Genetic and biological characterisation of a novel South African Plutella xylostella granulovirus (PlxyGV) isolate
- Authors: Abdulkadir, Fatima
- Date: 2014
- Subjects: Diamondback moth , Diamondback moth -- Control -- South Africa , Plutellidae -- Control -- South Africa , Baculoviruses , Cruciferae -- Diseases and pests -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4113 , http://hdl.handle.net/10962/d1013059
- Description: The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is an important pest of cruciferous crops worldwide. The prolonged use of synthetic chemical insecticides as a primary means of control has resulted in the development of resistance in pest populations. In addition, the pest has also evolved resistance to the bacterial insecticidal protein of Bacillus thuringiensis which is also widely used as a method of control. Baculoviruses are considered as effective alternatives to conventional methods of control when incorporated into integrated pest management (IPM) programmes. These viruses target the larval stages of insects, are generally host-specific and are safe for use in the environment. This study aimed to isolate a baculovirus from a laboratory-reared P. xylostella colony, characterise it genetically and then evaluate its virulence against neonate and fourth instar larvae. A laboratory colony of P. xylostella was established using pupae and asymptomatic larvae collected from a cabbage plantation outside Grahamstown in the Eastern Cape province of South Africa. The colony flourished in the laboratory due to prime conditions and availability of food. The duration of development from egg to adult was determined by observation and imaging of the various life stages. The mean developmental time from egg to adult was observed to be 14.59 ± 0.21 days. The population of the insects increased rapidly in number leading to overcrowding of the insect colony, and hence appearance of larvae with viral symptoms. Occlusion bodies (OBs) were extracted from symptomatic larval cadavers and purified by glycerol gradient centrifugation. Analysis of the purified OBs by transmission electron microscopy revealed the presence of a granulovirus which was named PlxyGV-SA. The virus isolate was genetically characterised by restriction endonuclease analysis of the genomic DNA, and PCR amplification and sequencing of selected viral genes. The complete genome sequence of a Japanese P. xylostella granulovirus isolate, PlxyGV-Japan, has been deposited on the GenBank database providing a reference strain for comparison with DNA profiles and selected gene sequences of PlxyGV-SA. BLAST analysis of the granulin gene confirmed the isolation of a novel South African PlxyGV isolate. Comparison of the restriction profiles of PlxyGV-SA with profiles of PlxyGV-Japan and other documented PlxyGV profiles obtained by agarose gel electrophoresis revealed that PlxyGV-SA is a genetically distinct isolate. The data obtained from the sequencing and alignment of ecdysteroid UDP-glucosyltransferase (egt), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes with those of PlxyGV-Japan also showed that PlxyGV-SA is a genetically different isolate. In order to determine the biological activity of PlxyGV-SA against neonate and fourth instar P. xylostella larvae, surface dose bioassays were conducted. The median lethal concentration of the virus required to kill 50% (LC₅₀) and 90% (LC₉₀) of the larvae was estimated by feeding insects with a range of doses. In addition, the time to kill 50% of the larvae (LT₅₀) was determined by feeding insects with the LC₉₀ concentration. Larval mortality was monitored daily until pupation. The data obtained from the dose response assays were subjected to probit analysis using Proban statistical software. The time response was determined using GraphPad Prism software (version 6.0). The LC₅₀ and LC₉₀ values for the neonate larvae were 3.56 × 10⁵ and 1.14 × 10⁷ OBs/ml respectively. The LT₅₀ was determined to be 104 hours. The neonate larvae were found to be more susceptible to infection than the fourth instar larvae with the same virus concentration. The concentrations used for the neonate larvae assay did not have a significant effect on the fourth instar as no mortality was recorded. This is the first study to describe a novel South African PlxyGV isolate and the results suggest that PlxyGV-SA has significant potential for development as an effective biopesticide for the control of P. xylostella in the field.
