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Enoch Y Park - One of the best experts on this subject based on the ideXlab platform.
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Versatility of chitosan/BmNPV Bacmid DNA nanocomplex as transfection reagent of recombinant protein expression in silkworm larvae
Biotechnology Letters, 2016Co-Authors: Tatsuya Kato, Sho Arai, Hirono Ichikawa, Enoch Y ParkAbstract:Objective To examine the feasibility of chitosan as an alternative transfection reagent candidate for protein expression in Bm5 cells and silkworm larvae using recombinant BmNPV Bacmid DNA. Result Chitosan 100 and recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) Bacmid DNA, in amino group/phosphate group (N/P) ratios of 0.1–10, were used for formation of chitosan/DNA nanocomplexes. The chitosan/BmNPV Bacmid DNA nanocomplexes showed higher specific activity of GFP_uv-β1,3- N -acetylglucosaminyltransferase 2 (β3GnT2) fusion protein (GGT2) expressed in silkworm larvae than DMRIE-C, a conventional silkworm transfection reagent. In particular, the composition of chitosan and BmNPV Bacmid DNA nanocomplexes formed by an N/P ratio of 8 or 10, respectively, showed the highest specific activity of β3GnT2 in the silkworm larvae hemolymph. In addition, three different proteins were expressed in silkworm larvae to the same extent using chitosan as that using DMRIE-C. Conclusion This is the first finding that chitosan/BmNPV Bacmid DNA nanocomplexes can rival the performance of commercially available transfection reagents for the expression of recombinant proteins in Bm5 cells and silkworm larvae.
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2016Co-Authors: Ssbiomed Centbmc Biotechnology, Tatsuya Kato, Makoto Nakajima, Makoto Ogata, Takakiyo Obara, Hirokazu Yagi, Koichi Kato, Taichi Usui, Enoch Y ParkAbstract:Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV Bacmid-injected silkworm larva
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Production of Rous sarcoma virus-like particles displaying human transmembrane protein in silkworm larvae and its application to ligand-receptor binding assay.
Journal of biotechnology, 2011Co-Authors: Yoshitaka Tsuji, Vipin Kumar Deo, Tatsuya Kato, Enoch Y ParkAbstract:Abstract Two types of Rous sarcoma virus (RSV) group-antigen protein (Gag) virus like particles (VLPs), full-length Gag (Gag701) and RSV protease domain (PR)-deleted mutant (Gag577) were expressed in silkworm larvae. Gag577 was secreted into hemolymph efficiently using wild type Bacmid (WT), cysteine protease-deficient Bacmid (CP − ), cysteine protease and chitinase-deficient Bacmid (CP − Chi − ) Bacmids, but comparatively Gag701 secretion levels were low. VLPs were purified on 10–60% (v/v) sucrose density gradient by ultracentrifugation and their structures confirmed under electron microscope. When hPRR and RSV Gag577 were co-expressed in silkworm larvae, human prorenin receptor (hPRR) was displayed on the surface of RSV VLPs, which was detected by Western blotting and immunoelectron microscopy. Moreover, binding of hPRR localized on the surface of VLPs to human prorenin was confirmed by ELISA. These results indicate that active hPRR was displayed on the surface of RSV VLPs, which can be utilized for drug discovery of hPRR blockers to prevent nephropathy. Moreover, this transmembrane protein display system using RSV Gag in silkworm larvae is applicable to expression of intact transmembrane proteins and binding assay of transmembrane proteins to its ligands, especially for transmembrane proteins which cannot be purified from membrane fractions in active states.
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Human insulin gene expressing with Bombyx mori multiple nucleopolyhedrovirus (BmMNPV) expression system
World Journal of Microbiology and Biotechnology, 2010Co-Authors: Wan-fu Yue, Enoch Y Park, Fang Zhou, J. Joe Hull, Yun-gen MiaoAbstract:Using human genomic DNA as a template, the human insulin gene was cloned and used to construct various reBmMNPVBacmids. Cysteine protease gene deletion (CPD-BmMNPV Bacmid) and cysteine protease- and chitinase-deficient (CPPD- BmMNPV Bacmid) baculoviruses were used to express both native and FLAG-tagged human insulin. Silkworm larvae were infected with the above recombinant Bacmid DNAs, and the expressed insulin was purified and identified from infected silkworm haemolymph. The highest expression was shown with the CPPD- BmMNPV Bacmid, which was about two times that of the wild type of reBmMNPVBacmid, reaching 15.827 ng/ml haemolymph.
