New Helicoverpa armigera Hbn cell line from larval hemocyte for baculovirus studies

A new cell line from the larval hemocytes of H. armigera was established in Grace's medium modified by adding lactalbumin hydrolysate and yeastolate (3.3g/l), and supplemented with fetal bovine serum (20%). The cell line was designated as NIV-HA-1195. The cell population at P-78 consisted mainly of epithelial-like cells (89.36%), fibroblast-like cells (8.31%) and giant cells (2.13%). The population doubling time was 96hr at P-8, 60hr at P-43. The chromosome number ranged from 45 to 200. The cell line is susceptible to the baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV), Spodoptera litura NPV and the homologous HaNPV. Isoenzyme profile and results of 16S rRNA heteroduplex analysis clearly indicated the species specificity of the new cell line.     

A new continuous cell line from larval hemocytes of Spodoptera litura (F.).

A new cell line has been established from larval hemocytes of the moth, S. litura (tobacco cut worm). It took 147 days to form a monolayer and one year for the first 17 passages. At present, the culture is at 86th passage level and is designated NIV-SU-1095. Three cell types could be distinguished, viz. plasmatocytes (53%), prohemocytes (36%) and granular hemocytes (11%). The chromosome number was very high, 74% metaphase cells showed more than 100 chromosomes. The cells could be cryopreserved. The cells were susceptible to the baculoviruses, Autographa californica nuclear polyhedrosis virus and S. litura nuclear polyhedrosis virus (SLNPV). Plaques could be observed on 7th post infection day with SLNPV. Six cloned cell lines have been developed of which clone II-1F was more sensitive to both the baculoviruses compared to the original cell line.     

A new cell line from the embryonic tissue of Helicoverpa armigera HBN. (Lepidoptera: Noctuidae)

A new cell line from the embryonic tissue of Helicoverpa armigera was established and designated as NIV-HA-197. It was maintained in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line at passage 20 had a heterogeneous population of cells consisting of mainly epithelial-like cells (70%), followed by fibroblast-like (27%), and multinucleated giant (3%) cells. The chromosome number ranged from 45 to 185. The growth curve at passage 40 showed a fivefold increase in cell number with a population-doubling time of approximately 60 h. The cell line was found infected with the microsporidium Nosema heliothids at passage 9. Using the antiprotozoan drug Metrogyl 400 and simultaneous heat treatment, the parasite was removed from the culture. The cell line can be cryopreserved for 30 mo. The species specificity of the new cell line was determined by studying the isoenzyme profile of four enzymes, viz., lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and glucose 6-phosphate dehydrogenase, and by heteroduplex analysis. Heteroduplex analysis was used to analyze the mitochondrial 16S ribosomal ribonucleic acid gene sequences along with the host insect gene sequences, and 100% homology was obtained, confirming the conspecificity of the cell line. The cell line was found to be susceptible to the baculoviruses Autographa californica multiple nucleopolyhedrovirus, Spodoptera litura multiple nucleopolyhedrovirus, and H. armigera single nucleopolyhedrovirus (HaSNPV). More than 90% of the cells were infected by HaSNPV on the seventh post infection day (PID), and 28.8 x 10(6) NPV/ml was yielded on the 10th PID. The in vitro-grown HaSNPV caused 100% mortality, when fed to the second instar H. armigera larvae, in 6 d. Cessation of feeding was observed on the second PID.     

A new Bombyx mori larval ovarian cell line highly susceptible to nucleopolyhedrovirus

Lepidopteran cell lines constitute the backbone for studying baculoviral biology in culturo and for baculovirus vector based recombinant protein expression systems. In the present study, we report establishment of a new continuous cell line designated as DZNU-Bm-1 from larval ovaries of the silkworm, Bombyx mori. The cells were grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat inactivated B. mori haemolymph at 25+/-1 degrees C. A large number of attached epithelial-like and round refractive cells migrated from the explants and multiplied in the primary cultures. Both type of cells were subcultured initially for a few passages but after 10 passages the round refractive cells dominated the population, which could be subcultured continuously using MGM-448 medium with 10% FBS. The population doubling time of cell line was about 42h at 25+/-1 degrees C. The cell populations were largely diploids and triploids, while a few tetraploids and hexaploids were also observed. DNA profiles using Inter Simple Sequence Repeat (ISSR)-PCR and Simple Sequence Repeat (SSR) loci established the differences between DZNU-Bm-1 cell line and most widely used BmN cell line and the B. mori W-chromosome specific sequences confirmed the origin of DZNU-Bm-1 cell line to be from female silkworm. When cells were infected with free nonoccluded B. mori nucleopolyhedrovirus (BmNPV), the cell line was found to be highly susceptible with 92-94% of the cells harbouring BmNPV and having an average of 20-23 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV based baculoviral expression system and also for studying in culturo virus replication.     

