 0.5x TBE gel solution 250 µl TEMED 250 µl 25% AMPS (after adding AMPS gel sets in ~5-10 min). Mix well. Commercially available Sequagel 80 ml gel solution 20 ml buffer solution 800 µl 10% AMPS (after adding AMPS gel sets in ~5-10 min). Mix well. Important note: the ambient temperature of the laboratory and the temperature used to store reagents will affect the rapidity of gel polymerisation, i.e. it will proceed more rapidly at higher temperatures. The amounts of gelling reagents (TEMED and AMPS) given above are for ambient PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 24 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel temperatures of approx. 20-22°C. For lower or higher temperature the amounts of AMPS and/or TEMED may be lowered or raised, respectively. Any reagents which are stored at 4°C should be allowed to equilibrate to ambient temperature before use. If you are unsure make up small volumes of gel solution and add varying amounts of gelling reagents. The gel should not set in less than 5 min and not take longer than 15 min. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 25 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Appendix II: Polymerase Chain Reaction Amplification and Cycle Sequencing of the 1D (VP1) Gene of Foot-and-Mouth Disease Viruses. Paper presented at the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Vienna, Austria, 19-22 September, 1994. POLYMERASE CHAIN REACTION AMPLIFICATION AND CYCLE SEQUENCING OF THE 1D (VP1) GENE OF FOOT-AND-MOUTH DISEASE VIRUSES N.J. Knowles and A.R. Samuel World and Community Reference Laboratory for Foot-and-Mouth Disease, Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom. Since 1987 the molecular epizootiology of foot-and-mouth disease (FMD) has largely been based on the comparison of genetic distances between virus isolates. Many molecular epizootiological studies have been published, or are awaiting publication, and include examples of all seven FMDV serotypes, viz. type O (Beck and Strohmaier, 1987; Knowles et al., 1988; Marquardt and Adam, 1989; Samuel et al., 1990a, b; Krebs et al., 1991; Armstrong et al., 1992; Marquardt and Krebs, 1992; Samuel et al., 1993; Lin et al., unpublished data), type A (Weddell et al., 1985; Beck and Strohmaier, 1987; Marquardt and Adam, 1988; Samuel et al., 1988; Carrillo et al., 1990; Armstrong et al., 1992; Armstrong et al., 1994), type C (Martínez et al., 1988; Piccone et al., 1988; Sobrino et al., 1989; Knowles and Samuel, 1990; Martínez et al., 1992), type SAT 1 (Dawe et al., 1994; Knowles et al., unpublished data), type SAT 2 (Vosloo et al., 1992; Knowles et al., unpublished data), type SAT 3 (Knowles et al., unpublished data) and type Asia 1 (Ansell et al., 1994a, b). Most of the data used for molecular epizootiological studies of FMD has been obtained by direct RNA sequencing (Knowles, 1990). This technique has a number of drawbacks including the need to obtain a relatively large quality of good quality viral RNA. The advent of polymerase chain reaction (PCR) amplification of reverse transcribed virus RNA promised the possibility of obtaining sequence data from relatively small amounts of RNA and without having to 'adapt' viruses to cell cultures. Determination of the nucleotide sequences of cDNA PCR products has not been straight forward owing to their double-stranded nature. However, recently a new sequencing technique has been developed - cycle sequencing. This is based on the amplification of dideoxy-sequencing products using a thermostable polymerase. This has two advantages, i) product extension takes place at a high temperature (72°C), meaning the double-stranded nature of the cDNA is no longer a problem and ii) the sequencing products themselves are amplified, meaning much less template cDNA is needed. Oligonucleotide primers We have developed four sets of oligonucleotide primers capable of amplifying the 1D (VP1) coding region of most FMD virus isolates belonging to serotypes O, A, C and Asia 1. The viruses sense (+) primers were designed using aligned 1C (VP3) sequences of the viruses shown in Table 1. The design of antisense (-) primer NK61 was based on aligned sequences of the 2B gene of types O, A, C and SAT 2 (data not shown). The internal sequencing primer, NK72, is identical to the FMDV universal primer described by Beck and Strohmaier (1987) and is also the primer used for most of the direct RNA sequencing studies in the laboratory. The locations of these primers on the FMDV genome are shown in Fig. 1. