应用荧光RT-PCR检测亚欧型口蹄疫病毒

根据口蹄疫病毒基因组的5’非翻译区序列比较保守的特点,采用软件设计亚欧型(A、O、C及Asia-1型)FMDV通用的引物和探针,经对反应条件和反应体系进行优化,建立了亚欧型FMDV的实时荧光RT-PCR检测方法。研究表明,该方法检测阳性质粒模板的线性范围为1.9×107-1.9×10拷贝,最低可检测19个拷贝。通过对FMDV各血清型(A、O、C、Asia-1及SAT-1,2,3型)的检测,证实该方法对亚欧型FMDV具有良好的特异性,能有效区分亚欧型和南非型FMDV感染。本研究为亚欧型口蹄疫的快速诊断和流行病学调查提供了新的方法。     

A serological survey for antibodies against foot-and-mouth disease virus (FMDV) in domestic pigs during outbreaks in Kenya

Foot-and-mouth disease (FMD) is endemic in Kenya and has been well studied in cattle, but not in pigs, yet the role of pigs is recognised in FMD-free areas. This study investigated the presence of antibodies against FMD virus (FMDV) in pigs sampled during a countrywide random survey for FMD in cattle coinciding with SAT 1 FMDV outbreaks in cattle. A total of 191 serum samples were collected from clinically healthy pigs in 17 districts. Forty-two of the 191 sera were from pigs vaccinated against serotypes O/A/SAT 2 FMDV. Antibodies against FMDV non-structural proteins were found in sera from 30 vaccinated and 71 non-vaccinated pigs, altogether 101/191 sera (53 %), and 91 % of these (92/101) also had antibodies measurable by serotype-specific ELISAs, predominantly directed against SAT 1 with titres of 10–320. However, only five high titres against SAT 1 in vaccinated pigs were confirmed by virus neutralisation test (VNT). Due to high degree of agreement between the two ELISAs, it was concluded that positive pigs had been infected with FMDV. Implications of these results for the role of pigs in the epidemiology of FMD in Kenya are discussed, and in-depth studies are recommended.     

Prevalence of seroreagents to FMDV in the cattle population in Poland: results of 9-year monitoring studies

The aim of this study was to evaluate the occurrence of antibodies to foot-and-mouth disease virus (FMDV) in sera of cattle in Poland. The examinations were performed using the virus neutralization (VN) test and ELISA methods: liquid-phase blocking ELISA (LPBE) and 3ABC-ELISA. During 1993-2001, about 681,000 samples of sera collected from animals held on the territory of Poland were tested. Of about 600,000 sera taken from animals exported to the European Union, 963 samples (0.16%) were found to be positive to FMDV types A, O and/or C. During 1996-2001 out of 85,000 sera tested as part of the national serological surveillance program for FMD, the FMDV antibodies were recorded in 51 (0.06%) samples. Persistence of FMD antibodies was observed in sera of cattle from the region around Zduńska Wola, which had been vaccinated annually during 1985-1985 with trivalent FMD vaccine. The results of the serological studies of 550 animals from this region indicates the presence of FMDV antibodies in sera of 240 (44%) cattle. A half-life of maternal antibodies in sera of calves born to seropositive dams was estimated; the highest level of FMDV antibodies was detected in sera taken from new-born calves aged 5-10 days. The level of FMDV antibodies in beestings of dams was highest during the first 10 hours after parturition; after 24 hours a significant decrease (3-5 times) was found and in two weeks post parturition FMDV antibodies were undetectable in the milk. It was established that all LPBE/VN positive sera of cattle exported from Poland, from the vaccination zone around Zduńska Wola as well as those tested as part of the national serological surveillance program for FMD, were taken exclusively from vaccinated animals or calves born to vaccinated dams.     

