Spread of recombinant Autographa californica nucleopolyhedrovirus in various tissues of silkworm Bombyx mori determined by real-time PCR

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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 373 (2008) 147–153 www.elsevier.com/locate/yabio

Spread of recombinant Autographa californica nucleopolyhedrovirus in various tissues of silkworm Bombyx mori determined by real-time PCR Yi Zhang

b

a,b,1

, Baozhong Tian b,c,1, Huanzhang Xia a, Tingqing Guo b, Jianyang Wang b, Shengpeng Wang d, Zhenguo Wei b,c, Changde Lu b,*

a School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Liaoning 110016, People’s Republic of China State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China c School of Material Engineering, Soochow University, Jiangsu 215006, People’s Republic of China d Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, People’s Republic of China

Received 19 July 2007 Available online 8 September 2007

Abstract A cassette harboring luciferase reporter driven by Bombyx mori A3 promoter was transferred to the bacmid AcDEGT to generate the recombinant virus AcNPVA3Luc (where Ac represents Autographa californica, NPV represents nucleopolyhedrovirus, and A3Luc represents the firefly luciferase reporter cassette driven by the A3 promoter). Recombinant baculovirus was injected into the hemocoele of newly ecdysed fifth instar larvae of the silkworm. The infection of virus in various silkworm tissues was determined by real-time PCR. The profile of viral infection showed that the copy number of recombinant AcNPV (rAcNPV) increased the fastest in the hemocyte, followed by the fat body, Malpighian tubule, middle gut, and silk gland. Detecting in nonpermissive strain silkworm showed that there was no significant difference in the entry of rAcNPV into all tested tissues. The difference in viral infection reflected mainly the big difference in replication of rAcNPV in various tissues of silkworm larvae. Real-time quantitative RT–PCR showed that it was due to the different expression of genes involved in viral DNA replication.  2007 Elsevier Inc. All rights reserved. Keywords: Real-time PCR quantification; Viral infection; Silkworm tissues; Replication of virus; rAcNPV

The silkworm Bombyx mori has been studied for its susceptibility to wild-type Autographa californica multiple nucleopolyhedrovirus (AcMNPV)2 and is recognized as *

Corresponding author. Fax: +86 21 54921011. E-mail address: [email protected] (C. Lu). 1 These authors contributed equally to the study and should be considered as joint first authors. 2 Abbreviations used: AcMNPV, Autographa californica multiple nucleopolyhedrovirus; rAcNPV, recombinant A. californica nucleopolyhedrovirus; EGFP, enhanced green fluorescent protein; FibH, fibroin heavychain gene; BmNPV, Bombyx mori nucleopolyhedrovirus; rBmNPV, recombinant B. mori nucleopolyhedrovirus; Luc, luciferase; PCNA, proliferating cell nuclear antigen; p143, DNA helicase; IE-1, immediately early protein-1; rRNA, ribosomal RNA; BV, budded recombinant virus; TCID50, tissue culture infectious dose 50; FBS, fetal bovine serum; Tm, melting temperature; cDNA, complementary DNA; GFP, green fluorescent protein; mRNA, messenger RNA. 0003-2697/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.09.004

being nonpermissive to AcMNPV infection either through oral ingestion or by intrahemocoelical injection [1–3]. In our previous work, we found that some strains of silkworm are permissive to recombinant A. californica nucleopolyhedrovirus (rAcNPV) [4]. Testing of a wide range of 31 strains of silkworm B. mori for intrahemocoelical rAcNPV infection led to the identification of 14 permissive strains and 17 nonpermissive strains, indicating that the intrahemocoelical infection of AcNPV to the silkworm is not a rare and isolated phenomenon [5]. Using rAcNPV vector, silk gland-specific secretory expression of the enhanced green fluorescent protein (EGFP) gene in the silkworm was achieved [6]. This was the first report about the silk gland bioreactor. rAcNPV has also been used as a gene transfer vector in investigations of tissue specificity of promoter activity [7,8] and in studies of the secretion

