Evolutionary phylodynamics of foot and mouth disease virus serotypes O and A circulating in Vietnam

Le et al BMC Veterinary Research (2016) 12:269 DOI 10.1186/s12917-016-0896-0 RESEARCH ARTICLE Open Access Evolutionary phylodynamics of foot-andmouth disease virus serotypes O and A circulating in Vietnam Van Phan Le1*, Thi Thu Hang Vu2, Hong-Quan Duong3, Van Thai Than4* and Daesub Song5 Abstract Background: Foot-and-mouth disease virus (FMDV) is one of the highest risk factors that affects the animal industry of the country The virus causes production loss and high ratio mortality in young cloven-hoofed animals in Vietnam The VP1 coding gene of 80 FMDV samples (66 samples of the serotype O and 14 samples of the serotype A) collected from endemic outbreaks during 2006–2014 were analyzed to investigate their phylogeny and genetic relationship with other available FMDVs globally Results: Phylogenetic analysis indicated that the serotype O strains were clustered into two distinct viral topotypes (the SEA and ME-SA), while the serotype A strains were all clustered into the genotype IX Among the study strains, the amino acid sequence identities were shared at a level of 90.1–100, 92.9–100, and 92.8–100% for the topotypes SEA, ME-SA, and genotype IX, respectively Substitutions leading to changes in the amino acid sequence, which are critical for the VP1 antigenic sites were also identified Our results showed that the studied strains are most closely related to the recent FMDV isolates from Southeast Asian countries (Myanmar, Thailand, Cambodia, Malaysia, and Laos), but are distinct from the earlier FMDV isolates within the genotypes Conclusions: This study provides important evidence of recent movement of FMDVs serotype O and A into Vietnam within the last decade and their genetic accumulation to be closely related to strains causing FMD in surrounding countries Keywords: Foot-and-mouth disease virus, Serotype O, Serotype A, Vietnam Background Foot-and-mouth disease virus (FMDV), causing footand-mouth disease (FMD), is a contagious virus affecting cloven-hoofed domestic (pig, cattle, goat, and sheep) and wild animals FMDV has been detected in >100 countries worldwide, mostly in Asia, Africa, and the Middle East [1] The FMD causes economic losses to the livestock population and reduces food security and economic development For this reason, FAO and OIE have launched a necessary strategy for global FMD control FMDV, a picornavirus, is the prototypical member of the Aphthovirus genus within the Picornaviridae family * Correspondence: letranphan@vnua.edu.vn; thai@cau.ac.kr Department of Microbiology and Infectious Disease, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi, Vietnam Department of Microbiology, Chung-Ang University College of Medicine, Seoul, South Korea Full list of author information is available at the end of the article The virus particle is about 25–30 nm in diameter and roughly spherical in shape [2] Similar to that of other picornaviruses, the FMDV genome organization consists of a large single open reading frame that encodes for the structure proteins, VP4, VP2, VP3, and VP1 (also known as 1A, 1B, 1C, and 1D, respectively), in which VP1, VP2, and VP3 are surface proteins; while VP4 is located internally FMDV is well identified as having seven immunological distinct serotypes, including serotype O, A, C, Asia 1, and the South African Territories (SAT) serotypes (including SAT1, SAT2, and SAT3) subsequently with numerous identified subtypes [3] The infection of a single viral serotype does not confer, in consequence, the full protection against the infection of other viral serotypes [4] The FMDV serotype A has been considered to be one of the most antigenically diverse among the seven serotypes [5, 6] The FMDV serotype A has been classified into 10 major genotypes © The Author(s) 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Le et al BMC Veterinary Research (2016) 12:269 (designated as I to X) based on the VP1 phylogenetic trees [5, 6] FMDV serotype O is classified into 11 topotypes, designated as Europe-South America (Euro-SA), Middle East-South Asia (ME-SA), Southeast Asia (SEA), Cathay (CHY), West Africa (WA), East Africa (EA-1), East Africa (EA-2), East Africa (EA-3), East Africa (EA-4), Indonesia-1 (ISA-1), and Indonesia-2 (ISA-2) [7, 8]; and FMDV serotype Asia is classified into seven genotypes (designated as I to VII) [9] FMD is an endemics and widespread disease in African, Asia, and its further spread into the FMD-free areas like American, Europe, and Australia is a direct threat [1] Among seven