In vitro and in vivo host range of Anticarsia Gemmatalis multiple nuclear polyhedrosis virus

Summary A clone of the wild type (wt) Anticarsia gemmatalis multiple nuclear polyhedrosis virus AgMNPV, derived from a geographical isolate (Hondrina, Brazil) and designated AgMNPV-CL4-3A1, was used to determine the host range of this virus in six established lepidopteran cell lines: Anticarsia gemmatalis (BCIRL-AG-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Heliothis virescens (BCIRL-HV-AM1), Helicoverpa armigera (BCIRL-HA-AM1), Trichoplusia ni (TN-CL1), Bombyx mori (BMN), and a coleopteran cell line Anthonomus grandis (BRL-AG-1). In addition, the in vivo host range of this clone was also assayed in larvae of Helicoverpa zea, Heliothis virescens, Trichoplusia ni, and the homologous species Anticarsia gemmatalis by probit analysis. On the basis of temporal studies of TCID₅₀ values, BCIRL-HV-AM1 cells gave the highest extracellular virus (ECV) titer (9.7×10⁶ TCID₅₀/ml) followed by BCIRL-HA-AM1 cells (8.3×10⁵ TCID₅₀/ml) and BCIRL-AG-AM1 cells (3.2×10⁵ TCID₅₀/ml). In addition, a low ECV titer of 1.37×10³ TCID₅₀/ml was detected from TN-CL1 cells 96 h postinoculation, while BRL-AG-1, BMN, and BCIRL-HZ-AM1 cells were nonpermissive to AgMNPV-CL4-3A1 on the basis of TCID₅₀ results. AgMNPV-CL4-3A1 and the wild type AgMNPV had similar restriction profiles that were different from wild type AcMNPV. The LC₅₀ values were 96.9, 564.6, 733.3, and 1.1×10⁴ occlusion bodies/cm² of diet for A. gemmatalis, Helicoverpa zea, Heliothis virescens, and T. ni, respectively. This article presents the results of research only. Mention of proprietary products in this article does not indicate endorsement or a recommendation for use by USDA-ARS. All programs and services of the USDA are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, marital status or handicap.     

Expression of the green fluorescent protein carried by Autographa Californica multiple nucleopolyhedrovirus in insect cell lines

Summary A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID₅₀ titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to Ac-MNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401+10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401+10% FBS (37.8×10⁶ TCID₅₀/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4×10⁶ TCID₅₀/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9×10⁶ TCID₅₀/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.     

Replication ofAutographa californica baculovirus (AcMNPV) in a coleopteran cell line

Summary A coleopteran cell line (AGE) derived from the cotton boll weevilAnthonomus grandis supported replication ofAutographa californica multiple nuclear polyhedrosis virus (AcMNPV). The titer of extracellular virus (ECV) and the number of occlusion bodies (OB) produced in AGE cells were approximately equal to those produced by aTrichoplusia ni cell line (TN-CL1), and the OB produced by both cell lines were equally infectious forT. ni larvae. The identity of the AGE cell line was established by chromosome and isoenzyme analyses.     

Relative effects of ultraviolet and visible light on the activities of corn earworm and beet armyworm (Lepidoptera: Noctuidae) nucleopolyhedroviruses

Five different combinations of fluorescent tubes (UV-B/UV-B, UV-B/UV-A, UV-A/ UV-A, UV-B/White, White/White) were used to determine relative effects of UV and visible light on the nucleopolyhedroviruses (NPV) of Helicoverpa zea and Spodoptera exigua. For both viruses, the greatest inactivation occurred with exposure to UV-B radiation. Both virus concentration and radiation exposure time influenced the rate and degree of inactivation. In the case of the UV-A/UV-A and White/White combinations inactivation occurred only with the longest exposure (24 h) and the lowest virus concentration (0.747 PIB/mm2). The NPV from H. zea was found to be more sensitive to UV radiation than the NPV from S. exigua.     

