An overview of control strategy and diagnostic technology for foot-and-mouth disease in China.

Foot-and-mouth disease (FMD) is one of most contagious animal diseases. It affects millions of cloven-hoofed animals and causes huge economic losses in many countries of the world. There are seven serotypes of which three (O, A and Asia 1) are endemic in China. Efficient control of FMD in China is crucial for the prevention and control of FMD in Asia and throughout the world. For the control of FMD, a powerful veterinary administration, a well-trained veterinary staff, a system of rapid and accurate diagnostic procedures and, in many countries, compulsory vaccination of susceptible animals are indispensable. This article strives to outline the Chinese animal disease control and prevention system, in particular for FMD, with the emphasis on diagnostic procedures applied in Chinese laboratories. In addition, new technologies for FMD diagnosis, which are currently in the phase of development or in the process of validation in Chinese laboratories, are described, such as lateral flow devices (LFD), Mab-based ELISAs, reverse transcription loop-mediated isothermal amplification (RT-LAMP) and gold nanopariticle immuno-PCR (GNP-IPCR).

Rapid typing of foot-and-mouth disease serotype Asia 1 by reverse transcription loop-mediated isothermal amplification.

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was rapidly used to detect serotype Asia 1 of foot-and-mouth disease virus (FMDV) within 45 min at 61°C. All FMDV serotype Asia 1 reference strains were positive by RT-LAMP, while other viruses such as FMDV serotypes O, C, A and classical swine fever virus, swine vesicular disease virus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus remained negative. Furthermore, FMDV sreotype Asia 1 positive samples were able to detect by RT-LAMP assay. This RT-LAMP assay may be suitable particularly for diagnosis of FMDV serotype Asia 1 infection in field stations.

Detection of foot-and-mouth disease virus RNA by reverse transcription loop-mediated isothermal amplification.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for foot-and-mouth disease virus (FMDV) RNA. The amplification was able to finish in 45 min under isothermal condition at 64°C by employing a set of four primers targeting FMDV 2B. The assay showed higher sensitivity than RT-PCR. No cross reactivity was observed from other RNA viruses including classical swine fever virus, swine vesicular disease, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus. Furthermore, the assay correctly detected 84 FMDV positive samples but not 65 FMDV negative specimens. The result indicated the potential usefulness of the technique as a simple and rapid procedure for the detection of FMDV infection.

Research in advance for FMD novel vaccines.

Foot-and-Mouth Disease (FMD), as a major global animal disease, affects millions of animals worldwide and remains the main sanitary barrier to the international and national trade of animals and animal products. Inactivated vaccination is the most effective measure for prevention of FMD at present, but fail to induce long-term protection and content new requires for production of FMD vaccines. As a number of Researchers hope to obtain satisfactory novel vaccines by new bio-technology, novel vaccines have been studied for more than thirty years. Here reviews the latest research progress of new vaccines, summarizes some importance and raises several suggestions for the future of FMD vaccine.

A highly sensitive detection for foot-and-mouth disease virus by gold nanopariticle improved immuno-PCR.

BACKGROUND: Foot-and-mouth disease (FMD) is one of the most contagious of all artiodactyl animal diseases, and its infection has an obvious ability to spread over long distances and to contribute to epidemics in FMD-free areas. A highly sensitive and specific method is required to detect FMDV. In this study, we evaluated the usefulness of a bio-barcode assay (BCA) technique for detecting clinical samples of FMDV. METHODS: Highly sensitive gold nanopariticle (GNP) improved immuno -PCR (GNP-IPCR) which derived from the bio-barcode assay (BCA) was designed for the detection of FMDV. The target viral particles were captured by a polyclonal antibody coated on ELISA microplate, followed by adding GNP which was dually modified with oligonucleotides and a FMDV specific monoclonal antibody (MAb) 1D11 to form a sandwiched immune complex. After the formation of immuno-complex, the signal DNA was released by heating, and consequently characterized by PCR and real time PCR. RESULTS: The detection limit of GNP-PCR could reach to 10 fg/ml purified FMDV particles, and the assay can detect clinical samples of FMDV with highly sensitivity, while detect limit of conventional ELISA is 100 ng/ml in this study. CONCLUSION: GNP-IPCR may provide a highly sensitive method for the detection of FMDV.

Development of a New RT-PCR with Multiple Primers for Detecting Southern African Territories Foot-and-mouth Disease Viruses.