serotypes of FMDV, serotype O and A have been distributed extensively and are responsible for outbreaks in Asia and Africa; the three SAT serotypes have been generally restricted in their distribution to Africa, and the serotype Asia has never been found outside of Asia [10, 11] Agriculture plays an important role in the national economy of Vietnam, in which animal production contributes about approximately 32% to the total GDP FMD is considered the most economically important infectious disease affecting domestic cattle, buffalo, and pigs The concurrent circulation of FMDV serotypes O, A, and Asia are detected in which the serotype O remains the most prevalent and is responsible for the highest numbers of outbreaks [12, 13] The FMDV serotype O and its outbreak in Vietnam were first described in academic research between 1996 and 2001 [14] The FMDV serotype Asia and A were subsequently identified in 2005 and 2009, respectively [12, 15] In 2008 and the first months of 2009, the FMDV serotypes O and A were reported to be the prevalent serotype and caused approximately 166 FMD outbreaks in 128 communes in 47 districts of 14 provinces throughout the country [16] Currently, limited information is available regarding the genetic characteristics and geographical distribution of the FMDV serotype O and A causing sporadic outbreaks in Vietnam In this study, the VP1 coding gene of 80 FMDV samples (66 samples of the serotype O and 14 samples of the serotype A) collected from endemic outbreaks during 2006–2014 were analyzed to investigate their phylogeny and genetic relationship with other available FMDVs globally These data will provide important evidence of recent movement of FMDVs serotype O and A into Vietnam within the last decade and their genetic characterization with strains causing FMD in neighboring countries Methods Sample collection and virus isolation A total of 80 FMD-positive samples were collected in a passive surveillance program from 19 provinces located in north and northern central Vietnam during 2006–2014 Page of 11 (Table 1, Fig 1) All the virus isolates were initially confirmed by FMDV antigen ELISA (WRL Pirbright, UK) and then isolated from the BHK-21 cell culture system with subsequence passages Briefly, BHK-21 cells were cultured in minimum essential medium (MEM; Gibco BRL, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS; Gibco BRL) and 0.1% Gentamicin (Gentamicin Reagent Solution, Gibco BRL) at 37 °C in a humidified atmosphere containing 5% CO2 The epithelial homogenate was centrifuged at 10,000 g for 10 min, and the supernatant was then filtered by using a 0.45-μm sterile syringe filter (Corning Costar, Corning, NY, USA) The filtered samples were inoculated into a monolayer of BHK-21 cells at 37 °C for h, followed by two washes with MEM media The infected cells were maintained in MEM media containing 2% FBS Infected cells were harvested after 2– days post-infection and were subsequently passed into the BHK-21 cells until cytopathic effects appeared The names of the isolates were assigned as follow: serotype, country code, laboratory record number, and the year of sample collection, and stored at −80 °C for further exam (Additional file 1) RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) The viral RNA was extracted from infected cell culture supernatants using the QIAamp viral RNA mini kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions The cDNA step was performed using the Superscript™ III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions The primer set of the VN-VP1F/VN-VP1R (VNVP1F: 5′-AGYGCYGGYAARGAYTTTGA-3′, VP1R: 5′CATGTCYTCYTGCATCTGGTT-3′) was used for the PCR-amplification of DNA fragments containing the 639 nt length of the VP1 coding region [17] Briefly, the reaction was carried out at 42 °C for 60 (reverse transcription), 35 cycles of 95 °C for (for denaturation), 52 °C for (for annealing), and 72 °C for (for extension), followed by 72 °C for 10 (for final extension) The PCR products were separated on 1.2% SeaKem LE agarose gel and viewed on a BioRad Gel Doc XR image-analysis system Nucleotide sequencing and sequence analysis Capsid VP1 is the most studied FMDV protein because of its significance for virus attachment and entry, protective immunity, and serotype specificity [3, 18, 19] The amplified capsid VP1 PCR products were either purified with QIAquick PCR purification kit or QIAquick gel extraction kit according to the manufacturer’s instructions (Qiagen) RT-PCR primers were used for the direct sequencing of internal gene segments by using Le et al BMC Veterinary Research (2016) 