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.

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.

Characterization of a fowl adenovirus 9 (FAdV-9) early promoter and its application in generating dual expression FAdV-9s.

The CMV immediate early promoter from the EGFP expression plasmid pEGFP-N1 was replaced with the very left end of the fowl adenovirus 9 (FAdV-9) genome (ntds 73-574) to demonstrate and delineate the promoter function of this sequence. Expression of an EGFP ORF which replaced ORF1 and ORF2 demonstrated that the native promoter can drive down stream foreign gene expression. Replacement of ORF1 and ORF2 with a bicistronic cassette, incorporating a 493 bp IRES from an Ontario strain of avian encephalomyelitis virus (AEV) separating an EGFP ORF and mCherry ORF allowed for expression of both ORFs from a recombinant FAdV. These results provide an additional platform for multivalent vaccines development based on a native FAdV-9 promoter and an avian virus IRES.

Mature viral cathepsin is required for release of viral occlusion bodies from Autographa californica multiple nucleopolyhedrovirus-infected cells.

Baculovirus-infected larvae release progeny viral occlusion bodies (OBs) to enable cyclical virus transmission to new hosts. The alphabaculovirus chitinase and cathepsin enzymes cause terminal liquefaction of host insect cadavers, aiding OB dispersal. The mechanism of cell lysis required to release the OBs is unclear but here we show Autographa californica multiple nucleopolyhedrovirus cathepsin protease activity is required for efficient release of the host tissue-degrading chitinase and cathepsin enzymes and critical for release of progeny OBs from virus-infected cells. Comparisons between viruses containing or lacking cathepsin indicate that cathepsin was necessary for OB release into cultured cell media or hemolymph of insects. In addition, pharmacological inhibition of cysteine protease activity in cells during infection blocked maturation of active cathepsin and OB release from infected cells. Together, these results suggest an important link between baculovirus-induced cell lysis, the concomitant maturation of cathepsin, and cellular release of chitinase, cathepsin and progeny OBs from cells.

Optimized Pre-Clinical Grade Production of Two Novel Lentiviral Vector Pseudotypes for Lung Gene Delivery.

Lung gene therapy requires efficient transduction of slow-replicating epithelia and stable expression of delivered transgenes in the respiratory tract. Lentiviral (LV) vectors have the ideal coding, expression, and transducing capacity required for gene therapy. A modified envelope glycoprotein from the Jaagsiekte Sheep Retrovirus, termed Jenv, is well suited for LV-mediated lung gene therapy due to its inherent lung tropism. Here, two novel Jenv-pseudotyped LVs that effectively transduce lung tissue and yield titers similar to the gold standard, vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped LVs, were generated. As the concentration efficiency of LVs was found to depend on envelope pseudotype, a large-scale production method tailored for Jenv-pseudotyped LVs was developed and the most appropriate method of concentration was determined. In contrast to VSVg and Ebola virus glycoprotein-pseudotyped LVs, ultracentrifugation through a sucrose cushion drastically reduced the yield of Jenv LVs, whereas polyethylene glycol precipitation and tangential flow filtration (TFF) proved to be more suitable methods for concentrating Jenv LVs. Importantly, pressure during TFF was found to be crucial for increasing LV recovery. Finally, a unique mouse model was developed to test the suitability of these novel Jenv-pseudotyped LVs for use in lung gene therapy applications.

ICTV Virus Taxonomy Profile: Nudiviridae.

Members of the family Nudiviridae are large dsDNA viruses with distinctive rod-shaped nucleocapsids and circular genomes of 96-232 kbp. Nudiviruses have been identified from a diverse range of insects and crustaceans and are closely related to baculoviruses. This is a summary of the International Committee on Taxonomy of Viruses Report on the taxonomy of the family Nudiviridae, which is available at ictv.global/report/nudiviridae.