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molecular chaperone assisted production of human α 1 4 n acetylglucosaminyltransferase in silkworm larvae using recombinant bmnpv Bacmids
Molecular Biotechnology, 2009Co-Authors: Makoto Nakajima, Tatsuya Kato, Shin Kanamasa, Enoch Y ParkAbstract:In this study, human α-1,4-N-acetylglucosaminyltransferase (α4GnT) fused with GFPuv (GFPuv-α4GnT) was expressed using both a transformed cell system and silkworm larvae. A Tn-pXgp-GFPuv-α4GnT cell line, isolated after expression vector transfection, produced 106 mU/ml of α4GnT activity in suspension culture. When Bombyx mori nucleopolyhedrovirus containing a GFPuv-α4GnT fusion gene (BmNPV-CP −/GFPuv-α4GnT) Bacmid was injected into silkworm larvae, α4GnT activity in larval hemolymph was 352 mU/ml, which was 3.3-fold higher than that of the Tn-pXgp-GFPuv-α4GnT cell line. With human calnexin (CNX) or human immunoglobulin heavy chain-binding protein (BiP, GRP78) coexpressed under the control of the ie-2 promoter, α4GnT activity in larval hemolymph increased by 1.4–2.0-fold. Moreover, when BmNPV-CP −/GFPuv-α4GnT Bacmid injection was delayed for 3 h after BmNPV-CP −/CNX injection, the α4GnT activity increased significantly to 922 mU/ml, which was 8.7-fold higher than that of the Tn-pXgp-GFPuv-α4GnT cell line. Molecular chaperone assisted-expression in silkworm larvae using the BmNPV Bacmid is a promising tool for recombinant protein production. This system could lead to large-scale production of more complex recombinant proteins.
Primitivo Caballero - One of the best experts on this subject based on the ideXlab platform.
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The ``11K'' gene family members sf68, sf95 and sf138 modulate transmissibility and insecticidal properties of Spodoptera frugiperda multiple nucleopolyhedrovirus
JOURNAL OF INVERTEBRATE PATHOLOGY, 2015Co-Authors: Ines Beperet, Oihane Simon, Miguel López-ferber, Trevor Williams, Primitivo CaballeroAbstract:The ``11K'' gene family is notable for having homologs in both baculoviruses and entomopoxviruses and is classified as either type 145 or type 150, according to their similarity with the ac145 or ac150 genes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). One homolog of ac145 (sf138) and two homologs of ac150 (sf68 and 495) are present in Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Recombinant Bacmids lacking sf68, sf95 or sf138 (Sf68null, Sf95null and Sf138null, respectively) and the respective repair Bacmids were generated from a Bacmid comprising the complete virus genome. Occlusion bodies (OBs) of the Sf138null virus were similar to 15-fold less orally infective to insects, which was attributed to a 100-fold reduction in ODV infectious titer. Inoculation of insects with Sf138null OBs in mixtures with an optical brightener failed to restore the pathogenicity of Sf138null OBs to that of the parental virus, indicating that the effects of sf138 deletion on OB pathogenicity were unlikely to involve an interaction with the gut peritrophic matrix. In contrast, deletion of sf68 and 495 resulted in a slower speed-of-kill by 9 h, and a concurrent increase in the yield of OBs. Phylogenetic analysis indicated that sf68 and sf95 were not generated after a duplication event of an ancestral gene homologous to the ac150 gene. We conclude that type 145 genes modulate the primary infection process of the virus, whereas type 150 genes appear to have a role in spreading systemic infection within the insect.
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Deletion of egt is responsible for the fast-killing phenotype of natural deletion genotypes in a Spodoptera frugiperda multiple nucleopolyhedrovirus population
Journal of invertebrate pathology, 2012Co-Authors: Oihane Simon, Miguel López-ferber, Trevor Williams, Primitivo CaballeroAbstract:Abstract A Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, includes fast-killing genotypes with deletions in the egt region. Four Bacmid based recombinants were constructed to determine the role of egt in this phenotype. SfdelF Bacmid encompassed the deletion found in the NIC-F genotype. Sfdel3AP2 Bacmid was constructed using the deletion reported in SfMNPV-3AP2 (Missouri, fast-killing isolate), whereas Sfdelegt and Sfdel27 Bacmids lacked the single genes egt and the adjacent sf27 gene, respectively. No significant differences were observed in occlusion body (OB) concentration–mortality metrics (LC 50 values) among the viruses. Larvae infected by NIC-B (a natural genotype with the largest genome), Sfbac (a Bacmid with NIC-B genome) and Sfdel27 survived significantly longer than insects infected by NIC-F, SfdelF, SfMNPV-3AP2, Sfdel3AP2 or Sfdelegt. Fast-killing viruses produced ∼6–13-fold fewer OBs/larva compared to other viruses tested. We conclude that deletion/disruption of egt is responsible for the fast-killing phenotypes of naturally-occurring genotypes in SfMNPV populations from Missouri and Nicaragua.