12:269 Page of 11 Table Origin of the serotypes O and A FMDVs sisolated in this study Year ofisolation Province Host Number of sample Type Topotype 2006 Vinh Phuc Buffalo O SEA Thai Nguyen Ha Noi Lang Son Son La 2007 2008 2009 2010 2013 2014 Buffalo O SEA Cattle O SEA Cattle O SEA Pig O SEA Cattle O SEA Pig O SEA Cattle O SEA Pig O SEA Buffalo O SEA Lai Chau Buffalo O SEA Thai Nguyen Cattle O SEA SEA Ha Tinh Cattle O Nghe An Buffalo A Genotype IX Cattle A Genotype IX Yen Bai Buffalo O SEA Son La Buffalo O SEA Pig O SEA Ha Giang Cattle O SEA Tuyen Quang Buffalo O SEA Nghe An Cattle O SEA Quang Ninh Buffalo O SEA Hoa Binh Buffalo O SEA Cattle O SEA Buffalo A Genotype IX Lang Son Buffalo O SEA Lao Cai Cattle O SEA Phu Tho Cattle O SEA Buffalo A Genotype IX Bac Can Buffalo A Genotype IX Cattle A Genotype IX Ha Giang Cattle A Genotype IX Quang Tri Buffalo A Genotype IX Son La Buffalo O SEA Lao Cai Buffalo O SEA Dien Bien Cattle O SEA SEA Tuyen Quang Buffalo O Yen Bai Cattle O SEA Buffalo O SEA Pig O ME-SA Bovine A Genotype IX Ha Noi Quang Tri Bovine O ME-SA Ha Tinh Bovine A Genotype IX Ha Noi Pig O ME-SA Ha Nam Pig O SEA Bovine O SEA Le et al BMC Veterinary Research (2016) 12:269 Page of 11 China Ha Giang Lai Chau Lao Cai Dien Bien Tuyen Quang Bac Can Lang Son Yen Bai Thai Nguyen Vinh Phuc Son La Ha Noi Phu Tho Quang Ninh Ha Nam Hoa Binh Nghe An Ha Tinh Laos Quang Tri Hue Gia Lai Cambodia Ho Chi Minh city Ca Mau Fig Map of Vietnam showing the provinces (red) from which the FMDV isolates were collected during outbreaks Le et al BMC Veterinary Research (2016) 12:269 Page of 11 2009-2010 2006-2007 O/VN/TN03/2006 GU582107 O/VN/TN125/2007 GU582111 O/VN/HN18/2006 GU582095 O/VN/VP38/2006 GU582096 O/VN/TN75/2006 GU582099 O/VN/LS08/2006 GU582105 O/VN/LS91/2006 GU582102 O/VN/TN85/2006 GU582094 O/VN/LC039/2007 GU582110 O/VN/SL06622/2006 GU582109 O/VN/SL22/2006 GU125647 O/VN/SL01/2006 GU125648 O/VN/SL601/2006 GU582101 O/VN/SL12/2006 GU582100 O/VN/SL6601/2006 GU582106 O/VN/SL21/2006 GU125649 O/VN/SL6622/2006 GU582108 O/VN/HN83/2006 GU582103 O/VN/SL607/2006 GU582104 O/VN/SL622/2006 GU582098 O/VN/SL07/2006 GU582097 SonLa-2/VIT/06 GQ855799 Hanoi/VIT/06 GQ855801 SonLa-3/VIT/06 GQ855800 100 SonLa-1/VIT/06 GQ855802 O/VN/TN089/2007 GU582112 88 O/VIT/4/2006 HQ116287 95 O/VIT/6/2006 HQ116289 99 O/VIT/5/2006 HQ116288 72 O/VIT/7/2006 HQ116290 99 O/VN/GL13/2006 GU125650 HaTinh/VIT/07 GQ855805 100 66 NgheAn/VIT/07 GQ855804 O/VN/HT016/2007 GU582113 O/VIT/6/2005 HQ116279 O/MYA/7/2002 DQ164928 93 O/MAY/6/2001 DQ164922 72 O/MAY/5/2001 DQ164921 56 O/LAO/4/2001 DQ164907 99 O/LAO/2/2001 EU667445 O/LAO/7/2003 EU667448 O/VN/LC169/2009 HM055510 50 O/VN/YB10/2010 HQ260720 O/VN/YB08/2010 HQ260718 100 O/VN/YB09/2010 HQ260719 87 O/VN/HB167/2009 HM055509 O/VIT/4/2005 HQ116278 96 94 O/TAI/4/99 AJ303536 65 O/MYA/5/99 DQ164926 95 O/MAY/3/2001 DQ164920 O/MYA/1/98 AJ303521 O/MYA/2/2000 DQ164927 O/VN/QN132/2009 GU582120 O/VN/HB162/2009 HM055498 O/VN/HB166/2009 HM055499 O/VN/YB106/2009 GU582117 O/VN/QN133/2009 GU582121 O/VN/HB138/2009 GU582122 O/VN/TQ06/2010 HQ260717 O/VN/LC03/2010 HQ260715 O/VN/TQ160/2009 HM055497 O/VN/DB04/2010 HQ260716 O/VN/HG128/2009 GU582119 O/VN/SL191/2009 HM055507 O/VN/SL192/2009 HM055508 O/VN/SL186/2009 HM055505 O/VN/YB176/2009 HM055503 O/VN/SL187/2009 HM055506 O/VN/SL145/2009 HM055496 O/VN/LS185/2009 HM055504 100 O/VN/NA79/2009 GU582114 O/VN/LC168/2009 HM055500 O/VN/SL109/2009 GU582118 O/VN/HB139/2009 HM055494 O/VN/SL144/2009 HM055495 O/VN/PT171/2009 HM055502 99 O/VN/PT170/2009 HM055501 O/VN/SL01/2010 HQ260713 O/VN/SL02/2010 HQ260714 O/VN/HN2/2014 KM588389 100 O/VN/HN3/2014 KM588390 O/VN/HN6/2014 KM588392 O/VN/HN5/2014 KM588391 O/CAM/1/98 AJ294908 100 O/CAM/2/98 AJ294909 65 O/CAM/6/89 AJ318827 88 O/CAM/11/94 AJ294906 94 O/VIT/7/97 AJ296328 100 O/LAO/1/98 EU667440 O/LAO/4/98 DQ164906 O/MYA/13/89 DQ164924 O/VIT/17/99 AJ318858 O/VIT/7/2002 HQ116273 O/VIT/6/2002 DQ165020 O/VIT/20/2002 DQ165029 57 O/VIT/9/2002 DQ165022 95 O/VIT/8/2002 DQ165021 O/VIT/10/2002 DQ165023 O/VIT/3/2005 HQ116277 O/IRQ/30/2000 AJ303499 100 O/VIT/14/2002 DQ165026 75 O/VIT/1/2003 DQ165030 O/VIT/17/2005 HQ116283 O/VIT/1/2004 DQ165032 64 O/GRE/27/96 AJ303491 77 O/SAU/100/94 AJ004660 95 O/NEP/46/95 AJ303523 88 O/JOR/3/96 AJ303510 O/SRL/2/97 AJ303531 96 O/VN/HN11/2014 KM588397 O/VN/HN12/2014 KM588398 O/VN/HN10/2014 KM588396 98 O/VN/HN7/2014 KM588393 O/VN/HN9/2014 KM588395 O/VN/HN2/2013 KM588384 98 O/VN/QT8/2013 KM588387 O/VN/HN8/2014 KM588394 88 100 O/VN/QT9/2013 KM588388 O/VN/QT7/2013 KM588386 O/VN/QT6/2013 KM588385 80 O/VIT/16/2002 DQ165027 O/VIT/2/2003 DQ165031 97 O/VIT/19/2002 DQ165028 