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 vi...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.展开更多
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 re...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.展开更多
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 accord...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.展开更多
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(epitope1),tandem repeat 200-213(epitope2(+2)) and the combinat...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(epitope1),tandem repeat 200-213(epitope2(+2)) and the combination of two epitopes(epitope1-2)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.展开更多
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 or...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.展开更多
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 indicat...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.展开更多
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 vect...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.展开更多
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....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 microneutralization 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 1:5×106 for C6 and 1:2×106 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.展开更多
In order to develop an anti-FMDV A Type monoclonal antibody (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produ...In order to develop an anti-FMDV A Type monoclonal antibody (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 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 7B11 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,Asia1 and C Type antigens.Their titers in abdomen liquor were 1:5×106 and 1:2×106,respectively.7B11 was found to be of subtype IgG1,8H4 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.展开更多
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...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.展开更多
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 ...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.展开更多
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,an...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.展开更多
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 expres...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.展开更多
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 st...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.展开更多
Receptors interaction protein 2(RIP2)is a specific adaptor molecule in the downstream of NOD2.The role of RIP2 during foot-and-mouth disease virus(FMDV)infection remains unknown.Here,our results showed that RIP2 inhib...Receptors interaction protein 2(RIP2)is a specific adaptor molecule in the downstream of NOD2.The role of RIP2 during foot-and-mouth disease virus(FMDV)infection remains unknown.Here,our results showed that RIP2 inhibited FMDV replication and played an important role in the activation of IFN-βand NF-κB signal pathways during FMDV infection.FMDV infection triggered RIP2 transcription,while it reduced the expression of RIP2 protein.Detailed analysis showed that FMDV 2B,2C,3C^(pro),and L^(pro) proteins were responsible for inducing the reduction of RIP2 protein.3C^(pro) and L^(pro) are viral proteinases that can induce the cleavage or reduction of many host proteins and block host protein synthesis.The carboxyl terminal 105-C114 and 135-C144 regions of 2B were essential for reduction of RIP2.Our results also showed that the N terminal 1-61 region of 2C were essential for the reduction of RIP2.The 2C-induced reduction of RIP2 was dependent on inducing the reduction of poly(A)-binding protein 1(PABPC1).The interaction between RIP2 and 2C was observed in the context of viral infection,and the residues 1-61 were required for the interaction.These data clarify novel mechanisms of reduction of RIP2 mediated by FMDV.展开更多
CpG DNA is DNA sequence that has immune stimulatory effects. Several lines of investigation over the past few years indicate that CpG DNA plays an important role in the induction of immune responses to DNA vaccines. I...CpG DNA is DNA sequence that has immune stimulatory effects. Several lines of investigation over the past few years indicate that CpG DNA plays an important role in the induction of immune responses to DNA vaccines. In this study, CpG DNA-containing synthetic oligodeoxynucleotide (CpG-ODN) was cloned into the eukaryotic expression plasmid encoding a fusion protein containing b- galactosidase from E. coli and immunogenic epitopes of foot- and-mouth disease virus (FMDV) type O, and the immune responses induced by the plasmid were assayed. The results showed that guinea pigs immunized with the recombinant plasmid containing CpG-ODN generated a higher level of FMDV-neutralizing antibody and a stronger T cell proliferative response and protection against viral challenge than those receiving the plasmid containing no CpG-ODN. Our study demonstrated that it is an effective route to enhance the efficacy of DNA vaccines by inserting exogenous CpG DNA into the plasmids, and the DNA vaccine developed here is a promising candidate to prevent FMDV infection.展开更多
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 FM...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).展开更多
Foot-and-mouth-disease virus(FMDV)replicates in epithelial cells.The restriction of FMDV RNA to the basal cell layer of epithelia suggests a possible link between FMDVreplication in vivo and the cell status.This paper...Foot-and-mouth-disease virus(FMDV)replicates in epithelial cells.The restriction of FMDV RNA to the basal cell layer of epithelia suggests a possible link between FMDVreplication in vivo and the cell status.This paper describes in vitro studies in which FMDV infection was investigated in cells that were held at various cell division phases using cell cycle inhibitors.The results suggest that when cells were arrested at the G_(1) or G_(1)/S phase,high levels of viral RNA were detected by quantitative real-time reverse transcription PCR and viral protein synthesis was observed by specific labeling techniques.In contrast,when cells were arrested at the G_(2)/M phase,reduced or no viral RNA synthesis was detected.展开更多
基金jointly supported by grants from National Natural Science Foundation of China(No.31060343)Innovative Talents in Science and Technology Project of Yunnan Province(2011HB035)
文摘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.
文摘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.
基金supported by the National Natural Science Foundation of China (30730068)the National High-Tech R&D Program of China (863 Program,2007AA100606)
文摘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.
文摘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(epitope1),tandem repeat 200-213(epitope2(+2)) and the combination of two epitopes(epitope1-2)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.
文摘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.
基金State Science and Technology Support Program (2006DAD06A03)Hi-tech Research and Development Program of China 863 (2006AA10A204).
文摘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.
基金Supported by NSFC-Joint Personnel Training Fund of Henan Province(U1204327)Special Fund for Construction of Provincial Key Laboratory in Henan Province(122300413217)
文摘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.
