斜纹夜蛾核多角体病毒DNA XbaI 4.0 kb片段锌指蛋白基因及重复序列分析

测定了斜纹夜蛾核多角体病毒 (Spodopteralituranucleopolyhedrovirus,SpltNPV)中山大学分离株基因组DNAXbaI4.0kb片段全序列 ,该片段包括一个锌指蛋白基因及三个区域的DNA重复序列 (SR1、SR2、SR3)。该锌指蛋白基因读码框为 2 196个核苷酸 ,编码 731个氨基酸的蛋白质 ,分子量为 83 .0 9kD ,该蛋白的等电点为 4.6 1。在其 5′非编码区内有一个杆状病毒早期启动子基序GAGT及一个TATA盒 ,在其终止密码的下游有 5个真核生物转录mRNA时poly(A)加尾信号AATAAA。在 2 2 3～ 2 41氨基酸残基之间有一个锌指蛋白基序 ,这一基序属于锌指蛋白基序中的C3HC4类 ,即环指 (Ringfinger)类基序。在 32 3～ 340氨基酸残基区域为一个核定位信号。该蛋白可能为一个高度折叠的蛋白质。在该片段中存在三个DNA重复序列区域 (SR1、SR2、SR3) ,其中SR1与SR3区域存在更大量的重复序列 ,SR1区域其中的一个重复序列长达 41bp ,SR1、SR3重复序列区域可能作为该病毒转录的增强子 ,或者作为DNA复制的起始点。     

Foraging in a pathogen reservoir can lead to local host population extinction: a case study of a Lepidoptera-virus interaction

In 1990, natural infestations of the polyphagous vapourer moth, Orgyia antiqua (Lepidoptera: Lymantriidae) in lodgepole pine plantations in northern Scotland, were studied to ascertain the role of host foraging behaviour on the prevalence of nucleopolyhedrovirus (NPV; Baculoviridae) infection in the population. Aerial dispersal of early instar larvae (L1–L3) from the tree canopy onto heather foliage at the forest understorey, with subsequent relocation back onto the tree as late-instar larvae (L4–L6) appeared to play a significant role in the development of a widespread virus epizootic in which approximately 80% of L4–L6 individuals succumbed to disease. Bioassays of foliage 1 year later showed that the distribution of NPV followed a pronounced vertical gradient through the forest canopy culminating in high concentrations of virus in the forest understorey. Experimental systems comprising potted pine trees positioned above heather bases showed that NPV infections could be acquired by early stage larvae following dispersal from the tree and feeding on the undercanopy vegetation, then translocated to the tree component for secondary transmission to susceptible tree-feeding individuals. Behavioural studies indicated that the tendency for first-, second- and third-instar larvae to disperse to the understorey was probably not influenced by larval density on the tree but was strongly dependent on larval instar. In contrast, the tendency for larvae to relocate from the understorey heather to the tree was affected by both larval density and larval instar, suggesting that both these factors may significantly affect virus acquisition, translocation and transmission in the host population. In the present study, the heather understorey appeared to act as a pathogen reservoir in which virus could persist between host generations. Spatial heterogeneity in virus distribution combined with host foraging behaviour (dispersal and feeding) resulted in the pathogen playing a major role in host population dynamics over an extended time period (3 years). The reservoir theory is supported by the observation that similar dynamics were not observed in O. antiqua populations at neighbouring sites which lacked understorey food plants. Received: 8 June 1998 / Accepted: 5 October 1998     

The Comparative Susceptibility of the Diamondback Moth Plutella xylostella and Some Other Major Lepidopteran Pests of Brassica Crops to a Range of Baculoviruses

