禽白血病病毒致病分子机制的研究进展

禽白血病(Avian leukosis,AL)是一类由禽白血病病毒(Avian leukosis virus,ALV)引起的禽类传染病。当机体感染ALV后,病毒侵入机体并在体内持续作用,从而诱导细胞损伤、肿瘤性病变形成和免疫抑制等。文章从ALV的感染复制、致瘤机制、免疫抑制机制及相关信号通路等方面对其致病分子机制进行了初步综述,为AL的进一步研究提供参考。     

昆虫杆状病毒囊膜蛋白F的研究进展

病毒入侵是病毒感染宿主细胞的第一步,涉及病毒与宿主细胞受体的结合、膜融合等过程,而这些过程是由病毒的囊膜蛋白所介导。已知昆虫杆状病毒中存在GP64和F 2种不同类型的囊膜蛋白。感染鳞翅目昆虫的核型多角体病毒(NPV)中的GroupⅠ组病毒同时具有F蛋白和GP64蛋白的同源物,但是F蛋白不是膜融合蛋白;GroupⅡ组NPV和颗粒病毒(GV)以及感染双翅目昆虫的病毒仅有F蛋白的同源物,而在感染膜翅目昆虫的病毒中,这2种蛋白质的同源物均不存在。F蛋白又可分为Fa和Fb 2类。在GroupⅡ组NPV中,Fa是出芽型病毒粒子的膜融合蛋白,能介导低p H依赖的膜融合;而在GroupⅠ组NPV中,Fb蛋白的功能目前尚不明确,其膜融合活性被认为在进化历程中被GP64所取代。本文重点综述昆虫杆状病毒囊膜蛋白F的研究进展。     

IFITM和IFIT家族基因的抗病毒机制研究进展

干扰素诱导的跨膜(Interferon-induced transmembrane,IFITM)蛋白家族和干扰素诱导(Interferon-induced proteins with tetratricopeptide repeats,IFIT)蛋白家族作为受干扰素所诱导的,执行抗病功能的家族,在免疫反应中扮演着至关重要的角色;特别是IFIT5是家禽体内存在唯一的IFIT家族成员。IFITM通过抑制病毒包膜与宿主细胞膜的融合,从而抑制病毒的入侵;而IFIT则是抑制病毒的转录与复制,阻断病毒繁殖,从而抑制病毒的感染。两者在生物体内协同发挥抗病毒功能,清除病毒,保护宿主细胞正常的生理活动。在数万年的进化中,病毒也进化出相应的机制来逃避免疫蛋白的抑制。文章就IFITM和IFIT基因家族抗病毒机制的研究进展进行总结,为之后的研究提供参考。     

病毒感染引发的鸡群免疫抑制

病毒感染可直接或间接损害免疫系统的细胞,从而引起鸡群免疫应答功能不全,产生免疫抑制,使机体免疫力低下,导致疫苗免疫失败以及继发感染发病率增加。主要介绍了MDV、REV、ALV、IBDV和CIAV等病毒引起鸡群免疫抑制的机理以及免疫抑制性病毒二重或多重感染对鸡群的危害,对如何防止和控制免疫抑制性病毒的感染进行了探讨。     

病毒感染引发的鸡群免疫抑制

病毒感染可直接或间接损害免疫系统的细胞，从而引起鸡群免疫应答功能不全，产生免疫抑制，使机体免疫力低下，导致疫苗免疫失败以及继发感染发病率增加。主要介绍了HDV、REV、ALV、IBDV和CIAV等病毒引起鸡群免疫抑制的机理以及免疫抑制性病毒二重或多重感染对鸡群的危害，对如何防止和控制免疫抑制性病毒的感染进行了探讨。     

麻疹病毒细胞受体研究进展

麻疹病毒是一种引起儿童急性呼吸道感染的传染病,属于副黏病毒科麻疹病毒属,同属的还包括犬瘟热病毒、牛瘟病毒等。细胞受体是病毒入侵易感细胞和启动感染的关键。目前,3种蛋白质分子——膜辅蛋白CD46、信号淋巴细胞激活因子SLAM和细胞黏附分子Nectin-4已经被证实是介导麻疹病毒入侵易感染细胞和启动感染的受体。论文综述了麻疹病毒3种细胞受体的研究进展,对深入了解病毒与宿主间的相互关系及有针对性地进行抗病毒药物、疫苗研制具有重大意义。     

