表达C亚型禽偏肺病毒糖蛋白和融合蛋白的重组新城疫病毒二价苗的构建及免疫效力评价
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摘要
新城疫和禽偏肺病分别是由新城疫病毒(Newcastle disease virus,NDV)和禽偏肺病毒(Avian metapneumovirus,aMPV)引起家禽呼吸道感染的急性高度传染性疾病,可在全世界范围内传播,其高发病率和致死率不仅会造成养禽业重大的经济损失,而且还严重威胁着食品安全,公共卫生及生物安全。目前,疫苗接种和严格的生物安全措施是控制NDV和aMPV疾病的主要措施,NDV弱毒株LaSota、B1、VG/GA可作为疫苗直接用于NDV疾病的防控,而aMPV却没有直接用于控制该病爆发的活疫苗弱毒株,现有疫苗的安全性和有效性仍有待于进一步提高。为了研发一种安全有效且经济实用的双价苗,因此,本研究以NDV弱毒株LaSota为疫苗载体,构建表达aMPV抗原蛋白融合蛋白(Fusion protein,F)和糖蛋白(Glycoprotein,G)的重组新城疫病毒,以获得可以抵抗NDV和aMPV的安全有效的双价苗。
为了获得表达aMPV C亚型融合蛋白F或糖蛋白G的重组新城疫病毒,该研究以新城疫病毒疫苗株LaSota为载体,首先将绿色荧光蛋白(Green fluorescence protein,GFP)作为报告基因插入到NDV基因组F-HN之间,利用反向遗传学技术构建表达GFP蛋白的重组新城疫病毒rLS/GFP。结果显示,LaSota可作为活疫苗载体安全稳定地表达GFP蛋白。进而构建了表达aMPV-C G蛋白的重组新城疫病毒rLS/aMPV-C G,并对其毒力、生物学特性及免疫保护率进行了评估。结果表明rLS/aMPV-C G的毒力在SPF(Specific-pathogen-free,SPF)鸡胚和1日龄雏鸡内轻微致弱,其生物学特性与LaSota株相似,并可完全抵新城疫病毒抗强毒株(NDV/CA02)的攻击,对aMPV-C有部分免疫保护。由此推测G蛋白的抗原性不强,产生的免疫力不足以完全保护aMPV-C的攻击,于是,该研究进一步构建表达aMPV-C双重抗原蛋白F和G的重组新城疫病毒。在rLS/GFP载体的基础上,以红色荧光蛋白(Red fluorescence protein,RFP)为另一报告基因插入到载体F基因末端,作为第二阅读框架,然后将内部核糖体进入位点IRES(Internal ribosome entry site ,IRES)插入F和RFP阅读框架之间用于启动RFP基因的表达,进而成功构建表达两个外源基因(RFP和GFP)的重组新城疫病毒rLS/I-RFP-GFP。在此基础上,构建了表达aMPV-C F和G蛋白的重组新城疫病毒rLS/aMPV-C F G,并对其进行免疫保护评估。结果显示,SPF火鸡免疫接种rLS/aMPV-C F G后可完全保护NDV/CA02的攻击,首免后60-80%的火鸡可诱导产生特异性aMPV-C抗体,二免后可达100%。用强毒aMPV-C攻击时,与对照组LaSota相比,其临床症状表现轻微,病毒在呼吸道内的检出率仅为20%,明显提高了对aMPV的保护率。
本研究结果表明,以LaSota为载体表达aMPV-C F和G蛋白的NDV重组疫苗,对NDV/CA02毒株完全保护,对aMPV有明显保护作用,可作为抵抗NDV和aMPV的候选双价疫苗,用于生产实践。同时,在本研究中所构建的重组NDV载体还可用于其他多价疫苗和基因治疗方面的研究,具有广泛的应用前景。
Virulent strains of Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) can cause serious respiratory diseases in poultry worldwide. These viruses are highly contagious and possess serious threats to food safety, public health, and biosecurity. The aMPV disease is usually accompanied by secondary bacterial infection that can increase high incidence and mortality, resulting in the significant economic losses to the poultry industry. Vaccination combined with strict biosecurity practices has been the recommendation for controlling both NDV and aMPV diseases in the field. Naturally occurring lentogenic NDV strains, such as B1, VG/GA, and LaSota strains, are routinely used as live vaccines throughout the world to prevent Newcastle disease. However, there are no low-virulence strains of aMPV that can be directly used as live vaccines. Live, attenuated aMPV vaccines appear to be effective against aMPV disease, but the stability and safety of these live attenuated vaccines remain a concern because of the potential of virulence revertance. Thus, there is a pressing need to develop a save, effective and economic bivalent vaccine that can be readily administered in the field to control these viral diseases. Therefore, this research proposed to develop NDV LaSota vaccine strain-based recombinant viruses that express aMPV fusion (F) protein and/or glycoprotein (G) as a bivalent vaccine against these viral diseases.