INTRODUCTION: The extremely high genetic variation and the continuously emerging variants of foot-and-mouth disease virus (FMDV) of Southern African Territory (SAT) serotypes including SAT1, SAT2, and SAT3 make it necessary to develop a new RT-PCR for general use for monitoring viruses based on the updated genome information. MATERIAL AND METHODS: A FMDV SAT-D8 one-step RT-PCR was established based on the 1D2A2B genes of the SAT serotype viruses with a multiplex primer set. FMDV A, O, C, and Asia 1 serotypes, other vesicular disease viruses, inactivated SAT viruses, and 125 bovine, ovine, caprine and porcine tissue samples collected from the Chinese mainland were included for evaluating the assay. RESULTS: The new RT-PCR was proven to be specific without cross-reactions with Eurasian FMDV, swine vesicular disease virus (SVDV), Seneca valley virus (SVV), or other common viral pathogens of cattle, sheep, goat, and pig. An around 257 bp-sized amplicon clearly appeared when the inactivated SAT viruses were detected. However, all 125 samples collected from FMDV-susceptible animals from the Chinese mainland which has not known SAT epidemics showed negative results. CONCLUSIONS: A FMDV SAT-D8 one-step RT-PCR is a promising method for primary screening for FMDV SAT serotypes.

The silent point mutations at the cleavage site of 2A/2B have no effect on the self-cleavage activity of 2A of foot-and-mouth disease virus.

The 2A region of the foot-and-mouth disease virus (FMDV) polyprotein is 18 amino acids in length, and 2A self-cleavage site (2A/2B) contains a conserved amino acid motif G2A/P2B. To investigate the synonymous codon usage for Glycine at the 2A/2B cleavage site of FMDV, 66 2A/2B1 nucleotide sequences were aligned and found that the synonymous codon usage of G2A is conserved and GGG was the most frequently used. To examine the role of synonymous codons for G2A in self-cleavage efficiency of 2A/2B, recombinant constructs which contains the chloramphenicol acetyltransferase protein (CAT) and enhanced green fluorescent protein (EGFP) linked by the FMDV 2A sequence with four synonymous codons for G2A were produced. The activities of all the F2As based plasmids were determined in CHO cells. The results showed that the synonymous codon usage patterns for G2A at the cleavage site (2A/2B) have no effect on the cleavage efficiency. This suggests that the synonymous codon usage of 2A peptide has no effect on the cleavage efficiency of FMDV 2A element.

Establishment and evaluation of stable cell lines inhibiting foot-and-mouth disease virus by RNA interference.

RNA interference (RNAi) has been proved to be a powerful tool for foot-and-mouth disease virus FMDV inhibition in vitro and in vivo. We established five stable baby hamster kidney 21 cell lines (BHK-21) containing five short hairpin RNAs (shRNAs) expression plasmids (p3D1shRNA, p3D2shRNA, p3D3shRNA, p3D4shRNA, and p3D5shRNA) targeting 3D gene of FMDV. Immunofluorescent assay, virus titration, and real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) were conducted to detect the effect of shRNAs on FMDV replication. After challenged with FMDV of O/CHA/99, two cell lines (p3D1shRNA and p3D4shRNA) showed a significant reduction in the synthesis of viral protein and RNA, accompanied by a sharp decrease in viral yield, and the inhibition could last for at least thirty passages. We developed an efficient procedure for the establishment and evaluation of stable cell lines for anti-FMDV research based on RNAi technology, which can be a candidate method for anti-FMDV research.

Immunity of foot-and-mouth disease serotype Asia 1 by sublingual vaccination.

Foot-and-mouth disease virus (FMDV) causes vesicular disease of cloven-hoofed animals, with severe agricultural and economic losses. Here we present study using a sublingual (SL) route with the killed serotype Asia 1 FMDV vaccine. Guinea pigs were vaccinated using a commercially available vaccine formulation at the manufacturer's recommended full, 1/4, and 1/16 antigen doses. Animals were challenged with homologous FMDV Asia1 strain at various times following vaccination. All control guinea pigs exhibited clinical disease, including fever, viremia, and lesions, specifically vesicle formation in feet. Animals vaccinated with the 1/16 and 1/4 doses were protected after challenge at days 7, 28, and 35 post vaccination. These data suggest that effective protection against foot-and-mouth disease can be achieved with 1/16 of the recommended vaccine dose using SL vaccination, indicating that the sublingual route is an attractive alternative for the administration of the FMDV vaccine.

The analysis of codon bias of foot-and-mouth disease virus and the adaptation of this virus to the hosts.

