Journal of Invertebrate Pathology 105 (2010) 8490

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Journal of Invertebrate Pathology

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Proteomic analysis of nucleopolyhedrovirus infection resistance in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)
Xiaoyong Liu a, Qin Yao a, Yong Wang b, Keping Chen a,*
a b

Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, Peoples Republic of China Department of Biological Technology, Jiangsu University, Zhenjiang 212013, Peoples Republic of China

a r t i c l e

i n f o

a b s t r a c t
Silkworm hemolymph is an important defense tissue to resist bacteria and virus infections. To study the response of silkworm hemolymph in the resistance of Bombyx mori L. nucleopolyhedrovirus (BmNPV), we constructed a near-isogenic silkworm line with BmNPV resistance using highly resistant and highly susceptible parental strains. In this paper, two-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization (MALDI)-mass spectrometry were employed to investigate the differences of protein patterns in the hemolymph of the highly resistant, highly susceptible and near-isogenic silkworm strains after BmNPV was administrated to the larvae. A comparison between the proteomes of these three silkworm strains led us to identify two differentially expressed proteins, beta-N-acetylglucosaminidase 2 and aminoacylase. The expression levels of these proteins were higher in the BmNPV resistant strains. 2010 Elsevier Inc. All rights reserved.

Article history: Received 4 February 2010 Accepted 10 May 2010 Available online 13 May 2010 Keywords: Silkworm Nucleopolyhedrovirus Near-isogenic line Two-dimensional gel electrophoresis Mass spectrometry Hemolymph Beta-N-acetylglucosaminidase 2

1. Introduction Bombyx mori is a commercially important insect for production of silk and recombinant proteins, and is also a good model lepidopteran (Goldsmith et al., 2005; Nagaraju and Goldsmith, 2002). Infection of B. mori larvae with Bombyx mori nucleopolyhedrovirus (BmNPV) reduces silk production, resulting in economic damage (Chen et al., 2003). Increased understanding of BmNPV resistance in the silkworm could reduce the loss in cocoon production, and facilitate the development of new strategies for pest control (Xue, 2005). Following per os inoculation of baculovirus, the midgut cells are infected, and the infection spreads from those initial foci to tracheoblasts, and thus to the haemocoel resulting in a fatal infection (Washburn et al., 1995, 2001). A number of studies have been conducted on insect resistance to baculoviruses (Sparks et al., 2008). However the mechanism of resistance to BmNPV is not fully elucidated. Insects lack adaptive immune responses (Hoffmann, 2003), but many host factors interact to determine the particular insect response to virus infection (Cory and Myers, 2003; Sparks et al., 2008). Those factors could be grouped into the following six categories. The rst category is the pH and substrate of the gut. A few antiviral proteins have been puried from the digestive juice of silkworm larvae, such as lipase, serine proteases (Ponnuvel et al., 2003; Nakazawa et al., 2004), and NADPH oxidoreductase (Selot
* Corresponding author. Fax: +86 511 88791923. E-mail address: kpchen@ujs.edu.cn (K. Chen). 0022-2011/$ - see front matter 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.05.007

et al., 2007). The second category is physical and physiological barriers. The third category is subcellular immune mechanisms. Apoptosis or programmed cell death is an important antiviral defense mechanism used by Lepidoptera (Narayan, 2004). Larvae resist baculovirus infection by selective apoptosis of infected midgut epithelial cells and by sloughing off infected cells from the midgut before they release virions into the haemocoel (Federici and Hice, 1997; Clem, 2001). The fourth category is cellular immune responses. Hemocytes in the haemocoel are recruited to the foci of infection, phagocytosing smaller microbes and encapsulating larger invaders, yet the mechanism is poorly understood (Popham et al., 2004; Lavine and Strand, 2002). In addition, immunosuppression of pestiferous moth larvae has revealed the likelihood of a cell-mediated antiviral immune response (Washburn et al., 2000). The fth category is humoral immunity. Antiviral activity from insect hemolymph has been described (Chernysh et al., 2002). The plasma phenoloxidase of Heliothis virescens exhibits antiviral activity against several vertebrate viruses in vitro (Ourth and Renis, 1993). In vitro incubation of Helicoverpa zea single capsid nucleopolyhedrovirus (HzSNPV) with plasma from H. virescens reduced the infectivity of the virus, and the enzyme phenoloxidase may act as an innate antiviral reagent (Popham et al., 2004). In addition, baculovirus infection increased hemolin expression, and hemolin might associate with virus in the hemolymph thereby slowing the progression of virus infection (Hirai et al., 2004). The sixth category consists of developmental, environmental and genetic factors (Watanabe, 2002; Engelhard and Volkman, 1995; Granados and Federici, 1986).

