家禽干扰素的分类及应用

干扰素是一类仅由脊椎动物单核细胞和淋巴细胞在受到病毒或其他诱生剂的作用时产生的一种具有抗病毒、抗肿瘤、免疫调节等多种生物学活性的分泌性可溶糖蛋白。随着基因工程方法的发展,外源表达干扰素已经进入了工业化生产阶段,并且大量投放市场,在医药、生物和农业等多个行业领域中广泛应用。近年来,随着人们对禽类干扰素研究的逐步深入,干扰素在一些禽类病毒性疾病及肿瘤性疾病治疗方面取得的良好进展。本文现就家禽干扰素的分类,家禽干扰素的抗病毒活性以及家禽基因工程重组干扰素的应用做以综述。     

禽类干扰素的研究进展

干扰素在抗病毒、抗肿瘤、免疫调节等方面的应用展现出了很多优势，尤其是基因工程重组技术的应用使干扰素在临床生产中的大规模使用成为可能。本文从干扰素的产生、分子结构、生物学功能等几个方面综述了近年来关于禽类干扰素的研究进展。     

重组鸡α-干扰素的表达及其生物活性的初步研究

为获得重组鸡α-干扰素并对其进行生物活性研究,本试验对重组大肠杆菌进行诱导表达,收集重组鸡α-干扰素包涵体后进行复性纯化及免疫印迹试验,并通过测定病毒EID₅₀,在接种H9亚型禽流感病毒、新城疫病毒和传染性支气管炎病毒的鸡胚中进行干扰素抗病毒活性试验,运用微变量细胞病变抑制法进行重组鸡α-干扰素抗病毒效价的测定。结果显示复性后重组鸡α-干扰素的免疫印迹试验成功获得了预期条带,在鸡胚中重组鸡α-干扰素具有显著的抗H9亚型禽流感病毒、新城疫病毒和传染性支气管炎病毒等活性作用。在CEF/VSV细胞系上测定重组鸡α-干扰素的抗病毒效价为1.5×2¹⁰ U/mg左右,高浓度重组鸡α-干扰素具有一定的细胞毒性。结果表明重组鸡α-干扰素蛋白具有免疫原性和抗原性,在鸡胚内具有较好的抑制或杀灭H9亚型禽流感病毒、新城疫病毒和传染性支气管炎病毒的效果,呈广谱性,抗病毒效价较高,为下一步抗病毒制品的创制奠定基础。     

这些新型兽用疫苗,你认识吗

<正> 1 基因工程疫苗1.1 重组活载体疫苗病毒活载体疫苗的研究较多,如用鸡痘病毒作为载体,表达成功的保护性抗原基因就有新城疫病毒、传染性法氏囊病病毒、马立克氏病病毒、禽网状内皮组织增生症病毒、狂犬病病毒、传染性支气管炎病毒、艾美尔球虫等。2005年12月24日,农业部在北京宣布,我国已成功研制出禽流感--新城疫重组二联活疫苗,并正式批准生产。1.2 基因工程亚单位疫苗已经制成并用于生产的主要有口蹄疫基因工程亚单位疫苗、鸡传染性法氏囊病基因工程亚单位疫苗,用于预防仔猪     

传染性支气管炎病毒疫苗研究进展

传染性支气管炎给我国规模化养鸡业造成巨大的经济损失,由此尝试研制重组鸡痘病毒传染性支气管炎病毒基因工程疫苗了解其免疫效应,以期获得高效而安全的基因工程疫苗。为更好地了解鸡传染性支气管炎的防治现状,文章从各种表达载体系统表达传染性支气管炎病毒蛋白的优缺点及其产生免疫保护效应的角度,对近年来传染性支气管炎病毒疫苗研究的发展状况做了简要概述,且针对传染性支气管炎病毒基因工程疫苗现存的问题,展望了将来的发展趋势。     

鸡传染性喉气管炎基因工程疫苗的研究与应用

论述了鸡传染性喉气管炎亚单位疫苗、基因缺失疫苗、重组病毒活载体疫苗、基因疫苗的研究和应用进展情况,表明应用基因工程疫苗免疫可以从根本上改变鸡传染性喉气管炎的防治现状,使传染性喉气管炎的根除成为可能。     