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Spread of rAcNPV in tissues of silkworm / Y. Zhang et al. / Anal. Biochem. 373 (2008) 147–153

of fibroin heavy-chain gene (FibH) of silkworm [9,10]. There are several advantages to using rAcNPV as a gene delivery vector in silkworm. First, the budded form of rAcNPV cannot infect silkworm larvae by oral ingestion, so it is safe to use in the laboratory. Second, the rAcNPV system is constructed using the ‘‘Bac-to-Bac’’ system, so it is simple and easy to select and purify the recombinant plaque. The Bac-to-Bac rAcNPV system is commercially available; although Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid has been developed [11], it is not commercially available. To facilitate further application of rAcNPV in the functional study of silkworm genes, and to construct a baculovirus-based silkworm bioreactor, it is important to know the spread of rAcNPV in various tissues of silkworm larvae post-intrahemocoelical injection of rAcNPV. The natural infection process of AcMNPV in its host insect, Trichoplusia ni, has been studied by immunohistochemistry and tracing of the expression of a reporter gene [12–14]. Infection of recombinant B. mori nucleopolyhedrovirus (rBmNPV) in silkworm larvae has also been investigated [15]. The rAcNPV that was used in our research is a budded form of virus; however, the spread of the budded form of rAcNPV in various tissues of susceptible strains of silkworm post-intrahemocoelical injection of rAcNPV remains unclear. In previous work, the methods for determining the multiplication of virus in different tissues of insects were either semiquantitative, such as dot hybridization, or indirect, such as the use of reporter gene, with low sensitivity [4,15]. The high sensitivity and accuracy of the realtime PCR method make it very useful to the studies of gene expression [16], determination of plasmid copy number in bacteria [17], determination of copy number of genes inserted in transgenic plants [18], bacteriophage titer assays [19], diagnostics in clinical microbiology [20], and the like. Real-time PCR has also been used in determining replication and release of baculovirus in culture cells [21] and in determining titer [22,23]. Therefore, we decided to use real-time PCR in the determination of copy numbers of rAcNPV genome in various tissues of B. mori larvae. In the current study, the recombinant virus AcNPVA3Luc, containing the firefly luciferase (Luc) reporter cassette driven by the A3 promoter, was injected into the hemocoele of newly ecdysed fifth instar larvae of silkworm. The Luc gene of rAcNPV were taken as an exogenous target gene, and the FibH gene of silkworm was taken as an intrinsic reference gene, for monitoring the replication of viral genome in different silkworm tissues. To observe different replications of rAcNPV in tissues of silkworm larvae, the expression of several viral genes, including proliferating cell nuclear antigen (PCNA), DNA helicase (p 143), and an early gene transactivator (immediately early protein-1 [IE-1]), were analyzed by real-time quantitative RT–PCR using highly expressed 28S ribosomal RNA (rRNA) as an intrinsic reference.

The spread of rAcNPV in various silkworm tissues is discussed. Materials and methods Plasmids, recombinant bacmids, and recombinant viruses A donor plasmid pFNA3Luc containing Luc expression cassette driven by the A3 promoter was constructed. First, a Luc gene was cut from pGL2basic (Promega, USA) with HindIII and HpaI and then inserted into the same sites in pFFa2 [4] to produce pFNLuc. Then the 671-bp A3 promoter of B. mori (positions 1764–2432 in the B. mori cytoplasmic actin [A3] gene BMU49854) was excised from pA3egfp [4] with BglII and HindIII and then ligated to the BamHI and HindIII sites of pFNLuc to yield pFNA3Luc. The construction was verified by DNA sequencing and restriction mapping. Donor plasmid pFNA3Luc was transferred to the bacmid AcDEGT [4] to generate the recombinant bacmid pBacAcNPVA3Luc. The bacmid pBacAcNPVA3Luc was identified with PCR (not shown). The purified bacmid was then used to transfect Sf9 culture cells with Cellfectin (Invitrogen, USA) to produce the budded recombinant virus (BV) AcNPVA3Luc. Generation and large-scale harvest of the recombinant baculovirus were carried out according to the instruction manual (Invitrogen) using the Sf9 cell line. Virus titer was determined by the tissue culture infectious dose 50 (TCID50) method described in our previous work [4]. Plasmids included pFNA3Luc and p5L, which contains the FibH gene [9] were used in constructing standard curves. Cell culture, B. mori inoculation, and dissection The Sf9 cells were maintained in Grace’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) at 27 C. B. mori strains 54A and Qingsong were provided by the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (China). B. mori larvae were routinely reared on Mulberry leaves at 27 C. An aliquot of 10 ll recombinant baculovirus (106 pfu) was injected into the hemocoele through the interval membrane of the abdominal segment of newly ecdysed fifth instar larvae. At each indicated time point (1, 2, 3, and 4 days postinjection), 10 larvae were randomly taken and dissected, and tissues from 10 larvae were put together as one sample at the time point. Briefly, one abdominal leg of each larva was cut to bleed, and hemocyte was collected from blood by centrifugation at 4000g for 5 min. After bleeding, the B. mori larvae were dissected, and the fat body, silk glands (also separated into anterior, middle, and posterior silk glands at the indicated time), Malpighian tubule, and middle gut tissues were collected after rinsing in buffer A (10 mM Tris–HCl [pH 8.0] and 50 mM NaCl) and stored at –70 C until use. Meanwhile, 10 uninjected larvae were dissected, and DNA of tissues was extracted as negative controls.