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Silkworm resistance to BmNPV was dened by the differences in 50% lethal dosage (LD50) values among silkworm populations (Chen et al., 2003). Watanabe discovered a silkworm strain with a BmNPV LD50 that was 800-fold higher than other silkworm strains (Watanabe, 1966). Previously we reported a BmNPV resistant silkworm strain NB and BmNPV susceptible strain 306 with LD50 difference up to 1000-fold (Chen et al., 2003). Based on their different genetic backgrounds, we established one near-isogenic line (NIL) with resistance to BmNPV. Near-isogenic lines have been established in many species through introgression (Busov et al., 2005), which is accomplished by repeatedly backcrossing a line carrying a gene of interest (donor parent) to a line having other properties (recurrent parent). After each cross, progeny that possess the phenotype of the target gene are selected. This process results in a line that carries a small portion of genomic sequences from the donor parent. Thus, the genetic background of the nearisogenic strain is similar to the recurrent parental strain. In the present study, the ninth near-isogenic line generation strain was established and compared to the resistant and susceptible parental strains (NB and 306, respectively). To investigate the character and mechanism of inherited resistance to BmNPV, we employed proteomic analysis to study the hemolymph proteins of a near-isogenic strain of silkworm with the susceptible, recurrent parent and the donor, non-isogenic resistant parent. Two proteins were differentially expressed in the resistant silkworm strains which may contribute to the observed difference in resistance to BmNPV. 2. Materials and methods 2.1. Silkworm parental strains BmNPV resistant silkworm strain NB (LD50 = 2.5 108 polyhedral/larvae) and BmNPV-susceptible silkworm strain 306 (LD50 = 3.4 105 polyhedral/larvae) were used in this study. These strains were preserved and reared in our laboratory. The LD50 of virus (BmNPV) was determined with fth instar larvae by oral inoculation with polyhedra. One hundred larvae were used for each infection and the experiments were repeated three times. The mortality of the infected larvae was observed within 10 days, and virus infection conrmed in the hemolymph with a microscope. The LD50 values were calculated by comparison of means analysis (SPSS Inc.) and further compared using an IndependentSamples T-Test. 2.2. Preparation of near-isogenic line The near-isogenic line was prepared in accordance with the method reported (Chen et al., 2003). To generate the BC9 line (LD50 = 2 108 polyhedra/larva), females of the susceptible strain 306 were crossed with males of the resistant strain NB. The offspring were selected for resistance to BmNPV administrated at 5 106 polyhedra/larva per os. Then the selected crossing progeny of NB 306 backcrossed with strain 306. The backcrosses were conducted for nine generations followed by two generations of self-crossing. 2.3. Sample preparation Silkworm larvae were raised with mulberry leaves under a 12 h light/12 h dark photoperiod. Then, BmNPV (5 106 viruses/larva) was administrated to the fth instar larvae per os. Because BmNPV occlusion bodies introduced per os enter the hemolymph at approximately 1224 h post inoculation (hpi) (Yao et al., 2005), hemolymph samples were collected at 22 hpi (n = 15 larvae per

