Comparative genomics of the Neodiprion sertifer nucleopolyhedrovirus from Turkey with the fewest ORFs among baculoviruses

Comparative genomics of the Neodiprion sertifer nucleopolyhedrovirus from Turkey with the fewest ORFs among baculoviruses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative genomics of the Neodiprion sertifer nucleopolyhedrovirus from Turkey with the fewest ORFs among baculoviruses Özgül DOĞAN, Mahir BUDAK, Melissa Şafak SALMAN, Ertan Mahir KORKMAZ This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3466248/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jan, 2024 Read the published version in Virus Genes → Version 1 posted 8 You are reading this latest preprint version Abstract The complete genome of the European pine sawfly Neodiprion sertifer nucleopolyhedrovirus was sequenced and characterized from next-generation sequencing data of the N. sertifer larva from Turkiye. This genome was comparatively analysed by previously reported genomes baculoviruses. The baculovirus phylogeny was reconstructed and the species boundary of the NeseNPV-TR was delineated using K2P distance. The length of the genome was 82,052 bp, with a G + C content of 33.28%. It contained 82 putative ORFs, including 33 baculovirus core genes, three lepidopteran baculovirus core genes, three non-conserved genes. It had five hrs with 20.6% overall mean distance on average. The pairwise K2P distances of lef-9 and polh genes were lower than the specified threshold value, while those of lef-8 , combinations of three genes and 33 genes were slightly higher between NeseNPV-TR and NeseNPV. The most variable genes were lef-2 , helicase , p40 , desmoplakin , p6.9 , vp91 and vp39 , while the most conserved were lef-8 , lef-9 , odv-e18 , pif2 and lef-5 among baculoviruses. The genome of NeseNPV-TR is smaller and contains the fewest ORFs among baculoviruses. Some of unassigned ORFs had conserved domains and hence, we suggest further investigation to determine their structural and functional roles. Phylogenetic analyses confirmed its position within Gammabaculovirus. The NeseNPV-TR can be considered as the same species with NeseNPV. The different divergence rates in the baculovirus core genes may be related with different selection pressures acting on the genes. The lower genetic diversity of Group I alphabaculoviruses is most probably due to recent emergence. Baculoviruses DNA viruses european pine sawfly gammabaculovirus genetic divergence nucleopolyhedrovirus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The family Baculoviridae (also known as baculoviruses) is a distinctive group of host-specific large DNA viruses that infect holometabolous insects in their larval stage, particularly some members of the orders Lepidoptera, Diptera and Hymenoptera (Miele et al. 2011 ). The members of this largest and most diverse family of DNA viruses are utilised as biological control agents in ecological pest management programs and/or as gene expression vectors in biotechnological applications (Szewczyk et al. 2006 ; Herniou and Jehle 2007 ; Assenberg et al. 2013 ). The baculovirus replication cycle generates two morphologically unique viral phenotypes including the occlusion body virus (ODV) and the budded virus (BV) regarding the difference in their envelope protein compositions. The morphology of occlusion bodies (OBs) allows the taxonomic classification of baculoviruses into nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) (Slack and Arif 2007 ; Rohrmann 2019 ; Chen et al. 2019 ). The baculoviruses are grouped under four genera based on their molecular characteristics and phylogenies: Deltabaculovirus (dipteran-specific NPV); Gammabaculovirus (hymenopteran-specific NPV); Betabaculovirus (lepidopteran-specific GV); Alphabaculovirus (lepidopteran-specific NPV) (Jehle et al. 2006a , b ). Alphabaculovirus can also be further divided into Group I and Group II based on their phylogenetic relationship and membrane fusion proteins (Zanotto et al. 1993 ; Bulach et al. 1999 ; Jehle et al. 2006a , b ; Wang et al. 2018 ). The rapidly growing number of the reported whole baculovirus genome has led to a deeper understanding of biology and evolution of baculoviruses. To date, the complete genomes for baculoviruses have been reported for approximately 100 host species in the public databases, representing mostly alphabaculoviruses and betabaculoviruses, but only three gammabaculoviruses, and one deltabaculovirus (NCBI, September 2022). A typical baculovirus genome is a double-stranded circular molecule packaged into rod-shaped nucleocapsid with a relatively conserved core gene content, despite a wide range in size from 81.755 kb ( Neodiprion lecontei NPV) to 178.733 kb ( Xestia c-nigrum GV) (NCBI, September 2023). Size discrepancies in the baculovirus genomes could be the result of gene loss/gain events which are most likely due to the frequent occurrence of horizontal gene transfer between the virus and its host (Gilbert et al. 2014 ). Although the core genes are peculiarly essential in many biological processes, including viral replication, transcription, structural composition, and oral infectivity (van Oers and Vlak 2007 ), the function of several core genes has still been unravelled. The information of these genomes can be comparatively utilised in two levels as the nucleotide/amino acid sequences or the structural features and architecture of genomes such as gene content, compositional features, genome size, genome organisation, homolog regions of replication and transcription processes (van Oers and Vlak 2007 ). These features requiring different evolutionary processes also allow us to understand better the baculovirus taxonomy, their phylogenetic relationships, evolutionary history, and life strategies such as host specificity and infectivity. For instance, a demarcating criterion used to distinguish baculovirus species has recently been proposed by researchers based on a total of 172 complete genomes (Jehle et al. 2006a , b ). In that criterion, if the pairwise evolutionary distance between single or concatenated late expression factor 8 ( lef-8 ), late expression factor 9 ( lef-9 ) (encode for subunits of the baculovirus RNA polymerase) and polh genes (encode polyhedrin ) or the homologous granulin of GVs, the major matrix protein of the occlusion bodies (OBs), which are the most conserved core genes of baculoviruses, under Kimura-2-parameter (K2P) is larger than 0.072, two viruses should be considered as different virus species. However, if the distance is smaller than 0.021, two (or more) baculoviruses should be considered within the same species. If the distance is between 0.021 and 0.072, more biological information should then be provided for species discrimination (Jehle et al. 2006a , b ; Wennmann et al. 2018 ). Among four baculovirus genera, the gammabaculovirus has the smallest genome (with 84.160 kb on average) comprising approximately 90 predicted open reading frames (ORFs) (Garcia-Maruniak et al. 2004 ; Lauzon et al. 2004 ; Duffy et al. 2006 ). So far, only three genomes of gammabaculovirus have been reported from merely the species of Neodiprion (Diprionidae: Symphyta), which are the most common defoliating sawfly insects of pine trees, suggesting undergone host-dependent evolution with their restricted hosts. They are Neodiprion lecontei NPV (NeleNPV) (Lauzon et al. 2004 ), Neodiprion sertifer NPV (NeseNPV) (Garcia-Maruniak et al. 2004 ) and Neodiprion abietis NPV (NeabNPV) (Duffy et al. 2006 ) and are used as bio-insecticide that infect the midgut cell of the Neodiprion larvae (Arif et al. 2011 ). Here, we sequenced and annotated the complete genome of a Neodiprion sertifer nucleopolyhedrovirus from Turkey (NeseNPV-TR). We compared it with the previously reported genomes of gammabaculovirus for a better understanding of the architecture and features of this baculovirus genome using current bioinformatics approaches. The proteins encoded by the ORFs were predicted in terms of the families, conserved domains and putative functions. We constructed phylogenies to estimate the phylogenetic position of NeseNPV-TR using a concatenated core gene dataset of 100 species from Baculoviridae. The pairwise distances between the members of gammabaculovirus were also calculated to delineate the species boundaries of the NeseNPV-TR of which genome sequence information was obtained, using the Kimura two-parameter distance. Finally, the core genes of all sequenced baculovirus genomes were compared based on nucleotide and amino acid sequences by calculating overall mean p-distances for each gene and genus in order to make inferences about baculovirus evolution. Materials and Methods DNA extraction, genome sequencing and assembly The virus genome data was generated through sequencing of the isolated total DNA of Neodiprion sertifer larvae collected from a pine tree in Gelenbe district of Turkey in 2014. The collected larvae were preserved in ethanol in the Entomology Collection of Cumhuriyet University, Sivas (ECCUS) and total genomic DNA was extracted from a whole larva by using DNeasy tissue kit (Qiagen, Hilden, Germany) following the instructions provided by the manufacturer. The quality and quantity of the extracted DNA were measured with a Qubit 4.0 fluorometer (Thermo Fisher) by using the Qubit double-strand High Sensitivity Assay kit and then stored at -20°C. The total genomic DNA extract was sequenced by Illumina HiSeq 2000 next-generation sequencing (NGS) platform using 250 bp paired-end reads, conducted at DONE Genetics Inc., Turkey. The quality of raw NGS reads was initially checked via FastQC v0.11.4 ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc ) and then filtered by fastp (Chen et al. 2018 ). The reference genome of the NeseNPV (Accession no. NC005905) was selected to generate the kraken2 database (Wood et al. 2019 ). The fastq file was searched against the kraken2 database to find short reads that are part of the viral genome via Kraken2. The awk tool in the linux was used to construct a new fastq file including short reads of the viral genome. Genome assembly of NeseNPV-TR was performed de novo using SPAdes v3.14.0 (Bankevich et al. 2012 ) from the new fastq file. A reference-based assembly was carried out employing Bowtie 2 (Langmead and Salzberg 2012 ) assembly algorithm using the genome of NeseNPV as reference. The generated assemblies by these two approaches were then aligned, manually compared, and finally compiled into a single contig. Genome annotation and organisation The open reading frames (ORFs) encoding proteins with 50 or longer amino acids were predicted using several different tools: (i) Prokka software (Seemann 2014 ) implemented in KBase (Arkin et al. 2018 ), (ii) Glimmer (Delcher et al. 1999 ) plugin implemented in Geneious R9 (Kearse et al. 2012 ), (iii) Benchmarking Universal Single-Copy Orthologs BUSCO v5.2.2 (Manni et al. 2021 ) tool with searches against baculoviridae_odb10.2020-11-26 dataset, and (iv) FGenesV0 which is available online at http://www.softberry.com/ . TTG (Leu) and GTG (Val) codons were also taken into account as alternative start codons in addition to standard ATG start codon. The identified ORFs were then compared with those of reference NeseNPV genome (Accession Number: NC005905). Homolog sequences of the ORFs were predicted by BLAST searches against UniProtKB and Swiss-Prot databases (Bateman et al. 2023 ). The families, conserved domains and putative functions of the proteins encoded by the ORFs were determined using Blast2GO (Conesa et al. 2005 ) tool via searches with InterPro (Paysan-Lafosse et al. 2023 ). The homologous repeated sequences ( hrs ) throughout the NeseNPV-TR genome were searched using Tandem Repeat Finder with the default parameters (Benson 1999 ). The annotated genome of NeseNPV-TR was finally visualized using Geneious R9. Nucleotide compositions of each gene and the entire genome were calculated separately by MEGA v6.0 (Tamura et al. 2013 ). Gene parity plots were constructed to assess the pairwise ORF synteny between NeseNPV-TR and the reported gammabaculovirus genomes using Microsoft Excel. Phylogenetic inference and genetic distance Phylogenetic analyses were performed using the nucleotide sequences of the 33 core genes and polyhedrin gene of the newly sequenced NeseNPV-TR genome (Table S3 ) and their homologous from the previously reported 99 reference baculovirus genomes which were downloaded from NCBI (Table S1 ). Culex nigripalpus NPV (Deltabaculovirus) (Afonso et al. 2001 ) was also included in analyses as an outgroup. Nucleotide sequences of each core gene were aligned individually using the “translation align” option with MAFFT algorithm (Katoh and Standley 2013 ) in Geneious R9. The gene alignments were concatenated with SequenceMatrix v.1.7.8 (Vaidya et al. 2011 ). Maximum Likelihood (ML) and Bayesian Inference (BI) approaches were used to confirm the phylogenetic position of the NeseNPV-TR and to infer the phylogenetic relationship between the available baculoviruses. The ML analysis was performed in IQ-TREE v2.1.4 (Minh et al. 2020 ) using default parameters and allowing it to automatically select the evolutionary model. Nodal support was estimated with 10000 bootstrap and fast approximate likelihood-based measures of branch supports (Shimodaira–Hasegawa [SH]-aLRT) with 1000 replicates under the fast bootstrapping option. For Bayesian Inference, the best model of evolution was selected using MEGA v6.0 setting branch swap filter as very strong. The best fitting model GTR + G + I (Table S4) was used in the subsequent Bayesian analysis. BI analyses were conducted using MrBayes v3.2.2 (Ronquist et al. 2012 ) under the unlinked branch lengths of each partition scheme with two independent runs of ten million generations with four Markov chains, sampling every 1000 generations. The stationarity for each run was assessed by evaluating the parameter files using Tracer v1.7 (Rambaut et al. 2018 ). After assessment, the first 25% of trees in each run were discarded as burn-in and a majority-rule consensus tree (BI tree) was generated from the remaining trees. The trees were finally visualised by FigTree v1.4.2 (Rambaut 2014 ). To species demarcation, pairwise K2P genetic distances between the NeseNPV-TR and previously reported gammabaculoviruses were estimated separately for both the concatenated 33 aligned core genes and the most conserved three individually aligned core genes ( lef-8 , lef-9 and polh ), using MEGA v6.0 with Kimura 2-parameter model (Kimura 1980 ) by treating the gaps in the alignment as “pairwise deletions”. In addition, overall mean p-distances for each gene and each genus were calculated using MEGA v6.0 at both the nucleotide and amino acid levels. Results and Discussion Genome characteristics of NeseNPV-TR Advances in sequencing technologies and decreasing costs have led to an increase in the number of baculoviruses for which genome sequence information has been obtained. Whole genome sequence information of 100 baculovirus isolates has been obtained and characterised so far (NCBI Reference Sequence Database, September 2023). Comparative analyses of these genomes helped us to infer baculovirus genome patterns and contributed to a better understanding of their evolution. Baculovirus genomes were highly variable in terms of genome length and organisation, gene content, repetitive sequence content and nucleotide composition (Table S1 ). The size of baculovirus genomes exhibited variability between 81,755 bp in NeleNPV (Lauzon et al. 2004 ) and 178,733 bp in XecnGV (Goto et al. 1998 ). Here, the newly sequenced and annotated complete genome of NeseNPV-TR (GenBank accession number: OP426321) was 82,052 bp in size, which was smaller than the previously reported genome of the NeseNPV (86,462 bp) (Garcia-Maruniak et al. 2004 ) and also exhibited relatively low overall pairwise identity (90.62%). The size difference was mainly resulted from the presence of a large indel comprising two ORFs coding hypothetical proteins (corresponding NeseORF18 and NeseORF19 in the reference genome), one direct and one homologous repeat regions (Table S2 ). The number of coding regions was variable among baculovirus genomes: from 82 ORFs in NeseNPV-TR genome to 183 ORFs in PsunGV genome (Table S1 ) (Li et al. 2021 ). The homologous repeats also varied among genomes; while some genomes had none, the EpapGV genome had the highest number of homologous repeats with 16 regions (Ferrelli et al. 2012 ). The genome of NeseNPV-TR has low G + C content, with an average of 33.28% (16.77% of G, 16.51% of C, 34.04% of A and 32.68% of T), which was similar to that of NeseNPV (33.76% G + C) and comparable to those of other reported baculoviruses (33.38% G + C in Nele-NPV and Neab-NPV), as well as fall in the range of those of reported baculoviruses [31.1% in OxocNPV (Wang et al. 2018 ) and 57.5% in LdMNPV (Krejmer-Rabalska et al. 2016 )] (Table S1 ). Gene content and features of NeseNPV-TR genome A total of 82 ORFs potentially encoding polypeptides consisting of 50 or more amino acids have been identified in the genome of NeseNPV-TR (Tables S2 and S7). The first nucleotide position of the genome was defined as the first adenine of the polyhedrin ( polh ) gene, known to be the major structural component for occlusion bodies (Ji et al. 2015 ). Thirty-nine of the identified ORFs in the genome of NeseNPV-TR encode functionally known proteins, whereas the remaining encodes hypothetical proteins whose functions are unknown (Tables S2 and S7). The ORFs were distributed on the both strands of the DNA across the genome. Fifty-two ORFs (63.41%) were in the clockwise direction and the remaining (36.59%) were in an anti-clockwise direction with respect to the direction of the polh gene (Fig. 1 ). A total of 76 ORFs, including those encoding hypothetical proteins, were shared between the newly sequenced genome of NeseNPV-TR and the reference genome of NeseNPV (Table S2 ). Out of 38 baculovirus core genes (Rohrmann 2019 ; Wang and Hu 2019 ), 33 were identified in the genome of NeseNPV-TR which are involved in the replication, transcription, structure, or infectivity/ODV structure. The five unidentified baculovirus core genes in the NeseNPV-TR genome were pif-7 (ac110), ac53 , ac78 , p18 ( ac93 ) and odv-e25 ( ac94 ), which are known to be related to infectivity or structure. In addition to the identified baculovirus core genes, three of the Lepidopteran baculovirus core genes known to be present in half of Gammabaculoviruses (Arif et al. 2011 ), ac8 (encoding polyhedrin), ac25 (encoding dbp) and ac37 (encoding late expression factor-11), have also been found in the NeseNPV-TR genome (Table S2 ). Five of the baculovirus core genes [ dnapol ( ac65 ), lef-1 ( ac14 ), lef-2 ( ac6 ), helicase ( ac95 ) and alk-exo ( ac133 )] and one lepidopteran baculovirus core gene [ dbp ( ac25 )] identified in the genome were associated with DNA replication processes (Table S3 ) (Rohrmann 2019 ). Of these core genes, lef-1 exhibits primase activity, while lef-2 gene serves as an accessory factor of primase by interacting with lef-1 and DNA (Mikhailov and Rohrmann 2002 ). The helicase gene encodes a protein that has both helicase and ATPase activity (McDougal and Guarino 2001 ), and dnapol encodes a DNA polymerase B family member polymerase with 3' to 5' exonuclease activity (Hang and Guarino 1999 ). The alk-exo core gene encodes alkaline exonuclease enzyme with 5′ to 3′ exonuclease activity that is involved in the maturation of the virus genomes and DNA recombination (Mikhailov et al. 2003 ). The lepidopteran baculovirus core gene dbp takes part in DNA replication and expression of the polh gene by binding to DNA (Quadt et al. 2007 ). The lef-3 and ie-1 genes, which are involved in genome replication in baculoviruses (Wang and Hu 2019 ), were absent in the NeseNPV-TR genome, similar to other gammabaculoviruses. Six baculovirus core genes [ lef-4 ( ac90 ), lef-5 ( ac99 ), lef-8 ( ac50 ), lef-9 ( ac62 ), p47 ( ac40 ) and vlf-1 ( ac77 )] and one lepidopteran core gene [ lef11 ( ac37 )] known to have an essential role in transcription (Lu and Miller 1995 ; Jin et al. 1998 ) were characterized in the NeseNPV-TR genome (Table S3 ). From these genes, lef-4 encodes the essential subunit of RNA polymerase and also the mRNA capping enzyme guanylyltransferase (Li and Guarino 2008 ; Wang and Hu 2019 ). lef-5 gene plays a role in the initiation of transcription by stimulating the activity of RNA polymerase complex and is crucial for productive infection (Su et al. 2011 ). The genes of lef-8 and lef-9 encoding the subunits of the RNA polymerase are critical for the late gene expression (Wang and Hu 2019 ). Although the exact role of p47 gene in the RNA polymerase complex is not known yet, it was reported that this gene is essential for late gene expression of late and very late gene promoters (Passarelli 2007 ). vlf-1 gene stimulates the high level expression of very late genes by interacting with the promoters of these genes (Mistretta and Guarino 2005 ) and also takes part in nucleocapsid assembly and packaging of DNA (Vanarsdall et al. 2006 ). The lepidopteran baculovirus core gene lef-11 promotes viral DNA replication (Dong et al. 2015 ) and supports expression of late genes (Todd et al. 1995 ). In addition to these core genes involved in transcription process, one non-conserved gene among baculovirus genomes named mtase1 (homologous to ac69 of Autographa californica NPV) was also characterized in the newly sequenced NeseNPV-TR genome (Table S2 and S3). mtase1 gene encodes an RNA Cap (Nucleoside-2-O)-Methyltransferase and stimulates late gene expression (Wu and Guarino 2003 ). This gene was also present in the previously reported NeseNPV reference genome, however it was absent in the other reported Neodiprion nucleopolyhedrovirus genomes (Lauzon 2006 ). Although the immediate-early genes ( ie-0 , ie-1 , ie-2 , and p38 ) trans-regulating early viral gene expression were not found in the NeseNPV-TR genome; lef-5 , lef-11 , and vlf-1 genes displaying homology to these genes have been identified as transcription genes (Olson et al. 2003 ). In addition to core genes associated with the replication or transcription processes, 22 core genes related to per os infectivity or structure have been characterized in the NeseNPV-TR genome (Table S3 ). Eight of these baculovirus core genes [ p74 ( pif0 ), pif1-6 and pif-8 ] encode the per os infectivity factors (PIFs) which are envelope proteins and form a stable protein complex. In similar to the other reported gammabaculoviruses, pif7 gene was absent in the NeseNPV-TR genome (Song et al. 2016 ). The complex constructed by PIFs takes part in the ODV binding to midgut cells and is resistant to proteolytic degradation in the insect midgut (Wang et al. 2019 ). The pif-8 gene, named vp91 ( ac83 ), is also involved in the nucleocapsid assembly process. In addition to this gene, it has also been reported that ac53 , vp1054 ( ac54 ), vp39 ( ac89 ), 38k ( ac98 ), p40 ( bv/odv-c42 or ac101 ), and odv-ec27 ( ac144 ) core genes play role in the process of nucleocapsid assembly (Wang and Hu 2019 ). The p40 gene takes part in actin polymerization, while the odv-ec27 in cell cycle arrest. It has been emphasised that the core genes gp41 (ac80) encoding a tegument protein and p33 (ac92) encoding sulfhydryl oxidase enzyme, as well as ac78 are involved in BV production and ODV morphogenesis (Wang and Hu 2019 ). The baculovirus core genes ac81 , p18 ( ac93 ), p48/p45 ( ac103 ), odv-ec43 ( ac109 ) and 49k ( ac142 ) found in the newly sequenced genome were related to BV production and ODV envelopment process, while the odv-e18 ( ac143 ) was only related to BV production (Wang and Hu 2019 ). The p6.9 ( ac100 ) gene encodes a protamine-like protein that functions in the condensation and packaging of viral DNA, an essential process for producing infectious viruses (Wang et al. 2010 ). Additionally, three additional genes related to per os infectivity or structure were also annotated in this newly sequenced genome (Table S2 and S3). Among these genes, polh encodes polyhedrin protein which is conserved in NPVs and is homologous to the granulin protein in GVs and constitutes the crystalline matrix of the occlusion body (Rohrmann 2019 ). A member of the iap gene family, characterized by having two conserved structural features, a zinc-binding domain known as RING finger and baculovirus IAP repeat (BIR) on the amino-terminus, was annotated in the NeseNPV-TR genome. Members of this gene family are known to be able to prevent apoptosis in host insect cells. The iap protein identified in the newly sequenced NeseNPV-TR genome exhibited 88.95% identity with its counterpart in the NeseNPV genome. The third one of these genes encode a trypsin-like serine protease that has been reported for all gammabaculovirus genomes (Ishimwe et al. 2015 ). Although the function of this protein has not been defined yet, it has been suggested that this enzyme may function in the release of OB from midgut-infected cells and their availability for the next replication cycle (Rohrmann 2019 ) or viral protein cleavage for per os infectivity (Slack et al. 2008 ). As well as the identified genes in the NeseNPV-TR genome, hypothetical/predicted proteins had striking structural and functional patterns (Table S7). The first of these was the orf3 protein contained an F-box domain similar to the lef-7 protein, enhancing the viral replication in baculoviruses, which is missing in the NeseNPV-TR genome. Furthermore, it was determined that the orf3 protein has a protein binding function (GO:0005515; Table S7). The orf9 protein is found to have a RING-type zinc-finger domain. It has been previously reported that several proteins such as ie2, iap1 and iap2 in baculoviruses contain RING domains (Imai et al. 2003 ) and the RING domains have been suggested to play a role as E3 ligases by binding to E2 ubiquitin-conjugating enzymes (Lorick et al. 1999 ; Vaux and Silke 2005 ). The InterProScan search results showed that the orf13 protein comprises pentapeptide repeat motif (Table S7). Pentapeptide repeat proteins (PRPs) were firstly identified in cyanobacteria, but are also found in almost all species. Albeit the functions of these repeats are unknown, it has been reported that members of this family have the ability to interact with DNA-binding proteins (Mérens et al. 2009 ). The searches revealed that the protein encoded by orf25 belongs to the DUF816 family of baculovirus proteins with unknown function. Surprisingly, two dsRNA-binding domains were detected in the orf44 protein of the NeseNPV-TR (Table S7). The presence of these domains was remarking because the innate immune response to viral infections in insects is mainly mediated by dsRNAs (Jayachandran et al. 2012 ) and as a result of co-evolution, insect viruses have also adapted a variety of approaches to overcome the barriers in their way of infection (Mehrabadi et al. 2015 ; Zhao et al. 2021 ). The dsRNA-binding domain-containing protein identified in the NeseNPV-TR genome might function as a viral suppressor of RNAi (VSR) by inhibiting the Dicer-2-mediated cleavage of dsRNA and also binding to siRNAs and blocking their loading into the RISC (Zhao et al. 2021 ). Additionally, orf45 protein of the NeseNPV-TR possesses a classic C2H2-type zinc-finger domain (Table S7). The gene ontology annotations indicated that the orf45 protein takes part in the regulation of the DNA-templated transcription biological process (GO:0006355; Table S7), and has RNA polymerase II-specific DNA-binding transcription factor activity (GO:0000981) and RNA-polymerase II cis-regulatory region sequence-specific DNA binding (GO: 0000978) molecular functions. orf60 encodes a small protein (~ 12 kDa) in the NeseNPV-TR genome that displayed 93.58% amino acid similarity to NeseORF67 and was a close relative of the Ac145/Ac150 proteins. This protein includes a conserved cysteine-rich region with six conserved cysteine residues featuring a C 1 X 13 C 2 X 5 C 3 X 7 C 4 X 12 C 5 X 7 C 6 motif, where C is a cysteine and X is any amino acid other than cysteine (Zhang et al. 2005 ). The orf60 protein is also predicted to localise to a membrane (GO: 0016020) and its conserved domain is found to have chitin-binding function (GO: 0008061), indicating that the protein, like its homologues, may localise to ODV envelopes and initiate the infection of midgut cells (Rohrmann 2019 ). Similar to the other reported gammabaculovirus genomes (Garcia-Maruniak et al. 2004 ; Lauzon et al. 2004 ; Lauzon 2006 ; Arif et al. 2011 ), three sequentially located hypothetical proteins (orf65, orf66 and orf67) were identified as homologous to regulators of chromosome condensation proteins (RCC1) of diverse insect species from a broad range of taxonomy (e.g. Coleoptera, Diptera, Hemiptera, Lepidoptera, Ephemeroptera, Hymenoptera) as the result of blast homology searches. Searches of these proteins against the NCBI CDD and InterPro databases revealed that they have conserved domains of the alpha-tubulin suppressor and RCC1 domain-containing protein conserved domain superfamily, and of the regulator of chromosome condensation 1/beta-lactamase-inhibitor protein II family, respectively (Table S7). RCC1 proteins are DNA-binding proteins involved in the regulation of chromosome condensation during the transcription or mRNA maturation process and act as a guanine nucleotide-dissociation stimulator (Lauzon 2006 ). Consistently, orf65 and orf66 proteins were assigned to have the molecular function of guanyl-nucleotide exchange factor activity (GO: 0005085). The existence of RCC1-like proteins in gammabaculovirus and the original proteins of RCC1 in various insect species may imply a conceivable horizontal transfer of this gene between viruses and their host (Lauzon 2006 ). The last striking feature of NeseNPV-TR hypothetical proteins was observed in the orf82 protein (Table S7). This protein was homologous to members of the RNA 2′-phosphotransferase KptA/Tpt1 family and its homologous were also reported only in all sequenced gammabaculovirus genomes (Lauzon 2006 ; Arif et al. 2011 ), but not in any other virus genomes. The members of this protein family are belong to ADP-ribosyltransferases (ARTs) that play a role in tRNA splicing and NAD metabolism, by transferring 2′-phosphate emerging from tRNA splicing and RNA ligation processes to NAD in the archaeo-eukaryotic lineages (de Souza and Aravind 2012 ). However, the functions of members of this family or homologues in gammabaculoviruses and bacteria lacking the tRNA-splice mechanism are not known yet. Although members of this protein family have not been reported in viruses, members of its superfamily, ARTs, have been reported in several virus species (Iyer et al. 2022 ). ARTs catalyse the ADP-ribosylation by transferring ADP-ribose to substrates from NAD + and this ribosylation is involved in a broad range of functions such as DNA damage, viral infection, intra- and extracellular signalling, protein biosynthesis, transcriptional regulation and cell death (Lüscher et al. 2022 ). Recent studies have shown that NAD+–ADPr (ADP-ribose) interactions are very crucial for antiviral immunity and counter-response of viruses against host immunity, and ADPr modifications are very common mechanism in DNA viruses to counter host systems (Iyer et al. 2022 ). The phosphotransferases characterized in the gammabaculoviruses might also be a counter-adaptation of the virus to evade host defence. Homologous repeats Another commonly observed feature in most baculovirus genomes is that they contain homologous repetitive regions, called hr s, with some shared characteristics (Rohrmann 2019 ). The hr s were found to be distributed across the genome and to have several tandem repeats of palindromes within direct repeats (Arif et al. 2011 ). They have been suggested both as transcriptional enhancers and origins of viral replication due to their symmetrical locations, high AT content and containing palindromic sequences (Cochran and Faulkner 1983 ; Rohrmann 2019 ). Five hr s ranging between 249 and 819 bp were defined in the NeseNPV-TR genome (Table 1 and Figure S1 ). The total length of these hr s was 2387 bp, corresponding to 2.91% of the genome. The average G + C content of the hr s was 51.32% in average, which was higher than that of the total genome (33.28%). They displayed high similarity with each other by having 20.6% overall mean distance (between 11.27% and 31.86% pairwise distance). The most diverse one was hr 5, with a range of from 22.55–31.86% p-distance from the others and was the furthest from the others in terms of genomic location (Table S8). Similar to those found in the NeseNPV-TR genome, hr s were also present in the reference NeseNPV genome. Six hr s were identified in the reference genome, with lengths varying between 396 to 794 bp, with an G + C content of 49.8% on average and an 18.1% overall mean p-distance (Garcia-Maruniak et al. 2004 ). The hr s of the NeseNPV-TR genome exhibited high nucleotide similarity with those of the reference genome, with 16.52% mean p-distance (0.49–31.86% pairwise distances) (Table S8). Similar to the pattern reported in the reference genome (Garcia-Maruniak et al. 2004 ), the baculovirus late/very late promoter motif (A/G/T)TAAG, which is usually not located inside the hr s in baculoviruses, was found in multiple copies within all of the NeseNPV-TR hr s (Figure S1 . Moreover, the GATA motif which has been reported as the host transcription factor binding site in baculoviruses (Krappa et al. 1992 ; Kogan and Blissard 1994 ), has also been detected in hr 1, hr 2 and hr 3 of NeseNPV-TR genome at overlapping positions with the (A/G/T)TAAG motifs (Figure S1 ). Although the exact role of hr s is not yet known, the promoter and transcription factor binding motifs found in the NeseNPV-TR genome may support their proposed role as transcriptional enhancers. Table 1 Structure of NeseNPV-TR homologous repeat regions ( hrs ) Name Length of repeat units/no. of times repeated Position at genome Repetitive region (total bp) G + C (%) hr1 110 bp/4.2 ; 219 bp/2.1 8631–9100 470 48.30 hr2 65 bp/7.4 11003–11483 481 52.61 hr3 110 bp/2.3 16578–16826 249 53.01 hr4 45 bp/18.8 17196–18014 819 52.75 hr5 44 bp/8.4 44668–45035 368 48.91 Gene parity The genome organizations of the newly sequenced NeseNPV-TR and reference genome were compared using gene parity plot analysis of homologous genes and ORFs. The gene arrangement of the NeseNPV-TR genome exhibited high collinearity with the reference genome with a few exceptions (Fig. 2 and Table S2 ). NeseORF10, NeseORF14, NeseORF15, NeseORF18, NeseORF27 and NeseORF76, which are known to encode hypothetical proteins in the reference genome, were absent in the newly sequenced genome. In addition, mtase1 encoded from the NeseNPV-TR genome corresponded to the concatenation of mtase1 and NeseORF6 encoded from the reference genome. Similarly, hypothetical protein encoded from the orf10 of NeseNPV-TR genome also corresponded to the concatenation of NeseORF12 and NeseORF13 of the reference genome. Instead of NeseORF4, NeseORF19, NeseORF21, NeseORF30 and NeseORF74 in the reference genome, there were various ORFs encoding different hypothetical proteins in the newly sequenced genome (Table S2 ). When the NeseNPV-TR genome is compared to NeleNPV and NeabNPV, it has been seen that although there are differences in several regions in their genome organization, the gene and ORF contents were widely shared between the genomes. In total, 67 and 64 ORFs of the NeseNPV-TR genome had homologs in NeleNPV and NeabNPV genomes, respectively (Table S2 ). The regions between the polyhedrin ( polh ) and DNA binding protein ( dbp ) genes were lack of conserved synteny between the three genomes. In addition, the region between orf23 and orf29 ( p6.9 gene) in the NeseNPV-TR genome was inverted in the NeleNPV (orf34 to orf28) and NeabNPV (orf37 to orf32) genomes (Fig. 2 and Table S2 ). In all three species, the organization of the second half of the genome was highly conserved compared to the first half (Fig. 2 and Table S2 ). Baculovirus phylogeny and species demarcation Early studies aiming to reveal baculovirus phylogeny have used nucleotide and amino acid sequences of highly conserved polyhedrin/granulin genes that form the occlusion body matrix in NPVs and GVs due to sequence availability (Herniou and Jehle 2007 ). However, in parallel with the developments in sequencing technologies and the decrease in costs, the number of baculovirus species for which genome sequence information is gathered increases and consequently, the use of multiple genes or the whole genome in phylogenetic analyses has become widespread. Here, the baculovirus phylogeny was tried to be revealed with two different approaches by using the concatenation of the nucleotide sequences of 33 core genes and polyhedrin/granulin genes (71227 nt in alignment) from 101 baculovirus genomes, including the NeseNPV-TR genome obtained in this study. Both BI and ML trees recovered almost the same tree topology (Fig. 3 and Figure S2 ), and BI tree was presented here (Fig. 3 ). The presence of four main clades was observed in the reconstructed trees, with the high support values. The tree topology was consistent with that obtained from previous studies (Jehle et al. 2006a ; Herniou and Jehle 2007 ), where the main clades consisted of alphabaculoviruses infecting lepidopteran insects, betabaculoviruses infecting lepidopteran insects, gammabaculoviruses infecting hymenopteran insects, and deltabaculoviruses infecting dipteran insects (Fig. 3 ). In addition, Group II alphabaculoviruses were found to be paraphyletic to Group I alphabaculoviruses in the tree, similar to the recovered trees by previous studies (Craveiro et al. 2013 ; Wang et al. 2018 ; Harrison and Rowley 2022 ; Ben Tiba et al. 2022 ). Finally, this phylogenetic