Electron microscope observations of Autographa californica (Noctuidae) nuclear polyhedrosis virus replication in the midgut of the saltmarsh caterpillar, Estigmene acrea (Arctiidae)

The nuclear polyhedrosis virus from Autographa californica was studied with the electron microscope in the midgut of the salt marsh caterpillar, Estigmene acrea. The results of the present study were compared with a previous study in which the same inoculum was fed to Spodoptera exigua. In Estigmene acrea polyhedra were produced, but virions were not occluded. Nonoccluded virions were found throughout the midgut cytoplasm and budding into the hemocoel. Within the cytoplasm, the rough endoplasmic reticulum was observed to contain paracrystalline proteinaceous bodies. Fibrous bodies and annulate lamellae were also found in the cytoplasm of infected cells.     

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.     

The replication of a cytoplasmic polyhedrosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) in Spodoptera frugiperda cells

A cytoplasmic polyhedrosis virus (CPV) from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) replicated in Spodoptera frugiperda cells. Low rates of infection were achieved, even at high multiplicities of infection and TCID₅₀ assays showed that there was negligible release of virus particles from infected cells. In an infected focus assay, based on formation of PIB, the dose-response data demonstrated that a single particle could initiate infection. No loss of infectivity occurred in virus preparations stored at 4°, ?20°, or ?90°C, but infectivity of virus stored at 20°C declined sharply. A small isometric virus contaminant was present in some CPV preparations and its interaction with the CPV is discussed. Limited CPV infection was achieved in Trichoplusia ni cells, but attempts to infect Aedes aegypti cells were unsuccessful.     

Oxygen uptake in tobacco budworm larvae (Heliothis virescens) infected with cytoplasmic polyhedrosis virus

We measured oxygen uptake in 3- to 13-day-old Heliothis virescens (tobacco budworm) larvae. The “test” group was infected with cytoplasmic polyhedrosis virus (CPV) while the “control” group was not. The healthy budworms had an average oxygen uptake of 7.64 μl of O₂/mg body weight, while those infected with CPV had an average uptake of 47.11 μl of O₂/mg body weight/hr. The weights of larvae from the two groups were likewise recorded. Budworms from the “control” group showed an average weight of 186.9 mg, while larvae infected with CPV had an average weight of 18.3 mg.     

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。     

Virus particle packaging in baculovirus and cytoplasmic polyhedrosis virus inclusion bodies

The structure of the inclusion bodies (IBs) of three multiply enveloped nuclear polyhedrosis viruses (MNPVs), one singly enveloped NPV (SNPV), two granulosis viruses (GVs) and one cytoplasmic polyhedrosis virus (CPV) were compared. A method was devised to calculate the numbers of virus particles and nucleocapsids in IBs using data from light microscopy and thin sections. The three MNPVs, from Agrotis segetum (English and Polish virus isolates) and Mamestra brassicae had similar concentrations of virus particles ranging from 17.3 to 19.6 per μm³ of IB. Plusia gamma SNPV had a higher density of 59.6 virus particles per μm³ of IB, which partly compensated for its having smaller IBs (mean volume 0.65 μm³) than the MNPVs (2.60–9.71 μm³). The English A. segetum MNPV isolate had the most nucleocapsids in each virus particle (mean, 4.04) and the largest IBs (mean volume, 9.71 μm³), giving 674 nucleocapsids per IB on average. The GVs, from A. segetum and Pieris brassicae, mainly contained one nucleocapsid per IB. P. gamma CPV IBs had a much higher density of virus particles than the baculoviruses (260 per μm³ compared with 17–60 per μm³). These data are discussed in relation to the biological properties of these viruses, and possible adaptational advantages of alternative IB designs are considered.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

A comparative study of three closely related cytoplasmic polyhedrosis viruses

A cytoplasmic polyhedrosis virus (CPV) from the pine caterpillar, Dendrolimus spectabilis, was compared with Japanese isolates of closely related viruses from the silkworm, Bombyx mori, and gypsy moth, Lymantria dispar. The sizes of the viral RNA genome segments were almost identical, although the CPVs from D. spectabilis and L. dispar could be distinguished from the silkworm virus by a small size difference (0.03 × 10⁶ daltons) in one segment. The same viruses were also distinguishable by RNA homology differences of 25–50% measured by the reannealing of ³H-labeled single-stranded viral messenger RNA (synthesized in vitro) to heat-denatured viral double-stranded RNA. Antigenic differences were also detected by gel immunodiffusion tests. CPVs of D. spectabilis and L. dispar were indistinguishable by these criteria.     