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 26 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Table 1. Foot-and-mouth disease virus sequences used for primer design. Virus Accession no.* Reference O1/Campos/Brazil/58 M95781 Jensen and Moore, unpub. O1/Kaufbeuren/FRG/66 X00871 Forss et al., 1984 O2/Brescia/Italy/47 M55287 Krebs et al., 1991 O/Syria/1/87 N/A† Samuel et al., unpub. A5/Spain/86 M72587 Saiz et al., 1991 A10/Holland/42 M20715-7 Thomas et al., 1988 A10/Argentina/61 X00429 Carroll et al., 1984 A12/119/UK/32 M10975 Robertson et al., 1985 A22/Iraq/24/64 N/A Bolwell et al., 1989 A24/Cruzeiro/Brazil/55 N/A Abrams, 1992 A32/Venezuela/70 N/A Knowles et al., unpub. C1/Germany/c.26 M90368 Martínez et al., 1992 C1/Oberbayern/FRG/60 X00130 Beck et al., 1983 C1/Santa Pau/Spain/70 (C-S8) N/A Sobrino et al., 1989 C1/Serra de Daró/Spain/81 (C-S15) N/A Sobrino et al., 1989 C2/Pando/Uruguay/44 M90367 Martínez et al., 1992 C3/Resende/Brazil/55 M90381 Martínez et al., 1992 C3/Indaial/Brazil/71 M90376 Martínez et al., 1992 C4/TDF/Argentina/66 M90372 Martínez et al., 1992 Asia 1/Pakistan/1/54 N/A Ansell et al., unpub. Asia 1/Tadzhikistan/USSR/64 N/A Sosnovtsev et al., 1989 Asia 1/L83 (Lebanon) U01207 Stram et al., 1994 Asia 1/Saudi Arabia/8/88 N/A Woodbury et al., 1994 * EMBL/GenBank accession number † not available RNA preparation Two methods were used i) phenol/chloroform extraction followed by cold ethanol precipitation (Knowles, 1990) and ii) QIAamp spin columns (Qiagen). Viral RNA was extracted from either i) 10% epithelium suspensions; ii) first passage in primary bovine thyroid cells; or iii) IB-RS-2 cell culture adapted virus. Reverse transcription Primer NK61 was used with either AMV or MMLV RTase (Boehringer Mannheim) in a standard RT reaction and extended for 30-60 min. at 42°C. Polymerase chain-reaction amplification The amplification cycles were preceded by heating the mix tube for 2 min. at 94°C and followed by heating for 5 min. at 72°C. Optimal PCR conditions for each primer pair are shown in Table 2. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 27 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Table 2. Optimum PCR conditions for each primer set Serotype Primer pair No. of cycles Denaturation (94°C) Annealing Extension (72°C) O ARS4/NK61 30 1 min 45 sec at 60°C 2 min A A-1C562/NK61 30 1 min 1 min at 55°C 1.5 min C C-1C536/NK61 25 1 min 30 sec at 60°C 2 min Asia 1 As1-1C505/NK61 30 1 min 1 min at 55°C 1.5 min The sequences of the primers and product lengths, when used with NK61, are shown in Table 3. A second primer located internally at the 1C (VP3) end of the product was sometimes used for sequencing. Alternatively these primers could be used with NK61 if the first PCR amplification was unsuccessful. Table 3. Oligonucleotide primers used for RT-PCR and cycle sequencing of foot-and-mouth disease viruses Primer designation* Primer sequence (5' 6 3') Product length (bp)† Positive sense primers O-1C124 (ARS4) ACCAACCTCCTTGATGTGGCT 1301 O-1C564 AATTACACATGGCAAGGCCGACGG 861 O-1C609 (Ovp3) TAGTGCTGGTAAAGACTTTGAGCT 816 A-1C562 TACCAAATTACACACGGGAA 863-866 A-1C612 TAGCGCCGGCAAAGACTTTGA 813-816 C-1C536 TACAGGGATGGGTCTGTGTGTACC 877-883 C-1C616 AAAGACTTTGAGCTCCGGCTACC 797-803 As1-1C505 TACACTGCTTCTGACGTGGC 908-914 As1-1C616 GGCAAGGACTTTGAGTTTCGC 797-803 Negative sense primers FMD-2B58 (NK61) GACATGTCCTCCTGCATCTG FMD-2A34 (NK72) GAAGGGCCCAGGGTTGGACTC * the primer name is constructed from the FMDV serotype followed by the gene and in subscript the location within the gene (alternative lab. name in parentheses). Universal FMD primers are designated FMD. † when used in PCR with FMD-2B58 (NK61). Post-PCR clean-up Residual oligonucleotide primers, dNTPs and enzyme were removed using Wizard™ PCR Preps (Promega) as per the manufacturer's protocol. Cycle sequencing Sequencing of part of the PCR fragments was performed using the fmol™ cycle sequencing kit PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 28 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel (Promega) employing 32P-end-labelled primers as per the manufacturers protocol. Primer NK72 was used for most of the sequencing although internal primers at the 1C (VP3) end of the product were sometimes also used (Table 3). Date analysis The computer software previously described by Knowles (1990) has been rewritten to run on IBM PC compatible microcomputers (the original programs ran on the Acorn Archimedes range). Each