Evolutionary trend of foot-and-mouth disease virus in Hong Kong

Foot-and-mouth disease is an endemic animal disease in Hong Kong. In this study, a total of 70 clinical specimens were collected from locally infected pigs from 2001 to 2010. Phylogenetic analysis of VP1 sequences reveal that all Hong Kong FMDV serotype O isolates are classified into three lineages: HK-A and HK-B in Cathay topotype, and HK-C in SEA topotype. Regression analysis projects that the time of divergence from the most recent common ancestor of HK-A and HK-B are 1964 ± 12 and 1987 ± 9 years respectively. Although HK-B shares a common ancestor with strains that caused outbreak in Taiwan and Philippines, there is no consolidated evidence demonstrating the order of introduction events among these regions. HK-C lineage is the latest FMDV isolated in Hong Kong. This virus is likely adopted from bovine into porcine. As local pigs confer immunity mainly against Cathay topotype viruses, introduction of HK-C viruses have led into an unexpectedly high severity and rapid spreading rate of the disease. A systematic surveillance and communication network is essential to provide accurate information in controlling the pandemics.     

Evaluation of cross-protection against three topotypes of serotype O foot-and-mouth disease virus in pigs vaccinated with multi-epitope protein vaccine incorporated with poly(I:C)

Epitope-based vaccines are always questioned for their cross-protection against the antigenically variable foot-and-mouth disease virus (FMDV). In this study, we proved the cross-protection effect of a multi-epitope vaccine incorporated with poly(I:C) against three topotypes of O type FMDV. A total of 45 naïve pigs were vaccinated with different doses of multi-epitope protein vaccine incorporated with poly(I:C). At 28 days post-vaccination, 45 vaccinated and 6 unvaccinated control pigs (two pigs for each group) were challenged with three topotypes of virulent O type FMDV, namely, O/Mya/98 (Southeast Asia topotype), O/HN/CHA/93 (Cathay topotype) and O/Tibet/CHA/99 (PanAsia topotype) strains. All unvaccinated pigs developed generalised FMD clinical signs. Results showed that all pigs (n = 15) conferred complete protection against the O/Mya/98 and O/HN/CHA/93 FMDV strains, 11 of which were protected against the O/Tibet/CHA/99 FMDV strain. The 50% protective dose values of the vaccine against the O/Mya/98, O/HN/CHA/93 and O/Tibet/CHA/99 FMDV strains were 15.59, 15.59 and 7.05, respectively. Contact challenge experiment showed that transmission occurred from the donors to the unvaccinated but not to vaccinated pigs. These results showed that vaccination with multi-epitope protein vaccine incorporated with poly(I:C) can efficiently prevent FMD in pigs.     

Role of apoptosis in the pathogenesis of Asian and South American foot-and-mouth disease viruses in swine

In this study, we performed experiments to evaluate the extend of the process of apoptotic cell death by foot-and-mouth disease virus (FMDV). Apoptosis can also occur in some virus-infected cells, and ability of viruses to either inhibit or promote apoptosis may influence the pathologic outcome of infection. In this study, to determine if apoptosis plays a role in the outcome of FMDV infection in swine, we evaluated apoptosis in diseased tissues collected from pigs inoculated with two different stains of FMDV (O1 Campos and O Taiwan). And host cell DNA fragmentation in diseased tissue from animals which were infected with either virus was evaluated by occurrence of a laddering pattern characteristic of apoptosis. Infection of cultured keratinocytes from swine tongue failed to demonstrate apoptosis in the first few hours of infection, suggesting that cell-to-cell correlation between viral antigen and apoptotic changes, e.g. cytokine secretions by immune system cells, could be critical to initiating apoptosis. Consistent with this finding, we were able to detect the pro-inflammatory cytokine TNF-alpha in diseased tissues. A clear difference in the pathogenicity of the two different FMDV isolates to pigs was not demonstrated in our study.     

云南边境O型口蹄疫监测及病毒VP1基因序列分析

口蹄疫是由口蹄疫病毒引起的主要侵袭偶蹄动物的一种急性热性高度接触性传染病。口蹄疫病毒为微RNA病毒科口蹄疫病毒属成员,存在7个不同血清型,病毒VP1蛋白抗原性差异是病毒血清型划分依据,而其编码基因(1D)核苷酸序列差异是同型病毒拓扑型(Topotype)或基因型鉴别依据。采用O/A/C/Asia-1多重RT-PCR技术,对2006年自云南边境地区采集的120份动物组织样品,进行口蹄疫病原监测,检出O型口蹄疫病毒阳性样品15份。对阳性样品中病毒VP1基因全序列进行扩增、纯化后,克隆至pMD18-T载体测序,并与已知代表性毒株进行比对及系统发育分析。结果发现:云南边境O型口蹄疫病毒阳性样品VP1基因核苷酸序列同源性介于77.3%~98.7%,可划分为3个不同的拓扑型或基因型:中东-南亚型(ME-SA)或泛亚型(PAN-Asia)、古典中国型(Cathay)、东南亚型(SEA)。部分样品VP1蛋白表位43位、154位关键性氨基酸位点存在变异。     