73 O/VIT/12/2002 DQ165024 84 O/VIT/8/2004 HQ116275 O/VIT/7/2005 HQ116280 O/VIT/7/2004 HQ116274 KhanhHoa/VIT/06 GQ855798 O/A/CHA/58 AJ131469 99 O/KEN/83/79 AJ303511 99 O/KEN/2/95 AJ303514 O/UGA/5/96 AJ296327 100 O/ALG/1/99 AJ303481 99 O/CIV/8/99 AJ303485 O/GHA/5/93 AJ303488 99 O/VIT/3/2004 DQ165034 89 O/VIT/11/2005 HQ116282 95 O/VIT/2/2004 DQ165033 72 ThuaThienHue/VIT/07 GQ855803 100 O/VIT/2/99 AJ318857 O/VIT/13/2002 DQ165025 66 O/HKN/16/96 AJ294923 59 O/1685/Moscow/Russia/95 AJ004680 100 O/TAW/81/97 AJ296321 74 O/TAW/83/97 AJ296322 81 O/PHI/7/96 AJ294926 99 O/VIT/3/97 AJ294930 O/VIT/1/2006 HQ116284 100 O/VIT/1/2008 HQ116291 O/VIT/2/2006 HQ116285 98 O/VIT/3/2006 HQ116286 99 O/VIT/9/2005 HQ116281 O/VIT/1/2005 HQ116276 100 O/GD/China/86 AJ131468 O/HKN/14/82 AJ294917 100 O/ISA/9/74 AJ303502 O/ISA/8/83 AJ303503 O/ISA/1/62 AJ303500 100 O/Caseros/ARG/82 M89900 O/Yrigoyen/ARG/82 Z21862 O1/Campos/Brazil/71 K01201 O1/Lombardy/ITL/46 M58601 SEA 2009, 2014 98 Mya-98 88 2013-2014 82 68 66 84 100 64 99 99 0.1 Fig (See legend on next page.) PanAsia ME-SA EA WA Cathay ISA Euro-SA A/LAO/36/2003 EU667455 (Out-group) Le et al BMC Veterinary Research (2016) 12:269 Page of 11 (See figure on previous page.) Fig Phylogenetic tree based on the complete nucleotide sequence of the VP1 coding region of type O FMDVs showing relationships between the study strains and other type O representatives worldwide The Vietnamese strains are marked in bold Numbers at nodes indicate the level of bootstrap support based on the neighbor-joining analysis of 1000 resampled datasets Only values above 50% are given A bar represents 0.1 substitutions per nucleotide position a BigDye terminator cycle sequencing kit and an automatic DNA sequencer (Model 3730, Applied Biosystems, Foster City, CA, USA) The obtained nucleotide and deduced amino acid sequences in this study were aligned using the ClustalX 2.1 program [20] and Lasergene software (DNASTAR; Madison, WI, USA) by using the parameters set against the corresponding FMD viral sequences from the NCBI GenBank Phylogenetic analysis The complete nucleotide sequences of the VP1 coding gene from the 80 FMDV samples examined in this study were compared against a representative VP1 coding gene from the available FMDV sequences in the GenBank database Phylogenetic trees were constructed by the neighbor-joining algorithm put into practiced with MEGA 6.06 software suite [21] The bootstrap resampling method with 1000 replicates was used to evaluate the topology of the phylogenetic tree The pairwise distance was calculated using MEGA 6.06 software package [21] Results Detection of FMDV genome and genome sequencing The viral RNA was sufficiently extracted from 80 FMDV samples (66 samples of the serotype O and 14 samples of the serotype A) (Table 1) The VP1 coding gene was successfully amplified by RT-PCR using the primer set as described above The full length sequence of the VP1 coding gene was determined by direct sequencing of the PCR amplicons Thereafter, the obtained nucleotide sequences have been deposited in the NCBI GenBank database under the accession numbers described in Additional file Genetic diversity and phylogenetic analyses of the VP1 coding gene For the FMDV serotype O, the nucleotide sequence identity among 66 FMDV serotype O isolates showed diversity at a level of 79.1–100% These strains shared nucleotide sequence identity at 100, 91.5–97.8, 86.2–100, 89.9–99.8, 94.2–99.3 and 83.8–99.8% in 2006, 2007, 2009, 2010, 2013 and 2014, respectively Moreover, phylogenetic analysis demonstrated that the VP1 coding gene of the FMDV serotype O was clustered into two distinct viral topotypes, the SEA (lineage Mya-98) and ME-SA (lineage PanAsia) (Fig 2) Most of the strains were clustered into the SEA topotype, while only the strains isolated between 2013 and 2014 were clustered into the ME-SA topotype (Fig 2) The study strains shared nucleotide identities at a level of 89.0–92.6 and 84.1–86.6% compared to the O/MYA/2/ 2000/SEA (SEA) and O/A/CHA/58 (ME-SA) prototype strains, respectively [7, 22] For FMDV serotype A, the VP1 nucleotide sequence identity among the study strains was 89.3–100% Phylogenetic analysis indicated that the VP1 coding gene of the 14 FMDV serotype A strains was classified within the genotype IX (topotype Asia), together with other Vietnamese strains isolated during 2004–2009 (Fig 3) The study strains showed nucleotide identity at a level of 87.9–91.1% compared to the A/TAI/118/87 prototype strain Moreover, these FMDV serotype A showed genetic diversity among the strains circulating in Vietnam in the 2004–2005, 