家蚕传染性软化病病毒（桐乡株）VP1基因片段的克隆及序列分析

以首株在中国分离到的家蚕传染性软化病病毒（Bombyx mori infectious flacherie virus,BmIFV）BmIFV-CHN001基因组为模板,扩增了编码主要结构蛋白的VP1基因。克隆测序后得到VP1基因片段906 bp。该序列与已发表的日本毒株相比,核苷酸序列的相似性为99.3%,编码氨基酸的相似性为100%,证明该毒株与家蚕传染性软化病病毒日本株的同源性较高。把BmIFV-CHN001的VP1序列与同属的另外6个昆虫小RNA病毒的结构蛋白进行序列比对,构建系统发育树,对其进化关系进行了初步分析,结果显示这7种病毒具有相近的亲缘关系,而BmIFV-CHN001与蜜蜂囊雏病毒的亲缘关系最近。     

云南家蚕核型多角体病毒分离株的毒力测定及系统进化分析

【目的】由家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)侵染家蚕Bombyx mori导致的核型多角体病(血液型脓病)在云南蚕区普遍发生,危害严重,给云南蚕业带来严重的经济损失。本研究旨在测定云南不同蚕区BmNPV分离株的毒力及了解BmNPV在云南的病害流行趋势,为监测和控制云南家蚕血液型脓病打下基础。【方法】通过BmNPV对家蚕5龄幼虫致死率和对家蚕BmN细胞毒力的测定评价了云南蚕区19个BmNPV分离株的致病力;对19个云南BmNPV分离株的bro-d基因进行克隆、测序及系统进化分析。【结果】BmNPV对家蚕5龄幼虫的致死率测定表明,云南不同地区BmNPV毒株对家蚕的口服感染力差异较大;细胞毒力测定结果显示不同BmNPV分离株的出芽型病毒粒子(budded virus, BV)滴度差异较大。进化分析表明,所获取来自云南的19个BmNPV分离株主要分为两个亚群,亚群I在云南省的东部、中部和西部地区均有分布,而亚群II主要集中分布于云南省北部地区。从地理分布图可以看出,亚群I所在地区多为亚热带,温度偏高;而亚群II所处的云南北部地区多为典型高原季风气候,温度相对偏低。【结论】云南蚕区存在丰富的BmNPV株系,各分离株对家蚕的毒力差异较大;云南BmNPV分离株的进化关系在一定程度上与地理气候密切相关。     

从广西蚕区分离的家蚕质型多角体病毒生物学特征研究

【背景】家蚕中肠型脓病是一种传染性强、危害大的病毒病,其病原为家蚕质型多角体病毒（Bombyx mori cytoplasmic polyhedrosis virus,BmCPV）,该病原抗逆性强、宿主域广,防控难度大。【目的】调查广西蚕区家蚕中肠型脓病发生情况,并研究家蚕质型多角体病毒的感染性、形态特征和分子鉴定,为养蚕生产中有效防控该病提供参考依据。【方法】通过外观和解剖观察病症与显微镜检验相结合,调查养蚕生产家蚕中肠型脓病的发生率;采用生物试验方法测定多角体病毒对家蚕的半数感染浓度（IC₅₀）;利用光学显微镜和扫描电镜观察多角体病毒的外部形态,利用透射电子显微镜观察多角体病毒的内部结构;采用PCR扩增和测序进行分子鉴定。【结果】广西蚕区家蚕中肠型脓病普遍存在,金城江蚕区和东兰蚕区的家蚕中肠型脓病平均发生率分别为6.06%和13.02%,最高发生率达到30.41%;分离获得2株BmCPV病原（暂命名为BmCPV-J和BmCPV-D）,它们的半数感染浓度（IC₅₀）分别为4.88×10³ PIBs/mL和1.63×10⁴ PIBs/mL,都具有很强的致病性;2株BmCPV的形态均为六角形多角体,大小有差异,多角体直径为1.0-3.4 μm;从2株BmCPV内部结构观察到球形病毒粒子,直径为30-50 nm,有刺状突起;可以扩增出BmCPV-J和BmCPV-D的RNA复制酶基因目的片段,BmCPV-J的目的片段序列与BmCPV参考株一致,而BmCPV-D的目的片段序列与参考株有2个碱基的差异。【结论】广西蚕区家蚕中肠型脓病危害严重,该病病原感染力强、分布广,多角体病毒具有典型的质型多角体病毒特征,属于家蚕质型多角体病毒。研究结果为养蚕生产中有效防控家蚕中肠型脓病提供重要依据。     