function within the package (called SeqProgs) was written as a separate program using either Power BASIC v.2.0a (Spectra Publishing) or Microsoft Professional BASIC v.7.01 (Microsoft). SeqProgs also interacts with various programs from the PHYLIP Phylogeny Package v.3.5c (Felsenstein, 1993). Essentially, the nucleotide sequences are stored in a number of ASCII (text) files (usually up to about 25 different sequences per file including a reference sequence); these files have the suffix name ALN. Each sequence may have multiple gel readings listed beneath a consensus sequence. The data input and deduction of consensus sequences is performed ’by hand’ using the MS-DOS editor. The program SPLIT can then be used to process the align files resulting in the extraction of the individual sequences into ASCII files (filename suffix: SEQ) and at the same time translate their amino acid sequences (filename suffix: PEP). Comparisons between any sequences in the database are performed in two basic ways; i) the program COMPARE processes a file containing a list of filenames (suffix: LST) and then compares each sequence on the list to a single sequence chosen from the database (’query’ sequence) and produces a sorted list, with those most closely related to the ’query’ sequence being at the top (filename suffix: CMP); ii) the program LINEUP also takes a list of filenames but then compares each sequence with every other sequence on the list producing a matrix file which can be used as the input file for programs within the PHYLIP package. Presently the programs KITSCH, FITCH and NEIGHBOR are used to calculate the phylogenetic trees; the latter program also includes the UPGMA method as an option. In the future it is planned to introduce new programs into the package to interact with other PHYLIP programs (e.g. DNADIST which produces distance matrices based on a number of different weighting methods) and with CLUSTAL V (Higgins et al., 1991) which can perform single and multiple sequence alignments. Results Examples of PCR amplification products from FMDV types O, A, C and Asia 1 are shown in Fig. 2. The PCR amplification was not always successful, although those proving negative could usually be amplified by using more RNA, more first strand cDNA or using a different RNA preparation. The sensitivity of the PCR has not been explored, however, it is not designed as a diagnostic PCR and it must be capable of amplifying as many different virus isolates as possible. For similar reason the PCR is also not type specific and, for example, some type O’s may be amplified by the type A primer set. This assay was successfully employed in the epidemiological investigations of the recent outbreaks of FMD type O in Greece. The PCR was carried out on seven samples (six 10% epithelium suspensions and one blood sample) received by the WRL in July 1994. These all proved negative (they were also negative in the typing ELISA). However, following isolation of virus on BTy cells from five of the samples, the PCR was carried out again and products were obtained. These were sequenced as described above and the results were obtained within 48 hours. The comparison of the Greek virus isolates and some other type O virus is shown in Fig. 3. PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 29 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Acknowledgements We would like to thank J.S. Thevasagayam and Lin Fengsheng who participated in this work while visiting this laboratory and D.M. Ansell and K.V. Marchant for valuable technical assistance. References Abrams, C. (1992). The biosynthesis of foot-andmouth disease virus empty capsids. Ph. D. Thesis, University of Reading. Ansell, D.M., Samuel, A.R., Carpenter, W.C. and Knowles, N.J. (1994a). Genetic relationships between foot-and-mouth disease type Asia 1 viruses. Epidemiology and Infection 112: 213-224. Ansell, D.M., Samuel, A.R. and Knowles, N.J. (1994b). Molecular epizootiology of foot-andmouth disease type Asia 1. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Vienna, Austria, 19-22 September, 1994. Ansell, D.M., Clarke, B.E., Samuel, A.R. and Knowles, N.J. The capsid-coding sequence of footand-mouth disease virus type Asia 1. Unpublished. Armstrong, R.M., Samuel, A.R., Knowles, N.J. and Uluturk, S. (1992). Genetic studies on foot-andmouth disease viruses isolated from samples collected in Turkey. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Berne, Switzerland. Rome: FAO, 1992. Armstrong, R.M., Samuel, A.R., Carpenter, W.C., Rama Kant and Knowles, N.J. (1994). A comparative study of serological and biochemical methods for strain differentiation of foot-and-mouth disease type A viruses. Veterinary Microbiology 39: 285-298. Beck, E. and Strohmaier, K. (1987). Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. Journal of Virology 61: 1621-9. Beck, E., Forss, S., Strebel, K., Cattaneo, R. and Feil, G. (1983). Structure of the FMDV translation initiation site and of the structural proteins. Nucleic Acids Research 11: 7873-7885. PROTOCOL.WPD Bolwell, C., Brown, A.L., Barnett, P.V., Campbell, R.O., Clarke, B.E., Parry, N.R., Ouldridge, E.J., Brown, F. and Rowlands, D.J. (1989). Host cell selection of antigenic variants of foot-and-mouth disease virus. Journal of General Virology 70: 4557. Carrillo, C., Dopazo, J., Moya, A., Gonzalez, M., Martínez, M.A., Saiz, J.C. and Sobrino, F. (1990). Comparison of vaccine strains and the virus causing the 1986 foot-and-mouth disease outbreak in Spain: epizootiological analysis. Virus Research 15: 45-56. Carroll, A.R., Rowlands, D.J. and Clarke, B.E. (1984). The complete nucleotide sequence of the RNA coding for the primary translation product of foot and mouth disease virus. Nucleic Acids Research 12: 2461-2472. Dawe, P.S., Flanagan, F.O, Madekurozwa, R.L., Sorensen, K.J., Anderson, E.C., Foggin, C.M., Ferris, N.P. and Knowles, N.J. (1994). Natural transmission of foot-and-mouth disease virus from African buffalo (Syncerus caffer) to cattle in a wildlife area of Zimbabwe. Veterinary Record 134: 230-232. Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle. Forss, S., Strebel, K., Beck, E. and Schaller, H. (1984). Nucleotide sequence and genome organization of foot-and-mouth disease virus. Nucleic Acids Research 12: 6587-6601. Higgins, D.G. Bleasby, A.J. and Fuchs, R. (1992). CLUSTAL V: improved software for multiple sequence alignment. Computer Applications in the Biosciences 8: 189-191. Jensen, M.J. and Moore, D.M. The nucleotide sequence of the untranslated region, L and P1 regions of the foot-and-mouth disease virus WRL-FMD: Molecular Epidemiology Group Page 30 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals serotype O1 Campos. Unpublished. Knowles, N.J. (1990). A method for direct nucleotide sequencing of foot-and-mouth disease virus RNA for epidemiological studies. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Lindholm, Denmark, 25-29 June, 1990, Appendix 06-112. Rome: FAO. Knowles, N.J. and Samuel, A.R. (1990). Molecular and antigenic analysis of foot-and-mouth disease type C viruses isolated from outbreaks in Italy during 1988 and 1989. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Lindholm, Denmark. Rome: FAO, 1990 : pp.122-8. Knowles, N.J., Marquardt, O. and Samuel, A.R. (1988). Antigenic and molecular characterization of isolates from recent outbreaks of foot-and-mouth disease virus in the Federal Republic of Germany. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Prague, Czechoslovakia. Rome: FAO, 1988: pp.149-55. Krebs, O., Berger, H.-G. and Marquardt, O. (1991). The capsid protein-coding sequence of foot-andmouth disease virus O2 Brescia. Archives of Virology 120: 135-143. Krebs, O., Berger. H,-G., Niedbalski, W. and Marquardt, O. (1991). Foot-and-mouth disease virus O1 Lombardy is biochemically related to O2 isolates. Virus Genes 5: 255-66. Marquardt, O. and Adam, K.-H. (1988). Sequences of capsid protein VP1 of two type A foot-and-mouth disease viruses. Virus Genes 2: 283-91. N.J. Knowles & A.R. Samuel Tierarztliche Umschau 47: 137-40. Martínez, M.A., Carrillo, C., Plana, J., Mascarella, R., Bergada, J., Palma, E.L., Domingo, E. and Sobrino, F. (1988). Genetic and immunogenic variations among closely related isolates of foot-and-mouth disease virus. Gene 62: 75-84. Martínez, M.A., Dopazo, J., Hernández, J., Mateu, M.G., Sobrino, F., Domingo, E. and Knowles, N.J. (1992). Evolution of the capsid protein genes of foot-and-mouth disease virus. Antigenic variation without accumulation of amino acid substitutions over six decades. Journal of Virology 66: 35573565. Piccone, M.E., Kaplan, G., Giavedoni, L., Domingo, E. and Palma, E.L. (1988). VP1 of serotype C foot-and-mouth disease viruses: long-term conservation of sequences. Journal of Virology 62: 1469-73. Robertson, B.H., Grubman, M.J., Weddell, G.N., Moore, D.M., Welsh, J.D., Fischer, T., Dowbenko, D.J., Yansura, D.G., Small, B. and Kleid, D.G. (1985). Nucleotide and amino acid sequence coding for polypeptides of foot-and-mouth disease virus type A12. Journal of Virology 54: 651-660. Saiz, J.C., Gonzalez, M.J., Borca, M.V., Sobrino, F. and Moore, D.M. (1991). Identification of neutralizing antigenic sites on VP1 and VP2 of type A5 foot-and-mouth disease virus, defined by neutralization resistant variants. Journal of Virology 65: 2518-2524. Samuel, A.R., Knowles, N.J. and Kitching, R.P. (1988). Serological and biochemical analysis of some recent type A foot-and-mouth disease virus isolates from the Middle East. Epidemiology and Infection 101: 577-90. Marquardt, O. and Adam, K.