The synonymous codon usage patterns of open reading frame (ORF) in foot-and-mouth disease virus (FMDV), the similarity degree of the synonymous codon usage between this virus and the hosts and the genetic diversities of FMDV ORFs and the viral functional genes in viral ORF have been investigated by some simply statistical analyses. As for the synonymous codon usage of FMDV, some over-represented and under-represented codons have a similar usage in all seven serotypes. 33 out of 59 synonymous codons are similarly selected between FMDV ORF and the hosts. It is interesting that the overall codon usage pattern of the strains of serotype O isolated from pigs is different with that of strains of the same serotype isolated from non-pig origin, suggesting that the factor of pigs takes part in the formation of codon usage of FMDV serotype O. Projection of codon usage of nine viral functional genes onto the two-dimensional map represents that even though viral functional genes have various genetic diversities and each gene is not separated from each other based on seven serotypes, the codon usage patterns of VP2, 2C, 3A, 3C and 3D genes belonging to serotype O strains isolated from pigs are different with those of the same serotype strains from non-pig origin. In addition, the interaction between GC12% and GC3% of viral functional genes indicates that the codon usage patterns of VP1, VP2, 2B, 3A, 3C and 3D genes are influenced by mutation pressure from virus. Furthermore, distribution plots of ENC value vs. GC3% for viral function genes indicate that mutation pressure is not the only factor in the formation of codon usage of these genes. The results suggest that both the mutation pressure from virus and the translation selection from the hosts take part in the evolution process of viral functional genes of FMDV.

The effects of the synonymous codon usage and tRNA abundance on protein folding of the 3C protease of foot-and-mouth disease virus.

The 3C protease of foot-and-mouth disease virus (FMDV) has a conserved amino acid sequence and is responsible for most cleavage in the viral polyprotein. The effects of the synonymous codon usage of FMDV 3C gene and tRNA abundance of the hosts on shaping different folding units (α-helix, ß-strand and the coil) in the 3C protease were analyzed based on the structural information of the FMDV 3C protease from Protein Data Bank (PDB: 2BHG) and 210 genes of 3C for all serotypes of FMDV. The strong correlation between some codons usage and the specific folding unit in the FMDV 3C protease is found. As for the effect of translation speed caused by tRNA abundance on the formation of folding units, the codon positions with lowly abundant tRNA scatter in the 3C gene and there is the obvious fluctuation of tRNA abundance locating in the transition boundaries from the ß-strand to the α-helix and the coil, respectively. However, codon positions with lowly abundant tRNA clustering into these boundaries are not found, suggesting that the adjustment of translation speed is likely also achieved by the single codon position with low tRNA abundance rather than a cluster. The observations can provide the information for insight into the role of the synonymous codon usage in the formation of 3C protease of FMDV and effect of the tRNA abundance of the hosts on this formation of protease.

Potential roles of synonymous codon usage and tRNA concentration in hosts on the two initiation regions of foot-and-mouth disease virus RNA.

The open reading frame of foot-and-mouth disease virus (FMDV) contains two authentic initiation codons and the second initiation codon is often selected in high frequency. In the study, we analyzed the effects of the host-cell synonymous codon usage and the overall tRNA concentration in the hosts on the region flanked by the two initiation codons (termed as the region 1) and the same length starting from the second initiation codon (defined as the region 2). We find that low-usage codons of hosts are more selected in the region 1 than the region 2; no obvious usage bias of codon with high C/G content exists in the region 1, and the latter part (ranging from the 13th codon position to the 28th codon position) of the region 1 generally contains the codon sites with the generally lower tRNA concentration than the counterpart of the region 2. The low-usage codons of the hosts with high selection in the region 1 and the cluster codon position with low tRNA concentration in the region 1 may serve as potential factors in decreasing the translation rate of the region 1 caused by initiation from the first start codon of FMDV.

The codon usage model of the context flanking each cleavage site in the polyprotein of foot-and-mouth disease virus.

To investigate the codon usage pattern of the contexts flanking 11 cleavage sites of foot-and-mouth disease virus (FMDV) polyprotein, the codon usage model of the corresponding codon position and the synonymous codon usage in the target contexts of 66 strains were characterized by two simple methods based on the relative synonymous codon usage value. The synonymous codons usage pattern was also compared between this virus and two species of hosts (cattle and domestic pig). It is indicated that FMDV bore a general resemblance to the hosts in terms of the synonymous codon usage pattern. This feature may help FMDV to utilize translational resources of host efficiently. The two amino acid residues constituting each cleavage site contain at least one conserved residue. It was noticed that the codon usage model with the strong bias appeared in some specific positions in the target contexts, and the under-represented synonymous codons, AUA for Ile, CUA for Leu, UUA for Leu and GUA for Val, are preferentially used in these positions. These under-represented synonymous codons likely play role in regulating the translation rate and influencing the secondary structure of the contexts flanking the cleavage sites.

