1 Departamento de Producción Agraria, Universidad Pública de Navarra, 31006 Pamplona, Spain
2 Laboratoire de Patologie Comparée, UMR 5087, INRA-CNRS-Université de Montpellier II, 30380 Saint Christol-Lez-Ales, France
Correspondence
Primitivo Caballero
pcm92{at}unavarra.es
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ABSTRACT |
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INTRODUCTION |
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Following consumption of occlusion bodies by the host insect and the liberation of occlusion-derived virions in the midgut, the principal steps of the baculovirus infection cycle involve entry into midgut columnar epithelial cells, the expression of viral early genes, DNA replication, late and very late gene expression, the production and release of budded virus, and occlusion body formation (Miller & Lu, 1997). The host range of any virus is determined by its ability to enter the cells of susceptible hosts, and then to replicate and produce new infectious virus particles. Although several mechanisms operate in conferring host cell specificity, little is known about the critical points at which the infection process is blocked in non-productive infection (Martin & Croizier, 1997
; Yanase et al., 1998b
). Elucidating the factors that are involved in determining the host range of baculoviruses is pertinent to understanding insectpathogen interactions and the application of baculoviruses as biopesticides.
Combining genomic elements from viruses possessing different host ranges offers a method of producing recombinant viruses with an extended host range (Kondo & Maeda, 1991). Blockage of Autographa californica NPV (AcMNPV) replication in Bombyx mori cells could be overcome by homologous recombination between the genomic DNA of AcMNPV and a 133 bp fragment of the helicase gene from B. mori NPV (BmNPV) (Croizier et al., 1994
). Martin & Croizier (1997)
investigated the infectivity of BmNPV in Spodoptera frugiperda (Sf) cell lines, non-permissive for BmNPV. They suggested that BmNPV virus progeny particles did not proliferate in cell culture due to a breakdown in cell-to-cell transmission in Sf9 cells.
The fall armyworm, S. frugiperda, the beet armyworm, Spodoptera exigua and the Egyptian cotton worm, Spodoptera littoralis, are polyphagous insects that regularly cause severe damage to a wide variety of crops in many parts of the world (Brown & Delhurst, 1975). The development of NPV-based biopesticides against these species has attracted attention for their potential implementation in integrated pest management programmes (Moscardi, 1999
).
The NPVs of S. frugiperda (SfMNPV), S. exigua (SeMNPV) and S. littoralis (SpliNPV) are virulent pathogens of their homologous hosts, but present a very variable response to heterologous hosts. Larvae of S. frugiperda and S. littoralis are considered non-permissive to SeMNPV, whereas larvae of S. exigua and S. littoralis are considered semi-permissive to SfMNPV, and all three Spodoptera species are permissive to SpliNPV (Murillo et al., 2003). It has been reported that SeMNPV is only capable of productive infection in S. exigua cell lines (Yanase et al., 1998b
). This virus can initiate replication in non-permissive insect cell lines including S. frugiperda, Spodoptera litura, S. littoralis, B. mori and Trichoplusia ni, but replication is restricted at various points, depending on the cell line (Yanase et al., 1998b
). Moreover, SeMNPV is capable of DNA replication in S. frugiperda cells co-infected with SeMNPV and AcMNPV (Yanase et al., 1998a
). SeMNPV and SfMNPV are closely related baculoviruses and their genomes present over 78 % identity (Tumilasci et al., 2003
). Despite their high degree of similarity, these two viruses have different host ranges. Studies of SeMNPV behaviour in S. frugiperda or S. littoralis hosts (considered to be non-permissive) or SfMNPV in S. exigua and S. littoralis hosts (considered to be semi-permissive), therefore represent an intriguing model to investigate the genetic determinants of baculovirus host specificity.
Most studies of baculovirus specificity have been performed in cell culture, particularly those related to SeMNPV. More realistic studies in vivo in non-permissive insects can reveal which step of the virus infection cycle is responsible for blocking replication, resulting in a non-productive infection. Is the entry into gut epithelial cells an important barrier for the virus? Or is it at the level of DNA replication or protein synthesis that virus propagation is impeded in heterologous hosts? Particularly for studies on virus entry, it is necessary to examine the behaviour of the virus in vivo.