-H. (1990). FMDV subtyping by sequencing VP1 genes. Advances in Veterinary Virology: Proceedings of the 1st Congress of the European Society for Veterinary Virology, Liege, 1989. Veterinary Microbiology 23: 175-83. Samuel, A.R., Knowles, N.J. and Kitching, R.P. (1990a). Preliminary molecular analysis of footand-mouth disease virus type O in the Middle East. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Lindholm, Denmark. Rome: FAO, 1990 : pp.133-8. Marquardt, O. and Krebs, O. (1992). Outbreaks of foot-and-mouth disease near Hannover in 1987 and 1989: evidence for two strains of virus. Samuel, A.R., Knowles, N.J. and Kitching, R.P. (1990b). Preliminary antigenic and molecular analysis of strains of foot-and-mouth disease virus PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 31 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals serotype O isolated from Saudi Arabia in 1988 and 1989. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-andMouth Disease, Lindholm, Denmark. Rome: FAO, 1990 : pp.139-45. N.J. Knowles & A.R. Samuel S.M. and Kitching, R.P. (1994). Analysis of mixed foot-and-mouth disease virus infections in Saudi Arabia: prolonged circulation of an exotic serotype. Epidemiology and Infection 112: 201-211. Samuel, A.R., Ansell, D.M., Rendle, R.T., Armstrong, R.M., Davidson, F.L., Knowles, N.J. and Kitching, R.P. (1993). Field and laboratory analysis of an outbreak of foot-and-mouth disease in Bulgaria in 1991. Revue scientifique et technique de l’Office International des Epizooties 12: 839-848. Sobrino, F., Martínez, M.A., Carrillo, C. and Beck, E. (1989). Antigenic variation of foot-and-mouth disease virus of serotype C during propagation in the field is mainly restricted to only one structural protein (VP1). Virus Research 14: 273-280. Sosnovtsev, S.V., Onishchenko, A.M., Petrov, N.A., Mamaeva, N.V., Kalashnikova, T.I., Perevozchikova, N.A., Ivanyushchenkov, V.N., Burdov, A.N. and Vasilenko, S.K. (1989). Primary structure of the gene of the VP1 protein of epidemic stomatitis virus of Asia 1 serotype. Molekulyarnaya Genetika, Mikrobiologiya i Virusologiya 12: 44-46. Stram, Y., Laor, O., Molad, T., Chai, D., Moore, D., Yadin, H. and Becker, Y. (1994). Nucleotide sequence of the P1 region of serotype Asia 1 foot-and-mouth disease virus. Virus Genes 8: 275278. Thomas, A.A.M., Woortmeijer, R.J., Puijk, W. and Barteling, S.J. (1988). Antigenic sites on foot-and-mouth disease virus type A10. Journal of Virology 62: 2782-2789. Vosloo, W., Knowles, N.J. and Thomson, G.R. (1992). Genetic relationships between southern African SAT-2 isolates of foot-and-mouth disease virus. Epidemiology and Infection 109: 547-58. Weddell, G.N., Yansura, D.G., Dowbenko, D.J., Hoatlin, M.E., Grubman, M.J., Moore, D.M. and Kleid, D.G. (1985). Sequence variation in the gene for the immunogenic capsid protein VP1 of foot-and-mouth disease virus type A. Proceedings of the National Academy of Sciences USA 82: 2618-22. Woodbury, E.L., Samuel, A.R., Knowles, N.J., Hafez, PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 32 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel pO-1C609 pO-1C564 pNK72 pNK61 pARS4 Type O pA-1C616 pNK72 pNK61 pA-1C562 Type A pC-1C616 pC-1C536 Type C pNK72 pNK61 pAs1-1C616 pNK72 pNK61 pAs1-1C505 Type Asia 1 Poly(C) VPg L 0 1A 1 1B 1C 2 1D 2 A 3 2B 3A 2C 4 3B 5 3C AAAAAAAAAn 3D 6 7 8 Kilobases Fig. 1. Foot-and-mouth disease virus genome map and PCR/sequencing primer locations Markers A/SAU/2/94 A/SAU/8/94 A/SAU/14/94 A/SAU/16/94 A/SAU/18/94 A/SAU/21/94 A/SAU/24/94 A10/Arg/61 A12/119/UK/32 Markers A/BRA/3/93 A/BRA/5/93 A24/Cruzeiro/BRA/55 Markers Markers O/EGY/2/93 O/EGY/3/93 FMDV type A O/EGY/1/93 O/EGY/2/89 O/JOR/6/93 O/JOR/5/93 O/JOR/3/93 O/SAU/31/92 O/SAU/1/92 Markers FMDV type O A-1C562/NK61 product (~866 bp) ARS4/NK61 product (1301 bp) C-1C536/NK61 product (~911 bp) Markers Asia1/NEP/19/86 