The susceptibility of larvae of the Diamondback moth (DBM), Plutella xylostella to infection by three baculoviruses was evaluated in the laboratory using a microdroplet feeding assay. The viruses tested were a granulovirus (GV), originally isolated in Taiwan from P. xylostella larvae (Px GV-Taiwan); the nucleopolyhedrovirus (NPV) from Galleria mellonella (Gm NPV), and the NPV from Autographa californica (Ac NPV). Neonate P. xylostella larvae were susceptible to infection by all three viruses. In an extensive series of bioassays carried out over a 21-month period, LD 50S for neonate DBM larvae ranged from 1.0-8.9 viral occlusion bodies (OB) for Px GV-Taiwan, and 9.5-30.2 OB for Gm NPV and Ac NPV. LT 50S for the three viruses ranged from 3.8-6.0 days at 27 C, with Gm NPV having a significantly shorter LT 50 than the other two viruses. Second and third instar larvae of P. xylostella were significantly less susceptible to infection by Px GV-Taiwan (LD 50 s ranging from 18-57 OB/larva) than were neonate larvae. Gm NPV also initiated infection in several other lepidopterous pest species that colonize brassica crops. In particular, neonate Crocidolomia binotalis larvae proved highly susceptible to Gm NPV, with mean LD 50 s ranging from 2.1 to 9.3 OB/larva and a mean LT 50 of 4.8 days at a dose of 8.08 OB. Heliothis virescens neonate larvae were also highly susceptible to Gm NPV (LD 50 , 7.1 OB), but Mamestra brassicae larvae were less so (LD 50 , 80-270 OB). The results of the bioassays suggest that Px GV-Taiwan is highly infective and could be developed as a selective microbial pesticide for DBM. While Gm NPV has a higher LD 50 in DBM larvae, its wider host range may be of considerable value in situations where DBM occurs on cruciferous crops together with a complex of other lepidopterous pests.     

Eicosanoids influence insect susceptibility to nucleopolyhedroviruses

Nine pharmaceutical inhibitors of eicosanoid biosynthesis (e.g., bromophenacyl bromide, clotrimazole, diclofenamic acid, esculetin, flufenamic acid, indomethacin, nimesulide, sulindac, tolfenamic acid) that increased the susceptibility of the gypsy moth, Lymantria dispar (L.), to the nucleopolyhedrovirus LdMNPV were tested against the beet armyworm Spodoptera exigua (Hübner), the corn earworm Helicoverpa zea (Boddie) and the fall armyworm Spodoptera frugiperda (J.E. Smith) and their respective NPVs to determine whether these compounds also alter the susceptibility of these insects. The susceptibility of the beet armyworm was increased by six inhibitors (bromophenacyl bromide, clotrimazole, diclofenic acid, esculetin, flufenamic acid, nimesulide). The susceptibility of the fall armyworm was increased by seven inhibitors, (bromophenacyl bromide, diclofenamic acid, esculetin, indomethacin, nimesulide, sulindac, tolfenamic acid), whereas the susceptibility of the corn earworm was increased by only one inhibitor (sulindac). The influence of the cyclooxygenase inhibitor, indomethacin was expressed in a concentration-related manner in beet armyworms. We infer from these findings that eicosanoids, including prostaglandins and lipoxygenase products, act in insect anti-viral defenses.     

Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms

We have developed a novel PCR-based assay for individual and simultaneous detection of three major pathogens (microsporidians, nucleopolyhedrovirus (NPV) and densovirus (DNV)) infecting the silkworm, Bombyx mori. Multiplex PCR, using three primer pairs, two of which were designed from the conserved regions of 16S small subunit ribosomal RNA gene of microsporidians, and polyhedrin gene of NPVs respectively, and a third primer pair designed from the internal sequences of B. mori DNVs (BmDNV), showed discrete and pathogen specific PCR products. The assay showed high specificity and sensitivity for the pathogenic DNA. Under optimized PCR conditions, the assay yielded a 794 bp DNA fragment from Nosema bombycis, 471 bp fragment from B. mori NPV (BmNPV) and 391 bp fragment from BmDNV. Further, this detection method was successfully applied to other silkworm species such as Antheraea mylitta and Samia cynthia ricini, in detecting same or similar pathogens infecting them. This method is a valuable supplement to the conventional microscopic diagnostic methods and can be used for the early detection of pathogens infecting silkworms. Furthermore it can assist research and extension centers for the safe supply of disease-free silkworms to farmers.     

