Genetic variation among African swine fever genotype II viruses, Eastern and Central Europe.
On January 24, 2014, the European Commission and the World Organisation for Animal Health received reports from Lithuanian authorities of 2 cases of ASF in wild boars. One of the infected animals was found in Salcininkai and the other in Varena, 5 km and 40 km, respectively, from the Belarus border (7). Then, on February 14 and 17, 2014, reports of 2 cases of ASF in wild boars were received from northeastern Poland (Sokolka County, Podlaskie Province). One of the infected animals in Poland was found in the municipality of Szudzialowo; the other was found in Kruszyniany, a forest area (8). The 2 wild boars in Poland were found dead [approximately equal to] 900 m and [approximately equal to] 200 m, respectively, from Poland's border with Belarus.
ASFV-positive clinical samples (spleen, kidney, lung, bone marrow) from the 4 infected wild boars were sent to the European Union reference laboratory for ASF, Centro de Investigacion en Sanidad Animal (CISAINIA), Madrid, Spain, for confirmatory testing and genetic characterization. After the presence of ASFV was confirmed in samples, initial genetic characterization was performed by using standardized genotyping procedures on virus DNA extracted directly from homogenized tissues and from bone marrow samples. These analyses included the C-terminal end of the p72 gene, the full sequence of the p54 gene, and the central variable region within the B602L gene (9). We also included in the study 21 genotype II ASFVs that were isolated from wild and domestic pigs in Russia and the Caucasus region during April 2007-June 2013 (Table).
We compared the nucleotide sequences obtained from the p72- and p54-based PCRs with those of previously described representative isolates (10). We used Clustal Omega (http://www.clustal.org/) to perform multiple sequence alignments. Minimum evolutiontrees, rooted at the midpoint, were constructed by using MEGA V6.0 (http://www.megasoftware.net/) with the p-distance nucleotide substitution model. The 2014 ASFVs from Lithuania (LT14/1482, LT14/1490) and Poland (Pol14/Sz and Pol14/Krus) clustered, as expected, within p72 genotype II (Figure 2) and showed 100% nucleotide identity with all compared ASFV isolates from eastern Europe across the 478-bp C-terminal p72 gene and the 558-bp full lengthp54 gene. We obtained the same result by sequencing the central variable region within the B602L gene, revealing 10 copies of amino acid tetramer repeats that were 100% identical and unique to those of the ASFV circulating in the Caucasus regions since 2007 (11).
Although the central variable region has proven useful for resolving epidemiologic complexities at the genotype (12), country (13), and region levels, additional genome markers are required to determine the origin and to map the spread of closely related ASFV isolates circulating in eastern Europe. Thus, we designed a set of primers, named ECO1A (5'-CCATTTATCCCCCGCTTTGG-3' binding site 172,270-172,290) and ECO1B (5'-TCGTCATCCTGAGACAGCAG-3' binding site 172,616-172,626), to amplify a 356-bp fragment located between the I73R and I329L genes and characterized by the presence of TRS (14). Primer binding sites were based on the genome of the ASFV from Georgia (GenBank accession no. FR682468.1). Using the same reaction conditions as used for full p54 gene amplification (10) and an annealing temperature of 60[degrees]C, we generated 367-bp amplicons from isolates from Ukraine, Belarus, Lithuania, and Poland. The estimated size of the remaining isolates from eastern Europe that were included in the study was 356 bp (data not shown). Nucleotide sequence analysis of the PCR products revealed that the size difference was caused by the insertion of an additional TRS (GGAATATATA) at nt 136 (Figure 3). All sequences generated in this study were submitted to GenBank under accession nos. KJ620028-51.