O型口蹄疫病毒不同基因型联合RT-PCR检测方法的建立

在比较国内外口蹄疫病毒(FMDV)流行毒株序列的基础上，设计了检测FMDV的PCR引物及O型FMDV不同基因型即中国型(Cathay)与乏亚株(PanAsia)的二联PCR引物。在确定单项RT-PCR反应条件的基础上，建立、优化了二联RT-PCR反应体系和条件，并进行了相关病毒检测试验。结果表明，建立的检测O型FMDV不同基因型中国型与泛亚株的二联RT-PCR，有效、特异且敏感，为FMD的诊断、流行病学调查及疫苗应用奠定了基础。     

Sequence analysis of the protein-coding regions of foot-and-mouth disease virus O/HK/2001

The nucleotide sequence of the protein-coding region of foot-mouth-disease virus (FMDV) strain O/HK/2001 was determined and compared with the sequences of other FMDVs that were registered in GenBank. The protein-coding region was 6966 nucleotides in length and encoded a protein of 2322 amino acid residues. Comparison of the nucleotide sequence and its deduced amino acid sequence with those of other isolates indicated that O/HK/2001 belonged to the Cathay topotype. A genomic coding region nucleotide sequence phylogenetic tree of several FMDV-O isolates showed that O/HK/2001 was most closely related to FMDV isolates found in Taiwan during 1997, and especially shared significant similarity to HKN/2002, suggesting that the virus causing outbreaks in Hong Kong was genetically most-closely related to that causing an outbreak of type O in Taiwan. Mutations in O/HK/2001 were revealed, including frequent substitutions in the VP1 and L proteins, and deletions involving 10 amino acid residues in the 3A protein. This study was undertaken to assess the regional variation of prevalent FMDV type O viruses and to establish a sequence database for FMDV molecular epidemiological investigation.     

Capsid coding sequences of foot-and-mouth disease viruses are determinants of pathogenicity in pigs

The surface exposed capsid proteins, VP1, VP2 and VP3, of foot-and-mouth disease virus (FMDV) determine its antigenicity and the ability of the virus to interact with host-cell receptors. Hence, modification of these structural proteins may alter the properties of the virus.In the present study we compared the pathogenicity of different FMDVs in young pigs. In total 32 pigs, 7-weeks-old, were exposed to virus, either by direct inoculation or through contact with inoculated pigs, using cell culture adapted (O1K B64), chimeric (O1K/A-TUR and O1K/O-UKG) or field strain (O-UKG/34/2001) viruses. The O1K B64 virus and the two chimeric viruses are identical to each other except for the capsid coding region.Animals exposed to O1K B64 did not exhibit signs of disease, while pigs exposed to each of the other viruses showed typical clinical signs of foot-and-mouth disease (FMD). All pigs infected with the O1K/O-UKG chimera or the field strain (O-UKG/34/2001) developed fulminant disease. Furthermore, 3 of 4 in-contact pigs exposed to the O1K/O-UKG virus died in the acute phase of infection, likely from myocardial infection. However, in the group exposed to the O1K/A-TUR chimeric virus, only 1 pig showed symptoms of disease within the time frame of the experiment (10 days). All pigs that developed clinical disease showed a high level of viral RNA in serum and infected pigs that survived the acute phase of infection developed a serotype specific antibody response. It is concluded that the capsid coding sequences are determinants of FMDV pathogenicity in pigs.     

Procedures for preventing transmission of foot-and-mouth disease virus (O/TAW/97) by people

The aim of this study was to determine personal hygiene protocols and animal avoidance periods needed to prevent transmission of FMDV (O/TAW/97). Forty-six, 9-week-old barrows free of FMDV were randomly allocated to five treatment groups and a control group. Investigators contacted and sampled FMDV-inoculated pigs for approximately 40 min and then contacted and sampled sentinel pigs after using no biosecurity procedures, washing hands and donning clean outerwear, or showering and donning clean outerwear. Personnel were sampled for nasal carriage of FMDV for 85.43 h. Contaminated personnel did not transmit FMDV to susceptible pigs after handwashing or showering, and donning clean outerwear. FMDV was transmitted when biosecurity procedures were not used. FMDV was not detected in nasal secretions of investigators. Thus, extended animal avoidance periods do not appear to be necessary to prevent transmission of FMDV (O/TAW/97) by people to pigs when organic material is removed through handwashing/showering and donning clean outerwear. This study supports similar findings in a previous publication using FMDV (O/UK/35/2001).     

Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies.

A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion (140S); Class II, restricted to 140S and the virion protein subunit (12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-1, A, O1 and C but neutralizing capacity was restricted to serotype Asia-1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.     

Molecular epidemiology of serotype O foot-and-mouth disease virus isolated from cattle in Ethiopia between 1979-2001

Partial 1D gene characterization was used to study phylogenetic relationships between 17 serotype O foot-and-mouth disease (FMD) viruses in Ethiopia as well as with other O-type isolates from Eritrea, Kenya, South and West Africa, the Middle East, Asia and South America. A homologous region of 495 bp corresponding to the C-terminus end of the 1D gene was used for phylogenetic analysis. This study described three lineages, viz. African/Middle East-Asia, Cathay and South American. Within lineage I, three topotypes were defined, viz. East and West Africa and the Middle East-Asia together with the South African isolate. The Ethiopian isolates clustered as part of topotype I, the East African topotype. Two clades (based on < 12 % nucleotide difference) A and B were identified within the East African isolates, with clade A being further classified into three significant branches, A1 (80% bootstrap support), A2 (89% bootstrap support) and A3 (94% bootstrap support). Clade B consisted of two Kenyan isolates. Within topotype I, the 17 Ethiopian isolates showed genetic heterogeneity between themselves with sequence differences ranging from 4.6-14 %. Lineage 2 and 3 could be equated to two significant topotypes, viz. Cathay and South America. Comparison of amino acid variability at the immunodominant sites between the vaccine strain (ETH/19/77) and other Ethiopian outbreak isolates revealed variations within these sites. These results encourage further work towards the reassessment of the type O vaccine strain currently being used in Ethiopia to provide protection against field variants of the virus.     

Study on the porcinophilic foot-and-mouth disease virus I. production and characterization of monoclonal antibodies against VP1

Monoclonal antibodies (MAbs) reported here were produced against the porcinophilic foot-and-mouth disease virus (FMDV) that caused the devastating swine disease on 1997 in Taiwan. A panel (25) of MAbs were found to react with VP1 of O/Taiwan/97 (O/97) by ELISA with various potencies. The biological identities of these VP1 reacting MAbs, such as neutralization activity, isotype and capability to distinguish between two serotype O FMDVs, O/97 and O/Taiwan/KM1/99 (O/99), were further analyzed. Eleven out of the total eighteen O/97 neutralizing MAbs were able to neutralize heterologous O/99. Eight O/97 neutralizing and five non-neutralizing MAbs could differentiate two serotype O FMDVs by immunofluorescence assay (IFA) implied that these thirteen MAbs recognized O/97 specific epitope(s). Furthermore, reactivities of the VP1 reacting MAbs with a 29 amino acids synthetic peptide (P29) representing the betaG-betaH loop of VP1 were analyzed by ELISA and fourteen were found positive. MAb clone Q10E-3 reacting strongest with VP1 and P29, neutralizing both but not differentiating two serotype O viruses suggested that the antibody binding site might involve the RGD motif and its C terminal conserved region on betaG-betaH loop. MAbs with diverse characters presented in this study were the first raised against porcinophilic FMDV. The complete set of MAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.     

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10⁹ CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID₅₀ of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 10⁹ CFU of p3D-NT56/S. cho were protected in 9 days.     

9株猪瘟分离毒株的致病特性

采用9株临床表现强、中、低致病特点的猪瘟野毒分离株第3代细胞毒作为种毒,按3mL/头剂量分别注射猪瘟抗原及抗体阴性猪,再用上一代传代猪发病后的血毒作为下一代接毒用种毒接种试验猪1头或2头,或上一代传代猪发病后,同圈放入1头或2头试验猪进行同居感染.如此进行,GDGZ1/95、BJCY1/96和JL1/94传至8代;FJFQ1/99传至7代;HeBHH1/95、HeNBY1/96、BJTX3/96、GXBH1/98和HeNXH2/98传至3代.结果显示:上述分离株传至3～5代过程中均表现出毒力增强的趋势,从3～5代传代到8(7)代的过程中毒力进一步增强并保持稳定,且均超过了标准石门强毒株的发病特点.所有试验猪均出现较典型的猪瘟临床症状,解剖后均表现出不同程度的病理变化,死后或解剖后的各种脏器经HCFA检查均为强阳性,这种现象进一步证实了猪是猪瘟病毒的敏感动物,各毒株之间毒力没有明显差异.对其中7株分离株传代血毒部分代次E2基因主要区域进行序列分析,结果仅GDGZ1/95株从F1～F8代中的F6代有2个核苷酸的差异,引起1个相应氨基酸的变异,其余毒株的不同代次没有碱基发生变异,初步说明猪瘟病毒基因型表现相对的稳定性.     