In this study, we report the effects of SeMNPV infection alone or in combination with SfMNPV or SpliNPV in three Spodoptera species. We determine the time-course of SeMNPV infection in the midgut (primary infection) and in the haemocoel (secondary infection) of heterologous hosts. We follow the early events of SeMNPV pathogenesis in S. frugiperda and S. littoralis and show that following primary infection, SeMNPV proliferation was blocked at the haemocoel transmission stage and virtually cleared.
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METHODS |
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These viruses were propagated in fourth instars of their respective homologous hosts by the droplet feeding method (Hughes & Wood, 1981; Caballero et al., 1992
; Muñoz et al., 1998
). Infected larvae were reared on formaldehyde-free diet and collected after death (47 days post-inoculation). Occlusion bodies (OBs) were extracted from dead diseased larvae by homogenizing insect corpses in sterile distilled water and filtering through a cheesecloth. OBs were washed twice with 0·1 % SDS and once with 0·1M NaCl and finally resuspended in bidistilled water. OB suspensions were quantified using a bacterial counting chamber and stored at 4 °C until use.
Inoculation of larvae.
To determine the infectivity of SfNIC, SeUS1 and SpliM2, larvae of S. frugiperda, S. exigua and S. littoralis were inoculated with all viruses per os or by intrahaemocoelic injection. Per os bioassays were performed by inoculating 50 newly moulted second instars by the droplet feeding method. Individual viruses (SfNIC, SeUS1 and SpliM2) as well as three mixtures of OBs (SeUS1/SfNIC, SeUS1/SpliM2 and SfNIC/SpliM2 in a ratio of 1 : 1, and SeUS1/SfNIC and SeUS1/SpliM2 in a ratio of 1 : 100 000) were used as inocula. For each virus inoculum, a single high concentration of OBs was used in order to cause high mortality, as observed by Murillo et al. (2003). Intrahaemocoelic injection bioassays were performed with 25 newly moulted fourth instars, using the same virus inocula and the appropriate concentration of OBs for fourth instar insects. The OB concentration used was approximately 108109 OBs ml1. Each larvae was injected with 8 µl occlusion derived virions (ODVs) obtained after alkali treatment of the corresponding concentration of OBs (1 : 1 : 5 OBs : 0·5 M Na2CO3 : H2O by volume) (López-Ferber et al., 2003
). Inoculated larvae were individually transferred to a 25 compartment Petri dish and provided with diet. Control larvae were treated identically with solutions not containing virus. All procedures were performed at 25±1 °C.
DNA extraction and endonuclease analysis.
OBs obtained from inoculated larvae were purified as described above. ODVs extraction from OBs was performed by incubation with SDS and Na2CO3 solution. DNA was extracted from ODVs by incubation with SDS and proteinase K, followed by phenol/chloroform extraction and alcohol precipitation (Croizier & Ribeiro, 1992; Muñoz et al., 1998
). The DNA concentration was estimated by agarose gel electrophoresis. For REN analysis, 2 µg viral DNA was mixed with 10 units of the restriction enzyme PstI (Amersham) and the mixture was incubated at 37 °C for 412 h. Reactions were stopped at 65 °C for 15 min and mixed with 4 µl loading buffer solution (0·25 %, w/v bromophenol blue, 40 %, w/v sucrose). Electrophoresis was performed using horizontal 1 % agarose gels in TAE (0·04 M Tris/acetate, 0·001 M EDTA, pH 8·0) and the DNA fragments were visualized by staining with ethidium bromide.
RNA extraction from larvae.
Total RNA was extracted following the manufacturer's instructions. Whole inoculated larvae used in detection of SeUS1, SfNIC and SpliM2 viral transcripts were triturated in 500 µl Trizol (Invitrogen) (1 ml/100 mg tissue). The suspension was then mixed with 200 µl chloroform, incubated for 10 min and centrifuged at 13 000 g for 15 min at 4 °C. The aqueous fraction was precipitated using 2-propanol, centrifuged at 13 000 g for 10 min at 4 °C and washed with 70 % ethanol. Purified RNA was resuspended in approximately 50 µl H2O, depending on the size of the final pellet observed. RNA solutions were incubated at 60 °C for 10 min to favour resuspension, and in some cases, it was necessary to freeze and thaw the samples several times. RNA was quantified by measuring absorbance at 260 nm in a spectrophotometer (Hitachi, model U-1100) and stored at 80 °C until used. All materials and reagents were previously sterilized and treated with diethyl pyrocarbonate to eliminate RNases.
