型内嵌合口蹄疫病毒全长感染性cDNA克隆的构建

【目的】近年来,O型口蹄疫的不断暴发严重危害了我国畜牧业的发展,其病原——O型口蹄疫病毒已演化出3种谱系:中国型猪毒系、泛亚系和缅甸98系。其中中国型猪毒系病毒高度嗜猪,对养猪业危害最大。目前应用的疫苗已不能有效保护中国型猪毒系变异株的流行,这给我国猪口蹄疫的防控带来了极大的困难。为了进一步发展免疫原性好、抗原谱广的猪O型口蹄疫疫苗候选株,本研究以O/HN/93现用疫苗毒株的感染性克隆为骨架,用流行的新猪毒系病毒的部分VP3和VP1基因(主要是替换VP1蛋白上的B-C环和G-H环)替换疫苗毒株的相应部分,构建了嵌合的FMDV全长cDNA克隆。【方法】线化的嵌合全长质粒和表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,体内转录拯救嵌合病毒。【结果】嵌合全长质粒转染BHK-21细胞36h后,出现明显的FMDV致细胞病变效应。对收获的病毒分别用RT-PCR、间接免疫荧光、电子显微镜观察结果证实成功拯救到嵌合的FMDV。拯救的病毒乳鼠致病性试验结果表明该拯救病毒对乳鼠的致病力减弱。该嵌合病毒的成功拯救为研制口蹄疫新型疫苗等奠定了基础。     

Evaluation of genetically engineered derivatives of a Chinese strain of foot-and-mouth disease virus reveals a novel cell-binding site which functions in cell culture and in animals

Adaptation of field isolates of foot-and-mouth disease virus (FMDV) to grow in cells in culture can result in changes in viral properties that include acquisition of the ability to bind to cell surface heparan sulfate (HS). After 13 passages on BHK cells to produce a vaccine, a Cathay topotype isolate of FMDV serotype O from China (O/CHA/90) extended its cell culture host range and bound to heparin-Sepharose, although it did not require cell surface HS as a receptor molecule. To understand these phenomena, we constructed chimeric viruses by using a type A(12) infectious cDNA and the capsid protein-coding regions of O/CHA/90 and its cell culture-adapted derivative (vac-O/CHA/90). Using a set of viruses derived from these chimeras by exchanging portions of the capsid-coding regions, we discovered that a group of amino acid residues that surround the fivefold axis of the icosahedral virion determine host range in cell culture and influence pathogenicity in pigs. These residues included aromatic amino acids at positions 108 and 174 and positively charged residues at positions 83 and 172 in protein 1D. To test if these residues participated in non-integrin-dependent cell binding, the integrin-binding RGD sequence in protein 1D was changed to KGE in two different chimeras. Evaluation of these KGE viruses indicated that growth in cell culture was not dependent on HS. One of these viruses was tested in pigs, where it produced a mild disease and maintained its KGE sequence. These results are discussed in terms of receptor utilization and pathogenesis of this important pathogen.     

Development of novel strategies to control foot-and-mouth disease: Marker vaccines and antivirals

Foot-and-mouth disease (FMD) is economically the most important viral-induced livestock disease worldwide. The disease is highly contagious and FMD virus (FMDV) replicates and spreads extremely rapidly. Outbreaks in previously FMD-free countries, including Taiwan, the United Kingdom, and Uruguay, and the potential use of FMDV by terrorist groups have demonstrated the vulnerability of countries and the need to develop control strategies that can rapidly inhibit or limit disease spread. The current vaccine, an inactivated whole virus preparation, has a number of limitations for use in outbreaks in disease-free countries. We have developed an alternative approach using a genetically engineered FMD subunit vaccine that only contains the portions of the viral genome required for virus capsid assembly and lacks the coding region for most of the viral nonstructural (NS) proteins including the highly immunogenic 3D protein. Thus, animals inoculated with this marker vaccine can readily be differentiated from infected animals using diagnostic assays employing the NS proteins not present in the vaccine and production of this vaccine, which does not contain infectious FMDV, does not require expensive high-containment manufacturing facilities. One inoculation of this subunit vaccine delivered in a replication-defective human adenovirus vector can induce rapid, within 7 days, and relatively long-lasting protection in swine. Similarly cattle inoculated with one dose of this recombinant vector are rapidly protected from direct and contact exposure to virulent virus. Furthermore, cattle given two doses of this vaccine developed high levels of FMDV-specific neutralizing antibodies, but did not develop antibodies against viral NS proteins demonstrating the ability of FMD subunit vaccinated animals to be differentiated from infected animals. To stimulate early protection prior to the vaccine-induced adaptive immune response we inoculated swine with the antiviral agent, type I interferon, and induced complete protection within 1 day. Protection can last for 3-5 days. The combination of the FMD marker vaccine and type I interferon can induce immediate, within 1 day, and long-lasting protection against FMD. Thus, this combination approach successfully addresses a number of concerns of FMD-free countries with the current disease control plan. By rapidly limiting virus replication and spread this strategy may reduce the number of animals that need to be slaughtered during an outbreak.     

