Baculoviruses are one of the largest and most diverse groups of viruses, and are phatogenic only for insects mostly of order Lepidoptera, Hymenoptera, and Coleoptera. The nuclear polyhedrosis virus isolated from Autographa californica (AcNPV), has a relatively broad host range(Ikonomou et al., 2003; Motohashi et al., 2005). So, the baculovirus expression system, AcNPV, is commonly applied for large scale expression for eukaryotic proteins in permissive cell lines or insects(Maeda, 1989). In recent years, this baculoviruses base vectors comes to be regarded as methods for foreign gene delivery and transient expression into mammalian cells in vivo and in vitro. The baculovirus is an especially strong host-specific virus that is a pathogenic insect virus, and it does not pose the threat of cross infection between species. (Rahman et al., 2002; Motohashi et al., 2005).

The baculoviruses used in the BEVS(baculovirus expression vector system) are AcNPV and BmNPV(Motohashi et al., 2005). Since developed expression systems based on Spodoptera frugiperda cultured cell lines and a recombinant AcNPV to produce human ¥á-interferon, AcNPV and BmNPV have been the two expression vectors that have been mostly used in BEVS to date(Smith et al., 1983).

Currently, Sf9, Sf21, and Tn368 cell lines are used, since these make the reproduction of AcNPV considerably easier. The AcNPVSf9 host system is widely used as a representative cassette in BEVS (Kondo et al., 1991; Maeda et al., 1993; Miller et al., 1995; Mishra et al., 1998). However, this cassette is costly and requires considerable effort in cell line maintenance and mass culture.

On the other hand, the use of B. mori as a host of BmNPV has been dubbed a “Bio factory” in the production of useful biological material. The mass rearing of the silkworm B. mori has already been established, therefore production is less costly, and the system also has excellent protein synthesis abilities. The production of proteins with high molecular weights is also possible (Motohashi et al., 2005). In veterinary medicine, feline interferon derived from BEVS utilizing B. mori has been marketed in Japan. The feline interferon expression in B. mori has been found to be 24,000 times higher than in E. coli, yeast, or cultured cells derived from monkey.

AcNPV hosted by A. californica has been used to infect cultured cells derived from B. mori, but they can fail to breed (Shikata et al., 1998; Yamao et al., 1999). Recently however, the existence of a B. mori strain that enabled the breeding of AcNPV has been reported (Guo et al., 2005; Lee et al., 2007). This has offered the opportunity for the development of a new material-producing cassette in combination with the mass rearing B. mori as a BEVS.

In this study, the differences in AcNPV proliferation between different strains of B. mori were examined, in order to develop a new useful biological material production system. B. mori bred by the Kyungpook National University Insect Genetic Resources Laboratory were used to determine breeding ability and the possibilities of developing a new mass protein production system using the AcNPV-B. mori system.

Seventy different strains from among the B. mori that are available at the Kyungpook National University were used in the study (Table 1). Virus was injected into 24-hour old larvae in the fifth instar after molting. A micro syringe (Hamilton, USA) with a 30 gauge needle was used to inject 10 μL of virus (AcNPV/BmA3-Luc) into the larvae to infect them. Hemocytes were collected 48 hours after virus injection to produce a hemolymph protein enabling the measurement of luciferase activation.

[Table 1] This study used 70 kinds of silkworm strain and features(ft.)

This study used 70 kinds of silkworm strain and features(ft.)

Recombinant AcNPV was produced using the luciferase gene as a reporter, using the Bac-to-Bac Baculovirus Expression System (Invitrogen, USA). In order to express the luciferase gene, an expression cassette containing the B. mori Actin3 promoter was digested from pBmActin3-Luc (Lee, 2003). The A3-Luc-polyA cassette was inserted into the pFastBac1 vector (Invitrogen) with the polyhedrin promoter and the SV40 polyadenylation signal removed to create pFBBmA3-Luc. Recombinant pFBBmA3-Luc was transformed to DH10Bac cells with a donor plasmid to establish AcNPV/BmA3-Luc, which contains the recombinant AcNPV. Recombinant AcNPV/BmA3-Luc was purified using the FlexiPrep kit (Amersham Pharmacia Biotech, USA), and then cultured and reproduced according to protocols of Invitrogen. The produced recombinant AcNPV/BmA3-Luc baculovirus concentration was determined, and they were stored at 4°C re until used (O’Reilly et al., 1992).