tree confirmed the location of the newly sequenced NeseNPV-TR within gammabaculoviruses, as sister group to the reference NeseNPV (Fig. 3 ). Previous studies have demonstrated that the use of nucleotide pairwise distances with K2P can be used as a criterion for baculovirus species (Jehle et al. 2006b ; Wennmann et al. 2018 ). When the K2P distance between two isolates is less than 0.021, they can be considered as the same species, while the distance between them is greater than 0.072, they can be considered as different species. If the values are between these two, additional information such as host range and genome organization is required for species discrimination. The K2P distances between NeseNPV-TR and the reference NeseNPV were calculated for the nucleotide sequences of lef-8 , lef-9 and polh genes individually, and also concatenated sequences of these three genes, as well as the combined 33 core genes identified in the NeseNPV-TR genome (Table S5). The pairwise K2P distances of lef-9 and polh genes were lower than the specified threshold value (0.0187 for lef-9 and 0.0165 for polh ), while those of lef-8 , combinations of three genes and 33 genes were slightly higher (0.0257 for lef-8 , 0.0221 for three genes combined and 0.0258 for the 33 genes combined) (Table S5); however, they were still too low than the threshold value to be considered these two samples as different species. The slightly high difference for the 33 genes combined here may be due to annotation differences, sequencing errors, differences in the sampling years between the genome obtained here and the reference genome, or missing genes. Genetic differentiation among baculoviruses Here, the overall mean p-distances for each baculovirus core gene (except for deltabaculovirus) were calculated based on both nucleotide and amino acid sequences (Fig. 4 and Table S6). When evaluated in terms of amino acid distances, the most variable genes were lef-2 , helicase , p40 , desmoplakin and vp91 , respectively, while the most conserved ones were p6.9 , lef-8 , lef-9 , odv-e18 and pif2 (Table S6). Interestingly, contrary to observed in the amino acid sequence, p6.9 was the most variable gene when nucleotide distances were considered followed by lef-2 , desmoplakin , vp91 , vp39 and p40 . The most conserved genes in terms of nucleotide distance were lef-8 , lef-9 , lef-5 , odv-e18 and pif2 , respectively (Fig. 4 and Table S6). In a study performed using 57 baculovirus genomes and 31 core genes, the most variable genes were found to be desmoplakin , helicase , lef-2 , p6.9 and vp91 , and the most conserved genes were pif2 , lef-9 , odv-e18 , lef-8 and ac81 , respectively (Miele et al. 2011 ). The reported results were largely consistent with those obtained here (Table S6). The variability observed in baculovirus core genes appears to be due to their approximately 300 million years co-evolutionary history with their hosts (Ikeda et al. 2015 ). The different levels of variability in core genes can also be explained by different evolutionary constraints acting on them. Here, the genes having the most variability seem to be related to nucleocapsid assembly ( p40 , vp91 ) and egress ( desmoplakin ), and replication ( lef-2 and helicase) , while the most conserved ones were related to transcription ( lef-8 , lef-9 and lef-5 ), per os infectivity ( pif-2 ) and BV production ( odv-e18 ). It has been reported in previous studies that helicase , one of the most variable genes, is an important host range factor (Thiem and Cheng 2009 ). High variability in other genes may also be shaped by host-dependent evolution and host ranges of baculoviruses. While the conserved core genes, lef-8 and lef-9 , encode the subunits of baculovirus RNA polymerase (Wang and Hu 2019 ), the lef-5 gene is directly involved in late transcription (Su et al. 2011 ). Another conserved gene, odv-e18 is crucial for the production of infectious BV and is necessary for interactions between the membrane and nucleocapsid in the nucleus (Blissard and Theilmann 2018 ). The last most conserved gene pif2 is involved in the binding of ODV onto midgut cells and is reported to have an essential role in the host specificity of baculoviruses (Song et al. 2016 ). The conservation level of these genes might be explained by their vital functions in transcription or infectivity given above. The overall mean p-distances for core genes based on both nucleotide and amino acid sequences were also evaluated within each genus (Fig. 5 ). When considering in terms of both nucleotide and amino acid distances, Group I alphabaculovirus (for nucleotide minimum: 0.1740 and maximum: 0.3414, for amino acid minimum: 0.1643 and maximum: 0.3837) and gammabaculovirus (for nucleotide minimum: 0.1915 and maximum: 0.3147, for amino acid minimum: 0.1545 and maximum: 0.4360) displayed lower genetic diversity, while Group II alphabaculovirus (for nucleotide minimum: 0.3059 and maximum: 0.4658, for amino acid minimum: 0.2724 and maximum: 0.5607) and betabaculovirus (for nucleotide minimum: 0.3310 and maximum: 0.4640, for amino acid minimum: 0.3013 and maximum: 0.5843) had higher (Fig. 5 ). In a previous comparative study of baculovirus genomes, it has been reported that Group I alphabaculoviruses and gammabaculoviruses have also lower diversity of gene content (Miele et al. 2011 ). The low diversity observed in gammabaculoviruses, both in terms of gene content and sequence, is most likely due to the limited number of sequenced genomes. However, considering the significant number of genome sequences in Group I alphabaculoviruses together with their position in the phylogenetic tree, the lower genetic diversity of this genus might be explained by their recent emergence and having less time to gain new sequences or accumulate mutations (Miele et al. 2011 ). Conclusion The sequencing and characterization of the complete genome of Neodiprion sertifer nucleopolyhedrovirus from Turkey and comparison with the other reported baculovirus genomes allowed us to denote several conclusions: (i) although it is smaller and contains the fewest ORFs among baculoviruses, the genome characteristics of the NeseNPV-TR are mostly consistent with the previously reported NeseNPV genome, (ii) the genome harbours 82 ORFs and the functions of 39 of them are known, while some of the remaining contain conserved domains and further studies are needed to reveal their structural and functional roles, (iii) phylogenetic analyses using 33 baculovirus core genes confirmed the position of NeseNPV-TR within the genus gammabaculovirus, (iv) Up to the pairwise K2P distances, the sample of which the genome sequence obtained can be considered as the same species with NeseNPV (v) the observation of different rates of divergence in the core genes can be explained by different selection pressures acting on the genes, and (vi) albeit lower genetic diversity of gammabaculoviruses appears to be due to limited number of sequenced genomes, that of Group I alphabaculoviruses is most probably due to recent emergence. More genome sequences (especially the representative of gammabaculoviruses) and further studies are needed to understand baculovirus genome patterns, evolutionary history and genetic diversity. Declarations Acknowledgements We thank our colleagues from the Department of Biology of the Ege University who provided the specimens. We are also grateful to Dr. Hasan H. BAŞIBÜYÜK (Department of Gerontology, Akdeniz University) and Dr. Merve Nur AYDEMİR for their contributions in data generation. Author contribution OD contributed to the generation and interpretation of data, and edited the manuscript. MB performed the analyses and contributed to interpretation of data. MŞS contributed to the generation of data and edition of the manuscript. EMK involved in the conceptualization, supervision, final interpretation of data, and editing of the manuscript. All authors read and approved the final manuscript. Funding This study was funded by TÜBİTAK (The Scientific and Technological Research Council of Turkey, grant number 112T418). Data availability The genome sequence generated in this study was deposited in the GenBank database under the accession number represented in the section of Methods. Competing interests The authors declare no competing interests. Ethical approval Research procedures in this study did not violate any relevant laws and regulations. No relevant ethics committee(s) or institution(s) were applicable for the materials used in this study. Conflict of interest The authors declare no conflict of interest References Afonso CL, Tulman ER, Lu Z, Balinsky CA, Moser BA, Becnel JJ, Rock DL, Kutish GF (2001) Genome sequence of a baculovirus pathogenic for Culex nigripalpus . J Virol 75:11157–11165. https://doi.org/10.1128/JVI.75.22.11157-11165.2001 Arif B, Escasa S, Pavlik L (2011) Biology and genomics of viruses within the genus Gammabaculovirus. Viruses 3:2214–2222. https://doi.org/10.3390/v3112214 Arkin AP, Cottingham RW, Henry CS, Harris NL, Stevens RL, Maslov S, Dehal P, Ware D, Perez F, Canon S, Sneddon MW, Henderson ML, Riehl WJ, Murphy-Olson D, Chan SY, Kamimura RT, Kumari S, Drake MM, Brettin TS, Glass EM, Chivian D, Gunter D, Weston DJ, Allen BH, Baumohl J, Best AA, Bowen B, Brenner SE, Bun CC, Chandonia J-M, Chia J-M, Colasanti R, Conrad N, Davis JJ, Davison BH, DeJongh M, Devoid S, Dietrich E, Dubchak I, Edirisinghe JN, Fang G, Faria JP, Frybarger PM, Gerlach W, Gerstein M, Greiner A, Gurtowski J, Haun HL, He F, Jain R, Joachimiak MP, Keegan KP, Kondo S, Kumar V, Land ML, Meyer F, Mills M, Novichkov PS, Oh T, Olsen GJ, Olson R, Parrello B, Pasternak S, Pearson E, Poon SS, Price GA, Ramakrishnan S, Ranjan P, Ronald PC, Schatz MC, Seaver SMD, Shukla M, Sutormin RA, Syed MH, Thomason J, Tintle NL, Wang D, Xia F, Yoo H, Yoo S, Yu D (2018) KBase: The United States department of energy systems biology knowledgebase. Nat Biotechnol 36:566–569. https://doi.org/10.1038/nbt.4163 Assenberg R, Wan PT, Geisse S, Mayr LM (2013) Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 23:393–402. https://doi.org/10.1016/j.sbi.2013.03.008 Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin A V., Sirotkin A V., Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021 Bateman A, Martin M-J, Orchard S, Magrane M, Ahmad S, Alpi E, Bowler-Barnett EH, Britto R, Bye-A-Jee H, Cukura A, Denny P, Dogan T, Ebenezer T, Fan J, Garmiri P, da Costa Gonzales LJ, Hatton-Ellis E, Hussein A, Ignatchenko A, Insana G, Ishtiaq R, Joshi V, Jyothi D, Kandasaamy S, Lock A, Luciani A, Lugaric M, Luo J, Lussi Y, MacDougall A, Madeira F, Mahmoudy M, Mishra A, Moulang K, Nightingale A, Pundir S, Qi G, Raj S, Raposo P, Rice DL, Saidi R, Santos R, Speretta E, Stephenson J, Totoo P, Turner E, Tyagi N, Vasudev P, Warner K, Watkins X, Zaru R, Zellner H, Bridge AJ, Aimo L, Argoud-Puy G, Auchincloss AH, Axelsen KB, Bansal P, Baratin D, Batista Neto TM, Blatter M-C, Bolleman JT, Boutet E, Breuza L, Gil BC, Casals-Casas C, Echioukh KC, Coudert E, Cuche B, de Castro E, Estreicher A, Famiglietti ML, Feuermann M, Gasteiger E, Gaudet P, Gehant S, Gerritsen V, Gos A, Gruaz N, Hulo C, Hyka-Nouspikel N, Jungo F, Kerhornou A, Le Mercier P, Lieberherr D, Masson P, Morgat A, Muthukrishnan V, Paesano S, Pedruzzi I, Pilbout S, Pourcel L, Poux S, Pozzato M, Pruess M, Redaschi N, Rivoire C, Sigrist CJA, Sonesson K, Sundaram S, Wu CH, Arighi CN, Arminski L, Chen C, Chen Y, Huang H, Laiho K, McGarvey P, Natale DA, Ross K, Vinayaka CR, Wang Q, Wang Y, Zhang J (2023) UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res 51:D523–D531. https://doi.org/10.1093/nar/gkac1052 Ben Tiba S, Laarif A, Wennmann JT, Bouslama T, Jehle JA (2022) Biological activity and genome composition of a Tunisian isolate of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV-Tun2). Egypt J Biol Pest Control 32:71. https://doi.org/10.1186/s41938-022-00568-w Benson G (1999) Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Res 27:573–580. https://doi.org/10.1093/nar/27.2.573 Blissard GW, Theilmann DA (2018) Baculovirus entry and egress from insect cells. Annu Rev Virol 5:113–139. https://doi.org/10.1146/annurev-virology-092917-043356 Bulach DM, Kumar CA, Zaia A, Liang B, Tribe DE (1999) Group II nucleopolyhedrovirus subgroups revealed by phylogenetic analysis of polyhedrin and DNA polymerase gene sequences. J Invertebr Pathol 73:59–73. https://doi.org/10.1006/jipa.1998.4797 Chen G, Fang Y, Yan Q, Li P, Wu L, Feng G (2019) The deficiency in nuclear localization signal of Neodiprion lecontei nucleopolyhedrovirus DNA polymerase prevents rescue of viral DNA replication and virus production in dnapol-null Autographa californica multiple nucleopolyhedrovirus. Virus Res 266:52–57. https://doi.org/10.1016/j.virusres.2019.04.005 Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890. https://doi.org/10.1093/bioinformatics/bty560 Cochran MA, Faulkner P (1983) Location of homologous DNA sequences interspersed at five regions in the baculovirus AcMNPV genome. J Virol 45:961–970. https://doi.org/10.1128/JVI.45.3.961-970.1983 Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676. https://doi.org/10.1093/bioinformatics/bti610 Craveiro SR, Melo FL, Ribeiro ZMA, Ribeiro BM, Báo SN, Inglis PW, Castro MEB (2013) Pseudoplusia includens single nucleopolyhedrovirus: genetic diversity, phylogeny and hypervariability of the pif-2 gene. J Invertebr Pathol 114:258–267. https://doi.org/10.1016/j.jip.2013.08.005 de Souza RF, Aravind L (2012) Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions. Mol Biosyst 8:1661–1677. https://doi.org/10.1039/c2mb05487f Delcher AL, Harmon D, Kasif S, White O, Salzberg SL (1999) Improved microbial gene identification with GLIMMER. Nucleic Acids Res 27:4636–41. https://doi.org/10.1093/nar/27.23.4636 Dong Z-Q, Hu N, Zhang J, Chen T-T, Cao M-Y, Li H-Q, Lei X-J, Chen P, Lu C, Pan M-H (2015) Oligomerization of baculovirus LEF-11 is involved in viral DNA replication. PLoS One 10:e0144930. https://doi.org/10.1371/journal.pone.0144930 Duffy SP, Young AM, Morin B, Lucarotti CJ, Koop BF, Levin DB (2006) Sequence analysis and organization of the Neodiprion abietis nucleopolyhedrovirus genome. J Virol 80:6952–6963. https://doi.org/10.1128/JVI.00187-06 Ferrelli ML, Salvador R, Biedma ME, Berretta MF, Haase S, Sciocco-Cap A, Ghiringhelli PD, Romanowski V (2012) Genome of Epinotia aporema granulovirus (EpapGV), a polyorganotropic fast killing betabaculovirus with a novel thymidylate kinase gene. BMC Genomics 13:548. https://doi.org/10.1186/1471-2164-13-548 Garcia-Maruniak A, Maruniak JE, Zanotto PMA, Doumbouya AE, Liu J-C, Merritt TM, Lanoie JS (2004) Sequence analysis of the genome of the Neodiprion sertifer nucleopolyhedrovirus. J Virol 78:7036–7051. https://doi.org/10.1128/JVI.78.13.7036-7051.2004 Gilbert C, Chateigner A, Ernenwein L, Barbe V, Bézier A, Herniou EA, Cordaux R (2014) Population genomics supports baculoviruses as vectors of horizontal transfer of insect transposons. Nat Commun 5:3348. https://doi.org/10.1038/ncomms4348 Goto C, Hayakawa T, Maeda S (1998) Genome organization of Xestia c-nigrum granulovirus. Virus Genes 16:199–210. https://doi.org/10.1023/a:1007972108026 Hang X, Guarino LA (1999) Purification of Autographa californica nucleopolyhedrovirus DNA polymerase from infected insect cells. J Gen Virol 80:2519–2526. https://doi.org/10.1099/0022-1317-80-9-2519 Harrison RL, Rowley DL (2022) The complete genome sequence of an alphabaculovirus from the brown tussock moth, Olene mendosa Hübner, expands our knowledge of lymantriine baculovirus diversity and evolution. Virus Genes 58:227–237. https://doi.org/10.1007/s11262-022-01899-4 Herniou EA, Jehle JA (2007) Baculovirus phylogeny and evolution. Curr Drug Targets 8:1043–1050. https://doi.org/10.2174/138945007782151306 Ikeda M, Hamajima R, Kobayashi M (2015) Baculoviruses: diversity, evolution and manipulation of insects. Entomol Sci 18:1–20. https://doi.org/10.1111/ens.12105 Imai N, Matsuda N, Tanaka K, Nakano A, Matsumoto S, Kang W (2003) Ubiquitin ligase activities of Bombyx mori nucleopolyhedrovirus RING finger proteins. J Virol 77:923–30. https://doi.org/10.1128/jvi.77.2.923-930.2003 Ishimwe E, Hodgson JJ, Clem RJ, Passarelli AL (2015) Reaching the melting point: degradative enzymes and protease inhibitors involved in baculovirus infection and dissemination. Virology 479–480:637–49. https://doi.org/10.1016/j.virol.2015.01.027 Iyer LM, Burroughs AM, Anantharaman V, Aravind L (2022) Apprehending the NAD+-ADPr-dependent systems in the virus world. Viruses 14:1977. https://doi.org/10.3390/v14091977 Jayachandran B, Hussain M, Asgari S (2012) RNA interference as a cellular defense mechanism against the DNA virus baculovirus. J Virol 86:13729–13734. https://doi.org/10.1128/JVI.02041-12 Jehle JA, Blissard GW, Bonning BC, Cory JS, Herniou EA, Rohrmann GF, Theilmann DA, Thiem SM, Vlak JM (2006a) On the classification and nomenclature of baculoviruses: a proposal for revision. Arch Virol 151:1257–66. https://doi.org/10.1007/s00705-006-0763-6 Jehle JA, Lange M, Wang H, Hu Z, Wang Y, Hauschild R (2006b) Molecular identification and phylogenetic analysis of baculoviruses from Lepidoptera. Virology 346:180–193. https://doi.org/10.1016/j.virol.2005.10.032 Ji X, Axford D, Owen R, Evans G, Ginn HM, Sutton G, Stuart DI (2015) Polyhedra structures and the evolution of the insect viruses. J Struct Biol 192:88–99. https://doi.org/10.1016/j.jsb.2015.08.009 Jin J, Dong W, Guarino LA (1998) The LEF-4 subunit of baculovirus RNA polymerase has RNA 5′-triphosphatase and ATPase activities. J Virol 72:10011–10019. https://doi.org/10.1128/JVI.72.12.10011-10019.1998 Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–80. https://doi.org/10.1093/molbev/mst010 Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–9. https://doi.org/10.1093/bioinformatics/bts199 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120. https://doi.org/10.1007/BF01731581 Kogan PH, Blissard GW (1994) A baculovirus gp64 early promoter is activated by host transcription factor binding to CACGTG and GATA elements. J Virol 68:813–822. https://doi.org/10.1128/JVI.68.2.813-822.1994 Krappa R, Behn-Krappa A, Jahnel F, Doerfler W, Knebel-Mörsdorf D (1992) Differential factor binding at the promoter of early baculovirus gene PE38 during viral infection: GATA motif is recognized by an insect protein. J Virol 66:3494–3503. https://doi.org/10.1128/jvi.66.6.3494-3503.1992 Krejmer-Rabalska M, Rabalski L, Skrzecz I, Szewczyk B (2016) Complete genome sequence of Lymantria dispar multiple nucleopolyhedrovirus isolated in Southwestern Poland. Microbiol Resour Announc 4. https://doi.org/10.1128/genomeA.01422-16 Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. https://doi.org/10.1038/nmeth.1923 Lauzon HAM (2006) Genomic comparison of Neodiprion sertifer and Neodiprion lecontei nucleopolyhedroviruses and identification of potential hymenopteran baculovirus-specific open reading frames. J Gen Virol 87:1477–1489. https://doi.org/10.1099/vir.0.81727-0 