2008–2009, and 2013 seasons by grouping into three distinct sub-clusters (Fig 3) Comparison of VP1 amino acid sequences The deduced amino acid sequences obtained from the VP1 gene segments were aligned and compared to investigate the consequences of the observed genetic characterization of FMDV serotype O and A in Vietnam during 2006–2014 For the FMDV serotype O, the O/MYA/2/2000/SEA and O/A/CHA/58 prototype strains were used as references Among the SEA topotype (lineage Mya-98), amino acid sequence identities significantly showed genetic variation of 90.1–100% within the Vietnamese strains and 89.6–93.4% to the O/MYA/2/2000 prototype strain Compared to the O/MYA/2/2000/SEA prototype strain, seven amino acid substitutions were detected at positions 14, 24, 51, 93, 111, 131, and 184 (Figs and 5) Among the ME-SA topotype (lineage PanAsia), the study strains shared the amino acid identity at a level of 92.9–100% and shared an identity with O/A/CHA/58 prototype strain at a level of 86.8–93.4% Eight amino acid substitutions were detected at positions 34, 56, 87, 127, 138, 139, 143, and 158 (Figs and 5) Notably, the change of the two amino acids at positions 45K-Q and 154K-R occurring at residues among the SEA topotypes, as well as the change of two amino acids at positions 43T-V and 208P-H occurring at residues among the ME-SA topotypes (Figs and 5) For the FMDV serotype A, the A/TAI/118/87 prototype strain was used as a reference strain for genotype IX Within the genotype IX (topotype Asia), the study strains shared 92.8–100% at the amino acid level and shared an identity with the A/TAI/118/87 prototype strain at level of 91.1–93.4% Compared to the A/TAI/ Le et al BMC Veterinary Research (2016) 12:269 Page of 11 A/VN/04/2009 GU582083 A/VIT/2/2009 HQ116378 A/VIT/4/2008 HQ116372 A/VIT/8/2009 HQ116384 A/VN/130/2009 GU582085 95 A/VN/131/2009 GU582086 A/VN/15/2009 GU582092 A/VIT/2/2008 HQ116370 A/VN/14/2009 GU582091 A/VIT/5/2008 HQ116373 A/VIT/8/2008 HQ116376 A/VN/09/2009 GQ406247 A/VIT/1/2009 HQ116377 A/VN/02/2009 GQ406248 A/VN/03/2009 GQ406249 A/VIT/3/2008 HQ116371 2008-2009 A/VN/11/2009 GQ406250 A/VIT/4/2009 HQ116380 A/VIT/3/2009 HQ116379 A/VIT/7/2008 HQ116375 A/VN/93/2008 GU582090 A/VIT/6/2008 HQ116374 A/VN/20/2009 GQ406252 A/VIT/7/2009 HQ116383 A/VIT/5/2009 HQ116381 A/VN/13/2009 GU582084 A/VN/89/2008 GU582087 A/VN/22/2009 GU582093 99 A/VN/16/2009 GQ406251 A/VIT/6/2009 HQ116382 A/VN/90/2008 GU582088 A/VN/91/2008 GU582089 A/VIT/10/2004 HQ116362 96 75 A/VIT/6/2004 HQ116360 A/VIT/4/2004 HQ116358 55 A/VIT/10/2005 HQ116366 100 A/VIT/8/2005 HQ116365 A/VIT/13/2005 HQ116367 2004-2005 A/VIT/12/2004 HQ116364 A/VIT/9/2004 HQ116361 98 A/VIT/5/2004 HQ116359 A/VIT/11/2004 HQ116363 51 100 A/VIT/14/2005 HQ116368 A/VIT/18/2005 HQ116369 A/VN/HN14/2013 KM588382 100 2013 A/VN/HT20/2013 KM588383 A/VN/HN13/2013 KM588381 A/MAY/2/2002 EU414533 A/TAI/118/87 EF208777 A/A8/Parma/ITL/50 EU553860 100 99 A/A7/GRE/50 EU553857 53 A/A24-Argentina/1965 AY593767 A/a24cruzeiro-iso71 AY593768 100 99 A/A24/Cruzeiro/Brazil/55 AJ251476 A/A10-Holland/1942 NC-011450 100 A/IND/55/86/1986 AF390655 A/a22iraq64-iso86 AY593763 89 A/a22iraq70-iso92 AY593764 100 A/a22iraq-95-iso95 AY593762 A/Talasskiy-A22 FJ623456 A/a22turkey-iso66 AY593765 94 76 A/IND/81/2000 AF390666 100 A/IND/80/2000 AF390665 A/IND/24/2001 AF390624 A/airan-iso105 AY593791 100 A/IRN/1/96 EF208771 A/NM/XZ/64 AJ131664 85 99 A/NM/EL/60 AJ131663 A/GS/LX/62 AJ131666 A/XJ/KT/58/III/AJ131665 A/IND/455/98 AF390650 98 100 A/IND/236/99 AF390622 A/IND/68/2001 AF390659 A/Nigde/TUR/11/07/97 EF126163 76 99 A/Samsun/TUR/8/08/96 EF126161 A/Samsun/TUR/10/06/96 EF126162 ASIA topotype 54 85 96 86 64 71 99 0.1 Fig (See legend on next page.) Genotype IX Genotype II Genotype V Genotype I Genotype IV Genotype VII Genotype X Genotype III Genotype VI Genotype VIII O/VN/TN85/2006/GU582094 (Out-group) Le et al BMC Veterinary Research (2016) 12:269 Page of 11 (See figure on previous page.) Fig Phylogenetic tree based on the complete nucleotide sequence of the VP1 coding region of type A FMDVs showing relationships between the study strains and other type A representatives worldwide The Vietnamese strains are marked in bold Numbers at nodes indicate the level of bootstrap support based on the neighbor-joining analysis of 1000 re-sampled datasets Only values above 50% are given A bar represents 0.1 substitutions per nucleotide position 118/87 prototype strain, four amino acid substitutions were detected at positions 138, 139, 153, and 195 (Fig 6) An amino acid changes in the antigenic sites noted at position 148S-P of the A/VN/130/2009 and