Multiple Origins of Foot-and-Mouth Disease Virus Serotype Asia 1 Outbreaks, 2003–2007

Viruses in 6 genetic groups have caused recent outbreaks in Asia.

F oot-and-mouth disease virus (FMDV) is an Aphthovirus within the family Picornaviridae that infects domestic and free-ranging cloven-hoofed mammals. The vi-rus occurs as 7 serotypes, and immunity after vaccination or after infection is type specific (1)(2)(3). Diversity is also apparent within serotypes, and phylogenetic studies have proved useful for tracing the origin of foot-and-mouth disease (FMD) outbreaks (4).
FMDV is highly contagious, and this, together with its ability to infect different hosts and to exist as multiple types and variants, makes FMD difficult to control and a severe constraint to international trade of livestock and their products. FMD is endemic to regions of South America and large areas of Africa and Asia, and it can readily cross international boundaries to cause epidemics in previously disease-free areas (5). High densities of ruminants and swine in Asia create potential reservoirs of virus maintenance and evolution not influenced by control measures. Intense trading of animals and their products from these reservoirs results in widespread dissemination of viruses within and outside this continent. Therefore, epidemiologic surveillance of FMD in Asia is essential for the timely detection of the emergence of new strains that could threaten neighboring countries (6) and for selecting the most appropriate vaccine strains for use and storage in emergency vaccine reserves (7).
Globally, FMDV serotypes O and the A are the most prevalent. However, Asia has its own unique serotype,  Figure 1; online Technical Appendix Table 1) prompted the current collaborative study to determine the relationships between viruses, with the goal of better understanding the origin of these Asia 1 disease outbreaks.

Viruses
Clinical samples containing FMDV Asia 1 were received from Afghanistan, China, Hong Kong, Iran, Kyrgyzstan, Mongolia, Myanmar, Pakistan, Russia, and Tajikistan by the Food and Agriculture Organisation World Reference Laboratory for FMD (WRLFMD), FGI All-Russian Research Institute for Animal Health (Russian Federation), Lanzhou Veterinary Research Institute (China), Project Directorate on FMD (India), Plum Island Animal Disease Center (USA), and Pakchong Regional Reference Laboratory for FMD (Thailand) (online Technical Appendix Table 2).

RNA Extraction, Reverse Transcription-PCR, and DNA Sequencing
RNA extraction, 1-step reverse transcription -PCR (RT-PCR), and DNA sequencing were performed as previously described (6), except that the primer annealing temperature in the RT-PCR was 55°C. The primers used for RT-PCR and DNA sequencing are listed in online Technical Appendix Table 3. Specific methods used by each laboratory can be obtained on request.

Phylogenetic Analysis
Sequences of these viruses were compared with complete VP1 sequences of Asia 1 viruses stored in the WRLF-MD database (n = 300) that have previously been published 2008 *Since 2005, countries are required to report a change in their foot-and-mouth epidemiologic situation only to the World Organisation for Animal Health.
(10, [16][17][18] or published in this article. Complete VP1 sequences were used to construct a midpoint-rooted neighbor-joining tree using the Kimura 2-parameter nucleotide substitution model as implemented in the program MEGA 4.0 (19). The robustness of the tree topology was assessed with 1,000 bootstrap replicates as implemented within the program. The topography of this tree was also checked by the maximum-parsimony (MEGA 4.0) and maximumlikelihood (TREE-PUZZLE 5.2) (20) methods, including a selection of isolates from each group to check the robustness of the topography. Subsequently, the sequences were ordered, based on their position in the neighbor-joining phylogenetic tree, and a matrix of percentage nucleotide differences was constructed by using MEGA 4.0. The matrix was imported into Excel 2007 (Microsoft Corporation, Redmond, WA, USA), and conditional formatting was used to identify relationships between sequences in the ranges 95%-100% and 90%-94.9%. The former value was used to group the most closely related virus sequences.