- Full Text:
- Date Issued: 2014
- Authors: Abdulkadir, Fatima
- Date: 2014
- Subjects: Diamondback moth , Diamondback moth -- Control -- South Africa , Plutellidae -- Control -- South Africa , Baculoviruses , Cruciferae -- Diseases and pests -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4113 , http://hdl.handle.net/10962/d1013059
- Description: The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is an important pest of cruciferous crops worldwide. The prolonged use of synthetic chemical insecticides as a primary means of control has resulted in the development of resistance in pest populations. In addition, the pest has also evolved resistance to the bacterial insecticidal protein of Bacillus thuringiensis which is also widely used as a method of control. Baculoviruses are considered as effective alternatives to conventional methods of control when incorporated into integrated pest management (IPM) programmes. These viruses target the larval stages of insects, are generally host-specific and are safe for use in the environment. This study aimed to isolate a baculovirus from a laboratory-reared P. xylostella colony, characterise it genetically and then evaluate its virulence against neonate and fourth instar larvae. A laboratory colony of P. xylostella was established using pupae and asymptomatic larvae collected from a cabbage plantation outside Grahamstown in the Eastern Cape province of South Africa. The colony flourished in the laboratory due to prime conditions and availability of food. The duration of development from egg to adult was determined by observation and imaging of the various life stages. The mean developmental time from egg to adult was observed to be 14.59 ± 0.21 days. The population of the insects increased rapidly in number leading to overcrowding of the insect colony, and hence appearance of larvae with viral symptoms. Occlusion bodies (OBs) were extracted from symptomatic larval cadavers and purified by glycerol gradient centrifugation. Analysis of the purified OBs by transmission electron microscopy revealed the presence of a granulovirus which was named PlxyGV-SA. The virus isolate was genetically characterised by restriction endonuclease analysis of the genomic DNA, and PCR amplification and sequencing of selected viral genes. The complete genome sequence of a Japanese P. xylostella granulovirus isolate, PlxyGV-Japan, has been deposited on the GenBank database providing a reference strain for comparison with DNA profiles and selected gene sequences of PlxyGV-SA. BLAST analysis of the granulin gene confirmed the isolation of a novel South African PlxyGV isolate. Comparison of the restriction profiles of PlxyGV-SA with profiles of PlxyGV-Japan and other documented PlxyGV profiles obtained by agarose gel electrophoresis revealed that PlxyGV-SA is a genetically distinct isolate. The data obtained from the sequencing and alignment of ecdysteroid UDP-glucosyltransferase (egt), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes with those of PlxyGV-Japan also showed that PlxyGV-SA is a genetically different isolate. In order to determine the biological activity of PlxyGV-SA against neonate and fourth instar P. xylostella larvae, surface dose bioassays were conducted. The median lethal concentration of the virus required to kill 50% (LC₅₀) and 90% (LC₉₀) of the larvae was estimated by feeding insects with a range of doses. In addition, the time to kill 50% of the larvae (LT₅₀) was determined by feeding insects with the LC₉₀ concentration. Larval mortality was monitored daily until pupation. The data obtained from the dose response assays were subjected to probit analysis using Proban statistical software. The time response was determined using GraphPad Prism software (version 6.0). The LC₅₀ and LC₉₀ values for the neonate larvae were 3.56 × 10⁵ and 1.14 × 10⁷ OBs/ml respectively. The LT₅₀ was determined to be 104 hours. The neonate larvae were found to be more susceptible to infection than the fourth instar larvae with the same virus concentration. The concentrations used for the neonate larvae assay did not have a significant effect on the fourth instar as no mortality was recorded. This is the first study to describe a novel South African PlxyGV isolate and the results suggest that PlxyGV-SA has significant potential for development as an effective biopesticide for the control of P. xylostella in the field.
