A baculovirus-mediated expression system for the analysis of HaSV RNA packaging
- Authors: Mendes, Adriano
- Date: 2012
- Subjects: RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4025 , http://hdl.handle.net/10962/d1004085 , RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Description: The Helicoverpa armigera stunt virus (HaSV) is a member of a family of small nonenveloped (+) ssRNA insect viruses currently known as the Tetraviridae. This family is unique in terms of the T=4 quasi-symmetry of its capsid particles and the unusually narrow host range and tissue tropism. Assembly of tetraviral particles has been well characterised and involves the combination of 240 copies of a single capsid precursor protein (VCap) into a procapsid followed by autoproteolytic cleavage to yield the major (β) and minor (γ) capsid subunits within the mature particle. HaSV has two genomic RNAs, RNA 1 encoding the replicase and RNA 2 encoding VCap and p17, the ORF of which lies upstream of and overlaping with the 5’ end of the VCap ORF. Prior to this study, Vlok (2009) used a plasmid expression system to study RNA packaging in HaSV VLPs assembled in Spodoptera frugiperda 9 (Sf9) cells co-expressing p17 and VCap. The study showed that the p17 ORF was required for the packaging of RNA 2 during capsid assembly but it was unclear whether p17 expression was required for packaging. In addition, expression from the transfected plasmids was sub-optimal affecting both the yield of VLPs and the detection of p17. The aim of this study was to use the plasmid system to test whether p17 expression was required for plasmid-derived VLP RNA packaging and then develop a baculovirus-mediated system to test this hypothesis. By using a plasmid in which the start codon of p17 was mutated, it was shown that p17 expression was required for RNA 2 packaging into plasmid-VLPs. For the baculovirus system, four recombinant baculoviruses based upon the pFastBac Dual expression system, were constructed. These included Bac20, expressing wild type RNA 2, Bac21, RNA 2 with p17 silenced, Bac23, RNA 2 and p17 expressed on a separate transcript and Bac24, RNA 2 with p17 silenced plus p17 expressed on a separate transcript. Assembly of VLPs was more efficient using the baculovirus expression system and p17 expression was observed in cells infected with Bac20, Bac23 and Bac24, but not Bac21. In contrast to the plasmid-VLPs, bac-VLPs did not require p17 for the encapsidation of RNA 2. In addition to RNA 2, Bac23 and Bac24 packaged the p17 mRNA transcribed separately from RNA 2. This insinuated that bac-VLPs may be packaging RNA non-selectively. It was proposed that p17 may play a role in packaging in an RNA-limiting environment (plasmid system) but functioned differently when viral RNA was in excess (baculovirus system). This data points to the importance of developing a replication system for the analysis of the packaging pathways of these viruses and this study has laid down the foundations for such a system in which RNA 1 and RNA 2 can be introduced into a single cell by means of a single recombinant virus.
- Full Text:
- Date Issued: 2012
- Authors: Mendes, Adriano
- Date: 2012
- Subjects: RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4025 , http://hdl.handle.net/10962/d1004085 , RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Description: The Helicoverpa armigera stunt virus (HaSV) is a member of a family of small nonenveloped (+) ssRNA insect viruses currently known as the Tetraviridae. This family is unique in terms of the T=4 quasi-symmetry of its capsid particles and the unusually narrow host range and tissue tropism. Assembly of tetraviral particles has been well characterised and involves the combination of 240 copies of a single capsid precursor protein (VCap) into a procapsid followed by autoproteolytic cleavage to yield the major (β) and minor (γ) capsid subunits within the mature particle. HaSV has two genomic RNAs, RNA 1 encoding the replicase and RNA 2 encoding VCap and p17, the ORF of which lies upstream of and overlaping with the 5’ end of the VCap ORF. Prior to this study, Vlok (2009) used a plasmid expression system to study RNA packaging in HaSV VLPs assembled in Spodoptera frugiperda 9 (Sf9) cells co-expressing p17 and VCap. The study showed that the p17 ORF was required for the packaging of RNA 2 during capsid assembly but it was unclear whether p17 expression was required for packaging. In addition, expression from the transfected plasmids was sub-optimal affecting both the yield of VLPs and the detection of p17. The aim of this study was to use the plasmid system to test whether p17 expression was required for plasmid-derived VLP RNA packaging and then develop a baculovirus-mediated system to test this hypothesis. By using a plasmid in which the start codon of p17 was mutated, it was shown that p17 expression was required for RNA 2 packaging into plasmid-VLPs. For the baculovirus system, four recombinant baculoviruses based upon the pFastBac Dual expression system, were constructed. These included Bac20, expressing wild type RNA 2, Bac21, RNA 2 with p17 silenced, Bac23, RNA 2 and p17 expressed on a separate transcript and Bac24, RNA 2 with p17 silenced plus p17 expressed on a separate transcript. Assembly of VLPs was more efficient using the baculovirus expression system and p17 expression was observed in cells infected with Bac20, Bac23 and Bac24, but not Bac21. In contrast to the plasmid-VLPs, bac-VLPs did not require p17 for the encapsidation of RNA 2. In addition to RNA 2, Bac23 and Bac24 packaged the p17 mRNA transcribed separately from RNA 2. This insinuated that bac-VLPs may be packaging RNA non-selectively. It was proposed that p17 may play a role in packaging in an RNA-limiting environment (plasmid system) but functioned differently when viral RNA was in excess (baculovirus system). This data points to the importance of developing a replication system for the analysis of the packaging pathways of these viruses and this study has laid down the foundations for such a system in which RNA 1 and RNA 2 can be introduced into a single cell by means of a single recombinant virus.