Spread of rAcNPV in tissues of silkworm / Y. Zhang et al. / Anal. Biochem. 373 (2008) 147–153

149

Sample preparation

Extraction of RNA and reverse transcription

After 300 ll of buffer A was added to the hemocyte pellets, the samples were homogenized. The supernatant was collected by centrifugation at 8000 rpm for 5 min at 4 C, and 200 ll of supernatant was extracted for 1 h with phenol and then was extracted three times with chloroform. The aqueous phase was retained as the DNA extract for real-time PCR assay. Appropriate volumes of buffer A (2 ml for 1 g tissue) were added to samples of fat body, silk gland, Malpighian tubule, and middle gut. The tissues were ground and then centrifuged at 8000 rpm for 5 min at 4 C. Then 250 ll of supernatant was collected and extracted with phenol and chloroform as for the hemocyte samples. UV absorption for every DNA extract was measured at a wavelength of 260 nm (DU7400, Beckman, USA). Every DNA extract was diluted with doubly distilled H2O to a specific concentration (see each experiment) and was subjected to real-time PCR.

Total RNA was extracted from various tissues of silkworm larvae at 24 h postinjection using TRNzol-A+ Total RNA Reagent (Tiangen Biotech, China) following the manufacturer’s instructions. Primary complementary DNA (cDNA) was synthesized in a final volume of 20 ll: 4 ll of 5 · reaction buffer, 1 lg of Total RNA, 0.5 mM of each dNTP, 25 U of RNasin (40 U/ll), 1 ll of 50 lM (dN)6, 2 ll of 10 lM oligo(dT15), and 200 U of M-MuLV reverse transcriptase (200 U/ll) (TaKaRa Biotechnology, China).

Primers for real-time PCR The Luc gene was taken as a viral gene for monitoring the multiplication of the rAcNPV genome in different B. mori tissues. The B. mori FibH gene was chosen as an internal control of the host genome. The amplifying fragments that we designed for all genes in real-time PCR were approximately 150 bp, and we made them as close as possible. PCR primers designed using Clone Manager software (version 7.01) are listed in Table 1. Because both the melting temperature (Tm) values for primers and the lengths of all amplified fragments were very similar, the PCR amplification efficiency with these primer sets should be the same. Primer pairs for real-time RT– PCR were designed as above, and their sequences, Tm values, and lengths of product are also listed in Table 1. The primer pairs were tested with several samples by PCR, and the PCR products were assayed by electrophoresis on a 6% polyacrylamide gel. Results showed that these pairs of primers were specific for their respective genes (not shown).

Real-time quantitative PCR protocol and treatment of realtime PCR data Real-time quantitative PCR was performed in a DNA Engine Option 2 thermal cycler (MJ Research, USA) using the SYBR Premix Ex Taq Kit (TaKaRa Biotechnology). The reaction volume was 30 ll. The real-time PCR protocol consisted of an initial denaturation at 95 C for 1 min, followed by 50 cycles: 95 C for 5 s, 55 C for 10 s, 72 C for 10 s, and 80 C for 2 s. According to the melting curves, the Tm values of PCR products were 82 and 83 C; fluorescence data were acquired at 80 C, a temperature above the melting temperature of nonspecific products [24]. At the end of PCR, a melting curve was produced by monitoring fluorescence continuously while slowly heating the samples from 50 to 95 C at intervals of 1 C. To the same sample, each target gene was amplified using a specific primer pair in separate PCR reactions. Serial dilutions of plasmid pFNA3Luc and p5L were used to construct standard curves, with the gene copy number being calculated using the equation of the standard curve. The ratio of copy numbers of the viral gene (Luc) to the host gene (FibH), (AcNPV/B. mori) was calculated by [C(T)Luc – C(T)FibH]/a = log(Luc/FibH), where a is the slope of the standard curve. Real-time quantitative RT–PCR was performed in the same instrument with the same protocol using products of reverse transcription as templates. Given the huge difference in transcriptional expression of target genes in various