棉铃虫核型多角体病毒ORF33基因的原核表达和在宿主细胞中亚细胞定位

对棉铃虫Helicoverpa ar migera核型多角体病毒HearSNPV的ORF33基因(ha33)进行克隆和原核表达,hass在E.coli中表达不完全,表达产物的大小为17kDa,小于预测的分子量28.4kDa。用纯化的原核表达产物免疫家兔,制备了多克隆抗体,应用多克隆抗体检测了HearSNPV感染的宿主细胞(HzAMI)中ORF33基因的表达,表达产物的分子量为31kDa。并通过共聚焦荧光显微镜方法,用多克隆抗体检测编码的蛋白在宿主细胞(HzAM1)中的亚细胞定位,发现ha33编码的蛋白存在于宿主细胞的细胞质中,并持续到感染后期。     

棉铃虫单粒包埋型核型多角体病毒极早期基因(ie-1)分析(英文)

克隆了棉铃虫Helicoverpaarmigera单粒包埋型核型多角体病毒 (HaSNPV)C1株基因组DNA ,并通过随机测序的方法测定了经XbaI酶切后的H片段的核苷酸全序列。序列比较和分析发现该片段中ORF1 3与苜蓿丫纹夜蛾Autographacalifornica多粒包埋型核型多角体病毒 (AcMNPV)基因组ORF1 47(ie 1 )同源。ie 1基因编码区全长 1 986bp ,根据推测的氨基酸序列 ,可编码 6 6 1个氨基酸残基组成的多肽 ,预计分子量为 76 .5kD。将所推导的HaSNPVIE 1氨基酸序列与其它已知的杆状病毒IE 1氨基酸序列进行比较 ,结果表明 ,HaSNPV和谷实夜蛾H .zea单粒包埋型核型多角体病毒IE 1氨基酸序列最为相似 ,同源性高达 98%。与AcMNPV、家蚕Bombyxmori核型多角体病毒 (BmNPV)、云杉卷叶蛾Choristoneurafu miferana多粒包埋型核型多角体病毒 (CfMNPV)、舞毒蛾Lymantriadispar多粒包埋型核型多角体病毒(LdMNPV)、黄杉毒蛾Orgyiapseudotsugata多粒包埋型核型多角体病毒 (OpMNPV)、甜菜夜蛾Spodopteraex igua多粒包埋型核型多角体病毒 (SeMNPV)、小菜蛾Plutellaxylostella颗粒体病毒 (PxGV)和Xestiac ni grum颗粒体病毒 (XcGV)的IE 1氨基酸序列同源性较低 ,分别为 2 3 %、2 3 %、2 3 %、2 5 %、2 3 %、1 4%、2 7%和 7%。根据氨基酸序列由GENETYX     

HzAm1细胞rpL13基因的序列分析及病毒感染对其转录水平的影响

从美洲棉铃虫细胞(HzAM1)中克隆了核糖体大亚基蛋白L13(RibosomalproteinL13,RpL13)的cDNA及其基因组DNA序列,并进行了序列分析。其编码框为666bp,无内含子,预测编码大小约为25kDa的蛋白。通过与其它15种动物的RpL13编码框序列进行进化分析,发现聚类结果与传统物种分类一致。转录研究表明,棉铃虫核型多角体病毒(Helicoverpaarmigerasinglenucleocapsidnucleopolyhedrovirus,HaSNPV)感染HzAm1后使rpL13在细胞内的转录水平降低,在病毒感染后96h,rpL13mRNA的拷贝数降到对照健康细胞的8%。     

In vitro host range of the Hz-1 nonoccluded virus in insect cell lines

A total of 13 insect cell lines spanning 4 orders (Lepidoptera, Coleoptera, Diptera, and Homoptera) were tested for their ability to replicate the nonoccluded virus Hz-1. Only the Lepidopteran cell lines supported replication of the virus with TN-CL1 and BCIRL-HZ-AM1 producing the highest titers of 2.4 × 10⁸ tissue culture infective dose (TCID)₅₀/ml and 2.0 × 10⁸ TCID₅₀/ml, respectively. A codling moth cell line (CP-169) was the only Lepidopteran cell line that did not replicate the virus and transfection of this cell line with Hz-1 DNA failed to replicate the virus. Also, transfection with DNA from a recombinant baculovirus carrying the red fluorescent protein gene (AcMNPVhsp70 Red) was not expressed in CP-169 cells. The replication cycle of Hz-1 in BCIRL-HZ-AM1 cells showed that this virus replicated rapidly starting at 16 h postinoculation (p.i.) and reaching a peak titer of 1.0 × 10⁸ TCID₅₀/ml 56 h postinoculation. Hz-1 when compared with several other baculoviruses has the widest in vitro host spectrum.     

