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Journal of General Virology (212), 93, 248 2489 DOI 1.199/vir..43836- A baculovirus isolated from wild silkworm encompasses the host ranges of Bombyx mori nucleopolyhedrosis virus and Autographa californica multiple nucleopolyhedrovirus in cultured cells Yi-Peng Xu, 1,2 Lin-Zhu Gu, 2 Yi-Han Lou, 2 Ruo-Lin Cheng, 2 Hai-Jun Xu, 2 Wen-Bing Wang 3 and Chuan-Xi Zhang 2 Correspondence Chuan-Xi Zhang chxzhang@zju.edu.cn 1 Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 3118, PR China 2 Institute of Insect Science, Zhejiang University, Hangzhou 3158, PR China 3 School of Life Science, Jiangsu University, Zhenjiang 21213, PR China Received 24 April 212 Accepted 3 July 212 A baculovirus, named BomaNPV S2, was isolated from a diseased larva of the wild silkworm, Bombyx mandarina. Notably, BomaNPV S2 exhibited a distinguishing feature in that its host range covered that of both Bombyx mori nucleopolyhedrosis virus (BmNPV) and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in cultured cells. It could replicate in cells of B. mori (Bm5 and BmN), Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn-5B1-4). However, occlusion-derived virions of BomaNPV S2 in B. mori cells contained only a single nucleocapsid, whereas they contained multiple nucleocapsids in Tn-5B1-4 cells. The complete genome sequence of BomaNPV S2, including predicted ORFs, was determined and compared with the genome sequence of its close relatives. The comparison results showed that most of the BomaNPV S2 genome sequence was shared with BmNPV (BmNPV T3) or BomaNPV S1, but several regions seemed more similar to regions of AcMNPV. This observation might explain why BomaNPV S2 covers the host ranges of BmNPV and AcMNPV. Further recombinant virus infection experiments demonstrated that GP64 plays an important role in BomaNPV S2 hostrange determination. INTRODUCTION Baculoviruses are arthropod-specific viruses that are particularly infectious to insects of the order Lepidoptera. So far, 58 baculovirus genomes have been sequenced completely, and the family Baculoviridae can be divided into four genera: Alphabaculovirus [lepidopteran-specific nucleopolyhedroviruses (NPVs)], Betabaculovirus (lepidopteran-specific granuloviruses), Gammabaculovirus (hymenopteran-specific NPVs) and Deltabaculovirus (dipteran-specific NPVs) (Jehle et al., 26; Miele et al., 211). Most baculoviruses have a unique biphasic life cycle, producing two virion phenotypes, the occlusion-derived virion (ODV) and the budded virion (BV). The former is used to establish the primary infection in the midgut of the host through an oral route, whilst the latter is responsible for systemic infection, spreading the virus from the midgut to other tissues (Keddie et al., 1989). The GenBank/EMBL/DDBJ accession number for the BomaNPV S2 genome determined in this study is JQ71499. Supplementary data are available with the online version of this paper. Over the past 3 years, baculoviruses have been applied widely as an efficient vector for the high-level expression of heterologous genes in cultured insect cells and larval insect hosts (Kost & Condreay, 1999; Possee, 1997). Baculoviruses have also been used as bioinsecticides to control insect pests in agriculture and forestry (Rohrmann, 211). However, a baculovirus often infects only a single insect or a small group of insects. Although Autographa californica multiple NPV (AcMNPV) has a wide larval host range of 33 lepidopteran species (Groener, 1986), it cannot replicate in Bombyx mori cell lines, which are permissive to B. mori NPV (BmNPV), a close relative of AcMNPV. Similarly, BmNPV cannot replicate in Spodoptera frugiperda or Trichoplusia ni cell lines, which are permissive to AcMNPV. The narrow host range of many baculoviruses is one of the limitations on their broader application. For instance, A. californica, one of the host insects of AcMNPV, is smaller and more difficult to rear than B. mori, so baculovirus expression systems based on AcMNPV are not suitable for the industrial production of foreign proteins in vivo. Thus, it is necessary to study the 248 43836 G 212 SGM Printed in Great Britain

