The third vaccine dose significantly reduces susceptibility to the B.1.1.529 (Omicron) SARS-CoV-2 variant.

The main coronavirus disease 2019 (COVID-19) vaccine formulations used today are mainly based on the wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein as an antigen. However, new virus variants capable of escaping neutralization activity of serum antibodies elicited in vaccinated individuals have emerged. The Omicron (B.1.1.529) variant caused epidemics in regions of the world in which most of the population has been vaccinated. In this study, we aimed to understand what determines individual's susceptibility to Omicron in a scenario of extensive vaccination. For that purpose, we collected nasopharynx swab (n = 286) and blood samples (n = 239) from flu-like symptomatic patients, as well as their vaccination history against COVID-19. We computed the data regarding vaccine history, COVID-19 diagnosis, COVID-19 serology, and viral genome sequencing to evaluate their impact on the number of infections. As main results, we showed that vaccination in general did not reduce the number of individuals infected by Omicron, even with an increased immune response found among vaccinated, noninfected individuals. Nonetheless, we found that individuals who received the third vaccine dose showed significantly reduced susceptibility to Omicron infections. A relevant evidence that support this finding was the higher virus neutralization capacity of serum samples of most patients who received the third vaccine dose. In summary, this study shows that boosting immune responses after a third vaccine dose reduces susceptibility to COVID-19 caused by the Omicron variant. Results presented in this study are useful for future formulations of COVID-19 vaccination policies.

Developments in the classification and nomenclature of arthropod-infecting large DNA viruses that contain pif genes.

Viruses of four families of arthropod-specific, large dsDNA viruses (the nuclear arthropod large DNA viruses, or NALDVs) possess homologs of genes encoding conserved components involved in the baculovirus primary infection mechanism. The presence of such homologs encoding per os infectivity factors (pif genes), along with their absence from other viruses and the occurrence of other shared characteristics, suggests a common origin for the viruses of these families. Therefore, the class Naldaviricetes was recently established, accommodating these four families. In addition, within this class, the ICTV approved the creation of the order Lefavirales for three of these families, whose members carry homologs of the baculovirus genes that code for components of the viral RNA polymerase, which is responsible for late gene expression. We further established a system for the binomial naming of all virus species in the order Lefavirales, in accordance with a decision by the ICTV in 2019 to move towards a standardized nomenclature for all virus species. The binomial species names for members of the order Lefavirales consist of the name of the genus to which the species belongs (e.g., Alphabaculovirus), followed by a single epithet that refers to the host species from which the virus was originally isolated. The common names of viruses and the abbreviations thereof will not change, as the format of virus names lies outside the remit of the ICTV.

Easily purified baculovirus/insect-system-expressed recombinant hepatitis B virus surface antigen fused to the N- or C-terminus of polyhedrin.

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.

Chikungunya virus produced by a persistently infected mosquito cell line comprises a shorter genome and is non-infectious to mammalian cells.

Although RNA viruses have high mutation rates, host cells and organisms work as selective environments, maintaining the viability of virus populations by eliminating deleterious genotypes. In serial passages of RNA viruses in a single cell line, most of these selective bottlenecks are absent, with no virus circulation and replication in different tissues or host alternation. In this work, Aedes aegypti Aag-2 cells were accidentally infected with Chikungunya virus (CHIKV) and Mayaro virus (MAYV). After numerous passages to achieve infection persistency, the infectivity of these viruses was evaluated in Ae. albopictus C6/36 cells, African green monkey Vero cells and primary-cultured human fibroblasts. While these CHIKV and MAYV isolates were still infectious to mosquito cells, they lost their ability to infect mammalian cells. After genome sequencing, it was observed that CHIKV accumulated many nonsynonymous mutations and a significant deletion in the coding sequence of the hypervariable domain in the nsP3 gene. Since MAYV showed very low titres, it was not sequenced successfully. Persistently infected Aag-2 cells also accumulated high loads of short and recombinant CHIKV RNAs, which seemed to have been originated from virus-derived DNAs. In conclusion, the genome of this CHIKV isolate could guide mutagenesis strategies for the production of attenuated or non-infectious (to mammals) CHIKV vaccine candidates. Our results also reinforce that a paradox is expected during passages of cells persistently infected by RNA viruses: more loosening for the development of more diverse virus genotypes and more pressure for virus specialization to this constant cellular environment.