Table 1 Primer pairs for real-time PCR primer P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12

Sequence 0

0

5 -CGGTAACGAGTCCATTGTAG-3 5 0 -CCTTGATGAGTGCTGTATCC-3 0 5 0 -AAACGCTGGGCGTTAATCAG-3 0 5 0 -TCGTCCCAGTAAGCTATGTC-3 0 5 0 -GACGCAATCGTAATCGCTATCC-3 0 5 0 -AGCGCAGCTCTGTACTGATG-3 0 5 0 -ACTCTCCGCTGTGGTTGTC-3 0 5 0 -GCCGCTAGTTTGGCCATTC-3 0 5 0 -TAAACTGGCCCACCACACC-3 0 5 0 -TGAGCAGCCTGTTGTGGAG-3 0 5 0 -CCCAGTGCTCTGAATGTCAAC-3 0 5 0 -AGATAGGGACAGTGGGAATCTC-3 0

Tm C

Target gene

length of product and Tm

57.80 57.80 57.80 57.80 60.07 59.85 59.72 59.72 59.72 59.72 59.97 60.07

FibH

147 bp, 83 C

Luc

145 bp, 83 C

PCNA

152 bp, 83 C

p143

148 bp, 83 C

IE-1

155 bp, 83 C

28s rRNA

150 bp, 82 C

150

Spread of rAcNPV in tissues of silkworm / Y. Zhang et al. / Anal. Biochem. 373 (2008) 147–153

tissues of B. mori larvae, to avoid the effect of template concentration on the efficiency of PCR, we adjusted the template amount by dilution of reverse transcription and made the threshold cycles in the 15–35 region. For the 28S rRNA assay, all samples were diluted by 105-fold. For the PCNA, p143, and IE-1 assays, the hemocyte sample was diluted by 102-fold. Samples from other tissues were diluted by 10-fold. Results and discussion Infection of rAcNPV in various tissues of B. mori larvae The copy number of the Luc gene in various tissues of silkworm strain 54A larvae injected with AcNPVA3Luc was determined, and the standard curve was constructed simultaneously with same amount of control silkworm DNA and using the plasmid pFNA3Luc as standard. The profile of the spread of AcNPVA3Luc in silkworm strain 54A is shown in Table 2. Results showed that rAcNPV can be detected in the hemocyte, fat body, and silk gland at 1 day postinjection. They indicated the entry and multiplication of rAcNPV in the hemocyte, fat body, and silk gland, albeit with different extents of spread. The copy number of rAcNPV in the hemocyte reached saturation at 2 days postinjection, whereas it needed 4 days in the fat body and was still increasing at 4 days postinjection in the silk gland. The profile of viral spread showed that rAcNPV increased the fastest in the hemocyte, followed by the fat body and silk gland. This is in agreement with our previous results determined by the dot blot method [4]. Our previous work showed that the viral titer in larval hemolymph dropped quickly after injection and then rose after 12 h postinjection and was maintained at the higher level after 24 h postinjection. According to the copy number of virus in different tissues, the early rising of viral titer in larval hemolymph was contributed mostly by the hemocyte and subsequently was also contributed by the fat body tissue. This is similar to the observation that direct injection of BV of BmNPV into the hemocoele of silkworm larvae resulted in initial infection and multiplication of the virus in the hemocyte [15].