膜联蛋白A2参与流感病毒感染的研究进展

流感病毒感染宿主细胞是一个病毒入侵、遗传物质释放、病毒基因组转录和复制、病毒组装和出芽的过程。这个过程无论对于病毒的生存还是病毒致病力都是非常重要的,而这一过程的完成,必须依赖宿主细胞蛋白的参与。宿主蛋白膜联蛋白A2(Annexin A2, ANXA2)是一个多功能蛋白,在内吞、胞吐等与囊膜病毒复制相关的生物膜活动中发挥重要作用,并且被证实能够参与许多病毒的感染过程。文章综述了ANXA2在流感病毒的入侵、复制、组装、出芽等致病过程中发挥的功能,以期为从分子水平上阐明流感病毒与宿主细胞相互作用的机制,揭示ANXA2在抗流感病毒感染中的潜在价值提供参考。     

苜蓿银纹夜蛾核型多角体病毒囊膜蛋白GP64与寄主细胞互作蛋白的初步鉴定

鉴定杆状病毒与宿主昆虫细胞间的互作蛋白,对于进一步研究病毒受体的特性与功能,理解病毒与宿主细胞的相互作用关系具有重要意义。为了阐明苜蓿银纹夜蛾核型多角体病毒(Ac MNPV)出芽型病毒粒子(BV)囊膜蛋白GP64是否与宿主细胞表面蛋白存在互作,利用蛋白酶K消化处理Tn细胞系的细胞膜蛋白,结果表明BV不能感染经300μg/m L蛋白酶K消化处理的Tn细胞,免疫荧光检测BV病毒粒子不能吸附于Tn细胞膜上。利用病毒覆盖蛋白印迹实验(VOPBA)及质谱初步鉴定的结果显示,Tn细胞膜上的翻译控制肿瘤蛋白(TCTP)是与BV囊膜蛋白GP64互作的候选蛋白。     

K亚群禽白血病病毒细胞受体的鉴定

禽白血病病毒(ALV)进入细胞启动感染依赖于其囊膜表面蛋白gp85与细胞受体的结合。K亚群ALV(ALV-K)是2012年从我国本地芦花鸡中分离到的一个ALV新亚群。为了鉴定ALV-K的细胞受体,本研究利用真核细胞表达了A和K亚群ALV gp85蛋白,并利用实验室前期拯救的重组病毒RCAS-A-GFP和RCAS-K-GFP进行病毒受体的交叉干扰试验,结果显示A亚群ALV(ALV-A)gp85蛋白能够抑制ALV-K的感染,同样ALV-Kgp85蛋白也能够抑制ALV-A的感染,存在受体交叉干扰现象,因而推测二者可能共用细胞受体。已有研究表明Tva是ALV-A的细胞受体,本研究利用Co-IP和Pull-down试验检测了ALV-Kgp85与Tva的互作,结果显示ALV-Kgp85蛋白能与可溶性的Tva(sTva)相互作用。进一步的流式细胞术结果显示ALV-Kgp85能够高效结合Tva,结合率在90%以上。为进一步验证Tva是否能介导ALV-K进入细胞,利用ALV的非易感细胞HEK293T进行了Tva受体重建试验,结果显示RCAS-K-GFP能够进入表达Tva的293T细胞并复制产生较强的绿色荧光。以上结果充分表明Tva也是ALV-K的细胞受体,能够介导ALV-K进入宿主细胞,启动其感染。该研究为阐明ALV-K侵入宿主细胞机制及抗病毒靶点筛选奠定了基础。     

犬瘟热研究进展

犬瘟热是由犬瘟热病毒引起的一种高发传染病,宿主范围包括大部分食肉目动物。犬瘟热病毒可以感染不同器官和组织的上皮细胞、间质细胞、神经内分泌细胞及造血干细胞,引发全身型或神经型的临床过程,并在中枢神经系统和淋巴组织中形成持续感染,免疫抑制和脱髓鞘性脑脊髓炎是代表性的病症,而淋巴细胞介导的细胞毒性作用的缺乏则与病毒在中枢神经系统的持续感染有关。犬瘟热发病机制与病理学的研究对于犬瘟热的免疫预防和临床诊治有重要的意义。     

非洲猪瘟病毒入侵细胞机制的研究进展

病毒进入宿主细胞的机制决定了病毒的趋向性和发病机理,病毒感染细胞包括吸附、穿入、脱壳、复制、组装及子代病毒颗粒的释放等步骤,对病毒入侵机制研究可以为开发有针对性的预防策略提供思路。非洲猪瘟病毒作为一种有囊膜双链DNA病毒,其入侵宿主细胞的机制与其他病毒有一定的相似之处,同样也有其特殊性。目前,人们对非洲猪瘟病毒与宿主细胞相互作用,特别是入侵机制的了解仍然非常有限。为此,本文就ASFV入侵细胞的有关研究进展进行了综述,以期为非洲猪瘟病毒致病机制相关研究提供参考。     

Sequential disruption of ALV host receptor genes reveals no sharing of receptors between ALV subgroups A,B, and J

Background: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus(ALV)subgroups using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A(TVA) and generate chicken cells resistant to infection by this virus.Results: CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely,overexpression of the wild-type TVA receptor(wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na~+/H~+ exchange 1(chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively.Conclusions: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry.     

