Regulation of multiple infection in alphabaculoviruses: critical factors that determine success

Abstract

The physical structure of multiple nucleopolyhedroviruses (family Baculoviridae, genus Alphabaculovirus) has been demonstrated to be a key factor in the maintenance of diversity in natural isolates during host infection and in situations of low pathogen density. The physical association of genomes in nucleopolyhedroviruses is also highly relevant when insects are infected by distinct species of these viruses. Co-occlusion of two different virus species within the same occlusion body (OB) was demonstrated in this thesis after co-infection with two closely related viruses, the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and a non-per os infective genotype of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). The phenomenon was also observed in infections involving two phylogenetically distant alphabaculoviruses, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and SfMNPV. In the latter case, co-envelopment of both viruses within the same occlusion-derived-virion (ODV) was observed, with approximately 50% of ODVs comprising genomes of both viruses following simultaneous inoculation of S. frugiperda larvae. However, infection of a cell/organism by two different alphabaculoviruses is not always possible. In S. frugiperda cells, successful infection by a second virus is only permitted within 20-24 hours following first infection. This exclusion to superinfection is a progressive process and involves both homologous and heterologous interference. Disruption of actin filaments by treatment with cytochalasin D (CD), a drug known to inhibit actin polymerization, resulted in the suppression of the exclusion and, consequently, permitted a successful second infection. A temporal window during which infection by two different virus species is possible was also observed when superinfections were performed in S. frugiperda larvae. Although total exclusion to a second infection was not detected in vivo, evidence of superinfection exclusion was observed. When larvae were first infected by AcMNPV and superinfected by SfMNPV, larval mortality, the presence of SfMNPV genomes in the progeny OBs, and the prevalence of ODVs containing only the second-infecting virus were lower than expected. Hence, superinfection exclusion prevents competition with a better adapted, i.e. more host-specific, competitor. The blockage was established in the early stages of infection, within 12 hours of inoculation of the first virus. The time interval between infections can be manipulated in order to produce mixed OBs with a particular viral proportion or prevalence of mixed ODVs. Similar OB pathogenicity (50% lethal concentration) and viral progeny production were observed for co-occluded OBs containing AcMNPV+SfMNPV and an equal mixture of OBs in S. frugiperda and S. exigua second instar larvae, whereas no positive or negative interactions between viruses were detected. This suggests that each virus infects host larvae in an independent manner, irrespective of their physical association in co-enveloped ODVs within co-occluded OBs or in mixtures of OBs of each virus.

The second part of the thesis focused on unique or unusual characteristics of SfMNPV. The largest and predominant genotype of a Nicaraguan isolate (SfMNPV-B) was completely sequenced and compared to other SfMNPV isolates that had been previously sequenced: SfMNPV-19 from Brazil and SfMNPV-3AP2 from the United States. A high degree of similarity was found among these sequences, with an overall nucleotide sequence identity of 99.35%. Selection pressure analysis revealed three genes that were potentially subjected to diversifying selection: sf49 (pif-3), sf57 (odv-e66a) and sf122. Both pif-3 and odv-e66a are known to be important during baculovirus primary infection, but sf122 had not been previously characterized. A selection of genes of SfMNPV-B were then functionally characterized. First, the sf32 unique gene, which has no homologs in other baculoviruses sequenced to date, was studied. Transcriptional analysis revealed that this is an early gene, which is in agreement with the presence of an early promoter detected in the sequence. Its deletion resulted in OBs 18% larger in diameter and containing 62% more genomic DNA. The latter was related to an increase of 17% observed in the number of nucleocapsids within ODVs, as the number of ODVs within OBs was not modified by the deletion. Insects infected by the deleted virus (Sf32null) produced 39% fewer OBs than those infected by the complete virus (Sfbac). Therefore, the SF32 protein could be involved in nucleocapsid organization during ODV assembly and occlusion. The sf68, sf95 and sf138 genes were selected for being homologs to the ac145 and ac150 genes of AcMNPV which, in turn, are members of the ¿11K¿ family of proteins, characterized for having members among baculoviruses and entomopoxviruses. A virus containing a deletion in the sf138 gene, homologous to ac145, was approximately 15-fold less pathogenic than the parental or repair viruses. This was related to a decrease of more than 100-fold in the ODV infective titre observed in vitro. Addition of an optical brightener to sf138-deleted virus OBs did not result in recovery of the pathogenic activity of OBs indicating that SF138 was unlikely to be involved in degradation of the insect peritrophic matrix. In contrast, viruses carrying a deletion of the sf68 and sf95 genes, homologs to ac150, were 9 hours slower-killing and produced 1.6-1.7 more OBs per larva. These results, together with previous publications, suggest that ac145-like genes may be important during the primary infection of alphabaculoviruses, whereas the ac150-like genes may have a role in the systemic spread of the infection. Both types of proteins appear to act in a host species-dependent manner.

Finally, the sf122 gene, identified previously as being subjected to diversifying selection, was functionally characterized. Transcriptional analysis revealed that this is a late gene. Its deletion severely compromised the biological activity of SfMNPV-B OBs, resulting in a 15-fold reduction in the oral pathogenicity, a slower speed-of-kill by 20 hours and a 3-fold reduction in the production of OBs per larva. The infective titer of ODVs of the deleted virus was decreased by 100-fold and, surprisingly, larvae that died following infection by the deleted virus did not liquefy. It is probably due to reduced expression of the chitinase and cathepsin viral genes observed in larvae infected by the deleted virus. Two different variants of the SF122 protein were identified in the SfMNPV sequences. Isolates containing the short variant produced partial liquefaction in the host population tested, whereas SfMNPV-B, that contains the long variant, produced total liquefaction. I conclude that the sf122 gene is essential for larval liquefaction and adaptation to the host species may be responsible for changes observed in the gene sequence between isolates. Part of the results obtained in this thesis have contributed to a European patent application (PCT/EP2013/069678), offering a method for the potential production of custom-designed biological insecticides, with low environmental impact, to control specific complexes of pests on agricultural crops or forests.