昆虫质型多角体病毒的研究进展

质型多角体病毒(Cytoplasmic polyhedrosis virus,CPV)隶属呼肠孤病毒科Reoviridae质型多角体病毒属Cypovirus,通常基因组由10个节段双链RNA构成。RNA分子量为3～27u。根据病毒基因组dsRNA片段在聚丙烯酰胺或琼脂糖凝胶中电泳图谱的差异,目前CPV已被分为19个电泳型。不同于呼肠孤病毒科其它成员,CPV为单层衣壳,而不是常见的双层衣壳结构,衣壳蛋白主要由衣壳蛋白、大突起蛋白及塔式突起蛋白组成。大部分质型多角体病毒引起昆虫慢性疾病,造成寄主死亡或适应性降低。随着RNA病毒基因序列测定技术的成熟,质型多角体病毒的序列测定方面取得较大进展,目前GenBank核苷酸序列数据库中已经公布了家蚕Bombyx mori CPV电泳型1两个株系(H株和I株)、舞毒蛾Lymantria dispar CPV电泳型1、舞毒蛾CPV电泳型14及粉纹夜蛾Trichoplusia ni CPV电泳型全基因组序列,为该病毒的进化与起源的研究提供更多的遗传信息。本文从结构功能、侵染特点、基因组特点及应用前景等方面综述了昆虫质型多角体病毒的研究进展。     

Effects of cytoplasmic polyhedrosis virus on diapausing Heliothis virescens

Laboratory studies were conducted to determine effects of cytoplasmic polyhedrosis virus on diapausing Heliothis virescens. Most virus-infected individuals died in the larval stage. Infected pupae yielded as many moths as healthy. Females from surviving virus-infected larvae produced fewer eggs than those from healthy larvae, but there was no statistical difference in longevity of adults between healthy and infected groups. Infected moths yielded lower than normal quantities of extracted fatty acids.     

An enzyme-linked immunosorbent assay for phytochrome

A double-antibody sandwich, enzyme-linked immunosorbent assay has been developed for phytochrome in Avena sativa L. cv. Saladin. An immunoglobulin fraction of rabbit antiserum raised to 118 kdalton phytochrome was used with alkaline phosphatase as the enzyme label. The assay detected as little as 0.2 ng phytochrome in extracts of dark-grown plant material. No evidence for specific or non-specific measurement of proteins other than phytochrome was found. The assay detected phytochrome in extracts of Avena grown in the light. Dilution curves for light-grown phytochrome extracts had a reduced slope and saturated at a lower level of enzyme activity than those for dark extracts. These differences were not caused by an inhibitor in extracts from light-grown plants. Phytochromes from dark- and light-grown plants may be immunologically different.     

单引物法扩增马尾松毛虫CPV基因组第8片段及其序列分析

本文利用T4 RNA连接酶将5'-磷酸、3'-氨基修饰的引物1连接到马尾松毛虫质型多角体病毒第8片段dsRNA的3'-OH端.经逆转录、退火、补齐形成全长双链cDNA.使用单一的互补引物2进行PCR扩增.扩增产物克隆在pMD18-T载体上.对重组子进行限制性内切酶分析及序列测定,结果表明,克隆片段全长330bp,S'端具有CPV-1型末端保守序列AGTAAA'端具有保守序列GTTAGCC.起始密码子从ATG位于38-40残基,终止密码子TAA位于1208～1210残基.推测S8片段编码390个氨基酸多肽,分子量为44kDa.与舞毒蛾质型多角体病(LdCPV)第8片段相比较,核苷酸和氨基酸同源性分别为97%和98%.与家蚕质型多角体病毒(BmCPV)第8片段相比较,核苷酸和氨基酸的同源性分别为83%和85%.与人的呼肠孤病毒第8片段比较没有明显的同源性.     

马尾松毛虫质型多角体病毒S4的cDNA克隆及序列分析

从马尾松毛虫质型多角体病毒湖南株中提取病毒核酸,回收、纯化第四片段S4,经RT-PCR扩增得到了S4的cDNA克隆并测定了其全序列。结果表明,S4全长由3262个碱基组成,包含一个编码1058个氨基酸的完整开放阅读框架。B1ast同源分析显示,DpCPV S4与LdCPVS4、BmCPVS4和RRSV S2编码蛋白氨基酸的同源性分别为94％、92％和22％。另外,氨基酸序列部分区域与Methanosarcina mazei Goel的甲基化转移酶有同源性,且序列中含有鸟苷酸转移酶活性位点。     

An enzyme-linked immunosorbent assay for detection of pyrene and related polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) can form DNA-binding compounds that show genotoxicity and carcinogenicity. Pyrene, as a PAH, was covalently linked to carrier protein bovine serum albumin and ovalbumin. A monoclonal antibody (McAb) was produced that showed high cross-reactivity values with chrysene (169.73%), benzo[a]pyrene (693.34%), benzo[a]anthracene (16.36%), and indeno[1,2,3-cd]pyrene (40.96%) and showed no significant cross-reactivity values with other homologues (<0.1%). A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of pyrene and some homologues in water samples. The detection limit of the assay was 65.08 pg ml⁻¹. The average recoveries of PAHs from tap water, lake water, and mineral water were 99.13, 99.74, and 99.19%, respectively, indicating that matrices of water samples do not interfere with the assay. The results demonstrated that the developed ELISA seems to be a potential method for monitoring of pyrene and some homologous PAHs in water samples.     