Role of nonstructural proteins 3A and 3B in host range and pathogenicity of foot-and-mouth disease virus

The genome of foot-and-mouth disease virus (FMDV) differs from that of other picornaviruses in that it encodes a larger 3A protein (>50% longer than poliovirus 3A), as well as three copies of protein 3B (also known as VPg). Previous studies have shown that a deletion of amino acids 93 to 102 of the 153-codon 3A protein is associated with an inability of a Taiwanese strain of FMDV (O/TAW/97) to cause disease in bovines. Recently, an Asian virus with a second 3A deletion (amino acids 133 to 143) has also been detected (N. J. Knowles et al., J. Virol. 75:1551-1556, 2001). Genetically engineered viruses harboring the amino acids 93 to 102 or 133 to 143 grew well in porcine cells but replicated poorly in bovine cells, whereas a genetically engineered derivative of the O/TAW/97 virus expressing a full-length 3A (strain A12) grew well in both cell types. Interestingly, a virus with a deletion spanning amino acid 93 to 144 also grew well in porcine cells and caused disease in swine. Further, genetically engineered viruses containing only a single copy of VPg were readily recovered with the full-length 3A, the deleted 3A (amino acids 93 to 102), or the "super" deleted forms of 3A (missing amino acids 93 to 144). All of the single-VPg viruses were attenuated in porcine cells and replicated poorly in bovine cells. The single-VPg viruses produced a mild disease in swine, indicating that the VPg copy number is an important determinant of host range and virulence. The association of VPg copy number with increased virulence in vivo may help to explain why all naturally occurring FMDVs have retained three copies of VPg.     

New Vaccine Design Based on Defective Genomes That Combines Features of Attenuated and Inactivated Vaccines

Background

New vaccine designs are needed to control diseases associated with antigenically variable RNA viruses. Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that inflicts severe economic losses. Although the current whole-virus chemically inactivated vaccine has proven effective, it has led to new outbreaks of FMD because of incomplete inactivation of the virus or the escape of infectious virus from vaccine production premises. We have previously shown that serial passages of FMD virus (FMDV) C-S8c1 at high multiplicity of infection in cell culture resulted in virus populations consisting of defective genomes that are infectious by complementation (termed C-S8p260).

Principal Finding

Here we evaluate the immunogenicity of C-S8p260, first in a mouse model system to establish a proof of principle, and second, in swine, the natural host of FMDV C-S8c1. Mice were completely protected against a lethal challenge with FMDV C-S8c1, after vaccination with a single dose of C-S8p260. Pigs immunized with different C-S8p260 doses and challenged with FMDV C-S8c1 either did not develop any clinical signs or showed delayed and mild disease symptoms. C-S8p260 induced high titers of both FMDV-specific, neutralizing antibodies and activated FMDV-specific T cells in swine, that correlated with solid protection against FMDV.

Conclusions

The defective virus-based vaccine did not produce detectable levels of transmissible FMDV. Therefore, a segmented, replication-competent form of a virus, such as FMDV C-S8p260, can provide the basis of a new generation of attenuated antiviral vaccines with two safety barriers. The design can be extended to any viral pathogen that encodes trans-acting gene products, allowing complementation between replication-competent, defective forms.     

Vaccines prepared from chimeras of foot-and-mouth disease virus (FMDV) induce neutralizing antibodies and protective immunity to multiple serotypes of FMDV.

The G-H loop of VP1 (residues 132 to 159) of foot-and-mouth disease virus (FMDV) is a prominent feature on the virion surface and has an important role in vaccine efficacy, generation of antigenic variants, and cell binding. Using an infectious cDNA of FMDV, we have constructed serotype A viruses in which the G-H loop has been substituted with the homologous sequences from serotype O or C. These chimeric viruses replicated to high titer and displayed plaque morphologies similar to those of wild-type viruses, demonstrating that the functions provided by the loop can be readily exchanged between serotypes. Monoclonal antibody analyses showed that epitopes contained within the loop were transferred to the chimeras and that epitopes encoded by the type A backbone were maintained. Chemically inactivated vaccines prepared from chimeric viruses induced antibodies in guinea pigs that neutralized both type A and either type O or type C viruses. Swine inoculated with the A/C chimera vaccine also produced cross-reactive antibodies, were protected from challenge with the type A virus, and partially protected against challenge with type C. These studies emphasize the importance of epitopes outside of the G-H loop in protective immunity in swine, which is a natural host of FMDV.     

Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease

We have previously shown that replication of foot-and-mouth disease virus (FMDV) is highly sensitive to alpha/beta interferon (IFN-alpha/beta). In the present study, we constructed recombinant, replication-defective human adenovirus type 5 vectors containing either porcine IFN-alpha or IFN-beta (Ad5-pIFNalpha or Ad5-pIFNbeta). We demonstrated that cells infected with these viruses express high levels of biologically active IFN. Swine inoculated with 10(9) PFU of a control Ad5 virus lacking the IFN gene and challenged 24 h later with FMDV developed typical signs of foot-and-mouth disease (FMD), including fever, vesicular lesions, and viremia. In contrast, swine inoculated with 10(9) PFU of Ad5-pIFNalpha were completely protected when challenged 24 h later with FMDV. These animals showed no clinical signs of FMD and no viremia and did not develop antibodies against viral nonstructural proteins, suggesting that complete protection from infection was achieved.     

Evaluation of Infectivity,Virulence and Transmission of FDMV Field Strains of Serotypes O and A Isolated In 2010 from Outbreaks in the Republic of Korea

Since the early 2000s outbreaks of foot-and-mouth disease (FMD) have been described in several previously FMD-free Asian nations, including the Republic of Korea (South Korea). One outbreak with FMD virus (FDMV) serotype A and two with serotype O occurred in South Korea in 2010/2011. The causative viruses belonged to lineages that had been spreading in South East Asia, far East and East Asia since 2009 and presented a great threat to the countries in that region. Most FMDV strains infect ruminants and pigs, as it happened during the outbreaks of FMDV serotype O in South Korea. Contrastingly, the strain of serotype A affected only ruminants. Based upon these findings, the intention of the work described in the current report was to characterize and compare the infectivity, virulence and transmission of both strains under laboratory conditions in cattle and pigs, by direct inoculation and contact exposure. As expected, FMDV serotype O was highly virulent in both cattle and swine by contact exposure and direct inoculation. Surprisingly, FMDV serotype A was highly virulent in swine, but was less infectious in cattle by contact exposure to infected swine or cattle. Interestingly, similar quantities of aerosolized FMDV RNA were detected during experiments with viruses of serotypes O and A. Specific virus-host interaction of A/SKR/2010 could affect the transmission of this strain to cattle, and this may explain in part the limited spread of the serotype A epizootic.     

Engineering Foot-and-Mouth Disease Viruses with Improved Growth Properties for Vaccine Development

Background

No licensed vaccine is currently available against serotype A foot-and-mouth disease (FMD) in China, despite the isolation of A/WH/CHA/09 in 2009, partly because this strain does not replicate well in baby hamster kidney (BHK) cells.

Methodology/Principal Findings

A novel plasmid-based reverse genetics system was used to construct a chimeric strain by replacing the P1 gene in the vaccine strain O/CHA/99 with that from the epidemic stain A/WH/CHA/09. The chimeric virus displayed growth kinetics similar to those of O/CHA/99 and was selected for use as a candidate vaccine strain after 12 passages in BHK cells. Cattle were vaccinated with the inactivated vaccine and humoral immune responses were induced in most of the animals on day 7. A challenge infection with A/WH/CHA/09 on day 28 indicated that the group given a 4-µg dose was fully protected and neither developed viremia nor seroconverted to a 3ABC antigen.

Conclusions/Significance

Our data demonstrate that the chimeric virus not only propagates well in BHK cells and has excellent antigenic matching against serotype A FMD, but is also a potential marker vaccine to distinguish infection from vaccination. These results suggest that reverse genetics technology is a useful tool for engineering vaccines for the prevention and control of FMD.     

Chimeric virus-like particles elicit protective immunity against serotype O foot-and-mouth disease virus in guinea pigs

Foot-and-mouth disease (FMD) is an acute and highly contagious disease caused by foot-and-mouth disease virus (FMDV) that can affect cloven-hoofed animal species, leading to severe economic losses worldwide. Therefore, the development of a safe and effective new vaccine to prevent and control FMD is both urgent and necessary. In this study, we developed a chimeric virus-like particle (VLP) vaccine candidate for serotype O FMDV and evaluated its protective immunity in guinea pigs. Chimeric VLPs were formed by the antigenic structural protein VP1 from serotype O and segments of the viral capsid proteins (VP2, VP3, and VP4) from serotype A. The chimeric VLPs elicited significant humoral and cellular immune responses with a higher level of anti-FMDV antibodies and cytokines than the control group. Furthermore, four of the five guinea pigs vaccinated with the chimeric VLPs were completely protected against challenge with 100 50% guinea pig infectious doses (GPID₅₀) of the virulent FMDV strain O/MAY98. These data suggest that chimeric VLPs are potential candidates for the development of new vaccines against FMDV.     

