家蚕二分浓核病毒感染家蚕后的释放动态研究

【目的】研究家蚕二分浓核病毒(Bm BDV)感染家蚕后不同时间段内释放到环境中的病毒量,为通过蚕沙快速检测病毒提供方法和依据。【方法】选取家蚕二分浓核病毒基因组节段VD1上的ns1基因和VD2上的ns3基因中的一小部分片段,分别克隆到p MD18-T载体上,制备标准品质粒,并用实时荧光定量PCR的方法绘制ns1和ns3的标准曲线。通过检测Bm BDV感染5龄家蚕后不同时间蚕沙中的病毒量来衡量释放到环境中的病毒的变化动态。【结果】在病毒感染家蚕第3天,大量的病毒随蚕沙释放到环境中,每微克蚕沙中约含1.38×106个VD1,6.54×105个VD2;从感染后第5天开始,释放到环境中的病毒量呈指数型增加;第7-8天病毒的释放量到达一个平台期,此时每微克蚕沙中约有2.12×107个VD1,1.34×107个VD2。普通PCR结果也反应出了类似的病毒释放动态。【结论】根据Bm BDV感染家蚕后的释放动态,推测病毒的复制周期约60 h。利用实时荧光定量PCR检测蚕沙中的Bm BDV,能简单、快速、准确地诊断病毒的感染。     

Isolation and reconstitution of the RNA replicase of the cytoplasmic polyhedrosis virus of silkworm,Bombyx mori

Summary After irradiation of the virus particles of CPV, the RNA replicase associated with the virion was isolated in the form of a genome-replicase complex with DEAE-Sephadex A-25 chromatography. This complex was then treated with Triton X-100 and purified by phosphocellulose column chromatography. The RNA replicase reconstituted with the doublestranded RNA of CPV showed both the enzyme activity of RNA polymerase and methyltransferase. The single-stranded RNA could not serve as the template for the RNA replicase. The role of the RNA replicase of CPV is discussed.     

Storage proteins in the silkworm,Bombyx mori

In the silkworm, Bombyx mori, two storage proteins named SP-1 and SP-2 were shown to decline in concentration in the haemolymph and increase in the fat body during the larval-pupal transformation, when protein granules are formed in the fat body at the same time as the degeneration of mitochondria and endoplasmic reticulum. At the larval-pupal ecdysis, in females the two proteins account for 60% of total fat body protein (80% of the soluble protein), while males have very little SP-1 and SP-2 comprises only 20% of the total fat body protein. The concentration of protein granules in the fat body cytoplasm is much greater in females than in males, and the granules in females have partially crystalline inner zones. This is different from males where granules with non-crystalline structure are most numerous.The properties of these proteins purified from pupal fat body are similar to those of Cecropia storage proteins and calliphorin, all of which have molecular weights of around 500,000 and are composed of subunits of mol. wt about 85,000. SP-1 differs from SP-2 by having an exceptionally high content of methionine, but much less glutamate, phenylalanine and tyrosine. SP-1 resembles another female-specific protein, vitellogenin and SP-2 resembles calliphorin in amino acid composition.From these results, it is concluded that SP-1 and SP-2 have storage roles and are deposited in protein granules.     

Antiviral proteins in midgut of the silkworm, Bombyx mori, fed on different food sources

Midgut proteins from the silkworms, Bombyx mori, fed on an artificial diet containing dried powder of the spiral blue-green alga, Spirulina platensis, had a very low fluorescence intensity. This indicated that chlorophyll in this alga is not utilizable for synthesizing the red fluorescent protein (RFP) which has been considered as an effective antiviral protein in silkworm midgut. Many electrophoretic bands of the midgut proteins from the mulberry-fed silkworms emitted red fluorescence under UV light. It is thus concluded that the RFP as a whole is more than one midgut protein. Enzymatic analyses showed that silkworms fed on mulberry leaves and artificial diets had similar protease activities, but they had different phospholipase C activities. Therefore, it is suggested that the antiviral activity of purified RFP in the silkworm gut juice is attributable to phospholipase C.