研究表明鸡传染性支气管炎病毒疫苗株与野毒株发生基因多重重组后产生与母本病毒不同特性的新病毒

正鸡传染性支气管炎(Infectious bronchitis,IB)是由鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)引起的鸡和其他禽类的一种急性、高度接触性、呼吸道传染性疫病,能够造成严重经济损失。病毒在世界范围内广泛存在。由于IBV的病毒聚合酶校对机制不全,病毒可通过基因突变和基因重组事件而产生新抗原型的毒株,新病毒一般无交叉保护性,因而用单一血清型或基因型的疫苗免疫控制该病比较困难。传染性支气管炎病毒频繁发生点突变或重组变异,传统疫苗的应用不能阻止该病持续发生。已有     

介绍几类兽用新疫苗

(一)基因工程疫苗1.重组活载体疫苗。病毒活载体疫苗研究得较多,如用鸡痘病毒作为载体,表达成功的保护性抗原基因就有新城疫病毒、传染性法氏囊病病毒、马立克氏病病毒、禽网状内皮组织增生症病毒、狂犬病病毒、传染性支气管炎病毒、艾美尔球虫等。2005年12月24日,农业部在北京     

重组牛干扰素γ对病毒性MDBK细胞病变的抑制作用

本实验研究了重组牛干扰素对５种病毒诱导ＭａｒｄｉｎＤａｒｂｙ牛肾细胞产生细胞病变（ＣＰＥ）的抑制作用。通过比较重组牛干扰素ｒ处理和未处理情况下５种病毒的ｌｏｇＴＣＩＤ５０,发现水泡性口炎病毒（ＶＳＶ）,牛呼吸道合胞病毒（ＢＲＳ）和副流感病毒３型对于重组牛干扰素有显著的敏感性,与对照相比,其ＴＣＩＤ５０分别增加１０００倍,７９４倍和３９８倍。牛腺病毒７型（Ａｄ－７）和传染性牛鼻气管炎病毒（ＩＢＲ）对重组牛干扰素的敏感性很弱,ＴＣＩＤ５０分别增长１．２５倍和１．５８倍。实验结果表明重组牛干扰素在治疗对其敏感性强的病毒性传染病方面具有潜在的应用价值。     

表达传染性法氏囊vp2基因的重组马立克病疫苗对SPF鸡和商品鸡的免疫保护试验

国内外很多学者都在进行着传染性法氏囊(Infectious bursal disease,IBD)各种基因工程疫苗的研制,IBDV-vp2基因在多种载体中得到表达并取得了较好的保护效力。本实验室已构建了表达IBDV-vp2基因的重组马立克氏病病毒。本研究对该重组马立克氏病病毒疫苗的遗传稳定性以及对SPF鸡和含有母源抗体的商品鸡的免疫保护作用进行了评价。结果表明,103 PFU和104 PFU剂量的重组病毒免疫后对SPF鸡抗传染性法氏囊标准强毒的保护率分别为53%和73%。104 PFU剂量的重组病毒免疫后对含有母源抗体的商品鸡抗传染性法氏囊标准强毒的保护率为87%,结果显示重组疫苗具有一定的应用前景。     

In situ production of interferon in tissues of chickens exposed as embryos to turkey herpesvirus and Marek's disease virus

Chicken eggs at embryonation day (ED) 18 or newly hatched chicks were inoculated with turkey herpesvirus (HVT), Marek's disease virus (MDV), or virus-free diluent and, at intervals after inoculation, tissue homogenates of virus-exposed and virus-free chickens or chicken embryos were examined for interferon (IFN) activity. Homogenates of lung, thymus and spleen specimens from chickens given HVT at ED 18 had IFN activity. Activity of IFN in the lungs was studied further. Homogenates of lung specimens from chickens exposed to HVT at hatching also had IFN activity, although the concentration of IFN was lower than that in chickens given HVT at ED 18. The pathogenic isolates of MDV (JM-MDV), but not the attenuated (Md11/75C-MDV) or nonpathogenic (SB1-MDV) isolates, inoculated at ED 18 also induced high lung IFN activity. Exposure to a combination of HVT and SB1-MDV induced IFN activity comparable with that in chickens given HVT alone. The IFN activity in homogenates of lung specimens from virus-exposed chickens was species specific and heat and pH stable, but was destroyed by trypsin treatment. Occasionally, low IFN activity also was detected in homogenates of tissue specimens from virus-free chickens or chicken embryos. This IFN activity could have been produced constitutively or may have been induced by substances (inducers) in the environment.     