- Full Text:
- Date Issued: 2014
The preparation of antigen for the generation of polyclonal antibodies against the capsid subunit, VP1, and the viral protease, 3Cpro, of Theiler's murine encephalomyelitis virus (TMEV)
- Authors: Moetlhoa, Boitumelo
- Date: 2014
- Subjects: Enteroviruses , Encephalomyelitis , Antigens
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4118 , http://hdl.handle.net/10962/d1013225
- Description: The Picornaviridae is a family of viruses of economic importance that have a major impact on human and animal health. Some of the major genera found in the Picornaviridae family are Enterovirus which includes Poliovirus (PV) and Human Rhinovirus (HRV), Cardiovirus which includes Theiler’s murine encephalomyelitis virus (TMEV) and Saffold virus (SAFV), Aphthovirus of which the Foot and Mouth disease virus (FMDV) is a member and Hepatovirus which includes Hepatitis A virus (HAV). Picornaviruses have a single stranded, positive sense RNA genome which is approximately 7.5-8.4 kb pairs in size. The picornavirus genome is translated into a large polyprotein and is proteolytically cleaved by viral proteases namely 2Apro, 3Cpro and 3CDpro into mature viral structural and non-structural polypeptides encoded by the P1, P2 and P3 domains. Picornaviruses utilise host cell machinery and cellular pathways for entry and uncoating, genome replication and capsid assembly. In our laboratory, we are studying the mechanisms by which TMEV interacts with host cell components and our recent research shows that molecular chaperones are required for a production infection. To follow up on this observation, the overall aim of this study was to prepare antigen for the generation of polyclonal antibodies against the TMEV VP1 and 3Cpro proteins. To this end, the TMEV VP1 and 3Cpro amino acid sequences were analysed to identify hydrophobic, hydrophilic and antigenic regions. Homology modelling was performed in order to predict linear B cell epitopes exposed on the surface of the protein structures. The full length coding sequences of VP1 and 3Cpro were selected for amplification by the PCR and cloning into pQE-80L for expression in a bacterial system. Time course induction studies of recombinant VP1 and 3Cpro showed that the proteins were maximally expressed at 6 hrs and 4 hrs respectively. Recombinant VP1 was solubilised using the detergent, Sarcosyl and purified by Nickel affinity chromatography under native conditions. Because recombinant VP1 co-purified with an unidentified protein, the pET expression system was used. Although no protein of the estimated size was observed by SDS-PAGE analysis in the time course induction study, Western analysis using anti-His6 (2) antibodies detected a signal of ~35 kDa. Solubility studies resulted in the presence of two protein bands in the insoluble fraction resolved between 35 and 40 kDa. Recombinant 3Cpro expressed in a bacterial system was predominantly present in the insoluble fraction. Treatment with Sarcosyl had no effect on the solubility of the recombinant protein and it was therefore purified under denaturing conditions using 8M urea. Following dialysis, 3Cpro was used for immunisation of rabbits. Crude anti-TMEV 3Cpro antibodies were able to detect as little as 107 ng of bacterially expressed antigen at a dilution of 1:100 000 by Western analysis. The presence of contaminating proteins was reduced using pre-cleared anti-TMEV 3Cpro antibodies. The antibodies were unable to detect virally expressed 3Cpro in BHK-21 cell lysate supernatant. In an attempt to determine whether TMEV 3Cpro is present in the insoluble fraction, anti-TMEV 3Cpro antibodies were tested using total protein prepared from infected and mock-infected cell lysates. Once again, no protein band the size of 3Cpro was detected. The antibodies were further tested for detection of 3Cpro in TMEV-infected cells by indirect immunofluorescence and confocal microscopy. A diffuse cytoplasmic and perinuclear distribution, as well as nuclear staining, was observed in infected BHK-21 cells. This staining pattern resembled that observed for the HRV, FMDV and EMCV 3Cpro in similar experiments. Further experiments are required to confirm specificity of these antibodies for virally-expressed 3Cpro by Western analysis and indirect immunofluorescence.