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Stability of a Spodoptera frugiperda Nucleopolyhedrovirus Deletion Recombinant during Serial Passage in Insects
Applied and environmental microbiology, 2009Co-Authors: Oihane Simon, Miguel López-ferber, Trevor Williams, Robert D. Possee, Primitivo CaballeroAbstract:The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome Bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null Bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null Bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null Bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.
George F. Rohrmann - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the Role of Very Late Expression Factor 1 in Baculovirus Capsid Structure and DNA Processing
Journal of virology, 2006Co-Authors: Adam L. Vanarsdall, Kazuhiro Okano, George F. RohrmannAbstract:Very late expression factor 1 (VLF-1) of Autographa californica multiple nucleopolyhedrovirus is a putative tyrosine recombinase and is required for both very late gene expression and budded virus production. In this report, we show that a vlf-1 knockout Bacmid was able to synthesize viral DNA at levels similar to that detected for a gp64 knockout Bacmid that served as a noninfectious control virus. Additionally, analysis of replicated Bacmid DNA by field-inversion gel electrophoresis indicated that VLF-1 is not required for synthesizing high-molecular-weight intermediates that could be resolved into unit-length genomes when cut at a unique restriction site. However, immunoelectron microscopic analysis revealed that in cells transfected with a vlf-1 knockout Bacmid, aberrant tubular structures containing the capsid protein vp39 were observed, suggesting that this virus construct was defective in producing mature capsids. In contrast, rescuing the vlf-1 knockout Bacmid construct with a copy of VLF-1 that carries a mutation of a highly conserved tyrosine (Y355F) was sufficient to restore the production of nucleocapsids with a normal appearance, but not infectious virus production. Furthermore, the results of a DNase I protection assay indicated that the DNA packaging efficiency of the VLF-1(Y355F) virus construct was similar to that of the gp64 knockout control. Finally, a recombinant virus containing a functional hemagglutinin epitope-tagged version of VLF-1 was constructed to investigate the association of VLF-1 with the nucleocapsid. Analysis by immunoelectron microscopy of Sf-9 cells infected with this virus showed that VLF-1 localized to an end region of the nucleocapsid. Collectively, these results indicate that VLF-1 is required for normal capsid assembly and serves an essential function during the final stages of the DNA packaging process.
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Characterization of a Baculovirus Lacking the Alkaline Nuclease Gene
Journal of virology, 2004Co-Authors: Kazuhiro Okano, Adam L. Vanarsdall, George F. RohrmannAbstract:The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) associates with the baculovirus single-stranded DNA binding protein LEF-3 and possesses both a 5′→3′ exonuclease and an endonuclease activity. These activities are thought to be involved in DNA recombination and replication. To investigate the role of AN in AcMNPV replication, the λ Red system was used to replace the an open reading frame with a chloramphenicol acetyltransferase gene (cat) and a Bacmid containing the AcMNPV genome in Escherichia coli. The AcMNPV an knockout Bacmid (vAcAN-KO/GUS) was unable to propagate in Sf9 cells, although an an-rescued Bacmid (vAcAN-KO/GUS-Res) propagated normally. In addition, the mutant did not appear to produce budded virions. These data indicated that an is an essential baculovirus gene. Slot blot and DpnI assays of DNA replication in Sf9 cells transfected with vAcAN-KO/GUS, vAcAN-KO/GUS-Res, and a wild-type Bacmid showed that the vAcAN-KO/GUS Bacmid was able to replicate to levels similar to those seen with the vAcAN-KO/GUS-Res and wild-type Bacmids at early stages posttransfection. However, at later time points DNA did not accumulate to the levels seen with the repaired or wild-type Bacmids. Northern analysis of Sf9 cells transfected with Bacmid vAcAN-KO/GUS showed that transcription of late and very late genes was lower at later times posttransfection relative to the results seen with wild-type and vAcAN-KO/GUS-Res Bacmids. These data suggest that the an gene might be involved in the maturation of viral DNA or packaging of the DNA into virions.