Asia1/AFG/5/72 Asia1/WBN/117/85 Asia1/CAM/1/90 Asia1/WBN/117/85 Asia1/NEP/1/85 Asia1/CAM/1/90 Asia1/MAY/13/92 Asia1/ISR/1/84 Asia1/TUR/15/73 Asia1/MAY/2/90 Asia1/KUW/2/79 Asia1/IND/12/76 Markers C/PHI/10/89 C/BAN/1/92 C/IND/12/82 C/IND/7/76 Markers C/PHI/3/90 C/IND/4/71 C/PHI/9/76 C/IND/3/83 Asia1/YEM/15/79 FMDV Asia 1 FMDV type C AS1-1C505/NK61 product (~911 bp) Fig. 2. Examples of PCR products of FMD viruses using conserved primer sets PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 33 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel O1/Kaufbeuren/FRG/66 O/Greece/1/94 O/Bulgaria/1/93 Bulgaria 1991 & 1993 O/Turkey/20/91 O/Saudi Arabia/35/90 O/Saudi Arabia/29/93 O/Egypt/1/93 OA/Saudi Arabia/34/92 O/Bulgaria/1/91 O/Egypt/4/93 O/Turkey/6/93 O/Bahrain/4/93 O/Israel/1/91 O/Jordan/6/93 O/Jordan/1/94 Greece 1994 O/Jordan/2/94 O/Iran/10/93 O/Saudi Arabia/45/94 O/Saudi Arabia/17/94 O/Israel/6/89 O/Jordan/1/89 O/Syria/1/89 O/Bahrain/9/88 O/Kuwait/3/88 O/Jordan/1/88 O/Turkey/5/90 O/Turkey/21/90 O/Syria/1/91 O/Saudi Arabia/3/91 O/Saudi Arabia/8/88 O/Saudi Arabia/3/89 O/Saudi Arabia/33/88 O/Israel/1/88 O/Saudi Arabia/7/91 O/Saudi Arabia/26/90 O/Italy/1/93 O/Italy/2/93 O/Italy/5/93 O/Italy/6/93 O/Israel/1/92 Italy 1993 O/Israel/3/92 O/Oman/3/91 O/Oman/54/91 O/Oman/58/91 O/Bahrain/2/91 18 16 14 12 10 8 6 4 2 0 Percentage nucleotide difference (positions 475-639 of VP1) Fig. 3. Nucleotide sequence relationships between FMD type O viruses which caused recent outbreaks in Europe and the Middle East PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 34 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel Appendix III: Recent Developments in the Epidemiology of Foot-and-Mouth Disease: Genetic Relationships Determined by Nucleotide Sequence Analysis. Paper published in: Foot-and-Mouth Disease Newsletter 1 (2): 13-19 (1994). Recent Developments in the Epidemiology of Foot-and-Mouth Disease: Genetic Relationships Determined by Nucleotide Sequence Analysis N.J. Knowles and A.R. Samuel World and Community Reference Laboratory for Foot-and-Mouth Disease, AFRC Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom. The advent of nucleotide sequence analysis has revolutionized the study of the epidemiology of virus infections. This technique has been used at the World Reference Laboratory (WRL) for Foot-and-Mouth Disease (FMD) since the end of 1987. The sequence database which has been generated has also been of great value to the Community Reference Laboratory (CRL) for FMD enabling the rapid identification of the likely origins of the last three FMD outbreaks in Europe. In July 1991, FMD type O appeared in Bulgaria and sequence comparisons eliminated the possibilities of either a laboratory escape of the virus or improperly inactivated vaccine. Furthermore genetically related type O viruses were found in the Middle East suggesting that locality as a probable origin (Samuel et al., 1993). In February 1993, FMD type O appeared in Southern Italy where it spread rapidly (Kitching, 1993). In the following month the disease appeared in Northern Italy, but was limited to two premises. Nucleotide sequence analysis again suggested the Middle East as an origin and additionally showed the virus responsible to be different from the earlier outbreak in Bulgaria. This analysis also confirmed that the outbreaks in Southern and Northern Italy were caused by the same virus (N.J. Knowles and A.R. Samuel, unpublished data; Kitching, 1993). Subsequently, in 1993, FMD type O again appeared in Bulgaria. Comparison with the earlier nucleotide sequences showed that the virus was identical to that causing the 1991 outbreak in Bulgaria (A.R. Samuel and N.J. Knowles, unpublished data; see front cover). In the WRL and CRL there are currently many different sequencing studies in progress aimed at elucidating the epidemiology of all seven serotypes of FMD. Some of these studies have been, or are about to be, published; we present here a resume of that work. FMD virus type O Further isolates of FMD virus type O have been examined from countries in the Middle East and North Africa. Virus isolates from Egypt and Jordan in 1993 are genetically related to a group of viruses that include isolates from Turkey in 1987, 1988 and 1991, Syria in 1987, Saudi Arabia in 1990, Bahrain in 1992, Iran in 1993 and Bulgaria in 1991 and 1993 (Fig. 1). The Egyptian isolates from 1993 are not closely related to the virus which caused epizootics in North Africa between 1989 and 1991 (Fig. 1). FMD virus type A Three serological and three biochemical methods have used to compare five field isolates of FMD virus from Western India with nine reference vaccine strains and five field isolates from other countries (Armstrong et al., 1994). The serological tests (liquid-phase ELISA and virus neutralization) were able to distinguish between the three reference vaccine strains examined, however, generally the five recent Indian field isolates reacted poorly with these vaccine strains. Analysis of monoclonal antibody (MAb) data was difficult to interpret PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 35 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel although generally the field isolate A/IND/5/87 reacted to a lesser extent with one of the MAb panels (A10/Holland/42) than the other four recent Indian isolates. The A22/Iraq/24/64 MAbs were generally less reactive, only reacting significantly with one of the reference vaccine strains. Polyacrylamide gel electrophoresis distinguished the reference vaccine strains from each other and from the field isolates. Additionally, one of the Indian isolates (A/IND/5/87) could be differentiated from the other four. Electrofocusing showed similarities between the reference vaccine strain A22/Iraq/24/64 and three of the Indian field isolates (A/IND/1/87, A/IND/2/87 and A/IND/3/87), however, A/IND/4/87 and A/IND/5/87 were distinct. Nucleotide sequencing showed that the isolates A/IND/1/87, A/IND/2/87 and A/IND/3/87 were very closely related to each other and related to A/IND/4/87, however, A/IND/5/87 was different. Foot-and-mouth disease type O is enzootic in Saudi Arabia, but type A only occurs occasionally. In 1986-87 type A appeared after an absence of 10 years (a single isolation of type A was made in imported stock for slaughter at an abattoir in 1984). Sequence analysis showed the virus responsible to be related, although not closely, to type A viruses from North-western India (Samuel et al., 1988; Fig. 2). The virus was eradicated following vaccination using one of the outbreak viruses (A/SAU/23/86). Type A viruses next appeared in Saudi Arabia in 1991 and, despite vaccination, continues to cause outbreaks. Sequence analysis has shown that the 1991-93 Saudi Arabian type A viruses are very different to those causing the 1986-87 outbreaks but are closely related to viruses circulating in Turkey between 1991-92 (Fig. 2). FMD virus type Asia 1 Ansell et al. (1994) determined the sequence of 165 nucleotides at the 3N end of the 1D (VP1) gene of 44 type Asia 1 FMD viruses. These strains had been isolated from throughout Asia between 1954-92. Analysis of the relationships between the virus genomes showed epidemiological links not previously evident, for example, between Bangladesh and Kuwait (Fig. 3). The origin of the only outbreak of FMD Asia 1 to have occurred in Europe, in Greece in 1984, was clarified; the nucleotide sequence of this virus was closely related to the sequences of viruses present in Israel, Lebanon and Bahrain between 1983-85. Variation in the region sequenced was not as great as that seen in the other FMD virus serotypes and all viruses shared greater than 85% nucleotide identity. Thus they considered all the virus isolates examined to belong to a single genotype. The above study was performed by directly sequencing FMD virus RNA (Knowles, 1990) and further studies are in progress using reverse transcription-polymerase chain reaction (RT-PCR) and cycle sequencing to examine a wider range of Asia 1 isolates. In another study (Woodbury et al., 1994) plaque purification of FMD type O viruses isolated from cattle in Saudi Arabia showed the presence of mixed serotype infections. Sixteen out of 31 samples collected between 1985 and 1991 also contained Asia 1 virus, a serotype which had previously only been isolated from a single outbreak in that country in 1980. Nucleotide sequences of the Asia 1 component of all these samples revealed little variation and showed that they were closely related to both a Russian lapinized vaccine virus strain (Asia 1/Tadzhikistan/64), and to a field isolate from Turkey (Asia 1/TUR/15/73). Although mixed FMD infections have been observed previously this is the first report of a serotype, considered to be exotic to a country, co-existing undetected for an extended period of time. Buffalo to cattle transmission of FMD virus Two recent studies (Dawe et al., 1994a,b) have demonstrated, in one case, the experimental