- Full Text:
- Date Issued: 2014
- Authors: Moetlhoa, Boitumelo
- Date: 2014
- Subjects: Enteroviruses , Encephalomyelitis , Antigens
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4118 , http://hdl.handle.net/10962/d1013225
- Description: The Picornaviridae is a family of viruses of economic importance that have a major impact on human and animal health. Some of the major genera found in the Picornaviridae family are Enterovirus which includes Poliovirus (PV) and Human Rhinovirus (HRV), Cardiovirus which includes Theiler’s murine encephalomyelitis virus (TMEV) and Saffold virus (SAFV), Aphthovirus of which the Foot and Mouth disease virus (FMDV) is a member and Hepatovirus which includes Hepatitis A virus (HAV). Picornaviruses have a single stranded, positive sense RNA genome which is approximately 7.5-8.4 kb pairs in size. The picornavirus genome is translated into a large polyprotein and is proteolytically cleaved by viral proteases namely 2Apro, 3Cpro and 3CDpro into mature viral structural and non-structural polypeptides encoded by the P1, P2 and P3 domains. Picornaviruses utilise host cell machinery and cellular pathways for entry and uncoating, genome replication and capsid assembly. In our laboratory, we are studying the mechanisms by which TMEV interacts with host cell components and our recent research shows that molecular chaperones are required for a production infection. To follow up on this observation, the overall aim of this study was to prepare antigen for the generation of polyclonal antibodies against the TMEV VP1 and 3Cpro proteins. To this end, the TMEV VP1 and 3Cpro amino acid sequences were analysed to identify hydrophobic, hydrophilic and antigenic regions. Homology modelling was performed in order to predict linear B cell epitopes exposed on the surface of the protein structures. The full length coding sequences of VP1 and 3Cpro were selected for amplification by the PCR and cloning into pQE-80L for expression in a bacterial system. Time course induction studies of recombinant VP1 and 3Cpro showed that the proteins were maximally expressed at 6 hrs and 4 hrs respectively. Recombinant VP1 was solubilised using the detergent, Sarcosyl and purified by Nickel affinity chromatography under native conditions. Because recombinant VP1 co-purified with an unidentified protein, the pET expression system was used. Although no protein of the estimated size was observed by SDS-PAGE analysis in the time course induction study, Western analysis using anti-His6 (2) antibodies detected a signal of ~35 kDa. Solubility studies resulted in the presence of two protein bands in the insoluble fraction resolved between 35 and 40 kDa. Recombinant 3Cpro expressed in a bacterial system was predominantly present in the insoluble fraction. Treatment with Sarcosyl had no effect on the solubility of the recombinant protein and it was therefore purified under denaturing conditions using 8M urea. Following dialysis, 3Cpro was used for immunisation of rabbits. Crude anti-TMEV 3Cpro antibodies were able to detect as little as 107 ng of bacterially expressed antigen at a dilution of 1:100 000 by Western analysis. The presence of contaminating proteins was reduced using pre-cleared anti-TMEV 3Cpro antibodies. The antibodies were unable to detect virally expressed 3Cpro in BHK-21 cell lysate supernatant. In an attempt to determine whether TMEV 3Cpro is present in the insoluble fraction, anti-TMEV 3Cpro antibodies were tested using total protein prepared from infected and mock-infected cell lysates. Once again, no protein band the size of 3Cpro was detected. The antibodies were further tested for detection of 3Cpro in TMEV-infected cells by indirect immunofluorescence and confocal microscopy. A diffuse cytoplasmic and perinuclear distribution, as well as nuclear staining, was observed in infected BHK-21 cells. This staining pattern resembled that observed for the HRV, FMDV and EMCV 3Cpro in similar experiments. Further experiments are required to confirm specificity of these antibodies for virally-expressed 3Cpro by Western analysis and indirect immunofluorescence.
- Full Text:
- Date Issued: 2014
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