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
Assembly of full-length cDNA, and heterologous expression, of Nudaurelia B virus RNA
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
- Full Text:
- Date Issued: 2001
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
- Full Text:
- Date Issued: 2001
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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