Lauzon HAM, Lucarotti CJ, Krell PJ, Feng Q, Retnakaran A, Arif BM (2004) Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome. J Virol 78:7023–35. https://doi.org/10.1128/JVI.78.13.7023-7035.2004 Li Y, Guarino LA (2008) Roles of LEF-4 and PTP/BVP RNA triphosphatases in processing of baculovirus late mRNAs. J Virol 82:5573–5583. https://doi.org/10.1128/JVI.00058-08 Li Y, Liu X, Tang P, Zhang H, Qin Q, Zhang Z (2021) Genome sequence and organization of the Mythimna (formerly Pseudaletia ) unipuncta granulovirus Hawaiian strain. Sci Rep 11:414. https://doi.org/10.1038/s41598-020-80117-3 Lorick KL, Jensen JP, Fang S, Ong AM, Hatakeyama S, Weissman AM (1999) RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc Natl Acad Sci U S A 96:11364–9. https://doi.org/10.1073/pnas.96.20.11364 Lu A, Miller LK (1995) The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. J Virol 69:975–982. https://doi.org/10.1128/jvi.69.2.975-982.1995 Lüscher B, Ahel I, Altmeyer M, Ashworth A, Bai P, Chang P, Cohen M, Corda D, Dantzer F, Daugherty MD, Dawson TM, Dawson VL, Deindl S, Fehr AR, Feijs KLH, Filippov D V., Gagné J-P, Grimaldi G, Guettler S, Hoch NC, Hottiger MO, Korn P, Kraus WL, Ladurner A, Lehtiö L, Leung AKL, Lord CJ, Mangerich A, Matic I, Matthews J, Moldovan G-L, Moss J, Natoli G, Nielsen ML, Niepel M, Nolte F, Pascal J, Paschal BM, Pawłowski K, Poirier GG, Smith S, Timinszky G, Wang Z-Q, Yélamos J, Yu X, Zaja R, Ziegler M (2022) ADP-ribosyltransferases, an update on function and nomenclature. FEBS J 289:7399–7410. https://doi.org/10.1111/febs.16142 Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM (2021) BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol 38:4647–4654. https://doi.org/10.1093/molbev/msab199 McDougal V V., Guarino LA (2001) DNA and ATP binding activities of the baculovirus DNA helicase P143. J Virol 75:7206–7209. https://doi.org/10.1128/JVI.75.15.7206-7209.2001 Mehrabadi M, Hussain M, Matindoost L, Asgari S (2015) The baculovirus antiapoptotic p35 protein functions as an inhibitor of the host RNA interference antiviral response. J Virol 89:8182–92. https://doi.org/10.1128/JVI.00802-15 Mérens A, Matrat S, Aubry A, Lascols C, Jarlier V, Soussy C-J, Cavallo J-D, Cambau E (2009) The pentapeptide repeat proteins MfpAMt and QnrB4 exhibit opposite effects on DNA gyrase catalytic reactions and on the ternary gyrase-DNA-quinolone complex. J Bacteriol 191:1587–1594. https://doi.org/10.1128/JB.01205-08 Miele SAB, Garavaglia MJ, Belaich MN, Ghiringhelli PD (2011) Baculovirus: molecular insights on their diversity and conservation. Int J Evol Biol 2011:1–15. https://doi.org/10.4061/2011/379424 Mikhailov VS, Okano K, Rohrmann GF (2003) Baculovirus alkaline nuclease possesses a 5′→3′ exonuclease activity and associates with the DNA-binding protein LEF-3. J Virol 77:2436–2444. https://doi.org/10.1128/JVI.77.4.2436-2444.2003 Mikhailov VS, Rohrmann GF (2002) Baculovirus replication factor LEF-1 is a DNA primase. J Virol 76:2287–2297. https://doi.org/10.1128/jvi.76.5.2287-2297.2002 Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, Lanfear R (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 37:1530–1534. https://doi.org/10.1093/molbev/msaa015 Mistretta T-A, Guarino LA (2005) Transcriptional activity of baculovirus very late factor 1. J Virol 79:1958–1960. https://doi.org/10.1128/JVI.79.3.1958-1960.2005 Olson VA, Wetter JA, Friesen PD (2003) The highly conserved basic domain I of baculovirus IE1 is required for hr enhancer DNA binding and hr -dependent transactivation. J Virol 77:5668–5677. https://doi.org/10.1128/JVI.77.10.5668-5677.2003 Passarelli AL (2007) Baculovirus RNA polymerase: activities, composition, and evolution. Virol Sin 22:94–107. https://doi.org/10.1007/s12250-007-0011-6 Paysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, Bileschi ML, Bork P, Bridge A, Colwell L, Gough J, Haft DH, Letunić I, Marchler-Bauer A, Mi H, Natale DA, Orengo CA, Pandurangan AP, Rivoire C, Sigrist CJA, Sillitoe I, Thanki N, Thomas PD, Tosatto SCE, Wu CH, Bateman A (2023) InterPro in 2022. Nucleic Acids Res 51:D418–D427. https://doi.org/10.1093/nar/gkac993 Quadt I, van Lent JWM, Knebel-Mörsdorf D (2007) Studies of the silencing of baculovirus DNA binding protein. J Virol 81:6122–6127. https://doi.org/10.1128/JVI.02768-06 Rambaut A (2014) FigTree v1.4.2, A Graphical Viewer of Phylogenetic Trees. Available from 〈http//tree.bio.ed.ac.uk/software/figtree/〉 Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst Biol 67:901–904. https://doi.org/10.1093/sysbio/syy032 Rohrmann GF (2019) Baculovirus Molecular Biology [Internet], 4th edn. Bethesda (MD): National Center for Biotechnology Information (US) Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard M a., Huelsenbeck JP (2012) Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542. https://doi.org/10.1093/sysbio/sys029 Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153 Slack J, Arif BM (2007) The baculoviruses occlusion-derived virus: virion structure and function. Adv Virus Res 69:99–165. https://doi.org/10.1016/S0065-3527(06)69003-9 Slack JM, Lawrence SD, Krell PJ, Arif BM (2008) Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection. J Gen Virol 89:2388–2397. https://doi.org/10.1099/vir.0.2008/002543-0 Song J, Wang X, Hou D, Huang H, Liu X, Deng F, Wang H, Arif BM, Hu Z, Wang M (2016) The host specificities of baculovirus per os infectivity factors. PLoS One 11:e0159862. https://doi.org/10.1371/journal.pone.0159862 Su J, Lung O, Blissard GW (2011) The Autographa californica multiple nucleopolyhedrovirus lef-5 gene is required for productive infection. Virology 416:54–64. https://doi.org/10.1016/j.virol.2011.04.019 Szewczyk B, Hoyos-Carvajal L, Paluszek M, Skrzecz I, Lobo de Souza M (2006) Baculoviruses — re-emerging biopesticides. Biotechnol Adv 24:143–160. https://doi.org/10.1016/j.biotechadv.2005.09.001 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. https://doi.org/10.1093/molbev/mst197 Thiem SM, Cheng X-W (2009) Baculovirus host-range. Virol Sin 24:436–457. https://doi.org/10.1007/s12250-009-3058-8 Todd JW, Passarelli AL, Miller LK (1995) Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J Virol 69:968–974. https://doi.org/10.1128/jvi.69.2.968-974.1995 Vaidya G, Lohman DJ, Meier R (2011) SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27:171–180. https://doi.org/10.1111/j.1096-0031.2010.00329.x van Oers M, Vlak J (2007) Baculovirus genomics. Curr Drug Targets 8:1051–1068. https://doi.org/10.2174/138945007782151333 Vanarsdall AL, Okano K, Rohrmann GF (2006) Characterization of the role of very late expression factor 1 in baculovirus capsid structure and DNA processing. J Virol 80:1724–1733. https://doi.org/10.1128/JVI.80.4.1724-1733.2006 Vaux DL, Silke J (2005) IAPs, RINGs and ubiquitylation. Nat Rev Mol Cell Biol 6:287–97. https://doi.org/10.1038/nrm1621 Wang J, Hou D, Wang Q, Kuang W, Zhang L, Li J, Shen S, Deng F, Wang H, Hu Z, Wang M (2018) Genome analysis of a novel Group I alphabaculovirus obtained from Oxyplax ochracea. PLoS One 13:e0192279. https://doi.org/10.1371/journal.pone.0192279 Wang M, Hu Z (2019) Advances in Molecular Biology of Baculoviruses. In: Insect Molecular Virology: Advances and Emerging Trends. Caister Academic Press Wang M, Tuladhar E, Shen S, Wang H, van Oers MM, Vlak JM, Westenberg M (2010) Specificity of baculovirus P6.9 basic DNA-binding proteins and critical role of the C terminus in virion formation. J Virol 84:8821–8828. https://doi.org/10.1128/JVI.00072-10 Wang X, Shang Y, Chen C, Liu S, Chang M, Zhang N, Hu H, Zhang F, Zhang T, Wang Z, Liu X, Lin Z, Deng F, Wang H, Zou Z, Vlak JM, Wang M, Hu Z (2019) Baculovirus per os infectivity factor complex: components and assembly. J Virol 93:e02053-18. https://doi.org/10.1128/JVI.02053-18 Wennmann JT, Keilwagen J, Jehle JA (2018) Baculovirus Kimura two-parameter species demarcation criterion is confirmed by the distances of 38 core gene nucleotide sequences. J Gen Virol 99:1307–1320. https://doi.org/10.1099/jgv.0.001100 Wood DE, Lu J, Langmead B (2019) Improved metagenomic analysis with Kraken 2. Genome Biol 20:257. https://doi.org/10.1186/s13059-019-1891-0 Wu X, Guarino LA (2003) Autographa californica nucleopolyhedrovirus orf69 encodes an RNA cap (nucleoside-2′- O)-methyltransferase. J Virol 77:3430–3440. https://doi.org/10.1128/JVI.77.6.3430-3440.2003 Zanotto PM, Kessing BD, Maruniak JE (1993) Phylogenetic interrelationships among baculoviruses: evolutionary rates and host associations. J Invertebr Pathol 62:147–164. https://doi.org/10.1006/jipa.1993.1090 Zhang J-H, Ohkawa T, Washburn JO, Volkman LE (2005) Effects of Ac150 on virulence and pathogenesis of Autographa californica multiple nucleopolyhedrovirus in noctuid hosts. J Gen Virol 86:1619–1627. https://doi.org/10.1099/vir.0.80930-0 Zhao S, Kong X, Wu X (2021) RNAi-based immunity in insects against baculoviruses and the strategies of baculoviruses involved in siRNA and miRNA pathways to weaken the defense. Dev Comp Immunol 122:104116. https://doi.org/10.1016/j.dci.2021.104116 Additional Declarations No competing interests reported. Supplementary Files FigureS1.pdf Figure S1. Homologous repeats ( hr s) of NeseNPV-TR. The late/ very late promoter motif (A/G/T)TAAG and the potential GATA factor-binding site are underlined with red and boxed, respectively. FigureS2.pdf Figure S2. Phylogenetic tree of baculoviruses constructed under ML approach using the dataset of combination of the 33 core genes and polh / gran genes. Culex nigripalpus NPV (deltabaculovirus) used as outgroup. Only bootstrap values lower than 100 in ML were shown. Supplementarytables.xlsx Cite Share Download PDF Status: Published Journal Publication published 19 Jan, 2024 Read the published version in Virus Genes → Version 1 posted Editorial decision: Revision requested 10 Dec, 2023 Reviews received at journal 06 Nov, 2023 Reviewers agreed at journal 03 Nov, 2023 Reviewers agreed at journal 30 Oct, 2023 Reviewers invited by journal 29 Oct, 2023 Submission checks completed at journal 20 Oct, 2023 Editor assigned by journal 20 Oct, 2023 First submitted to journal 19 Oct, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3466248","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":241564733,"identity":"2f9c6406-cac8-4de2-b7ee-9ad85856c144","order_by":0,"name":"Özgül DOĞAN","email":"","orcid":"","institution":"Sivas Cumhuriyet University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Özgül","middleName":"","lastName":"DOĞAN","suffix":""},{"id":241564734,"identity":"8f8e7db3-15ab-4680-86db-b7a766a741d3","order_by":1,"name":"Mahir BUDAK","email":"","orcid":"","institution":"Sivas Cumhuriyet University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mahir","middleName":"","lastName":"BUDAK","suffix":""},{"id":241564735,"identity":"eb267f27-d9b5-4091-8836-318fd4d26a34","order_by":2,"name":"Melissa Şafak SALMAN","email":"","orcid":"","institution":"Sivas Cumhuriyet University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Melissa","middleName":"Şafak","lastName":"SALMAN","suffix":""},{"id":241564736,"identity":"00e1a716-7dc9-46ad-86c3-7a54ce91ae15","order_by":3,"name":"Ertan Mahir KORKMAZ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIie3RwUrDMBjA8S8E5uXrem1Rpo8QKPYyWF6lENDLLkPoxYM9ZZcOr+tjDCHnjsB6CfU6KKjDF9CbF8FKcZe1244D8ychBPKjJQGw2U4xBCBJvbr1pM3+t94RxE+2hDYEDxGWN2fhIHFRrz6z8YgHhbl+msiXC1Y85vARa+DneSvxZ1JkCyVoaMZhlck7ZEYDmZcasB+1EvaMAdko2gvXGFaOjJCtBVBH1qTjz3hDHjCY/5HXd6Dfewhz0oAslPaYt/0KBUr2EM+sBMlUwTxzE1dYRugbwZZpeYtoOm4sFZrM1D13p1pVGEe8Xyw3b1/xcHCWtpPdrnKAenS/5G6XyfFnbTab7X/0A2JmVsCbXF0oAAAAAElFTkSuQmCC","orcid":"","institution":"Sivas Cumhuriyet University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ertan","middleName":"Mahir","lastName":"KORKMAZ","suffix":""}],"badges":[],"createdAt":"2023-10-19 10:44:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3466248/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3466248/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11262-024-02050-1","type":"published","date":"2024-01-19T15:00:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":45118750,"identity":"922d23df-d484-49da-aeec-5f4c959485e3","added_by":"auto","created_at":"2023-10-23 23:43:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":962827,"visible":true,"origin":"","legend":"\u003cp\u003eComplete genome organization of NeseNPV-TR. Blue and red arrows represent the identified ORFs and homolog regions (hrs), respectively. Blue and green lines inside the circle are shown as G+C% and A+T%, respectively.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3466248/v1/d291fcb3e66f27bf29616865.png"},{"id":45116683,"identity":"efb9bba9-39f0-449f-a8e4-4a8599829ed3","added_by":"auto","created_at":"2023-10-23 23:35:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54627,"visible":true,"origin":"","legend":"\u003cp\u003eGene-parity plots of NeseNPV-TR and NeseNPV (a), NeleNPV(b), and NeabNPV(c). The presence and relative positions of genes (black tiles) are in pairwise comparisons.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3466248/v1/5f0abe53b2336944838bd731.png"},{"id":45116685,"identity":"bd387df9-7046-40fc-9461-74192a510f54","added_by":"auto","created_at":"2023-10-23 23:35:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":784531,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of baculoviruses constructed under BI approach using the dataset of combination of the 33 core genes and \u003cem\u003epolh\u003c/em\u003e/\u003cem\u003egran\u003c/em\u003e genes. \u003cem\u003eCulex nigripalpus NPV\u003c/em\u003e (deltabaculovirus) used as outgroup. Only support values lower than 1.0 in BI were shown.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3466248/v1/20d6c7b6e9205c652a8db26f.png"},{"id":45116689,"identity":"1ad2f57c-c1de-4b9a-9353-8d71455e1ab2","added_by":"auto","created_at":"2023-10-23 23:35:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1430597,"visible":true,"origin":"","legend":"\u003cp\u003eBaculovirus core gene variability. Histograms show overall mean p-distances for each gene based on amino acid and nucleotide sequences. The grey coloured histograms show the amino acid distances, while the anthracite coloured ones show the nucleotide distances.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3466248/v1/d5234676a9365ff0470a1c1a.png"},{"id":45116684,"identity":"74b805cd-eaf5-4690-a0c4-9c9e6aad4583","added_by":"auto","created_at":"2023-10-23 23:35:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":72178,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic variability of the baculovirus core genes by genus. The overall mean p-distances for core genes within each genus based on both nucleotide (a) and amino acid (b) sequences were shown with boxplots. The green boxplot refers to genetic variability within Group I alphabaculoviruses, the orange boxplot refers to genetic variability within Group II alphabaculoviruses, the purple boxplot refers to genetic variability within betabaculoviruses and the pink boxplot refers to genetic variability within gammabaculoviruses.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3466248/v1/581dbfd8e8d2cd4defcf8755.png"},{"id":49978796,"identity":"e5c25559-1348-4952-ba57-523cac10ca7f","added_by":"auto","created_at":"2024-01-22 15:09:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1258948,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3466248/v1/0d840cd3-bb07-47fc-ac17-599916b34b6a.pdf"},{"id":45116686,"identity":"a91c6370-ca20-4c1b-9d1a-2ba6adcdccb3","added_by":"auto","created_at":"2023-10-23 23:35:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1550003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1. \u003c/strong\u003eHomologous repeats (\u003cem\u003ehr\u003c/em\u003es) of NeseNPV-TR. The late/ very late promoter motif (A/G/T)TAAG and the potential GATA factor-binding site are underlined with red and boxed, respectively.\u003c/p\u003e","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3466248/v1/5a98ef2d0ac73278536bb1ef.pdf"},{"id":45116690,"identity":"5bfa631c-d2fb-4a3f-bc53-60c7b62857aa","added_by":"auto","created_at":"2023-10-23 23:35:35","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4866761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2. \u003c/strong\u003ePhylogenetic tree of baculoviruses constructed under ML approach using the dataset of combination of the 33 core genes and \u003cem\u003epolh\u003c/em\u003e/\u003cem\u003egran\u003c/em\u003e genes. \u003cem\u003eCulex nigripalpus NPV\u003c/em\u003e (deltabaculovirus) used as outgroup. Only bootstrap values lower than 100 in ML were shown.\u003c/p\u003e","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3466248/v1/9d98615e3f2840443f54f9f7.pdf"},{"id":45116687,"identity":"5dfeefa9-c201-4a89-9a10-db62057a7691","added_by":"auto","created_at":"2023-10-23 23:35:35","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":56784,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3466248/v1/b9aea76570471b59e95848fa.