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

Host range expansion by recombination of the baculoviruses Bombyx mori nuclear polyhedrosis virus and Autographa californica nuclear polyhedrosis virus.

The mechanisms of host specificity of nuclear polyhedrosis viruses (NPVs) (Baculoviridae) were analyzed after coinfection of Bombyx mori NPV (BmNPV) and one of four distinct groups of Spodoptera litura NPV (SlNPV), including an Autographa californica NPV (AcNPV) variant (S. Maeda, Y. Mukohara, and A. Kondo, J. Gen. Virol. 71:2631-2639, 1990), into various lepidopteran cell lines. Replication of BmNPV in nonpermissive cells (TN-386, SF-21, and CLS-79) was induced by coinfection with AcNPV but not with the other three SlNPV groups. These induced progeny NPVs were plaque purified in BmN cells, which are susceptible to only BmNPV, and characterized. Most of these isolates did not replicate in the cell lines in which they were produced, indicating the existence of a helper function of AcNPV for BmNPV replication in nonpermissive cells. Some of these isolates, however, were able to replicate in cell lines nonpermissive to BmNPV, indicating the appearance of a new virus with wider host specificity. DNA restriction endonuclease analysis showed that the isolates exhibiting wider host range were recombinant viruses between the parents, AcNPV and BmNPV, resulting from various types of crossovers of relatively large areas of their genomes. Expansion of host range was also observed in larvae.     

Rapid detection of multiple nucleopolyhedroviruses using polymerase chain reaction.

A technique using the polymerase chain reaction (PCR) was developed for detection of the nucleopolyhedrovirus (NPV) polyhedrin gene. The amino acid sequences of the polyhedrin gene were compared in twenty-six NPVs. A highly conserved DNA sequence within the coding region of the polyhedrin gene was targeted for amplification. One pair of degenerate PCR primers was designed to produce fragments of about 430 bp. The NPVs detected by this technique were Autographa californica NPV, Bombyx mori NPV, Hyphantria cunea NPV, Spodoptera exigua NPV, S. litura NPV, and Lymantria dispar NPV. This technique would be useful in monitoring the distribution of NPVs and release of the wild type and recombinant NPVs.     

科云牌棉铃虫核型多角体病毒生物农药的规模化生产和应用

昆虫病毒生物农药是纯天然无公害生物农药产品,本文介绍了中国科学院动物研究所同河南省济源白云实业有限公司合作,成功开发并商业化棉铃虫病毒生物农药的基本情况。我们开发的棉铃虫病毒生物农药注册商标"科云牌",包括两个产品:科云牌5 000亿PIB/g棉铃虫病毒原粉和科云牌600亿PIB/g棉铃虫病毒水分散粒剂。原粉产品已出口到欧洲,制剂产品用于棉铃虫防治,2006至2007年在棉田累计推广应用10万hm~2次。     

细胞松弛素D对棉铃虫核型多角体病毒复制和装配的影响

杆状病毒感染引起宿主细胞肌动蛋白骨架的构象变化 ,使之形成缆绳结构 .棉铃虫核型多角体病毒 (HaNPV)的衣壳蛋白也能使宿主昆虫的肌动蛋白发生凝聚 ,用细胞松弛素D抑制宿主肌动蛋白形成纤丝结构 ,病毒感染Hz AM1,空斑计数表明 ,0 1μg/ml细胞松弛素D可使棉铃虫核型多角体病毒的增殖下降 10 4倍 ,细胞松弛素D浓度增高到 0 5 μg/ml则测不到子代病毒粒子 .Western印迹分析表明 ,细胞松弛素D并不影响受染细胞中肌动蛋白的含量 .斑点印迹 (dotblot)也表明 ,病毒DNA的合成也没有受到影响 ,推测宿主细胞的肌动蛋白纤丝结构与病毒的复制有关 .在电子显微镜下观察超薄切片发现 ,在 0 5 μg/ml细胞松弛素D处理细胞中形成的病毒粒子形态与正常形态明显不同 ,提示细胞松弛素D抑制HaNPV的增殖是由于抑制病毒组装成完整有感染性的病毒粒子 .从而可以认为宿主昆虫细胞的丝状肌动蛋白对子代病毒的复制和组装是必需的 .     

