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 expressio...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.展开更多
Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombin...Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombinant empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production. A plasmid containing the capsid precursor P12A and protease 3C sequences of foot-and-mouth disease virus (FMDV) was constructed and used to compare transient and stable expression in mammalian cells. When BHK-21 cells were transfected with the recombinant vector, protease 3C cleaved the capsid precursor P12A into the structural proteins VP0, VP1 and VP3. A sucrose gradient demonstrated that the structural proteins assembled into different subviral particles. Attempts to generate a stable cell line only allowed isolating low-level-expressing clones, probably due to the effect of protease 3C on the cells. Moreover, the recombinant protein yield achieved in transient expression assays was much higher than the one achieved in stable expression assays. Results indicate that mammalian cells are a good strategy to produce recombinant FMDV subviral particles. However, the alternative approach of transient gene expression in scalable systems should be used instead of the standard method that involves the generation of a stable cell line.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
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
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
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
The present study describes the frequency of Foot and Mouth Disease （FMD） virus serotypes （O, A and Asia-l） in major regions （all provinces） of Pakistan using Indirect Sandwich ELISA. Also, spatial distribution ...The present study describes the frequency of Foot and Mouth Disease （FMD） virus serotypes （O, A and Asia-l） in major regions （all provinces） of Pakistan using Indirect Sandwich ELISA. Also, spatial distribution of various FMD serotypes and their comparison is discussed. A total of 590 samples （Epithelial tissue） have been analyzed during a period of five years （2005-2009）. Out of 590 samples, 180 were found positive, giving an overall confirmation of FMDV about 33.2 %. Of the prevalent serotypes, FMDV ＇O＇ serotype caused most outbreaks （20.7 %）, followed by serotype A （6.6 %） and serotype Asia-1 （4.6 %） while there was no positive case of type ＇C＇. The study clearly showed that the disease was more frequent in the agro-climatic zones than in hilly areas. Based on the data of 590 samples （〉50 outbreaks）, the overall prevalence of FMDV in cattle and buffaloes in Pakistan was 33.2 %, while in cattle alone, it was 37.1%, higher than in buffalo （28.7 %）. There were eight cases of mixed serotypes infection, indicating the presence of endemic state of disease. Another significant feature was the change over time. In phase-I （2005-2007）, there was an overall prevalence of 29.4 %, while the occurrence of the serotype O, A and Asia-1 was 20.4 %, 2.9 % and 4.7 %, respectively. During phase-II （2008-2009）, the overall prevalence was 59.21%, while those of serotype O, A and Asia-1 were 22.4 %, 31.6 % and 4.0 %, respectively. This clearly indicated a shift from serotype O to A, which may help to explain the occurrence of more severe outbreaks, despite vaccination.展开更多
The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to...The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.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 (FMD),caused by foot-and-mouth disease virus (FMDV),is considered one of the most important viral diseases of cloven-hoofed animals,causing severe economic losses in affected regions of the worl...Foot-and-mouth disease (FMD),caused by foot-and-mouth disease virus (FMDV),is considered one of the most important viral diseases of cloven-hoofed animals,causing severe economic losses in affected regions of the world.Three serotypes (A,O,and Asia 1) of FMDV have been identified in China since 1958.In addition,the occurrence of novel subtypes within these serotypes has made the epidemiology of FMDV more complicated over the last few years.In this review,we summarize the history and the current epidemiological situation in China,genetic diversity (e.g.,quasispecies dynamics,antigenic heterogeneity,and functional constraints),intertypic recombination,and the evidence for positive selection of different FMDV serotypes.We also assess these genetic data to understand the origin,evolution,and transmission of FMDV,the findings of which may be useful in developing control measures for future epidemics.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
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
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
The intact open reading frame (ORF) of foot-and-mouth disease virus (FMDV) Asia I/XJ strain was am- plified by RT-PCR and inserted into the transfer vector pVL1393 to generate plasmid pVL-ORF. Bm-N cells were transfec...The intact open reading frame (ORF) of foot-and-mouth disease virus (FMDV) Asia I/XJ strain was am- plified by RT-PCR and inserted into the transfer vector pVL1393 to generate plasmid pVL-ORF. Bm-N cells were transfected with pVL-ORF and linearized Bm-BacPAK6 DNA, and the recombinant silkworm baculovirus Bm-ORF containing the full ORF of FMDV was obtained. The results of indirect im- munofluorescence assay (IFA) showed that Bm-ORF could be expressed efficiently in Bm-N cell. After inoculating the early 5th instar larvae of silkworm, the polyprotein of FMDV could be detected by sandwich ELISA and empty capsid-like particles could be observed under the electron microscope. Expression products from silkworm were used as the antigen to immunize the cattle. The specific an- tibody was induced in all vaccinated animals. The immunized cattle were challenged with the virulent FMDV Asia I/XJ strain, two of the four cattle were completely protected and clinical symptoms were alleviated and delayed in the others. The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.展开更多
基金Supported by the Science and Technology Foundation from Science&Technology Department of Guangxi Autonomous Region(0779001)~~
文摘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.
