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     

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。     

马尾松毛虫CPV基因组第5片段的cDNA克隆及其序列分析

通过差速离心从感染的马尾松毛虫幼虫虫体中提取质型多角体病毒.碱解法提纯病毒粒子,1%琼脂糖凝胶分离基因组dsRNA,回收纯化第五片段(S5).根据同源性设计五对引物,经RT-PCR,最终获得五个亚克隆片断,测序拼接后,得到S5全长.片断全长2851个核苷酸,包括一个2643个核苷酸的开放阅读框.推测DpCPV S5基因编码了881个氨基酸长的多肽,分子量为100.3kDa,与舞毒蛾质型多角体病毒(LdCPV-1)和家蚕质型多角体病毒(BmCPV)比较,核苷酸和氨基酸的同源性都很高.进一步分析,利用几种CPV序列绘制了系统进化树,对病毒的分类和进化做了探讨研究.     

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。     

烟青虫质型多角体病毒的形态及某些理化特性

本文对烟青虫(Heliothis assulta)质型多角体病毒(CPV)的形态大小以及某些理化特性进行了研究。烟青虫CPV多角体为正五角形的十二面体,大小为0.8-4.6μm;CPV 粒子为外形呈六角形或球形的廿面体,大小为62nm。CPV多角体蛋白的主要多肽为一种,分子量23000,为非糖蛋白。 用SDS-酚法提取的CPV基因,经Rnase I和Dnase I处理后在1%琼脂糖凝胶上电泳,结果表明 其基因是双链RNA,并由10个基因片段组成。各片段大小为0.3-2.68X10⁶,总分子量为15.85x10⁶。本文所报道的烟青虫质型多角体病毒在国内外尚属首次。     

不同世代及中肠病变类型的松毛虫单头幼虫多角体含量

不同世代及中肠病变类型的松毛虫单头幼虫多角体含量许再福（浙江农业大学植保系,杭州３１００２９）戴冠群（华南农业大学植保系,广州５１０６４２）关键词马尾松毛虫,质型多角体病毒,多角体含量,世代,病变中肠源于日本赤松毛虫质型多角体病毒（Ｄｅｎｄｒｏｌｉｍ...     

山东省几种林果害虫病毒记述

近几年来,山东省一些林场的赤松毛虫自然发生流行性病毒病,当地进行人工喷洒病毒防治松毛虫大面积试验获得良好效果。继后我们又发现山东省其它一些林果业害虫也有类似的自然死亡现象,现将采集到的几种昆虫病毒进行研究鉴定,简报如下。 1.赤松毛虫(Dendrolimus spectabilis Butler)细胞质型多角体病毒(CPV)及颗粒体病毒(GV) 1973年以来,山东省沂南县东风林场的赤松毛虫经常发生流行病而大量死亡;经调查此病分为萎     

马尾松毛虫CPV基因组第5片段的cDNA克隆及其序列分析

通过差速离心从感染的马尾松毛虫幼虫虫体中提取质型多角体病毒。碱解法提纯病毒粒子,1％琼脂糖凝胶分离基因组dsRNA,回收纯化第五片段(S5)。根据同源性设计五对引物,经RT-PCR,最终获得五个亚克隆片断,测序拼接后,得到S5全长。片断全长2851个核苷酸,包括一个2643个核苷酸的开放阅读框。推测DpCPVS5基因编码了881个氨基酸长的多肽,分子量为100．3kDa,与舞毒蛾质型多角体病毒(LACPV—1)和家蚕质型多角体病毒(BmCPV)比较,核苷酸和氨基酸的同源性都很高。进一步分析,利用几种CPV序列绘制了系统进化树,对病毒的分类和进化做了探讨研究。     

文山松毛虫质型多角体病毒S8片段cDNA克隆与原核表达

文山松毛虫质型多角体病毒(DpwCPV)S8片段被克隆和测序,该片段全长1332bp,编码390个氨基酸组成的分子量大约为43kDa的蛋白P44.根据本实验室测定出的马尾松毛虫质型多角体病毒(DpCPV)基因组全序列,设计引物,扩增出文山松毛虫质型多角体病毒S8部分片段,并亚克隆出p44基因序列,然后将p44基因序列cDNA克隆到表达载体pET-28a中,构建成表达质粒pET-S8,用IPTG诱导大肠杆菌BL21,经SDS-PAGE证明p44基因在大肠杆菌中获得成功表达,并对其编码蛋白序列进行了分析.     

