Molecular Epidemiology of An Asian Pandemic	
	Strain of
Foot-&-Mouth Disease 
Type ‘O’ Virus	
World Reference Laboratory for Foot-&-Mouth Disease,
Institute for Animal Health,
Pirbright Laboratory,
Pirbright,
Woking,
Surrey,
GU24 0NF,
UK.

INTRODUCTION

The OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRLFMD) is established within the high security laboratory at:
The Institute for Animal Health, Pirbright, U.K.
Annually, over 400 samples are received for diagnosis of foot-and-mouth disease (FMD) from throughout the FMD endemic region, which is predominantly Africa, Asia and parts of South America. There are seven serotypes of FMD virus; SAT 1, SAT 2 and SAT 3 restricted to Africa, Asia 1 restricted to Asia and O, A and C present in Africa, Asia and South America and occasionally Europe. In each of the last five years, serotype O has been isolated from over 60% of the positive FMD samples received.

Foot-and-mouth disease virus (FMDV) is a member of the genus Aphthovirus within the family Picornaviridae, and has a genome consisting of a single strand of positive sense RNA. The virus has a relatively high mutation rate, potentially changing, on a random basis, between one and eight nucleotides per replication cycle. This tendency towards genetic variability has been utilized to individually characterize strains of FMD virus, and track their movement across international borders. A region of approximately 200 nucleotides of the 1D gene, which codes for the major structural protein VP1, was selected for genomic comparison between strains of FMD virus. Sequence differences between two isolates of less than 4% indicates a recent common origin, whereas differences of 15% or more signify geographical isolation over many years. Isolates with greater than 85% nucleotide sequence identity have been placed within groups or topotypes, which tend to be restricted in their geographical distribution. 

FMD causes an acute disease of pigs and wild and domestic ruminants such as cattle, water buffalo, sheep, goats and deer. It can cause high mortality in young animals and production losses in adults, and is considered the single most important constraint to trade in live animals and animal products. Movement of FMDV is predominantly associated with the legal and illegal movement of infected animals, although it can spread in animal products, and, under the right conditions, up to 250 km as an aerosol.

We describe here a strain of serotype O FMD virus which we have identified as spreading from India westward into Saudi Arabia and then throughout the Middle East and into Europe, and eastward through Nepal and Bhutan to mainland China, Taiwan Province of China (POC), Japan and South Korea. 

MATERIALS AND METHODS

Total RNA was extracted either directly from epithelial suspensions or from cell culture grown virus using a commercial kit (RNeasy, Qiagen) and an RT-PCR was performed as described by Knowles and Samuel (1994) using Primers ARS4 and NK61. The PCR amplicons were partially sequenced using a commercial kit (fmol, Promega) and an automated sequencer (ALFexpress II, Pharmacia) with Cy5-labelled primers NK72, 1D296F, 1C564F and ARS4. UPGMA trees were constructed using the PHYLIP package (Felsenstein, 1993). All these techniques are fully described in a laboratory manual (Knowles and Samuel, 1998) which is downloadable from our website:
http://www.iah.bbsrc.ac.uk/virus/Picornaviridae/Aphthovirus/fmdv.htm

RESULTS & DISCUSSION

The pandemic virus was first isolated from outbreaks of FMD in northern India in 1990. It spread into Saudi Arabia, probably with the live trade in sheep and goats. Saudi Arabia imports over 6 million live animals annually to supportthe requirements of the Muslim festivals. During 1994 the virus spread within Saudi Arabia and then moved into neighbouring countries so that by 1996 it had reached Turkey. >From Turkey it crossed the Evros river, probably with illegally moved sheep, into Greece, where it caused 39 reported outbreaks. The same virus also caused an outbreak in Bulgaria. During the following two years major outbreaks of FMD were reported from Iran, Iraq, Syria, Israel, Lebanon, Jordan and the Arabian Peninsular all due to the same strain of type O FMD virus (Fig. 1).