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Résumé Les protéines de l'intestin moyen de Bombyx mori, nourri avec un aliment artificiel contenant de la poudre séchée de l'algue bleue spiralée, Spirulina pratensis, avaient une très faible fluorescence. Cesi indique que la chlorophylle a de cette algue n'est pas utilisable pour la synthèse de la protéine fluorescente rouge (RFP), considérée comme une protéine antivirale efficace de l'intestin moyen. Après séparation par électrophorèse sur gel de polyacrylamine DISC plusieurs bandes de la protéine de l'intestin moyen de B. mori élevé sur mûrier émettaient une fluorescence rouge en lumière U.V. On peut conclure que RFP est un complexe plutôt qu'une protéine particulière de l'intestin moyen. Les analyses enzymatiques ont montré que la phospholipase C et non les protéases ont une activité antivirale dans la RFP purifiée provenant de vers à soie nourris à partir de mûrier ou sur régime artificiel.

     

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.     

Purification and characterization of membrane-bound alkaline proteases from midgut tissue of the silkworm, Bombyx mori

Membrane-bound alkaline proteases from the midgut epithelia of the silkworm, Bombyx mori, were solubilized with 1% Lubrol-WX, at pH 11.2. They were purified by gel filtration on Sepharose 6B and Ultrogel AcA-202 columns and a preparative polyacrylamide gel electrophoresis. Two proteases, caseinolytic (6B3-Tc) and benzoyl-arginine-p-nitroanilide-lytic (6B3-Tb) were obtained. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis. These enzymes showed high pH optima, 11.2, and pI values, above 11, and were extremely stable over a wide range of pH. The Km values for 6B3-Tb and Tc were 0.476 mM and 2.5 mg/ml respectively. Hammarsten casein and mulberry leaf protein were rapidly hydrolyzed by Tc, whereas the hydrolytic activity of Tb for Azocoll was higher than that of Tc. The protease Tb was strongly inhibited by diisopropylfluorophosphate, p-chloromercuribenzoate, benzamidine, leupeptin, and soybean trypsin inhibitor; Tc was inhibited by diisopropylfluorophosphate, tosyl phenylalanine chloromethylketone and chymostatin, but not by tosyl lysine chloromethylketone, p-chloromercuribenzoate, or iodoacetamide. The molecular weights of the proteases were estimated to be 12,800 (Tb) and 13,300 (Tc) by Sephacryl S-300 gel filtration. The amino acid analyses showed that both proteases contain a large number of acidic amino acids but a relatively small number of basic amino acids.     

Alkaline proteases in the midgut tissue and digestive fluid of the silkworm,Bombyx mori

In larvae of Bombyx mori proteolytic activity was found in the midgut tissue and digestive fluid. The pH-activity curves of both proteases were very similar and optimal activity was about pH 11.2. The reduction in activity at 50°C, for 10 min was about 80% in digestive fluid protease, and about 40% in tissue protease. HgCl₂ and DFP strongly inhibited protease activity, and the influence was greater in digestive fluid than in the midgut tissue. Most of the tissue proteases was found to be membrane bound enzyme from results of differential centrifugation and column experiments.     

Cellular localization and proposed function of midgut trehalase in the silkworm larva, Bombyx mori

A sorbitol density gradient analysis with the aid of several marker enzymes demonstrated that midgut trehalase of the silkworm larvae. Bombyx mori, was localized in the microsomal membranes, but not in mitochondria, lysosomes and microvilli at the apical surface. Electron microscopic examination showed that trehalase-enriched membrane fraction consisted of heterogeneous mixtures of membrane vesicles derived from the endoplasmic reticulum and plasma membrane parts other than the microvillus membrane. The enzyme-histochemical stains of trehalase activity on the midgut section could be detected only at the basal surface of the epithelium against haemocoel. Such a specific localization was further confirmed by immunohistochemistry with the peroxidase-conjugated antibody technique. Thus, it is concluded that midgut trehalase of silkworm larvae is situated on the plasma membrane at the basal surface of the epithelium. An intact preparation of midgut incubated in vitro in the medium containing [(14)C]trehalose could hydrolyse trehalose into glucose and take it up into the cell, although some glucose was liberated into the medium when incubated for extended periods. These results suggest that midgut trehalase plays a physiological role in utilization of haemolymph trehalose not in nutrient absorption.     