家蚕浓核病毒 (中国镇江株)在不同感受性宿主体内的复制

家蚕浓核病毒中国镇江株是一株双生浓核病毒(bidensovirus)。其宿主感染后的病症与典型的家蚕浓核病毒(BmDNV-1伊那株)表现相似,病蚕软化,中肠的圆筒型细胞呈浓核症。该病毒的最大特点是基因组中含有二套DNA分子(VD1,VD2),这两种核酸分子以单链( VD1,-VD1, VD2,-VD2)线型方式被分开包装在各自的衣壳蛋白中,成为四种病毒体,而且它自身编码DNA聚合酶。有部分蚕品种对该病毒表现完全抗性,即不发病。分别对敏感性家蚕品种(华八35)和抗性家蚕品种(秋丰d)的幼虫进行经口接种病毒。在接种后,从2h到96h分9个时间点,对中肠组织进行取样。以家蚕细胞质肌动蛋白A3(actinA3)基因作为参比基因,用来标定取样组织细胞数。针对VD1和VD2分别设计特异引物,用荧光定量PCR的方法分别检测各个时间点的样品中的病毒基因组VD1和VD2拷贝数。结果表明:无论是在感性还是在抗性宿主体内,家蚕浓核病毒中国株的基因组VD1和VD2在各时间点拷贝数相近,表现出VD1和VD2是同步复制的;病毒侵入两种宿主中肠的初始量(接种后2h)基本相等,每个细胞约为6~10拷贝数。在敏感性宿主体内病毒感染过程表现为潜伏期,指数增长期,平台期。从接种后2h到12h为病毒潜伏期;12h到36h为指数增长期,倍增时间为1·71h,大约扩增15次;36h到96h为平台期,进入平台期病毒的拷贝数达到20万个。在抗性宿主体内病毒处于一种极低水平的增殖,从添毒后2h的6~10拷贝数到96h的150~200拷贝数,病毒复制倍增时间分别为3h和12h,大约扩增5次。推测家蚕对浓核病毒中国株的抗病性,只是一种慢性的带毒不发病的表现。     

日本血吸虫28kDGST基因在家蚕细胞和幼虫中的表达

应用修饰的家蚕核型多角体病毒为载体在家蚕细胞和幼虫中表达日本血吸虫中国大陆株２８ＫＤ胱甘肽Ｓ－转移酶基因。Ｓｏｕｔｈｅｒｎ杂交证实该基因被插入到病毒基因组中的正确位置。在家蚕培养细胞中的产量为０．７７ｍｇ,在家蚕幼虫中则超过５ｍｇ／条蚕。     