Identification and Characterization of the Nucleolar Localization Signal of Autographa californica Multiple Nucleopolyhedrovirus LEF5.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 5 (LEF5) is highly conserved in all sequenced baculovirus genomes and plays an important role in production of infectious viral progeny. In this study, nucleolar localization of AcMNPV LEF5 was characterized. Through transcriptome analysis, we identified two putative nucleolar proteins, Spodoptera frugiperda nucleostemin (SfNS) and fibrillarin (SfFBL), from Sf9 cells. Immunofluorescence analysis demonstrated that SfNS and SfFBL were localized to the nucleolus. AcMNPV infection resulted in reorganization of the nucleoli of infected cells. Colocalization of LEF5 and SfNS showed that AcMNPV LEF5 was localized to the nucleolus in Sf9 cells. Bioinformatic analysis revealed that basic amino acids of LEF5 are enriched at residues 184 to 213 and may contain a nucleolar localization signal (NoLS). Green fluorescent protein (GFP) fused to NoLS of AcMNPV LEF5 localized to the nucleoli of transfected cells. Multiple-point mutation analysis demonstrated that amino acid residues 197 to 204 are important for nucleolar localization of LEF5. To identify whether the NoLS in AcMNPV LEF5 is important for production of viral progeny, a lef5-null AcMNPV bacmid was constructed; several NoLS-mutated LEF5 proteins were reinserted into the lef5-null AcMNPV bacmid with a GFP reporter. The constructs containing point mutations at residues 185 to 189 or 197 to 204 in AcMNPV LEF5 resulted in reduction in production of infectious viral progeny and occlusion body yield in bacmid-transfected cells. Together, these data suggested that AcMNPV LEF5 contains an NoLS, which is important for nucleolar localization of LEF5, progeny production, and occlusion body production.IMPORTANCE Many viruses, including human and plant viruses, target nucleolar functions as part of their infection strategy. However, nucleolar localization for baculovirus proteins has not yet been characterized. In this study, two nucleolar proteins, SfNS and SfFBL, were identified in Sf9 cells. Our results showed that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection resulted in redistribution of the nucleoli of infected cells. We demonstrated that AcMNPV late expression factor 5 (LEF5) could localize to the nucleolus and contains a nucleolar localization signal (NoLS), which is important for nucleolar localization of AcMNPV LEF5 and for production of viral progeny and yield of occlusion bodies.

The U3 and Env Proteins of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Both Contribute to Tissue Tropism.

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are small-ruminant betaretroviruses that share high nucleotide and amino acid identity, utilize the same cellular receptor, hyaluronoglucosaminidase 2 (Hyal2) for entry, and transform tissues with their envelope (Env) glycoprotein; yet, they target discrete regions of the respiratory tract-the lung and nose, respectively. This distinct tissue selectivity makes them ideal tools with which to study the pathogenesis of betaretroviruses. To uncover the genetic determinants of tropism, we constructed JSRV-ENTV chimeric viruses and produced lentivectors pseudotyped with the Env proteins from JSRV (Jenv) and ENTV (Eenv). Through the transduction and infection of lung and nasal turbinate tissue slices, we observed that Hyal2 expression levels strongly influence ENTV entry, but that the long terminal repeat (LTR) promoters of these viruses are likely responsible for tissue-specificity. Furthermore, we show evidence of ENTV Env expression in chondrocytes within ENTV-infected nasal turbinate tissue, where Hyal2 is highly expressed. Our work suggests that the unique tissue tropism of JSRV and ENTV stems from the combined effort of the envelope glycoprotein-receptor interactions and the LTR and provides new insight into the pathogenesis of ENTV.

Fowl adenovirus 9 ORF19, a lipase homolog, is nonessential for virus replication and is suitable for foreign gene expression.

Fowl adenovirus 9 (FAdV-9) has one of the largest genomes (45 kb) so far sequenced from all adenoviruses studied. Genus-specific genes located within the early (E) regions at the right and left ends of the viral genome have unknown functions except for ORF8 (Gam-1 gene), ORF22 and ORF1 (dUTPase gene). ORF19, located at the right end of the genome (nts 34,220-36,443), is predicted to encode a lipase protein and its homologs are also found in all FAdV genomes so far sequenced. The role of ORF19 in virus replication and virulence is unknown. To study ORF19 and explore its potential as a locus for foreign gene insertion, we generated one ORF19-deleted mutant virus (rFAdV-9Δ19-SwaI) and three FAdV-9Δ19-based recombinant viruses replacing ORF19 as follows: rFAdV-9Δ19-CAT and enhanced-green fluorescent protein (EGFP) cassette (CMV promoter-EGFP-poly A) in a rightward (rFAdV-9Δ19-EGFP-R) and leftward orientation (rFAdV-9Δ19-EGFP-L). All recombinant viruses were stable after three passages. In chicken hepatoma cells, rFAdV-9Δ19-SwaI, rFAdV-9Δ19-CAT and rFAdV-9Δ19-EGFP-R replicated at titers similar to that of the wild-type virus, whilst rFAdV-9Δ19-EGFP-L replicated at a much lower titer. Interestingly, FAdV-9Δ19-SwaI replicated at higher titers in cells and in embryonated eggs, respectively than those of wild-type and recombinant viruses. These observations suggest ORF19 is nonessential for replication and can be used as a novel cloning site for engineering FAdV-9-based recombinant viruses and rFAdV-9Δ19-SwaI could be used to determine its role for virus replication in vivo.