A baculovirus host range covers BmNPV and AcMNPV host-range determination of baculoviruses for their broader application. In addition, it is important to elucidate baculovirus host specificity for their safe application as biopesticides. Therefore, many studies have focused on the baculovirus host range. So far, the GP64/67 (Kadlec et al., 28; Monsma et al., 1996; Oomens & Blissard, 1999; Rahman & Gopinathan, 23; Zhou & Blissard, 28), helicase (Argaud et al., 1998; Croizier et al., 1994; Maeda et al., 1993), HCF-1 (Lu & Miller, 1995; 1996), HRF-1 (Chen et al., 1998; Du & Thiem, 1997; Thiem, 1997; Thiem et al., 1996), LEF-7 (Chen & Thiem, 1997; Lu & Miller, 1995; Morris et al., 1994) and P35 and IAP (Birnbaum et al., 1994; Bump et al., 1995; Clem et al., 1991; Crook et al., 1993; Eckelman et al., 26; Hershberger et al., 1992) proteins are all thought to be associated with the baculovirus host range. Here, we have described a wild baculovirus that was infectious to S. frugiperda (Sf9) and T. ni (Tn-5B1-4) cell lines and to B. mori cell lines. This virus was isolated from Bombyx mandarina and was named BomaNPV S2 to distinguish it from the previously reported baculovirus BomaNPV S1 (Xu et al., 21). The genome of BomaNPV S2 was sequenced completely and compared with other closely related baculoviruses, and GP64 was determined to be at least partially responsible for its host-range expansion. (a) (b) NC Infected Bm5 OBs OB (c) (d) OB Infected Tn-5B1-4 OBs Fig. 1. Infection of Bm5 and Tn-5B1-4 cells by BomaNPV S2. (a, c) Bm5 and Tn-5B1-4 cells infected with BomaNPV S2. (b, d) Occlusion bodies (OBs) in the nuclei of infected Bm5 and Tn- 5B1-4 cells observed by transmission electron microscopy (TEM), showing the presence of single (b) or several (d) nucleocapsids (NCs). Bars, 1 mm. NC RESULTS Infection of BomaNPV S2 in Bm5, Tn-5B1-4 and Sf9 cell lines BomaNPV S2 could successfully infect Bm5, Tn-5B1-4 (High Five) and Sf9 cells (Figs 1 and S1, available in JGV Online), but there were more OBs in Bm5 nuclei than in Tn- 5B1-4 nuclei (Fig. 1a, c). The OBs in the nuclei of Bm5 cells also appeared rougher and contained fewer nucleocapsids than those in Tn-5B1-4 cells (Fig. 1b, d). In addition, ODVs in the OBs contained only one nucleocapsid [single NPV (SNPV)] in Bm5 cells (Fig. 1b) but up to several nucleocapsids (MNPV) in Tn-5B1-4 cells (Fig. 1d). BV growth curve in Bm5 and Tn-5B1-4 cells BV production of BomaNPV S2 in cultured cells (Bm5 and Tn-5B1-4) was compared with BmNPV T3 and racmnpvegfp, a recombinant AcMNPV expressing the egfp gene (Fig. 2). The results showed that BomaNPV S2 yielded approximately ten times more BVs than BmNPV T3 in Bm5 cells. Similarly, BomaNPV S2 produced five to ten times more BVs than racmnpv-egfp in Tn-5B1-4 cells. Taken together, these results indicated that BomaNPV S2 has an advantage over BmNPV T3 and AcMNPV in BV production in cultured cells. Genome sequence analysis The BomaNPV S2 genome consisted of 129 646 nt, with 4.4 mol% G+C content (GenBank accession no. log 1 (TCID 5 ml 1 ) 9 8 7 6 5 4 3 2 1 9 8 7 6 5 4 3 2 1 (a) Bm5 1 2 (b) Tn-5B1-4 3 4 5 6 7.5 1 2 3 4 5 6 Time p.i. (days) Fig. 2. BVs growth curve of BomaNPV S2 in Bm5 and Tn-5B1-4 cells. (a) Growth of BomaNPV S2 compared with BmNPV T3 in Bm5 cells. X, BmNPV T3; m, BomaNPV S2. (b) Growth of BomaNPV S2 compared with racmnpv-egfp in Tn-5B1-4 cells. &, racmnpv-egfp; m, BomaNPV S2. http://vir.sgmjournals.org 2481