A/VN/131/ 2009 strains (Fig 6) Discussion The sequences of the VP1 coding gene are widely used to identify and characterize FMDV lineages and sub-lineages [23] In addition, the VP1 capsid protein is the most helpful protein to investigate the relationship between different isolates of the FMDV because of its significance for viral attachment and entry, protective immunity, and serotype specificity [18, 19] In this study, we used the VP1 coding region of the FMDV serotype O and type A isolated from the north and northern central regions of Vietnam during Amino acid Strain name O/MYA/2/2000 (ref) O/VN/SL07/2006, n=13 O/VN/TN75/2006, n=1 O/VN/VP38/2006, n=1 O/VN/TN03/2006, n=1 O/VN/LC039/2007, n=1 O/VN/TN089/2007, n=1 O/VN/TN125/2007, n=1 O/VN/HT016/2007, n=1 O/VN/HB138/2009, n=1 O/VN/HB139/2009, n=1 O/VN/HB162/2009, n=1 O/VN/HB167/2009, n=1 O/VN/QN132/2009, n=1 O/VN/YB176/2009, n=1 O/VN/TQ160/2009, n=1 O/VN/SL144/2009, n=1 O/VN/SL191/2009, n=9 O/VN/PT170/2009, n=1 O/VN/LC168/2009, n=1 O/VN/LC169/2009, n=1 O/VN/TQ06/2010, n=1 O/VN/LC03/2010, n=1 O/VN/DB04/2010, n=1 O/VN/SL01/2010, n=1 O/VN/SL02/2010, n=1 O/VN/YB08/2010, n=3 O/VN/HN2/2014, n=1 O/VN/HN3/2014, n=1 O/VN/HN5/2014, n=1 O/VN/HN6/2014, n=1 O/A/CHA/58 (ref) O/VN/HN2/2013, n=1 O/VN/QT6/2013, n=1 O/VN/QT7/2013, n=1 O/VN/QT8/2013, n=1 O/VN/QT9/2013, n=1 O/VN/HN7/2014, n=1 O/VN/HN8/2014, n=1 O/VN/HN9/2014, n=1 O/VN/HN10/2014, n=1 O/VN/HN11/2014, n=1 O/VN/HN12/2014, n=1 2006–2014 for the determination of their phylogeny and genetic relationships with other available Vietnamese and global FMDV strains in the NCBI GeneBank database Importantly, the 19 provinces enrolled in this study have historically been highly affected by FMDVs and share both a border and a trade of animals with the highly affected FMDV countries of China, Laos, Cambodia, and Thailand [9, 14, 16] The FMDV serotype O is the most prevalent out of the seven serotypes that circulate in many parts of the world The FMD outbreaks of type O viruses were first identified in 1987 from FMD outbreaks in Europe by analysis of its nucleotide sequence [24] Based on the accumulation of VP1 genome sequences, 10 topotypes of FMDV serotype O were designated as the Euro-SA, ME-SA, SEA, CHY, WA, EA-1, EA-2, EA-3, ISA-1, and ISA-2 The topotypes 10 20 30 40 50 60 70 80 90 100 | | | | | | | | | | | | | | | | | | | | TTSTGESADPVTATVENYGGETQVQRRHHTDISFILDRFVKVTPKDQINVLDLMQTPPHTLVGALLRTATYYFADLEVAVKHEGDLTWVPNGAPEAALDN T TT .V Q L S N .A T E L S N .A T E L S N .A T E L S N .A T E L S N .A T E .T TT .V Q L S N .A T E .T TT .V Q D L S N .A T E .T .T .V Q L S N .A E .TD T .V Q L .A G .T G T .V Q L .A G .T .T .V Q L .A G .T .APT S.V.Q V S L .A D .T .T .V Q L .A G .T .S.T .V Q LNQ AA.N S A G .T .T .V Q L .A G .T .T .V V .Q L .A G .T .T .V Q L .A G .T .T .V V .Q L .A G .I Q L .A G .T .T .V S L .A D .T .T .V Q L .A T G .T .T .V Q L .A G .T .T .V QG L .A G .T .T .V Q L .A G .T .T .V Q L .A G .T .T .V S L .A D .A .T .V LN S K .A T E .A .T .V L S A T E .RS T .V L S A T E .A .T .V L S A T E TTSPGESADPVTATVENYGGETQVQRRQHTDVSYILDRFVKVTPKDQINVLDLMQIPAHTLVGALLRTATYYFADLEVAVKHKGNLKWVPNGAPETALDN T R F T E T T R .F T.V T T A F T E T T F T E T TD F T E T C TD R F T E T T F T E T T R F T G E T GR G.E D T R F T E T T R F T E T T .I T .R F T E T Fig Deduced amino acid sequences of the VP1 proteins (aa 1-100) of the type O FMDVs in this study Only sequences different from the consensus are shown Strains with similar profiles of the antigenic site are grouped together Similar amino acid sequences of the two reference trains, the O/MYA/2/2000 strain (for the SEA group) and the O/A/CHA/58 strain (for the ME-SA group), are shadowed The VP1 antigenic sites are at amino acid position 43, 44, 45, 144, 148, 149, 154, and 208 The “n” represents the strains with similar profiles to the antigenic site Le et al BMC Veterinary Research (2016) 12:269 Amino acid Strain name O/MYA/2/2000 (ref) O/VN/SL07/2006, n=13 O/VN/TN75/2006, n=1 O/VN/VP38/2006, n=1 O/VN/TN03/2006, n=1 O/VN/LC039/2007, n=1 O/VN/TN089/2007, n=1 O/VN/TN125/2007, n=1 O/VN/HT016/2007, n=1 O/VN/HB138/2009, n=1 O/VN/HB139/2009, n=1 O/VN/HB162/2009, n=1 O/VN/HB167/2009, n=1 O/VN/QN132/2009, n=1 O/VN/YB176/2009, n=1 O/VN/TQ160/2009, n=1 O/VN/SL144/2009, n=1 O/VN/SL191/2009, n=9 O/VN/PT170/2009, n=1 O/VN/LC168/2009, n=1 O/VN/LC169/2009, n=1 O/VN/TQ06/2010, n=1 O/VN/LC03/2010, n=1 O/VN/DB04/2010, n=1 O/VN/SL01/2010, n=1 O/VN/SL02/2010, n=1 O/VN/YB08/2010, n=3 O/VN/HN2/2014, n=1 O/VN/HN3/2014, n=1 O/VN/HN5/2014, n=1 O/VN/HN6/2014, n=1 O/A/CHA/58 (ref) O/VN/HN2/2013, n=1 O/VN/QT6/2013, n=1 O/VN/QT7/2013, n=1 O/VN/QT8/2013, n=1 