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Characterization of a baculovirus with a deletion of vlf-1.
Virology, 2004Co-Authors: Adam L. Vanarsdall, Kazuhiro Okano, George F. RohrmannAbstract:Very late expression factor (VLF-1) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for high levels of expression of the very late genes p10 and polh, and evidence suggests VLF-1 may also be involved in viral DNA replication. In this study, investigations determined whether VLF-1 is essential for viral DNA replication by generating a vlf-1 knockout Bacmid containing the AcMNPV genome through homologous recombination in Escherichia coli. Additionally, a vlf-1 repair Bacmid was constructed by transposing the vlf-1 ORF and native promoter region into the polh locus of the vlf-1 knockout Bacmid. After transfecting these virus constructs into Spodoptera frugiperda (Sf-9) cells, the vlf-1 knockout Bacmid was unable to produce a viral infection while the repair Bacmid propagated at wild-type levels. Experiments were performed to conclude whether the vlf-1 knockout phenotype was due to a defect in viral DNA synthesis or late gene transcription. Southern blot analyses determined that the vlf-1 knockout Bacmid was able to replicate viral DNA but only to about one-third the level of wild-type or rescued controls. In addition, virion DNA was not detected in the supernatant of transfected cells, indicating that the DNA synthesized by the mutant virus was unable to assemble into virions that bud out of the cells. Analysis of viral gene transcription confirmed that late gene transcription was not affected by the vlf-1 knockout but transcription of the very late gene p10 was substantially reduced.
Oihane Simon - One of the best experts on this subject based on the ideXlab platform.
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The ``11K'' gene family members sf68, sf95 and sf138 modulate transmissibility and insecticidal properties of Spodoptera frugiperda multiple nucleopolyhedrovirus
JOURNAL OF INVERTEBRATE PATHOLOGY, 2015Co-Authors: Ines Beperet, Oihane Simon, Miguel López-ferber, Trevor Williams, Primitivo CaballeroAbstract:The ``11K'' gene family is notable for having homologs in both baculoviruses and entomopoxviruses and is classified as either type 145 or type 150, according to their similarity with the ac145 or ac150 genes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). One homolog of ac145 (sf138) and two homologs of ac150 (sf68 and 495) are present in Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Recombinant Bacmids lacking sf68, sf95 or sf138 (Sf68null, Sf95null and Sf138null, respectively) and the respective repair Bacmids were generated from a Bacmid comprising the complete virus genome. Occlusion bodies (OBs) of the Sf138null virus were similar to 15-fold less orally infective to insects, which was attributed to a 100-fold reduction in ODV infectious titer. Inoculation of insects with Sf138null OBs in mixtures with an optical brightener failed to restore the pathogenicity of Sf138null OBs to that of the parental virus, indicating that the effects of sf138 deletion on OB pathogenicity were unlikely to involve an interaction with the gut peritrophic matrix. In contrast, deletion of sf68 and 495 resulted in a slower speed-of-kill by 9 h, and a concurrent increase in the yield of OBs. Phylogenetic analysis indicated that sf68 and sf95 were not generated after a duplication event of an ancestral gene homologous to the ac150 gene. We conclude that type 145 genes modulate the primary infection process of the virus, whereas type 150 genes appear to have a role in spreading systemic infection within the insect.
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Deletion of egt is responsible for the fast-killing phenotype of natural deletion genotypes in a Spodoptera frugiperda multiple nucleopolyhedrovirus population
Journal of invertebrate pathology, 2012Co-Authors: Oihane Simon, Miguel López-ferber, Trevor Williams, Primitivo CaballeroAbstract:Abstract A Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, includes fast-killing genotypes with deletions in the egt region. Four Bacmid based recombinants were constructed to determine the role of egt in this phenotype. SfdelF Bacmid encompassed the deletion found in the NIC-F genotype. Sfdel3AP2 Bacmid was constructed using the deletion reported in SfMNPV-3AP2 (Missouri, fast-killing isolate), whereas Sfdelegt and Sfdel27 Bacmids lacked the single genes egt and the adjacent sf27 gene, respectively. No significant differences were observed in occlusion body (OB) concentration–mortality metrics (LC 50 values) among the viruses. Larvae infected by NIC-B (a natural genotype with the largest genome), Sfbac (a Bacmid with NIC-B genome) and Sfdel27 survived significantly longer than insects infected by NIC-F, SfdelF, SfMNPV-3AP2, Sfdel3AP2 or Sfdelegt. Fast-killing viruses produced ∼6–13-fold fewer OBs/larva compared to other viruses tested. We conclude that deletion/disruption of egt is responsible for the fast-killing phenotypes of naturally-occurring genotypes in SfMNPV populations from Missouri and Nicaragua.