the copy number of the Luc gene was measured using primers P3 and P4, the copies of the FibH gene were measured simultaneously under the same conditions using primers P1 and P2. The time course of the ratio of Luc/FibH in various tissues is shown graphically in Fig. 1. The viral copy number in cells was the result of a dynamic balance of virus entry, multiplication in the cells, and release of virus to the hemolymph at the same time as the cellular genomic DNA was synthesizing continuously. The slope of the time curves showed the relative increase of rAcNPV to cellular genomic DNA. It was observed that the increase of viral copies was much faster than the increase of cellular DNA in the hemocyte, followed by the fat body cells, and the increase of the ratio of virus genome to host genome was the lowest in the silk gland. The amplification of genomic DNA in the silk gland was very fast during the fifth instar of silkworm larvae, especially in the posterior silk gland. However, the increase of rAcNPV was still faster than the increase of cellular genomic DNA. This ratio also showed the kinetic infection of recombinant AcNPV in different tissues of silkworm larvae. The curves showed that in the hemocyte the increase of virus was faster than that occurring in the fat body or silk gland. The copy number of recombinant virus reached saturation by multiplication at 2 days postinjection in the hemocyte. In contrast, in the fat body the increase of virus was less and an exponential increase phase could be seen at 2 days postinjection. In the silk gland, viral replication started to increase rapidly at 3 to 4 days postinjection. The ratio of rAcNPV genome to host genome was highest at approximately 90-fold in the hemocyte. The genome of silkworm is 4.5 · 108 bp, whereas the genome of AcNPV is 1.34 · 105 bp [25], so even though the copy of viral genome was 100-fold higher than that of host genome, it was only 3% of the total DNA. Thus, the profile of relative dosage was close to that obtained by absolute measurement.

100 80

The relative dosages of virus genome to host genome in various tissues of silkworm larvae were measured. When Table 2 Kinetics of AcNPVA3Luc infection in different tissues of B. mori larvae Tissue

1 day postinjection

2 days postinjection

3 days postinjection

4 days postinjection

Hemocyte Fat body Silk gland

172,683 24,238 595

1,991,042 152,605 2,004

1,160,806 1,069,995 16,589

1,340,708 1,366,567 74,798

Note. Data are averages of three measurements. Data show copy numbers of virus DNA within 0.0018 A260 unit of DNA extracts.

Luc/FibH

Relative dosage of rAcNPV to host genome

60 40 20 0 0

1

2

3

4

5

Day Fig. 1. Time course of copy ratio of luciferase to fibroin heavy-chain genes (Luc/FibH) in various tissues of B. mori. , hemocytes; j, fat body; m, silk gland.

Spread of rAcNPV in tissues of silkworm / Y. Zhang et al. / Anal. Biochem. 373 (2008) 147–153

rAcNPV can enter and replicate in various tissues of silkworm The copy numbers of AcNPVA3Luc in the Malpighian tubule, middle gut, and different parts of the silk gland (anterior, middle, and posterior) were determined. Fig. 2 shows the copy numbers of AcNPVA3Luc in 0.0018 A260 unit of total DNA in different tissues of silkworm larvae at 4 days postinjection. Results showed that recombinant virus can enter and replicate in all kinds of silkworm tissues analyzed. (Our primary assay showed that rAcNPV can enter and replicate in ovary and testis as well [data not shown].) Interestingly, the viral copies in 0.0018 A260 unit of total DNA was the highest in the anterior silk gland, followed by the fat body, hemocyte, middle silk gland, Malpighian tubule, middle gut, and posterior silk gland. The viral copy number measured in a certain amount of total DNA not only was dependent on the multiplication rate of viral genome in different tissues but also was affected by the proliferation of cells and replication of cellular genomic DNA in various tissues. The simultaneous increase in cellular DNA and viral DNA causes a decrease in the copy number of recombinant virus in a given amount of total DNA. Results from this work showed that the viral copy number in a certain amount of total DNA was the lowest in the posterior silk gland, yet it was the highest in the anterior silk gland. The difference of the viral copy number in a certain amount of total DNA in two parts of the silk gland reached approximately 50 times. During the larval stage, the genomic DNA in the cells of the silk gland is amplified dramatically by a factor of tens of thousands, corresponding to the enlargement of silk gland cells until the third day of the fifth instar [26]. Early work indicated that the amplification of cellular DNA in anterior silk gland cells is lower than that in posterior silk gland cells. The overall DNA synthesis from the diploid value is estimated to correspond to 18 to 19 endomitotic cycles in the nuclei of the posterior silk gland, as compared with only 13 endomitotic cycles in those of the anterior silk gland [27]. The increase of cellular genomic DNA is 32 to 64 times greater, corresponding to the replication of five