赤松毛虫(Dendrolimus spectabilis)质型多角体病毒经卵传递的研究

昆虫病毒的经卵传递,对于昆虫种群中病毒病的自然发生和传播有着重要意义,有关这方面的工作,前人已有一些报道,作者在利用质型多角体病毒(CPV)防治赤松毛虫工作中,对赤松毛虫CPV经卵传递的现象进行了观察,报道如后。 材料和方法 CPV系中国林科院林科所提供。将CPV悬液浸湿松枝,室内晾干后饲喂6—7龄幼虫,室外选择虫口密度较大的林地,在幼虫6—7龄时用CPV悬液喷雾防治。分别收取室内和防治区松树上的虫蛹,单个置于灭菌玻璃容器中,成虫羽化后,检查蛹便有无多角体。解剖部分成虫观察肠道病变,检查中肠有无多角体,另部分成虫置养虫笼中产卵,幼虫孵化后,单个饲养于灭菌容器中,逐日检查记载子代病死虫数。用扫描电镜检查雌蛾外生殖器和卵的表面有无多角体。     

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.     

Persistence of insect viruses in field populations of alfalfa insects

The persistence of viruses of five insects was observed in alfalfa fields. The insects were Autographa californica, Colias eurytheme, Pseudaletia unipuncta, Spodoptera exigua, and Trichoplusia ni. The isolated viruses were the granulosis (GV), the cytoplasmic-polyhedrosis (CPV), and the nuclear-polyhedrosis (NPV) viruses. The viruses persisted in the soil, on the alfalfa foliage, and in alternate hosts. In the soil, the viruses persisted even during the winter months when no foliage remained on the plants. Alfalfa sprouts harboring virus-infected larvae of C. eurytheme and S. exigua produced virus infections in larvae of these insects, but those with larvae of A. californica and P. unipuncta did not cause virus infection. The GVs and CPVs isolated from these insects were transmitted to nearly all of the other four species, but the NPVs appeared to be host specific.     

Host-Specific Parvovirus Evolution in Nature Is Recapitulated by In Vitro Adaptation to Different Carnivore Species

Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that >95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range.     

The study of orthopoxvirus genes for Kelch-like proteins. II. Construction of cowpox virus variants with targeted gene deletions

Integrative plasmids p delta C, p delta D, and p delta G were designed to contain a selective marker beyond the region of homology to virus DNA and to allow construction of recombinant cowpox viruses (CPV) that lack C18L, D11L, or G3L coding for kelch-like proteins. CPV mutants lacking one (C18L, D11L, or G3L), two (D11L/G3L or C18L/D11L), or three (D11L/G3L/C18L, that is, all) kelch-like protein genes of the left variable region of the virus genome were obtained. Impaired reproduction was observed for the triple mutant. Pocks produced by the triple mutant and the original virus differed in size and morphology. In addition, the two CPV variants differed in destructive changes caused in the chorioallantoic membrane of chicken embryos.     

Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus.

Analysis of canine parvovirus (CPV) isolates with a panel of monoclonal antibodies showed that after 1986, most viruses isolated from dogs in many parts of the United States differed antigenically from the viruses isolated prior to that date. The new antigenic type (designated CPV type 2b) has largely replaced the previous antigenic type (CPV type 2a) among virus isolates from the United States. This represents the second occurrence of a new antigenic type of this DNA virus since its emergence in 1978, as the original CPV type (CPV type 2) had previously been replaced between 1979 and 1981 by the CPV type 2a strain. DNA sequence comparisons showed that CPV types 2b and 2a differed by as few as two nonsynonymous (amino acid-changing) nucleotide substitutions in the VP-1 and VP-2 capsid protein genes. One mutation, resulting in an Asn-Asp difference at residue 426 in the VP-2 sequence, was shown by comparison with a neutralization-escape mutant selected with a non-CPV type 2b-reactive monoclonal antibody to determine the antigenic change. The mutation selected by that monoclonal antibody, a His-Tyr difference in VP-2 amino acid 222, was immediately adjacent to residue 426 in the three-dimensional structure of the CPV capsid. The CPV type 2b isolates are phylogenetically closely related to the CPV type 2a isolates and are probably derived from a common ancestor. Phylogenetic analysis showed a progressive evolution away from the original CPV type. This pattern of viral evolution appears most similar to that seen in some influenza A viruses.     