- Full Text:
- Date Issued: 2012
Enumeration of insect viruses using microscopic and molecular analyses: South African isolate of cryotophlebia leucotreta granulovirus as a case study
- Authors: Dhladhla, Busisiwe I R
- Date: 2012
- Subjects: Baculoviruses , Insects -- Viruses , Molecular genetics , Microbial genomics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10318 , http://hdl.handle.net/10948/d1008395 , Baculoviruses , Insects -- Viruses , Molecular genetics , Microbial genomics
- Description: Baculoviruses have been used as biocontrol agents to control insect pests in agriculture since the 1970s. Out of the fifteen virus families known to infect insects, baculoviruses offer the greatest potential as insect biopesticides, due to their high host specificity which makes them extremely safe to humans, other vertebrates, plants and non-target microorganisms. They comprise of two genera: nucleopolyhedroviruses (NPVs) and granuloviruses (GVs). The South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA) which is infectious for the false codling moth (FCM), Thaumatotibia leucotreta, (Meyrick) (Lepidoptera: Tortricidae), has been successfully developed into two commercial biopesticides; Cryptogran® and Cryptex®, for the control of FCM in citrus crops. The current method of enumeration used for CrleGV-SA virus particles in routine experiments during the production of the GV as biopesticides, is dark field microscopy. However, due to the small size of GVs (300-500 nm in length), the technique is not easy to perform on these viruses, and no systemic comparison has been made of potential alternative methods. Therefore, the main objective of this study was to develop a quantitative enumeration method for CrleGV-SA occlusion bodies (OBs) which is accurate, reliable, and feasible, and compare the developed methods of enumeration to the current method. Purified and semi-purified CrleGV-SA viral stocks were prepared for enumeration studies using spectrophotometry, dark field microscopy, scanning electron microscopy (SEM) and real time qPCR. Spectrophotometry was found to be an unreliable method for enumeration of GVs in the production, standardisation, and quality control of biopesticides. Dark field microscopy and SEM were found to be accurate, and statistically comparable (p = 0.064) enumeration techniques. qPCR is currently being optimised for the enumeration of GVs. This technique was demonstrated to generate accurate standard curves for absolute quantification of virus particles for pure and semi-pure virus preparations. qPCR offers the greatest potential as an accurate enumeration method because it is not affected by contamination with non-biological contaminating debris, nor by other biological material due to the specificity of PCR primers. Further work is required to fully develop qPCR as an enumeration method for GVs. However, dark field microscopy has been successfully validated as an enumeration method. SEM, which has a high resolution compared to light microscopy, has an added advantage over dark field microscopy, which is to distinguish virus particles in semi-pure viral stock preparations during counting. Therefore, SEM currently provides the most unambiguous and feasible enumeration method for GVs in both purified and semi-purified virus samples.
- Full Text:
- Date Issued: 2012
- Authors: Dhladhla, Busisiwe I R
- Date: 2012
- Subjects: Baculoviruses , Insects -- Viruses , Molecular genetics , Microbial genomics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10318 , http://hdl.handle.net/10948/d1008395 , Baculoviruses , Insects -- Viruses , Molecular genetics , Microbial genomics
- Description: Baculoviruses have been used as biocontrol agents to control insect pests in agriculture since the 1970s. Out of the fifteen virus families known to infect insects, baculoviruses offer the greatest potential as insect biopesticides, due to their high host specificity which makes them extremely safe to humans, other vertebrates, plants and non-target microorganisms. They comprise of two genera: nucleopolyhedroviruses (NPVs) and granuloviruses (GVs). The South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA) which is infectious for the false codling moth (FCM), Thaumatotibia leucotreta, (Meyrick) (Lepidoptera: Tortricidae), has been successfully developed into two commercial biopesticides; Cryptogran® and Cryptex®, for the control of FCM in citrus crops. The current method of enumeration used for CrleGV-SA virus particles in routine experiments during the production of the GV as biopesticides, is dark field microscopy. However, due to the small size of GVs (300-500 nm in length), the technique is not easy to perform on these viruses, and no systemic comparison has been made of potential alternative methods. Therefore, the main objective of this study was to develop a quantitative enumeration method for CrleGV-SA occlusion bodies (OBs) which is accurate, reliable, and feasible, and compare the developed methods of enumeration to the current method. Purified and semi-purified CrleGV-SA viral stocks were prepared for enumeration studies using spectrophotometry, dark field microscopy, scanning electron microscopy (SEM) and real time qPCR. Spectrophotometry was found to be an unreliable method for enumeration of GVs in the production, standardisation, and quality control of biopesticides. Dark field microscopy and SEM were found to be accurate, and statistically comparable (p = 0.064) enumeration techniques. qPCR is currently being optimised for the enumeration of GVs. This technique was demonstrated to generate accurate standard curves for absolute quantification of virus particles for pure and semi-pure virus preparations. qPCR offers the greatest potential as an accurate enumeration method because it is not affected by contamination with non-biological contaminating debris, nor by other biological material due to the specificity of PCR primers. Further work is required to fully develop qPCR as an enumeration method for GVs. However, dark field microscopy has been successfully validated as an enumeration method. SEM, which has a high resolution compared to light microscopy, has an added advantage over dark field microscopy, which is to distinguish virus particles in semi-pure viral stock preparations during counting. Therefore, SEM currently provides the most unambiguous and feasible enumeration method for GVs in both purified and semi-purified virus samples.
- Full Text:
- Date Issued: 2012
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