151

to six endomitotic cycles. Therefore, dilution of viral DNA by cellular DNA can explain the difference in viral copy number in these two parts of the silk gland. Regarding the fat body, Malpighian tubule, and middle gut, the copy numbers of virus in a certain amount of total DNA were less than that in the anterior silk gland and more than that in the posterior silk gland. Cells in these tissues divided continually in fifth instar larvae as well. The complete cell cycle in normal cells should be longer than the endomitotic cycle, which is lacking the G2/M phase, in the nuclei of the posterior gland; thus, the increase of cellular DNA in these tissues might be slower than that in the posterior silk gland. From this point of view, the extent of rAcNPV infection in the silk gland might be more than what is observed in Table 1. The natural route of AcMNPV (or BmNPV) infection is oral ingestion of viral polyhedra by the host larvae, and the infection spreads from the middle guts through the tracheae to the hemocyte and fat body as well as other tissues. It has been found that the infected region was mostly localized to the cells associated with the tracheae [12–15]. The virus multiplication has not been observed in the anterior silk gland, which lacked the associated tracheae [15]. Results in the current work showed that the budded form of rAcNPV can enter the hemocyte, fat body, Malpighian tubule, middle gut, anterior silk gland, middle silk gland, and posterior silk gland directly from the hemolymph. Furthermore, the copy number of rAcNPV in the anterior silk gland was definite. Mechanism research showed that the entry of the budded form of virus into insect cells is via endocytosis followed by low pH-induced fusion of a viral envelope protein, GP64, with the endosomal membrane, thereby allowing viral entry into the cytoplasm and nucleus [1,28,29]. The entry of rAcNPV into the anterior silk gland directly from the hemolymph could occur through the interaction between the virus and the anterior silk gland surface. Failure to observe the fluorescence of green fluorescent protein (GFP) in the anterior silk gland [15] could be due to the low expression of GFP in the anterior silk gland. Budded virus is the phenotype of baculovirus that spreads infection within the host, and budding is one of major stages in the life cycle of baculovirus (including AcMNPV and BmNPV); no more than 16% of the intracellular virus copies bud from the insect cells [21]. Because an insect larva has an open circulatory system, when a high level of viral titer is established in the hemolymph, the spread of virus becomes explosive, so the spread of virus through the tracheae to other tissues might be the early event. Entrance of rAcNPV into various tissues was similar, but the replication level was different

Fig. 2. Genome numbers of AcNPVA3Luc in different tissues of B. mori larvae at 4 days postinjection. 1, hemocytes; 2, fat body; 3, Malpighian tubule; 4, middle gut; 5, anterior silk gland; 6, middle silk gland; 7, posterior silk gland. For real-time PCR assay, 0.0018 A260 unit of DNA extracts was used.

Our previous work showed that rAcNPV cannot replicate in the nonpermissive silkworm strain Qingsong [5]. To observe the entry of rAcNPV into various tissues of silkworm larvae only, AcNPVA3Luc was injected into

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Spread of rAcNPV in tissues of silkworm / Y. Zhang et al. / Anal. Biochem. 373 (2008) 147–153

Table 3 Transcriptional expression of viral PCNA, p143, and IE-1 in various tissues of B. mori strain 54A larvae at 24 h postinjection ([mRNA/28S rRNA] · 104) Tissue

Hemocyte

Fat body

Anterior silk gland

Middle silk gland

Posterior silk gland

Malpighian tubule

Middle gut

PCNA p143 IE-1

6,579 8,211 14,960

4.371 6.723 8.601

0.855 1.657 1.227

0.422 0.744 0.446

0.082 0.204 0.137

2.528 4.480 4.131

0.519 0.526 0.776

Note. Data are averages of at least three measurements.

