Virion-associated viral fibroblast growth factor stimulates cell motility

AbstractThe Autographa californica M nucleopolyhedrovirus (AcMNPV) viral fibroblast growth factor (vFGF) has functional parallels to cellular FGFs. Deletion of the AcMNPV vfgf has no obvious phenotype in cell culture but delays the time of insect death. Here, we determined vFGF production during virus infection. vFGF was detected at 24 hours post infection and through the remainder of the infection cycle. Since vFGF is thought to be a secreted membrane-binding protein and virions acquire an envelope derived from the cell membrane, we examined virions for the presence of vFGF using microscopy, flow cytometry, and affinity chromatography. We found that vFGF associated with virions. Furthermore, budded virus carrying vFGF had more affinity to heparin than vFGF-deficient budded virus, consistent with the affinity of FGFs for heparan sulfate proteoglycans. Although the function of virion-associated vFGF is not clear, we found that virion-associated vFGF stimulated cell motility and affected virus attachment

Analysis and functional annotation of expressed sequence tags from the fall armyworm Spodoptera frugiperda

BACKGROUND: Little is known about the genome sequences of lepidopteran insects, although this group of insects has been studied extensively in the fields of endocrinology, development, immunity, and pathogen-host interactions. In addition, cell lines derived from Spodoptera frugiperda and other lepidopteran insects are routinely used for baculovirus foreign gene expression. This study reports the results of an expressed sequence tag (EST) sequencing project in cells from the lepidopteran insect S. frugiperda, the fall armyworm. RESULTS: We have constructed an EST database using two cDNA libraries from the S. frugiperda-derived cell line, SF-21. The database consists of 2,367 ESTs which were assembled into 244 contigs and 951 singlets for a total of 1,195 unique sequences. CONCLUSION: S. frugiperda is an agriculturally important pest insect and genomic information will be instrumental for establishing initial transcriptional profiling and gene function studies, and for obtaining information about genes manipulated during infections by insect pathogens such as baculoviruses

Infection pattern and transmission potential of chikungunya virus in two New World laboratory-adapted Aedes aegypti strains

Citation: Dong, S. Z., Kantor, A. M., Lin, J. Y., Passarelli, A. L., Clem, R. J., & Franz, A. W. E. (2016). Infection pattern and transmission potential of chikungunya virus in two New World laboratory-adapted Aedes aegypti strains. Scientific Reports, 6, 13. doi:10.1038/srep24729Chikungunya virus (CHIKV) is an emerging mosquito-borne virus belonging to the Togaviridae, which is transmitted to humans by Aedes aegypti and Ae. albopictus. We describe the infection pattern of CHIKV in two New World Ae. aegypti strains, HWE and ORL. Both mosquito strains were susceptible to the virus but showed different infection patterns in midguts and salivary glands. Even though acquisition of a bloodmeal showed moderate levels of apoptosis in midgut tissue, there was no obvious additional CHIKV-induced apoptosis detectable during midgut infection. Analysis of expression of apoptosis-related genes suggested that CHIKV infection dampens rather than promotes apoptosis in the mosquito midgut. In both mosquito strains, the virus was present in saliva within two days post-oral infection. HWE and ORL mosquitoes exhibited no salivary gland infection barrier; however, only 60% (HWE) to 65% (ORL) of the females had released the virus in their saliva at one week post-oral acquisition, suggesting a salivary gland escape barrier. CHIKV induced an apoptotic response in salivary glands of HWE and ORL mosquitoes, demonstrating that the virus caused pathology in its natural vector

Genetic Requirements for Homologous Recombination in Autographa californica Nucleopolyhedrovirus

It is known that baculovirus infection promotes high-frequency recombination between its genomes and plasmid DNA during the construction of recombinant viruses for foreign gene expression. However, little is known about the viral genes necessary to promote homologous recombination (HR). We developed an assay to identify viral genes that are necessary to stimulate HR. In this assay, we used two plasmids containing extensive sequence homology that yielded a visible and quantifiable phenotype if HR occurred. The plasmids contained the green fluorescent protein gene (gfp) that was mutated at either the N or the C terminus and a viral origin of DNA replication. When the plasmids containing these mutant gfp genes were transfected into insect cells alone or together, few green fluorescent protein (GFP)-positive cells were observed, confirming that the host cell machinery alone was not able to promote high levels of HR. However, if viral DNA or viral genes involved in DNA replication were cotransfected into cells along with the mutant gfp-containing plasmids, a dramatic increase in GFP-positive cells was observed. The viral genes ie-1, ie-2, lef-7, and p35 were found to be important for efficient HR in the presence of all other DNA replication genes. However, ie-1 and ie-2 were sufficient to promote HR in the absence of other viral genes. Recombination substrates lacking a viral origin of replication had similar genetic requirements for recombination but were less dependent on ie-1. Interestingly, even though HR was stimulated by the presence of a viral origin of DNA replication, virally stimulated HR could proceed in the presence of the DNA synthesis inhibitor aphidicolin

