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Baculoviruses are circular double-stranded DNA viruses that infect insects and are widely used as the baculoviral expression vectors (BEVs), which provide a eukaryotic milieu for heterologous expression. The most frequently used vector is based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, purification of recombinant proteins produced using BEVs is laborious, time-consuming, and often expensive. Numerous strategies have been explored to facilitate purification of heterologous proteins, such as fusion with occlusion body (OBs)-forming proteins like polyhedrin (Polh). Baculoviruses produce OBs in the late stages of infection to protect the virion in the cellular environment, and the main protein responsible for OB formation is Polh. In this study, we investigated the effect of fusing the gene that encodes the surface antigen (S-HBsAg) of hepatitis B virus (HBV) to either the N- or C-terminus of the AcMNPV Polh. The production of recombinant viruses and recombinant proteins was confirmed, and the ability to form chimeric S-HBsAg-containing OBs was accessed by light and scanning electron microscopy of infected cells. The fusion was found to affect the shape and size of the OBs when compared to wild-type OBs, with the N-terminal fusion producing less-amorphous OBs than the C-terminal construct. In addition, the N-terminal construct gave higher levels of expression than the C-terminal construct. Quantitative and qualitative immunoassays with human serum or plasma antibodies against HBsAg showed that the two forms of the antigen reacted differently. Although both reacted with the antibody, the N-terminal fusion protein reacted with more sensitivity (2.27-fold) and is therefore more suitable for quantitative assays than the C-terminal version. In summary, the BEVs represents a promising tool for the production of reagents for the diagnosis of HBV infection. [ABSTRACT FROM AUTHOR]

In a comparative analysis of genome sequences from isolates of the baculovirus Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) from Brazil and Guatemala, we identified a subset of isolates possessing chimeric genomes. We identified six distinct phylogenetically incongruous regions (PIRs) dispersed in the genomes, of between 279 and 3345 bp in length. The individual PIRs possessed high sequence similarity among the affected ChinNPV isolates but varied in coverage in some instances. The donor for four of the PIRs implicated in horizontal gene transfer (HGT) was identified as Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), an alphabaculovirus closely related to ChinNPV, or another unknown but closely related virus. BLAST searches of the other two PIRs returned only ChinNPV sequences, but HGT from an unknown donor baculovirus cannot be excluded. Although Chrysodeixis includens and Trichoplusia ni are frequently co-collected from soybean fields in Brazil, pathogenicity data suggest that natural coinfection of C. includens larvae with ChinNPV and TnSNPV is probably uncommon. Additionally, since the chimeric ChinNPV genomes with tracts of TnSNPV sequence were restricted to a single monophyletic lineage of closely related isolates, a model of progressive restoration of the native DNA sequence by recombination with ChinNPV possessing a fully or partially non-chimeric genome is reasonable. However, multiple independent HGT from TnSNPV to ChinNPV during the evolution of these isolates cannot be excluded. Mortality data suggest that the ChinNPV isolates with chimeric genomes are not significantly different in pathogenicity towards C. includens when compared to most other ChinNPV isolates. Exclusion of the PIRs prior to phylogenetic analysis had a large impact on the topology of part of the maximum-likelihood tree, revealing a homogenous clade of three isolates (IB, IC and ID) from Paraná state in Brazil collected in 2006, together with an isolate from Guatemala collected in 1972 (IA), comprising the lineage uniquely affected by HGT from TnSNPV. The other 10 Brazilian ChinNPV isolates from Paraná, Mato Grosso, and Minas Gerais states showed higher variability, where only three isolates from Paraná state formed a monophyletic group correlating with geographical origin. [ABSTRACT FROM AUTHOR]