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Appendix 12
Table 3. List of primers and sequences used in this study.
Primer Virus Sense Gene Primer Seq. (5'-3')
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pNK61 Universal Negative P2B GACATGTCCTCCTGCATCTG
pNK72 Universal Negative P2A GAAGGGCCCAGGGTTGGACTC
pARS4 Type O Positive P1C ACCAACCTCCTTGATGTGGCT
1C562 Type A Positive P1C TGGTTGGAGGAACTACACCGA
Results and Discussion
The nucleotide sequences of type O and A viruses used in this study and previously sequenced viruses were plotted as dendograms and shown in Figures 1 and 2. Viruses that have a difference of about 5% or less in nucleotide sequence were considered as closely related ( Samuel et al., 1988). All type A viruses sequenced in this study were closely related to each other and A/Iran/96 virus group. A/Iran/96 was first detected in Turkey at the end of 1997 (A/TUR/12/97). The nucleotide difference between these viruses and other type A viruses isolated previously from Turkey was over 18% (Aktas, 1998). The results of this study showed that type A viruses isolated from Turkey between 2000 and 2002 were belonging to A/Iran/96 group. The close genetic relationship between field isolates and the Turkish vaccine strain (same as A/TUR/40/98) and r values detected by antigenic characterisation ELISA, shown in brackets in Figure 1 and Table 3 ), confirmed that the current type A vaccine strain was suitable for Turkey.
The sequencing results of type O viruses isolated from Turkey between 2000 and 2002 showed that there were three different genetic sublineages circulating in Turkey during this period. Similar result was found in a previous study (Aktas, 1998). Although these viruses were genetically different from each other, all of these type O FMD viruses could be considered as belonging to a single genotype since nucleotide differences between these viruses were less than 15% (Samuel et al., 1997). On the other hand antigenic study showed that these viruses were closely related to the vaccine strain, O Manisa 1969 (Table 3).
Table 1. List of type A viruses used in this study.
Viruses Geographical origin Date Reference
A24//BRA/55 Brasil 1955 Knowles et all. (1999) A10/HOL/42 A/IRQ/24/64 Iraq A/IND/17/77 India 1964 Knowles N.J. (1999) 1977 Tosh, et all.(2000)
A/IRN/87 Iran 1987 Marquardt & Adam (1988)
A/IRN/17/96 Iran A/MCD/3/96 Macedonia
1996 Pirbright 1996 Pirbright A/SAU/23/99 Saudi Arabia 1999 Pirbright A/TUR/12/73 Antalya ,Turkey 1973 Aktas,S., (1998) A/TUR/2/80 Van ,Turkey 1980 Aktas,S., (1998) A/TUR/7/85 Sivas ,Turkey 1985 Aktas,S., (1998) A/TUR/3/89 Ankara, Turkey 1989 Aktas,S., (1998) A/TUR/9/91 Afyon, Turkey 1991 Aktas,S., (1998) A/TUR/3/92 Kayseri ,Turkey 1992 Aktas,S., (1998) A/TUR/3/95 Kastamonu ,Turkey 1995 Aktas,S., (1998) A/TUR/8/96 Samsun, Turkey 1996 Aktas,S., (1998) A/TUR/11/97 Nigde Turkey 1997 Aktas,S., (1998) A/TUR/12/97 Kutahya ,Turkey 1997 Aktas,S., (1998) A/TUR/25/98 Ankara,Turkey 1998 Aktas,S., (1998) A/TUR/40/98 Izmit, Turkey 1998 Aktas,S., (1998) A/TUR/1/00 Malatya Turkey 2000 This work A/TUR/2/00 Kırıkkale Turkey 2000 This work A/TUR/3/01 Malatya Turkey 2001 This work A/TUR/4/01 Manisa Turkey 2001 This work A/TUR/5/01 Sivas Turkey 2001 This work A/TUR/6/01 Afyon, Turkey, 2001 This work A/TUR/7/01 Bolu, Turkey 2001 This work A/TUR/8/02 Bingol, Turkey 2002 This work A/TUR/9/02 Kars ,Turkey 2002 This work
Table 2. List of type O viruses used in this study
Virus Geographical origin Date Reference
O/GRE/27/96 Evros,Greece 1998 Samuel & Knowles (2001) O/IRQ/30/00 Iraq 2000 Samuel & Knowles (2001) O/IRN/7/94 Iran 1994 Samuel & Knowles (2001) O/Manisa/TUR/69 Manisa,Turkey 1969 Aktas & Samuel (2000) O/TUR/10/94 Nilufer, Bursa, Turkey 1994 Samuel & Knowles (2001) O/TUR/1/96 Kesan, Edirne, Turkey 1996 Samuel & Knowles (2001) O/TUR/1/00 Konya, Turkey 2000 This work O/TUR/2/00 Nigde, Turkey 2001 This work O/TUR/3/01 Mudurnu, Bolu 2001 This work O/TUR/4/02 Sivas, Turkey 2002 This work
Table 3. r values obtained against Turkish vaccine strains.
A/TUR/6/01 0.66 A/TUR/7/01 0.75 A/TUR/9/01 1 A/TUR/1/02 >1 A/TUR/2/02 0.5 A/TUR/3/02 1 A/TUR/4/02 0.35 A/TUR/5/02 0.75 O/TUR/1/01 0.5 O/TUR/2/01 0.5 O/TUR/3/01 1
References:
Aktas, S. (1998). Molecular epidemiology of FMD types O and A in Turkey. PhD Thesis, University of Reading.
Felsenstain, J., 1993. PHYLIP- Phylogeny inference package, version 3.5c. University of Washington, Seattle, WA, USA.
Knowles, N.J.,Samuel, A.R.(1998). RT-PCR and sequencing protocols for the molecular epidemiology of exotic diseases of animals. IAH, Pirbright Laboratory, Surrey, UK.
Marquardt, O. and Adam, K.-H. (1988). Sequences of capsid protein VP1 of two type A footand-mouth disease viruses. Virus Genes, 2: 283-291.
Samuel, A.R., Knowles, N.J. and Kitching, R.P. (1988). Serological and biochemical analysis of some recent type A FMDV isolates from the Middle East. Epidemiology and Infection, 101, 577590.
Samuel, A.R., Knowles, N.J., Kitching, R.P. and Hafez, S.M. (1997). Molecular analysis of FMD type O viruses isolated in Saudi Arabia between 1983 and 1995. Epidemiology and Infection, 119, 381-389.
Thompson ,J.D., Gibson,T.J., Plewniak,F., Jeanmougin,F. and Higgins,D.G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 24:4876-4882.
A/TUR/7/01
( 0.75)
A/TUR/4/01
A/TUR/9/02
A/TUR/6/01
( 0.66)
type A field isolates and the vaccine strains.
A/TUR/5/01
A/IRN/17/96
A/TUR/2/00
A/TUR/25/98
A/TUR/40/98
A/TUR/12/97
A10/Hol/42
A24/Cr/Bra/55
A/MCD/3/96
A22/IND/17/77
A/TUR/02/80
AMahmatlı
0.1
A/TUR/12/73
A/TUR/11/97
A/TUR/08/96
A/TUR/3/95
A/SAU/23/99
A/TUR/03/92
A/TUR/09/91
A/IRN/87
O/IRQ/30/00
O/TUR/3/01
(1)
O/TUR/1/00
0.1
Figure 2. Dendogram depicting relationships between FMDV type O field isolates and the vaccine strain.