家蚕微孢子虫海藻糖酶3的表达、定位及功能

【目的】本研究旨在初步明确家蚕微孢子虫Nosema bombycis海藻糖酶3(NbTre3)的功能,为家蚕Bombyx mori微粒子病的防治提供理论依据和线索。【方法】通过PCR扩增NbTre3,构建原核表达载体pET28a-NbTre3;经IPTG诱导在大肠杆菌Escherichia coli中表达重组蛋白NbTre3,Western blot检测目的蛋白;Ni柱亲和层析法对重组蛋白NbTre3进行纯化,用获得的NbTre3免疫新西兰兔制备多克隆抗体;利用间接免疫荧光技术对成熟家蚕微孢子虫中的NbTre3进行定位;qRT-PCR检测家蚕微孢子虫感染家蚕5龄起蚕后不同时间中肠中NbTre3的转录水平;通过分别注射siRNA-1, siRNA-2和siRNA-3进行RNAi,qRT-PCR检测RNAi后不同时间感染家蚕微孢子虫的家蚕5龄起蚕中肠中NbTre3和16S rRNA的转录水平。【结果】成功纯化并获得重组目的蛋白NbTre3,大小约为34 kD。免疫新西兰兔后,收集血清,纯化获得NbTre3多克隆抗体,经Western blot鉴定正确。间接免疫荧光结果显示NbTre3主要分布在成熟家蚕微孢子虫孢原质中。qRT-PCR结果表明,家蚕微孢子虫感染后6 h时家蚕5龄起蚕中肠中NbTre3的表达量最高;siRNA抑制NbTre3的表达后,家蚕微孢子虫16S rRNA的转录水平没有明显的变化。【结论】结果提示NbTre3可能在家蚕微孢子虫感染初期的发芽过程中发挥重要的作用。     

传染性法氏囊病病毒多聚蛋白基因在家蚕中的表达

将传染性法氏囊病病毒(IBDV)细胞致弱株(JD1株)的基因组A节段基因重组于家蚕杆状病毒转移载体pAcHLT-C中,获得的重组转移载体pAcHLT-C-A与线性化病毒Bm-BacPAK6 DNA共转染家蚕培养细胞,获得重组病毒BacPAK-A。DIG标记的DNA点杂交证实重组病毒基因组中含有A节段基因,重组病毒感染家蚕5龄幼虫进行表达, ELISA和Western blotting等结果表明多聚蛋白基因在蚕体内得到了表达,表达产物具有免疫反应性,表达量在感染后5～6 d达到最高。家蚕生物反应器表达IBDV多聚蛋白具有我国的资源优势,为今后研制低成本、实用化的IBDV基因工程疫苗打下基础。     

一种家蚕类浓核病毒的组织病理学特征

本文从家蚕病蚕中分离到一种家蚕类浓核病毒（BmDNV-Like）,对它的组织病理学研究表明:该病毒首先寄生家蚕中肠柱状细胞,继而引起其细胞核的膨大和破裂;组织原位杂交结果表明该病毒既能在家蚕中肠柱状细胞中增殖,也能在中肠的杯形细胞中增殖,甚至在感染后期能在家蚕幼虫的大部分组织细胞中感染和增殖。     

家蚕对马尾松毛虫质型多角体病毒的敏感性

用虫体克隆技术,对马尾松毛虫质型多角体病毒湖南株（DpCPV-HN）进行了分离纯化,鉴定为质型多角体病毒1型。以家蚕春蕾×镇珠杂种F₁代及自交的F₂代4或5日龄幼虫进行毒力测定,以纯化的家蚕质型多角体病毒对F₁代幼虫的毒力测定为对照。结果表明：家蚕品种春蕾×镇珠对家蚕质型多角体病毒敏感,马尾松毛虫质型多角体病毒湖南株能引起其感染发病;马尾松毛虫质型多角体病毒湖南株感染家蚕品种春蕾×镇珠F₁代幼虫和F₂代幼虫28天后的半致死剂量（LD₅₀）分别为885个和18个CPB(质多角体),前者为后者的49倍。马尾松毛虫质型多角体病毒湖南株感染后的家蚕,其结茧率、化蛹率、羽化率、全茧量、茧层量和单蛾产卵数均有所下降,全茧量、茧层量、茧层率和单蛾产卵数与病毒感染剂量之间无显著关联。     