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative genomics of the Neodiprion sertifer nucleopolyhedrovirus from Turkey with the fewest ORFs among baculoviruses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe family Baculoviridae (also known as baculoviruses) is a distinctive group of host-specific large DNA viruses that infect holometabolous insects in their larval stage, particularly some members of the orders Lepidoptera, Diptera and Hymenoptera (Miele et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The members of this largest and most diverse family of DNA viruses are utilised as biological control agents in ecological pest management programs and/or as gene expression vectors in biotechnological applications (Szewczyk et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Herniou and Jehle \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Assenberg et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The baculovirus replication cycle generates two morphologically unique viral phenotypes including the occlusion body virus (ODV) and the budded virus (BV) regarding the difference in their envelope protein compositions. The morphology of occlusion bodies (OBs) allows the taxonomic classification of baculoviruses into nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) (Slack and Arif \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rohrmann \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The baculoviruses are grouped under four genera based on their molecular characteristics and phylogenies: Deltabaculovirus (dipteran-specific NPV); Gammabaculovirus (hymenopteran-specific NPV); Betabaculovirus (lepidopteran-specific GV); Alphabaculovirus (lepidopteran-specific NPV) (Jehle et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003eb\u003c/span\u003e). Alphabaculovirus can also be further divided into Group I and Group II based on their phylogenetic relationship and membrane fusion proteins (Zanotto et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Bulach et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Jehle et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe rapidly growing number of the reported whole baculovirus genome has led to a deeper understanding of biology and evolution of baculoviruses. To date, the complete genomes for baculoviruses have been reported for approximately 100 host species in the public databases, representing mostly alphabaculoviruses and betabaculoviruses, but only three gammabaculoviruses, and one deltabaculovirus (NCBI, September 2022). A typical baculovirus genome is a double-stranded circular molecule packaged into rod-shaped nucleocapsid with a relatively conserved core gene content, despite a wide range in size from 81.755 kb (\u003cem\u003eNeodiprion lecontei\u003c/em\u003e NPV) to 178.733 kb (\u003cem\u003eXestia c-nigrum\u003c/em\u003e GV) (NCBI, September 2023). Size discrepancies in the baculovirus genomes could be the result of gene loss/gain events which are most likely due to the frequent occurrence of horizontal gene transfer between the virus and its host (Gilbert et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although the core genes are peculiarly essential in many biological processes, including viral replication, transcription, structural composition, and oral infectivity (van Oers and Vlak \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), the function of several core genes has still been unravelled. The information of these genomes can be comparatively utilised in two levels as the nucleotide/amino acid sequences or the structural features and architecture of genomes such as gene content, compositional features, genome size, genome organisation, homolog regions of replication and transcription processes (van Oers and Vlak \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These features requiring different evolutionary processes also allow us to understand better the baculovirus taxonomy, their phylogenetic relationships, evolutionary history, and life strategies such as host specificity and infectivity. For instance, a demarcating criterion used to distinguish baculovirus species has recently been proposed by researchers based on a total of 172 complete genomes (Jehle et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003eb\u003c/span\u003e). In that criterion, if the pairwise evolutionary distance between single or concatenated \u003cem\u003elate expression factor 8\u003c/em\u003e (\u003cem\u003elef-8\u003c/em\u003e), \u003cem\u003elate expression factor 9\u003c/em\u003e (\u003cem\u003elef-9\u003c/em\u003e) (encode for subunits of the baculovirus RNA polymerase) and \u003cem\u003epolh\u003c/em\u003e genes (encode \u003cem\u003epolyhedrin\u003c/em\u003e) or the homologous \u003cem\u003egranulin\u003c/em\u003e of GVs, the major matrix protein of the occlusion bodies (OBs), which are the most conserved core genes of baculoviruses, under Kimura-2-parameter (K2P) is larger than 0.072, two viruses should be considered as different virus species. However, if the distance is smaller than 0.021, two (or more) baculoviruses should be considered within the same species. If the distance is between 0.021 and 0.072, more biological information should then be provided for species discrimination (Jehle et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Wennmann et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong four baculovirus genera, the gammabaculovirus has the smallest genome (with 84.160 kb on average) comprising approximately 90 predicted open reading frames (ORFs) (Garcia-Maruniak et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Lauzon et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Duffy et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). So far, only three genomes of gammabaculovirus have been reported from merely the species of \u003cem\u003eNeodiprion\u003c/em\u003e (Diprionidae: Symphyta), which are the most common defoliating sawfly insects of pine trees, suggesting undergone host-dependent evolution with their restricted hosts. They are \u003cem\u003eNeodiprion lecontei\u003c/em\u003e NPV (NeleNPV) (Lauzon et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), \u003cem\u003eNeodiprion sertifer\u003c/em\u003e NPV (NeseNPV) (Garcia-Maruniak et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and \u003cem\u003eNeodiprion abietis\u003c/em\u003e NPV (NeabNPV) (Duffy et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and are used as bio-insecticide that infect the midgut cell of the \u003cem\u003eNeodiprion\u003c/em\u003e larvae (Arif et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Here, we sequenced and annotated the complete genome of a \u003cem\u003eNeodiprion sertifer nucleopolyhedrovirus\u003c/em\u003e from Turkey (NeseNPV-TR). We compared it with the previously reported genomes of gammabaculovirus for a better understanding of the architecture and features of this baculovirus genome using current bioinformatics approaches. The proteins encoded by the ORFs were predicted in terms of the families, conserved domains and putative functions. We constructed phylogenies to estimate the phylogenetic position of NeseNPV-TR using a concatenated core gene dataset of 100 species from Baculoviridae. The pairwise distances between the members of gammabaculovirus were also calculated to delineate the species boundaries of the NeseNPV-TR of which genome sequence information was obtained, using the Kimura two-parameter distance. Finally, the core genes of all sequenced baculovirus genomes were compared based on nucleotide and amino acid sequences by calculating overall mean p-distances for each gene and genus in order to make inferences about baculovirus evolution.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction, genome sequencing and assembly\u003c/h2\u003e \u003cp\u003eThe virus genome data was generated through sequencing of the isolated total DNA of \u003cem\u003eNeodiprion sertifer\u003c/em\u003e larvae collected from a pine tree in Gelenbe district of Turkey in 2014. The collected larvae were preserved in ethanol in the Entomology Collection of Cumhuriyet University, Sivas (ECCUS) and total genomic DNA was extracted from a whole larva by using DNeasy tissue kit (Qiagen, Hilden, Germany) following the instructions provided by the manufacturer. The quality and quantity of the extracted DNA were measured with a Qubit 4.0 fluorometer (Thermo Fisher) by using the Qubit double-strand High Sensitivity Assay kit and then stored at -20\u0026deg;C. The total genomic DNA extract was sequenced by Illumina HiSeq 2000 next-generation sequencing (NGS) platform using 250 bp paired-end reads, conducted at DONE Genetics Inc., Turkey. The quality of raw NGS reads was initially checked via FastQC v0.11.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.babraham.ac.uk/projects/fastqc\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.babraham.ac.uk/projects/fastqc\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and then filtered by fastp (Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The reference genome of the NeseNPV (Accession no. NC005905) was selected to generate the kraken2 database (Wood et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003efastq\u003c/em\u003e file was searched against the kraken2 database to find short reads that are part of the viral genome via Kraken2. The awk tool in the linux was used to construct a new fastq file including short reads of the viral genome. Genome assembly of NeseNPV-TR was performed \u003cem\u003ede novo\u003c/em\u003e using SPAdes v3.14.0 (Bankevich et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) from the new \u003cem\u003efastq\u003c/em\u003e file. A reference-based assembly was carried out employing Bowtie 2 (Langmead and Salzberg \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) assembly algorithm using the genome of NeseNPV as reference. The generated assemblies by these two approaches were then aligned, manually compared, and finally compiled into a single contig.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGenome annotation and organisation\u003c/h2\u003e \u003cp\u003eThe open reading frames (ORFs) encoding proteins with 50 or longer amino acids were predicted using several different tools: (i) Prokka software (Seemann \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) implemented in KBase (Arkin et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), (ii) Glimmer (Delcher et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) plugin implemented in Geneious R9 (Kearse et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), (iii) Benchmarking Universal Single-Copy Orthologs BUSCO v5.2.2 (Manni et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) tool with searches against baculoviridae_odb10.2020-11-26 dataset, and (iv) FGenesV0 which is available online at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.softberry.com/\u003c/span\u003e\u003cspan address=\"http://www.softberry.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. TTG (Leu) and GTG (Val) codons were also taken into account as alternative start codons in addition to standard ATG start codon. The identified ORFs were then compared with those of reference NeseNPV genome (Accession Number: NC005905). Homolog sequences of the ORFs were predicted by BLAST searches against UniProtKB and Swiss-Prot databases (Bateman et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The families, conserved domains and putative functions of the proteins encoded by the ORFs were determined using Blast2GO (Conesa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) tool via searches with InterPro (Paysan-Lafosse et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The homologous repeated sequences (\u003cem\u003ehrs\u003c/em\u003e) throughout the NeseNPV-TR genome were searched using Tandem Repeat Finder with the default parameters (Benson \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The annotated genome of NeseNPV-TR was finally visualized using Geneious R9. Nucleotide compositions of each gene and the entire genome were calculated separately by MEGA v6.0 (Tamura et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Gene parity plots were constructed to assess the pairwise ORF synteny between NeseNPV-TR and the reported gammabaculovirus genomes using Microsoft Excel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic inference and genetic distance\u003c/h2\u003e \u003cp\u003ePhylogenetic analyses were performed using the nucleotide sequences of the 33 core genes and polyhedrin gene of the newly sequenced NeseNPV-TR genome (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) and their homologous from the previously reported 99 reference baculovirus genomes which were downloaded from NCBI (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). \u003cem\u003eCulex nigripalpus\u003c/em\u003e NPV (Deltabaculovirus) (Afonso et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) was also included in analyses as an outgroup. Nucleotide sequences of each core gene were aligned individually using the \u0026ldquo;translation align\u0026rdquo; option with MAFFT algorithm (Katoh and Standley \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) in Geneious R9. The gene alignments were concatenated with SequenceMatrix v.1.7.8 (Vaidya et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Maximum Likelihood (ML) and Bayesian Inference (BI) approaches were used to confirm the phylogenetic position of the NeseNPV-TR and to infer the phylogenetic relationship between the available baculoviruses. The ML analysis was performed in IQ-TREE v2.1.4 (Minh et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) using default parameters and allowing it to automatically select the evolutionary model. Nodal support was estimated with 10000 bootstrap and fast approximate likelihood-based measures of branch supports (Shimodaira\u0026ndash;Hasegawa [SH]-aLRT) with 1000 replicates under the fast bootstrapping option. For Bayesian Inference, the best model of evolution was selected using MEGA v6.0 setting branch swap filter as very strong. The best fitting model GTR\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I (Table S4) was used in the subsequent Bayesian analysis. BI analyses were conducted using MrBayes v3.2.2 (Ronquist et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) under the unlinked branch lengths of each partition scheme with two independent runs of ten million generations with four Markov chains, sampling every 1000 generations. The stationarity for each run was assessed by evaluating the parameter files using Tracer v1.7 (Rambaut et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). After assessment, the first 25% of trees in each run were discarded as burn-in and a majority-rule consensus tree (BI tree) was generated from the remaining trees. The trees were finally visualised by FigTree v1.4.2 (Rambaut \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo species demarcation, pairwise K2P genetic distances between the NeseNPV-TR and previously reported gammabaculoviruses were estimated separately for both the concatenated 33 aligned core genes and the most conserved three individually aligned core genes (\u003cem\u003elef-8\u003c/em\u003e, \u003cem\u003elef-9\u003c/em\u003e and \u003cem\u003epolh\u003c/em\u003e), using MEGA v6.0 with Kimura 2-parameter model (Kimura \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) by treating the gaps in the alignment as \u0026ldquo;pairwise deletions\u0026rdquo;. In addition, overall mean p-distances for each gene and each genus were calculated using MEGA v6.0 at both the nucleotide and amino acid levels.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGenome characteristics of NeseNPV-TR\u003c/h2\u003e \u003cp\u003eAdvances in sequencing technologies and decreasing costs have led to an increase in the number of baculoviruses for which genome sequence information has been obtained. Whole genome sequence information of 100 baculovirus isolates has been obtained and characterised so far (NCBI Reference Sequence Database, September 2023). Comparative analyses of these genomes helped us to infer baculovirus genome patterns and contributed to a better understanding of their evolution. Baculovirus genomes were highly variable in terms of genome length and organisation, gene content, repetitive sequence content and nucleotide composition (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The size of baculovirus genomes exhibited variability between 81,755 bp in NeleNPV (Lauzon et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and 178,733 bp in XecnGV (Goto et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Here, the newly sequenced and annotated complete genome of NeseNPV-TR (GenBank accession number: OP426321) was 82,052 bp in size, which was smaller than the previously reported genome of the NeseNPV (86,462 bp) (Garcia-Maruniak et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and also exhibited relatively low overall pairwise identity (90.62%). The size difference was mainly resulted from the presence of a large indel comprising two ORFs coding hypothetical proteins (corresponding NeseORF18 and NeseORF19 in the reference genome), one direct and one homologous repeat regions (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The number of coding regions was variable among baculovirus genomes: from 82 ORFs in NeseNPV-TR genome to 183 ORFs in PsunGV genome (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) (Li et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The homologous repeats also varied among genomes; while some genomes had none, the EpapGV genome had the highest number of homologous repeats with 16 regions (Ferrelli et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The genome of NeseNPV-TR has low G\u0026thinsp;+\u0026thinsp;C content, with an average of 33.28% (16.77% of G, 16.51% of C, 34.04% of A and 32.68% of T), which was similar to that of NeseNPV (33.76% G\u0026thinsp;+\u0026thinsp;C) and comparable to those of other reported baculoviruses (33.38% G\u0026thinsp;+\u0026thinsp;C in Nele-NPV and Neab-NPV), as well as fall in the range of those of reported baculoviruses [31.1% in OxocNPV (Wang et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and 57.5% in LdMNPV (Krejmer-Rabalska et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)] (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGene content and features of NeseNPV-TR genome\u003c/h2\u003e \u003cp\u003eA total of 82 ORFs potentially encoding polypeptides consisting of 50 or more amino acids have been identified in the genome of NeseNPV-TR (Tables S2 and S7). The first nucleotide position of the genome was defined as the first adenine of the \u003cem\u003epolyhedrin\u003c/em\u003e (\u003cem\u003epolh\u003c/em\u003e) gene, known to be the major structural component for occlusion bodies (Ji et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thirty-nine of the identified ORFs in the genome of NeseNPV-TR encode functionally known proteins, whereas the remaining encodes hypothetical proteins whose functions are unknown (Tables S2 and S7). The ORFs were distributed on the both strands of the DNA across the genome. Fifty-two ORFs (63.41%) were in the clockwise direction and the remaining (36.59%) were in an anti-clockwise direction with respect to the direction of the \u003cem\u003epolh\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of 76 ORFs, including those encoding hypothetical proteins, were shared between the newly sequenced genome of NeseNPV-TR and the reference genome of NeseNPV (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Out of 38 baculovirus core genes (Rohrmann \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang and Hu \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), 33 were identified in the genome of NeseNPV-TR which are involved in the replication, transcription, structure, or infectivity/ODV structure. The five unidentified baculovirus core genes in the NeseNPV-TR genome were \u003cem\u003epif-7\u003c/em\u003e (ac110), \u003cem\u003eac53\u003c/em\u003e, \u003cem\u003eac78\u003c/em\u003e, \u003cem\u003ep18\u003c/em\u003e (\u003cem\u003eac93\u003c/em\u003e) and \u003cem\u003eodv-e25\u003c/em\u003e (\u003cem\u003eac94\u003c/em\u003e), which are known to be related to infectivity or structure. In addition to the identified baculovirus core genes, three of the Lepidopteran baculovirus core genes known to be present in half of Gammabaculoviruses (Arif et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), \u003cem\u003eac8\u003c/em\u003e (encoding polyhedrin), \u003cem\u003eac25\u003c/em\u003e (encoding dbp) and \u003cem\u003eac37\u003c/em\u003e (encoding late expression factor-11), have also been found in the NeseNPV-TR genome (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Five of the baculovirus core genes [\u003cem\u003ednapol\u003c/em\u003e (\u003cem\u003eac65\u003c/em\u003e), \u003cem\u003elef-1\u003c/em\u003e (\u003cem\u003eac14\u003c/em\u003e), \u003cem\u003elef-2\u003c/em\u003e (\u003cem\u003eac6\u003c/em\u003e), \u003cem\u003ehelicase\u003c/em\u003e (\u003cem\u003eac95\u003c/em\u003e) and \u003cem\u003ealk-exo\u003c/em\u003e (\u003cem\u003eac133\u003c/em\u003e)] and one lepidopteran baculovirus core gene [\u003cem\u003edbp\u003c/em\u003e (\u003cem\u003eac25\u003c/em\u003e)] identified in the genome were associated with DNA replication processes (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) (Rohrmann \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Of these core genes, \u003cem\u003elef-1\u003c/em\u003e exhibits primase activity, while \u003cem\u003elef-2\u003c/em\u003e gene serves as an accessory factor of primase by interacting with \u003cem\u003elef-1\u003c/em\u003e and DNA (Mikhailov and Rohrmann \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The \u003cem\u003ehelicase\u003c/em\u003e gene encodes a protein that has both helicase and ATPase activity (McDougal and Guarino \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and \u003cem\u003ednapol\u003c/em\u003e encodes a DNA polymerase B family member polymerase with 3' to 5' exonuclease activity (Hang and Guarino \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The \u003cem\u003ealk-exo\u003c/em\u003e core gene encodes alkaline exonuclease enzyme with 5\u0026prime; to 3\u0026prime; exonuclease activity that is involved in the maturation of the virus genomes and DNA recombination (Mikhailov et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The lepidopteran baculovirus core gene \u003cem\u003edbp\u003c/em\u003e takes part in DNA replication and expression of the \u003cem\u003epolh\u003c/em\u003e gene by binding to DNA (Quadt et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The \u003cem\u003elef-3\u003c/em\u003e and \u003cem\u003eie-1\u003c/em\u003e genes, which are involved in genome replication in baculoviruses (Wang and Hu \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), were absent in the NeseNPV-TR genome, similar to other gammabaculoviruses. Six baculovirus core genes [\u003cem\u003elef-4\u003c/em\u003e (\u003cem\u003eac90\u003c/em\u003e), \u003cem\u003elef-5\u003c/em\u003e (\u003cem\u003eac99\u003c/em\u003e), \u003cem\u003elef-8\u003c/em\u003e (\u003cem\u003eac50\u003c/em\u003e), \u003cem\u003elef-9\u003c/em\u003e (\u003cem\u003eac62\u003c/em\u003e), \u003cem\u003ep47\u003c/em\u003e (\u003cem\u003eac40\u003c/em\u003e) and \u003cem\u003evlf-1\u003c/em\u003e (\u003cem\u003eac77\u003c/em\u003e)] and one lepidopteran core gene [\u003cem\u003elef11\u003c/em\u003e (\u003cem\u003eac37\u003c/em\u003e)] known to have an essential role in transcription (Lu and Miller \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Jin et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) were characterized in the NeseNPV-TR genome (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). From these genes, \u003cem\u003elef-4\u003c/em\u003e encodes the essential subunit of RNA polymerase and also the mRNA capping enzyme guanylyltransferase (Li and Guarino \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wang and Hu \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003elef-5\u003c/em\u003e gene plays a role in the initiation of transcription by stimulating the activity of RNA polymerase complex and is crucial for productive infection (Su et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The genes of \u003cem\u003elef-8\u003c/em\u003e and \u003cem\u003elef-9\u003c/em\u003e encoding the subunits of the RNA polymerase are critical for the late gene expression (Wang and Hu \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although the exact role of \u003cem\u003ep47\u003c/em\u003e gene in the RNA polymerase complex is not known yet, it was reported that this gene is essential for late gene expression of late and very late gene promoters (Passarelli \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). \u003cem\u003evlf-1\u003c/em\u003e gene stimulates the high level expression of very late genes by interacting with the promoters of these genes (Mistretta and Guarino \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and also takes part in nucleocapsid assembly and packaging of DNA (Vanarsdall et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The lepidopteran baculovirus core gene \u003cem\u003elef-11\u003c/em\u003e promotes viral DNA replication (Dong et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and supports expression of late genes (Todd et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In addition to these core genes involved in transcription process, one non-conserved gene among baculovirus genomes named \u003cem\u003emtase1\u003c/em\u003e (homologous to \u003cem\u003eac69\u003c/em\u003e of \u003cem\u003eAutographa californica\u003c/em\u003e NPV) was also characterized in the newly sequenced NeseNPV-TR genome (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and S3). \u003cem\u003emtase1\u003c/em\u003e gene encodes an RNA Cap (Nucleoside-2-O)-Methyltransferase and stimulates late gene expression (Wu and Guarino \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). This gene was also present in the previously reported NeseNPV reference genome, however it was absent in the other reported \u003cem\u003eNeodiprion\u003c/em\u003e nucleopolyhedrovirus genomes (Lauzon \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Although the immediate-early genes (\u003cem\u003eie-0\u003c/em\u003e, \u003cem\u003eie-1\u003c/em\u003e, \u003cem\u003eie-2\u003c/em\u003e, and \u003cem\u003ep38\u003c/em\u003e) trans-regulating early viral gene expression were not found in the NeseNPV-TR genome; \u003cem\u003elef-5\u003c/em\u003e, \u003cem\u003elef-11\u003c/em\u003e, and \u003cem\u003evlf-1\u003c/em\u003e genes displaying homology to these genes have been identified as transcription genes (Olson et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to core genes associated with the replication or transcription processes, 22 core genes related to \u003cem\u003eper os\u003c/em\u003e infectivity or structure have been characterized in the NeseNPV-TR genome (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Eight of these baculovirus core genes [\u003cem\u003ep74\u003c/em\u003e (\u003cem\u003epif0\u003c/em\u003e), \u003cem\u003epif1-6 and pif-8\u003c/em\u003e] encode the \u003cem\u003eper os\u003c/em\u003e infectivity factors (PIFs) which are envelope proteins and form a stable protein complex. In similar to the other reported gammabaculoviruses, \u003cem\u003epif7\u003c/em\u003e gene was absent in the NeseNPV-TR genome (Song et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The complex constructed by PIFs takes part in the ODV binding to midgut cells and is resistant to proteolytic degradation in the insect midgut (Wang et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003epif-8\u003c/em\u003e gene, named \u003cem\u003evp91\u003c/em\u003e (\u003cem\u003eac83\u003c/em\u003e), is also involved in the nucleocapsid assembly process. In addition to this gene, it has also been reported that \u003cem\u003eac53\u003c/em\u003e, \u003cem\u003evp1054\u003c/em\u003e (\u003cem\u003eac54\u003c/em\u003e), \u003cem\u003evp39\u003c/em\u003e (\u003cem\u003eac89\u003c/em\u003e), \u003cem\u003e38k\u003c/em\u003e (\u003cem\u003eac98\u003c/em\u003e), \u003cem\u003ep40\u003c/em\u003e (\u003cem\u003ebv/odv-c42\u003c/em\u003e or \u003cem\u003eac101\u003c/em\u003e), and \u003cem\u003eodv-ec27\u003c/em\u003e (\u003cem\u003eac144\u003c/em\u003e) core genes play role in the process of nucleocapsid assembly (Wang and Hu \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003ep40\u003c/em\u003e gene takes part in actin polymerization, while the \u003cem\u003eodv-ec27\u003c/em\u003e in cell cycle arrest. It has been emphasised that the core genes \u003cem\u003egp41\u003c/em\u003e (ac80) encoding a tegument protein and \u003cem\u003ep33\u003c/em\u003e (ac92) encoding sulfhydryl oxidase enzyme, as well as \u003cem\u003eac78\u003c/em\u003e are involved in BV production and ODV morphogenesis (Wang and Hu \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The baculovirus core genes \u003cem\u003eac81\u003c/em\u003e, \u003cem\u003ep18\u003c/em\u003e (\u003cem\u003eac93\u003c/em\u003e), \u003cem\u003ep48/p45\u003c/em\u003e (\u003cem\u003eac103\u003c/em\u003e), \u003cem\u003eodv-ec43\u003c/em\u003e (\u003cem\u003eac109\u003c/em\u003e) and \u003cem\u003e49k\u003c/em\u003e (\u003cem\u003eac142\u003c/em\u003e) found in the newly sequenced genome were related to BV production and ODV envelopment process, while the \u003cem\u003eodv-e18\u003c/em\u003e (\u003cem\u003eac143\u003c/em\u003e) was only related to BV production (Wang and Hu \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003ep6.9\u003c/em\u003e (\u003cem\u003eac100\u003c/em\u003e) gene encodes a protamine-like protein that functions in the condensation and packaging of viral DNA, an essential process for producing infectious viruses (Wang et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Additionally, three additional genes related to \u003cem\u003eper os\u003c/em\u003e infectivity or structure were also annotated in this newly sequenced genome (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and S3). Among these genes, \u003cem\u003epolh\u003c/em\u003e encodes polyhedrin protein which is conserved in NPVs and is homologous to the granulin protein in GVs and constitutes the crystalline matrix of the occlusion body (Rohrmann \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A member of the \u003cem\u003eiap\u003c/em\u003e gene family, characterized by having two conserved structural features, a zinc-binding domain known as RING finger and baculovirus IAP repeat (BIR) on the amino-terminus, was annotated in the NeseNPV-TR genome. Members of this gene family are known to be able to prevent apoptosis in host insect cells. The iap protein identified in the newly sequenced NeseNPV-TR genome exhibited 88.95% identity with its counterpart in the NeseNPV genome. The third one of these genes encode a trypsin-like serine protease that has been reported for all gammabaculovirus genomes (Ishimwe et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Although the function of this protein has not been defined yet, it has been suggested that this enzyme may function in the release of OB from midgut-infected cells and their availability for the next replication cycle (Rohrmann \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or viral protein cleavage for \u003cem\u003eper os\u003c/em\u003e infectivity (Slack et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs well as the identified genes in the NeseNPV-TR genome, hypothetical/predicted proteins had striking structural and functional patterns (Table S7). The first of these was the orf3 protein contained an F-box domain similar to the lef-7 protein, enhancing the viral replication in baculoviruses, which is missing in the NeseNPV-TR genome. Furthermore, it was determined that the orf3 protein has a protein binding function (GO:0005515; Table S7). The orf9 protein is found to have a RING-type zinc-finger domain. It has been previously reported that several proteins such as ie2, iap1 and iap2 in baculoviruses contain RING domains (Imai et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and the RING domains have been suggested to play a role as E3 ligases by binding to E2 ubiquitin-conjugating enzymes (Lorick et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Vaux and Silke \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The InterProScan search results showed that the orf13 protein comprises pentapeptide repeat motif (Table S7). Pentapeptide repeat proteins (PRPs) were firstly identified in cyanobacteria, but are also found in almost all species. Albeit the functions of these repeats are unknown, it has been reported that members of this family have the ability to interact with DNA-binding proteins (M\u0026eacute;rens et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The searches revealed that the protein encoded by \u003cem\u003eorf25\u003c/em\u003e belongs to the DUF816 family of baculovirus proteins with unknown function. Surprisingly, two dsRNA-binding domains were detected in the orf44 protein of the NeseNPV-TR (Table S7). The presence of these domains was remarking because the innate immune response to viral infections in insects is mainly mediated by dsRNAs (Jayachandran et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and as a result of co-evolution, insect viruses have also adapted a variety of approaches to overcome the barriers in their way of infection (Mehrabadi et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The dsRNA-binding domain-containing protein identified in the NeseNPV-TR genome might function as a viral suppressor of RNAi (VSR) by inhibiting the Dicer-2-mediated cleavage of dsRNA and also binding to siRNAs and blocking their loading into the RISC (Zhao et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, orf45 protein of the NeseNPV-TR possesses a classic C2H2-type zinc-finger domain (Table S7). The gene ontology annotations indicated that the orf45 protein takes part in the regulation of the DNA-templated transcription biological process (GO:0006355; Table S7), and has RNA polymerase II-specific DNA-binding transcription factor activity (GO:0000981) and RNA-polymerase II cis-regulatory region sequence-specific DNA binding (GO: 0000978) molecular functions. \u003cem\u003eorf60\u003c/em\u003e encodes a small protein (~\u0026thinsp;12 kDa) in the NeseNPV-TR genome that displayed 93.58% amino acid similarity to NeseORF67 and was a close relative of the Ac145/Ac150 proteins. This protein includes a conserved cysteine-rich region with six conserved cysteine residues featuring a C\u003csup\u003e1\u003c/sup\u003eX\u003csub\u003e13\u003c/sub\u003e C\u003csup\u003e2\u003c/sup\u003eX\u003csub\u003e5\u003c/sub\u003e C\u003csup\u003e3\u003c/sup\u003eX\u003csub\u003e7\u003c/sub\u003e C\u003csup\u003e4\u003c/sup\u003eX\u003csub\u003e12\u003c/sub\u003e C\u003csup\u003e5\u003c/sup\u003eX\u003csub\u003e7\u003c/sub\u003eC\u003csup\u003e6\u003c/sup\u003e motif, where C is a cysteine and X is any amino acid other than cysteine (Zhang et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The orf60 protein is also predicted to localise to a membrane (GO: 0016020) and its conserved domain is found to have chitin-binding function (GO: 0008061), indicating that the protein, like its homologues, may localise to ODV envelopes and initiate the infection of midgut cells (Rohrmann \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similar to the other reported gammabaculovirus genomes (Garcia-Maruniak et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Lauzon et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Lauzon \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Arif et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), three sequentially located hypothetical proteins (orf65, orf66 and orf67) were identified as homologous to regulators of chromosome condensation proteins (RCC1) of diverse insect species from a broad range of taxonomy (e.g. Coleoptera, Diptera, Hemiptera, Lepidoptera, Ephemeroptera, Hymenoptera) as the result of blast homology searches. Searches of these proteins against the NCBI CDD and InterPro databases revealed that they have conserved domains of the alpha-tubulin suppressor and RCC1 domain-containing protein conserved domain superfamily, and of the regulator of chromosome condensation 1/beta-lactamase-inhibitor protein II family, respectively (Table S7). RCC1 proteins are DNA-binding proteins involved in the regulation of chromosome condensation during the transcription or mRNA maturation process and act as a guanine nucleotide-dissociation stimulator (Lauzon \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Consistently, orf65 and orf66 proteins were assigned to have the molecular function of guanyl-nucleotide exchange factor activity (GO: 0005085). The existence of RCC1-like proteins in gammabaculovirus and the original proteins of RCC1 in various insect species may imply a conceivable horizontal transfer of this gene between viruses and their host (Lauzon \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The last striking feature of NeseNPV-TR hypothetical proteins was observed in the orf82 protein (Table S7). This protein was homologous to members of the RNA 2\u0026prime;-phosphotransferase KptA/Tpt1 family and its homologous were also reported only in all sequenced gammabaculovirus genomes (Lauzon \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Arif et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), but not in any other virus genomes. The members of this protein family are belong to ADP-ribosyltransferases (ARTs) that play a role in tRNA splicing and NAD metabolism, by transferring 2\u0026prime;-phosphate emerging from tRNA splicing and RNA ligation processes to NAD in the archaeo-eukaryotic lineages (de Souza and Aravind \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, the functions of members of this family or homologues in gammabaculoviruses and bacteria lacking the tRNA-splice mechanism are not known yet. Although members of this protein family have not been reported in viruses, members of its superfamily, ARTs, have been reported in several virus species (Iyer et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). ARTs catalyse the ADP-ribosylation by transferring ADP-ribose to substrates from NAD\u0026thinsp;+\u0026thinsp;and this ribosylation is involved in a broad range of functions such as DNA damage, viral infection, intra- and extracellular signalling, protein biosynthesis, transcriptional regulation and cell death (L\u0026uuml;scher et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recent studies have shown that NAD+\u0026ndash;ADPr (ADP-ribose) interactions are very crucial for antiviral immunity and counter-response of viruses against host immunity, and ADPr modifications are very common mechanism in DNA viruses to counter host systems (Iyer et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The phosphotransferases characterized in the gammabaculoviruses might also be a counter-adaptation of the virus to evade host defence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHomologous repeats\u003c/h2\u003e \u003cp\u003eAnother commonly observed feature in most baculovirus genomes is that they contain homologous repetitive regions, called \u003cem\u003ehr\u003c/em\u003es, with some shared characteristics (Rohrmann \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003ehr\u003c/em\u003es were found to be distributed across the genome and to have several tandem repeats of palindromes within direct repeats (Arif et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). They have been suggested both as transcriptional enhancers and origins of viral replication due to their symmetrical locations, high AT content and containing palindromic sequences (Cochran and Faulkner \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Rohrmann \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Five \u003cem\u003ehr\u003c/em\u003es ranging between 249 and 819 bp were defined in the NeseNPV-TR genome (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The total length of these \u003cem\u003ehr\u003c/em\u003es was 2387 bp, corresponding to 2.91% of the genome. The average G\u0026thinsp;+\u0026thinsp;C content of the \u003cem\u003ehr\u003c/em\u003es was 51.32% in average, which was higher than that of the total genome (33.28%). They displayed high similarity with each other by having 20.6% overall mean distance (between 11.27% and 31.86% pairwise distance). The most diverse one was \u003cem\u003ehr\u003c/em\u003e5, with a range of from 22.55\u0026ndash;31.86% p-distance from the others and was the furthest from the others in terms of genomic location (Table S8). Similar to those found in the NeseNPV-TR genome, \u003cem\u003ehr\u003c/em\u003es were also present in the reference NeseNPV genome. Six \u003cem\u003ehr\u003c/em\u003es were identified in the reference genome, with lengths varying between 396 to 794 bp, with an G\u0026thinsp;+\u0026thinsp;C content of 49.8% on average and an 18.1% overall mean p-distance (Garcia-Maruniak et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The \u003cem\u003ehr\u003c/em\u003es of the NeseNPV-TR genome exhibited high nucleotide similarity with those of the reference genome, with 16.52% mean p-distance (0.49\u0026ndash;31.86% pairwise distances) (Table S8). Similar to the pattern reported in the reference genome (Garcia-Maruniak et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), the baculovirus late/very late promoter motif (A/G/T)TAAG, which is usually not located inside the \u003cem\u003ehr\u003c/em\u003es in baculoviruses, was found in multiple copies within all of the NeseNPV-TR \u003cem\u003ehr\u003c/em\u003es (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Moreover, the GATA motif which has been reported as the host transcription factor binding site in baculoviruses (Krappa et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Kogan and Blissard \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), has also been detected in \u003cem\u003ehr\u003c/em\u003e1, \u003cem\u003ehr\u003c/em\u003e2 and \u003cem\u003ehr\u003c/em\u003e3 of NeseNPV-TR genome at overlapping positions with the (A/G/T)TAAG motifs (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Although the exact role of \u003cem\u003ehr\u003c/em\u003es is not yet known, the promoter and transcription factor binding motifs found in the NeseNPV-TR genome may support their proposed role as transcriptional enhancers.