ICTV Virus Taxonomy Profile: Baculoviridae.

The family Baculoviridae comprises large viruses with circular dsDNA genomes ranging from 80 to 180 kbp. The virions consist of enveloped, rod-shaped nucleocapsids and are embedded in distinctive occlusion bodies measuring 0.15-5 µm. The occlusion bodies consist of a matrix composed of a single viral protein expressed at high levels during infection. Members of this family infect exclusively larvae of the insect orders Lepidoptera, Hymenoptera and Diptera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Baculoviridae, which is available at www.ictv.global/report/baculoviridae.

Fowl Adenovirus 4 (FAdV-4)-Based Infectious Clone for Vaccine Vector Development and Viral Gene Function Studies.

Fowl adenovirus 4 (FAdV-4) is associated with economically important poultry diseases. Recent studies of fully sequenced genomes of FAdV-4 isolates suggest potential genomic regions associated with virulence and amenable for manipulation and vector development. Direct manipulation of viral genomes is cumbersome, as opposed to that of infectious clones-viral genomes cloned into plasmid or cosmid vectors. In this work, we generated an infectious clone, pFAdV-4 ON1, containing the entire viral genome of a nonpathogenic FAdV-4 (ON1 isolate). pFAdV-4 ON1 was used for targeted deletion of open reading frames (ORFs) 16 and 17 and replacement with the enhanced green fluorescence protein (EGFP) expression cassette to generate recombinant viruses. These viruses were viable, and EGFP was expressed in infected cells. Their replication, however, was significantly reduced with respect to that of the wild-type virus. These observations suggest the potential utility of FAdV-4 as a vaccine vector and the importance of ORFs 16 and 17 for virus replication at wild-type levels. To our knowledge, this is the first report of an infectious clone based on the FAdV-4 genome, and our results demonstrate its utility for studies of virulence determinants and as a platform for either vaccine or gene delivery vectors.

The N Terminus of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Is Required for Efficient Viral DNA Replication and Virus and Occlusion Body Production.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) plays a crucial role in viral DNA synthesis, and the N terminus (residues 1 to 186) is highly conserved in the baculovirus DNApol family. However, the functional role of the N terminus of DNApol has not yet been characterized. Here we report a functional analysis of the AcMNPV DNApol N terminus. We truncated the DNApol N terminus to construct truncation mutants Bac-GFP-PolΔ64, Bac-GFP-PolΔ110, and Bac-GFP-PolΔ186, which lack 64, 110, and 186 N-terminal residues, respectively. Although the truncation mutants rescued viral DNA synthesis and infectious virus production, the level of DNA replication decreased, and Bac-GFP-PolΔ64, Bac-GFP-PolΔ110, and Bac-GFP-PolΔ186 showed 10-fold, 89-fold, and 891-fold reductions in infectious viral yield compared to that of the wild-type repair virus, respectively. Production of occlusion bodies was compromised for all truncation mutants. Further bioinformatic analysis showed that the first 64 amino acids (aa) at the extreme N terminus contains a conserved α(-helix)-ß(-sheet)-ß-ß secondary-structure region, and further downstream sequence from aa 67 to 186 is comprised of four conserved sequence motifs. Multiple alanine point substitutions in the α-ß-ß-ß structure region or the four sequence motifs in the N terminus impaired viral DNA replication and resulted in reduction of virus yield and occlusion body production. Together, our results suggested that the secondary structure and four conserved motifs within the N terminus of AcMNPV DNApol are important for viral DNA synthesis, infectious virus yield, and production of occlusion bodies.IMPORTANCE DNA polymerase (DNApol) is highly conserved in all baculoviruses and is required for viral DNA replication. The N terminus is one of the highly conserved regions of baculovirus DNApols. Our results showed that the N terminus of baculovirus DNA polymerase plays an important role in efficient viral DNA synthesis and infectious virus yield and production of occlusion bodies. We identified five features, including a highly conserved secondary structure and four conserved amino acid motifs, in the AcMNPV DNApol N terminus, all of which are important for efficient viral DNA synthesis, infectious virus yield, and production of occlusion bodies.