家蚕幼虫不同龄期体内主要解毒酶及其基因表达的性别差异

为了研究家蚕Bombyx mori性别与抗性的关系,本研究采用Thermo酶活性测定法和实时荧光定量PCR法,比较不同龄期雌雄家蚕起蚕体内解毒酶活性及其基因的表达差异。结果表明：解毒酶及其基因在雌雄个体之间均存在明显差异,谷胱甘肽S-转移酶活性及其基因BmGSTe5的表达量在1～3龄表现为雌蚕高于雄蚕,4龄和5龄则表现相反,其中4龄雄蚕酶活性和基因表达量分别是雌蚕的3.65倍和5.11倍,推测与雄蚕精巢在4龄初迅速发育有关。雄蚕乙酰胆碱酯酶活性在2～4龄分别是雌蚕的1.48,1.34和1.40倍;乙酰胆碱酯酶基因Bm-ace1在2龄和3龄性别差异不明显,Bm-ace2在2～4龄为雄性高于雌性,雄性分别是雌性的1.75,2.17和2.40倍,提示Bm-ace2对性别间该酶活性的差异影响较大。羧酸酯酶活性在2龄和3龄雄蚕较高,分别是雌蚕的1.23和1.87倍,4～5龄雌蚕较高,分别是雄蚕的1.23和1.22倍;羧酸酯酶基因BmCarE-5和BmCarE-10在性别间的差异规律均与酶活性相反,即羧酸酯酶表现为较高的基因表达量对应较低的酶活性,推测在家蚕体内可能存在“羧酸酯酶突变”现象。结果为研究基因表达产物的修饰方式及其功能开辟了新的研究路径,为家蚕性别与抗药性的研究提供了重要参考。     

The three-dimensional structure of Infectious flacherie virus capsid determined by cryo-electron microscopy

Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 Å. The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 Å and a single shell thickness of 15 Å. The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The “Rossmann canyon” is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed.     

Thermal therapy of the flacherie virus disease in the silkworm,Bombyx mori

An effective method of thermal therapy to fifth-instar silkworm larva (Bombyx mori) has been developed for the control of the flacherie virus disease. Fifth-instar larvae, which were infected with the flacherie virus in their fourth instar, were reared at 27°C for 5 days and then transferred to 37°C for 1–3 days. Such larvae were able to form normal cocoons. The basis for the thermal therapy appeared to be: (1) the discharge of the virus-infected goblet cells into the midgut lumen and out with the feces and (2) the escape of the newly regenerated goblet cells from infection and virus multiplication.     

Characteristics of structural proteins of infectious flacherie virus from the silkworm, Bombyx mori

Structural proteins and the characteristics of infectious flacherie virus (IFV) purified from the silkworm, Bombyx mori, are described. The purified IFV had four major structural proteins, which were detected only in high concentration gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a few minor ones. Molecular weights of the major proteins were 35,200 (VP 1), 33,000 (VP 2), 31,200 (VP 3), and 11,600 (VP 4), and numbers per virion were 62, 57, 54, and 31, respectively. Amino acid compositions of VP 1, VP 2, and VP 3 were similar to each other but that of VP 4 was somewhat different. By isoelectric focusing and two-dimensional electrophoresis, high resolution of the structural proteins was obtained with silver staining. The isoelectric points of the four major proteins were determined as 7.7(VP 1), 6.7(VP 2), 4.8(VP 3), and 5.5(VP 4). This work is the first report on insect picornaviruses that presents some discriminative properties of each viral protein that was compared to those of mammalian picornaviruses.     

Detection of an internal translation activity in the 5′ region of Bombyx mori infectious flacherie virus

The 5′ untranslated region plays an important role in positive-sense single-stranded RNA virus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation and is applied to simultaneously express several proteins. Infectious flacherie virus (IFV) is a positive-sense single-stranded RNA virus; however, the IRES function is still not proved. To investigate whether the sequences of IFV contain IRES activity, a series of bicistronic reporter (DsRed and enhanced green fluorescent protein) recombinant baculoviruses were constructed to infect the insect cells and silkworm using the Bombyx mori baculovirus expression system. Results showed that the upstream 311, 323, 383, 551, and 599 nt have IRES activity except for the 155-nt region in BmN cells. More importantly, the tetraloop structure containing region between 551 and 599 nt appeared to be responsible for the enhanced IRES activity in different insect cell lines and silkworm. These results indicated that the IRES activity is not species specific and tissue specific. Therefore, our findings may provide the basis for the simultaneous expression of two or various different genes under the same promoter in baculovirus expression system.     