Total RNA from SeUS1-, SfNIC- and SpliM2-infected Spodoptera larvae was extracted at 6, 12 and 24 h post-inoculation (p.i.). Total RNA from the midgut and the haemocoel of mock-infected and SeUS1-infected fourth instar Spodoptera larvae was extracted at 24, 48, 72, 120, 144 and 168 h p.i. and from the pupae of S. frugiperda and S. littoralis as described previously.
Detection of viral transcripts.
RT-PCR was performed to detect gene expression in insect larvae and pupae, and to determine the presence or absence of SfNIC, SeUS1 and SpliM2 gene transcripts. After treatment with DNase, equivalent amounts of RNA (0·6 µg) were used in each reaction. To verify the absence of contaminant DNA in the samples, a PCR was performed on all RNA samples. RT-PCR was performed in two different steps. First, cDNA synthesis was performed using the Improm-II reverse transcriptase (Promega) and the internal reverse oligonucleotides specific to the viral genes described in Table 1, according to the manufacturer's instructions. An aliquot of the reaction (1/4) was then subjected to PCR amplification with a Taq DNA polymerase (Bioline) and the forward and reverse primer mixture for each gene (Table 1
). PCR products were analysed in 1 % agarose gels. A 100 bp marker ladder (Invitrogen), containing fragments of 0·12·6 kb in size, was used for size determination. DNA fragments were stained with ethidium bromide, visualized in a UV transilluminator, photographed and examined using the Molecular Analyst program (Bio-Rad).
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Detection of SfNIC, SeUS1 and SpliM2 DNA in infected and co-infected larvae.
The presence of viral DNA was investigated by PCR (Sambrook et al., 1989) with total DNA extracted as described previously. Volumes of 0·1 µl of the DNA solution were used for each reaction (approx. 10 ng). PCR was performed to detect SfNIC, SeUS1 and/or SpliM2 DNA in larvae inoculated with SfNIC, SeUS1 or SpliM2, or with a virus mixture (SfNIC/SeUS1, SeUS1/SpliM2 or SfNIC/SpliM2) as described previously. Infected larvae were sampled immediately following death (56 days) in the case of S. exigua and at the same moment for the other species, whether or not they had died of polyhedrosis disease. Specific primers were used for the SfNIC and SeUS1 ie-0 genes, and the SpliM2 egt gene (Table 1
). PCR products were analysed in 1 % agarose gels and visualized with ethidium bromide as described above.
Quantification of NPV transcripts and genomic DNA.
The detection of viral transcripts was performed by RT-PCR using RNA obtained at various intervals post-infection, as described above. For the semi-quantitative detection of viral genomic DNA, a PCR was performed using template DNA obtained from infected larvae at 56 days p.i. or co-infected larvae. The RT-PCR and PCR products were analysed in 1 % agarose gels, visualized with ethidium bromide and digitally photographed at a resolution of 19·7 pixels cm1. The relative intensities of the RT-PCR and PCR products were estimated by densitometric analysis, using the Scion Image PC program (Scion Corporation, USA). The results of densitometric analyses are presented graphically.
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RESULTS |
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SfNIC also produced a fatal infection in all three Spodoptera species following consumption or injection of inocula. In S. frugiperda the infection produced the typical signs and symptoms of an NPV disease, whereas in S. littoralis and S. exigua the symptoms were atypical. For example, a very low number of OBs were produced per larva and the infected larvae did not liquefy at the end of the infection process in these hosts. The low number of OBs extracted from infected larvae of heterologous hosts made it impossible to examine the viral DNA profile by REN analysis. In contrast, while SeUS1 produced a fatal infection in its homologous host, S. exigua, the larvae of S. frugiperda and S. littoralis were resistant to SeUS1. Heterologous larvae remained healthy, even after oral inoculation with a high concentration of OBs or injection of a high concentration of ODVs into the haemocoel. No OBs of SeMNPV were visualized in the haemocoel of inoculated heterologous hosts by optical microscopy.