猪口蹄疫病毒多抗原表位的原核表达与纯化

利用限制性酶切从重组质粒pShuttle-CMV-VP中得到猪O型口蹄疫病毒VP1(21-60)-(141-160)-(200-213)位氨基酸的基因。将此多抗原表位基因克隆至原核高效表达载体pET43.1 a(+),在E.coliBL21中用IPTG诱导表达了含有猪口蹄疫病毒多抗原表位的融合蛋白,并用镍柱亲和层析法获得了纯化蛋白。W estern-b lot结果表明融合蛋白可被猪O型口蹄疫病毒标准阳性血清所识别,从而为进一步研究FMDV多表位抗原的免疫特性和诊断方法奠定了基础。     

Quantitative analysis of foot-and-mouth disease virus RNA duration in tissues of experimentally infected pigs

Quantitative analysis of the duration of foot-and-mouth disease virus (FMDV) RNA in tissues was carried out in pigs experimentally infected with FMDV O UKG 34/2001 and O SKR 1/2000. The results showed that the viral RNA was still detectable in cervical lymph nodes, mandibular lymph nodes and tonsils collected from both inoculated and contact pigs at 28 days post infection. There was no detectable viral RNA in the soft palate or pharynx, which are thought to be tissue sites for viral persistence in cattle. Further study is needed to clarify whether this difference has significance in terms of viral clearance in pigs.     

Are Low‐Density Granulocytes the Major Target Cells of Classical Swine Fever Virus in the Peripheral Blood?

The target cells of classical swine fever (CSF) virus in the peripheral blood of pigs infected with recent field isolates from Germany were studied. Eight weaned pigs were inoculated oronasally with the CSF virus field isolate Visbek/Han 95 and three weaners were inoculated with the isolate Losten/Freese 98. All pigs showed severe clinical signs typical of CSF and died or had to be euthanized between 9 and 24 days post‐infection (dpi). The first cells in the peripheral blood which became infected with CSF virus were mixed granulocytes (a combination of low‐ and high‐density granulocytes). These cells yielded the highest infectivity for PK 15 cell cultures. On day 7 post‐infection, the peripheral blood mononuclear cell (PBMC) fraction was virus positive, while the peripheral blood leucocyte (PBL), peripheral blood T lymphocyte (PBT) and high‐density granulocyte fractions were either negative or their infectivity was lower than the infectivity of the PBMC fraction. These results indicate that PBMC contain more virus‐positive cells than other fractions of leucocytes. These findings may also have diagnostic implications for the detection of CSF virus in blood samples. Because PBMC showed the highest infectivity in the early stages of CSF, it should be the sample of choice for CSF virus isolation.     

Increased humoral antibody response of foot-and-mouth disease virus vaccine in growing pigs pre-treated with poly-γ-glutamic acid

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.     

Phylodynamic reconstruction of O CATHAY topotype foot-and-mouth disease virus epidemics in the Philippines

Reconstructing the evolutionary history, demographic signal and dispersal processes from viral genome sequences contributes to our understanding of the epidemiological dynamics underlying epizootic events. In this study, a Bayesian phylogenetic framework was used to explore the phylodynamics and spatio-temporal dispersion of the O CATHAY topotype of foot-and-mouth disease virus (FMDV) that caused epidemics in the Philippines between 1994 and 2005. Sequences of the FMDV genome encoding the VP1 showed that the O CATHAY FMD epizootic in the Philippines resulted from a single introduction and was characterised by three main transmission hubs in Rizal, Bulacan and Manila Provinces. From a wider regional perspective, phylogenetic reconstruction of all available O CATHAY VP1 nucleotide sequences identified three distinct sub-lineages associated with country-based clusters originating in Hong Kong Special Administrative Region (SAR), the Philippines and Taiwan. The root of this phylogenetic tree was located in Hong Kong SAR, representing the most likely source for the introduction of this lineage into the Philippines and Taiwan. The reconstructed O CATHAY phylodynamics revealed three chronologically distinct evolutionary phases, culminating in a reduction in viral diversity over the final 10 years. The analysis suggests that viruses from the O CATHAY topotype have been continually maintained within swine industries close to Hong Kong SAR, following the extinction of virus lineages from the Philippines and the reduced number of FMD cases in Taiwan.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0090-y) contains supplementary material, which is available to authorized users.