The NDV LaSota vaccine strain was chosen as a vector to generate recombinant viruses in this study. Firstly, a reporter gene, green fluorescence protein (GFP), was inserted into the intergenic region between F and HN genes of NDV genome, and successfully rescued a recombinant virus expressing GFP by using a reverse genetics approach. Secondly, the recombinant LaSota virus expressing the aMPV-C G protein was generated. Subsequently, the recombinant virus was characterized in vitro and in vivo. The results showed that the recombinant virus, rLS/aMPV-C G, was slightly attenuated in vivo, yet maintained similar growth dynamics, cytopathic effects, and virus titers in vitro when compared to the parental LaSota virus. Vaccination of turkeys with rLS/aMPV-C G induced complete protection against velogenic NDV challenge and partial protection against pathogenic aMPV-C challenge. The results, suggest that expression of the aMPV-C G protein alone is not sufficient to provide full protection against an aMPV-C infection. Expression of additional aMPV-C immunogenic protein(s) may be needed to induce stronger protective immunity against the aMPV-C disease. Thirdly, to prove whether the LaSota vaccine can express two foreign genes, another reporter gene, the red fluorescence protein (RFP), was added into the rLS/GFP vector. The RFP and an internal ribosome entry site sequence (IRES) were inserted into the F gene end as a second open reading frame (ORF). A recombinant NDV LaSota virus that expressed both RFP and GFP was rescued. Based on this rLS/I-RFP-GFP construct. Finally, the NDV recombinant virus expressing the aMPV-C F and G protein was successfully generated by replacing the RFP and GFP with the F and G genes, respectively. This recombinant virus vectoring the aMPV-C F and G displayed similar growth dynamics, virus titer and lentogenic pathotype as the parental LaSota strain. SPF turkeys vaccinated with rLS/aMPV-C F G were completely protected against the challenge with a virulent NDV strain, NDV/CA02. About 60-80% of rLS/aMPV-C G vaccinated birds developed an aMPV-C specific antibody response following the first vaccination and all birds seroconverted after the booster. After challenge with the pathogenic aMPV-CO virus, vaccinated turkeys displayed milder clinical signs and significantly less virus shedding in tracheal tissues than the control birds that were vaccinated with the parental LaSota strain.
In summary, the results demonstrated that vaccination of SPF turkeys with the NDV LaSota vaccine strain-based recombinant virus expressing the aMPV-C F and G proteins provided complete protection against the virulent NDV/CA02 strain challenge and significant protection against the pathogenic aMPV-C challenge. Thus, this recombinant virus could be used as a bivalent vaccine against NDV and aMPV-C diseases. In addition, the recombinant NDV LaSota vaccine vector can be used to develop multivalent vaccines against other avian viral diseases and may also have potential applications in gene therapy or anti- cancer research.?
为了获得表达aMPV C亚型融合蛋白F或糖蛋白G的重组新城疫病毒,该研究以新城疫病毒疫苗株LaSota为载体,首先将绿色荧光蛋白(Green fluorescence protein,GFP)作为报告基因插入到NDV基因组F-HN之间,利用反向遗传学技术构建表达GFP蛋白的重组新城疫病毒rLS/GFP。结果显示,LaSota可作为活疫苗载体安全稳定地表达GFP蛋白。进而构建了表达aMPV-C G蛋白的重组新城疫病毒rLS/aMPV-C G,并对其毒力、生物学特性及免疫保护率进行了评估。结果表明rLS/aMPV-C G的毒力在SPF(Specific-pathogen-free,SPF)鸡胚和1日龄雏鸡内轻微致弱,其生物学特性与LaSota株相似,并可完全抵新城疫病毒抗强毒株(NDV/CA02)的攻击,对aMPV-C有部分免疫保护。由此推测G蛋白的抗原性不强,产生的免疫力不足以完全保护aMPV-C的攻击,于是,该研究进一步构建表达aMPV-C双重抗原蛋白F和G的重组新城疫病毒。在rLS/GFP载体的基础上,以红色荧光蛋白(Red fluorescence protein,RFP)为另一报告基因插入到载体F基因末端,作为第二阅读框架,然后将内部核糖体进入位点IRES(Internal ribosome entry site ,IRES)插入F和RFP阅读框架之间用于启动RFP基因的表达,进而成功构建表达两个外源基因(RFP和GFP)的重组新城疫病毒rLS/I-RFP-GFP。在此基础上,构建了表达aMPV-C F和G蛋白的重组新城疫病毒rLS/aMPV-C F G,并对其进行免疫保护评估。结果显示,SPF火鸡免疫接种rLS/aMPV-C F G后可完全保护NDV/CA02的攻击,首免后60-80%的火鸡可诱导产生特异性aMPV-C抗体,二免后可达100%。用强毒aMPV-C攻击时,与对照组LaSota相比,其临床症状表现轻微,病毒在呼吸道内的检出率仅为20%,明显提高了对aMPV的保护率。
本研究结果表明,以LaSota为载体表达aMPV-C F和G蛋白的NDV重组疫苗,对NDV/CA02毒株完全保护,对aMPV有明显保护作用,可作为抵抗NDV和aMPV的候选双价疫苗,用于生产实践。同时,在本研究中所构建的重组NDV载体还可用于其他多价疫苗和基因治疗方面的研究,具有广泛的应用前景。