Detection of NPV-specific transcripts in Spodoptera larvae
RT-PCR analysis of total RNA extracted from virus-inoculated larvae showed that each of the three NPVs (SfNIC, SeUS1 and SpliM2) initiated an infection in all three host species tested (S. frugiperda, S. exigua and S. littoralis). Following oral inoculation with OBs, SfNIC-ie-0 or SpliM2-egt transcripts were detected at 12 and 24 h p.i. at a similar level in all three host species (Fig. 1a and b). SeUS1-ie-0 transcripts were also detected in S. exigua at 6 h p.i., S. frugiperda at 24 h p.i. and S. littoralis at 12 h p.i., signalling the onset of infection by SeUS1 in each host species (Fig. 1c
). However, in S. exigua the transcription level increased between 6 and 24 h p.i., whereas in S. frugiperda and S. littoralis the transcription of this gene was delayed and the quantity of transcript detected by RT-PCR was markedly lower (Fig. 1c
).
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Viral progeny in single and double virus-infected larvae
The effect of the homologous NPV on the replication of heterologous NPVs was assessed. Viral DNA produced in S. exigua, S. frugiperda and S. littoralis larvae infected by single NPVs, used as controls, or co-infected with combinations of SeUS1, SfNIC and SpliM2, was analysed by PCR (Fig. 4). The quantity of SeUS1 DNA detected by PCR in larvae of S. frugiperda or S. littoralis co-infected with SeUS1/SfNIC or SeUS1/SpliM2 was 3·1 and 3·0 times greater, respectively, by per os inoculation (Fig. 4a
) and 1·6 and 2·2 times greater by intrahaemocoelic inoculation (Fig. 4b
), compared to larvae inoculated with SeUS1 alone. However, the reverse was not true; we did not observe a clear increase in the quantity of SfNIC or SpliM2 DNA in heterologous larvae when co-infected with the homologous viruses, as observed for SeUS1. In S. exigua larvae, the quantity of SfNIC DNA was 1·8 and 1·2 times lower by per os and intrahaemocoelic inoculation, respectively, compared to S. exigua larvae inoculated with SfNIC alone (Fig. 4a and b
). In contrast, in S. littoralis larvae double-infected with SfNIC/SpliM2, the quantity of SfNIC DNA was 2·1 times lower by per os inoculation (Fig. 4a
), but 4·7 times greater by intrahaemocoelic inoculation (Fig. 4b
), compared to S. littoralis inoculated via each route with SfNIC alone. In S. exigua larvae co-infected with SpliM2/SeUS1 by both routes, the abundance of SpliM2 DNA decreased slightly (
1·3 times), compared to the abundance of SpliM2 DNA in S. exigua larvae inoculated with SpliM2 alone (Fig. 4a and b
). In S. frugiperda co-infected orally with SpliM2/SfNIC the abundance of SpliM2 DNA was 3·6 times greater than in S. frugiperda larvae inoculated with SpliM2 alone, but did not change in larvae inoculated by intrahaemocoelic injection (Fig. 4b
). In general, DNA replication of SeUS1 in heterologous hosts, such as S. frugiperda and S. littoralis, increased when co-infected with the homologous viruses. Conversely, the DNA replication of wide-host-range viruses in heterologous hosts competed with homologous viruses, resulting in either an increase or a decrease, depending on the hosts and the inoculation route.
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In the second inoculation, S. exigua, S. frugiperda or S. littoralis larvae were inoculated with OBs of SfNIC/SeUS1 or SpliM2/SeUS1 at a ratio of 1 : 100 000. A REN analysis with PstI was performed with the DNA from the OBs obtained after inoculation of each host species. We observed similar REN patterns to those observed with 1 : 1 inoculation in all cases (data not shown); we could not detect the SeUS1 virus in co-infected larvae despite its being inoculated in a quantity 105 times greater than that of the homologous virus. When interpreting these results, it should be remembered that RT-PCR studies were performed on larval tissues or haemolymph at 48 h p.i., whereas DNA for REN analysis was extracted from OBs harvested from dead larvae.
