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

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. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0896-0) contains supplementary material, which is available to authorized users.


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.
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.
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 1 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 1 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 1 and A were subsequently identified in 2005 and 2009, respectively [12,15]. In 2008 and the first 2 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.

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 (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% CO 2 . 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 1 h, followed by two washes with MEM media. The infected cells were maintained in MEM media containing 2% FBS. Infected cells were harvested after 2-4 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 (VN-VP1F: 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 min (reverse transcription), 35 cycles of 95°C for 1 min (for denaturation), 52°C for 1 min (for annealing), and 72°C for 1 min (for extension), followed by 72°C for 10 min (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  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].

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 1.  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).

Comparison of VP1 amino acid sequences
The deduced amino acid sequences obtained from the VP1 gene segments were aligned and compared to investigate Notably, the change of the two amino acids at positions 45 K-Q and 154 K-R occurring at residues among the SEA topotypes, as well as the change of two amino acids at positions 43 T-V and 208 P-H occurring at residues among the ME-SA topotypes (Figs. 4 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. 8 118/87 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 148 S-P of the A/VN/130/2009 and A/VN/131/ 2009 strains (Fig. 6).