Hemocytes were collected from 200 ¥ìL of hemolymph from virus infected individuals by centrifugation at 3000 rpm for five minutes. Seventy μL of cell solution (25 mM Tris-phosphate pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane- N,N,N’,N’-tetraacetic acid, 10% Glycerol and 1% Triton X-100) was added to the collected hemocytes and gently mixed for 30 minutes. The cell suspension was centrifuged at 10,000 rpm for 1 minute at 4°C and 20 μL of the supernatant and 50 μL of luciferase (Promega, USA) were mixed, after which the luciferase activity was measured (Lumistar Galaxy, BMG).

Recombinant AcNPV/BmA3-Luc was propagated using insect cells (Invitrogen, Carlsbad, CA, USA). Sf9 insect cells were cultured in Grace’s insect medium (Gibco-BRL, USA) with 10% fetal bovine serum (FBS, Invitrogen) at 27°C.

cDNA was synthesized from 1 μg of total RNA. Using a high capacity cDNA archive kit (Applied Bio systems, USA), PCR was performed on the obtained cDNA. A gene specific primer was made in order to partially amplify the luciferase gene inserted into the recombinant AcNPV (Fig. 1). One μL of cDNA was used of 30 amplification cycles, seconds at 94°C, 30 seconds at 48°C, and 1 minute at 72°C. Real-time PCR reaction mixture including the designed primer and SYBR green (TaKaRa) were used to apply the PCR. (Applied Biosystem 7300 Real-time PCR system). The amplification consisted of 10 seconds at 94°C, followed by 40 cycles of 95°C for 5 seconds, 62°C for 32 seconds and then a final cycle of 95°C for 5 seconds, 62°C for 32 seconds and 72°C for 30 seconds. 18S rRNA was used as a control group.

[Fig. 1] Construction of AcNPV/BmA3-Luc bacmid using the Bac-to-Bac baculovirus expression system.

Dot blot analysis was performed by dotting 500 ng of extracted DNA from the hemocytes and fat cells onto Hybond N⁺ membranes (Amersham Pharmacia Biotech), and fixing with a UV-cross linker (Spectrolinker XL-100, USA). The luciferase gene obtained by PCR was labeled with radioisotopes (³²P-dCTP) (Perkin Elmer) and used as a probe. The membranes were pre-hydrated in a hybridization solution without the probe for two hours at 68°C, and then hydrated with the probe in an hybridization solution overnight at 68°C. The film was then washed in 2X SSC, 0.1X SSC and exposed onto a film at −70°C.

The differences in the susceptibility of the 70 strains of B. mori to AcNPV infection were studied. Two-day old larvae of the 5th instar were infected with recombinant AcNPV/BmA3-Luc, at 1 × 10⁴ pfu . Hemocytes were collected two days after infection, and the luciferase activity was measured. The differences between strains revealed that they fell into one of three groups (Fig. 2), high-permissive strains (HPS), middle-permissive strains (MPS), and low-permissive strains (LPS), according to the luciferase activity levels. There were six strains classified as HPS (activity greater than 3 × 10⁵ RLU), 21 strains classified as LPS (activity less than 2 × 10³ RLU), and the remaining 43 strains were classified as MPS (Table 2). Among the HPS, S35, S37, and S151, all showed higher than average activity, and among the LPS, K43, S11, and W31, all showed less than average activity. It can be concluded that permissiveness of B. mori was not related to blood color or larval spots, but to the strain used.

[Fig. 2] Screening for the AcNPV high-permissive host range strain in 70 different silkworm. The luciferase activity achieved by the infection of the recombinant AcNPV in larval hemocyte was assayed at 48 hrs post- infection.