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.     

Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine

Background

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines.

Methodology and Principal Findings

A recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD₅₀ (50% bovine protective dose) test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD₅₀ per dose.

Conclusion

The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.     

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV “O” 100 GPID₅₀ challenge virus.

     

3A蛋白104–115位氨基酸缺失口蹄疫A型标记病毒的构建

【目的】构建一株含3A非结构蛋白104–115位氨基酸缺失的口蹄疫A型标记病毒,分析其生物学特性和发展标记疫苗的潜力。【方法】采用融合PCR技术,在当前流行毒株A/Sea-97/CHA/2014全长感染性克隆p QAHN中引入3A104–115位氨基酸的缺失,构建全长重组质粒。全长质粒经NotI线化后转染表达T7RNA聚合酶的稳定细胞系,拯救标记病毒。RT-PCR、序列分析、间接免疫荧光和Western blotting鉴定标记病毒。噬斑表型和一步生长曲线分析标记病毒的生物学特性,并用实验室开发的针对3A优势表位(AEKNPLE)的阻断ELISA方法分析其区分亲本和标记病毒感染的动物。【结果】成功拯救到一株含3A 104–115位氨基酸缺失的口蹄疫A型标记病毒,3A表位的缺失没有影响标记病毒的噬斑表型和一步生长曲线。3A单抗阻断ELISA可以明显区分标记病毒和亲本病毒感染的动物。【结论】本研究构建的3A蛋白104–115位氨基酸缺失的标记病毒可以作为发展口蹄疫鉴别诊断疫苗的候选毒株,用于我国未来口蹄疫A型的有效防控。     

Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus

ABSTRACT: BACKGROUND: Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV. METHODS: Two serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice. RESULTS: Three amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain. CONCLUSIONS: These results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine.     

A simple and rapid colloidal gold-based immunochromatogarpic strip test for detection of FMDV serotype A

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test, affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody, and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip, the capture antibody was laid on a sample pad, the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C, Swine vesicular disease (SVD), Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically, the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple, easy and fast for clinical testing on field sites; no special instruments and skills are required, and the result can be obtained within 15 min. To our knowledge, this is the first rapid immunochromatogarpic assay for serotype A of FMDV.     

嵌合O/GSLX/CHN/2010株S片段的基因工程口蹄疫病毒的生物学特性

【目的】研究嵌合口蹄疫病毒(Foot-and-mouth disease virus,FMDV) O/GSLX/CHN/2010株S片段的基因工程病毒的生物学特性。【方法】运用反向遗传操作技术,将FMDV O/GSLX/CHN/2010的S片段嵌合进2株同谱系FMDV的感染性克隆骨架,拯救得到两株基因工程嵌合病毒rGDexc、rGZexc;进而从病毒复制和病毒对乳鼠的致病力方面,对嵌合病毒、亲本基因工程病毒以及FMDV O/GSLX/CHN/2010株进行了评价。【结果】FMDV O/GSLX/CHN/2010株复制较慢,对乳鼠致病力较差。嵌合病毒和亲本基因工程病毒的复制能力、对乳鼠的致病力明显高于FMDV O/GSLX/CHN/2010株。【结论】研究表明FMDV O/GSLX/CHN/2010的S片段不能独自决定该病毒较差的复制能力和较弱的乳鼠致病力。这一发现为进一步研究O/GSLX/CHN/2010的致弱机制以及S片段对病毒生物学特性的影响奠定了基础。     

Development of a Foot-and-Mouth Disease Virus Serotype A Empty Capsid Subunit Vaccine Using Silkworm (Bombyx mori) Pupae

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that inflicts severe economic losses in the livestock industry. In 2009, FMDV serotype A caused outbreaks of FMD in cattle in China. Although an inactivated virus vaccine has proven effective to control FMD, its use may lead to new disease outbreaks due to a possible incomplete inactivation of the virus during the manufacturing process. Here, we expressed the P1-2A and the 3C coding regions of a serotype A FMDV field isolate in silkworm pupae (Bombyx mori) and evaluated the immunogenicity of the expression products. Four of five cattle vaccinated with these proteins developed high titers of FMDV-specific antibody and were completely protected against virulent homologous virus challenge with 10,000 50% bovine infectious doses (BID(50)). Furthermore, the 50% bovine protective dose (PD(50)) test was performed to assess the bovine potency of the empty capsid subunit vaccine and was shown to achieve 4.33 PD(50) per dose. These data provide evidence that silkworm pupae can be used to express immunogenic FMDV proteins. This strategy might be used to develop a new generation of empty capsid subunit vaccines against a variety of diseases.