Figures 2 and 3 show phylogenetic trees (UPGMA) comparing either partial or complete VP1 sequences, respectively. For purposes of clarity Figure 2 only shows a proportion of the virus isolates that we have identified as belonging to the PanAsian lineage. The success of this virus can be measured by the observation that it has now replaced all other strains circulating in the Middle East. It not only caused large-scale disease when it first arrived, but it has persisted, and in 1999 it was responsible for outbreaks of FMD on at least three of the large Saudi Arabian dairy farms. The cattle on these farms are regularly vaccinated with well produced vaccines which laboratory tests indicate should protect the cattle. In spite of vaccination and high security surrounding the farms, the virus was able to gain entry and cause clinical disease. The eastward spread of the strain is less certain. In 1993 and 1994 it was recovered from samples submitted from Nepal and in 1998 from Bhutan. During 1998 and 1999 there have been outbreaks of FMDin China (Yunnan Province in July 1998; Tibet, Hainan Province and Fujian Province in May 1999). Sequence analysis of three viruses from Tibet (O/CHA/1/99, O/CHA/2/99 and O/CHA/3/99) and one from Hainan (O/CHA/4/99) shows that these outbreaks were caused by the same virus. The first outbreak in Taiwan was on an island (Kinmen) belonging to Taiwan within a kilometre of the Chinese mainland. Cattle were moved from this island to the main island of Taiwan before FMD had been suspected, and initially infection was detected in Tainan prefecture. In January 2000 cattle were affected in Yunlin and Chiayi prefectures and in February 2000, goats were affected in Kaoshiung and Changhwa prefectures. In March 2000, FMD type O appeared in South Korea and Japan and sequence analysis revealed the PanAsian strain to be responsible.

Towards the end of 1999 it became clear that the PanAsian virus was moving into South East Asia, where a different FMDVtype O topotype normally exists. By April 2000 all mainland South East Asian countries, with the exception of Myanmar, had outbreaks due to the new strain. It has recently been reported that a severe outbreak of FMD type O in pigs in the Ussuriysk district of eastern Russia, was also caused by this new strain (report to OIE). 

The extent of this spread is unique for one strain of FMD virus, and its presence in all recent samples from the Middle East indicates that it has out-competed the other strains and topotypes of FMD virus previously established in these areas. Its appearance in South Korea and Japan, previously free of the disease since 1934 and 1908, respectively shows that this strain is capable of spreading to countries where strict measures are undertaken to exclude importation of animal diseases. Whether this fitness to survive is related to its ability to cause more severe clinical disease, as has been indicated from reports from the Middle East, or an increased ability to transmit between susceptible species, whether cattle, sheep or pigs, is not clear. However, its spread across most of Asia and into Europe demonstrates how a newly evolved virus with a competitive advantage can become established, in spite of controls at international borders to prevent the spread of animal disease. The presence of FMD in a previously free country can seriously interfere with the local and export trade in susceptible animals and their products. The appearance in Taiwan Province of China in 1997 of a pig adapted strain of FMD virus has cost the country over 1.5 billion dollars annually in lost export markets, apart from the expenditure on the control programme. A large outbreak of FMD in northern Europe or USA would result in losses exceeding 100 billion dollars. It is not surprising that many nations view with anxiety the threat of economic terrorism based on the use of rapidly spreading biological agents targeted at farm animal. The emergence of this competitively advantaged strain of FMD virus, and its spread within the territory bounded by Greece in the West and Japan in the east, provides an example of the economic damage that can result.

REFERENCES

Felesenstein , J. PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington,

Seattle, 1993.

Knowles, N.J. and Samuel, A.R.(1994). Polymerase chain reaction amplification and cycle sequencing of the 1D(VP1) gene of foot-and-mouth disease viruses. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Vienna, Austria, 19-22 September, 1994.

Knowles, N.J. and Samuel, A.R. (1998). "RT-PCR and Sequencing Protocols for the Molecular Epidemiology of Exotic Virus Diseases of Animals".

Institute for Animal Health, Pirbright. 
http://www.iah.bbsrc.ac.uk/virus/Picornaviridae/Aphthovirus/Protocol.pdf

ACKNOWLEDGEMENTS

We would like to thank Drs. Qi-Zu Zhao and Qing-Ge Xie (Lanzhou Veterinary Research Institute, Lanzhou, People's Republic of China) and Dr. Toru Kanno (National Institute of AnimalHealth, Kodaira, Japan) for providing sequence data prior to publication. We also thank Paul Davies and David Ansell for valuable technical assistance. This work was supported by the Ministry of Agriculture, Fisheries and Food, UK.

Fig. 1. Conjectured spread of the India-90 (PanAsia) lineage of the South Asian topotype of FMDV-O

Sorry - unable to copy map etc.