Y.-P. Xu and others JQ71499). Compared with BmNPV T3 (GenBank accession no. NC_1962), BomaNPV S1 (GenBank accession no. NC_12672) and AcMNPV (GenBank accession no. NC_1623), most of the BomaNPV S2 DNA sequence was found to be shared with BmNPV (BmNPV T3) or BomaNPV S1, but several regions, including cg3, pif-1 p24, alk-exo p74 and Ac149 ie-2, were more similar to those of AcMNPV (Figs 3 and 4). Moreover, BomaNPV S2 was most closely related to BomaNPV S1 than BmNPV T3. These results suggested that BomaNPV S2 might be the ancestor of BmNPV and AcMNPV or a natural recombinant virus of BmNPV S1 and AcMNPV. The BomaNPV S2 genome had 141 ORFs encoding predicted proteins of.5 aa, of which four ORFs encoded predicted products with,6 aa (Table S1). Eight homologous regions (hr2l, hr2r, hr3, hr4a, hr4b, hr4c, hr5 and hr1) were also found in this genome, containing 11, 3, 4, 6, 2, 1, 6 and 7 repeat units, respectively (Fig. 4). ORF content and relatedness As shown in Table S1 and Fig. 4, most of the BomaNPV S2 ORFs were very similar to those of BmNPV ( 95 % predicted amino acid identity), whilst only 21 ORFs showed higher identity to AcMNPV (Table 1). Additionally, ten ORFs (vp91, vp15, cg3, p4, Ac12, chitinase, pp34, alk-exo, odv-e56 and ptp) seemed to be the heterozygotes of BmNPV and AcMNPV, making it hard to determine their relatedness; two other ORFs (ubiquitin and gp16) were identical to those of BmNPV or AcMNPV, with 1 % predicted amino acid identity in BmNPV and AcMNPV. ORFs that showed a lower predicted amino acid identity to those of BmNPV than AcMNPV, or where it was difficult to determine relatedness, might be associated with the BomaNPV S2 host range. Recombinant BmNPVs containing different BomaNPV S2 genome regions and their infection in Tn-5B1-4 cells To determine which gene plays a key role in the hostrange expansion of BomaNPV S2, different BomaNPV S2 genome regions that were less similar to BmNPV were cloned and inserted into a BmNPV bacmid (BmBacmid) for testing. Some ORFs, such as vp15, me53, ie-1 and odve56 (Table S1), which seemed to be in the transition regions of recombination of BmNPV and AcMNPV in BomaNPV S2, were also taken into consideration. The Pk2 gene of BomaNPV S2, which was much more identical to AcPk2, was left out, because PK2 of a hybrid virus (a recombinant virus of AcMNPV and BmNPV in our laboratory) that has a similar host range to BomaNPV S2 is BmNPV-like. Overall, 23 ORFs were divided into nine regions: H1 (Ac124 lef-7 chitinase), H2 (v-cath gp64/67 p24), H3 (alk-exo p94), H4 (p35 hr5 p26 p1), H5 (p74 me53), H6 (ie-1 odv-e56), H7 (Ac149 Ac15 ie-2), H8 (pif-1 Ac121 Ac122) and H9 (vp15 cg3). After amplification by PCR using specific primers (Table S2), these nine regions were cloned into a BmBacmid to produce rbmbacmid-h1 rbmbacmid-h9, respectively (Fig. S2). These recombinant BmBacmids were then transfected into Bm5 cells. All the recombinant viruses were propagated successfully in Bm5 cells. For preliminary assessment of the roles of the chosen sequences in expanding the host range BomaNPV S2, the recombinant viruses were used to infect Tn-5B1-4 cells, which are non-permissive to BmNPV. Using a recombinant BmBacmid containing the egfp gene (rbmbacmid-efgp) as a control, the number of viral DNA copies in Tn-5B1-4 cells infected with the different recombinant viruses was assessed and compared using quantitative PCR (qpcr). BomaNPV S1 126 875 5 BmNPV T3 128 413 5 2 4 6 8 1 129 646 BomaNPV S2 2 4 6 8 1 129 646 BomaNPV S2 AcMNPV 133 894 5 2 4 6 8 1 129 646 BomaNPV S2 Fig. 3. Comparison of the genomic DNA of BomaNPV S2 with that of BomaNPV S1, BmNPV T3 and AcMNPV. The dot matrices were generated by BLASTn (http://blast.ncbi.nlm.nih.gov/blast.cgi). 2482 Journal of General Virology 93