Umatilla virus genome sequencing and phylogenetic analysis: identification of stretch lagoon orbivirus as a new member of the Umatilla virus species

The genus Orbivirus, family Reoviridae, includes 22 species of viruses with genomes composed of ten segments of linear dsRNA that are transmitted between their vertebrate hosts by insects or ticks, or with no identified vectors. Full-genome sequence data are available for representative isolates of the insect borne mammalian orbiviruses (including bluetongue virus), as well as a tick borne avian orbivirus (Great Island virus). However, no sequence data are as yet available for the mosquito borne avian orbiviruses.We report full-length, whole-genome sequence data for Umatilla virus (UMAV), a mosquito borne avian orbivirus from the USA, which belongs to the species Umatilla virus. Comparisons of conserved genome segments 1, 2 and 8 (Seg-1, Seg-2 and Seg-8) - encoding the polymerase-VP1, sub-core 'T2' protein and core-surface 'T13' protein, respectively, show that UMAV groups with the mosquito transmitted mammalian orbiviruses. The highest levels of sequence identity were detected between UMAV and Stretch Lagoon orbivirus (SLOV) from Australia, showing that they belong to the same virus species (with nt/aa identity of 76.04%/88.07% and 77.96%/95.36% in the polymerase and T2 genes and protein, respectively). The data presented here has assisted in identifying the SLOV as a member of the Umatilla serogroup. This sequence data reported here will also facilitate identification of new isolates, and epidemiological studies of viruses belonging to the species Umatilla virus.     

Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo.

Canine parvovirus (CPV) emerged as an apparently new virus during the mid-1970s. The origin of CPV is unknown, but a variation from feline panleukopenia virus (FPV) or another closely related parvovirus is suspected. Here we examine the in vitro and in vivo canine and feline host ranges of CPV and FPV. Examination of three canine and six feline cell lines and mitogen-stimulated canine and feline peripheral blood lymphocytes revealed that CPV replicates in both canine and feline cells, whereas FPV replicates efficiently only in feline cells. The in vivo host ranges were unexpectedly complex and distinct from the in vitro host ranges. Inoculation of dogs with FPV revealed efficient replication in the thymus and, to some degree, in the bone marrow, as shown by virus isolation, viral DNA recovery, and Southern blotting and by strand-specific in situ hybridization. FPV replication could not be demonstrated in mesenteric lymph nodes or in the small intestine, which are important target tissues in CPV infection. Although CPV replicated well in all the feline cells tested in vitro, it did not replicate in any tissue of cats after intramuscular or intravenous inoculation. These results indicate that these viruses have complex and overlapping host ranges and that distinct tissue tropisms exist in the homologous and heterologous hosts.     

用裂解气液色谱法鉴定昆虫包涵体病毒的初步研究

用Shimadzu GC-9A气相色谱仪和PyR-2A管式炉裂解器,对11株昆虫包涵体病毒进行了裂解气相色谱鉴定,通过对“指纹图”的分析,既可明显地区分GV、NPV和CPV包涵体病毒彼此间的差异,亦可较好的区别不同分离株间的异同,实验结果初步证明,用裂解气液色谱法分析、鉴定昆虫包涵体病毒是可行的。     

中国松毛虫属八个种和亚种亲缘关系的DNA指纹证据

利用DNA指纹谱方法探讨了中国松毛虫属8个种和亚种〔马尾松毛虫Dendrolimus punctatus punctatus (Walker),德昌松毛虫D. punctatus tehchangensis Tsai et Liu,文山松毛虫D. punctatus wenshanensis Tsai et Liu,思茅松毛虫D. kikuchii Matsumura,赤松毛虫D. spectabilis Butler,油松毛虫D. tabulaeformis Tsai et Liu,落叶松毛虫D. superans (Butler),云南松毛虫D. houi Lajonquiere之间的亲缘关系。13个随机引物在8种松毛虫中共检测到168个多态分子标记。分析表明,这8个种间的遗传距离的变化范围为0.3780～0.7360;马尾松毛虫与其亚种德昌松毛虫的遗传距离最近,为0.3780,与其另一亚种文山松毛虫以及油松毛虫的遗传距离次之,皆为0.5233;赤松毛虫、落叶松毛虫、云南松毛虫与马尾松毛虫的遗传距离则再次之,分别为0.6362,0.6770和0.6944;与马尾松毛虫遗传距离最远的是思茅松毛虫,为0.7360。8种松毛虫间具体的亲缘关系为: (D. superans (D. tabulaeformis (D. p. wenshanensis (D. p. tehchangensis, D. p. punctatus)(D. Kikuchii (D. spectabilis, D. houi))。