Molecular characterization of a putative new cavemovirus isolated from wild chicory (Cichorium intybus).

A putative new virus with sequence similarity to members of the genus Cavemovirus in the family Caulimoviridae was identified in wild chicory (Cichorium intybus) by next-generation sequencing (NGS). The putative new virus was tentatively named "chicory mosaic cavemovirus" (ChiMV), and its genome was determined to be 7,775 nucleotides (nt) long with the typical genome organization of cavemoviruses. ORF1 encodes a putative coat protein/movement polyprotein (1,278 aa), ORF2 encodes a putative replicase (650 aa), and ORF3 encodes a putative transactivator factor (384 aa). The first two putative proteins have 46.2% and 68.7% amino acid sequence identity to the CP/MP protein (YP_004347414) and replicase (YP_004347415), respectively, of sweet potato collusive virus (SPCV). ORF3 encodes a protein with 38.5% amino acid sequence identity to the putative transactivator factor (NP_056849) of cassava vein mosaic virus (CsVMV). The new putative viral genome and those of three cavemoviruses (epiphyllum virus 4 [EpV-4], SPCV, and CsVMV) differ by 24-27% in the nt sequence of the replicase gene, which exceeds the species demarcation cutoff (>20%) for the family.

Mosaic genome evolution and phylogenetics of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and virulence of seven new isolates from the Brazilian states of Minas Gerais and Mato Grosso.

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.

A novel cypovirus found in a betabaculovirus co-infection context contains a poxvirus immune nuclease (poxin)-related gene.

The cassava hornworm Erinnyis ello ello (Lepidoptera: Sphingidae) is an important pest in Brazil. This insect feeds on host plants of several species, especially Manihot esculenta (cassava) and Hevia brasiliensis (rubber tree). Cassava hornworm outbreaks are quite common in Brazil and can cause great impact over crop production. Granulare and polyhedral-shaped occlusion bodies (OBs) were observed in extracts of dead E. ello larvae from rubber-tree plantations by light and scanning electron microscopy (SEM), suggesting a mixed infection. The polyhedral-shaped OB surface revealed indentations that resemble those found in cypovirus polyhedra. After OB nucleic acid extraction followed by cDNA production and Illumina deep-sequencing analysis, the results confirmed for the presence of a putative novel cypovirus that carries ten segments and also a betabaculovirus (Erinnyis ello granulovirus, ErelGV). Phylogenetic analysis of the predicted segment 1-enconded RdRP showed that the new cypovirus isolate is closely related to a member of species Cypovirus 2, which was isolated from Inachis io (Lepidoptera: Nymphalidae). Therefore, we named this new isolate Erinnyis ello cypovirus 2 (ErelCPV-2). Genome in silico analyses showed that ErelCPV-2 segment 8 (S8) has a predicted amino acid identity of 35.82 % to a hypothetical protein of betabaculoviruses. This putative protein has a cGAMP-specific nuclease domain related to the poxvirus immune nucleases (poxins) from the 2',3'-cGAMP-degrading enzyme family.

Identification and genome sequencing of RNA viruses in the eucalyptus snout beetle Gonipterus spp. (Coleoptera: Curculionidae).

The genomes of two putative new RNA viruses (macula-like virus and bunya-like virus) were identified in total RNA extracted from dead eucalyptus snout beetles (Gonipterus spp.) from a laboratory colony. However, only bunya-like virus was detected in field-collected insects. The macula-like virus has a monopartite single-stranded RNA genome that contains three open reading frames (ORFs) encoding an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), protein with unknown function. The bunya-like virus genome was predicted to consist of two RNA segments: a large segment (L) encoding a single protein (RdRp) and a small segment (S) encoding a putative nucleocapsid protein.