A baculovirus host range covers BmNPV and AcMNPV ie-2 Ac15 Ac149 alk-exo p24 gp64 v-cath chitinase lef-7 Ac124 pk2 Ac122 Ac121 Ac12 pif-1 p94 p35 p26 p1 p74 hr5 gp16 hr4c hr4b hr4a 1 hr1129646 BomaNPV S2 129 646 bp hr3 hr2l hr2r Ubiquitin cg3 BmNPV = AcMNPV 99 > %ID 95 to both BmNPV and AcMNPV 95 > %ID 85 to both BmNPV and AcMNPV %ID = 1 to BmNPV %ID = 1 to AcMNPV 1 > %ID 99 to BmNPV 1 > %ID 99 to AcMNPV 99 > %ID 95 to BmNPV 99 > %ID 95 to AcMNPV 95 > %ID 9 to BmNPV 9 > %ID 85 to BmNPV Fig. 4. Predicted amino acid sequence comparison of the ORFs of BomaNPV S2 with those of BmNPV and AcMNPV. Different colours represent different degrees of predicted amino acid identity (ID) for ORFs of BomaNPV S2 compared with BmNPV or AcMNPV, as indicated. Table 1. Percentage of predicted amino acid identity (ID) of the ORFs of BomaNPV S2 compared with those of BomaNPV S1, BmNPV T3 and AcMNPV C6 BomaNPV S2 ORF Name Length (aa) BomaNPV S1 BmNPV T3 AcMNPV C6 Length (aa) ID (%) Length (aa) ID (%) Length (aa) ID (%) 71 cg3 265 267 95.5 267 94.8 264 96.3 96 pif-1 53 527 86.1 527 85.9 53 96.8 97 Ac12 82 82 93.9 82 95.1 82 98.8 97a Ac121 58 57 93.1 57 87.9 58 98.3 98 Ac122 62 61 91.9 61 9.3 62 1 99 gcn2/pk2 215 225 9.7 225 91.1 215 99.1 1 Ac124 247 244 88.3 244 87.9 247 1 11 lef-7 226 227 81.7 227 81.3 226 1 12 Chitinase 551 551 96.6 552 96.2 551 98.6 13 v-cath 323 323 97.2 323 96.6 323 99.1 14 gp64/67 531 53 95.3 53 95.1 53 99.4 15 p24 195 195 94.4 195 94.4 198 97.5 19 alk-exo 419 42 96.4 42 96.4 419 98.6 11 p94 83 56 1.5 7 1.7 83 99.9 111 p35 299 299 9.7 299 9.7 299 1 112 p26 24 24 93.8 24 93.8 24 1 113 p1 94 7 64.9 7 66 94 98.9 114 p74 (pif) 645 645 91.5 645 91.2 645 98.9 125 Ac149 17 16 86.9 16 86.9 17 99.1 126 Ac15 99 115 64.7 115 64.7 99 1 127 ie-2 48 422 73.1 422 72.9 48 99.5 http://vir.sgmjournals.org 2483

Y.-P. Xu and others Fig. 5 shows that rbmbacmid-h2 produced significantly more viral DNA in Tn-5B1-4 cells than the other recombinant BmBacmids. Consistent with this, Tn-5B1-4 cells infected with rbmbacmid-h2 showed a viral cytopathic effect, displaying no spindle and having enlarged nuclei, whilst cells infected with the other recombinant BmBacmids appeared normal. These results indicated that the H2 region containing v-cath gp64/67 p24 might be related to the host range of BomaNPV S2. Infectivity of rbmbacmid-egfps2gp64 and racbacmid-egfps264 As GP64 has been reported to play important roles in BV infection (Blissard & Wenz, 1992; Hefferon et al., 1999; Monsma et al., 1996; Oomens & Blissard, 1999), and the above result showed that the H2 (v-cath gp64/67 p24) region might be related to the host range of BomaNPV S2, we wanted to determine whether GP64 played a role in the host-range expansion of this virus. The BomaNPV S2 gp64 gene was cloned into BmNPV and AcMNPV bacmids to yield rbmbacmid-egfps2gp64 and racbacmid-egfps2gp64, respectively (Fig. 6). The two recombinant viruses were then used to infect Tn-5B1-4 or B. mori BmN cells at an m.o.i. of 1. The results showed that rbmbacmid-egfps2gp64 could infect Tn-5B1-4 cells, and racbacmid-egfps2gp64 could infect BmN cells successfully, whilst the control virus, rbmbacmid-egfp, failed to replicate in Tn-5B1-4 cells, and the other control virus, racbacmid-egfp, failed to infect BmN cells (Fig. 7). Because the egfp gene is under control of the promoter of the very late viral polyhedrin gene (polh), the expression of egfp represents expression of this very late viral gene. Green fluorescence emission in infected cells thus reflected successful viral infection. Although green fluorescence in Tn-5B1-4 cells infected with rbmbacmid-egfps2- gp64 or BmN infected with racbacmid-egfps2gp64 was much less bright than in BmN cells infected with rbmbacmid-egfp or Tn-5B1-4 cells infected with racbacmid-egfp, the increase in the number of cells that emitted green fluorescence indicated the low