Introduction

Foot-and-mouth (FMD) is an acute, febrile, highly contagious infection of ungulates induced by the Foot-and-mouth virus (FMDV), with a worldwide distribution, high morbidity, and rapid spread, its outbreaks and epidemics not only directly menace livestock production, but also the losses incurred from international trade embargo are incalculable [1, 2, 3].

The causative agent of FMD is FMDV, a member of the genus Aphthovirus, family Picornaviridae [4]. FMDV particles are composed of 60 copies of each of four capsid proteins termed VP1, VP2, VP3 and VP4. The VP1 is surface exposed and contains the major antigenic determinants of the virus, and VP1 contains two immunogenic sites at amino acid positions 140–160 (the G–H loop) and at residues 200–213 (the C-terminus region) and known to elicit neutralizing antibodies [5, 6]. Variation at critical amino acid residues within the G–H loop and C-terminus regions contributes to the antigenic variation of FMDV [7]. In addition, VP1 contains a highly conserved sequence called the arginine–glycine–aspartic acid (RGD) motif that is involved in cell attachment [8]. FMDV mutation studies have revealed that selection arising from immunologic pressure is the major factor causing virus mutation, which tends to occur in the VP1 gene, and so numerous researchers have focused their attention on the VP1 gene in conducting FMD molecular epidemiological surveys [9, 10].

The present study was undertaken to assess the regional variation of prevalent FMDV type O viruses in pigs in Hong Kong and to establish a sequence database for FMDV molecular epidemiological investigation.

Material and methods

Cell cultures and virus isolations

FMDV was isolated from vesicles on the hoof and lip of swine on some pig farms in Hong Kong during 2001–2002. The tissue sample was used to infect baby hamster kidney (BHK-21) cells cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal calf serum (FCS) as described previously [11]. All virus isolation was done in a biosafety laboratory.

RNA extraction and RT–PCR

Cultures with distinct CPE were repeatedly freeze-thawed thrice, and RNA from the 250 μl cell suspension extracted with TRIZOL LS kit (Invitrogen Co.) by following the procedure described on the instruction pamphlet. Virus cDNA was synthesized by using the RNA from isolates as template, Oligo dT as primers and employing the cDNA synthesizing kit (Invitrogen Co.). After completion of the reaction, PCR amplification was done using a pair of specific primers [12] (P1: 5′-ACCACCTCTGCGGGTGATGAGTC-3′ and P2: 5′-CAGAAGCTGTTGCTTTGCGGGTG-3′), and by utilizing the Expand High Fidelity PCR System (Roche Co.). The reaction conditions were: first denaturing at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 1 min; finally extension at 72°C for 10 min.

Cloning and sequence analysis

The RT–PCR product was directly cloned into pGEM-T Easy vector (Promega Co.) and two discrete positive clones were selected for each isolate to provide for nucleotide sequencing. Sequencing was performed on an AB1377 sequencer using M13±universal primer. Sequence analysis of VP1 gene of O/HKN/3/01, O/HKN/5/01, O/HKN/12/01, O/HKN/7/02 and O/HKN/10/02 with those of reference FMDV strains O/HKN/7/96 (GenBank accession AJ294922), O/HKN/20/96 (AJ294924), O/HKN/1/99 (AJ294925), O/HKN/10/99 (AJ318836), O/HKN/16/96 (AJ294923), O/HKN/12/91 (AJ294921), Taoyuan-113 (AF095872), Kaohsiung-190 (AF095885), Chunhwa-188 (AF095883), Taichung-186 (AF095882), Nantow-089 (AF095868), TAW/83/97 (AJ296322), TAW/81/97 (AJ296321), Miaoli-165 (AF095879), O/HKN/6/83 (AJ294919), O/HKN/33/77 (AJ294916), O/HKN/21/70 (AJ294911), O/HKN/3/75 (AJ294915), O/HKN/19/73 (AJ294913), O/HKN/1/73 (AJ294912), O/HKN/14/82 (AJ294917), HK/93 (AJ131470), GD/China/86 (AJ131468), TAI/4/99 (AJ303536), O/HKN/17/82 (AJ294918), O/MAY/2/2000 (AJ318846), O/CAM/2/2000 (AJ318828), O/TAW/2/99 (AJ294927), O/1734/RUS/2000 (AJ318850), O/SKR/1/2000 (AJ318854), O/JPN/2000 (AB079061), UKG/35/2001 (AJ539141), O/SKR/2000 (AF428246), SKR/AS/2002 (AY114146), O/MOG/2000 (AJ318847), O/SAU/38/98 (AJ318852), O/IRQ/26/2000 (AJ318841), O/BAR/8/98 (AJ318825), O1/Manisa/Turkey/69 (AJ251477), O/TUR/6/98 (AJ318855), TAN/7/98 (AJ296320) and O1/Lujan/Arg/83 (AJ308706) were performed using LaserGene Biocomputing Software Package (DNASTAR, 1997). In brief, the entire VP1 gene nucleotide sequences were maximally aligned using MegAlign program. The phylogenetic tree was conducted using CLUSTAL algorithm.