Characterization of a bacteriophage with broad host range against strains of Pseudomonas aeruginosa isolated from domestic animals.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen and one of the leading causes of nosocomial infections. Moreover, the species can cause severe infections in cystic fibrosis patients, in burnt victims and cause disease in domestic animals. The control of these infections is often difficult due to its vast repertoire of mechanisms for antibiotic resistance. Phage therapy investigation with P. aeruginosa bacteriophages has aimed mainly the control of human diseases. In the present work, we have isolated and characterized a new bacteriophage, named Pseudomonas phage BrSP1, and investigated its host range against 36 P. aeruginosa strains isolated from diseased animals and against P. aeruginosa ATCC strain 27853. RESULTS: We have isolated a Pseudomonas aeruginosa phage from sewage. We named this virus Pseudomonas phage BrSP1. Our electron microscopy analysis showed that phage BrSP1 had a long tail structure found in members of the order Caudovirales. "In vitro" biological assays demonstrated that phage BrSP1 was capable of maintaining the P. aeruginosa population at low levels for up to 12 h post-infection. However, bacterial growth resumed afterward and reached levels similar to non-treated samples at 24 h post-infection. Host range analysis showed that 51.4% of the bacterial strains investigated were susceptible to phage BrSP1 and efficiency of plating (EOP) investigation indicated that EOP values in the strains tested varied from 0.02 to 1.72. Analysis of the phage genome revealed that it was a double-stranded DNA virus with 66,189 bp, highly similar to the genomes of members of the genus Pbunavirus, a group of viruses also known as PB1-like viruses. CONCLUSION: The results of our "in vitro" bioassays and of our host range analysis suggested that Pseudomonas phage BrSP1 could be included in a phage cocktail to treat veterinary infections. Our EOP investigation confirmed that EOP values differ considerably among different bacterial strains. Comparisons of complete genome sequences indicated that phage BrSP1 is a novel species of the genus Pbunavirus. The complete genome of phage BrSP1 provides additional data that may help the broader understanding of pbunaviruses genome evolution.

Cell-line-dependent crystal morphology and sublocalization of the Thyrinteina arnobia cypovirus polyhedrin expressed from a recombinant baculovirus.

We describe an unexpected feature observed for the heterologous expression of the Thyrinteina arnobia cypovirus polyhedrin from a recombinant baculovirus infection in different insect cell lines. The in cellulo-formed crystals varied in size and shape depending on the cell line. Crystals formed in Trichoplusia ni-derived cells were cubic (0.1-2 µm) and localized in both the nucleus and cytoplasm, whereas those formed in Spodoptera frugiperda-derived cells were ovate and ellipsoidal (0.1-3 µm) and also localized in both the nucleus and cytoplasm. The molecular basis for differences in the morphology, size, and location of cypovirus occlusion bodies is unclear, and cellular proteins might play a role in their formation and location.

Nanopore sequencing of a novel bipartite New World begomovirus infecting cowpea.

A new bipartite begomovirus (family Geminiviridae) was detected on cowpea (Vigna unguiculata) plants exhibiting bright golden mosaic symptoms on leaves under field conditions in Brazil. Complete consensus sequences of DNA-A and DNA-B components of an isolate of the virus (PE-088) were obtained by nanopore sequencing and confirmed by Sanger sequencing. The genome components presented the typical genomic organization of New World (NW) begomoviruses. Pairwise sequence comparisons revealed low levels of identity with other begomovirus species previously reported infecting cowpea around the world. Phylogenetic analysis using complete sequences of DNA-A components revealed that the closest relatives of PE-088 (85-87% nucleotide sequence identities) were three legume-infecting begomoviruses from Brazil: bean golden mosaic virus, macroptilium common mosaic virus and macroptilium yellow vein virus. According to the current classification criteria, PE-088 represents a new species in the genus Begomovirus, tentatively named as cowpea bright yellow mosaic virus (CoBYMV).

Production of GP64-free virus-like particles from baculovirus-infected insect cells.