基金State Key Projects of Transgene Program(No.2011ZX08011-0042009ZX08007-008B2009ZX08006-002B)
文摘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 microneutralization 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 1:5×106 for C6 and 1:2×106 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.
基金State Key Projects of Transgene Program(2011ZX08011-0042009ZX 08007- 008B2009ZX08006-002B)
文摘In order to develop an anti-FMDV A Type monoclonal antibody (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 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 7B11 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,Asia1 and C Type antigens.Their titers in abdomen liquor were 1:5×106 and 1:2×106,respectively.7B11 was found to be of subtype IgG1,8H4 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.
基金The national high technology research and development program of China 863 (2006AA10A204)The national science and technology pillar program (2006BAD06A17)
文摘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.
文摘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.
基金supported by grants from the National Natural Science Foundation of China (Nos. 31302106, 31260616, and 31602035)the National Key Research and Development Program of China (Nos. 2016YFD0500901 and 2017YFD0500903)
文摘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.
基金the financial support of the National Infrastructure of Natural Resources for Science and Technology Program(No.2011-572)the National Natural Sciences Foundation of China(No.31370185)+2 种基金the National Basic Research Program of China(No.2011CB504800)to Prof.Congyi Zhengthe Fundamental Research Funds for the Central Universities(2042018kf0241)the National Science and Technology Infrastructure Grants(NSTI-CR15 and NSTI-CR16)to Dr.Chao Shen.
文摘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.
基金Supported by the National Key Basic Research Program of China (Grant No. 2005CB523201)National High-Tech Research and Development Program of China (Grant No. 2006BAD06A03)
文摘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.
基金This work was supported by Grants from the National Key R&D Programme of China(No.2017YFD0501103 and 2017YFD0501800)the Key Development and Research Foundation of Yunnan(No.2018BB004)the Chinese Academy of Agricultural Science and Technology Innovation Project(CAAS-XTCX2016011-01 and Y2017JC55).
文摘Receptors interaction protein 2(RIP2)is a specific adaptor molecule in the downstream of NOD2.The role of RIP2 during foot-and-mouth disease virus(FMDV)infection remains unknown.Here,our results showed that RIP2 inhibited FMDV replication and played an important role in the activation of IFN-βand NF-κB signal pathways during FMDV infection.FMDV infection triggered RIP2 transcription,while it reduced the expression of RIP2 protein.Detailed analysis showed that FMDV 2B,2C,3C^(pro),and L^(pro) proteins were responsible for inducing the reduction of RIP2 protein.3C^(pro) and L^(pro) are viral proteinases that can induce the cleavage or reduction of many host proteins and block host protein synthesis.The carboxyl terminal 105-C114 and 135-C144 regions of 2B were essential for reduction of RIP2.Our results also showed that the N terminal 1-61 region of 2C were essential for the reduction of RIP2.The 2C-induced reduction of RIP2 was dependent on inducing the reduction of poly(A)-binding protein 1(PABPC1).The interaction between RIP2 and 2C was observed in the context of viral infection,and the residues 1-61 were required for the interaction.These data clarify novel mechanisms of reduction of RIP2 mediated by FMDV.
文摘CpG DNA is DNA sequence that has immune stimulatory effects. Several lines of investigation over the past few years indicate that CpG DNA plays an important role in the induction of immune responses to DNA vaccines. In this study, CpG DNA-containing synthetic oligodeoxynucleotide (CpG-ODN) was cloned into the eukaryotic expression plasmid encoding a fusion protein containing b- galactosidase from E. coli and immunogenic epitopes of foot- and-mouth disease virus (FMDV) type O, and the immune responses induced by the plasmid were assayed. The results showed that guinea pigs immunized with the recombinant plasmid containing CpG-ODN generated a higher level of FMDV-neutralizing antibody and a stronger T cell proliferative response and protection against viral challenge than those receiving the plasmid containing no CpG-ODN. Our study demonstrated that it is an effective route to enhance the efficacy of DNA vaccines by inserting exogenous CpG DNA into the plasmids, and the DNA vaccine developed here is a promising candidate to prevent FMDV infection.
基金supported by grants from the National Science and Technology Ministry (2015BAD12B04)National Natural Sciences Foundation of China (No.31302118,31502042 and 31402179)+2 种基金the Gansu Science Foundation for Distinguished Young Scholars (no.145RJDA328)the International Atomic Energy Agency (16025/R0)the Key technologies R&Dprogram of Gansu Province (1302NKDA027)
文摘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).
基金This work was mainly supported by the Biotechnology and Biological Sciences Research Council(China partnership award and project BB/E003435/1),UK.
文摘Foot-and-mouth-disease virus(FMDV)replicates in epithelial cells.The restriction of FMDV RNA to the basal cell layer of epithelia suggests a possible link between FMDVreplication in vivo and the cell status.This paper describes in vitro studies in which FMDV infection was investigated in cells that were held at various cell division phases using cell cycle inhibitors.The results suggest that when cells were arrested at the G_(1) or G_(1)/S phase,high levels of viral RNA were detected by quantitative real-time reverse transcription PCR and viral protein synthesis was observed by specific labeling techniques.In contrast,when cells were arrested at the G_(2)/M phase,reduced or no viral RNA synthesis was detected.