Results

Virus isolation

Five isolates producing obvious Cytopathic effect (CPE) in BHK-21 cells were obtained, and given the names O/HKN/3/01, O/HKN/5/01, O/HKN/12/01, O/HKN/7/02 and O/HKN/10/02. All of five isolates did not show CPE after 72 h at the passage, but exhibited obvious CPE at the 3rd passage.

Sequence analysis

The entire VP1 gene nucleotide sequences of the five isolates examined were 639 nt long, coding for 213 amino acids, and they all belonged to type O FMDV. Sequence identity between the five FMDV type O isolates was high (98.3–99.7%), and these isolates also displayed high sequence identity (95%) with type O FMDV, O/HKN/7/96, O/HKN/20/96, O/HKN/1/99 and O/HKN/10/99.

The VP1 nucleotide sequences of selected FMDV type O viruses isolated from outbreaks in the Chinese mainland, Hong Kong and Taiwan and other countries (i.e., United Kingdom, Tanzania, South Korea, Japan, Cambodia, Mongolia, Malaysia, Iraq, Russia, Saudi Arabia, Bahrain, Turkey, Thailand and Argentina) were used to construct a phylogenetic tree. All strains of selected FMDV were divided into 3 groups (Fig. 1). The first group comprises isolates exclusively from Hong Kong (1991–2002) and Taiwan (1997). The second group comprises isolates mainly from Hong Kong (1970–1993) and the Chinese mainland (1986). The third group comprises a wide range of isolates mainly from United Kingdom, Tanzania, Taiwan, South Korea, Japan, Cambodia, Mongolia, Malaysia, Iraq, Russia, Saudi Arabia, Bahrain, Hong Kong, Turkey, Thailand and Argentina. The second group is more closely related to the first group than to the third (Fig. 1). The five isolates examined shared the greatest similarity at the nucleotide level with FMD type O viruses isolated from localized outbreaks in Hong Kong during 1991–1999 and with isolates from a major FMD outbreak in Taiwan in 1997. In contrast, a relatively low identity level, below 85%, was seen in comparison with FMDV type O viruses, e.g., TAI/4/99, O/HKN/17/82, O/MAY/2/2000, O/CAM/2/2000, O/TAW/2/99, O/1734/RUS/2000, O/SKR/l/2000, O/JPN/2000, UKG/35/2001, O/SKR/2000, SKR/AS/2002, O/MOG/2000, O/SAU/38/98, O/IRQ/26/2000, O/BAR/8/98, O1/Manisa/Turkey/69, O/TUR/6/98, TAN/7/98 and O1/Lujan/Arg/83.

Fig. 1
figure 1

Phylogenetic tree showing genetic relationship between selected type O FMD virus isolates at 1–639 region of the VP1 gene. The dendrogram was obtained using the MegAlign program of the DNASTAR package

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Comparison of deduced amino acid sequence

Deduced ammo acid sequence analysis of the major VP1 protein antigen epitope of type O FMDV demonstrated that amino acid mutation of the five isolates chiefly occurred within the sites 133–144, and 195–200, and key amino acids sites at 148, 149, 154 and 208 were all relatively conserved, however, although no change appeared at the arginine–glycine–aspartate (RGD) sequence involved in viral attachment to cell, much mutation occurred in the amino acids at the sites 133–144 (Fig. 2).

Fig. 2
figure 2

Comparison of amino acid sequence in 133–160 and 200–213 region of VP1 polypeptide of type O FMDV strains. Sequence dissimilarity is indicated in single letter code. Dash (–) indicates sequence identity in relation to the isolate obtained in this study (O/HKN/3/0l), and dots (.) represent region not sequenced. ‘X’ indicates sequence not determined. The domain involved in the formation of locus for the attachment of virus to cell host are marked □

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Discussion

During 2001–2002, five FMDV type O viruses with nearly identical VP1 gene sequences (O/HKN/3/01, O/HKN/5/01, O/HKN/12/01, O/HKN/7/02 and O/HKN/10/02) were isolated from localized outbreaks in Hong Kong.