Host immune-related gene responses against highly pathogenic avian influenza virus infection in vitro differ among chicken cell lines established from different organs

Highly pathogenic avian influenza virus (HPAIV) induces acute disease in chickens causing high mortality and morbidity and is a major threat to poultry industries in Southeast Asian countries. The mechanisms of disease manifestation and host innate immune responses against HAPIV in chickens are not well understood. In this study, we examined virus replication and host gene expressions in four chicken cell lines in vitro to elucidate the impact of host innate immune responses against viral replication. It was demonstrated that viral replication efficiencies were different depending on the cell line. The viral replication appeared to be affected by the basal expression of IFN related genes. The expression of immune-related genes against the viral infection also varied in a cell line dependent manner. In non-immune derived cell lines, but not in immune derived cell lines, the expression of the CCL5 and CCL20 genes were induced by HPAIV infection. Reverse genetics HPAIV, with internal genes from avirulent avian influenza, reduced virus replication and affected immune-related gene expression in a cell line dependent manner. These results suggest the possibility that differential immune responses in different cell types in local tissues could modulate the consequences of HPAIV infection in chickens.     

Molecular biology of avian infectious laryngotracheitis virus

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes an economically important chicken disease, which results in delayed growth, reduced egg production, and also frequently in death of the animals. After acute infection of the upper respiratory tract, the virus can establish latency in the central nervous system, and subsequent reactivations can lead to infection of naive chickens. For prevention of ILT, conventionally attenuated live vaccines are available. However, these vaccine strains are genetically not characterized, and reversions to a virulent phenotype occur. Although molecular analyses of ILTV are hampered by the lack of an optimal cell culture system, the complete nucleotide sequence of the ILTV genome has recently been elucidated, and several ILTV recombinants lacking nonessential, but virulence determining genes have been constructed. Animal trials indicated that genetically engineered stable gene deletion mutants are safe alternatives to the current vaccine strains. Furthermore, since live ILTV vaccines are suitable for fast and inexpensive mass administration, they are promising as vectors for immunogenic proteins of other chicken pathogens. Thus, immunization with ILTV recombinants expressing avian influenza virus hemagglutinin was shown to protect chickens against ILT and fowl plague. Using monospecific antisera and monoclonal antibodies several virion proteins of ILTV have been identified and characterized. Since they include immunogenic envelope glycoproteins, these results can contribute to the improvement of virus diagnostics, and to the development of marker vaccines.     

利用反向遗传技术拯救H9N2重组病毒

为研制高效特异的H9N2亚型AI疫苗,选取我国具有代表性的毒株A/chicken/Shanghai/10/01(H9N2)(简称SH10),以12质粒系统为基础,利用反向遗传操作技术构建了1株表面基因由SH10提供、内部基因由12质粒系统提供的重组病毒SH/PR8,为新型疫苗的研制提供了新的毒株。     

Protection against infectious laryngotracheitis by in ovo vaccination with commercially available viral vector recombinant vaccines

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is mainly controlled through biosecurity and by vaccination with live-attenuated vaccines. The chicken embryo origin (CEO) vaccines, although proven to be effective in experimental settings, have limited efficacy in controlling the disease in dense broiler production sites due to unrestricted use and poor mass vaccination coverage. These factors allowed CEO vaccines to regain virulence, causing long lasting and, consequently, severe outbreaks of the disease. A new generation of viral vector fowl poxvirus (FPV) and herpesvirus of turkey (HVT) vaccines carrying ILTV genes has been developed and such vaccines are commercially available. These vaccines are characterized by their lack of transmission, lack of ILTV-associated latent infections, and no reversion to virulence. HVT-vectored ILTV recombinant vaccines were originally approved for subcutaneous HVT or transcutaneous (pox) delivery. The increased incidence of ILTV outbreaks in broiler production sites encouraged the broiler industry to deliver the FPV-LT and HVT-LT recombinant vaccines in ovo. The objective of this study was to evaluate the protection induced by ILTV viral vector recombinant vaccines after in ovo application in 18-day-old commercial broiler embryos. The protection induced by recombinant ILTV vaccines was assessed by their ability to prevent clinical signs and mortality; to reduce challenge virus replication in the trachea; to prevent an increase in body temperature; and to prevent a decrease in body weight gain after challenge. In this study, both recombinant-vectored ILTV vaccines provided partial protection, thereby mitigating the disease, but did not reduce challenge virus loads in the trachea.     