treatment) in the presence of protease inhibitor (Protease Inhibitor Cocktail, Amresco) and phenylthiourea (nal concentration of 2 lg/ml). The hemolymph was also collected from silkworms without infection of BmNPV as control. Hemolymph samples from each one of 15 larvae were collected and pooled for each strain, and triplicate samples were used for analysis. 2.4. Two-dimensional gel electrophoresis (2-DE) Hemolymph containing hemocytes (0.8 mL) was combined with one-fourth volume of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.2% Bio-Lyte pH 310 (Bio-Rad), 1 mM PMSF, 2 mM EDTA, and 65 mM dithiothreitol, pH 8.5). The solutions were sonicated for 3 min followed by centrifugation at 35,000g for 30 min at 4 C. The protein concentration was determined by Bradford assay (Bradford, 1976). Isoelectric focusing electrophoresis was carried out with 17 cm (pH 310) IPG strips at 20 C according to the manufacturers instructions (Bio-Rad). Briey, the strips were rehydrated under 50 V for 13 h. and isoelectric focusing was programmed at a gradient mode. The IPG strips were focused for 1 h at progressively increasing voltages, 250 V, 1000 V, 4000 V and 10,000 V, respectively, then continued at 10,000 V until reaching a total of 60 kVh. The focused strips were equilibrated in buffer with 6 M urea, 0.375 M TrisHCl, 20% glycerol, 2% SDS and a trace amount of bromophenol blue, and subsequently treated by DTT and iodoacetamide. The treated strips were transferred onto 12% SDSpolyacrylamide gels running at 5 W per gel for 15 min and 15 W per gel until the bromophenol blue dye reached the bottom of the gel. The gels were stained with Coomassie blue. The gels were scanned using an Image scanner and the image analysis was conducted with PDQuest licensed by Bio-Rad. Approximately 0.8 mg protein was loaded onto gels and each pooled sample was run three times to achieve reproducibility. Students t-tests were used to determine signicant differences. 2.5. In-gel digestion Protein spots were excised manually using a hand-held pipette with a trimmed polypropylene tip and digested by trypsin according to Mirza et al., 2000. Briey, excised spots were destained and cleaned by shaking the gels with water, 50 mM ammonium bicarbonate, 50% acetonitrile, and 100% acetonitrile, respectively. After reduction with dithiothreitol and derivatization with iodoacetamide, the gel pieces were treated with freshly prepared trypsin (Promega) solution (20 lg/ml in 40 mM ammonium bicarbonate/ 10% acetonitrile) and incubated at 37 C overnight. The gels were extracted with 5% formic acid in 1:1 (v/v) water/acetonitrile. 2.6. Mass spectrometry analysis Peptide mixture (1 ll) was mixed with an equal volume of 10 mg/ml a-cyano-4-hydroxycinnamic acid (Sigma) saturated with 50% acetonitrile in 0.1% triuoroacetic acid, and analyzed by Matrix-Assisted Laser Desorption/IonizationTime-of-Flight (MALDI-TOF) Mass Spectrometer (Bruker, Ultraex tof/tof), to acquire spectra with a mass range from 1000 to 4000 Da. External calibration was performed with standard peptides. The matrix and the autolytic peaks of trypsin served as internal standards for mass calibration. 2.7. Protein identication Proteins were identied by comparing the masses of peptides to theoretical tryptic peptides of the NCBI protein database and a self-constructed silkworm EST translated database. Peptide mass

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ngerprinting (PMF) was performed by the MASCOT program licensed by Matrix Science Ltd. 2.8. Beta-N-acetylglucosaminidase activity assays Beta-N-acetylglucosaminidase activity was measured from hemolymph of the fth instar using 4-Nitrophenyl-N-acetyl-Nacetyl-beta-D-glucosaminide (PNP-NAG, Sigma) as substrate. The reaction mixture contained 20 ll of hemolymph, 0.1 M phosphate buffer at pH 5.6 and 0.2 mM PNP-NAG in a total volume of 2 ml. After incubation at 37 C for 10 min, the reaction was terminated by the addition of 2 ml of 0.5 M NaOH, and the concentration of released p-nitrophenol (pNP) was determined by OD405 using a molar extinction coefcient of 8.8 103 l/ (mol cm2). One enzyme unit (U) was dened as 1 mol of PNP released per min at 37 C. The beta-N-acetylglucosaminidase activities were measured thirty times using the hemolymph from different fth instars for each strain of NB, 306 and near-isogenic line BC9. 3. Results 3.1. Comparative analysis by two-dimensional gel electrophoresis At least 360 different protein spots were identied in hemolymph using 2-DE (Fig. 1). Because strains NB and BC9 were resistant to BmNPV compared to strain 306, spots having similar intensity in samples from the infected and uninfected resistant strains NB and BC9 were compared to the similarly treated susceptible 306 strain. Differences were observed in the protein expression proles for the resistant and susceptible strains infected with BmNPV (panes A and B of Fig. 1). Three-dimensional computational reconstructions of the protein spots contained in panes A and B of Fig. 1 show increased expression of two proteins, labeled 1 and 2 (Fig. 2). Proteins at two spots were found expressed more than 5-fold in strain NB and BC9 compared to strain 306. The normalized spot quantity (ppm OD Area) of proteins (1 and 2) and the average intensity were indicated in Fig. 3. And the average intensity ratios of protein spots (1 and 2): Spot No 1. NB/