Generation and characterization of a fowl adenovirus 9 dual-site expression vector.

Fowl adenoviruses (FAdVs) are widely considered as excellent platforms for vaccine development and gene therapy. We improved on our right-end partial TR-2 deleted or a left-end 2.3 kb deleted vectors by developing a single, dual-site delivery vector. We demonstrated that, in addition to ORF11, the right end ORF17 is also dispensable. To further improve the capacity and flexibility of the FAdV-9 based vector system, we generated an infectious recombinant FAdV-9 dual-site expression clone lacking 1.9 kb of the left end and replaced with mCherry under the control of a native promoter, and 3.6 kb of the right-end replaced with an EGFP expression cassette. Five intermediate FAdmid clones were successfully constructed: a) pFAdV-9Δ0-2RED (mCherry replacing the left end 2.2 kb ORF0 to 2); b) pFAdV-9RED (mCherry replacing the left end 1.9 kb ORF1 to 2); c) pFAdV-9Δ17 (deletion of ORF17 and 393 bp downstream untranslated region); d) pFAdV-9GFP (EGFP expression cassette replacing the right end 3.6 kb) and e) pFAdV-9Dual (both mCherry in the left end and the EGFP expression cassette in the right end of our vector). Our novel FAdV-9 dual-site vaccine vector, produced infectious virus and expressed either one or both mCherry and EGFP.

Identification and expression analysis of multiple small heat shock protein genes in spruce budworm, Choristoneura fumiferana (L.).

Fifteen small heat shock protein (sHSP) genes were identified from spruce budworm, Choristoneura fumiferana (L.), an important native forest pest in North America. The transcript levels of each CfHSP were measured under non-stress conditions in all life stages from egg to adult and in five different larval tissues. CfHSP transcript levels showed variation during development, with highest levels in adults and lowest in eggs. Most CfHSP transcripts are highly expressed in larval fat body and Malpighian tubules; two CfHSPs display extremely high expression in the head and epidermis. Upon heat stress, nine CfHSP genes are significantly upregulated, increasing by 50- to 2500-fold depending on developmental stage and tissue type. Upon starvation, eight CfHSPs are upregulated or downregulated, whereas six others retain constant expression. These results suggest that CfHSPs have important and multiple roles in spruce budworm development and in response to heat stress and starvation.

Foreign gene expression and induction of antibody response by recombinant fowl adenovirus-9-based vectors with exogenous promoters.

Fowl adenoviruses (FAdVs) are promising vectors for poultry vaccines and gene therapy. The commonly used human cytomegalovirus (CMV) promoter in recombinant FAdV-9 viruses (recFAdV-9s) leads to foreign gene expression that elicits an antibody response. Despite its strength, studies have shown that the CMV promoter is prone to silencing by methylation hampering the in vivo application of vectors containing this promoter. Therefore, to improve our virus vector system and circumvent potential limitations of silencing, we engineered recFAdV-9s with foreign gene expression cassettes carrying the CMV enhancer/chicken ß-actin (CAG) or the human elongation factor 1 alpha (EF1α) promoters with or without the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE). Chicken hepatoma cells (CH-SAH) infected with recFAdV-9s carrying either CAG or EF1α promoters expressed higher levels of foreign protein than those infected with recFAdV-9 carrying the CMV promoter. Incorporation of the WPRE element rendered lower gene expression regardless of promoter type. Surprisingly, most chickens inoculated with recFAdV-9 containing the CMV promoter had the highest antibody response to foreign protein compared to other promoters. Our findings suggest the importance of promoter selection for candidate virus vector vaccines based on humoral immune response rather than foreign protein expression levels in vitro.