transmission of FMD virus type SAT 2 from African buffalo (Syncerus caffer) to cattle and in the other a case of PROTOCOL.WPD WRL-FMD: Molecular Epidemiology Group Page 36 RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals N.J. Knowles & A.R. Samuel natural transmission of type SAT 1 from African buffalo to cattle. Nucleotide sequencing played a key role in determining the origin of the viruses in both cases. Four female cattle and three male African buffalo which were free of FMD virus were held together on an island in Lake Kariba, Zimbabwe. The buffalo were experimentally infected with FMD virus type SAT 2, showed generalised disease and became virus carriers. While the buffalo were in the acute phase of disease the susceptible contact cattle did not show lesions, no virus was recovered from them nor did they develop serum antibodies. However, five months later the cattle showed severe FMD. Direct nucleotide sequencing of the virus used to infect the buffalo and of the virus from the in-contact cattle showed that the two isolates were almost identical. The results suggest that in nature it is possible for transmission from buffalo to cattle to occur under the influence of factors not yet defined and that there was very little change in the nucleotide sequence of the virus during the carrier period of five months. An outbreak of FMD occurred during April, 1991 in a trypanosomiasis sentinel cattle herd in the Rifa River area of the Hurungwe hunting blocks, to the east of Lake Kariba in Zimbabwe (Dawe et al., 1994a). Despite having been vaccinated bi-annually for the previous five years the disease was severe. Viruses isolated from the affected animals were typed as FMD virus type SAT 1. Free-living African buffalo which had been using the same watering place as the affected cattle were sampled and FMD type SAT 1 virus was isolated. Partial nucleotide sequencing of the gene coding for the capsid protein 1D was performed for one of the viruses isolated from cattle and for two of the viruses isolated from buffalo. Comparison of these sequences demonstrated a close relationship between all three virus isolates. Since no other cattle were present in this area and no SAT 1 outbreaks had occurred in Zimbabwe since 1989, it was concluded that transmission from buffalo to cattle had taken place. References Ansell, D.M., Samuel, A.R., Carpenter, W.C. and Knowles, N.J. (1994). Genetic relationships between foot-and-mouth disease type Asia 1 viruses. Epidemiology and Infection 112: 213-224. Armstrong, R.M., Samuel, A.R., Carpenter, W.C., Rama Kant and Knowles, N.J. (1994). A comparative study of serological and biochemical methods for strain differentiation of foot-and-mouth disease type A viruses. Veterinary Microbiology 39: 285298. Dawe, P.S., Flanagan, F.O, Madekurozwa, R.L., Sorensen, K.J., Anderson, E.C., Foggin, C.M., Ferris, N.P. and Knowles, N.J. (1994). Natural transmission of foot-and-mouth disease virus from African buffalo (Syncerus caffer) to cattle in a wildlife area of Zimbabwe. Veterinary Record 134: 230-232. Dawe, P.S., Sorensen, K.J., Ferris, N.P., Barnett, I.T.R., Armstrong, R.M. and Knowles, N.J. (1994). Experimental transmission of foot-and-mouth disease virus from carrier African buffalo (Syncerus caffer) to cattle in Zimbabwe. Veterinary Record 134: 211-215. Kitching, R.P. (1993). Foot and mouth disease in Italy. Community Reference Laboratory Newsletter PROTOCOL.WPD 1 (1): 3-5. Knowles, N.J. (1990). A method for direct nucleotide sequencing of foot-and-mouth disease virus RNA for epidemiological studies. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Lindholm, Denmark, 2529 June, 1990, Appendix 06-112. Rome: FAO. Samuel, A.R., Knowles, N.J. and Kitching, R.P. (1988). Serological and biochemical analysis of some recent type A foot-and-mouth disease virus isolates from the Middle East. Epidemiology and Infection 101: 577-590. Samuel, A.R., Ansell, D.M., Rendle, R.T., Armstrong, R.M., Davidson, F.L., Knowles, N.J. and Kitching, R.P. (1993). Field and laboratory analysis of an outbreak of foot-and-mouth disease in Bulgaria in 1991. Revue scientifique et technique de l’Office International des Epizooties 12: 839-848. Woodbury, E.L., Samuel, A.R., Knowles, N.J., Hafez, S.M. and Kitching, R.P. (1994). Analysis of mixed foot-and-mouth disease virus infections in Saudi Arabia: prolonged circulation of an exotic serotype. Epidemiology and Infection 112: 201-211. WRL-FMD: Molecular Epidemiology Group Page 37