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Stability of a Spodoptera frugiperda Nucleopolyhedrovirus Deletion Recombinant during Serial Passage in Insects
Applied and environmental microbiology, 2009Co-Authors: Oihane Simon, Miguel López-ferber, Trevor Williams, Robert D. Possee, Primitivo CaballeroAbstract:The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome Bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null Bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null Bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null Bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.
Miguel López-ferber - One of the best experts on this subject based on the ideXlab platform.
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The ``11K'' gene family members sf68, sf95 and sf138 modulate transmissibility and insecticidal properties of Spodoptera frugiperda multiple nucleopolyhedrovirus
JOURNAL OF INVERTEBRATE PATHOLOGY, 2015Co-Authors: Ines Beperet, Oihane Simon, Miguel López-ferber, Trevor Williams, Primitivo CaballeroAbstract:The ``11K'' gene family is notable for having homologs in both baculoviruses and entomopoxviruses and is classified as either type 145 or type 150, according to their similarity with the ac145 or ac150 genes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). One homolog of ac145 (sf138) and two homologs of ac150 (sf68 and 495) are present in Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Recombinant Bacmids lacking sf68, sf95 or sf138 (Sf68null, Sf95null and Sf138null, respectively) and the respective repair Bacmids were generated from a Bacmid comprising the complete virus genome. Occlusion bodies (OBs) of the Sf138null virus were similar to 15-fold less orally infective to insects, which was attributed to a 100-fold reduction in ODV infectious titer. Inoculation of insects with Sf138null OBs in mixtures with an optical brightener failed to restore the pathogenicity of Sf138null OBs to that of the parental virus, indicating that the effects of sf138 deletion on OB pathogenicity were unlikely to involve an interaction with the gut peritrophic matrix. In contrast, deletion of sf68 and 495 resulted in a slower speed-of-kill by 9 h, and a concurrent increase in the yield of OBs. Phylogenetic analysis indicated that sf68 and sf95 were not generated after a duplication event of an ancestral gene homologous to the ac150 gene. We conclude that type 145 genes modulate the primary infection process of the virus, whereas type 150 genes appear to have a role in spreading systemic infection within the insect.
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Deletion of egt is responsible for the fast-killing phenotype of natural deletion genotypes in a Spodoptera frugiperda multiple nucleopolyhedrovirus population
Journal of invertebrate pathology, 2012Co-Authors: Oihane Simon, Miguel López-ferber, Trevor Williams, Primitivo CaballeroAbstract:Abstract A Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, includes fast-killing genotypes with deletions in the egt region. Four Bacmid based recombinants were constructed to determine the role of egt in this phenotype. SfdelF Bacmid encompassed the deletion found in the NIC-F genotype. Sfdel3AP2 Bacmid was constructed using the deletion reported in SfMNPV-3AP2 (Missouri, fast-killing isolate), whereas Sfdelegt and Sfdel27 Bacmids lacked the single genes egt and the adjacent sf27 gene, respectively. No significant differences were observed in occlusion body (OB) concentration–mortality metrics (LC 50 values) among the viruses. Larvae infected by NIC-B (a natural genotype with the largest genome), Sfbac (a Bacmid with NIC-B genome) and Sfdel27 survived significantly longer than insects infected by NIC-F, SfdelF, SfMNPV-3AP2, Sfdel3AP2 or Sfdelegt. Fast-killing viruses produced ∼6–13-fold fewer OBs/larva compared to other viruses tested. We conclude that deletion/disruption of egt is responsible for the fast-killing phenotypes of naturally-occurring genotypes in SfMNPV populations from Missouri and Nicaragua.
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Stability of a Spodoptera frugiperda Nucleopolyhedrovirus Deletion Recombinant during Serial Passage in Insects
Applied and environmental microbiology, 2009Co-Authors: Oihane Simon, Miguel López-ferber, Trevor Williams, Robert D. Possee, Primitivo CaballeroAbstract:The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome Bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null Bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null Bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null Bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.