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DISCUSSION |
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The regulation of 100 or more open reading frames is required to accomplish productive infection by SeMNPV; this is highly complex and involves sequential and coordinated expression of immediately early, early, late and very late genes (Lu & Miller, 1997). In the cascade of gene expression, successive stages of virus replication are dependent on the correct expression of genes from the preceding stages. The transcription of four temporally distinct SeUS1 genes was studied in order to determine the moment at which the virus cycle is blocked. The kinetics of SeUS1 transcription in the three Spodoptera species observed by RT-PCR indicated that the virus was actively attempting to replicate in heterologous hosts, although at a much lower level than in its homologous host. The positive replication signal obtained with the late genes chitinase and polyhedrin indicated that all genes required for virus replication in heterologous hosts (Kool et al., 1994
) were present in the SeUS1 genome. It is likely that the genes responsible for SeUS1 replication are homologous with those present in SfNIC or SpliM2, since SfNIC and SeUS1 are closely related. The low level of SeUS1 replication activity in heterologous hosts, as determined by the relatively low levels of SeUS1 gene transcription, could be due to a small number of infected cells producing high levels of virus transcripts, rather than a large number of cells producing very low levels, as has been suggested for BmNPV in Sf9 cell lines (Martin & Croizier, 1997
). An alternative hypothesis to account for the low level of SeUS1 gene expression observed in heterologous hosts is that the viral particles budding from infected cells may be unable to infect neighbouring cells, as observed with cells infected with gp64 Autographa californica multicapsid NPV (AcMNPV) (Monsma et al., 1996
). The occurrence of DNA replication and the appearance of an increasing quantity of polh transcripts over time are consistent with S. frugiperda and S. littoralis being semi-permissive species for SeUS1 replication. The lower transcriptional activity of SeUS1 in heterologous hosts could explain why S. frugiperda and S. littoralis have so far been considered as non-permissive species for SeMNPV.
A further experiment was performed to determine whether SeUS1 is able to produce secondary infections in heterologous hosts. Very late gene expression was studied in the midgut and haemolymph of SeUS1-infected Spodoptera larvae. SeUS1 polh gene expression was detected in the midgut and the rest of the body indicating that SeUS1 can transmit BVs to neighbouring cells and produce secondary infection in heterologous hosts. In addition, when S. frugiperda or S. littoralis were each co-infected with SeUS1 and their respective homologous virus, DNA replication of SeUS1 increased with respect to infections of SeUS1 alone. In contrast, by REN analysis we could not observe any characteristic bands of a secondary (heterologous) virus in co-infected larvae, suggesting that REN analysis was probably not sufficiently sensitive to detect low levels of replication in heterologous hosts. We did, however, observe the presence of numerous other bands at low concentrations, probably due to the use of wild-type isolates (SeUS1 and SfNIC) that comprised various genotypic variants. Alternatively, the heterologous infection did not reach the OB production stage. Experiments are in process using pure genotypes in order to detect possible recombination between viruses or genotypic mixtures. However, PCR analysis performed with co-infected larvae indicated that non-permissive virus DNA replication, such as SeUS1, in heterologous hosts (S. frugiperda and S. littoralis), increased when co-infected with the respective homologous viruses. This suggests that, SfNIC and SpliM2 assist the replication of SeUS1. The origin of DNA replication in SeMNPV, the non-homologous region (hr), replicates in AcMNPV co-infected cells at low levels (Heldens et al., 1997), a phenomenon that may have also occurred in S. frugiperda and S. littoralis larvae. Kamita et al. (2003)
reached similar conclusions after observing high-frequency recombination between two types of BmNPV in Sf9 cells, a weakly permissive insect cell line, higher than that observed between BmNPV and AcMNPV. High frequencies of recombination indicated that the replication of BmNPV DNAs occurred actively in this cell line (Kamita et al., 2003
). After demonstrating that the replication of SeMNPV in S. frugiperda cells was improved by co-infection with AcMNPV, Yanase et al. (1998a)
suggested that SeMNPV may use the transcripts of AcMNPV for replication in co-infected cells.