[Table 2] The results of silkworm host range against the propagation of recombinant AcNPV.

The results of silkworm host range against the propagation of recombinant AcNPV.

To study the reproduction of AcNPV in B. mori in more detail, we examined and compared strains S37, an HPS, and strain S39, an LPS. After the fifth instar, the growth pattern and the proliferation of the virus following infection was investigated by measuring the luciferase activity. With HPS S37, luciferase activity began to increase 24 h after virus injection (AcNPV/ BmA3-Luc 1 × 10⁵ pfu), and it reached its maximum after 48 h, regardless of the growth stage (Fig. 3). Luciferase activity in LPS S39 was relatively low regardless of its growth stage or the time from virus injection(Fig. 4). The results obtained from using a less concentrated virus injection (2 × 10³ pfu) are shown in Figures 5 and 6. In these cases, luciferase activity in HPS 37 differed depending on the growth stage. These groups showed its maximum proliferation 72 h after the injection. It took 24 h more than the high concentrate virus of 1× 10⁵ pfu (Fig. 5). LPS did not show any luciferase activity (Fig. 6).

[Fig. 3] Time-course of luciferase activity in 5th instar larva of HPS (S37) infected with 1X105 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting. 24, 48, 72hr, respectively.

[Fig. 4] Time-course of luciferase activity in 5th instar larva of LPS (S39) infected with 1X105 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting, 24, 48, 72hr, respectively.

[Fig. 5] Time-course of luciferase activity in 5th instar larva of HPS (S37) infected with 2X103 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting, 24, 48, 72hr, respectively.

[Fig. 6] Time-course of luciferase activity in 5th instar larva of LPS (S39) infected with 2X103 AcNPV/BmA3-Luc. A~D represents the virus injection time after 4th molting, just molting, 24h, 48h, 72h, respectively.

Dot blotting was performed to verify virus proliferation in the B. mori tissue infected with recombinant AcNPV/BmA3-Luc. HPS S10 and LPS S39 were injected with AcNPV/BmA3-Luc 24 h after the fifth instar. The presence of viral DNA was confirmed in the hemocytes of the HPS. The dot blots of the DNA extracted 24 h after injection were hazy due to the small amounts of DNA present, confirming that the proliferation of the virus was low. However, clear dots were observed 48, 72, and 96 h after injection, confirming that viral DNA proliferation increased over time. Viral DNA in the fat cells of strain S37 was very low. Viral DNA was not observed in hemocytes and fat cells from the LPS (Fig. 7).

[Fig. 7] Viral DNA in tissues of AcNPV/BmA3-LUC infected silkworm larvae. Total DNA extracted at the indicated times of post infection (shown on the right) from tissues, hemocyte(hemo) and fatbody(fat), of silkworm larvae. HSP and LPS in the upper indicates the high and low permissive strain, respectiviely. The amount of 0.5ug DNA was dotted and hybridized with 32P-dCTP labeled luciferase DNA. Dots on the first lane, M, indicated luciferase DNA (amounts are shown on the left) used as a standard.

Dot blotting of recombinant viral DNA showed that virus proliferation was high in the HPS. In order to confirm this result, viral RNA expression within different tissues was studied using RT-PCR and real-time PCR. HPS S10 and LPS S39 were used The luciferase gene was observed in hemocytes, fat cells, the mid-gut, and the gonads. It was observed beginning 48 h after infection in blood cells and reached its maximum level within 72 h The expression level of luciferase in fat cells, the mid-gut, and the gonads was less than that seen in hemocytes, and viral RNA began to be observed after 72 h, 24 h later than in hemocytes (Fig. 8A). The luciferase gene was only detected in hemocytes inthe LPS(Fig. 8B). The real time PCR results corresponded with those from the RT-PCR. The luciferase gene was observed in all tissues from the HPS. only observed in hemocytes, and it reached its maximum levels within 48 h. Proliferation of the luciferase gene was not detected in tissues other than hemocytes (Fig. 9).