家蚕核型多角体病毒水平转移基因分析

为了探讨杆状病毒基因组的遗传进化模式,文章利用家蚕核型多角体病毒(BmNPV)和其宿主家蚕全基因组数据,进行了全基因组的同源性搜索和系统进化分析,结果显示,BmNPV的几丁质酶(Chi)基因、凋亡抑制蛋白3(IAP3)基因和尿苷二磷酸葡萄糖转移酶(UGT)基因为水平转移基因。这3个基因都来源于其宿主昆虫。通过核苷酸组成、密码子偏好性、选择压力等基因特征分析,发现BmNPV水平转移基因与其基因组序列存在明显差异,进一步验证水平转移基因的外源性。对3个水平转移基因的功能分析发现它们有利于杆状病毒在宿主昆虫中的侵染与繁殖,并提高杆状病毒在昆虫中的生存能力。     

Arginine kinase is highly expressed in a resistant strain of silkworm (Bombyx mori, Lepidoptera): Implication of its role in resistance to Bombyx mori nucleopolyhedrovirus

A gene encoding Bombyx mori arginine kinase (BmAK) has been indentified differentially expressed in the midguts of Bombyx mori strain NB which is resistant to nucleopolyhedrovirus (BmNPV), strain 306 which is susceptible to NPV and a near isogenic line BC(8) with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). In this study, we characterized the expression profiles of BmAK using RT-PCR and real-time quantitative PCR. The expression level of BmAK fluctuated in various developing stage and various tissue. Remarkably, the expression level of BmAK increased more than 10-fold 24 hours post inoculation (h p.i.) of NPV in strain NB and BC(8), while such increment was abraded in strain 306 although the basal expression level of BmAK in strain 306 was higher than that of strain NB and BC(8). Western blotting analysis using polyclonal antibody against BmAK verified such observation, and immunofluoresence analysis indicated for the first time that BmAK was mainly located to the cytoplasm or some structures in cytoplasm. These findings suggest that arginine kinase is involved in the antiviral process of Bombyx mori larvae against NPV infection.     

Mass production of polyhedral occlusion bodies of NPV of Helicoverpa armigera in relation to dose,age and larval weight

A significant difference was noticed in the yield of polyhedral occlusion bodies (POBs) in various larval instars of H. armigera when three different doses of the nuclear polyhedrosis virus (NPV) were administered. The yield of POBs from a single larva ranged from 0.35 x 10(6) to 25033.33 x 10(6) with a mean of 18422.33 x 10(6) for fourth instar inoculated. Positive correlation existed between larval weight and number of POBs recovered. The regression analysis indicated POBs recovered responded with predictable manner to the weight of different larval instars and the various concentration of virus administered. The medium lethal time increased in the instars of the larva advanced with a minimum of 3.5 and maximum of 8 days in the first and fifth instars respectively.     

Molecular Dissection of Bombyx mori Nucleopolyhedrovirus orf8 Gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and often eoevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus (NPV) and its homologues are found only in group I NPV genomes. The Autographa californica NPV Acl6 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that Bm8/Ac 16 interacts with baculoviral and cellular proteins. Bm8/Ac 16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bin8 function. Ac16 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group INPVs.     