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStructure of NeseNPV-TR homologous repeat regions (\u003cem\u003ehrs\u003c/em\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength of repeat units/no. of times repeated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePosition at genome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRepetitive region\u003c/p\u003e \u003cp\u003e(total bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehr1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110 bp/4.2 ; 219 bp/2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8631\u0026ndash;9100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehr2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65 bp/7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11003\u0026ndash;11483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e481\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehr3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110 bp/2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16578\u0026ndash;16826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehr4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45 bp/18.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17196\u0026ndash;18014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e819\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehr5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44 bp/8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44668\u0026ndash;45035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGene parity\u003c/h2\u003e \u003cp\u003eThe genome organizations of the newly sequenced NeseNPV-TR and reference genome were compared using gene parity plot analysis of homologous genes and ORFs. The gene arrangement of the NeseNPV-TR genome exhibited high collinearity with the reference genome with a few exceptions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). NeseORF10, NeseORF14, NeseORF15, NeseORF18, NeseORF27 and NeseORF76, which are known to encode hypothetical proteins in the reference genome, were absent in the newly sequenced genome. In addition, \u003cem\u003emtase1\u003c/em\u003e encoded from the NeseNPV-TR genome corresponded to the concatenation of \u003cem\u003emtase1\u003c/em\u003e and NeseORF6 encoded from the reference genome. Similarly, hypothetical protein encoded from the orf10 of NeseNPV-TR genome also corresponded to the concatenation of NeseORF12 and NeseORF13 of the reference genome. Instead of NeseORF4, NeseORF19, NeseORF21, NeseORF30 and NeseORF74 in the reference genome, there were various ORFs encoding different hypothetical proteins in the newly sequenced genome (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). When the NeseNPV-TR genome is compared to NeleNPV and NeabNPV, it has been seen that although there are differences in several regions in their genome organization, the gene and ORF contents were widely shared between the genomes. In total, 67 and 64 ORFs of the NeseNPV-TR genome had homologs in NeleNPV and NeabNPV genomes, respectively (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The regions between the polyhedrin (\u003cem\u003epolh\u003c/em\u003e) and DNA binding protein (\u003cem\u003edbp\u003c/em\u003e) genes were lack of conserved synteny between the three genomes. In addition, the region between orf23 and orf29 (\u003cem\u003ep6.9\u003c/em\u003e gene) in the NeseNPV-TR genome was inverted in the NeleNPV (orf34 to orf28) and NeabNPV (orf37 to orf32) genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In all three species, the organization of the second half of the genome was highly conserved compared to the first half (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBaculovirus phylogeny and species demarcation\u003c/h2\u003e \u003cp\u003eEarly studies aiming to reveal baculovirus phylogeny have used nucleotide and amino acid sequences of highly conserved polyhedrin/granulin genes that form the occlusion body matrix in NPVs and GVs due to sequence availability (Herniou and Jehle \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, in parallel with the developments in sequencing technologies and the decrease in costs, the number of baculovirus species for which genome sequence information is gathered increases and consequently, the use of multiple genes or the whole genome in phylogenetic analyses has become widespread. Here, the baculovirus phylogeny was tried to be revealed with two different approaches by using the concatenation of the nucleotide sequences of 33 core genes and polyhedrin/granulin genes (71227 nt in alignment) from 101 baculovirus genomes, including the NeseNPV-TR genome obtained in this study. Both BI and ML trees recovered almost the same tree topology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), and BI tree was presented here (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The presence of four main clades was observed in the reconstructed trees, with the high support values. The tree topology was consistent with that obtained from previous studies (Jehle et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e; Herniou and Jehle \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), where the main clades consisted of alphabaculoviruses infecting lepidopteran insects, betabaculoviruses infecting lepidopteran insects, gammabaculoviruses infecting hymenopteran insects, and deltabaculoviruses infecting dipteran insects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, Group II alphabaculoviruses were found to be paraphyletic to Group I alphabaculoviruses in the tree, similar to the recovered trees by previous studies (Craveiro et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Harrison and Rowley \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ben Tiba et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Finally, this phylogenetic tree confirmed the location of the newly sequenced NeseNPV-TR within gammabaculoviruses, as sister group to the reference NeseNPV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that the use of nucleotide pairwise distances with K2P can be used as a criterion for baculovirus species (Jehle et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e; Wennmann et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). When the K2P distance between two isolates is less than 0.021, they can be considered as the same species, while the distance between them is greater than 0.072, they can be considered as different species. If the values are between these two, additional information such as host range and genome organization is required for species discrimination. The K2P distances between NeseNPV-TR and the reference NeseNPV were calculated for the nucleotide sequences of \u003cem\u003elef-8\u003c/em\u003e, \u003cem\u003elef-9\u003c/em\u003e and \u003cem\u003epolh\u003c/em\u003e genes individually, and also concatenated sequences of these three genes, as well as the combined 33 core genes identified in the NeseNPV-TR genome (Table S5). The pairwise K2P distances of \u003cem\u003elef-9\u003c/em\u003e and \u003cem\u003epolh\u003c/em\u003e genes were lower than the specified threshold value (0.0187 for \u003cem\u003elef-9\u003c/em\u003e and 0.0165 for \u003cem\u003epolh\u003c/em\u003e), while those of \u003cem\u003elef-8\u003c/em\u003e, combinations of three genes and 33 genes were slightly higher (0.0257 for \u003cem\u003elef-8\u003c/em\u003e, 0.0221 for three genes combined and 0.0258 for the 33 genes combined) (Table S5); however, they were still too low than the threshold value to be considered these two samples as different species. The slightly high difference for the 33 genes combined here may be due to annotation differences, sequencing errors, differences in the sampling years between the genome obtained here and the reference genome, or missing genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGenetic differentiation among baculoviruses\u003c/h2\u003e \u003cp\u003eHere, the overall mean p-distances for each baculovirus core gene (except for deltabaculovirus) were calculated based on both nucleotide and amino acid sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table S6). When evaluated in terms of amino acid distances, the most variable genes were \u003cem\u003elef-2\u003c/em\u003e, \u003cem\u003ehelicase\u003c/em\u003e, \u003cem\u003ep40\u003c/em\u003e, \u003cem\u003edesmoplakin\u003c/em\u003e and \u003cem\u003evp91\u003c/em\u003e, respectively, while the most conserved ones were \u003cem\u003ep6.9\u003c/em\u003e, \u003cem\u003elef-8\u003c/em\u003e, \u003cem\u003elef-9\u003c/em\u003e, \u003cem\u003eodv-e18\u003c/em\u003e and \u003cem\u003epif2\u003c/em\u003e (Table S6). Interestingly, contrary to observed in the amino acid sequence, \u003cem\u003ep6.9\u003c/em\u003e was the most variable gene when nucleotide distances were considered followed by \u003cem\u003elef-2\u003c/em\u003e, \u003cem\u003edesmoplakin\u003c/em\u003e, \u003cem\u003evp91\u003c/em\u003e, \u003cem\u003evp39\u003c/em\u003e and \u003cem\u003ep40\u003c/em\u003e. The most conserved genes in terms of nucleotide distance were \u003cem\u003elef-8\u003c/em\u003e, \u003cem\u003elef-9\u003c/em\u003e, \u003cem\u003elef-5\u003c/em\u003e, \u003cem\u003eodv-e18\u003c/em\u003e and \u003cem\u003epif2\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table S6). In a study performed using 57 baculovirus genomes and 31 core genes, the most variable genes were found to be \u003cem\u003edesmoplakin\u003c/em\u003e, \u003cem\u003ehelicase\u003c/em\u003e, \u003cem\u003elef-2\u003c/em\u003e, \u003cem\u003ep6.9\u003c/em\u003e and \u003cem\u003evp91\u003c/em\u003e, and the most conserved genes were \u003cem\u003epif2\u003c/em\u003e, \u003cem\u003elef-9\u003c/em\u003e, \u003cem\u003eodv-e18\u003c/em\u003e, \u003cem\u003elef-8\u003c/em\u003e and \u003cem\u003eac81\u003c/em\u003e, respectively (Miele et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The reported results were largely consistent with those obtained here (Table S6). The variability observed in baculovirus core genes appears to be due to their approximately 300\u0026nbsp;million years co-evolutionary history with their hosts (Ikeda et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The different levels of variability in core genes can also be explained by different evolutionary constraints acting on them. Here, the genes having the most variability seem to be related to nucleocapsid assembly (\u003cem\u003ep40\u003c/em\u003e, \u003cem\u003evp91\u003c/em\u003e) and egress (\u003cem\u003edesmoplakin\u003c/em\u003e), and replication (\u003cem\u003elef-2\u003c/em\u003e and \u003cem\u003ehelicase)\u003c/em\u003e, while the most conserved ones were related to transcription (\u003cem\u003elef-8\u003c/em\u003e, \u003cem\u003elef-9\u003c/em\u003e and \u003cem\u003elef-5\u003c/em\u003e), per os infectivity (\u003cem\u003epif-2\u003c/em\u003e) and BV production (\u003cem\u003eodv-e18\u003c/em\u003e). It has been reported in previous studies that \u003cem\u003ehelicase\u003c/em\u003e, one of the most variable genes, is an important host range factor (Thiem and Cheng \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). High variability in other genes may also be shaped by host-dependent evolution and host ranges of baculoviruses. While the conserved core genes, \u003cem\u003elef-8\u003c/em\u003e and \u003cem\u003elef-9\u003c/em\u003e, encode the subunits of baculovirus RNA polymerase (Wang and Hu \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the \u003cem\u003elef-5\u003c/em\u003e gene is directly involved in late transcription (Su et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Another conserved gene, \u003cem\u003eodv-e18\u003c/em\u003e is crucial for the production of infectious BV and is necessary for interactions between the membrane and nucleocapsid in the nucleus (Blissard and Theilmann \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The last most conserved gene \u003cem\u003epif2\u003c/em\u003e is involved in the binding of ODV onto midgut cells and is reported to have an essential role in the host specificity of baculoviruses (Song et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The conservation level of these genes might be explained by their vital functions in transcription or infectivity given above.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe overall mean p-distances for core genes based on both nucleotide and amino acid sequences were also evaluated within each genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). When considering in terms of both nucleotide and amino acid distances, Group I alphabaculovirus (for nucleotide minimum: 0.1740 and maximum: 0.3414, for amino acid minimum: 0.1643 and maximum: 0.3837) and gammabaculovirus (for nucleotide minimum: 0.1915 and maximum: 0.3147, for amino acid minimum: 0.1545 and maximum: 0.4360) displayed lower genetic diversity, while Group II alphabaculovirus (for nucleotide minimum: 0.3059 and maximum: 0.4658, for amino acid minimum: 0.2724 and maximum: 0.5607) and betabaculovirus (for nucleotide minimum: 0.3310 and maximum: 0.4640, for amino acid minimum: 0.3013 and maximum: 0.5843) had higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In a previous comparative study of baculovirus genomes, it has been reported that Group I alphabaculoviruses and gammabaculoviruses have also lower diversity of gene content (Miele et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The low diversity observed in gammabaculoviruses, both in terms of gene content and sequence, is most likely due to the limited number of sequenced genomes. However, considering the significant number of genome sequences in Group I alphabaculoviruses together with their position in the phylogenetic tree, the lower genetic diversity of this genus might be explained by their recent emergence and having less time to gain new sequences or accumulate mutations (Miele et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe sequencing and characterization of the complete genome of \u003cem\u003eNeodiprion sertifer\u003c/em\u003e nucleopolyhedrovirus from Turkey and comparison with the other reported baculovirus genomes allowed us to denote several conclusions: (i) although it is smaller and contains the fewest ORFs among baculoviruses, the genome characteristics of the NeseNPV-TR are mostly consistent with the previously reported NeseNPV genome, (ii) the genome harbours 82 ORFs and the functions of 39 of them are known, while some of the remaining contain conserved domains and further studies are needed to reveal their structural and functional roles, (iii) phylogenetic analyses using 33 baculovirus core genes confirmed the position of NeseNPV-TR within the genus gammabaculovirus, (iv) Up to the pairwise K2P distances, the sample of which the genome sequence obtained can be considered as the same species with NeseNPV (v) the observation of different rates of divergence in the core genes can be explained by different selection pressures acting on the genes, and (vi) albeit lower genetic diversity of gammabaculoviruses appears to be due to limited number of sequenced genomes, that of Group I alphabaculoviruses is most probably due to recent emergence. More genome sequences (especially the representative of gammabaculoviruses) and further studies are needed to understand baculovirus genome patterns, evolutionary history and genetic diversity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank our colleagues from the Department of Biology of the Ege University who provided the specimens. We are also grateful to Dr. Hasan H. BAŞIB\u0026Uuml;Y\u0026Uuml;K (Department of Gerontology, Akdeniz University) and Dr. Merve Nur AYDEMİR for their contributions in data generation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOD contributed to the generation and interpretation of data, and edited the manuscript. MB performed the analyses and contributed to interpretation of data. MŞS contributed to the generation of data and edition of the manuscript. EMK involved in the conceptualization, supervision, final interpretation of data, and editing of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by T\u0026Uuml;BİTAK (The Scientific and Technological Research Council of Turkey, grant number 112T418).