Fig. 2. The difference between two proteins from panes A and B. The circle indicate two different proteins, (A) protein No. 1 is different within six treatments and (B) protein No. 2 is different within six treatments. Panes A and B are enlarged from Fig. 1. The lower panels show their corresponding 3D views.

306 = 5.57, NBBmNPV/306BmNPV = 6.90, BC9/306 = 5.73, BC9 BmNPV/306BmNPV = 6.69. Spot No. 2 NB/306 = 15.58, NB BmNPV/306BmNPV = 14.11, BC9/306 = 8.43, BC9BmNPV/306 BmNPV = 8.03. The two spots were quantied by densitometry analysis as shown in Fig. 3, which indicated that the amount of protein at the two spots was signicantly higher in strain NB and BC9 (p < 0.001).

Fig. 1. The protein expression prole of hemolymph in silkworm strain NB (highly resistant strain), 306 (highly susceptible strain) and BC9 (the ninth generation NIL). NB BmNPV, NB strain with BmNPV administrated; 306BmNPV, 306 strain with BmNPV administrated; BC9BmNPV, BC9 strain with BmNPV administrated; panes A and B, show protein spots within the two areas that were expressed differently between BmNPV resistant strains and the susceptible strain 306 and which are magnied in Fig. 2.

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3.2. Mass spectral data analysis The identied two protein spots were excised from 2-DE gels and analyzed by MALDI-TOF mass spectrometry. Fig. 4 shows the mass spectrometry data of the two identied proteins. Details of masses and peptides are listed in Table 1. Protein 1 was identied as silkworm beta-N-acetylglucosaminidase 2 with the NCBI accession number of gi|139004977. Protein 2 was identied as silkworm aminoacylase protein with the NCBI accession number of gi|114052174. Their Mascot scores were as high as 157 and 102, respectively (p < 0.05). 3.3. Beta-N-acetylglucosaminidase activity assays The beta-N-acetylglucosaminidase activity in BmNPV resistant strains NB and the near-isogenic line BC9 was signicantly higher than that in the BmNPV susceptible strain 306 (Students t-tests; p < 0.05), with activity of 1.976 0.17 U/l, 1.862 0.14 U/l and 1.402 0.21 U/l, respectively (Fig. 5). These results are consistent with the nding that hemolymph isolated from strains NB and BC9 possessed greater quantities of beta-N-acetylglucosaminidase protein (Figs. 2 and 3). 4. Discussion Silkworm strains and cell lines have been employed for studying silkworm resistance to BmNPV in many labs (Bao et al., 2009). Previously we reported a highly resistant strain and a highly susceptible strain (Chen et al., 2003). Herein, we constructed a ninth generation near-isogenic line with resistance to BmNPV. In theory, the minor difference between their genomes included all or part of the genes contributing to the BmNPV resistance. Using the aforementioned silkworm strains, we had identied some genes and molecular markers related to BmNPV resistance (Xu et al., 2005; Yao et al., 2003). In addition, the protein expression prole from NILs hemolymph was more similar to that of the susceptible parental strain than that of the highly resistant strain.

Fig. 3. The diagrams demonstrate changes in normalized spot quantity of the protein spots within six treatments. NB, highly resistant strain; NBBmNPV, NB strain with BmNPV administrated; 306, highly susceptible strain; 306BmNPV, 306 strain with BmNPV administrated; BC9, the ninth generation NIL; BC9BmNPV, BC9 strain with BmNPV administrated; Protein No. 1 is above and Protein No. 2 below.

Fig. 4. Peptide mass ngerprinting of two protein spots extracted from 2-DE gels. MALDI-TOF MS analysis of tryptic digest. (1) Beta-N-acetylglucosaminidase 2. (2) Aminoacylase.