腺胃病变型鸡传染性支气管炎病毒的分离鉴定

从１９９７年在辽宁省大连市某鸡场发生的一种以腺胃肿大为特征的鸡的传染病病例中收集腺胃组织（Ｄ９７１）接种ＳＰＦ鸡胚,传至１３代,具有规律性死亡和典型胚胎病变特征,ＥＩＤ５０为１０－６．４８／０．２ｍｌ。Ｄ９７１毒株感染ＳＰＦ鸡胚尿囊液经负染电镜观察,可见直径为８０～１２０ｎｍ,有囊膜和纤突的冠状病毒粒子。Ｄ９７１毒株经卵磷脂酶Ｃ处理后能凝集鸡红细胞,并能特异的被Ｄ９７１多抗所抑制。用反转录聚合酶链反应获得了Ｄ９７１毒株免原（Ｓ１）基因ｃＤＮＡ,经１％琼脂糖凝胶电泳可见１．７ｋｂ的特征性条带。用Ｄ９７１毒株接种ＳＰＦ鸡,能引起与自然病例相似的病理变化,并分离到病毒。用Ｄ９７１毒株可直接感染ＳＰＦ鸡胚成纤维细胞。结果证实从腺胃病变型ＩＢ鸡群中分离的Ｄ９７１毒株是冠状病毒科ＩＢＶ成员。     

J亚群与E亚群禽白血病自然重组病毒的全基因组序列分析

为了解我国东北地区部分养鸡场禽白血病病毒(ALV)的基因组序列特征及其变异情况,本研究从具有典型血管瘤病变的禽白血病发病鸡中分离到一株J亚群ALV(ALV-J)命名为JL0901,并进行了全基因测序.将该序列与已发表的ALV-J毒株序列进行比较研究,结果表明JL0901基因组的gag和pol基因相对保守,而env基因和3'端非编码区(3'UTR)的变异较大.对JL0901的env基因核苷酸序列进一步分析发现,在其gp85基因和gp37基因交界位置发生J亚群和E亚群ALV重组现象.本研究证实国内鸡群中存在J亚群和其他亚群ALV的自然重组现象,并表明国内ALV已出现新的变异趋势.     

1株E亚群禽白血病病毒的分离鉴定及遗传进化分析

E亚群禽白血病病毒（ALV-E）是指存在于鸡染色体中的内源性逆转录病毒基因组DNA或片段。具有转录活性的ALV-E既会对鸡的生产性能（体重和产蛋率）产生负面影响，又能从抗体水平干扰对外源性ALV的鉴别诊断。为对黑龙江省某鸡场内一禽白血病病毒RT-PCR阳性病料进行病毒分离鉴定及分析其基因组特征和遗传进化情况，通过分子生物学、病毒形态学及全基因组序列测定方法对病毒培养物进行鉴定和分析，结果显示，该分离株可在CEF细胞盲传至第9代，电镜下可观察到近似球形、直径约为80 nm，并具有囊膜和纤突结构的病毒粒子，将其命名为HLJE2020株。序列分析结果显示，其全基因组序列中的gag和pol基因相对保守，LTR和env基因与ALV-E同属一个进化分支，而gp85基因则与ALV-E和ALV-B均具有较高相似性，遗传进化分析显示在ALV-E和ALV-B间出现一个单独的分支，结合RDPv.4和SimPlot软件分析结果，推测该毒株gp85基因可能存在E亚群AF229株与B亚群SDAU09C1株的重组现象。本研究为了解禽白血病病毒基因组遗传演化情况提供数据资料，并为ALV的防控提供参考和依据。     

Effects of interferon-γ knockdown on vaccine-induced immunity against Marek’s disease in chickens

Interferon (IFN)-γ has been shown to be associated with immunity to Marek’s disease virus (MDV). The overall objective of this study was to investigate the causal relationship between IFN-γ and vaccine-conferred immunity against MDV in chickens. To this end, 3 small interfering RNAs (siRNAs) targeting chicken IFN-γ, which had previously been shown to reduce IFN-γ expression in vitro, and a control siRNA were selected to generate recombinant avian adeno-associated virus (rAAAV) expressing short-hairpin small interfering RNAs (shRNAs). An MDV challenge trial was then conducted: chickens were vaccinated with herpesvirus of turkey (HVT), administered the rAAAV expressing shRNA, and then challenged with MDV. Tumors were observed in 4 out of 10 birds that were vaccinated with HVT and challenged but did not receive any rAAAV, 5 out of 9 birds that were administered the rAAAV containing IFN-γ shRNA, and 2 out of 10 birds that were administered a control enhanced green fluorescent protein siRNA. There was no significant difference in MDV genome load in the feather follicle epithelium of the birds that were cotreated with the vaccine and the rAAAV compared with the vaccinated MDV-infected birds. These results suggest that AAAV-based vectors can be used for the delivery of shRNA into chicken cells. However, administration of the rAAAV expressing shRNA targeting chicken IFN-γ did not seem to fully abrogate vaccine-induced protection.