Caroline Knox

Effects of Foot-and-Mouth Disease Virus Nonstructural Proteins on the Structure and Function of the Early Secretory Pathway: 2BC but Not 3A Blocks Endoplasmic Reticulum-to-Golgi Transport

ABSTRACT Infection of cells by picornaviruses leads to the generation of intracellular membrane vesicles. The expression of poliovirus (PV) 3A protein causes swelling of the endoplasmic reticulum (ER) and inhibition of protein trafficking between the ER and the Golgi apparatus. Here, we report that the nonstructural proteins of a second picornavirus, foot-and-mouth disease virus (FMDV), also perturb the secretory pathway. FMDV proteins 3A, 2B, 2C, and 2BC expressed alone in cells were recovered from crude membrane fractions, indicating membrane association. Immunofluorescence microscopy showed that 3A was located in a reticular structure and 2B was located in the ER, while 2C was located in both the ER and the bright punctate structures within the Golgi apparatus. 2BC gave punctate cytoplasmic staining and also caused accumulation of ER proteins in large vesicular structures located around the nuclei. The effect of the FMDV proteins on the trafficking of the vesicular stomatitis virus glycoprotein (G protein) from the ER to the cell surface was determined. Unlike its PV counterpart, the 3A protein of FMDV did not prevent trafficking of the G protein to the cell surface. Instead, surface expression of the G protein was blocked by 2BC, with retention of the G protein in a modified ER compartment staining for 2BC. The results suggest that the nonstructural proteins of different picornaviruses may vary in their ability to perturb the secretory pathway. Since FMDV 2BC can block the delivery of proteins to the cell surface, it may, as shown for PV 3A, play a role in immune evasion and contribute to the persistent infections observed in ruminants.

Inhibition of the Secretory Pathway by Foot-and-Mouth Disease Virus 2BC Protein Is Reproduced by Coexpression of 2B with 2C, and the Site of Inhibition Is Determined by the Subcellular Location of 2C

ABSTRACT Infection of cells with picornaviruses can lead to a block in protein secretion. For poliovirus this is achieved by the 3A protein, and the consequent reduction in secretion of proinflammatory cytokines and surface expression of major histocompatibility complex class I proteins may inhibit host immune responses in vivo. Foot-and-mouth disease virus (FMDV), another picornavirus, can cause persistent infection of ruminants, suggesting it too may inhibit immune responses. Endoplasmic reticulum (ER)-to-Golgi apparatus transport of proteins is blocked by the FMDV 2BC protein. The observation that 2BC is processed to 2B and 2C during infection and that individual 2B and 2C proteins are unable to block secretion stimulated us to study the effects of 2BC processing on the secretory pathway. Even though 2BC was processed rapidly to 2B and 2C, protein transport to the plasma membrane was still blocked in FMDV-infected cells. The block could be reconstituted by coexpression of 2B and 2C, showing that processing of 2BC did not compromise the ability of FMDV to slow secretion. Under these conditions, 2C was located to the Golgi apparatus, and the block in transport also occurred in the Golgi apparatus. Interestingly, the block in transport could be redirected to the ER when 2B was coexpressed with a 2C protein fused to an ER retention element. Thus, for FMDV a block in secretion is dependent on both 2B and 2C, with the latter determining the site of the block.

Overcrowding of false codling moth, Thaumatotibia leucotreta (Meyrick) leads to the isolation of five new Cryptophlebia leucotreta granulovirus (CrleGV-SA) isolates.

False codling moth, Thaumatotibia leucotreta (Meyrick) is a serious pest of economic importance to the South African fruit industry. As part of sustainable efforts to control this pest, biological control options that involve the application of baculovirus-based biopesticides such as Cryptogran and Cryptex (both formulated with a South African isolate of Cryptophlebia leucotreta granulovirus, CrleGV-SA) are popularly used by farmers. In order to safeguard the integrity of these biopesticides as well as protect against any future development of resistance in the host, we conducted a study to bioprospect for additional CrleGV isolates as alternatives to existing ones. Using overcrowding as an induction method for latent infection, we recovered five new CrleGV isolates (CrleGV-SA Ado, CrleGV-SA Mbl, CrleGV-SA Cit, CrleGV-SA MixC and CrleGV-SA Nels). Single restriction endonuclease (REN) analysis of viral genomic DNA extracted from purified occlusion bodies showed that isolates differed in their DNA profiles. Partial sequencing of granulin and egt genes from the different isolates and multiple alignments of nucleotide sequences revealed the presence of single nucleotide polymorphisms (SNPs), some of which resulted in amino acid substitutions in the protein sequence. Based on these findings as well as comparisons with other documented CrleGV isolates, we propose two phylogenetic groups for CrleGV-SA isolates recovered in this study.

Heat shock protein 40 (Hsp40) plays a key role in the virus life cycle

The heat shock proteins (Hsps) are a diverse subset of molecular chaperones that generally promote the proper folding of proteins after translation and also prevent their aggregation during cellular stress. Paradoxically, cellular chaperones might perform important antiviral functions for host cells, yet, at the same time, might be beneficial for virus replication. Among them, Hsp40 is a specialized co-chaperone that has recently received much attention for its crucial role in both constitutive cellular functions and virus pathogenicity. The aim of this review is to raise awareness of its importance in the life cycles of a wide range of viruses.