88 Table 1 Peptide masses for PMF of proteins 1 and 2. Spot Protein name no. 1 Beta-Nacetylglucosaminidase 2

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Monoisotopic masses 2193.3049, 1754.9930, 1770.9865, 1450.8539, 919.5635, 1889.0690, 1018.6360, 1812.0475, 1805.0795, 956.5242, 860.5331, 1408.8531, 1536.9370, 1321.8099, 1193.7017, 1366.8173, 1002.5591, 1600.9570, 2523.3054, 2679.4734, 1466.8529, 2018.1964, 1669.9135, 795.0262, 944.4905

Matched peptides K.GAIWPRPQMQSIEIPYYK.F, K.VMDHDCPILSNAVQR.S, K.VMDHDCPILSNAVQR.S, R.DMLRIASPYVNR.N, R.IASPYVNR.N, R.NAPQQVLDDDTYDGPLK.S, R.SSSIWGILR.G, R.GLESWTHLFHLSDNR.D, R.DQLHINKGEVHDFPR.Y, K.GEVHDFPR.Y, R.GLLVDTSR.H, R.LGAYHETLIYTK.K, R.LGAYHETLIYTKK.D, K.KDIQTVIDYAR.N, K.DIQTVIDYAR.N, R.VIPEFDVPGHTR.S, K.DSTYTFLR.E, R.ELFHEVQALFPDR.Y, R.YIHIGGDEVDLDCWESNPEFK.R, R.YIHIGGDEVDLDCWESNPEFKR.Y, K.GNEVYEMLNILR.A, R.ASHQLIYSSGWYLDHLK.T, K.TGGDWTEFFNKDPR.D, R.LWGHESQAAYQVYSR.L, R.LEEHTCR.M K.SDPSVSTLQNYLR.I, R.SVHPNVDYNECINFLK.N, K.IGLQVQVVEPLPK.K, K.SVGIQYIEAVR.R, K.SVGIQYIEAVRR.L, R.TVHLSFVPDEEIGGDTGMGK.F, R.TVHLSFVPDEEIGGDTGMGK.F, K.NMNVGFALDEGVASPNDDYLVFNGER.I, K.NMNVGFALDEGVASPNDDYLVFNGER.I, K.SGHGSLLLPDNCGEK.L, R.YIIDKFMDLR.Q, R.IALNVDLKEFENMIQK.W, K.EFENMIQK.W, K.WCTEAGR.G, K.DPYTTPTQLDDANIYWK.A, K.QTAQELR.M, R.MSIKPQTFTGGTDSR.Y, R.MSIKPQTFTGGTDSR.Y

Aminoacylase

1479.7985, 1949.0435, 1419.8822, 1234.6853, 1390.7955, 2089.1299, 2105.1343, 2843.4856, 2859.5122, 1583.7773, 1329.7029, 1921.1141, 1054.5076, 879.4034, 2041.0735, 845.4709, 1625.8475, 1641.8622

Fig. 5. The beta-N-acetylglucosaminidase activity of hemolymph in silkworm strain NB (highly resistant strain), 306 (highly susceptible strain) and BC9 (the ninth generation NIL). : The enzyme activity in NB and BC9 was signicantly higher than that in 306 (p < 0.05).

BmNPV initially infectes the midgut cells of silkworm and is subsequently detectable in hemolymph by real time PCR within 24 h (Yao et al., 2005). Resistance to BmNPV could be attributed to the rst line of defense, the midgut cell apical membrane. But in our previous studies, it was found that the virus proliferates much faster in the hemolymph of the susceptible strain when BmNPV virions were injected into hemolymph (unpublished data). Therefore, hemplymph also contributes to BmNPV resistance. Therefore, in the present study we compared the protein expression proles of the hemolymph of BmNPV resistant strains and a susceptible strain. In addition, the BmNPV resistance related protein maybe expressed only on virus infection, so we studied the protein expression proles with or without virus infection. Through this work, we identied two proteins expressed differently in hemolymph between resistant and susceptible strains, which might contribute to BmNPV resistance. Moreover, beta-N-acetylglucosaminidase activity assay demonstrated that enzyme activity of the protein was much higher in silkworm strains NB and BC9 than that in strain 306. Some of the beta-N-acetylglucosaminidase activity was associated with membranes in Sf9 and other lepidopteron insect cells (Altmann et al., 1995; Tomiya et al., 2006). Beta-N-acetylglucosaminidase showed broad substrate specicity, and it cleaves terminal N-acetylglucosamine residues from the a-3 and a-6 branches