Pathology and ultrastructure of Hz-2V infection in the agonadal female corn earworm,Helicoverpa zea

The pathology and ultrastructure of the reproductive tract of Hz-2V-infected female corn earworm moths, Helicoverpa zea, were studied. The identity of malformed reproductive tissues found in virus-infected moths was determined by examining these tissues in moths that were infected with the virus at different life stages. Malformation of reproductive tissues in the progeny of virus-infected female moths was first observed by 3 days post-pupation (dpp), indicating that virus replication had altered the differentiation of these tissues very early on in their development. The ultrastructure of the grossly malformed agonadal reproductive tissues from insects aged 3-10dpp revealed the absence of the cuticular lining found in the oviducts of normal moths, and the proliferation of epithelial cells in these infected oviduct tissues. In addition, large quantities of virus were found aggregated into a large mass in the lumen of the malformed cervix bursa of 10dpp agonadal female pharate adult moths. Prior to eclosion, the virus in the cervix bursa was observed separated into spherical masses, which are thought to exude through the ductus bursa and collect over the vulva, forming a viral "waxy plug" that is likely to play an important role in virus transmission.     

Influence of the host plant on occluded virus production and lethal infectivity of a baculovirus

Intra- and inter-specific effects of cotton, soybean, and clover on the time until death of Helicoverpa zea (Boddie) and Heliothis virescens (F.) larvae lethally infected with H. zea nucleopolyhedrovirus (HzSNPV) were evaluated in the laboratory. In the first test, on second instar only, the time until death of lethally infected larvae of both species differed with the plant tissues (vegetative or reproductive) and plant species. The total viral activity produced per larva in LC(50) units (occluded viral bodies (OBs) per larva/LC(50) in OBs/mm(2) of diet surface) was greater from H. virescens larvae fed vegetative than reproductive tissues of all host plants, but from H. zea virus production was greater only when fed vegetative tissue of soybean. In a second test that compared second and fourth instar H. virescens on cotton, total viral activity from larvae treated in both instars was greater when fed vegetative than reproductive tissues. Results of these tests suggest that the ability of host plants to influence baculovirus disease is more complex than previously believed. When examining the epizootic potential of a baculovirus, more attention must be given to the effects of the host plant on the insect-virus interactions.     

Effect of Temperature Oscillation on Insect Cell Growth and Baculovirus Replication

Temperature oscillation can enhance cell viability of sf9 insect cells and baculovirus production of occlusion bodies (OB) and extracellular virus (ECV) compared with constant temperature in stationary culture and suspension culture. The optimal oscillation range was 24 to 28°C. At this temperature oscillation, the viability of uninfected and infected sf9 cells can be maintained much longer than at 28°C. Although the rate of virus infection was a little low at 24 to 28°C, the final cell infectivity was similar to that at a constant temperature of 28°C. The production of OB was increased from 13.4 to 17.4/cell in stationary culture and from 13.9/cell to 18.1/cell in suspension culture. The titer of ECV was increased from 87 to 114 PFU/cell in stationary culture and from 79 to 114 PFU/cell in suspension culture.     

Effects of virus concentration and ultraviolet irradiation on the activity of corn earworm and beet armyworm (Lepidoptera: Noctuidae) nucleopolyhedroviruses

Laboratory studies were initiated to determine the relationship between virus concentration and radiation-caused inactivation of NPVs from Helicoverpa zea (HzSNPV) and Spodoptera exigua (SeMNPV). In the laboratory, a UV-B/UV-A system was used for inactivation studies. For both viruses inactivation was dependent upon both length of UV exposure and virus concentration. At all virus concentrations HzSNPV was more sensitive to UV than SeMNPV. In the field HzSNPV was used and virus persistence was significantly affected by virus concentration (i.e., inactivation was inversely related to virus concentration).     