昆虫小RNA病毒研究进展

昆虫小RNA病毒依据基因组结构特点分为家蚕软化病毒组、蟋蟀痹病毒组和豌豆蚜病毒组。昆虫小RNA病毒蛋白质翻译具有独特性：不依赖于帽子结构,不需要启始因子eIF1、eIF1A和帽子结合蛋白eIF4E;而且蟋蟀痹病毒组和豌豆蚜病毒组结构蛋白翻译从内部核糖体进入位点独立启始,启始密码子是CUU。该文还简介了昆虫小RNA病毒复制机理及与其它动、植物小RNA病毒或类小RNA病毒的亲缘关系等的研究进展。     

Properties of flacherie virus of the silkworm, Bombyx mori

The flacherie virus of the silkworm, Bombyx mori, was isolated from infected larvae reared under aseptic conditions. Two types of infectious particles, tentatively designated FVS I and FVS II, were separated by density gradient centrifugation. Some properties of the separated particles were investigated. Electron micrographs showed that FVS I and FVS II were spherical particles with diameters of 27 ± 2 nm and 22 ± 2 nm, respectively. The sedimentation coefficients of FVS I and FVS II were 180 S and 134 S, respectively. It was concluded from experiments of incorporation of ³H-uracil inoculated into diseased larvae at late stage of flacherie disease that the nucleic acid of FVS II was RNA. The two types of particles were present in Sakaki and Wadayama strains of flacherie virus.     

Oral administration of milk fermented with Lactococcus lactis subsp. cremoris FC protects mice against influenza virus infection

Aims: To evaluate the protective effects of oral administration of milk fermented with a Lactococcus strain against influenza virus (IFV) infection in a mouse model. Methods and Results: Milk fermented with exopolysaccharide‐producing Lactococcus lactis subsp. cremoris (L. cremoris) FC was orally administered to BALB/c mice for 12 days. Mice were intranasally infected with IFV A/New Caledonia/20/99 (H1N1) on day 8, and survival was determined for 14 days after IFV infection. Survival rate and body weight loss after IFV infection in the L. cremoris FC fermented milk‐administered group were significantly improved compared with those in the control group. In the unfermented milk‐administered group, survival rate was not improved, whereas body weight loss was slightly improved compared with that in the control group. The mean virus titre in the lung of the L. cremoris FC fermented milk‐administered group 3 days after infection was significantly decreased compared with that in the control group. Conclusions: These results suggest that oral administration of milk fermented with L. cremoris FC protects mice against IFV infection. Significance and Impact of the Study: These results demonstrate that oral administration of milk fermented with exopolysaccharide‐producing Lactococcus strains might protect host animals against IFV infection.     

Intranasally administered Lactobacillus gasseri TMC0356 protects mice from H1N1 influenza virus infection by stimulating respiratory immune responses

We conducted a study to evaluate the possibility that intranasal administration of a new probiotic strain Lactobacillus gasseri TMC0356 (TMC0356) may protect host animals from influenza virus (IFV) infection, which was indicated by enhanced respiratory immune responses in a mouse model. After 3 days of exposure to TMC0356, BALB/c mice were intranasally infected with IFVA/PR/8/34 (H1N1). Lung cells were isolated from the tested mice and evaluated for cytotoxicity against YAC-1 cells. After intranasal treatment with TMC0356, mice showed a lower morbidity and higher survival rate compared to control mice (P < 0.05). The cytotoxicity of lung cells isolated from mice after intranasal treatment against YAC-1 cells was statistically higher than that of lung cells isolated from control mice (P < 0.05). Intranasal administration of TMC0356 significantly increased mRNA expression of interleukin (IL)-1β, tumor necrosis factor, IL-10, and monocyte chemotactic protein-1 (P < 0.01). These results suggest that intranasal administration of TMC0356 may protect the host animal from IFV infection. They also indicate that TMC0356 can enhance respiratory cell-mediated immune responses of host animals characteristically with up-regulated activation of lung natural killer cells. Further studies will evaluate the possible role of the immune stimulatory effects of TMC0356 within the protective effects of this bacterium against IFV, as observed in the present study.