Autographa californica Multiple Nucleopolyhedrovirus Ac92 (ORF92, P33) Is Required for Budded Virus Production and Multiply Enveloped Occlusion-Derived Virus Formation▿

The Autographa californica multiple nucleopolyhedrovirus orf92 (p33), ac92, is one of 31 genes carried in all sequenced baculovirus genomes, thus suggesting an essential function. Ac92 has homology to the family of flavin adenine dinucleotide-linked sulfhydryl oxidases and is related to the ERV/ALR family of sulfhydryl oxidases. The role of ac92 during virus replication is unknown. Ac92 was associated with the envelope of both budded and occlusion-derived virus (ODV). To investigate the role of Ac92 during virus replication, an ac92-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration and plaque assays showed no virus spread in ac92-knockout bacmid DNA-transfected insect cells. Deletion of ac92 did not affect viral DNA replication. However, ac92-knockout bacmid DNA-transfected cells lacked multiply enveloped occlusion-derived nucleocapsids; instead, singly enveloped nucleocapsids were detected. To gain insight into the requirement for sulfhydryl oxidation during virus replication, a virus was constructed in which the Ac92 C155XXC158 amino acids, important for sulfhydryl oxidase activity, were mutated to A155XXA158. The mutant virus exhibited a phenotype similar to that of the knockout virus, suggesting that the C-X-X-C motif was essential for sulfhydryl oxidase activity and responsible for the altered ODV phenotype

A typical baculovirus replication cycle.

<p>Nucleocapsids are produced in the nucleus of infected cells, but two forms of enveloped virions are produced. The first, budded virus (BV), is formed when single nucleocapsids exit the nucleus and bud from the cell, acquiring an envelope from the plasma membrane. The BV attachment and fusion protein, either GP64 or F depending on the type of baculovirus, is concentrated at one end of the virion in structures that are visible by electron microscopy, known as peplomers. Later, the second type of virion, called occlusion-derived virus (ODV), is formed when nucleocapsids obtain an envelope from the inner nuclear membrane. ODV become embedded in large proteinaceous crystals that are primarily composed of a single viral protein called polyhedrin, and are known as occlusion bodies. The occlusion bodies remain within the nucleus until they are liberated by cell lysis. The ODV of some baculoviruses, known as multiple nucleopolyhedroviruses (MNPVs), contain multiple nucleocapsids within a single-enveloped virus particle, as is illustrated here. In nature, the baculovirus replication cycle begins when a susceptible insect larva consumes viral occlusion bodies contaminating their food source. The occlusion bodies dissolve in the highly alkaline environment of the larval midgut, releasing ODV, which attach to the microvillar membranes of midgut epithelial cells. Attachment and fusion occurs via the PIF proteins, found in the ODV envelope. Infected midgut epithelial cells produce BV, which bud from the basal side and infect tracheal epithelial cells. Infection of tracheal cells is thought to be one mechanism that allows BV to escape across the midgut basal lamina (BL) and spread infection throughout the insect. The majority of tissues become infected, producing large amounts of ODV and BV. The infected insects liquefy after death, allowing dispersal of occlusion bodies and promoting subsequent infections.</p

Transcriptional Reprogramming of Autographa Californica Multiple Nucleopolyhedrovirus Chitinase and Cathepsin Genes Enhances Virulence

The baculoviral chitinase (CHIA) and cathepsin (V-CATH) enzymes promote terminal insect host liquefaction, which aids viral progeny dissemination. Recombinant Autographa californica nucleopolyhedrovirus (AcMNPV)-derived viruses were previously generated with reprogrammed chiA transcription by replacing the native promoter with the AcMNPV polyhedrin (polh) or core protein (p6.9) promoter sequences, but of both these chiA-reprogrammed viruses lacked v-cath transcription and V-CATH enzymatic activity. Here, we report that dual p6.9/polh promoter reprogramming of the adjacent chiA/v-cath genes resulted in modulated temporal transcription of both genes without impacting infectious budded virus production. These promoter changes increased CHIA and V-CATH enzyme activities in infected Spodoptera frugiperda-derived cultured cells and Trichoplusia ni larvae. In addition, larvae infected with the dual reprogrammed virus had earlier mortalities and liquefaction. This recombinant baculovirus, lacking exogenous genomic elements and increased chiA/v-cath expression levels, may be desirable for and amenable to producing enhanced baculovirus-based biopesticides