Cloning and Prokaryotic Expression of VP1 Gene of Foot-and-Mouth Disease Virus (FMDV) Type O

According to the complete genome of foot-and-mouth disease virus(FMDV)type O,a pair of special primers was designed to amplify VP1 gene.The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG.Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight.Then the product was purified by GSTrap FF columns.The product was detected through Western-blot that showed the protein has antigenicity.It provided fundamental data and materials for further investigation on diagnosis method of FMDV.

Prokaryotic Expression of P1 Gene of Type Asia1 Foot and Mouth Disease Virus(FMDV)and the Preparation of Its Antiserum

[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gene cloning techniques,and then cloned into pET-32a（＋）plasmid;subsequently the recombinant plasmid was transformed into E.coli BL21（DE3）;after the IPTG induction and protein purification,SDS-PAGE analysis was carried out;the ultrasonic wave was use to lyse the cultivated recombinant strain,and after the isolation and purification,this fusion protein was utilized to immunize New Zealand rabbits so as to prepare P1 protein antiserum.[Result]The positive clones were obtained;SDS-PAGE result showed that the target band was appeared at 105 kD;Western blot analysis showed that the antisera could bind to the expressed P1 fusion protein specifically;the ELISA titer of the rabbit anti-FMDV-P1 sera was approximately 1∶5 120.[Conclusion]This study had provided foundations for FMDV serological diagnostic methods and genetically engineered vaccine.

B Cell Epitopes within VP1 of Type O Foot-and-mouth Disease Virus for Detection of Viral Antibodies

In this study, the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141-160 （epitopel）, tandem repeat 200-213 （epitope2 （＋2）） and the combination of two epitopes （epitopel-2） was genetically cloned into the prokaryotic expression vector PPRoExHTb and pGEX4T-1, respectively. VP1 and the fused epitopes GST-E1, GST-E2 （＋2） and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity. Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test. For VP1-ELISA and all the epitope ELISAs, there were clear distinctions between the FMDV-positive and the FMDV-negative samples. Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A, C and Asia1 did not occur. The relative sensitivity and specificity for the GST-E1 ELISA, GST-E2 （＋2）, GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%, 95.0% and 90%, 100% and 81.8%, 96.6% and 80.9% respectively. This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.

Effect of amino acid mutation at position 127 in 3A of a rabbitattenuated foot-and-mouth disease virus serotype Asia1 on viral replication and infection

An amino acid mutation(R127→I) in the 3A non-structural protein of an FMDV serotype Asia1 rabbit-attenuated ZB strain was previously found after attenuation of the virus. To explore the effects of this mutation on viral replication and infection, the amino acid residue isoleucine(I) was changed to arginine(R) in the infectious cDNA clone of the rabbit-attenuated ZB strain by sitedirected mutagenesis, and the R127-mutated virus was rescued. BHK monolayer cells and suckling mice were inoculated with the R127-mutated virus to test its growth property and pathogenicity, respectively. The effects of the R127 mutation on viral replication and virulence were analyzed. The data showed that there was a slight difference in plaque morphology between the R127-mutated and wild-type viruses. The growth rate of the mutated virus was lower in BHK-21 cells and its virulence in suckling mice was also attenuated. This study indicates that the R127 mutation in 3A may play an important role in FMDV replication in vitro and in pathogenicity in suckling mice.

Amplification and Characterization of Bull Semen Infected Naturally with Foot-and-mouth Disease Virus Type Asia1 by RT-PCR

To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group.

Single amino acid substitution of VP1 N17D or VP2 H145Y confers acid-resistant phenotype of type Asia1 foot-and-mouth disease virus

Infection by foot-and-mouth disease virus(FMDV) is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. However, dissociation of the FMDV 146S particle in mildly acidic conditions renders inactivated foot-and-mouth disease(FMD) vaccines much less effective. Type Asia1 FMDV mutants with increased resistance to acid inactivation were selected to study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of FMDV. Sequencing of capsid-coding regions revealed four amino acid replacements(VP1 N17D, VP2 H145Y, VP2 G192D, and VP3 K153E) in the viral population of the acid-selected 10th passage. We performed single or combined mutagenesis using a reverse genetic system, and our results provide direct experimental evidence that VP2 H145Y or VP1 N17D substitution confers an acid-resistant phenotype to type Asia1 FMDV.