speed of spread of the virus. This might be due to the existence of the original gp64 gene in the recombinant viruses, which might interfere with the function of the foreign gp64. rbmbacmid-egfps2gp64 BVs propagated in Tn-5B1-4 cells and racbacmid-egfps2gp64 BVs propagated in BmN cells were further used to infect Tn- 5B1-4 and BmN cells, respectively. The results confirmed that cell-to-cell infection occurred for the two recombinant viruses. In summary, these results demonstrated that GP64 is related to host-range determination of BomaNPV S2. DISCUSSION BomaNPV S2 showed a difference in OBs following infection of Bm5 and Tn-5B1-4 cells. BomaNPV S2 was an SNPV in Bm5 cells but an MNPV in Tn-5B1-4 cells, indicating that Tn-5B1-4 cells are more favourable than Bm5 cells for producing ODVs containing more than one nucleocapsid. Compared with one-nucleocapsid ODVs, multinucleocapsid ODVs have an advantage in causing systemic infection because, after penetrating the cytoplasmic membrane, some nucleocapsids from multinucleocapsid ODVs may stay in the cytoplasm rather than entering into the nucleus and may get repackaged into BVs to infect tracheal cells, initiating systemic infection (Engelhard et al., 1994; Flipsen et al., 1995; Granados & Lawler, 1981; Washburn et al., 1995, 1999, 23). Additionally, more single-nucleocapsid ODVs are required to achieve the same final mortality as multinucleocapsid ODVs (Washburn et al., 1999). These observations can partially explain why MNPVs have wider individual host ranges than SNPVs. However, only 13 MNPVs have been completely sequenced, all belonging to the genus Alphabaculovirus, and are restricted to the lepidopteran NPVs, indicating that MNPVs are evolutionarily younger than SNPVs (Washburn et al., 1999). According to this hypothesis, the host range of BomaNPV S2 might expand from B. mandarina (or B. mori) tot. ni and S. frugiperda. This hypothesis appears to be supported by the genome structure of BomaNPV S2, most of which was BmNPV-like sequence and less of which was AcMNPV-like. The host-range expansion of BomaNPV S2 was at the expense of a deformity or reduction in OBs in the nuclei of cultured cells. The OBs produced by BomaNPV S2 in the nuclei of Bm5 cells had rough surfaces and contained only a few ODVs, but BomaNPV S2 yielded fewer OBs in Tn- 5B1-4 than in Bm5 cells. The question of whether these Viral genome copies in 1 ng total DNA 4. 1 6 3.5 1 6 3. 1 6 2.5 1 6 2. 1 6 1.5 1 6 1. 1 6.5 1 6 cd egfp H1 e c a d a bcda H2 H3 ab a H4 b d a ab d a a d H5 H6 H7 H8 a abc H9 Fig. 5. Viral genome copies in Tn-5B1-4 cells infected with different recombinant virus. H1 H9 represent rbmbacmid-h1 rbmbacmid- H9, respectively. rbmbacmid-efgp (egfp) was used as a control. The number of genome copies in 1 ng total DNA was assessed at 2 (open columns) and 3 (shaded columns) days p.i. Different lower-case letters above columns of the same colour indicate statistically significant differences (P,.5), analysed by PASW Statistics 18 through Duncan s test (one-way analysis of variance). 2484 Journal of General Virology 93

A baculovirus host range covers BmNPV and AcMNPV (a) (b) (i) (ii) 75 5 25 rbmbacmid -egfps2gp64 (iii) (iv) 75 5 25 racbacmid -egfps2gp64 Fig. 6. (a) PCR identification of rbmbacmid-egfps2gp64 and racbacmid-egfps2gp64 with M13 primers. A DNA ladder (bp) is shown in the left lane. (b) Bm5 cells and Tn-5B1-4 cells infected with rbmbacmid-egfps2gp64 and racbacmidegfps2gp64, respectively. Panels (i) and (ii) show Bm5 cells infected with rbmbacmid-egfps2gp64, whilst panels (iii) and (iv) show Tn-5B1-4 cells infected with racbacmid-egfps2gp64. Green fluorescence in cells indicates that the egfp gene was successfully expressed and thus that the recombinant viruses had successfully infected the cells. Magnification, ¾5. observations