A betabaculovirus DNA polymerase cannot substitute for the DNA polymerase of the alphabaculovirus Autographa californica nucleopolyhedrovirus.

DNA polymerase (DNApol) is present in all baculoviruses and plays a crucial role in viral DNA replication. Previously we showed that the DNApol of the alphabaculovirus group II Spodoptera litura nucleopolyhedrovirus (SpltNPV) could partially substitute for the DNApol of a group I alphabaculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, it is not known if a betabaculovirus DNApol could subsititute for the alphabaculovirus DNApol in AcMNPV. In this report, DNApol of the betabaculovirus Pieris rapae granulovirus (PiraGV) was inserted into a dnapol-null AcMNPV bacmid, creating Bac-AcΔpol:PrPol. The repair virus did not spread to neighboring cells; virus growth curve and real-time PCR revealed that the PiraGV dnapol substitution abrogated AcMNPV DNA replication and virus production. Immunofluorescence microscopy showed that PiraGV DNApol could be expressed and localized to the nucleus. Collectively, our results suggested that the alphabaculovirus AcMNPV DNApol could not be replaced by a DNApol from the betabaculovirus, PiraGV.

The complete mitogenome of the Emerald Ash Borer (EAB), Agrilus planipennis (Insecta: Coleoptera: Buprestidae).

The complete mitogenome of the Emerald Ash Borer (EAB, Agrilus planipennis) was obtained by gleaning mitochondrial sequences from whole-genome Illumina sequencing data. The circular genome has 15,942 base pairs and contains 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and an A-T-rich region. All PCGs begin with ATN codons. The nucleotide composition is highly asymmetric (31.65% A, 40.25% T, 17.39% G, 10.71% C), with an overall A-T content of 71.9%. Phylogenetic analysis based on insect mitogenomes indicated that EAB is closely related to other Buprestoidea species, clustering most closely with Chrysochroa fulgidissima.

Molecular characterization of pathogenic and nonpathogenic fowl aviadenovirus serotype 11 isolates.

Fowl aviadenoviruses, many of which are of importance in veterinary medicine, are classified into 5 species. In this study, a pathogenic isolate and a nonpathogenic isolate of fowl aviadenovirus serotype 11 (FAdV-11) of species Fowl aviadenovirus D were characterized. Growth rates were analyzed for the 2 isolates, showing notable differences. The complete genomic sequences of the viruses were fully determined and were analyzed. The genomes of the 2 isolates showed 98.1% sequence identity and revealed 6 nonsynonymous mutations between the Ontario isolates. Two of the 6 mutations were also found in the sequences of recently published pathogenic Chinese fowl aviadenovirus 11 isolates, suggesting potential molecular markers that could be associated with pathogenesis. Deletions were found in the L5 region within the overlapping coding sequences for the 100, 22, and 33 kDa proteins, and these were found in only the nonpathogenic isolates. This molecular pattern was identified in FAdV-9, another nonpathogenic FAdV-D species virus. Furthermore, the tandem repeat regions varied dramatically; the pathogenic isolates contained a reduced number of tandem repeats compared with the nonpathogenic isolates. Lastly, a protein produced early in infection was analyzed using bioinformatics to determine its role in disease. This study highlights several candidate molecular determinants of avian adenovirus genomes related to pathogenicity.

Characterization and functional studies of fowl adenovirus 9 dUTPase.

Deoxyuridine 5'-triphosphate pyrophosphatase (dUTPase), a ubiquitous enzyme that catalyzes the hydrolysis of dUTP to dUMP and found in many viruses, has yet to be identified in fowl adenovirus 9 (FAdV-9). By a multiple alignment of dUTPase amino acid sequences, FAdV-9 ORF1 contained the five conserved motifs that define the protein family, and encoded a functional dUTPase. Moreover, transcription and protein expression patterns were characterized, indicating that dUTPase was transcribed from 2h post-infection (h.p.i.) and translated from 6h.p.i., and both continued to the late phase of virus infection. An HA-tagged dUTPase recombinant virus was generated, and dUTPase was found to be localized in both the cytoplasm and nucleus in chicken hepatoma cells (CH-SAH). A dUTPase knockout virus was generated and compared with the wild-type virus, showing that dUTPase upregulated the expression of type I interferons, but was not required for viral DNA or virus replication in CH-SAH cells.