Inapparent infection ofDiatraea grandiosella byHeliothis virescens cytoplasmic polyhedrosis virus

The terminal stage of infection with cytoplasmic polyhedrosis viruses (CPVs) is formation of crystal-like inclusion bodies (polyhedra) in host insects. The degree of susceptibility of larvae to CPV, based on light microscopy and presence of polyhedra, varies with the host species.Heliothis virescens (F.) andSpodoptera exigua (Hübner) are highly susceptible to CPV. In CPV treatedDiatraea grandiosella (Dyar), polyhedra were absent in all 400 + insects examined with light and electron microscopy. However,H. virescens larvae became infected when fed haemolymph ofD. grandiosella larvae or pupae (36±10 days post treatment) developed from CPV-treated larvae. No difference in pathology was observed betweenH. virescens larvae infected with CPV polyhedra and haemolymph fromD. grandiosella. This study provides evidence thatD. grandiosella can serve as a symptomless (no occlusion bodies) carrier of a CPV which is fully expressed inH. virescens species. The observation is interesting because it reveals a potentially important aspect of the epizootiology of this insect virus.     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Incidence and influence on baculovirus activity

A small RNA virus infectious to Trichoplusia ni larvae (TRV) was observed as a contaminant of several Autographa californica nuclear polyhedrosis virus preparations (AcMNPV). The extent of contamination in various AcMNPV preparations was studied by means of serial enrichment passages through T. ni larvae and enzyme-linked immunosorbent assay (ELISA). TRV could not be detected by ELISA in the original preparation of AcMNPV polyhedra prepared in 1968 even after five enrichment passages. Antibody inactivation offers a possible prophylactic method against TRV but temperature inactivation (55°C) does not. Although TRV reduced larval weight, it had little or no effect on bioassays of AcMNPV to T. ni and Heliothis virescens.     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Gross pathology and infectivity

The pathology and infectivity of an RNA virus infectious to Trichoplusia ni larvae was investigated. The enzyme-linked immunosorbent assay (ELISA) and weight depression were used as criteria for virus concentration in larval homogenates and live larvae, respectively. Infected larvae were severely stunted, weighing as little as 13 times less than uninfected individuals of the same age, yet appeared normal morphologically. The virus was found to cause only slight mortality at high concentrations. Infected larvae displayed the pathological stunting response down to a dose of 0.1 ng of virus. Larvae infected with doses 100 times lower did not show the weight response but such inapparent infections were detectable by ELISA. Because of these subtle gross pathological symptoms, particularly at low levels of infection, infected individuals could easily remain unde-tected in a group-reared colony.     

Nucleoprotein-based indirect enzyme-linked immunosorbent assay(indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays(ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in antiEbola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G(Ig G) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.     

Comparison of bioassay and enzyme-linked immunosorbent assay for quantification of Spodoptera frugiperda nuclear polyhedrosis virus in soil

Standard curves with known amounts of Spodoptera frugiperda nuclear polyhedrosis virus (NPV) in soil were established with a bioassay and with an enzyme-linked immunosorbent assay (ELISA). The bioassay detected as few as 4 × 10⁴ polyhedral inclusion bodies (PIB)/g sandy soil and <10 PIB/g soils with large amounts of silt or clay. The ELISA detected as few as 360 PIB/g in all three soil types, and absorbance values were inversely related to the amount of clay. Results of the bioassay and ELISA were significantly (P < 0.01) correlated for natural NPV from field samples of silt (R = 0.961) and sandy soil (R = 0.723). Soil samples from Louisiana pastures and corn fields contain up to 7.6 × 10⁴ PIB/g, and 2× 10⁴ PIB/g are commonly present.     