are due to changes in some genes encoding ODV-associated proteins needs to be addressed. To compensate, BomaNPV S2 produced more BVs than BmNPV T3 (in Bm5 cells) or racbacmid-egfp (in Tn- 5B1-4 cells). This may result from a distribution balance of nucleocapsids in ODVs and BVs. Based on equal DNA replication, a decreasing number of nucleocapsids in one virion phenotype will induce an increasing number of nucleocapsids in the other. Accordingly, BomaNPV S2 generated many more BVs than BmNPV T3 or racbacmidegfp in cultured cells, regardless of its genomic divergence from BmNPV or AcMNPV. As this is the first report concerning a wild baculovirus covering the host ranges of BmNPV and AcMNPV in cultured cells, it is necessary and meaningful to reveal how its host range is determined. Sequences of BomaNPV S2 that showed high identity to AcMNPV must be associated with the host range of BomaNPV S2. Some sequences that were not easy to classify as BmNPV or AcMNPV, such as ORFs odv-e56, cg3 and vp15, may also contribute to the BomaNPV S2 host range. Based on the results of the recombinant BmBacmid infection experiments, the H2 region (v-cath gp64/67 p24) of BomaNPV S2 may be related to its host range. Further infection experiments showed that rbmbacmid-egfps2gp64 could infect Tn-5B1-4 cells, a non-permissive cell line for BmNPV, and that racbacmid-egfps2gp64 could infect BmN cells, a nonpermissive cell line for AcMNPV. However, v-cath or p24 from BomaNPV S2 could not achieve the same results (data not shown). These results imply that GP64 plays an important role in BomaNPV S2 host-range determination. GP64 is required for the lepidopteran NPVs of group I baculoviruses to propagate successfully in their hosts. It plays a vital role in viral binding to the cell, membrane fusion during viral entry into the host cell through endocytosis and BV budding from the surface of infected cells (Blissard & Wenz, 1992; Hefferon et al., 1999; Monsma et al., 1996; Oomens & Blissard, 1999). Although AcMNPV and BmNPV are closely related and their GP64 proteins are very similar, AcMNPV GP64 (AcGP64) shows stronger functions than BmNPV GP64 (BmGP64) in low-ph-triggered membrane fusion and virus entrance into the nuclei of nonpermissive cells (Boyce & Bucher, 1996; Hofmann et al., 1995; Katou et al., 26, 21; van Loo et al., 21). Compared with the GP64 proteins of BomaNPV S1, BmNPV T3 and AcMNPV, BomaNPV S2 GP64 is most similar to AcGP64, except for a mutation in domain III (D382G), another mutation in the transmembrane domain http://vir.sgmjournals.org 2485

Y.-P. Xu and others 2 days p.i. 3 days p.i. 5 days p.i. 7 days p.i. rbmbacmidegfps2gp64 infecting Tn-5B1-4 cells rbmbacmidegfp infecting Tn-5B1-4 cells racbacmidegfps2gp64 infecting BmN cells racbacmidegfp infecting BmN cells Fig. 7. Infectivity of rbmbacmid-egfps2gp64 in Tn-5B1-4 cells and racbacmid-egfps2gp6 in BmN cells. Green fluorescence in cells indicates that the egfp gene was successfully expressed and thus that the recombinant viruses had successfully infected the cells. Magnification, ¾25. (I523V) and an additional amino acid (T528) in the C- terminal domain (CTD) (Fig. S3), according to the structure of GP64 determined by Kadlec et al. (28). Therefore, in this study, rbmbacmid-egfps2gp64 and racbacmid-egfps2- gp64 could propagate in Tn-5B1-4 cells and B. mori cells, respectively, indicating that BomaNPV S2 GP64 (BomaS2- GP64) plays a role in BomaNPV S2 host-range determination. These three site differences between BomaS2GP64 and AcGP64 may need to be evaluated further. As the removal of GP64 CTD results in a dramatic reduction in budding efficiency (Oomens & Blissard, 1999), the functions of the CTD of AcGP64 and BomaS2GP64 could be compared. The I523V mutation, located near the CTD, might not influence the functions