the hemocoele of newly ecdysed fifth instar larvae of nonpermissive silkworm strain Qingsong. The ratios of viral genome to host genome in various tissues were determined quantitatively. Results showed that the copies of virus were very low in all silkworm tissues at 4 days postinjection of AcNPVA3Luc and that the rAcNPV/B. mori ratios in most tissues were similar at 24 h postinjection (data not shown). These results indicated that the entry of rAcNPV into different tissues was similar; therefore, the difference of viral copy numbers in various tissues of permissive strain larvae was due mainly to the different multiplications of rAcNPV in cells of different tissues. Then the expression of two components of viral DNA replication machinery, PCNA and p143, was analyzed by real-time quantitative RT–PCR using 28S rRNA as an intrinsic reference. On the 24-h post-intrahemocoelical injection of AcNPVA3Luc into silkworm larvae of strain 54A, total RNA was extracted from different tissues. Real-time quantitative RT–PCR was performed. The 28S rRNA was highly expressed in cells; after diluting by 105fold, the mean threshold cycle was 27.71 for the blood sample, whereas the mean threshold cycles were 17 to 20 for other tissue samples. The expression level of PCNA and p143 genes of rAcNPV was normalized to the expression level of 28S rRNA in each sample. As shown in Table 3, viral PCNA and p143 displayed a high expression level in the hemocyte, a mild level in the fat body and Malpighian tubule, and a weak level in the middle gut and silk gland. The expressional levels of PCNA and p143 reflected the different replication levels of rAcNPV in various tissues of silkworm larvae and were in agreement with copy number measurements in various tissues of silkworm larvae. The onset of viral DNA replication depends on the expression of viral early gene transactivator, and we also assayed the transcriptional expression of IE-1 of rAcNPV in various tissues of silkworm larvae by real-time quantitative RT– PCR. The ratio of IE-1 messenger RNA (mRNA) to 28S rRNA in various tissues showed the same trends as those of PCNA and p143 in different tissues (Table 3). The higher expression of viral genes in the hemocyte supported the replication of viral DNA; the increase of copy number of viral genome caused more expression of viral genes. At 24 h postinjection, the expression of viral genes and the replication of viral DNA reached a very high level in the hemocyte, began to increase in the fat body, and was still very low in the silk gland. In the hemocyte, the copy numbers of viral mRNAs (PCNA, p143, and IE-1) had already reached the same order of magnitude as 28S rRNA at 24 h

postinjection. If the viral mRNA increased further, the synthesis of viral protein would slow down due to the shortage of ribosome; thus, the multiplication of rAcNPV would slow down as well. On the other hand, the viruses released from the hemocyte as the budded form. That is why we saw the copy number reach saturation in hemocytes after 2 days of injection of rAcNPV. A real-time PCR method proved to be a sensitive, accurate, and convenient method for measuring viral infection in various tissues of silkworm. The effect of varying the concentration of total DNA on the amplification efficiency was assayed. We found that when more than 0.6 ng/ll silkworm DNA was added to the reaction, efficiency of PCR amplification of plasmid DNA is affected by competition by silkworm DNA. So, it is important to refer to a standard curve that was constructed with same amount of control silkworm DNA. In real-time quantitative RT–PCR, to avoid the effect of template on the efficiency of PCR, the template amount was adjusted by dilution of reverse transcripts with doubly distilled H2O. Acknowledgments We thank Guozheng Zhang from the Sericultural Research Institute, Chinese Academy of Agricultural Sciences, for kindly providing silkworms. This work was supported by grants from the National Natural Science Foundation of China to Changde Lu (30370326 and 30470350). References [1] M.M. Rahman, K.P. Gopinathan, Analysis of host specificity of two closely related baculoviruses in permissive and nonpermissive cell lines, Virus Res. 93 (2003) 13–23. [2] M. Shikata, H. Shibata, M. Sakurai, Y. Sano, Y. Hashimoto, T. Matsumoto, The ecdysteroid UDP–glucosyltransferase gene of Autographa californica nucleopolyhedrovirus alters the moulting and metamorphosis of a non-target insect, the silkworm Bombyx mori (Lepidoptera, Bombycidae), J. Gen. Virol. 79 (1998) 1547–1551. [3] M. Yamao, N. Katayama, H. Nakazawa, M. Yamakawa, Y. Hayashi, S. Hara, K. Kamei, H. Mori, Gene targeting in the silkworm by use of a baculovirus, Genes Dev. 13 (1999) 511–516. [4] T.Q. Guo, J.Y. Wang, X.Y. Guo, S.P. Wang, C.D. Lu, Transient in vivo gene delivery to the silkworm Bombyx mori by EGT-null recombinant AcNPV using EGFP as a reporter, Arch. Virol. 150 (2005) 93–105. [5] T. Guo, S. Wang, X. Guo, C. Lu, Productive infection of Autographa californica nucleopolyhedrovirus in silkworm Bombyx mori strain Haoyue due to the absence of a host antiviral factor, Virology 341 (2005) 231–237.

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