Yield, biological activity, and field performance of a wild-type Helicoverpa nucleopolyhedrovirus produced in H. zea cell cultures

This paper reports studies of the in vitro production of a virus from Helicoverpa armigera (HaSNPV) and its possible use as a specific Helicoverpa/Heliothis larvicide. Growth kinetics of Helicoverpa zea (H. zea) cells and virus occlusion body yields were compared in three SF900II-based media, namely, SF900II (serum-free), SF900II + 1% serum, or SF900II + 10% serum. Viable cell densities were usually higher in the media supplemented with serum than in the serum-free medium; however, in the serum-free medium, cell diameters were 1.7 times greater (i.e., individual cell volumes were five times larger). Both volumetric production of virus occlusion bodies and production per cell were higher in the serum-free medium than in the media supplemented with serum. However, the infectivity of the occlusion bodies from the serum-free medium was less than that with those from the medium supplemented with 10% serum, when compared in bioassays employing newly hatched larvae. The infectivity of the in vitro produced occlusion bodies was also less than that of in vivo produced occlusion bodies in a commercially available virus product, GemStar. High levels of infection of H. armigera larvae obtained in a preliminary field assessment on preflowering tomatoes using the in vitro produced occlusion bodies indicated the suitability of the in vitro process for biopesticide production.     

Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.) (Lepidoptera: Noctuidae)

A multicapsid nucleopolyhedrovirus (ThorMNPV) that was co-isolated with a single nucleocapid ThorSNPV from mixed infected larvae of Thysanoplusia orichalcea L. (Lepidoptea: Noctuidae) is characterized. Scanning electron microscopy of ThorMNPV showed a dodecahedral-shaped occlusion body (OB). The occluded virions contained one to as many as eight nucleocapsids/virion. Virion band profiles in gradient centrifugation were consistent in at least 10 rounds of centrifugation from different virion sample preparations. The ThorMNPV had high virulence to third instar Trichoplusia ni and Pseudoplusia includens with LD50 values of 17 and 242OBs per larva, respectively. However, ThorMNPV did not cause mortality in Spodoptera exigua, Spodoptera frugiperda, Spodoptera eridania, Anticarsia gemmatalis, and Helicoverpa zea. ThorMNPV replicates in cells of various tissues such as the fat body and tracheal epithelium cells. T. ni High 5 cells were permissive to ThorMNPV in terms of infection and viral DNA transfection, but SF-21 was less permissive and the infection process was slower. Production of OBs by ThorMNPV in the nuclei of SF-21 was not well pronounced. The genome size of ThorMNPV was estimated to be 136 kb. The polyhedrin gene open reading frame (ORF) was cloned and completely sequenced. The promoter sequence is identical to that of Autographa californica MNPV. Phylogenetic analyses using partial sequences of the polh, lef-8, and lef-9 revealed that ThorMNPV is a member of the Group I NPVs and is related but distinct from the AcMNPV/Rachiplusia ou NPV/Bombyx mori NPV cluster.     

Infectivity studies of a new baculovirus isolate for the control of the diamondback moth (Plutellidae: Lepidoptera)

This study describes a new baculovirus isolate recovered from infected larvae of the diamondback moth, Plutella xylostella (L.), and identified as a multiple nucleopolyhedrovirus (MNPV). The plaque purified isolate designated as PxMNPVCL3 was found to be pathogenic to P. xylostella, Heliothis virescens (F.), Trichoplusia ni (Hübner), H. subflexa (Guenée), Helicoverpa zea (Boddie), Spodoptera exigua (Hübner), and S. frugiperda (J. E. Smith) larvae in decreasing order of susceptibility. The LC50 for diamondback moth, the most susceptible, was 6 occlusion bodies (OB)/cm2, whereas the most resistant species, namely S. frugiperda, was 577 OB/cm2. PxMNPVCL3 was more pathogenic to diamondback moth by 3-4 log cycles as compared with 2 broad-spectrum baculoviruses, namely Autographa california (alfalfa looper) MNPV and Anagrapha falcifera (celery looper) MNPV. The 3 baculoviruses were compared with each other and characterized by restriction endonuclease (REN) analysis, hybridization, and neutralization tests. Fragmentation profiles generated by REN showed that the 3 baculoviruses shared some fragments in common. Hybridization studies employing digoxigenin labeled PxMNPVCL3 DNA as a probe revealed the close but distinct relationship of these 3 viruses. Neutralization tests confirmed the hybridization studies, namely that the 3 viruses although genetically similar are distinguishable from each other.     

