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
Development of experimental systems for studying the biology of Nudaurelia capensis ß virus
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Date Issued: 2005
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
Characterisation of the genome of Nudaurelia Omega Virus
- Authors: Cox, Dermot
- Date: 1995
- Subjects: Imbrasia cytherea , RNA , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4023 , http://hdl.handle.net/10962/d1004083 , Imbrasia cytherea , RNA , Insects -- Viruses
- Description: Nudaurelia co virus (Nco V) is a small RNA virus belonging to the Family Tetraviridae. Nco V was successfully isolated from field collected larvae of the pine emperor moth, Nudaurelia cytherea capensis. By polyacrylamide gel electrophoresis-it was possible Jo determine the size of the capsid proteins. Anti-NcoV antiserum was raised by inoculating a rabbit with purified virus. RNA was extracted from the purified virus using a phenol\chloroform extraction procedure. It was possible to separate the viral RNA into its constituent species using sucrose density gradient centrifugation. The sizes of both species of RNA was accurately determined by agarose gel electrophoresis. These sizes corresponded to the replicative form of the RNA which was extracted from infected host tissue. The absence of a poly(A) tract on the RNA was shown through poly(U) sepharose chromatography. Cell-free translation of the viral RNA elucidated the sizes of proteins encoded in vitro in a rabbit reticulocyte lysate system. Optimal conditions for in vitro translation of Nco V were determined for a range of conditions. Immunoprecipitaion of viral encoded proteins with anti-Nco V antiserum suggested that the putative coat protein of the virus was encoded by RNA 2, as a precursor polypeptide which underwent posttranslational cleavage. Reverse transcription - polymerase chain reaction (RT -PCR) was used to successfully produce a radiolabelled probe which could detect dot-blotted viral RNA. The efficacy of this probe in detecting the presence of Nco V RNA in infected tissue was also tested.
- Full Text:
- Date Issued: 1995
- Authors: Cox, Dermot
- Date: 1995
- Subjects: Imbrasia cytherea , RNA , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4023 , http://hdl.handle.net/10962/d1004083 , Imbrasia cytherea , RNA , Insects -- Viruses
- Description: Nudaurelia co virus (Nco V) is a small RNA virus belonging to the Family Tetraviridae. Nco V was successfully isolated from field collected larvae of the pine emperor moth, Nudaurelia cytherea capensis. By polyacrylamide gel electrophoresis-it was possible Jo determine the size of the capsid proteins. Anti-NcoV antiserum was raised by inoculating a rabbit with purified virus. RNA was extracted from the purified virus using a phenol\chloroform extraction procedure. It was possible to separate the viral RNA into its constituent species using sucrose density gradient centrifugation. The sizes of both species of RNA was accurately determined by agarose gel electrophoresis. These sizes corresponded to the replicative form of the RNA which was extracted from infected host tissue. The absence of a poly(A) tract on the RNA was shown through poly(U) sepharose chromatography. Cell-free translation of the viral RNA elucidated the sizes of proteins encoded in vitro in a rabbit reticulocyte lysate system. Optimal conditions for in vitro translation of Nco V were determined for a range of conditions. Immunoprecipitaion of viral encoded proteins with anti-Nco V antiserum suggested that the putative coat protein of the virus was encoded by RNA 2, as a precursor polypeptide which underwent posttranslational cleavage. Reverse transcription - polymerase chain reaction (RT -PCR) was used to successfully produce a radiolabelled probe which could detect dot-blotted viral RNA. The efficacy of this probe in detecting the presence of Nco V RNA in infected tissue was also tested.
- Full Text:
- Date Issued: 1995
Physico-chemical and substructural studies on Nudaurelia capensis β virus
- Authors: Struthers, J Keith
- Date: 1974
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4075 , http://hdl.handle.net/10962/d1007327 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: From Introduction: The pine emperor moth, Nudaurelia cytherea capensis Stoll is an insect which, during the larval stage, causes extensive defoliation of the pine tree, Pinus radiata in the Cape province. These insects are susceptible to a virus disease, which on occasions causes large scale mortality. Five nonoccluded viruses have been shown to infect the pine emperor moth, and of these, one found in the greatest concentration, Nudaurelia capensis β virus (NβV) has been characterised to the greatest extent. This virus has been shown to contain RNA, to be isometric with a diameter of 36 mm, and to have a molecular weight of 16 million. The virus occurs in all stages of the insect's development, and by fluorescent antibody staining has been shown to develop in the cytoplasm of the host's cells. There have in recent years been a number of reports describing nonoccluded RNA viruses which appear to be similar to NβV. These are the viruses isolated from the moths Gonometa podocarpi and Antheraea eucalypti, and the one from the citrus red mite, Panonychus citri. These viruses have not been as extensively characterised as NβV, so the extent of the similarity between them and NβV is not known. However it would appear as if their discovery collectively heralds the emergence of a distinct new grouping within the nonoccluded RNA viruses of insects. This work reports the isolation and further characterisation of N. capensis β virus, its protein and nucleic acid.
- Full Text:
- Date Issued: 1974
- Authors: Struthers, J Keith
- Date: 1974
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4075 , http://hdl.handle.net/10962/d1007327 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: From Introduction: The pine emperor moth, Nudaurelia cytherea capensis Stoll is an insect which, during the larval stage, causes extensive defoliation of the pine tree, Pinus radiata in the Cape province. These insects are susceptible to a virus disease, which on occasions causes large scale mortality. Five nonoccluded viruses have been shown to infect the pine emperor moth, and of these, one found in the greatest concentration, Nudaurelia capensis β virus (NβV) has been characterised to the greatest extent. This virus has been shown to contain RNA, to be isometric with a diameter of 36 mm, and to have a molecular weight of 16 million. The virus occurs in all stages of the insect's development, and by fluorescent antibody staining has been shown to develop in the cytoplasm of the host's cells. There have in recent years been a number of reports describing nonoccluded RNA viruses which appear to be similar to NβV. These are the viruses isolated from the moths Gonometa podocarpi and Antheraea eucalypti, and the one from the citrus red mite, Panonychus citri. These viruses have not been as extensively characterised as NβV, so the extent of the similarity between them and NβV is not known. However it would appear as if their discovery collectively heralds the emergence of a distinct new grouping within the nonoccluded RNA viruses of insects. This work reports the isolation and further characterisation of N. capensis β virus, its protein and nucleic acid.
- Full Text:
- Date Issued: 1974
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