of GP64, because Ile and Val are both hydrophobic amino acids, differing only in a methyl group. The D382G mutation in domain III of GP64, from an acidic to a hydrophobic amino acid, may have effects on the structure and function of GP64. As the predicted secondary structure suggests (Fig. S4), the D382G mutation will smooth the turning between the r-sheet and s-sheet of domain III, described by Kadlec et al. (28). The extent to which D382G affects the structure and functions of GP64 requires further elucidation. The Acgp64-like gene makes BomaNPV S2 virions better able to penetrate the cytoplasmic membrane and enter into the nucleus, but the reporter gene (egfp) driven by the polh promoter in the BomaS2GP64 recombinant virus (rbm- Bacmid-egfpS2gp64 or racbacmid-egfps2gp64) was expressed at a much lower level in the virus non-permissive cells than in the permissive cells, indicating that the expression of very late genes in non-permissive cells is partly limited. Thus, some other factors besides GP64 may be associated with BomaNPV S2 host-range determination. In addition to GP64, the viral helicase has also been shown to affect the host range of AcNPV and BmNPV and has been applied to construct host-range-expanded hybrid baculoviruses for the expression of foreign genes (Argaud et al., 1998; Croizier et al., 1994; Deo et al., 26; Kamita & Maeda, 1997; Maeda et al., 1993; Wu et al., 24). BomaS2 helicase is 99.8 % identical to BomaS1 helicase (Table S1), with two mutations, D291N and D3N. The former mutation seems to be more important because this site of AcMNPV helicase is also asparagine. Whether this mutation is related to the host range of BomaNPV S2 needs further confirmation. The host range of BomaNPV S2 may not be determined by a single gene but by a set of genes, as non-overlapping host ranges between BmNPV and AcMNPV could be attributed to the differential expression of their genes in the permissive and non-permissive cell lines. AcMNPV has more genes expressed in non-permissive cells (BmN cells) 2486 Journal of General Virology 93

A baculovirus host range covers BmNPV and AcMNPV than BmNPV in Sf9 cells, and the expression of BmNPV genes is compromised at the transcriptional level (Iwanaga et al., 24). These results demonstrate the importance of the timely expression of viral genes during infection, as successful infection depends on the cascade regulation of gene expression in host cells. However, the viral gene expression profile in Tn-5B1-4 cells inoculated with BmNPV is not the same as that in Sf9 cells (Yamagishi et al., 23), even though they are both non-permissive to BmNPV. Consequently, gp64-null BmNPV with Acgp64 can yield progeny virions in Tn-5B1-4 cells but not in Sf9 cells (Katou et al., 26), implying that GP64 plays more roles than its function in viral entry into the membrane and budding of infectious BVs, and that different hosts may have different defence models to resist viral infection. Therefore, further studies comparing the gene expression profiles of BmN, Sf9 and Tn-5B1-4 cells when infected with BmNPV, AcMNPV and BomaNPV S2 will be worthwhile. METHODS Viruses and cell lines. BomaNPV S2 was originally isolated from a diseased larva of B. mandarina in Suzhou, Jiangsu Province, China. BomaNPV S2 was plaque purified and then propagated in BmN B. mori cells. B. mori cells (Bm5 orbmn), S. frugiperda cells (Sf9) and T. ni cells (High Five Tn-5B1-4) were maintained at 27 uc in TC-1 or TNM- FH medium supplemented with 1 % (v/v) FBS. Viral DNA extraction. OBs from diseased larvae were collected and purified by sucrose-gradient centrifugation. The purified OBs were then incubated in polyhedra lysis buffer (.1 M Na 2 CO 3,.1 M NaCl, ph 1.5) for 1 2 h. SDS was then added to the lysis buffer to a concentration of.5 % and the viral genomic