Results
The phylogenetic analysis of the complete VP1 gene sequences from isolates of serotype Asia 1 characterized in this study showed that recent viruses (isolated during 2003-2007) belonged to 6 different groups (I-VI) ( Figure  2; online Technical Appendix Figure 1). These groups were defined by members of a group having 95%-100% nucleotide identity (online Technical Appendix Figure 2). All groups were supported by bootstrap values of 80%-100% (online Technical Appendix Figure 1) and were found by using alternative phylogenetic algorithms (maximum parsimony and maximum likelihood) (data not shown). Most virus groups were monophyletic. However, 1 group (VI) fell into 3 distinct lineages (a, b, c) and appeared to be ancestral to group II viruses (Figure 3, panel B). This grouping was also evident from the percentage identity matrix, in which the values between viruses in group VI and those in group II were 91.8%-95.9% (online Technical Appendix Figure 2). Relationships between groups II, III, and VI and between group IV and some unnumbered groups were also evident (online Technical Appendix Figure 2).   (Figure 3, panel B) and from partial VP1 sequences (data not shown) (10,12). These data suggest that this epidemic may have originated in Pakistan. Previously, Asia 1 epidemics occurred in 1973 and 1983-1985. In 1973, the virus spread through Iran and Turkey without any traceable origin (10) (Figure 3, panel A), and in 1983-1985, genetically closely related viruses were found in many Middle Eastern countries, including Armenia, Azerbaijan, Bahrain, Georgia, Greece, Israel, and Lebanon (represented in online Technical Appendix Figure 1 by LEB/83 and GRE/1/84). However, the ultimate source of this virus strain was also not established (4,10). Surprisingly, FMD isolates collected in Pakistan in 2003 and 2005 (group VIa) were closely related to PAK/2/98, which had been isolated 5-7 years earlier, with 0.3% and 0.0% nucleotide differences, respectively ( Figure 3, panel B). These differences would be consistent with a laboratory escape, use of an improperly inactivated vaccine, or laboratory contamination.
Group III contained only viruses that were collected in  Appendix Figure 1). The latter virus clustered with Indian virus isolates, suggesting a possible introduction into Myanmar from the west. In addition, in Myamar, several viruses belonging to 2 sublineages of group IV were detected in a relatively short period (1997-2000 and 2005; Figure  3, panel A), which implies that either multiple lineages are present or that multiple introductions have been made into that country. data). Five of these sequences belonged to group IV and 2 belonged to group V (Figure 3, panel A), indicating movements of viruses between China and Southeast Asia and the presence of group V viruses in a more southerly distribution than has previously been reported.

Discussion
This phylogenetic study demonstrates that the viruses from groups II and V that have been responsible for FMD outbreaks in China appear to have spread large distances in a short time, although the means is unknown. The possibility of spread of viruses of these 2 groups beyond the border where they have been detected must be considered as a potential risk. Furthermore, the close relationships between some recent and older isolates within group V (India 1976- In Asia, vaccination against FMD varies from country to country; generally, only cattle and water buffalo are vaccinated. Various vaccine strains are used in the region, and vaccines are produced either by large pharmaceutical companies or by national or regional FMD vaccine laboratories. Vaccine matching studies are performed in various FMD reference laboratories on an ad hoc basis, and reference reagents for all the vaccine strains are not always available. This situation requires improvement. These studies suggest rapid spread of FMD viruses across Asia, but the means by which the viruses are moved has rarely been determined. The spread of some of these FMDV Asia 1 lineages across large parts of Asia, and occasionally outside Asia, demonstrates the continuing need for active surveillance to be improved in Asia to provide real-time monitoring of virus evolution and to disclose more effectively the links between outbreaks. The means of virus transport needs also to be defined, taking into consideration the role played by large antelope populations in central Asia. This information is needed as a prerequisite for further development of regional control programs. India, Pakistan, and China, with their large livestock populations, are expected to play a major role in FMD control in this part of the world. This work was supported by the Department for Environment, Food and Rural Affairs, UK (Reference Laboratory Contract and Research Grant numbers SE2921 and SE2935). Virus isolation and serotyping at the Institute for Animal Health (IAH) was also supported by Defra under a Reference Laboratory Contract. The work conducted at the IAH was registered by the British Standards Institution to ISO 9001:2000 (certificate number FS 60875).
Dr Valarcher is a veterinary clinician and virologist. His research interests focus on infectious diseases of livestock and their control.