We conclude that SeMNPV replication in heterologous hosts requires certain helper functions from SfMNPV or SpliNPV, presumably by sharing some viral factors. Other examples of recombination between viruses or helper functions have demonstrated an increase in DNA replication or the extension of a virus host range. A recombinant AcMNPV bearing a small region of the p143 gene from BmNPV replicated in a BmN cell line, whereas the original AcMNPV was incapable of replication in these cells (Maeda et al., 1993; Croizier et al., 1994
). The replacement or modification of certain NPV genes with those of other NPVs resulted in an extended host range or improved DNA replication, suggesting that these domains might interact with host-specific factors. Other genes that influence baculovirus host range include late transcription factors and apoptotic suppressors. AcMNPV was capable of productive infection in a Lymantria dispar (Ld) cell line in the presence of the hrf-1 gene product from LdMNPV (Thiem et al., 1996
; Thiem, 1997
). Recently Zhang et al. (2002)
demonstrated that a host apoptotic response to virus infection reduced AcMNPV cell-to-cell transmission of infection in S. litura larvae, apoptosis representing a host-range limiting factor for AcMNPV infection. The molecular mechanisms involved in apoptosis signalling are still unknown. Potential stimuli consist of shut-off of RNA synthesis, viral DNA replication and viral gene expression (Clem & Miller, 1993
; Prikhod'ko & Miller, 1996
; Miller, 1997
; Clem, 2001
), and it is possible that several factors are involved in triggering programmed cell death (LaCount & Friesen, 1997
).
In the present study, time-course experiments revealed that the quantity of polh transcripts in the midgut or haemocoel of SeUS1-infected heterologous hosts decreased significantly at 72120 h p.i. and subsequently almost disappeared at 168 h p.i., whereas a progressive increase was seen in the homologous host. None of the heterologous larvae succumbed to polyhedrosis disease. Similar results were observed when non-permissive hosts, Manduca sexta and Helicoverpa zea were inoculated with an AcMNPV recombinant virus expressing the lacZ gene (Washburn et al., 1996, 2000
). These authors suggested that a cellular immune response was responsible for clearance of a potentially fatal infection of AcMNPV in non-permissive hosts. In both species, primary infection of midgut columnar cells by AcMNPV began at the same time as in permissive hosts, and secondary infections in midgut-associated tracheae were revealed by optical microscope observation of lacZ expression. However, in heterologous hosts, a decline in the number of infection foci of AcMNPV was detected. By 72 h p.i., haemocytes surround infected cells and haemocyte aggregations transformed infected cells into melanized capsules. These infections failed to spread and were ultimately cleared. The time-course of SeUS1 infection in heterologous Spodoptera hosts is similar to that described by Washburn et al. (1996
, 2000)
, although the reporter gene technique they used was far less sensitive than the RT-PCR method that we used. Between 24 and 72 h p.i. SeUS1-polh transcription increased but after 72 h p.i. the infection declined and disappeared. It appears that S. frugiperda and S. littoralis larvae exhibit an immune response to SeUS1 infection. However, due to the technique of midgut dissection we employed, we cannot exclude the possibility that some of the cells intimately associated with the insect midgut, such as tracheal cells, may also have contributed to the results observed in RT-PCR analysis of viral transcripts from midgut tissue.
In conclusion, SpliM2 is able to infect, replicate and produce progeny OBs in all the Spodoptera species tested. In contrast, SfNIC is lethal to heterologous hosts S. exigua and S. littoralis but infected larvae do not melt, liquefy and melanize. The determination of the factors or mechanisms that induce such responses in heterologous hosts infected by SfNIC was not analysed. We could not determine the factors or mechanisms that induce such responses in heterologous hosts infected by SfNIC. Finally, SeUS1 is able to replicate in heterologous hosts and, in addition, all genes required for SeUS1 replication are present in the SeUS1 genome, as the virus infection cycle was observed. However, gene expression is significantly lower in heterologous hosts. It seems that anti-viral responses (apoptosis or cellular immune response) of the heterologous hosts appear to play an important role in the specificity of SeUS1. However, SeUS1 was not blocked at an obvious point during the infection cycle, but declined gradually over time and eventually disappeared. We therefore conclude that entry and the primary virus infection cycle are not the principal determinants for SeUS1 infection of heterologous Spodoptera species. Experiments are in progress to determine the mechanisms involved in SeUS1 specificity and which step(s) of the virus cycle are inhibited in heterologous hosts. The system described in this study, SeMNPV, SfMNPV and SpliNPV and their respective hosts, represents a useful model for studying the determinants of baculovirus host range. Such studies can also provide a basis for host-range risk assessment applied to the development of natural and recombinant baculovirus bioinsecticides.
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ACKNOWLEDGEMENTS |
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Received 7 April 2004;
accepted 18 June 2004.
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