DNA was extracted using phenol/chloroform. Viral genomic DNA sequencing and determination. Viral genomic DNA was amplified using a REPLI-g Mini kit (Qiagen), sheared into 2 3 bp fragments by ultrasonication and sequenced by Solexa technology using a Solexa 1G Genome Analyser. Ambiguous regions were amplified by PCR using specific primers (Table S3). The PCR products were sequenced directly or cloned into a pmd18-t vector (Takara) and then sequenced. DNA and protein sequence analysis. The nucleotide composition of the genomic DNA and predicted ORFs were analysed using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), Softberry GENE Finding in Viruses (http://www.linux1.softberry.com/berry.phtml) and Lasergene software. Alignments of proteins encoding homologous ORFs were carried out using the ClustalX2 and MEGALIGN programs. The protein secondary structure was predicted using ANTHEPROT software. Virions observed by TEM. Cells infected with viruses for 3 4 days were fixed in 2.5 % glutaraldehyde for 1 h at 4 uc. The cells were then collected by centrifugation and washed three times with PBS. The cell pellet was then embedded in 1 % low-melting-point agarose to aid subsequent steps, comprising fixation with 1 % osmium tetroxide, dehydration with graded ethanol (15 1 %) and acetone, infiltration in graded Spurr s resin (5 1 %) and polymerization for 16 h at 7 uc. Ultrathin sections were stained with uranyl acetate and lead citrate. Samples were observed using a JEM-123 transmission electron microscope (JEOL). BV growth curve. Bm5 and Tn-5B1-4 cells cultured in 24-well plates were infected with BVs of BomaNPV S2 at an m.o.i. of 1. BmNPV T3 and racmnpv-egfp were compared with BomaNPV S2 in infecting Bm5 and Tn-5B1-4 cells, respectively. After a 1 h incubation period, the inoculum was replaced with fresh medium. The supernatant was then harvested at various time intervals post-infection (p.i.). The BV titre was determined by an end-point dilution assay (TCID 5 ). Production of recombinant baculovirus. The construction of recombinant baculovirus was performed following the manual of the Bac-to-Bac baculovirus expression system (Invitrogen). Target DNA fragments including the viral gene promoters were amplified from BomaNPV S2 DNA and cloned into the pmd18-t vector (or pgem- T Easy vector) and then inserted into the multiple cloning site of pfastbac1 (or pbachtegfpt) to construct a recombinant donor plasmid. The egfp gene in the pbachtegfpt vector is under control of the promoter of the very late viral polh gene. After transformation with the recombinant donor plasmid, Escherichia coli DH1Bac clones with the recombinant virus bacmid were obtained and identified by PCR with the M13F/M13R primer pair (59-GTAAAACGACGGC- CAGT-39/59-AACAGCTATGACCATG-39) (Fig. S5). Monolayers of insect cells (~1 5 cells in a 35 mm diameter plate) were transfected with 1 mg recombinant bacmid DNA using stearylamine liposome (Wang et al., 1996; Zhang & Wu, 1998). The recombinant virus was harvested at 5 days post-transfection and propagated further in cells to obtain high-concentration virus stocks. qpcr analysis of viral DNA replication. To assess the viral DNA replication of various recombinant viruses in cultured cells, qpcr was used to quantify the viral genomic DNA. Tn-5B1-4 cells were cultured in 35 mm dishes and incubated with various recombinant BmBacmids inserted with different regions of the BomaNPV S2 genome at an m.o.i. of 5. Incubation was performed as described above. Cells were harvested at 2 and 3 days p.i., and total intracellular viral DNA was extracted using a Universal Genomic DNA Extraction kit version 3. (Takara) coupled with RNase A treatment. The DNA solution of 5 ml was diluted ten times for qpcr analysis. 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