[Fig. 8] Expression profile of luciferase gene monitored at tissues of AcNPV/BmA3-Luc infected (A) HSP (high-permissive strain), (B) LSP (low-permissive strain) larvae by RT-PCR. Total RNA extracted at the indicated times of post infection (shown on the upper) from tissues. B. mori cytoplasmic actin3 was used as a control.

[Fig. 9] Expression profile of luciferase gene monitored at tissues of silkworm larvae by Real-time PCR. the white box indicate the high-permissive strain (HSP) and grey box indicate the low-permissive stain (LPS). , Hemocyte; , fat body; , mid-gut; , gonad; , gonad; , silk glad. 18s RNA wre quantified by densitometric scanning.

Functional development of function, the synthesis of active substances, and genes, of insects are receiving more attention in life science the utilization of insects is increasing. The useful production of biological material using insect tissues is increasing, by taking advantage of insect behavior and their specific characteristics (Tamura et al., 2000). The utilization of the silkworm, B. mori, in the production of biologically useful material is particularly prevalent (Miller et al., 1988; Hiyoshi et al., 2007).

B. mori is currently the only insect that can be evaluated scientifically as a biological manufacturer, with a wealth of background information available, and the ability to be mass reared. The process of protein synthesis (including post-translational modification; acetylation, methylation, glycosylation, phosphorylation) using BEVS in B. mori is particularly similar to that in mammals, thus enabling protein production that is not feasible using E. coli or yeast. The production rate is also several hundred times higher than what can be achieved using in vitro expression systems (Luckow et al., 1993; O’Reilly et al., 1992).

AcNPV infection of B. mori-derived cells is already been known to be possible; however, the proliferation of AcNPV within B. mori cells has not been studied. The recent report of a B. mori system that enabled infection and proliferation of AcNPV (Guo et al., 2005) led us to undertake this study in order to establish a biological material production system. We used 70 different strains of B. mori from Kyungpook National University to investigate the proliferation of AcNPV, and we confirmed that there are distinct differences in AcNPV proliferation depending on the strain utilized (Fig. 2). In some strains, a high rate of AcNPV proliferation was observed (the HPS group), while it was much lower in other strains (the LPS group Proliferation was up to 500 times greater in HPS strains compared to LPS strains (Fig. 2). The results coinsides well with the results of previously done studies (Morris and Miller, 1993; Ikeda et al., 2001) using B. mori derived cell, Bm-N4, Bm5 as AcNPV host.

It has been reported previously that the proliferation of B. mori AcNPV is related to the virus concentration (Shikata et al., 1998; Yamao et al., 1999). To investigate this, we compared the proliferation of B. mori AcNPV in both HPS and LPS, both in terms of the duration since injection, and the initial concentration of virus used. The results showed that maximal luciferase activity was achieved in 48 h using 1 × 10⁵ pfu recombinant AcNPV/BmA3-Luc, and in 72 h using 2 × 10³ pfu (Figs. 3, 5). It took at least 24 more hours for the LPS to reach the maximum proliferation level. We undertook these experiments as it is essential to establish the most suitable concentration of AcNPV to use for its effective utilization in biological material production.

Virus proliferation in B. mori tissues was found to be the highest in hemocytes with less proliferation in fat cells, the midgut, gonads, and silk thread. The time needed for proliferation to be detected was different for each tissue. Virus proliferation began to be detected after 24 h in hemocytes, but not until after 72 h in fat cells, the mid-gut, and gonads(Figs. 7, 8, 9). Therefore, it would be the most economical and effective to produce biological material with B. mori hemocytes via recombinant virus DNA proliferation and RNA expression.

B. mori has recently been recognized as a viable mass producer of large molecular proteins, and studies are being performed in order to maximize its utility as a protein producer. Our results demonstrate that it is possible to establish a new cassette of AcNPV–B. mori, in addition to the already developed BmNPV-B. mori cassette. The identification of the HPS for AcNPV proliferation in this study will contribute to the effectiveness of biological material production using the AcNPV–B. mori system.