Establishment and characterization of a continuous cell line from pupal ovaries of Japanese oak silkworm Antheraea yamamai Guerin-Meneville

Pupal ovaries of the wild oak silkworm Antheraea yamamai Guerin-Meneville were cultured in MGM-448 (Modified Grace Medium-448) medium containing 10% fetal bovine serum. After the primary culture was set up in 1988, a continuous cell line was obtained in 1991, designated as NISES-Anya-0611 (Anya-0611). The population doubling time was 54 hrs. and 19 min. at 96 passages and 88 hrs. and 29 min. at 387 passages. Spindle-shaped and spherical cells coexisted in the cell group. The cell line karyotype line was typical of lepidopteran cell lines, consisting of numerous small chromosomes. The cell line was distinguished from other lepidopteran cell lines by comparing malic enzyme, phosphoglucose isomerase, phosphoglucose mutase, and isocitric dehydrogenase isozyme patterns. The cell line was highly infected to the Antheraea yamamai nuclear polyhedrosis virus (Anya NPV). The luciferase gene of recombinant Bm NPV (BmNPVP6ETL) was able to express in the cell line, too, so that luciferase recombinant products were able to be detected in the cell body and in supernatant. The Anya NPV clone group was isolated on the cell seat using plaque purification.     

Silkworm, Bombyx mori larvae expressed the spider silk protein through a novel Bac-to-Bac/BmNPV baculovirus

Abstract:  The silkworm has become an ideal multicellular eukaryotic model system for basic research. The major advantages of expressing foreign genes in silkworm larvae are the low cost of feeding, the extremely high levels of expression achievable compared with expression in cell lines and increased safety because the baculovirus is noninfectious to vertebrates. In this study, we used a recently developed Bombyx mori Nucleopolyhedrovirus (BmNPV) bacmid to express the spider flagelliform silk gene in silkworm larvae. The recombinant bacmid baculoviruses (rBacmid/BmNPV/Flag) were introduced into the first-day larvae of the fifth instar by subcutaneous injection. The worms presented symptoms typical of NPV infection from 72 h after injection compared with control. The haemolymph was collected from the infected larvae 120 h post-infection and the recombinant 6× His-tagged Flag protein was purified by the Ni-NTA spin kit under denaturing conditions with 8  m urea. A 37.0-kDa protein was visualized both in rBacmid/BmNPV/Flag-infected haemolymph and eluting fraction. The results showed that the Bac-to-Bac/BmNPV baculovirus expression system is an efficient tool to express the target gene in silkworm larvae, which takes only 7–10 days for generating recombinant baculovirus, compared with the traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses.     

转座子介导的多角体基因表达及提高病毒粒子的感染效率

现行的杆状病毒表达外源基因的方法是将外源基因取代病毒中的多角体基因,因而得到的重组杆状病毒感染活体时不能经口感染,只能进行针刺注射,效率低且易引起活体感染其他疾病。将家蚕核型多角体病毒(Bombyx mor inucleopolyhedrovirus,BmNPV)中的多角体基因(polyhedrin,poly)及其启动子片段克隆到转座子载体pigA3GFP中,将其与辅助质粒pHA3PIG利用脂质体介导法导入家蚕细胞中,经过多次筛选获得稳定的转基因家蚕细胞。之后先将BmPAK6(含LacZ)及BmGFP(含GFP)重组病毒分别感染转基因细胞,再将得到的重组病毒经口感染5龄家蚕幼虫。结果显示,重组杆状病毒可以经口感染家蚕幼虫。这些研究表明来自于转基因家蚕细胞的poly基因表达产物可以提高重组杆状病毒经口感染家蚕率,为解决杆状病毒表达系统中重组病毒不能经口感染家蚕幼虫的问题提供新思路。     

Cloning and partial characterization of a gene in Bombyx mori homologous to a human adiponectin receptor

In this study, we report the cloning and characteristics of an adiponectin-like receptor gene from Bombyx mori (BmAdipoR) with highly conserved deduced amino-acid sequences and similar structure to the human adiponectin receptor (AdipoR). Structural analysis of the translated cDNA suggested it encoded a membrane protein with seven transmembrane domains. BmAdipoR was found to be expressed in multiple tissues and highly expressed in Malpighian tubules, fat body and testis. BmNPV (Bombyx mori nucleopolyhedrovirus) bacmid system combined with confocal microscopy revealed that BmAdipoR was targeted to the cell membrane. We also found that infection with BmNPV did not have an effect on BmAdipoR mRNA quantity in the midgut of susceptible Bombyx mori strain (306) at 48 h, but BmAdipoR mRNA quantity increased significantly at 72 h. We concluded that BmAdipoR gene was a membrane protein ubiquitously expressed in Bombyx mori tissues and that its expression was altered by treating with BmNPV.