Development and characterization of monoclonal antibodies to subgroup A avian leukosis virus

Avian leukosis virus subgroup A (ALV‐A) is a retrovirus which infects egg‐type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV‐A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV‐A env‐gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV‐A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV‐A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV‐A.     

鸡传染性支气管炎病毒受体研究进展

鸡传染性支气管炎是一种世界性分布的严重影响养鸡业的高度接触性传染病,其病原是一种最早发现的冠状病毒鸡传染性支气管炎病毒(IBV)。IBV入侵细胞的受体是目前IBV研究中的热点之一。已知IBV囊膜上的纤突蛋白(S)是宿主组织和细胞嗜性及致病性的主要决定因素,S蛋白通常断裂为S1和S2两部分,S1与宿主细胞膜上的细胞受体相结合,在S2的作用下病毒囊膜与宿主细胞膜融合,IBV与受体结合之后于中性或微碱性pH时发生构象改变,从而获得病毒进入宿主细胞的能力。对于IBV的受体研究主要集中于IBV可能使用的几种功能性受体,包括氨肽酶N、唾液酸、硫酸乙酰肝素等。     

新城疫病毒囊膜糖蛋白生物学特性的研究进展

新城疫是当今全球范围内最严重的禽类传染病之一。其病原新城疫病毒是单股负链RNA病毒,编码NP、P、M、F、HN和L 6种结构蛋白,其中最主要的是位于囊膜上的F和HN两种糖蛋白。F蛋白具有使病毒囊膜与宿主细胞膜融合,致使病毒穿入宿主细胞膜的作用,是决定病毒毒力的关键因子;HN蛋白具有血凝素和神经氨酸酶两种活性,这两种活性对于NDV侵染细胞具有重要的作用。这两种糖蛋白不仅对NDV的毒力及致病性方面起着决定性作用,而且也是诱导产生保护性抗体的蛋白。作者主要对这两种蛋白的结构与功能作一概述。     

High virus titer in feather pulp of chickens infected with subgroup J avian leukosis virus

Subgroup J avian leukosis viruses (ALVs), which are a recombinant virus between exogenous and endogenous ALVs, can spread by either vertical or horizontal transmission. Exogenous and endogenous ALVs can be detected in feather pulp. In this study, virus titers in feather pulp of chickens infected with subgroup J ALV were compared with those of plasma and cloacal swab. All of the broiler chickens inoculated with subgroup J ALV at 1 day old were positive for virus from feather pulp during the experimental period of between 2 wk and 8 wk of age. Virus titers in feather pulp of some broiler chickens infected with subgroup J ALV were very high, ranging from 10(7) to 10(8) infective units per 0.2 ml. Virus titers in feather pulp were usually the highest among the samples of plasma, cloacal swab, and feather pulp tested. In another experiment in which layer chickens were inoculated with subgroup J ALV at 1 day old, virus was detected in feather pulp from 2 wk until 18 wk of age, and virus persisted longer in feather pulp than in plasma. Almost all of the layer chickens tested were positive for virus by polymerase chain reaction (PCR) with DNA extracted from feather pulp samples at 2, 4, and 10 wk of age, and the PCR from feather pulp was more sensitive than virus isolation from plasma, cloacal swab, and feather pulp. All above results indicate that samples of feather pulp can be useful for virus isolation and PCR to confirm subgroup J ALV infection.     

动物3种DNA病毒在宿主细胞内形态发生比较

对猪细小病毒(PPV)、牛疱疹病毒2型(BHV2)和犬腺病毒1型(CAV1)3种动物DNA病毒在宿主细胞内的增殖、释放方式以及所致细胞结构的变化,通过电镜进行了观察比较.(1)这3种动物DNA病毒的复制和装配过程均发生在细胞核内,以毒浆结构(Viroplast)或核内包涵体为增殖场所和物质基础,但并非都形成结晶样结构.(2)有囊膜的BHV2,其核壳体在细胞核内装配完成后,从核内膜上以出芽方式获得囊膜,然后进入核周池,聚集的病毒使核外膜向胞质方向隆起,形成病毒性包涵体而脱离核外膜,并逐渐向细胞膜的方向移动,最后从细胞膜的破损处以病毒包涵体形式释放到细胞间隙.而无囊膜的CAV1,核壳体在细胞核内装配完成后,从细胞核膜破损处或细胞核崩解后进入细胞质,待整个细胞崩解后才能释放出来.无囊膜的PPV,在核壳体装配完成后,成堆地以病毒流的方式,从扩张的核孔释放到细胞质中,待细胞崩解后再释放出来.(3)3种病毒增殖时,宿主细胞的固有细胞器,如线粒体、内质网以及溶酶体等均出现不同程度的超微结构变化,并能诱导宿主细胞出现一些新的结构,除毒浆结构外,还有管状结构、细纤维样结构、周期性结构和髓膜样结构等,其中周期性结构仅见于BHV2感染.     