The retroviral Gag protein is frequently used to generate 'virus-like particles' (VLPs) for a variety of applications. Retroviral Gag proteins self-assemble and bud at the plasma membrane to form enveloped VLPs that resemble natural retrovirus virions, but contain no viral genome. The baculovirus expression vector system has been used to express high levels of the retroviral Gag protein to produce VLPs. However, VLP preparations produced from baculovirus-infected insect cells typically contain relatively large concentrations of baculovirus budded virus (BV) particles, which are similar in size and density to VLPs, and thus may be difficult to separate when purifying VLPs. Additionally, these enveloped VLPs may have substantial quantities of the baculovirus-encoded GP64 envelope protein in the VLP envelope. Since VLPs are frequently produced for vaccine development, the presence of the GP64 envelope protein in VLPs, and the presence of Autographa californica multicapsid nucleopolyhedrovirus BVs in VLP preparations, is undesirable. In the current studies, we developed a strategy for reducing BVs and eliminating GP64 in the production of VLPs, by expressing the human immunodeficiency virus type 1 gag gene in the absence of the baculovirus gp64 gene. Using a GP64null recombinant baculovirus, we demonstrate Gag-mediated VLP production and an absence of GP64 in VLPs, in the context of reduced BV production. Thus, this approach represents a substantially improved method for producing VLPs in insect cells.

A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein.

The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage.

Discovery of two small circular ssDNA viruses associated with the whitefly Bemisia tabaci.

The complete genome sequences of two novel small circular DNA viruses isolated from sweet-potato whiteflies collected in central-West (AdDF) and Southeast (AdO) regions of Brazil were determined by Next Generation Sequencing (NGS), and confirmed by cloning and Sanger sequencing. The genomes are 2,199 and 2,211 nt-long, respectively, encoding a putative coat protein (CP) and a replication-associated protein (Rep) and showing a genomic organization typical of viruses from the family Genomoviridae. Phylogenetic analysis with deduced amino acid sequences of Rep indicates that the virus from AdO is closely related to other members of the genus Gemycircularvirus, while the virus from AdDF is distantly related to other genomovirus. It was thus classified in a putative new genus, for which the name "Gemybolavirus" is proposed. These new genomoviruses are tentatively named "Bemisia associated gemybolavirus AdDF", and "Bemisia associated gemycircularvirus AdO".

Erratum to: Discovery of two small circular ssDNA viruses associated with the whitefly Bemisia tabaci.

The complete genome sequences of two novel small circular DNA viruses isolated from sweet-potato whiteflies collected in Central-West (AdDF) and Southeast (AdO) regions of Brazil were determined by Next Generation Sequencing (NGS), and confirmed by cloning and Sanger sequencing. The genomes are 2,199 and 2,211 nt-long, respectively, encoding a putative coat protein (CP) and a replication-associated protein (Rep) and showing a genomic organization typical of viruses from the family Genomoviridae. Phylogenetic analysis with deduced amino acid sequences of Rep indicates that the virus from AdO is closely related to other members of the genus Gemycircularvirus, while the virus from AdDF is related to those of the genus Gemyduguivirus. These new genomoviruses are tentatively named bemisia-associated genomovirus AdO and bemisia-associated genomovirus AdDF.

The genome sequence of Condylorrhiza vestigialis NPV, a novel baculovirus for the control of the Alamo moth on Populus spp. in Brazil.

Condylorrhiza vestigialis (Lepidoptera: Cambridae), commonly known as the Brazilian poplar moth or Alamo moth, is a serious defoliating pest of poplar, a crop of great economic importance for the production of wood, fiber, biofuel and other biomaterials as well as its significant ecological and environmental value. The complete genome sequence of a new alphabaculovirus isolated from C. vestigialis was determined and analyzed. Condylorrhiza vestigialis nucleopolyhedrovirus (CoveNPV) has a circular double-stranded DNA genome of 125,767bp with a GC content of 42.9%. One hundred and thirty-eight putative open reading frames were identified and annotated in the CoveNPV genome, including 38 core genes and 9 bros. Four homologous regions (hrs), a feature common to most baculoviruses, and 19 perfect and imperfect direct repeats (drs) were found. Phylogenetic analysis confirmed that CoveNPV is a Group I Alphabaculovirus and is most closely related to Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Choristoneura fumiferana DEF multiple nucleopolyhedrovirus CfDEFMNPV. The gp37 gene was not detected in the CoveNPV genome, although this gene is found in many NPVs. Two other common NPV genes, chitinase (v-chiA) and cathepsin (v-cath), that are responsible for host insect liquefaction and melanization, were also absent, where phylogenetic analysis suggests that the loss these genes occurred in the common ancestor of AgMNPV, CfDEFMNPV and CoveNPV, with subsequent reacquisition of these genes by CfDEFMNPV. The molecular biology and genetics of CoveNPV was formerly very little known and our expectation is that the findings presented here should accelerate research on this baculovirus, which will facilitate the use of CoveNPV in integrated pest management programs in Poplar crops.