O/VN/QT9/2013, n=1 O/VN/HN7/2014, n=1 O/VN/HN8/2014, n=1 O/VN/HN9/2014, n=1 O/VN/HN10/2014, n=1 O/VN/HN11/2014, n=1 O/VN/HN12/2014, n=1 Page of 11 110 120 130 140 150 160 170 180 190 200 210 | | | | | | | | | | | | | | | | | | | | | | TTNPTAYHKAPLTRLALPYTAPHRVLATVYNGNCKYAEGSLTNVRGDLQVLAQKAARPLPTSFNYGAIKATRVTELLYRMKRAETYCPRPLLAVHPNEARHKQKIVAPVKQSL P N R P E A P N R SP E A P N R P .S .E A P N R.T P E A P N R P E A P N R P N E A F P N R P E A P N R RV E N Y P N K G.P E Q W P N K G.P E Q W P A N K G.P .I .E Q W P N K G E SA P N K G.P T .E Q W P N K G.P E Q W P N K G.L E Q W P N K G.P E Q W P N K G.P E Q W P N K G.P E Q W P N K G.P E Q W P N K G E SA A .P N K G.P E Q W P N FK G.P EM Q W P N K G.P E Q W A P N K G.P E Q W P N K G.P E Q W P N K G E SA P N K E QS W P N K E F QS W P N K E QS W P N K E QS W TTNPTAYHKAPLTRLALPYTAPHRVLGTVYNGNCKYGVGPVTKVRGDLQVLAQKAARSLPTSFNYGAIKATRVTELLYRMKRAETYCPRPLLAIHPSAARHKQKIVAPGKRLL A G.R AS N TP T Q .G R .V.Q A AS N T .E V.Q A .R AS N T .E V.Q A .R AS N T .E V.Q A .R AS N T .E .G V.Q A D.R AS.M.N TP T P .E R .V.Q A .R AS N T .E V.Q K A D.R AS.M.N TP T K .Q .E R .V.Q A G.R ASSM.N TP T Q F RET HVRE A G.R AS.M.N TP T Q .E R .V.Q A G.R AS.M.N TP T Q .E R .V.Q Fig Deduced amino acid sequences of the VP1 proteins (aa 101-213) of the type O FMDVs in this study Only sequences different from the consensus are shown Strains with similar profiles of the antigenic site are grouped together Similar amino acid sequences of the two reference trains, the O/MYA/2/2000 strain (for the SEA group) and the O/A/CHA/58 strain (for the ME-SA group), are shadowed The VP1 antigenic sites are at amino acid position 43, 44, 45, 144, 148, 149, 154, and 208 The “n” represents the strains with similar profiles to the antigenic site Amino acid Strain name A/TAI/118/87 (ref) A/VN/89/2008 A/VN/90/2008 A/VN/91/2008 A/VN/93/2008 A/VN/04/2009 A/VN/13/2009 A/VN/14/2009 A/VN/15/2009 A/VN/22/2009 A/VN/130/2009 A/VN/131/2009 A/VN/HN13/2013 A/VN/HN14/2013 A/VN/HT20/2013 10 20 30 40 50 60 70 80 90 100 | | | | | | | | | | | | | | | | | | | | TTATGESADPVTTTVENYGGETQAQRRHHTDVSFIMDRFVRIQHVSPTHVIDLMQTHQHGLVGALLRAATYYFSDLEIVVRHTGNLTWVPNGAPEAALSN -P M .N R D - .V Q.KP M .N R D .V V Q.KP M .N R D V Q.KP M .N R D .V R .L Q.KP M .N R D .V R .L Q.KP M .N R D .T Q.KP I I .N .N .T Q.KP I I .N .N .T Q.KP I I .N .N Amino acid Strain name A/TAI/118/87 (ref) A/VN/89/2008 A/VN/90/2008 A/VN/91/2008 A/VN/93/2008 A/VN/04/2009 A/VN/13/2009 A/VN/14/2009 A/VN/15/2009 A/VN/22/2009 A/VN/130/2009 A/VN/131/2009 A/VN/HN13/2013 A/VN/HN14/2013 A/VN/HT20/2013 110 120 130 140 150 160 170 180 190 200 210 | | | | | | | | | | | | | | | | | | | | | | TSNPTAYHKEPFTRLALPYTAPHRVLATVYNGTSKYSGSVTRRGDLGSLAARVAAQLPASFNFGAIRATEIQELLVRMKRAELYCPRPLLAVEVSSQDRHKQKIIAPAKQLL A APA L .Y .T APA L .Y .T APA L .Y .T R APA E .L .Y .T .A APA L .Y .T Y .A APA L .Y .T .A AP L .Y .T APA V.L .Y .T S APA L .Y .T .A APA .P L .Y .T S .A APA .P L P Y .T S P A APTP L .T .F P A APTP L .T .F P A APTP L .T .F Fig Deduced amino acid sequences of the VP1 proteins of the type A FMDVs in this study Only sequences different from the consensus are shown Strains with similar profiles of the antigenic site are grouped together The VP1 antigenic sites are at amino acid position 43, 44, 45, 144, 148, 149, 154, and 208 Le et al BMC Veterinary Research (2016) 12:269 ME-SA and SEA highly affect China, the Indian subcontinent (India, Pakistan, Bangladesh, Sri Lanka, Nepal, and Bhutan), and Southeast Asian countries (Myanmar, Thailand, Cambodia, Malaysia, Laos, and Vietnam) The Vietnamese FMDV serotype O fell within the ME-SA (lineage Mya-98), SEA (lineage PanAsia), and Cathay topotypes in which the isolates form distinct genetic sublineages and are distant from the prototype isolates These findings highlight that these topotypes might have adapted in recent years to circulate in Vietnam The FMDV serotype A has been reported in all FMDV infected areas around the world Based on the phylogeny analysis of the VP1 capsid genes, the global FMDV serotype A is divided into 10 major genotypes (designated as I to X) with over 15% nucleotide divergence [5, 6] In Vietnam, these viruses were reported to be predominant during the outbreaks between 2008 and 2009 in the northern central regions of the country [16] Genetic characterization of six serotype A strains isolated from the northern central regions of Vietnam revealed that the Vietnamese FMDV serotype A strains were all clustered into the genotype IX (topotype ASIA) and shared close relation to the recent FMDV serotype A strains isolated in Laos, Thailand, and Malaysia [12] The