Amino acids at the 132–157 and 200–213 sites on the VP1 protein of type O FMDV contain important epitopes of the virus, among which the amino acids sites 144 (Val), 148 (Leu), 154 (Lys) and 208 (Pro) are the most critical, their alteration more or less affecting antigenic loci and corresponding monoclonal antibody reactivity [13]. Amino acid alignment of the five 2001–2002 Hong Kong viruses examined revealed that no changes occurred in these critical amino acid sites (Fig. 2). The RGD cell attachment site at amino acid position 145–147, also remained intact.

From a phylogenetic analysis of the complete VP1 nucleotide sequences of type O FMDV the five isolates (O/HKN/3/01, O/HKN/5/01, O/HKN/12/01, O/HKN/7/02 and O/HKN/10/02) appear to be most closely related to type, O FMDVs found in Hong Kong during 1991, 1996 and 1999, e.g., O/HKN/12/91, O/HKN/16/96, O/HKN/1/99, O/HKN/10/99, O/HKN/7/96.and O/HKN/20/96. This indicates that five isolates examined share a common ancestor with FMDV isolates that occurred previously in Hong Kong, and reflects that type O FMDV persists in an endemic low level that occasionally erupts into a localized epidemic under the specific natural environment of Hong Kong. Similarly, the five isolates examined appear to be most closely related to FMDV isolates from Taiwan in 1997, e.g., Taoyuan-113, Kaohsiung-190, Chunhwa-188, Taichung-186, Nantow-089, TAW/83/97, TAW/81/97 and Miaoli-165 (Fig. 1). And so it is possible that FMDV isolates in Hong Kong at least share a relatively recent common ancestor with the Taiwan 1997 FMDV type O isolates. Thus, it is possible that the large FMD outbreak in pigs in Taiwan during 1997 was a contributory factor in the occurrence of the disease in Hong Kong from 1999 to 2002. However, live pigs are not imported from Taiwan and the import of pork products is negligible. In addition, there is no correlation between the number and severity of FMD outbreaks in Hong Kong and Taiwan. It is believed that FMD might be transmitted by contaminated animal products, mechanical transfer involving people and vehicles, as well as airborne transmission over long distances. The persistence of FMD in Hong Kong has complex contributing factors such as asymptomatic carriers, host species, frequency of animal and human transport, the importation of animals and food products from affected areas, climate and weather patterns.

From the phylogeny of VP1 nucleotide sequences it can be seen that the isolates that infected cattle in 1996, O/HKN/7/96 and O/HKN/20/96, are most closely related to FMDV type O isolates from swine causing FMD during 2001–2002, with which it shares about 95% sequence similarity at the nucleotide level. It is possible that the small population of feral Asian water buffalo and South China yellow cattle in Hong Kong may act as a reservoir of FMDV. Pigs do not ordinarily become carriers following recovery from FMDV infection, and domestic cattle can shed viable FMDV particles for 6–24 months whilst African buffalo can become lifelong carriers. Whether this fitness of FMDV to survive is related to its ability to cause severe clinical disease, or an increased ability to transmit between susceptible species, whether they are feral buffalo, cattle, or pigs, is not clear.

Vaccination against FMD is an acknowledged means of controlling outbreaks of the disease. Emergency vaccination coupled with rapid culling was useful in limiting the spread of FMD [9]. Due to the multiple serotypes of FMDV in circulation, identification of the serotype affecting any one region is required in order to select the most appropriate antigens for inclusion in a vaccine preparation [14, 15]. The important immunogenic site of type O FMDV is the VP1 surface antigen encoded by the 1D region. Analysis of the VP1 major epitope of type O FMDV has revealed that although the critical amino acids on the antigenic epitope of prevalent type O FMDV isolates and those of the recommended FMD vaccine currently in use in Hong Kong includes antigens derived from type O FMDV 01/Manisa/Turkey/69 are basically unchanged, the VP1 gene nucleotide sequence of prevalent isolates in Hong Kong shows relatively low homology with vaccine virus 01/Manisa/Turkey/69. If the sequence homology of VP1 gene of FMDV should be directly related quantitatively to immunogenicity, it would be necessary to conduct serological testing and, if the antigenicity was found to differ, the vaccine virus should be replaced. Taking into consideration the high mutation rate of type O FMDV and the advanced experience in vaccine development, it would be advisable for maximal efficacy to select vaccine strains that are as closely as possible related in antigenicity with prevalent isolates in Hong Kong.