The Autographa californica M nucleopolyhedrovirus ac79 gene encodes an early gene product with structural similarities to UvrC and intron-encoded endonucleases that is required for efficient budded virus production

The Autographa californica M nucleopolyhedrovirus (AcMNPV) orf79 (ac79) gene is a conserved gene in baculoviruses and shares homology with genes in ascoviruses, iridoviruses, and several bacteria. Ac79 has a conserved motif and structural similarities to UvrC and intron-encoded endonucleases. Ac79 is produced at early times during infection and concentrates in the nucleus of infected cells at late times, suggesting a cellular compartment-specific function. To investigate its function, an ac79-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration assays showed that budded virus (BV) production was reduced in the ac79-knockout virus compared to control viruses, following either virus infection or the transfection of bacmid DNA. The ac79-knockout virus-infected cells produced plaques smaller than those infected with control ac79-carrying viruses. No obvious differences were observed in viral DNA synthesis, viral protein accumulation, or the formation of occlusion bodies in ac79-knockout and control viral DNA-transfected cells, indicating progression into the late and very late phases of viral infection. However, comparative analyses of the amounts of BV genomic DNA and structural proteins in a given quantity of infectious virions suggested that the ac79-knockout virus produced more noninfectious BV in infected cells than the control virus. The structure of the ac79-knockout BV determined by transmission electron microscopy appeared to be similar to that of the control virus, although aberrant capsid protein-containing tubular structures were observed in the nuclei of ac79-knockout virus-infected cells. Tubular structures were not observed for ac79 viruses with mutations in conserved endonuclease residues. These results indicate that Ac79 is required for efficient BV production.     

WOUND HEALING AND COLLAGEN FORMATION : VI. The Origin of the Wound Fibroblast Studied in Parabiosis

Healing skin wounds were studied in a series of parabiotic rats. The femurs of one parabiont of each pair were shielded whilst both animals were given 800 r from a Co⁶⁰ source. The animals were wounded 3 days after irradiation. Each animal with partially shielded marrow was then given tritiated thymidine intraperitoneally daily while the cross-circulation was arrested by clamping. After the thymidine-³H had cleared the blood, the clamp was released. Animals were sacrificed, and wounds were prepared for radioautography 1, 2, and 6 days after wounding. In the wounds of the shielded animals thymidine-³H was observed in epidermis, endothelium, leukocytes, fibroblasts, and mast cells. Only neutrophilic leukocytes, monocytes, and lymphocytes were labeled, as determined by light and electron microscope radioautography, in the wounds of each nonshielded parabiont. None of the many fibroblasts present were found to contain label in the wounds of the nonshielded parabionts through the 6 day period. These observations provide further evidence that wound fibroblasts do not arise from hematogenous precursors and, therefore, must arise from adjacent connective tissue cells.     

Further characterization of the gonad-specific virus of corn earworm, Helicoverpa zea

The gonad-specific virus (GSV) is a DNA virus infecting the reproductive tracts of adults of both sexes of the corn earworm, Helicoverpa zea, causing severe tissue deformities leading to sterility. Atypical occlusion bodies containing large concentrations of virions embedded in a granular matrix were seen in the lumen of the oviduct and the bursa copulatrix of infected females. The virus, transmitted by both sexes, was successfully propagated in vivo and in tissue culture. The GSV genome is about 225 kb in size, with no apparent similarity to the nucleopolyhedrovirus type species, AcMNPV, genomic DNA, as determined by Southern hybridization. PCR amplification of GSV genomic DNA with primers derived from the highly conserved polyhedra gene of several baculoviruses indicated no similarity. GSV at 10(-2) female equivalents (based on virus obtained from the bursa copulatrix and oviducts of one infected female) injected into a newly emerged female and mated to a normal male resulted in >95% agonadal progeny. However, at lower doses, some of the adult progeny looked normal but apparently carried a low level of the virus that could be responsible for sustenance of infection in a given colony, as well as in nature.