Contribution of a parasitoid species to multiplication and transmission of a multiple nucleopolyhedrovirus in caterpillars

Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and Microplitis pallidipes are both used as biocontrol agents of the beet armyworm (Spodoptera exigua). However, it has not been determined how beet armyworms respond when these agents interact. Here, we studied the effects of M. pallidipes on virus multiplication and transmission using quantitative detection of SeMNPV. Our results indicated that parasitoids promoted virus multiplication in caterpillars (10⁵ copies per caterpillar) and that it was more advantageous when the M. pallidipes oviposited one day prior to infection with NPV. Interestingly, SeMNPV was transmitted by M. pallidipes in four ways. Transmission efficiency was higher for parasitoids whose body surfaces were contaminated with NPV, and for parasitoids ovipositing on NPV-infected caterpillars, than for those emerging from NPV-infected caterpillars, or feeding on mixtures of honey, water and NPV. Our study reveals that parasitoids do affect the proliferation and transmission of NPV in caterpillars and suggests that M. pallidipes could be used to strengthen the effectiveness of SeMNPV as a biocontrol agent.     

Current Status of Deltabaculoviruses, Cypoviruses and Chloriridoviruses Pathogenic for Mosquitoes

There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridoviruses (MIVs) (Iridoviridae: Chloriridovirus). Baculoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This review presents an overview of the current status and recent advancements in understanding the biology and molecular features of mosquito pathogenic viruses.     

Current status of <Emphasis Type="Italic">Deltabaculoviruses,Cypoviruses</Emphasis> and <Emphasis Type="Italic">Chloriridoviruses</Emphasis> pathogenic for mosquitoes

There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridoviruses (MIVs) (Iridoviridae: Chloriridovirus). Baculoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This review presents an overview of the current status and recent advancements in understanding the biology and molecular features of mosquito pathogenic viruses.      

Structural divergence among genomes of closely related baculoviruses and its implications for baculovirus evolution

Baculoviruses are members of a large, well-characterized family of dsDNA viruses that have been identified from insects of the orders Lepidoptera, Hymenoptera, and Diptera. Baculovirus genomes from different virus species generally exhibit a considerable degree of structural diversity. However, some sequenced baculovirus genomes from closely related viruses are structurally very similar and share overall nucleotide sequence identities in excess of 95%. This review focuses on the comparative analysis of partial and complete nucleotide sequences from two groups of closely related baculoviruses with broad host ranges: (a) group I multiple nucleopolyhedroviruses (MNPVs) from a cluster including Autographa californica (Ac)MNPV, Rachiplusia ou (Ro)MNPV, and Plutella xylostella (Plxy)MNPV; and (b) granuloviruses (GVs) from a cluster including Xestia c-nigrum (Xecn)GV and Helicoverpa armigera (Hear)GV. Even though the individual viruses in these clusters share high nucleotide sequence identities, a significant degree of genomic rearrangement (in the form of insertions, deletions, and homologous recombination resulting in allelic replacement) is evident from alignments of their genomes. These observations suggest an important role for recombination in the early evolution and biological characteristics of baculoviruses of these two groups.     

Effects of long-term storage on the stability of OpMNPV DNA contained in TM Biocontrol-1

Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) DNA was extracted from samples representing 10 lots of TM Biocontrol-1 stored at -10 degrees C for 5-15 years and digested with the restriction enzymes BglII, PstI, and SalI. DNA from the OpMNPV virus strain (MEM-75-STANDARD) used to produce the TM Biocontrol-1 lots was also extracted and digested. No restriction fragment length polymorphisms were observed in any of the samples and there was no evidence of DNA degradation. This indicates that long-term cold storage of TM Biocontrol-1 had no adverse effect on the quality of the OpMNPV DNA. In addition to the expected >23 kb OpMNPV DNA, extracts from lots 4a, 5b, and 6 contained 10 additional nucleic acid segments, ranging in size from 0.9 to 4.2 kb. The electrophoretic profile of these segments was characteristic of O. pseudotsugata cypovirus (OpCPV). RNase A/DNase I treatment showed that the nucleic acid contaminants were composed of RNA, suggesting that lots 4a, 5b, and 6 contained OpCPV as well as OpMNPV. Bioassay results have shown that there is a decrease in efficacy of stored TM-biocontrol-1, but this did not appear to be directly correlated with the length of time in storage.