Baculovirus-based strategies for the management of insect pests: a focus on development and application in South Africa

There is growing concern among governments, scientists, agricultural practitioners and the general public regarding the negative implications of widespread synthetic chemical pesticide application for the control of crop pests. As a result, baculovirus biopesticides are gaining popularity as components of integrated pest management (IPM) programmes in many countries despite several disadvantages related to slow speed of kill, limited host range and complex large scale production. In South Africa, baculoviruses are incorporated into IPM programmes for the control of crop pests in the field, and recent bioprospecting has led to the characterisation of several novel isolates with the potential to be formulated as commercial products. This contribution will provide an overview of the use of baculoviruses against insect pests in South Africa, as well as research and development efforts aimed at broadening their application as biocontrol agents. Challenges faced by researchers in developmental projects as well as potential users of baculoviruses as biopesticides in the field are also discussed.

Human defined antigenic region on the nucleoprotein of Crimean-Congo hemorrhagic fever virus identified using truncated proteins and a bioinformatics approach.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne viral zoonosis widely distributed in Africa, Asia and eastern Europe. In this study, amino acid sequence data for the CCHFV nucleoprotein (NP) was used to identify potential linear epitopic regions which were subsequently included in the design of large and small truncated recombinant NP antigens and peptide libraries. Two truncated recombinant CCHFV NP antigens were prepared based on results of prediction studies to include epitopic regions and exclude hydrophobic regions that could influence protein expression and solubility. Serum samples were collected from acute and convalescent patients. An IgG antibody response was detected in 16/16 samples tested using the large recombinant NP-based ELISA and in 2/16 using the small recombinant NP-based ELISA. A total of 60 peptides covering predicted epitopic regions of the NP were synthesized and peptide NRGGDENPRGPVSR at amino acid position 182-195, reacted with 13/16 human serum samples. In summary, functional assays are required to determine the biological activity of predicted epitopes for development of peptide based assays for antibody detection. Bacterially expressed complete NP antigens have previously been shown to be useful tools for antibody detection. Truncation of the antigen to remove the hydrophobic C terminus had no impact on the ability of the antigen to detect IgG antibody in human sera. The results indicate that the region from amino acids 123 to 396 includes a highly antigenic region of the NP with application in development of antibody detection assays.

The isolation and genetic characterisation of a South African strain of Phthorimaea operculella granulovirus, PhopGV-SA.

The Phthorimaea operculella granulovirus (PhopGV) is considered a promising biopesticide that can be incorporated into integrated pest management programmes for sustainable control of the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), a major pest of solanaceous crops in sub-tropical and tropical regions worldwide. Several PhopGV isolates recovered from geographically different insect populations have been genetically characterised, and the full genome of the Tunisian PhopGV-1346 isolate has been sequenced, providing a reference strain for comparison of novel isolates. Here we report the identification and genetic characterisation of a South African PhopGV isolate recovered from a P. operculella colony held under laboratory conditions. Transmission electron microscopy examination of purified occlusion bodies together with analysis of granulin and late expression factor-8 (lef-8) gene sequences confirmed the identity of the virus as PhopGV. The sequenced ecdysteroid UDP-glucosyltransferase (egt) gene was 1353nt in length, placing PhopGV-SA in egt group II. Finally, a phylogenetic analysis using a range of egt sequences grouped PhopGV-SA together with the Kenyan, Ecuadorian, Indonesian and Colombian isolates. The results are discussed with reference to the possible origin of PhopGV-SA, and provide a platform for future studies involving virulence evaluation against geographically different P. operculella populations with a view to biopesticide development.

Comparison of the Biology of Geographically Distinct Populations of the Citrus Pest, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), in South Africa

Baculovirus biopesticides are an important component of integrated pest management programmes worldwide. One such example is the Cryptophlebia leucotreta granulovirus (CrleGV) which is used for the control of false codling moth, Thaumatotibia (= Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae), a pest of citrus and other crops in South Africa. A potential problem associated with constant application of viral biopesticides is the differing susceptibility to the virus observed between different geographic populations of the insect host. This could be related to a number of factors, including biological performance and fitness of the target insect population. This study compared a variety of phenotypic traits between geographically distinct T. leucotreta populations collected from the Addo, Marble Hall, Citrusdal and Nelspruit regions of South Africa, and reared under laboratory conditions for several generations. Traits including pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and developmental time were used as parameters to measure biological performance and fitness. Insects from the Citrusdal region of the Western Cape exhibited significantly lower pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and the longest duration in larval and pupal development compared to the other colonies investigated. This is the first study to report differences in the performance of laboratory reared T. leucotreta from different geographic locations, and the findings may have important implications for the application of viral biopesticides for the control of this pest in South Africa.

Heterogeneity in virulence relationships between Cryptophlebia leucotreta granulovirus isolates and geographically distinct host populations: lessons from codling moth resistance to CpGV-M

In South Africa, the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV) is used to control the citrus pest Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). However, the risk associated with continuous application of a single active ingredient is resistance. In order to manage resistance should it occur in field populations of T. leucotreta in South Africa, five new CrleGV genotypes from geographically distinct insect populations which were shown to exhibit some degree of phenotypic variation were recovered and genetically characterized. In droplet bioassays using seven CrleGV isolates against five T. leucotreta populations, some isolates were found to show higher virulence to some host populations than others. There were marked differences in the LD50 values of isolates and the number of occlusion bodies required per larva ranged between 0.79 and 3.12. The significance of these findings with respect to the application of CrleGV biopesticides and the management of resistance is discussed.

Morphological and Genetic Characterization of a South African Plutella xylostella Granulovirus (PlxyGV) Isolate

Plutella xylostella (L.) (Lepidoptera: Plutellidae), also known as diamondback moth, is a destructive insect pest of cruciferous crops (Talekar & Shelton 1993; Shelton 2004). The pest occurs wherever its host plants are cultivated and the global annual cost of damage and control is estimated to be US