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
The development of a baculovirus expression system for the production of Helicoverpa armigera stunt virus capsids for use in the encapsidation of foreign molecules
- Mosisili, Kekeletso Mpho Thakane
- Authors: Mosisili, Kekeletso Mpho Thakane
- Date: 2003
- Subjects: Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4088 , http://hdl.handle.net/10962/d1007700 , Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Description: The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.
- Full Text:
- Date Issued: 2003
- Authors: Mosisili, Kekeletso Mpho Thakane
- Date: 2003
- Subjects: Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4088 , http://hdl.handle.net/10962/d1007700 , Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Description: The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.
- Full Text:
- Date Issued: 2003
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