of biantennary N-glycan substrate and hydrolyzes chitotriose to N-acetylglucosamine monomers (Aumiller et al., 2006; Okada et al., 2007; Geisler et al., 2008). Nucleopolyhedrovirus exists in two structurally and functionally distinct virion phenotypes during the infection cycle, the occlusion-derived virion (ODV) and the budded virion (BV). The BV mediates movement of the viral infection from the midgut to other tissues and propagation of the infection from cell to cell within the infected animal. BV enters cells via receptor-mediated endocytosis, GP64 is essential for cell-to-cell transmission of the virus in cell culture and in infected animals (Monsma et al., 1996). GP64 serves two major roles during virus entry. First, GP64 is involved in host cell receptor binding. Second, GP64 mediates the low-pH-triggered membrane fusion activity necessary for release of the nucleocapsid into the cytosol during entry by endocytosis (Zhou and Blissard, 2008). Gp64 is known to be phosphorylated, acylated, and glycosylated in baculovirusinfected insect cells (Oomens et al., 1995). It had been reported previously that N-glycosylation of AcMNPV GP64 is important for its intracellular transport and fusogenic activity, as well as for BV infectivity (Jarvis and Garcia, 1994; Jarvis and Finn, 1995). And mutational analysis of the N-linked glycans on Autographa californica nucleopolyhedrovirus GP64 indicated that the amounts of infectious progeny produced by AcMNPV mutants lacking one, two, or three N-linked glycans on GP64 were about 10- to 100-fold lower than wild-type levels (Jarvis et al., 1998). BmNPVGP64 was located in the membrane and cytoplasm of the infected host cells. The production of infectious budded virus and the fusion activity were reduced when glycosylation of GP64 was inhibited (Rahman and Gopinathan, 2003). Several lines of evidence indicate that glycosylation of viral envelope proteins is a molecular determinant for virus replication and infectivity (Scherret et al., 2001; Shi et al., 2007; Shirato et al., 2004; Risatti et al., 2007). Previous studies showed that beta-N-acetylglucosaminidase accounts for differences in glycosylation of inuenza virus hemagglutinin expressed in insect cells from a baculovirus vector (Wagner et al., 1996). And cellular beta-N-acetylglucosaminidase expressed in baculovirus-infected TN5 cells results in depletion of N-acetylglucosamine residues and this can be reversed using specic inhibitors of betaN-acetylglucosaminidase (Watanabe et al., 2002). So we speculate that the excessive expression of beta-N-acetylglucosaminidase in hemolymph of the resistant silkworm can disturb the N-linked glycans of GP64 protein on the cell membrane which is an essential for initiating secondary infections, and thus reduce the

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reproduction of infectious viruses. The gene of silkworm beta-Nacetylglucosaminidase 2 was reported (Accession No. AB 286958) (Okada et al., 2007). Another identied protein, aminoacylase is a class of enzymes involved in hydrolysis of N-acetylated proteins. N-terminal acetylation of proteins is a widespread and highly conserved process that is involved in protection and stability of proteins (Polevoda and Sherman, 2003). It is interesting to note the Gp64 is also acylated. The actual roles of beta-N-acetylglucosaminidase 2 and aminoacylase in BmNPV resistance require further studies. There are no obvious differences in the expression levels of these two proteins between infected and uninfected larvae of the same strain. This suggests that expression levels of the identied proteins are continous high in the resistant strains and are not induced by viral infection. Elevated, but not induced activity of these enzymes suggests that it may protect from initial infection. Since the silkworm resistant strain under investigation is a near-isogenic line, not completely isogenic, to the susceptible strain, the different protein expression proles observed might be due to the remaining genetic differences between the near-isogenic line and the recurrent parent 306, rather than due to the BmNPV resistance factors. Acknowledgments This work was supported by the National Basic Research Program of China under Grant No. 2005CB121000, the National High Technology Research and Development Program of China under Grant No. 2008AA10Z145, the Agricultural High-tech Project of Jiangsu under Grant No. BE2008379, and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China under Grant No. 06KJD180042. References
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