O型和Asia1型口蹄疫病毒感染RT-PCR鉴别诊断方法的建立

根据GenBank中O型和Asia1型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)的vp3、vp1和2A基因序列,并与其它血清型FMDV的对应基因序列进行比较,设计用于扩增O型和Asia1型FMDV vp1基因的特异性引物,建立O型和Asia1型FMDV RT-PCR鉴别诊断方法。本方法首先用通用型引物进行RT-PCR,确定是否为FMDV感染,然后用特异性引物鉴别O型或Asia1型FMDV的感染。用vp1基因序列分析进行符合性试验,验证了该方法所具有的特异性和敏感性。本方法可用于O型和Asia1型FMD的快速诊断及流行病学调查。     

Implementation in Australia of molecular diagnostic techniques for the rapid detection of foot and mouth disease virus

OBJECTIVE: To evaluate and implement rapid molecular diagnostic techniques for the detection of foot and mouth disease virus (FMDV) suitable for use in Australia. DESIGN: Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks. RESULTS: Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3. CONCLUSION: Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia.     

口蹄疫病毒RT-PCR检测与血清型鉴别

针对编码非结构蛋白的3D基因合成一对引物进行口蹄疫病毒RT-PCR扩增,不同血清型病毒的RNA样本均显现一条457bp的目的带,与预期设计的长度相符合。在敏感性试验中,O型、A型和AsiaⅠ型病毒的最小RNA检出量分别为0.8ng、8ng和8ng。根据GenBank发表的口蹄疫病毒VP1和2A基因序列,采用多重RT-PCR鉴别口蹄疫病毒血清型,O型、A型和AsiaⅠ型病毒的特异性扩增片段分别为200bp、340bp和500bp。对9份乳鼠感染病料进行检测,确诊为O血清型口蹄疫病毒感染。     

Detection of Foot and Mouth Disease and Porcine Reproductive and Respiratory Syndrome Viral Genes Using Microarray Chip

Two viral pathogens, namely, porcine reproductive and respiratory syndrome virus (PRRSV) and foot and mouth disease virus (FMDV), were selected as models for multiple pathogen detection in a cDNA microarray. Two signature regions selected from ORF2 (around 500 bp) and ORF5 (around 600 bp) of PRRVS (America serotype), and one signature region from structural genes VP1 (around 500 bp) of FMDV type O were designed and spotted on a nylon membrane. For PCR sensitivity study, the cloned FMDV–VP1 template could be diluted to near one copy and its PCR product was still detectable in gel electrophoresis. In the microarray detection, the labelling FMDV probes (3 mg/ml) could be diluted 320 times and still maintained a visible colour when hybridized with the chip. Using the mixing primers, the microarray chip demonstrated rapid and accurate detection of the specific genes. To our knowledge, this preliminary study is the first example reported applying the long signature sequences to the multiple pathogen detection in cDNA microarray.     

Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus

A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMD diagnosis.     

口蹄疫灭活疫苗抗原稳定性研究进展

口蹄疫是一种感染牛、羊和猪等偶蹄动物、具有高度传染性的动物疫病。疫苗免疫是控制该病的关键措施之一,口蹄疫灭活疫苗在口蹄疫流行地区广泛使用。灭活疫苗中,完整口蹄疫病毒粒子(146S粒子)是至关重要的免疫抗原,它的数量和稳定性决定了疫苗的免疫效果。不同血清型,甚至同型不同毒株的口蹄疫病毒粒子稳定性不同,146S粒子在一定温度、酸、碱条件下容易分解为五聚体(12S粒子),导致疫苗免疫效力大幅下降。近年来口蹄疫病毒结构及其稳定性的分子基础研究取得了一定进展,为研究口蹄疫病毒稳定性提高疫苗质量,开发新型口蹄疫空衣壳疫苗提供了重要的理论基础。本文简要介绍了口蹄疫病毒结构基础、稳定性研究方法和研究进展。为同行了解口蹄疫病毒结构与免疫的关系,评价新型疫苗提供一些参考信息。     

Detection of foot-and-mouth disease virus by competitive ELISA using a monoclonal antibody specific for the 12S protein subunit from six of the seven serotypes.

Foot-and-mouth disease (FMD) prevention and control programs are dependent upon rapid, reliable diagnostic procedures. The widely used FMD diagnostic complement fixation (CF) procedures require a specific antiserum for each of the seven FMDV serotypes making the tests both cumbersome and difficult to standardize. An FMD diagnostic, monoclonal antibody based inhibition-ELISA procedure was developed. The test uses a single monoclonal antibody (MAb) that reacts with all European and South American FMDV isolates examined. The procedure detects a highly conserved epitope on the 12S protein subunit of FMDV which appears to be common to all FMDV's with the exception of the South African Territories 2 serotype. The results indicate that the sensitivity of this test is greater than CF and approaches that of virus isolation.     