The Complete Genomic Sequence of a Foot-and-Mouth Disease Virus Isolated from the Swine

The complete genomic sequence of foot-and-mouth disease virus (FMDV) Chinese strain OH/CHA/99 was determined. The 8040 nt sequence and the deduced amino acid sequence werecompared with FMDV sequences published. The results showed that OH/CHA/99 shared highersequence homology with OTYTW/97, indicating their close genetic relationship. However,the strain had lower sequence identity with O1/Kaufbeuren/66 strain. Besides, largedeletions in 3A coding region were observed in OH/CHA/99. It was shown that the poly (A)tail of OH/CHA/99 had 56 As at least.

Development of a Synthetic Peptide ELISA Assay for the Detection of Foot-and-Mouth Disease Virus Nonstructural Protein Antibodies

In order to develop an ELISA assay with synthetic peptides for the detection of antibody to the nonstructural proteins of foot-and-mouth disease virus, specific peptides were synthesized by a solid-phase method according to FMDV NSPs B-cell epitopes, and were conjugated to carrier protein BSA. An ELISA system was developed to detect FMDV NSPs antibody with the conjugated proteins as the coating antigen. The optimal coating concentration of the antigen was determined as 2.5 μg mL-1. The comparative study of this assay with UBI NSP ELISA kit and national commercial 3ABC ELISA kit in the detection of 199 serum samples showed that they were very coincident, and the identity rates were 96.48 and 97.48%, respectively. The development of ELISA using the synthetic peptides as coating antigen is specific, reproducible, stable, and easy, and can be used to differentiate FMDV infected pigs from immunized pigs.

Comparison of Three ELISA Kits for the Differentiation of Foot-and-mouth Disease Virus-infected from Vaccinated Animals

研究被执行验证工具包为 FMDV 的区别在中国开发了的 3 FMDV 3ABC-I-ELISA 感染并且种牛痘的动物。从天真、种牛痘的牛以及从被感染了的牛的 sera 的集合被商业诊断工具包对 FMDV 的 nonstructural 蛋白质(NSP ) 为抗体测试， Ceditest 吗？FMDV-NS (Ceditest？工具包) ， UBI？FMDV NONSTRUCTURAL 蛋白质 ELISA 方向插入(UBI？工具包) 并且一个 FMDV 3ABC-I-ELISA 工具包在 Lanzhou 发展了兽医研究院。三个工具包的测试参数(敏感和特性) 被决定，并且从 FMD 3ABC-I-ELISA 工具包获得的结果与从二个外国工具包获得了那相比。结果显示巧合在 FMDV 3ABC-I-ELISA 和 Ceditest 之间评价吗？工具包 98.05% 是，并且在 FMDV 3ABC-I-ELISA 和 UBI 之间的巧合率？工具包是 94.4% ；两 Ceditest 的敏感？并且 FMDV 3ABC-I-ELISA 工具包是 100% 。然而， UBI 的敏感？工具包仅仅是 81.8% 。与从天真或种牛痘的非感染的动物的 sera，所有测试的特性超过了 90% 。关键词 Foot-and-mouth 疾病病毒(FMDV )- 非结构的蛋白质 3ABC - ELISA CLC 数字 S852.65 基础项目：国家鈥 ? 73 鈥 ? 研究工程(G199901190， 2005CB23201 ) ；国际原子能机构(国际原子能机构)(10697/R2 )

Review of Air Dispersion Modelling Approaches to Assess the Risk of Wind-Borne Spread of Foot-and-Mouth Disease Virus

Foot-and-mouth disease virus (FMDV) is one of the most economically serious veterinary pathogens due to its negative effects on livestock and its highly infectious nature via a variety of transmission paths through oral and inhalation routes. Measures to enhance outbreak management can be designed according to analytical results predicted by mathematical models for wind-borne dispersion, an important path of virus transmission. Accurate atmospheric dispersion models are useful tools for properly determining risk management plans, while inaccurate models may conversely lead to accidental loss in two possible ways. Overly strict measures, e.g., slaughter for too wide an area, can cause severe economic difficulties, including irreversible loss of business operations for a number of farms. On the contrary, inestimable loss potentially caused by lax controls is a persistent threat. In this paper, available modelling procedures for forecasting the spread of FMDV, which have been used since the 1970s, each having its advantages and limitations, are reviewed for the purpose of ensuring suitable application in various conditions of any future emergency cases.

Preparation and Characterization of Monoclonal Antibodies against VP1 Protein of Foot-and-mouth Disease Virus O/China99

Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.