Current available molecular data derived by using standardized genotyping procedures (9) have indicated the presence of only 1 ASFV variant. That variant belongs to p72 genotype II, which has been circulating in eastern European countries since the introduction of ASFV into Georgia in 2007 (11). In agreement with those findings, results from our analysis of the 3 independent regions included in the classical genotyping showed that sequences for ASFV isolates from Lithuania and Poland were 100% homologous with those for ASFVs from eastern Europe. However, the long-term presence of ASFV in Russia and the Caucasus regions and the rapid spread of the virus to neighboring countries highlight the need for finding additional ASFV genome markers capable of discriminating among circulating virus isolates so that we may better determine their source and evolution.
The whole-genome sequence analysis of ASFV has identified some regions that contain tandem repeat arrays that have proven useful for discriminating between closely related ASFVs (15). Thus, the approach described in our study focused on analysis of the TRS in the intergenic region between the I73R and I329L genes at the right end of the genome (14). The results showed that the viruses from Poland and Lithuania had a TRS insertion identical to that present in ASFV isolates from Belarus and Ukraine. This TRS insertion was absent in the remaining viruses from eastern Europe, including those obtained in Tver Oblast, Russia, in 2012 and in Georgia in 2007. These molecular data, together with the epidemiologic findings, confirmed that the ASFVs detected in Poland and Lithuania most probably originated from Belarus. However, knowledge of the epidemiology of ASF and a full understanding of the evolution and spread of ASFV in this region require additional sequence analysis of ASFVs currently circulating in Russian regions bordering Belarus and Ukraine.
Our results show the genetic variability among ASFVs circulating in eastern Europe and describe a new method that can be useful for distinguishing between closely related ASFV isolates. Such genetic data are essential for determining the source and studying the evolution of ASFV isolates and to fully elucidate the spread of ASFV in the eastern and central European countries.
We appreciate the intellectual and practical contributions of our colleagues at the National Reference Laboratories for ASF in Poland, Lithuania, Belarus, and Ukraine and at the European Union reference laboratory for ASF. We are especially grateful to Elena Martin and Alicia Simon for technical assistance and Irene Iglesias for map generation.
The study was funded by the European Union Seventh Framework Program under the ASFORCE (Targeted Research Effort on African Swine Fever) project (grant no. 311931) and the European Union reference laboratory for ASF (grant no. UE-1 LR PPA/03).
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Author affiliations: Centro de Investigacion en Sanidad Animal (CISA-INIA), Madrid, Spain (C. Gallardo, J. Fernandez-Pinero, V. Pelayo, R. Nieto, P. Fernandez-Pacheco, C. Perez, A. Soler, M. Arias); National Institute of Veterinary Virology and Microbiology, Pokrov, Russia (I. Gazaev, D. Kolvasov); National Veterinary Research Institute, Pulawy, Poland (I. Markowska-Daniel); National Food and Veterinary Risk Assessment Institute, Vilnius, Lithuania (G. Pridotkas); Belarusian State Veterinary Centre, Minsk, Belarus (S. Bokhan); and State Research Institute of Laboratory Diagnostic and Veterinary Sanitary Expertise, Kiev, Ukraine (O. Nevolko, Z. Drozhzhe)
Dr Gallardo is the laboratory research coordinator of the European and Food and Agricultural Organization of the United Nations Reference Laboratory for African swine fever at CISAINIA. Her research work mainly focuses on molecular characterization, epidemiology and diagnosis of ASF.