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。     

Investigation of serum protein profiles in sheep naturally infected with foot-and-mouth disease virus

This study was carried out to determine serum protein profiles in naturally infected sheep with foot-and-mouth disease virus (FMDV). The study material consisted of twelve healthy and 36 sheep with foot-and-mouth disease (FMD). FMD had been diagnosed on the basis of clinical findings and results of serological examination. Serotypes serologically detected in the FMDV-infected sheep were as follows: O (n = 11), A (n = 8) and mixed infection with serotypes O, A and Asia-1 (n = 17).The total protein, albumin and globulin concentrations as well as Albumin/Globulin ratio were slightly different among the groups (P < 0.05). Three protein bands of 66 kDa, 45 kDa and 20 kDa were remarkable. Moderate differences were determined between healthy and infected sheep for proportion of distribution in serum proteins. In conclusion, serum protein concentrations and serum protein profiles were slightly changed and no specific serum protein profile occurred in sheep infected with either O or A or in sheep mixed infected with the O and A and Asia-1 serotypes of FMDV compared to healthy ones.     

Construction of a multiple targeting RNAi plasmid that inhibits target gene expression and FMDV replication in BHK-21 cells and suckling mice

Foot-and-mouth disease (FMD) is a highly contagious disease that afflicts cloven-hoofed animals. The etiological agent of FMD is foot-and-mouth disease virus (FMDV). The VP1 gene of FMDV is essential during the life cycle of the virus and plays a key role in the attachment of the virus to susceptible cells. We constructed a plasmid, pCWN11, that expresses siRNAs multiple-targeting the VP1 genes of FMDV. We evaluated the gene silencing efficiency of the plasmid using an enhanced green fluorescent protein (EGFP) reporter system in BHK-21 cells. The antiviral potential of the plasmid in BHK-21 cells and suckling mice were investigated. The results indicate that cotransfection of pCWN11 with any one of three serotypes VP1-EGFP plasmids resulted in a reduction in the EGFP signal relative to the control. Moreover, the antiviral potential induced by pCWN11 was evident during challenge with one FMDV isolate of either serotype O (HKN/2002) or serotype Asia I (YNBS/58), and the inhibition extended to almost 40 h. Furthermore, subcutaneous injection of pCWN11 in the neck made suckling mice significantly less susceptible to FMDV serotype O and Asia I.     

Evaluation of foot and mouth vaccination for yak (<Emphasis Type="Italic">Bos grunniens</Emphasis>) in Pakistan

In northern Pakistan, many farming communities rely on domestic yak (Bos grunniens) as a principle source of income. A 2006 participatory disease surveillance report from this region indicated that foot-and-mouth disease (FMD) is the most prevalent annual disease of yak. Our objectives of this study were to determine exposure levels of yak to FMD virus; implement a vaccination program based on current, regional FMD virus serotypes and subtypes; and quantify immune responses following vaccination. Blood samples were used to determine pre-vaccination exposure of animals to FMD virus by antibody presence to non-structural proteins of FMD virus using a 3-ABC trapping indirect ELISA. Vaccine used consisted of FMD serotypes ‘O’ (PanAsia-2), ‘A’ (Iran-05), and ‘Asia-1’ (Shamir), but changed later during the study to match newly circulating viruses in the country (‘O’-PanAsia-2; ‘A’-Turk-06 and Asia-1-Sindh-08). Three hundred sixty-three blood samples were tested from selected villages to determine pre-vaccination FMD virus exposure in yak with an average of 37.7%. Immune responses from initial vaccination and booster dose 30 days later showed clear protective levels (as mean percent inhibition) of antibodies against structural proteins of serotypes ‘O,’ ‘A,’ and ‘Asia-1.’ These responses remained above threshold positive level even at day 210 following initial vaccination. Results of sero-surveillance and anecdotal information of repeated FMD outbreaks demonstrate the persistence of FMD virus of yak in northern Pakistan. Laboratory results and field observations clearly indicated that yak can be protected against FMD with a good quality vaccine with FMD serotype(s) matching current, regionally circulating FMD virus.     