Generation of Monoclonal Antibodies against Non-structural Protein 3AB of Foot-and-Mouth Disease Virus

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus （FMDV）, BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells. Two hybridoma monoclonal antibodies （mAbs） cell lines against the 3AB protein of foot-and-mouth disease virus （FMDV） were obtained, named C6 and E7 respectively. The mieroneutralization titer was 1：1024 for mAb C6, and 1：512 for E7. Both mAbs contain kappa light chains, and were of subclass IgG2b. In order to define the mAbs binding epitopes, the reactivity of these mAbs against FMDV were examined by indirect ELISA. The results showed that both mAbs can react with FMDV, but had no cross-reactivity with Swine Vesicular Disease （SVD） antigens. The titers in abdomen liquor were 1：5×10^6 for C6 and 1：2×10^6 for E7. In conclusion, the mAbs obtained from this study are specific for the detection of FMDV, can be used for etiological and immunological researches on FMDV, and have potential use in diagnosis and future vaccine designs.

Develope Monoclonal Antibody against Foot-and-mouth Disease Virus A Type

In order to develop an anti-FMDV A Type monoclonal antibody （mAb）, BABL/c mice were immunized with FMDV A type. Monoclonal antibodies （mAbs） 7B 11 and 8H4 against Foot-and-mouth disease virus （FMDV） serotype A were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with A/AV88. The microneutralization titer of the mAbs 7Bll and 8H4 were 1024 and 512, respectively. Both mAbs contain kappa light chainS, the mAbs were IgG1. In order to define the mAbs binding epitopes, the reactivity of these mAbs against A Type FMDV, were examined using indirect ELISA, the result showed that both mAbs reacted with A Type FMDV. These mAbs may be used for further vaccine studies, diagnostic methods, prophylaxis, etiological and immunological research on FMDV. Characterization of these ncindicated that prepared anti-FMDV A mAbs had no cross-reactivity with Swine Vesicular Disease （SVD） or FMDV O, Asial and C Type antigens. Their titers in abdomen liquor were 1：5×10^6 and 1：2×10^6, respectively. 7B 11 was found to be of subtype IgGb 8H4 was classified as IgG2b subtype. The mAbs prepared in this study, are specific for detection of FMDV serotype A, and is potentially useful for pen-side diagnosis.

Expression,Purification and Activity Detection of Structural Protein VP1 of Foot-and-Mouth Disease Virus Serotype A

The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.

Selection of Highly Susceptible Cell Lines to Foot and Mouth Disease Virus Infection

Different non-established cultures were examined to find those that showed high sensitivity to Foot and Mouth Disease Virus (FMDV). Ovine kidney cultures showed high sensitivity to types A, O and C, and were suitable to detect viral infection in samples of animal, as well as cell culture origin. The level of detection in this system was up to ten times higher than in BHK21 cells, which were commonly used for FMDV isolation and production. Viral production levels in ovine kidney cultures ranged from similar to twice as high as in BHK21. Ovine kidney cultures maintained these characteristics for at least 18 passages, allowing their use as an alternative system for FMDV diagnoses.

Phylogenetic analyses and antigenic characterization of foot-and-mouth disease virus PanAsia lineage circulating in China between 1999 and 2023

Foot-and-mouth disease(FMD)is one of the most important transboundary animal diseases caused by foot-andmouth disease virus(FMDV),leading to significant economic losses worldwide.Thefirst report of PanAsia lineage of FMDV in China was in 1999.Since 2011,18 outbreaks attributed to PanAsia lineage viruses have been reported across 7 provinces or municipality in China.Phylogenetic analysis indicated that these PanAsia strains were clustered into three distinct clades(clade 1,clade 2,and clade 3),with nucleotide homology ranging from 91.4%to 100%.The outbreaks of FMD caused by clade 1 strains occurred around 1999 when this lineage was prevalent globally.Clade 2 strains dominated from 2011 to 2013,while clade 3 strains were prevalent during 2018–2019,sharing only 93%homology with clade 2 strains and 91%with clade 1 strains.Tracing analysis showed that these outbreaks represented 3 distinct introductions of PanAsia viruses into China.Virus neutralization tests(VNT)have demonstrated that current commercial vaccines are effective to protect susceptible animals against these strains(r1>0.3).However,the growing demand for livestock has promoted animal movement and encouraged the exchange of products,services,and materials between countries,thereby heightening the risk of exotic strain incursions.Therefore,it is imperative to reinforce border controls and limit animal movements among various Asian countries continually to reduce the risk of new transboundary diseases,such as FMD incursion.Additionally,PanAsia-2 strains need to be taken seriously to prevent its incursions,and the relevant vaccines against PanAsia-2 strains need to be stockpiled in preparation for any possible incursion.

Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus

Foot-and-mouth disease virus(FMDV) is the causative agent of a highly contagious disease in livestock. The viral proteinaseL^(rop) of FMDV is involved in pathogenicity, and mutation of theL^(rop) SAP domain reduces FMDV pathogenicity in pigs. To determine the gene expression profiles associated with decreased pathogenicity in porcine cells, we performed transcriptome analysis using next-generation sequencing technology and compared differentially expressed genes in SK6 cells infected with FMDV containingL^(rop) with either a wild-type or mutated version of the SAP domain. This analysis yielded 1,853 genes that exhibited a ≥ 2-fold change in expression and was validated by real-time quantitative PCR detection of several differentially expressed genes. Many of the differentially expressed genes correlated with antiviral responses corresponded to genes associated with transcription factors, immune regulation, cytokine production, inflammatory response, and apoptosis. Alterations in gene expression profiles may be responsible for the variations in pathogenicity observed between the two FMDV variants. Our results provided genes of interest for the further study of antiviral pathways and pathogenic mechanisms related to FMDV L^(rop).

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

Foot-and-mouth disease virus(FMDV)can infect domestic and wild cloven-hoofed animals.The non-structural protein 3D plays an important role in FMDV replication and pathogenesis.However,the interaction partners of 3D,and the effects of those interactions on FMDV replication,remain incompletely elucidated.In the present study,using the yeast two-hybrid system,we identified a porcine cell protein,DEAD-box RNA helicase 1(DDX1),which interacted with FMDV 3D.The DDX1-3D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay(IFA)in porcine kidney 15(PK-15)cells.DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses.However,the roles of DDX1 during FMDV infection remain unclear.Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner.In addition,DDX1 stimulated IFN-p activation in FMDV-infected cells.Together,our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.

Comparative Transcriptome Analysis Reveals Different Host Cell Responses to Acute and Persistent Foot-and-Mouth Disease Virus Infection

Foot-and-mouth disease virus(FMDV) rapidly causes cytopathic effects in susceptible cells. Incomplete viral clearance during the acute infection leads to persistent infection. The relationship between host gene expression and the persistent infection remains unclear. In this study, we analyzed the transcriptome profiles of BHK-21 cells acutely and persistently infected with FMDV to identify differences in gene expression. GO and KEGG enrichment analyses showed that the 8,378 differentially expressed genes were significantly enriched in categories including metabolism, biosynthesis, ribosome function, and endocytosis. In persistently infected BHK-21 cells, ribosome-and translation-related genes were significantly down-regulated. There were more differentially expressed immune-related genes during persistent infection than during acute infection. Two hundred and seventy-four genes were differentially expressed in both acutely and persistently infected BHK-21 cells. Among these genes, heat shock protein family B member 1(Hspb1) knockdown significantly inhibited FMDV replication. Our research provides a basis for further research to understand the mechanisms of persistent FMDV infection including the genes involved in FMDV replication.

Engineering infectious foot-and-mouth disease virus in vivo from a full-length genomic cDNA clone of the A/AKT/58 strain

Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructed that contained three point-mutants [A174G and A308G (not present in pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58 strain of foot-and-mouth disease virus. These two viruses were rescued by co-transfection of pCA/FMDV with pCT7RNAP, which can express T7 RNA polymerase in BHK-21 cell-lines, or by transfection of the in vitro transcribed RNA. Their biological properties were analyzed for their antigenicity, virulence in suckling-mice (LD50) and growth kinetics in BHK-21 cells. The in vivo rescued viruses showed high pathogenicity for 3-day-old unweaned mice (LD50=10?7.5). However, the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicity for suckling-mice (LD50=10?6), and the in vivo transcribed RNA recovered from pCA/FMDV co-transfected with pCT7RNAP showed no significant differences from the wild type virus. These data showed that recovery of the infectious foot-and-mouth disease virus directly from the use of in vivo techniques was better than from in vitro methods. Furthermore, the reverse genetic procedure technique was simplified to a faster one-step procedure based on co-transfection with pCT7RNAP. These results suggest that in vivo RNA tran- scripts may be more valuable for engineering recombinant foot-and-mouth disease virus than in vitro RNA transcripts, and may contribute to further understanding of the biological properties, such as replication, maturation and quasispecies, of the foot-and-mouth disease virus.