文摘Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombinant empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production. A plasmid containing the capsid precursor P12A and protease 3C sequences of foot-and-mouth disease virus (FMDV) was constructed and used to compare transient and stable expression in mammalian cells. When BHK-21 cells were transfected with the recombinant vector, protease 3C cleaved the capsid precursor P12A into the structural proteins VP0, VP1 and VP3. A sucrose gradient demonstrated that the structural proteins assembled into different subviral particles. Attempts to generate a stable cell line only allowed isolating low-level-expressing clones, probably due to the effect of protease 3C on the cells. Moreover, the recombinant protein yield achieved in transient expression assays was much higher than the one achieved in stable expression assays. Results indicate that mammalian cells are a good strategy to produce recombinant FMDV subviral particles. However, the alternative approach of transient gene expression in scalable systems should be used instead of the standard method that involves the generation of a stable cell line.
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.
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.
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Food & Agriculture Organization FMD Project"Progressive Control of Foot and Mouth Disease in Pakistan(GCP/PAK/123/USA)the FAO (GTFS/INT/907/ITA) and EU(SLSP) funded projects
文摘The present study describes the frequency of Foot and Mouth Disease （FMD） virus serotypes （O, A and Asia-l） in major regions （all provinces） of Pakistan using Indirect Sandwich ELISA. Also, spatial distribution of various FMD serotypes and their comparison is discussed. A total of 590 samples （Epithelial tissue） have been analyzed during a period of five years （2005-2009）. Out of 590 samples, 180 were found positive, giving an overall confirmation of FMDV about 33.2 %. Of the prevalent serotypes, FMDV ＇O＇ serotype caused most outbreaks （20.7 %）, followed by serotype A （6.6 %） and serotype Asia-1 （4.6 %） while there was no positive case of type ＇C＇. The study clearly showed that the disease was more frequent in the agro-climatic zones than in hilly areas. Based on the data of 590 samples （〉50 outbreaks）, the overall prevalence of FMDV in cattle and buffaloes in Pakistan was 33.2 %, while in cattle alone, it was 37.1%, higher than in buffalo （28.7 %）. There were eight cases of mixed serotypes infection, indicating the presence of endemic state of disease. Another significant feature was the change over time. In phase-I （2005-2007）, there was an overall prevalence of 29.4 %, while the occurrence of the serotype O, A and Asia-1 was 20.4 %, 2.9 % and 4.7 %, respectively. During phase-II （2008-2009）, the overall prevalence was 59.21%, while those of serotype O, A and Asia-1 were 22.4 %, 31.6 % and 4.0 %, respectively. This clearly indicated a shift from serotype O to A, which may help to explain the occurrence of more severe outbreaks, despite vaccination.
基金Supported by Joint Funds of the NSFC and Henan Province(U1204327)Henan Provincial Key Laboratory Construction(122300413217)
文摘The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.
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 National High Tech-nology Research and Development Program of China (2011AA10A211)Ministry of Agriculture of China for Animal Disease Control in Border Areas (201003008)the Science and Technology Program of Lanzhou,Gansu Province,China (2010-1-157)
文摘Foot-and-mouth disease (FMD),caused by foot-and-mouth disease virus (FMDV),is considered one of the most important viral diseases of cloven-hoofed animals,causing severe economic losses in affected regions of the world.Three serotypes (A,O,and Asia 1) of FMDV have been identified in China since 1958.In addition,the occurrence of novel subtypes within these serotypes has made the epidemiology of FMDV more complicated over the last few years.In this review,we summarize the history and the current epidemiological situation in China,genetic diversity (e.g.,quasispecies dynamics,antigenic heterogeneity,and functional constraints),intertypic recombination,and the evidence for positive selection of different FMDV serotypes.We also assess these genetic data to understand the origin,evolution,and transmission of FMDV,the findings of which may be useful in developing control measures for future epidemics.
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.
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 the National 863 Project of China (Grant No. 2006AA02Z440) Chinese Funding for Social Public Interests (Grant No. 2005DIB4J041)
文摘The intact open reading frame (ORF) of foot-and-mouth disease virus (FMDV) Asia I/XJ strain was am- plified by RT-PCR and inserted into the transfer vector pVL1393 to generate plasmid pVL-ORF. Bm-N cells were transfected with pVL-ORF and linearized Bm-BacPAK6 DNA, and the recombinant silkworm baculovirus Bm-ORF containing the full ORF of FMDV was obtained. The results of indirect im- munofluorescence assay (IFA) showed that Bm-ORF could be expressed efficiently in Bm-N cell. After inoculating the early 5th instar larvae of silkworm, the polyprotein of FMDV could be detected by sandwich ELISA and empty capsid-like particles could be observed under the electron microscope. Expression products from silkworm were used as the antigen to immunize the cattle. The specific an- tibody was induced in all vaccinated animals. The immunized cattle were challenged with the virulent FMDV Asia I/XJ strain, two of the four cattle were completely protected and clinical symptoms were alleviated and delayed in the others. The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.