Foot-and-mouth disease in camelids: a review

Foot-and-mouth disease (FMD) in South American camelids, in dromedaries and Bactrians is reviewed. Recent well-executed experimental studies in New World camels indicate that, although the llama and alpaca can be infected with FMD virus (FMDV) by direct contact, they are not very susceptible and do not pose a risk in transmitting FMD to susceptible animal species. They do not become FMDV carriers. Reports on FMD in dromedaries are, however, conflicting. Serological investigations in Africa and the United Arab Emirates (UAE) on thousands of camel sera were negative and experimental infections have been conducted on only a few dromedaries with one serotype and in one country. The design and execution of most of these experiments were poor and therefore the conclusions are questionable. From these investigations, it seems that dromedaries can contract the disease after experimental infection and through close contact with FMD diseased livestock, but do not present a risk in transmitting FMD to susceptible animals. They do not become FMDV carriers. Recent reports from Mongolia describe similar FMD lesions in Bactrian camels. However, so far no samples have tested positive for FMD. To clarify the situation in Bactrians, samples from suspected clinical cases should be tested because other viral vesicular diseases cannot be distinguished from FMD. Thus, further research on the epidemiology of FMD in camelids is necessary. This would include large-scale serological investigations and experimental infections with different FMD serotypes in connection with susceptible contact animals. The Office International des Epizooties (OIE) Code chapter on FMD includes camelids as being susceptible species to FMD, giving the impression that they are similar to cattle, sheep, goats and pigs in their potential involvement in the epidemiology of FMD. This is clearly not the case, and this issue should be re-addressed by the relevant authorities.     

Differentiation of foot-and-mouth disease-infected pigs from vaccinated pigs using antibody-detecting sandwich ELISA

The presence of serum antibodies for nonstructural proteins of the foot-and-mouth disease virus (FMDV) can differentiate FMDV-infected animals from vaccinated animals. In this study, a sandwich ELISA was developed for rapid detection of the foot-and-mouth disease (FMD) antibodies; it was based on an Escherichia coli-expressed, highly conserved region of the 3ABC nonstructural protein of the FMDV O/TW/99 strain and a monoclonal antibody derived from the expressed protein. The diagnostic sensitivity of the assay was 98.4%, and the diagnostic specificity was 100% for na?ve and vaccinated pigs; the detection ability of the assay was comparable those of the PrioCHECK and UBI kits. There was 97.5, 93.4 and 66.6% agreement between the results obtained from our ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies for nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in bovine sera. Furthermore, the absence of cross-reactions generated by different antibody titers against the swine vesicular disease virus and vesicular stomatitis virus (VSV) was also highlighted in this assay's specificity.     

Direct contact transmission of three different foot-and-mouth disease virus strains in swine demonstrates important strain-specific differences

A novel direct contact transmission model for the study of foot-and-mouth disease virus (FMDV) infection of swine was utilized to investigate transmission characteristics of three FMDV strains belonging to serotypes A, O and Asia1. Each strain demonstrated distinct transmission characteristics and required different exposure times to achieve successful contact transmission. While a 4h exposure was sufficient for strain A24 Cruzeiro (A24Cru), both O1 Manisa and Asia1 Shamir transmission required 18h or more. Viral excretion levels from donors (for all three strains) and virus present in room air (for A24Cru and O1 Manisa) were evaluated and associated with clinical signs and observed transmission pattern. Although all directly inoculated donor animals showed acute FMD, A24Cru had the highest levels of viral shedding in saliva and nasal swabs followed by O1 Manisa and Asia1 Shamir. Virus levels in room air were higher and were detected longer for A24Cru than for O1 Manisa. These results provide direct evidence for important strain-specific variation in transmission characteristics and emphasize the need for thorough evaluation of different FMDV viral strains using a well defined contact transmission methodology. This information is critical for vaccine and biotherapeutic efficacy testing, pathogenesis and disease modeling of FMDV transmission.