Detection and characterization of avian leukosis virus in Marek's disease vaccines

Avian leukosis virus (ALV) infection in chickens is known to induce increased mortality, tumors, delayed growth, and suboptimal egg production. Countries importing specified pathogen-free eggs, vaccines, and poultry breeding stock require freedom of infection or contamination with ALV in such products among other avian pathogens. Recently, ALV was found as a contaminant in a limited number of commercial poultry vaccines, even after routine quality assurance procedures cleared the vaccines for commercialization. The contaminated vaccines were promptly withdrawn from the market, and no direct detrimental effects were reported in poultry vaccinated with such vaccines. We describe herein the characterization in vitro of the contaminant viruses. All exogenous viruses detected in four vaccine lots belong to subgroup A of ALV based on cell receptor interaction, subgroup-specific polymerase chain reaction (PCR), envelope gene sequencing, and virus neutralization. A combination of thermal treatment and serial dilutions of the contaminated vaccines facilitated detection of contaminating ALVs in cell culture coupled with antigen-capture enzyme-linked immunosorbent assay. Subgroup-specific PCR readily detected ALV-A directly in the contaminated vaccines but not in naive vaccines or cell controls. Our methods are proposed as complementary procedures to the currently required complement fixation for avian leukosis test for detection of ALV in commercial poultry vaccines.     

Identification and characterization of hens transmitting avian leukosis virus (ALV) to their embryos by ELISAs for detecting infectious ALV, ALV antigens and antibodies to ALV.

A total of 72 White Leghorn grandparent hens was examined by ELISA for avian leukosis virus (ALV), ALV antigens and anti-ALV antibodies to identify and characterize the hens transmitting ALV to their embryos (transmitters) by using fertilized eggs. These hens were divided into 3 groups as no antibody and non-viremic (NANV) (49 hens), antibody-positive and non-viremic (APNV) (21 hens) and no antibody and viremic (NAV) (2 hens) by testing the sera for the presence of ALV and anti-ALV antibody. Egg albumen and embryos were tested for the presence of ALV and ALV antigens. As a result, no ALV was detected in both albumen and embryos in the NANV group. On the other hand, all albumen samples collected repeatedly from 3 hens of the APNV group and 2 hens of the NAV group contained infectious ALV, although the infectivity differed with the individual. Also, these 5 hens produced infected embryos at varying frequencies. However, on AP hen which shed neither ALV nor ALV antigens into the albumen produced an infected embryo at a lower rate. These results indicate that testing for infectious ALV in albumen from a newly laid egg per hen is effective to identify the transmitters to some extent. When virus titers in each of 8 tissue samples from the 6 transmitting hens were determined, the highest virus titers were found in washing from the ampulla of the oviducts in most of the shedders, suggesting that embryo infection is closely correlated with ALV produced at the oviduct, but not with ALV transferred from the other parts of the body.     

犬冠状病毒分子生物学研究进展

犬冠状病毒 ( CCV )基因组为一 2 8～3 2 kb大小的单股正链 RNA,基因在病毒自身RNA聚合酶的存在下、以先导引物转录机制进行转录 ,不同的病毒基因组和亚基因组翻译产生除病毒依赖 RNA的 RNA聚合酶以外 ,还产生其它病毒的结构蛋白和非结构蛋白 ,其中有相对比较保守、参与病毒核衣壳形成、介导宿主细胞免疫的核衣壳蛋白 ( N ) ;在病毒出芽、组装和成熟中起重要作用的膜蛋白 ( M) ;能刺激机体产生中和抗体 ,且易变异的纤突蛋白 ( S) ,另外 S蛋白还决定着病毒血清型、感染力 ,同时通过识别 ,并与病毒宿主细胞受体相互作用决定病毒感染的宿主范围 ;参与病毒组装的小膜蛋白 ( E)和其它病毒蛋白。 CCV还可识别猫氨肽酶 N( f APN)并与之相互作用