FMDV serotype A strains in this study were also clustered into the genotype IX, together with the strains isolated between 2004 and 2009 Interestingly, the strains isolated in 2013 clustered into a single sub-cluster and showed distance from previous isolates These results indicated the genetic variation of the FMDV serotype A strains and their persistent circulation in Vietnam, particularly in the north and northern central regions Available since the early 1900s, the FMDV vaccine continues to plays a significant role in the protection of animals against FMDV-related morbidity and mortality; however, the vaccinated individuals could only be protected for a specific serotype and/or subtype, and this protection is only valid for a short term [25] The concurrent circulation of FMD outbreaks of the FMDV serotype O, A, and Asia and its genetic variation may apply pressure for the selection of effective vaccine strains to prevent and control FMDV outbreaks in Vietnam [15] These points also suggest that more FMDV surveillance studies will be necessary in order to evaluate the genetic relationship and efficacy of the current used vaccines against different FMDV serotypes circulating in Vietnam Conclusions This study provides valuable information on the genetic variation among the FMDVs serotype O and A circulating in Vietnam during 2006–2014, and likely indicates transmission between neighboring countries in Southeast Asia, such as Myanmar, Thailand, Cambodia, Malaysia, and Laos Page 10 of 11 Additional file Additional file 1: Vietnamese serotypes O and FMDVs used in this study (DOCX 33 kb) Abbreviations CHY: Cathay; EA-1: East Africa 1; EA-2: East Africa 2; EA-3: East Africa 3; EA-4: East Africa 4; ELISA: Enzyme-linked immunosorbent assay; Euro-SA: Europe-South America; FMDV: Foot-and-mouth disease virus; ISA-1: Indonesia-1; ISA-2: Indonesia-2; ME-SA: Middle East-South Asia; RT-PCR: Reverse transcription polymerase chain reaction; SAT: South African Territories; SEA: Southeast Asia; WA: West Africa Funding This work was supported by the Vietnam National Project under the Project Code No: SPQG.05b.01 Availability of data and materials We provided data of the Vietnamese type O and type A FMDVs separately as Additional file 1, the nucleotide information of the study strains and reference strains can be found from GenBank databases, and TreeBase (Study Accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S20003 Reviewer access URL: http://purl.org/phylo/treebase/phylows/study/TB2:S20003?x-accesscode=55af947d9fc7ab8b432f637bf12f988a&format=html) Authors’ contributions VPL and VTT conceived and designed the proposal VPL and TTHV performed the experiments VPL, VTT, TTHV, HQD, and DS participated in analyzing the data VTT, HQD, and VPL wrote the paper All authors have read and approved the final manuscript Competing interest The authors declare that they have no competing interests Consent for publication Not applicable Ethics approval No specific experiments on animals were conducted to perform the study so no ethical approval was required Author details Department of Microbiology and Infectious Disease, Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi, Vietnam Research and Development Laboratory, Rural Technology Development JSC, Hung Yen, Vietnam 3Institute of Research and Development, Duy Tan University, Danang, Vietnam 4Department of Microbiology, Chung-Ang University College of Medicine, Seoul, South Korea 5College of Pharmacy, Korea University, Sejong, South Korea Received: 12 March 2016 Accepted: 22 November 2016 References OIE-FAO The Global Foot and Mouth Disease Control Strategy Strengthening Animal Health System through Improved Control of Major Diseases pp43 FAO, Rome, Italy 2012 Fry EE, Lea SM, Jackson T, Newman JW, Ellard FM, Blakemore WE, et al The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex EMBO J 1999;18(3):543–54 Carrillo C, Tulman ER, Delhon G, Lu Z, Carreno A, 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Development of footand -mouth disease virus strain characterisation a review Trop Anim Health Prod 1989;21(3):153–66 Kitching RP Global epidemiology and prospects for control of foot- andmouth disease. .. Meeting of the OIE Sub-Commission for Foot and Mouth Disease in South-East Asia, Kota Kinabalu, Sabah, Malaysia, 9–13 March 2009 Jamal SM, Ferrari G, Hussain M, Nawroz AH, Aslami AA, Khan E, et al... Buffalo O SEA Cattle O SEA Cattle O SEA Pig O SEA Cattle O SEA Pig O SEA Cattle O SEA Pig O SEA Buffalo O SEA Lai Chau Buffalo O SEA Thai Nguyen Cattle O SEA SEA Ha Tinh Cattle O Nghe An Buffalo