00 Viruses isolated and sequenced by WRLFMD (IAH-Pirbright)
00 Viruses isolated and sequenced at IVRI-Mukteswar (India)
99
99
95-98
97-99
98 93-96
95
99 99
99-2000
99-2000
99 94-99
96
96
96-99
95-98
97-99
97-99 99-2000 95-99
98-99
98-99

First recorded occurrences

India 1990
Nepal 1993
Saudi Arabia 1994
Malaysia 1995
Yemen 1995
Lebanon 1995
Israel 1995
Kuwait 1996
Turkey 1996
Bulgaria 1996
Greece 1996
Armenia 1996
Georgia 1997
Bahrain 1997
United Arab Emirates 1997
Iran 1997
Bhutan 1998
Jordan 1998
Syria 1998
People's Republich of China 1999
Taiwan Province of China 1999
Qatar 1999
Iraq 1999
Thailand 1999
Vietnam 1999
Cambodia 1999
Laos 2000
Malaysia 2000
Japan 2000
South Korea 2000
Russia 2000
2000
2000
2000
90-99
99-2000

00 Viruses isolated and sequenced at Lanzhou Veterinary Research Institute (P.R. China)
00 Virus sequenced at NIAH, Kodaira (Japan)
2000
00 Virus sequenced at ARRIAH, Vladimir (Russia)
2000 97
96
96

Fig. 2. Phylogenetic tree showing the relationships between partial VP1 sequences of
viruses belonging to the PanAsian lineage with other FMD type O viruses.

O/ALG/1/99
O/ALG/2/99
O/ALG/3/99
O/MOR/2/99
O/MOR/11/99
O/CIV/8/99
O/GNA/6/99
O/GNA/10/99
O/TUN/1/99
O/TUN/5/99
O/BAR/2/97
O/IRN/5/97
O/KUW/3/97
O/KUW/4/97
O/UAE/7/97
O/IRN/15/97
O/IRN/16/97
O/ISR/1/96
O/SAU/2/95
O/SAU/5/95
O/SAU/14/95
O/SAU/20/95
O/SAU/28/95
O/YEM/8/95
O/KUW/1/96
O/JOR/3/96
O/SAU/2/97 O/LEB/1/98
O/LEB/3/98
O/BAR/8/98 O/IRN/20/98
O/IRN/8/99
O/IRN/12/98
O/IRN/1/99
O/MAY/1/2000
O/MAY/2/2000
O/MAY/3/2000
O/SKR/1/2000
O/SKR/2/2000
O/TAW/1/99
O/TAW/2/99
O/TAW/3/99
O/BHU/1/98
O/BHU/2/98
O/LAO/1/2000
O/LAO/2/2000
O/CAM/2/2000
O/CAM/4/2000
O/VIT/17/99
O/JPN/A/2000
O/JPN/B/2000
O/BHU/6/98
O/IRQ/2/99
O/SAU/7/99
O/IRQ/9/99 O/QTR/3/99
O/ISR/2/99
O/ISR/3/99
O/ISR/5/99
O/LEB/9/98
O/LEB/12/98
O/LEB/14/98
O/LEB/17/98
O/SAU/36/98
O/SAU/38/98
O/SAU/41/98
O/SAU/25/98
O/YEM/3/98
O/YEM/6/98
O/SAU/1/99
O/SAU/2/99
O/ISR/A/99
O/YEM/15/98
O1/Manisa/TUR/69
O/PAK/1/98
O/ERI/1/96
O/ETH/8/94
O/YEM/4/95
O/CAM/1/98
O/VIT/2/97 O1/Kaufbeuren/FRG/66
O/HKN/1/99
O/HKN/10/99
O/VIT/2/99
O/TAW/81/97
O/TAW/83/97
O/VIT/3/97
O/PHI/5/99

Percentage nucleotide difference (nt 469-639 of VP1)

West African topotype
South Asian topotype
South-east Asian topotype
European / South American topotype
Hong Kong topotype
0 2 4 6 8 10 12 14 16 18 20
18.9%
O/ALG/1/99
O/BHU/1/98
O/CAM/2/2000
O/Tibet/CHA/1/99
O/Tibet/CHA/2/99
O/Tibet/CHA/3/99
O/TAW/2/99
O/Hainan/CHA/4/99
O/IRN/9/99
O/SKR/1/2000
O/JPN/A/2000
O/MAY/2/2000
O1/Manisa/TUR/69
O/TUR/6/98
O/CAM/6/99
O/VIT/2/97
O/HKN/1/99
O/TAW/81/97
O/TAW/4/99
O/VIT/3/97
O1/Kaufbeuren/FRG/66
O1/Lombardy/ITL/46
O2/Brescia/ITL/47
ALG, Algeria
BHU, Bhutan
CAM, Cambodia
CHA, China
FRG, Fed. Rep. of Germany
HKN, Hong Kong
IRN, Iran
ITL, Italy
JPN, Japan
MAY, Malaysia
SKR, South Korea
TAW, Taiwan POC
TUR, Turkey
VIT, Vietnam
20.3%

Percentage nucleotide difference (complete VP1)
0 2 4 6 8 10 12 14 16 18 20

Fig. 3. Phylogenetic tree showing the relationships between the complete
VP1-coding sequences of some of the FMD type O viruses studied.
Nick J. Knowles and Alan R. Samuel

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