Selective diminishment of virus-specific translatable mRNA in the densonucleosis virus-infected midgut of the silkworm, Bombyx mori, reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of virus-specific translatable mRNAs was examined in the larvae of the silkworm. Bombyx mori, infected with Bombyx densonucleosis virus type 2. The results showed that incubation of infected larvae at 35 degrees C resulted in a rapid and selective decrease in the virus-specific translatable mRNAs which preexisted in the infected midgut. Upon temperature-shift from 35 degrees to 25 degrees C, the virus-specific translatable mRNAs became clearly observed within 12 hr. These results indicate that a supraoptimal temperature restricts the accumulation of virus-specific translatable mRNAs. Both rapid decay and suppressed synthesis might be responsible for the selective restriction of virus-specific translatable mRNA accumulation at a supraoptimal temperature.     

Developmental changes in midgut trehalase activity and its localization in the silkworm, Bombyx mori

The changes in trehalase activity and its localization in the midgut of the silkworm, Bombyx mori, were studied during larval-pupal-adult development. Trehalase activity in larval midgut epithelium increased with the larval growth, reached a maximum level at the middle of the fifth instar, and then decreased gradually. Trehalase activity in larval midgut was found in the epithelial tissue but not in the digestive juice or the midgut contents.The trehalase activity in the whole midgut started to rise at the onset of spinning and increased abruptly at larval-pupal ecdysis to reach an extremely high level 3 days later. This high activity was maintained throughout the subsequent pharate adult development and dropped suddenly at emergence. The midgut trehalase activity during pupal-adult development was mainly found in the midgut contents but scarcely any in the epithelium.Subcellular distribution of midgut trehalase depended upon larval-pupal-adult development. The activity was concentrated in a precipitate fraction of the epithelium until the middle of the fifth instar. During larval-pupal development, however, the activity increased in the soluble fraction with a concomitant decrease in the precipitate fraction. Almost all the trehalase activity in pupal and pharate adult midgut was recovered in the soluble fraction of the midgut contents. The data are discussed from a viewpoint of the histolysis.     

A polyhedrosis virus forming polyhedra in midgut-cell nucleus of silkworm, Bombyx mori. II. Chemical nature of the virion

A number of tumorlike lesions of the ctenidia and mantle of the freshwater bivalve Dreissena polymorpha are described. The lesions are not considered to be neoplasms and are similarly not attributable to any of the known parasites of Dreissena. No visible signs of external injury to the affected organs could be detected and it is considered that the granulomas are the result of the hemocyte response of the animal to traumatic irritation or to bacterial infection. The bacteria may be of primary or merely of secondary importance. In the extreme cases examined the granulomas comprised internally a mass of epithelioid cells and granulocytes, surrounded by a proliferation of fibroblasts. Intense inflammatory hyperplasia characterises the surrounding tissues which results, in extreme cases, in distortion of the ctenidial filaments and disturbance to the ciliary feeding currents.Such structures are rarely reported upon in molluscs and throw light on the processes involved in the response to injury in these animals.     

Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm,Bombyx mori,reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.     

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.     

Effect of high temperature on the development of nuclear polyhedrosis virus in the silkworm,Bombyx mori

Effect of a high temperature on the development of nuclear polyhedrosis and nuclear polyhedrosis virus (NPV) was studied employing pupae and isolated pupal abdomens of the silkworm, Bombyx mori. It was shown that pupae inoculated with an NPV and incubated at 35°C survived longer than those incubated at 25°C. At lower dosages of virus, pupae at 35°C escaped death from NPV. When inoculated pupae were incubated at 35°C for varying periods and then transferred to 25°C, the longer the pupae had been kept at 35°C the longer they survived. In contrast, when inoculated pupae were transferred from 25° to 35°C, the longer the pupae had been kept at 25°C the sooner after inoculation they died. Essentially the same results were obtained in isolated abdomens which were in an arrested state of development, excluding the possibility that observed thermal inhibition of viral diseases is dependent upon the altered developmental processes at high temperatures. Virus titration experiments showed that, under experimental conditions utilized, no detectable accumulation of infectious NPV was present in abdomens inoculated with an NPV and incubated at 35°C. When inoculated abdomens were shifted up from 25° to 35°C at 3 days postinoculation, NPV accumulation was inhibited almost immediately, and when inoculated abdomens were shifted down from 35° to 25°C, infectious NPV started to accumulate as early as 1 day after the shift. It was also shown that the pattern of infectious NPV accumulation and that of nucleic acid increase in infected abdomens gave a rough correlation. These results indicate that the thermal inhibition of viral diseases is attributed, at least in part, to the restricted accumulation of infectious progeny and suggest that the virus replication mechanism itself is more sensitive to high temperatures than that related to other events necessary for viral replication to be initiated.