Address for correspondence: Carmina Gallardo, CISA-INIA. Ctra Algete el Casar s/n. 28130 Valdeolmos, Madrid, Spain; email: firstname.lastname@example.org
Table. African swine fever virus isolates from eastern Europe selected for a study of the genetic variation among genotype II viruses in eastern and central Europe, 2007-2014 * Isolate Source country, area Host Abk07 Georgia, Abkhazia Republic, Gulripish DP Arm07 Armenia, Dilijan DP Che07 Russia, Chechnya Republic, Shatoysky EWB Az08D Azerbaijan, Qebele District DP Az08B Azerbaijan, Qebele District DP Ing08 Russia, Ingushetia Republic, Sunzhensky EWB Oren08 Russia, Orenburg Oblast, Chernorechye DP NO08/Av Russia, Republic of North Osetia, Vladikawkaz DP NO08/Ap Russia, Republic of North Osetia, Prigorodni DP Dagestan09 Russia, Dagestan Republic, Tarumovsky, EWB District StPet09 Russia, Leningradskaya Oblast, Kirovsky DP Kalmykia09 Russia, Republic of Kalmykia, Yashaltinsky DP district Rostov09 Russia, Rostov Oblast, Krasnosulinsky District DP Tver0511/Torjo Russia, Tver Oblast, Torjo DP Tver0312/Novo Russia, Novozavidovskii, Tver region DP Tver0312/Torjo Russia, Torjo, Tver region EWB Tver0712/Les Russia, Lesnoi, Tver region DP Ukr12/Zapo Ukraine, Zaporozhye region DP Tver0812/Bolo Russia, Bologovskii, Tver region EWB Tver1112/Zavi Russia, Zavidovo, Tver region EWB Bel13/Grodno Belarus, Grodno region, Lelyukinskiy District DP of Ivye LT14/1490 Lithuania, Salcininkai District Municipality EWB LT14/1482 Lithuania, Alytus County, Varena District EWB Municipality Pol14/Sz Poland, Szudzialowo, Sokolka County, EWB Podlaskie Province Pol14/Krus Poland, Kruszyniany, Sokolka County, EWB Podlaskie Province GenBank accession no. Onset of Isolate outbreak p72 gene P54 gene CVR Abk07 2007 Jul 04 JX857509 JX857495 JX857523 Arm07 2007 Aug 07 JX857508 JX857494 JX857522 Che07 2007 Dec 04 JX857510 JX857496 JX857524 Az08D 2008 Jan 22 JX857515 JX857501 JX857529 Az08B 2008 Jan 22 JX857516 JX857502 JX857530 Ing08 2008 Jul 21 JX857511 JX857497 JX857525 Oren08 2008 Jul 10 JX857512 JX857498 JX857526 NO08/Av 2008 Jul 18 JX857513 JX857499 JX857527 NO08/Ap 2008 Jul 21 JX857514 JX857500 JX857528 Dagestan09 2009 Sep 11 JX857517 JX857503 JX857531 StPet09 2009 Oct 01 JX857520 JX857506 JX857534 Kalmykia09 2009 Oct 10 JX857519 JX857505 JX857533 Rostov09 2009 Oct 20 JX857518 JX857504 JX857532 Tver0511/Torjo 2011 May 31 KJ627208 KJ627186 KJ627197 Tver0312/Novo 2012 Mar 14 KJ627212 KJ627190 KJ627201 Tver0312/Torjo 2012 Mar 28 KJ627211 KJ627189 KJ627200 Tver0712/Les 2012 Jul 16 KJ627210 KJ627188 KJ627199 Ukr12/Zapo 2012 Jul 30 JX857521 JX857507 JX857535 Tver0812/Bolo 2012 Aug 15 KJ627209 KJ627187 KJ627198 Tver1112/Zavi 2012 Nov 20 KJ627214 KJ627191 KJ627202 Bel13/Grodno 2013 Jun 19 KJ627215 KJ627192 KJ627203 LT14/1490 2014 Jan 21 KJ627216 KJ627193 KJ627204 LT14/1482 2014 Jan 21 KJ627217 KJ627194 KJ627205 Pol14/Sz 2014 Feb 14 KJ627218 KJ627195 KJ627206 Pol14/Krus 2014 Feb 17 KJ627219 KJ627196 KJ627207 * CRV, central variable region; DP, domestic pig; EWB, European wild boars.
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|Author:||Gallardo, Carmina; Fernandez-Pinero, Jovita; Pelayo, Virginia; Gazaev, Ismail; Markowska-Daniel, Iwo|
|Publication:||Emerging Infectious Diseases|
|Date:||Sep 1, 2014|
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