单引物法扩增马尾松毛虫CPV基因组第8片段及其序列分析

本文利用T4 RNA连接酶将5'－磷酸,3'-氨基修饰的引物1连接到马尾松毛虫质型多角体病毒第8片段dsRNA的3'-OH端,经逆转录,退火,补齐形成全长双链cDNA。使用单一的互补引物2进行PCR 增,扩增产物克隆在pMD18-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长330bp,S'端具有CPV－1型末端保守序列AGTAAA'端具有保守序列GTTAGCC。起始密码子从ATG位于38－40残基,终止密码子TAA位于1208－1210残基。推测S8片段编码390年氨基酸多肽,分子量为44kDa。与舞毒蛾质多角体病（LdCPV)第8片段相比较,核苷酸和氨基酸同源性分别为97％和98％。与家蚕质型多角体病毒（BmCPV）第8片段相比较,核苷酸和氨基酸的同源性分别为83％和85％。与人的呼肠孤病毒第8片段比较没有明显的同源性。     

松毛虫CPV不同分离株的基因组电泳图谱

综述了松毛虫CPV不同分离株基因组电泳图谱的研究状况。松毛虫CPV是松毛虫Dedrolimusspp .的重要病原微生物 ,在松毛虫灾害治理中起着重要作用。病毒基因组电泳图谱是CPV重要的分类依据和研究基础。到目前为止 ,全世界范围共分离报道了 1 0株不同的松毛虫CPV ,基因组电泳图谱研究表明CPV -1型是松毛虫CPV的主要类型。PAGE分析显示 ,松毛虫CPV不同分离株中至少存在有 3种不同CPV -1的型内变异 ,它们有时以纯一型或混合型的形式出现。特别需要指出的是中国马尾松毛虫CPV分离株 ,其存在有 2种不同形态的病毒多角体 ,通过蔗糖密度梯度离心可以分为上下 2条带 ,上带多角体为锥形 ,基因组dsRNA电泳图谱显示 1 3条带 ,不属于目前已确定的 1 4种电泳型中的任何一种 ;下带多角体为六边形 ,基因组为纯合的CPV -1型 ;另外在不同时期不同地方感染马尾松毛虫也分离得到了纯合型的马尾松毛虫CPV -型     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum

An enzyme-linked immunosorbent assay (ELISA) was developed and evaluated to detect equine antisperm antibodies (ASA) in horse serum. Six maiden mares between 12 and 18 mo of age were immunized with stallion sperm cells (SC group, N=2), seminal plasma (SP group, N=2), or phosphate-buffered saline (PBS) as a control (C group, N=2). Horses received a second injection of the same antigen 2 wk after the first. Blood was collected weekly for 10 wk after initial immunization and again at Week 15. Serum ASA levels (IgG and IgA) were measured by ELISA using two assay systems, one containing stallion SC as the plate antigen and another containing SP.

In horses immunized with SC, peak IgG levels were detected by ELISA during Wk 2 and 3 after first injection using either plate antigen. The antibody levels persisted through Week 5 and then slowly declined until Week 15. Horses immunized with SP had IgG levels that did not differ from control horses using either ELISA plate antigen. The only significant elevation in serum IgA ASA occured during Week 5 after initial immunization and only in mares immunized with SC as detected by ELISA using SC as the plate antigen. Attachment of ASA to stallion spermatozoa was confirmed by an indirect immunofluorescence assay.     

The infectivity of a nuclear polyhedrosis virus of the velvetbean caterpillar for eight noctuid hosts

Eight species of noctuid larvae were tested for susceptibility to a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis. Velvetbean caterpillar larvae were highly susceptible to crude preparations of polyhedral inclusion bodies (PIBs; LD₅₀ = 4.7 PIBs/larva), but preparations of purified polyhedra were much less effective against these larvae (LD₅₀ = 319.7 PIBs/larva). Of seven other noctuid species tested, only Heliothis virescens was as susceptible to the virus as A. gemmatalis. High dosages were required to kill Heliothis zea, Trichoplusia ni, Pseudoplusia includens, and Spodoptera ornithogalli. Plathypena scabra and Spodoptera frugiperda were not susceptible.     

Bionanocapsule-based enzyme-antibody conjugates for enzyme-linked immunosorbent assay

Macromolecules that can assemble a large number of enzyme and antibody molecules have been used frequently for improvement of sensitivities in enzyme-linked immunosorbent assays (ELISAs). We generated bionanocapsules (BNCs) of approximately 30 nm displaying immunoglobulin G (IgG) Fc-binding ZZ domains derived from Staphylococcus aureus protein A (designated as ZZ-BNC). In the conventional ELISA using primary antibody and horseradish peroxidase-labeled secondary antibody for detecting antigen on the solid phase, ZZ-BNCs in the aqueous phase gave an approximately 10-fold higher signal. In Western blot analysis, the mixture of ZZ-BNCs with secondary antibody gave an approximately 50-fold higher signal than that without ZZ-BNCs. These results suggest that a large number of secondary antibody molecules are immobilized on the surface of ZZ-BNCs and attached to antigen, leading to the significant enhancement of sensitivity. In combination with the avidin-biotin complex system, biotinylated ZZ-BNCs showed more significant signal enhancement in ELISA and Western blot analysis. Thus, ZZ-BNC is expected to increase the performance of various conventional immunoassays.