Virulent strains of Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) can cause serious respiratory diseases in poultry worldwide. These viruses are highly contagious and possess serious threats to food safety, public health, and biosecurity. The aMPV disease is usually accompanied by secondary bacterial infection that can increase high incidence and mortality, resulting in the significant economic losses to the poultry industry. Vaccination combined with strict biosecurity practices has been the recommendation for controlling both NDV and aMPV diseases in the field. Naturally occurring lentogenic NDV strains, such as B1, VG/GA, and LaSota strains, are routinely used as live vaccines throughout the world to prevent Newcastle disease. However, there are no low-virulence strains of aMPV that can be directly used as live vaccines. Live, attenuated aMPV vaccines appear to be effective against aMPV disease, but the stability and safety of these live attenuated vaccines remain a concern because of the potential of virulence revertance. Thus, there is a pressing need to develop a save, effective and economic bivalent vaccine that can be readily administered in the field to control these viral diseases. Therefore, this research proposed to develop NDV LaSota vaccine strain-based recombinant viruses that express aMPV fusion (F) protein and/or glycoprotein (G) as a bivalent vaccine against these viral diseases.
The NDV LaSota vaccine strain was chosen as a vector to generate recombinant viruses in this study. Firstly, a reporter gene, green fluorescence protein (GFP), was inserted into the intergenic region between F and HN genes of NDV genome, and successfully rescued a recombinant virus expressing GFP by using a reverse genetics approach. Secondly, the recombinant LaSota virus expressing the aMPV-C G protein was generated. Subsequently, the recombinant virus was characterized in vitro and in vivo. The results showed that the recombinant virus, rLS/aMPV-C G, was slightly attenuated in vivo, yet maintained similar growth dynamics, cytopathic effects, and virus titers in vitro when compared to the parental LaSota virus. Vaccination of turkeys with rLS/aMPV-C G induced complete protection against velogenic NDV challenge and partial protection against pathogenic aMPV-C challenge. The results, suggest that expression of the aMPV-C G protein alone is not sufficient to provide full protection against an aMPV-C infection. Expression of additional aMPV-C immunogenic protein(s) may be needed to induce stronger protective immunity against the aMPV-C disease. Thirdly, to prove whether the LaSota vaccine can express two foreign genes, another reporter gene, the red fluorescence protein (RFP), was added into the rLS/GFP vector. The RFP and an internal ribosome entry site sequence (IRES) were inserted into the F gene end as a second open reading frame (ORF). A recombinant NDV LaSota virus that expressed both RFP and GFP was rescued. Based on this rLS/I-RFP-GFP construct. Finally, the NDV recombinant virus expressing the aMPV-C F and G protein was successfully generated by replacing the RFP and GFP with the F and G genes, respectively. This recombinant virus vectoring the aMPV-C F and G displayed similar growth dynamics, virus titer and lentogenic pathotype as the parental LaSota strain. SPF turkeys vaccinated with rLS/aMPV-C F G were completely protected against the challenge with a virulent NDV strain, NDV/CA02. About 60-80% of rLS/aMPV-C G vaccinated birds developed an aMPV-C specific antibody response following the first vaccination and all birds seroconverted after the booster. After challenge with the pathogenic aMPV-CO virus, vaccinated turkeys displayed milder clinical signs and significantly less virus shedding in tracheal tissues than the control birds that were vaccinated with the parental LaSota strain.
In summary, the results demonstrated that vaccination of SPF turkeys with the NDV LaSota vaccine strain-based recombinant virus expressing the aMPV-C F and G proteins provided complete protection against the virulent NDV/CA02 strain challenge and significant protection against the pathogenic aMPV-C challenge. Thus, this recombinant virus could be used as a bivalent vaccine against NDV and aMPV-C diseases. In addition, the recombinant NDV LaSota vaccine vector can be used to develop multivalent vaccines against other avian viral diseases and may also have potential applications in gene therapy or anti- cancer research.?
引文
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