An enzyme-linked immunosorbent assay for the detection of cytoplasmic polyhedrosis virus

An enzyme-linked immunosorbent assay (ELISA) for the detection of Euxoa scandens cytoplasmic polyhedrosis virus (EsCPV) is described. Antisera to EsCPV, produced in rabbits and guinea pigs, are specific to EsCPVs when used in an indirect assay. This indirect assay approach permits the detection of homologous antigens at a concentration of about 1 μg/ml; however, this procedure is not suitable to test large numbers of unpurified specimens. For this type of analysis we used a double antibody sandwich assay which can detect 10 ng/ml of homologous antigen in unpurified material without nonspecific reactions. This assay is used to diagnose EsCPV infections in field and laboratory studies.     

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。     

A new cytoplasmic polyhedrosis virus from the salt-marsh mosquito, Aedes cantator (Diptera: Culicidae)

The ultrastructure, development, and histopathology of a new cytoplasmic polyhedrosis virus of Aedes cantator are described. Virus particles measure 70 nm in diameter, are icosahedral in shape, and consist of a central electron-dense core surrounded by a capsid with six projections. Occlusion bodies are irregular in size (0.5–3.0 μm) and shape and contain several virus particles. Virus particles are assembled within an interconnecting network of fine filaments and are occluded by the deposition of a proteinaceous crystal around groups of mature virus particles within a virogenic stroma. Infections are confined to cells of the cardia, gastric ceca, and posterior portion of the midgut, which hypertrophy and frequently lyse. Infected larvae die during the fourth larval instar or as pupae. The prevalence of infection in natural field populations is less than 1%.     

Resistance to a nuclear polyhedrosis virus in the light-brown apple moth Epiphyas postvittana (Lepidoptera: Tortricidae)

Measurements were made of the relative susceptibility to a nuclear polyhedrosis virus of three populations of light-brown apple moth Epiphyas postvittana: a resistant laboratory strain (CAN), a susceptible laboratory strain (BAR), and a field population. CAN was found to be 50 times more resistant than BAR and 160 times more resistant than the field line. Experiments on hybrid crosses of resistant and susceptible strains showed that resistance is genetically determined. This serves as a warning of the possible selection of virus-resistant strains of insect pests, where viral insecticides are being used in the field.     

自同安钮夜蛾幼虫分离的一种核型多角体病毒

对在林间导致同安钮夜蛾Anua indiscriminate Moore幼虫死亡的一种病原物进行了分离和鉴定,并对该病原物进行了室内毒力测定。结果表明：该病原物为核型多角体病毒, 定名为同安钮夜蛾核型多角体病毒(AiNPV)。其对同安钮夜蛾幼虫的致死浓度LC₅₀和LC₉₀分别为3.4×10⁴和3.8×10⁷ PIB/mL。     

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。     

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。     

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     

Comparison of the time-mortality response of Heliothis zea to 14 isolates of Heliothis nuclear polyhedrosis virus

Twelve singly embedded isolates (SEV) and two multiply embedded isolates (MEV) of nuclear polyhedrosis viruses from Heliothis larvae were compared by time-mortality assays in neonate H. zea larvae. The isolates could be separated into six groups based on differences in the 50% survival time (ST₅₀) values. Isolates with identical restriction endonuclease (REN) profiles did not differ significantly in their ST₅₀ values, whereas isolates with several different REN cleavage sites also had significantly different ST₅₀ values. With the exception of one isolate from India, the singly embedded isolates acted faster than the multiply embedded isolates.