Characteristics of codon usage bias in two regions downstream of the initiation codons of foot-and-mouth disease virus.

The mechanism of utilization of alternative two AUGs in foot-and-mouth disease virus (FMDV) is still unknown to date. In this study, the characteristics of codon usage bias (CUB) of the region between the two AUGs (the region-La) and of the same-sized region behind the second AUG (the region-Lb) in 94 different FMDV RNA sequences were analyzed using relative synonymous codon usage (RSCU) values. The results indicated that many codons with negative CUB were preferentially used in the region-La. There were two conserved residues (Thr and Cys) on the 4th and 6th residue positions of the region-La. The conserved residues had a general tendency to choose synonymous codons with negative CUB. Although most positions in the region-La did not contain conserved residues, many positions tended to use codons with negative CUB in this region. Among these codons, the majority belonged to the amino acids containing synonymous codons with clearly positive and negative CUB, including Asp, Val, Ile, Leu, Thr, Ala, Ser, Asn and Arg. The presence of many codons with negative CUB in the region-La might impair the efficiency of the first AUG selection. The phylogenetic incongruence of the region-La and the region-Lb implied that intertypic recombination played an important role in the evolution of FMDV. Furthermore, due to the existence of more positions with positive CUB and more widespread phylogenetic incongruence in the region-Lb than the region-La, a probable relationship between the degree of CUB and the evolution of the two target regions was revealed.

Analysis of synonymous codon usage in foot-and-mouth disease virus.

In this study, we calculate the relative synonymous codon usage (RSCU) values and codon usage bias (CUB) values to carry out a comparative analysis of codon usage pattern for open reading frames (ORFs) among 85 samples which belong to all seven serotypes of foot-and-mouth disease virus (FMDV). Although the degree of CUB for ORFs is a relatively slight, there is a significant variation for CUB among different serotypes, which is mainly determined by codon usage pattern depending on RSCU. By comparison with RSCU values for all samples, although RSCU values fail to show the relationship of specific-lineage serotype, there are two main genetic populations existing in FMDV, namely (i) serotypes Asia 1, A, C & O; (ii) serotypes SAT 1, 2 & 3. This interesting characteristic may be formed by the mechanism of RNA virus recombination. The analysis of quantitative & qualitative evaluation based on CUB indicates interesting characteristic of codon usage, which suggests that more FMDV genome diversity may exist in specific-lineage serotypes rather than exist randomly. Furthermore, the relationship between amino acids and codon usage pattern indicates that mutation pressure rather than translational selection in nature is the important determinant of the codon usage bias observed. Our work might give some sight into some characteristics of FMDV ORF and some evolutionary information of this virus.

Preparation and characterization of neutralizing monoclonal antibodies against FMDV serotype O with synthetic peptide antigen.

A short linear peptide was designed according to the antigenic site analysis of VP1 protein of foot-and-mouth virus (FMDV) serotype O and synthesized as the peptide immunogen. The peptide, which covers the region from amino acid 133 to 160 of VP1 of FMDV, was linked to the N-terminal cysteine and conjugated with the carrier protein of keyhole limpet hemocyanin (KLH). Normal 6- to 8-week-old BALB/c mice were immunized with the 20 µg dose conjugated peptide antigen four times. The splenocytes from the immunized mice were fused with SP2/0 mouse myeloma cells, and positive hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and subcloned four times with limiting dilution. Five stable hybridoma cell lines, designated as 4F9, 1B11, 1E10, 1D4, and 4B8, were obtained. Isotyping of all obtained MAbs indicated that the MAbs of 4F9, 1E10, and 4B8 belonged to IgG2b; the 1B11 and 1D4 belonged to IgG1 and IgM, respectively. The micro-neutralization test indicated that the MAbs of 4F9, 4B8, and 1B11 were capable of neutralizing FMDV serotype O with neutralization indices ranging from 1.81 to 2.11. These results suggest that linear synthetic peptide conjugate can elicit antibodies against native FMDV virus and can be used as an alternative immunogen for production of MAbs with exact epitope.