A betabaculovirus encoding a gp64 homolog.

BACKGROUND: A betabaculovirus (DisaGV) was isolated from Diatraea saccharalis (Lepidoptera: Crambidae), one of the most important insect pests of the sugarcane and other monocot cultures in Brazil. RESULTS: The complete genome sequence of DisaGV was determined using the 454-pyrosequencing method. The genome was 98,392 bp long, which makes it the smallest lepidopteran-infecting baculovirus sequenced to date. It had a G + C content of 29.7% encoding 125 putative open reading frames (ORF). All the 37 baculovirus core genes and a set of 19 betabaculovirus-specific genes were found. A group of 13 putative genes was not found in any other baculovirus genome sequenced so far. A phylogenetic analysis indicated that DisaGV is a member of Betabaculovirus genus and that it is a sister group to a cluster formed by ChocGV, ErelGV, PiraGV isolates, ClanGV, CaLGV, CpGV, CrleGV, AdorGV, PhopGV and EpapGV. Surprisingly, we found in the DisaGV genome a G protein-coupled receptor related to lepidopteran and other insect virus genes and a gp64 homolog, which is likely a product of horizontal gene transfer from Group 1 alphabaculoviruses. CONCLUSION: DisaGV represents a distinct lineage of the genus Betabaculovirus. It is closely related to the CpGV-related group and presents the smallest genome in size so far. Remarkably, we found a homolog of gp64, which was reported solely in group 1 alphabaculovirus genomes so far.

The genome sequence of Pseudoplusia includens single nucleopolyhedrovirus and an analysis of p26 gene evolution in the baculoviruses.

BACKGROUND: Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated. RESULTS: The complete genome of PsinSNPV was sequenced (Roche 454 GS FLX - Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double-stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins. CONCLUSIONS: PsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide.

Functional characterization of hesp018, a baculovirus-encoded serpin gene.

The serpin family of serine proteinase inhibitors plays key roles in a variety of biochemical pathways. In insects, one of the important functions carried out by serpins is regulation of the phenoloxidase (PO) cascade - a pathway that produces melanin and other compounds that are important in insect humoral immunity. Recent sequencing of the baculovirus Hemileuca sp. nucleopolyhedrovirus (HespNPV) genome revealed the presence of a gene, hesp018, with homology to insect serpins. To our knowledge, hesp018 is the first viral serpin homologue to be characterized outside of the chordopoxviruses. The Hesp018 protein was found to be a functional serpin with inhibitory activity against a subset of serine proteinases. Hesp018 also inhibited PO activation when mixed with lepidopteran haemolymph. The Hesp018 protein was secreted when expressed in lepidopteran cells and a baculovirus expressing Hesp018 exhibited accelerated production of viral progeny during in vitro infection. Expression of Hesp018 also reduced caspase activity induced by baculovirus infection, but caused increased cathepsin activity. In infected insect larvae, expression of Hesp018 resulted in faster larval melanization, consistent with increased activity of viral cathepsin. Finally, expression of Hesp018 increased the virulence of a prototype baculovirus by fourfold in orally infected neonate Trichoplusia ni larvae. Based on our observations, we hypothesize that hesp018 may have been retained in HespNPV due to its ability to inhibit the activity of select host proteinases, possibly including proteinases involved in the PO response, during infection of host insects.