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequence generated in this study was deposited in the GenBank database under the accession number represented in the section of Methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch procedures in this study did not violate any relevant laws and regulations. No relevant ethics committee(s) or institution(s) were applicable for the materials used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAfonso CL, Tulman ER, Lu Z, Balinsky CA, Moser BA, Becnel JJ, Rock DL, Kutish GF (2001) Genome sequence of a baculovirus pathogenic for \u003cem\u003eCulex nigripalpus\u003c/em\u003e. J Virol 75:11157\u0026ndash;11165. https://doi.org/10.1128/JVI.75.22.11157-11165.2001\u003c/li\u003e\n\u003cli\u003eArif B, Escasa S, Pavlik L (2011) Biology and genomics of viruses within the genus Gammabaculovirus. Viruses 3:2214\u0026ndash;2222. https://doi.org/10.3390/v3112214\u003c/li\u003e\n\u003cli\u003eArkin AP, Cottingham RW, Henry CS, Harris NL, Stevens RL, Maslov S, Dehal P, Ware D, Perez F, Canon S, Sneddon MW, Henderson ML, Riehl WJ, Murphy-Olson D, Chan SY, Kamimura RT, Kumari S, Drake MM, Brettin TS, Glass EM, Chivian D, Gunter D, Weston DJ, Allen BH, Baumohl J, Best AA, Bowen B, Brenner SE, Bun CC, Chandonia J-M, Chia J-M, Colasanti R, Conrad N, Davis JJ, Davison BH, DeJongh M, Devoid S, Dietrich E, Dubchak I, Edirisinghe JN, Fang G, Faria JP, Frybarger PM, Gerlach W, Gerstein M, Greiner A, Gurtowski J, Haun HL, He F, Jain R, Joachimiak MP, Keegan KP, Kondo S, Kumar V, Land ML, Meyer F, Mills M, Novichkov PS, Oh T, Olsen GJ, Olson R, Parrello B, Pasternak S, Pearson E, Poon SS, Price GA, Ramakrishnan S, Ranjan P, Ronald PC, Schatz MC, Seaver SMD, Shukla M, Sutormin RA, Syed MH, Thomason J, Tintle NL, Wang D, Xia F, Yoo H, Yoo S, Yu D (2018) KBase: The United States department of energy systems biology knowledgebase. Nat Biotechnol 36:566\u0026ndash;569. https://doi.org/10.1038/nbt.4163\u003c/li\u003e\n\u003cli\u003eAssenberg R, Wan PT, Geisse S, Mayr LM (2013) Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 23:393\u0026ndash;402. https://doi.org/10.1016/j.sbi.2013.03.008\u003c/li\u003e\n\u003cli\u003eBankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin A V., Sirotkin A V., Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455\u0026ndash;477. https://doi.org/10.1089/cmb.2012.0021\u003c/li\u003e\n\u003cli\u003eBateman A, Martin M-J, Orchard S, Magrane M, Ahmad S, Alpi E, Bowler-Barnett EH, Britto R, Bye-A-Jee H, Cukura A, Denny P, Dogan T, Ebenezer T, Fan J, Garmiri P, da Costa Gonzales LJ, Hatton-Ellis E, Hussein A, Ignatchenko A, Insana G, Ishtiaq R, Joshi V, Jyothi D, Kandasaamy S, Lock A, Luciani A, Lugaric M, Luo J, Lussi Y, MacDougall A, Madeira F, Mahmoudy M, Mishra A, Moulang K, Nightingale A, Pundir S, Qi G, Raj S, Raposo P, Rice DL, Saidi R, Santos R, Speretta E, Stephenson J, Totoo P, Turner E, Tyagi N, Vasudev P, Warner K, Watkins X, Zaru R, Zellner H, Bridge AJ, Aimo L, Argoud-Puy G, Auchincloss AH, Axelsen KB, Bansal P, Baratin D, Batista Neto TM, Blatter M-C, Bolleman JT, Boutet E, Breuza L, Gil BC, Casals-Casas C, Echioukh KC, Coudert E, Cuche B, de Castro E, Estreicher A, Famiglietti ML, Feuermann M, Gasteiger E, Gaudet P, Gehant S, Gerritsen V, Gos A, Gruaz N, Hulo C, Hyka-Nouspikel N, Jungo F, Kerhornou A, Le Mercier P, Lieberherr D, Masson P, Morgat A, Muthukrishnan V, Paesano S, Pedruzzi I, Pilbout S, Pourcel L, Poux S, Pozzato M, Pruess M, Redaschi N, Rivoire C, Sigrist CJA, Sonesson K, Sundaram S, Wu CH, Arighi CN, Arminski L, Chen C, Chen Y, Huang H, Laiho K, McGarvey P, Natale DA, Ross K, Vinayaka CR, Wang Q, Wang Y, Zhang J (2023) UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res 51:D523\u0026ndash;D531. https://doi.org/10.1093/nar/gkac1052\u003c/li\u003e\n\u003cli\u003eBen Tiba S, Laarif A, Wennmann JT, Bouslama T, Jehle JA (2022) Biological activity and genome composition of a Tunisian isolate of \u003cem\u003eSpodoptera littoralis\u003c/em\u003e nucleopolyhedrovirus (SpliNPV-Tun2). Egypt J Biol Pest Control 32:71. https://doi.org/10.1186/s41938-022-00568-w\u003c/li\u003e\n\u003cli\u003eBenson G (1999) Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Res 27:573\u0026ndash;580. https://doi.org/10.1093/nar/27.2.573\u003c/li\u003e\n\u003cli\u003eBlissard GW, Theilmann DA (2018) Baculovirus entry and egress from insect cells. Annu Rev Virol 5:113\u0026ndash;139. https://doi.org/10.1146/annurev-virology-092917-043356\u003c/li\u003e\n\u003cli\u003eBulach DM, Kumar CA, Zaia A, Liang B, Tribe DE (1999) Group II nucleopolyhedrovirus subgroups revealed by phylogenetic analysis of polyhedrin and DNA polymerase gene sequences. J Invertebr Pathol 73:59\u0026ndash;73. https://doi.org/10.1006/jipa.1998.4797\u003c/li\u003e\n\u003cli\u003eChen G, Fang Y, Yan Q, Li P, Wu L, Feng G (2019) The deficiency in nuclear localization signal of Neodiprion lecontei nucleopolyhedrovirus DNA polymerase prevents rescue of viral DNA replication and virus production in dnapol-null Autographa californica multiple nucleopolyhedrovirus. Virus Res 266:52\u0026ndash;57. https://doi.org/10.1016/j.virusres.2019.04.005\u003c/li\u003e\n\u003cli\u003eChen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884\u0026ndash;i890. https://doi.org/10.1093/bioinformatics/bty560\u003c/li\u003e\n\u003cli\u003eCochran MA, Faulkner P (1983) Location of homologous DNA sequences interspersed at five regions in the baculovirus AcMNPV genome. J Virol 45:961\u0026ndash;970. https://doi.org/10.1128/JVI.45.3.961-970.1983\u003c/li\u003e\n\u003cli\u003eConesa A, G\u0026ouml;tz S, Garc\u0026iacute;a-G\u0026oacute;mez JM, Terol J, Tal\u0026oacute;n M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674\u0026ndash;3676. https://doi.org/10.1093/bioinformatics/bti610\u003c/li\u003e\n\u003cli\u003eCraveiro SR, Melo FL, Ribeiro ZMA, Ribeiro BM, B\u0026aacute;o SN, Inglis PW, Castro MEB (2013) Pseudoplusia includens single nucleopolyhedrovirus: genetic diversity, phylogeny and hypervariability of the pif-2 gene. J Invertebr Pathol 114:258\u0026ndash;267. https://doi.org/10.1016/j.jip.2013.08.005\u003c/li\u003e\n\u003cli\u003ede Souza RF, Aravind L (2012) Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions. Mol Biosyst 8:1661\u0026ndash;1677. https://doi.org/10.1039/c2mb05487f\u003c/li\u003e\n\u003cli\u003eDelcher AL, Harmon D, Kasif S, White O, Salzberg SL (1999) Improved microbial gene identification with GLIMMER. Nucleic Acids Res 27:4636\u0026ndash;41. https://doi.org/10.1093/nar/27.23.4636\u003c/li\u003e\n\u003cli\u003eDong Z-Q, Hu N, Zhang J, Chen T-T, Cao M-Y, Li H-Q, Lei X-J, Chen P, Lu C, Pan M-H (2015) Oligomerization of baculovirus LEF-11 is involved in viral DNA replication. PLoS One 10:e0144930. https://doi.org/10.1371/journal.pone.0144930\u003c/li\u003e\n\u003cli\u003eDuffy SP, Young AM, Morin B, Lucarotti CJ, Koop BF, Levin DB (2006) Sequence analysis and organization of the Neodiprion abietis nucleopolyhedrovirus genome. J Virol 80:6952\u0026ndash;6963. https://doi.org/10.1128/JVI.00187-06\u003c/li\u003e\n\u003cli\u003eFerrelli ML, Salvador R, Biedma ME, Berretta MF, Haase S, Sciocco-Cap A, Ghiringhelli PD, Romanowski V (2012) Genome of Epinotia aporema granulovirus (EpapGV), a polyorganotropic fast killing betabaculovirus with a novel thymidylate kinase gene. BMC Genomics 13:548. https://doi.org/10.1186/1471-2164-13-548\u003c/li\u003e\n\u003cli\u003eGarcia-Maruniak A, Maruniak JE, Zanotto PMA, Doumbouya AE, Liu J-C, Merritt TM, Lanoie JS (2004) Sequence analysis of the genome of the Neodiprion sertifer nucleopolyhedrovirus. J Virol 78:7036\u0026ndash;7051. https://doi.org/10.1128/JVI.78.13.7036-7051.2004\u003c/li\u003e\n\u003cli\u003eGilbert C, Chateigner A, Ernenwein L, Barbe V, B\u0026eacute;zier A, Herniou EA, Cordaux R (2014) Population genomics supports baculoviruses as vectors of horizontal transfer of insect transposons. Nat Commun 5:3348. https://doi.org/10.1038/ncomms4348\u003c/li\u003e\n\u003cli\u003eGoto C, Hayakawa T, Maeda S (1998) Genome organization of Xestia c-nigrum granulovirus. Virus Genes 16:199\u0026ndash;210. https://doi.org/10.1023/a:1007972108026\u003c/li\u003e\n\u003cli\u003eHang X, Guarino LA (1999) Purification of Autographa californica nucleopolyhedrovirus DNA polymerase from infected insect cells. J Gen Virol 80:2519\u0026ndash;2526. https://doi.org/10.1099/0022-1317-80-9-2519\u003c/li\u003e\n\u003cli\u003eHarrison RL, Rowley DL (2022) The complete genome sequence of an alphabaculovirus from the brown tussock moth, \u003cem\u003eOlene mendosa\u003c/em\u003e H\u0026uuml;bner, expands our knowledge of lymantriine baculovirus diversity and evolution. Virus Genes 58:227\u0026ndash;237. https://doi.org/10.1007/s11262-022-01899-4\u003c/li\u003e\n\u003cli\u003eHerniou EA, Jehle JA (2007) Baculovirus phylogeny and evolution. Curr Drug Targets 8:1043\u0026ndash;1050. https://doi.org/10.2174/138945007782151306\u003c/li\u003e\n\u003cli\u003eIkeda M, Hamajima R, Kobayashi M (2015) Baculoviruses: diversity, evolution and manipulation of insects. Entomol Sci 18:1\u0026ndash;20. https://doi.org/10.1111/ens.12105\u003c/li\u003e\n\u003cli\u003eImai N, Matsuda N, Tanaka K, Nakano A, Matsumoto S, Kang W (2003) Ubiquitin ligase activities of Bombyx mori nucleopolyhedrovirus RING finger proteins. J Virol 77:923\u0026ndash;30. https://doi.org/10.1128/jvi.77.2.923-930.2003\u003c/li\u003e\n\u003cli\u003eIshimwe E, Hodgson JJ, Clem RJ, Passarelli AL (2015) Reaching the melting point: degradative enzymes and protease inhibitors involved in baculovirus infection and dissemination. Virology 479\u0026ndash;480:637\u0026ndash;49. https://doi.org/10.1016/j.virol.2015.01.027\u003c/li\u003e\n\u003cli\u003eIyer LM, Burroughs AM, Anantharaman V, Aravind L (2022) Apprehending the NAD+-ADPr-dependent systems in the virus world. Viruses 14:1977. https://doi.org/10.3390/v14091977\u003c/li\u003e\n\u003cli\u003eJayachandran B, Hussain M, Asgari S (2012) RNA interference as a cellular defense mechanism against the DNA virus baculovirus. J Virol 86:13729\u0026ndash;13734. https://doi.org/10.1128/JVI.02041-12\u003c/li\u003e\n\u003cli\u003eJehle JA, Blissard GW, Bonning BC, Cory JS, Herniou EA, Rohrmann GF, Theilmann DA, Thiem SM, Vlak JM (2006a) On the classification and nomenclature of baculoviruses: a proposal for revision. Arch Virol 151:1257\u0026ndash;66. https://doi.org/10.1007/s00705-006-0763-6\u003c/li\u003e\n\u003cli\u003eJehle JA, Lange M, Wang H, Hu Z, Wang Y, Hauschild R (2006b) Molecular identification and phylogenetic analysis of baculoviruses from Lepidoptera. Virology 346:180\u0026ndash;193. https://doi.org/10.1016/j.virol.2005.10.032\u003c/li\u003e\n\u003cli\u003eJi X, Axford D, Owen R, Evans G, Ginn HM, Sutton G, Stuart DI (2015) Polyhedra structures and the evolution of the insect viruses. J Struct Biol 192:88\u0026ndash;99. https://doi.org/10.1016/j.jsb.2015.08.009\u003c/li\u003e\n\u003cli\u003eJin J, Dong W, Guarino LA (1998) The LEF-4 subunit of baculovirus RNA polymerase has RNA 5\u0026prime;-triphosphatase and ATPase activities. J Virol 72:10011\u0026ndash;10019. https://doi.org/10.1128/JVI.72.12.10011-10019.1998\u003c/li\u003e\n\u003cli\u003eKatoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772\u0026ndash;80. https://doi.org/10.1093/molbev/mst010\u003c/li\u003e\n\u003cli\u003eKearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647\u0026ndash;9. https://doi.org/10.1093/bioinformatics/bts199\u003c/li\u003e\n\u003cli\u003eKimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111\u0026ndash;120. https://doi.org/10.1007/BF01731581\u003c/li\u003e\n\u003cli\u003eKogan PH, Blissard GW (1994) A baculovirus gp64 early promoter is activated by host transcription factor binding to CACGTG and GATA elements. J Virol 68:813\u0026ndash;822. https://doi.org/10.1128/JVI.68.2.813-822.1994\u003c/li\u003e\n\u003cli\u003eKrappa R, Behn-Krappa A, Jahnel F, Doerfler W, Knebel-M\u0026ouml;rsdorf D (1992) Differential factor binding at the promoter of early baculovirus gene PE38 during viral infection: GATA motif is recognized by an insect protein. J Virol 66:3494\u0026ndash;3503. https://doi.org/10.1128/jvi.66.6.3494-3503.1992\u003c/li\u003e\n\u003cli\u003eKrejmer-Rabalska M, Rabalski L, Skrzecz I, Szewczyk B (2016) Complete genome sequence of \u003cem\u003eLymantria dispar\u003c/em\u003e multiple nucleopolyhedrovirus isolated in Southwestern Poland. Microbiol Resour Announc 4. https://doi.org/10.1128/genomeA.01422-16\u003c/li\u003e\n\u003cli\u003eLangmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357\u0026ndash;359. https://doi.org/10.1038/nmeth.1923\u003c/li\u003e\n\u003cli\u003eLauzon HAM (2006) Genomic comparison of \u003cem\u003eNeodiprion sertifer \u003c/em\u003eand\u003cem\u003e Neodiprion lecontei \u003c/em\u003enucleopolyhedroviruses and identification of potential hymenopteran baculovirus-specific open reading frames. J Gen Virol 87:1477\u0026ndash;1489. https://doi.org/10.1099/vir.0.81727-0\u003c/li\u003e\n\u003cli\u003eLauzon HAM, Lucarotti CJ, Krell PJ, Feng Q, Retnakaran A, Arif BM (2004) Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome. J Virol 78:7023\u0026ndash;35. https://doi.org/10.1128/JVI.78.13.7023-7035.2004\u003c/li\u003e\n\u003cli\u003eLi Y, Guarino LA (2008) Roles of LEF-4 and PTP/BVP RNA triphosphatases in processing of baculovirus late mRNAs. J Virol 82:5573\u0026ndash;5583. https://doi.org/10.1128/JVI.00058-08\u003c/li\u003e\n\u003cli\u003eLi Y, Liu X, Tang P, Zhang H, Qin Q, Zhang Z (2021) Genome sequence and organization of the Mythimna (formerly \u003cem\u003ePseudaletia\u003c/em\u003e) unipuncta granulovirus Hawaiian strain. Sci Rep 11:414. https://doi.org/10.1038/s41598-020-80117-3\u003c/li\u003e\n\u003cli\u003eLorick KL, Jensen JP, Fang S, Ong AM, Hatakeyama S, Weissman AM (1999) RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc Natl Acad Sci U S A 96:11364\u0026ndash;9. https://doi.org/10.1073/pnas.96.20.11364\u003c/li\u003e\n\u003cli\u003eLu A, Miller LK (1995) The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. J Virol 69:975\u0026ndash;982. https://doi.org/10.1128/jvi.69.2.975-982.1995\u003c/li\u003e\n\u003cli\u003eL\u0026uuml;scher B, Ahel I, Altmeyer M, Ashworth A, Bai P, Chang P, Cohen M, Corda D, Dantzer F, Daugherty MD, Dawson TM, Dawson VL, Deindl S, Fehr AR, Feijs KLH, Filippov D V., Gagn\u0026eacute; J-P, Grimaldi G, Guettler S, Hoch NC, Hottiger MO, Korn P, Kraus WL, Ladurner A, Lehti\u0026ouml; L, Leung AKL, Lord CJ, Mangerich A, Matic I, Matthews J, Moldovan G-L, Moss J, Natoli G, Nielsen ML, Niepel M, Nolte F, Pascal J, Paschal BM, Pawłowski K, Poirier GG, Smith S, Timinszky G, Wang Z-Q, Y\u0026eacute;lamos J, Yu X, Zaja R, Ziegler M (2022) ADP-ribosyltransferases, an update on function and nomenclature. FEBS J 289:7399\u0026ndash;7410. https://doi.org/10.1111/febs.16142\u003c/li\u003e\n\u003cli\u003eManni M, Berkeley MR, Seppey M, Sim\u0026atilde;o FA, Zdobnov EM (2021) BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol 38:4647\u0026ndash;4654. https://doi.org/10.1093/molbev/msab199\u003c/li\u003e\n\u003cli\u003eMcDougal V V., Guarino LA (2001) DNA and ATP binding activities of the baculovirus DNA helicase P143. J Virol 75:7206\u0026ndash;7209. https://doi.org/10.1128/JVI.75.15.7206-7209.2001\u003c/li\u003e\n\u003cli\u003eMehrabadi M, Hussain M, Matindoost L, Asgari S (2015) The baculovirus antiapoptotic p35 protein functions as an inhibitor of the host RNA interference antiviral response. J Virol 89:8182\u0026ndash;92. https://doi.org/10.1128/JVI.00802-15\u003c/li\u003e\n\u003cli\u003eMérens A, Matrat S, Aubry A, Lascols C, Jarlier V, Soussy C-J, Cavallo J-D, Cambau E (2009) The pentapeptide repeat proteins MfpAMt and QnrB4 exhibit opposite effects on DNA gyrase catalytic reactions and on the ternary gyrase-DNA-quinolone complex. J Bacteriol 191:1587\u0026ndash;1594. https://doi.org/10.1128/JB.01205-08\u003c/li\u003e\n\u003cli\u003eMiele SAB, Garavaglia MJ, Belaich MN, Ghiringhelli PD (2011) Baculovirus: molecular insights on their diversity and conservation. Int J Evol Biol 2011:1\u0026ndash;15. https://doi.org/10.4061/2011/379424\u003c/li\u003e\n\u003cli\u003eMikhailov VS, Okano K, Rohrmann GF (2003) Baculovirus alkaline nuclease possesses a 5\u0026prime;\u0026rarr;3\u0026prime; exonuclease activity and associates with the DNA-binding protein LEF-3. J Virol 77:2436\u0026ndash;2444. https://doi.org/10.1128/JVI.77.4.2436-2444.2003\u003c/li\u003e\n\u003cli\u003eMikhailov VS, Rohrmann GF (2002) Baculovirus replication factor LEF-1 is a DNA primase. J Virol 76:2287\u0026ndash;2297. https://doi.org/10.1128/jvi.76.5.2287-2297.2002\u003c/li\u003e\n\u003cli\u003eMinh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, Lanfear R (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 37:1530\u0026ndash;1534. https://doi.org/10.1093/molbev/msaa015\u003c/li\u003e\n\u003cli\u003eMistretta T-A, Guarino LA (2005) Transcriptional activity of baculovirus very late factor 1. J Virol 79:1958\u0026ndash;1960. https://doi.org/10.1128/JVI.79.3.1958-1960.2005\u003c/li\u003e\n\u003cli\u003eOlson VA, Wetter JA, Friesen PD (2003) The highly conserved basic domain I of baculovirus IE1 is required for hr enhancer DNA binding and hr -dependent transactivation. J Virol 77:5668\u0026ndash;5677. https://doi.org/10.1128/JVI.77.10.5668-5677.2003\u003c/li\u003e\n\u003cli\u003ePassarelli AL (2007) Baculovirus RNA polymerase: activities, composition, and evolution. Virol Sin 22:94\u0026ndash;107. https://doi.org/10.1007/s12250-007-0011-6\u003c/li\u003e\n\u003cli\u003ePaysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, Bileschi ML, Bork P, Bridge A, Colwell L, Gough J, Haft DH, Letunić I, Marchler-Bauer A, Mi H, Natale DA, Orengo CA, Pandurangan AP, Rivoire C, Sigrist CJA, Sillitoe I, Thanki N, Thomas PD, Tosatto SCE, Wu CH, Bateman A (2023) InterPro in 2022. Nucleic Acids Res 51:D418\u0026ndash;D427. https://doi.org/10.1093/nar/gkac993\u003c/li\u003e\n\u003cli\u003eQuadt I, van Lent JWM, Knebel-Mörsdorf D (2007) Studies of the silencing of baculovirus DNA binding protein. J Virol 81:6122\u0026ndash;6127. https://doi.org/10.1128/JVI.02768-06\u003c/li\u003e\n\u003cli\u003eRambaut A (2014) FigTree v1.4.2, A Graphical Viewer of Phylogenetic Trees. Available from 〈http//tree.bio.ed.ac.uk/software/figtree/〉\u003c/li\u003e\n\u003cli\u003eRambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst Biol 67:901\u0026ndash;904. https://doi.org/10.1093/sysbio/syy032\u003c/li\u003e\n\u003cli\u003eRohrmann GF (2019) Baculovirus Molecular Biology [Internet], 4th edn. Bethesda (MD): National Center for Biotechnology Information (US)\u003c/li\u003e\n\u003cli\u003eRonquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, H\u0026ouml;hna S, Larget B, Liu L, Suchard M a., Huelsenbeck JP (2012) Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539\u0026ndash;542. https://doi.org/10.1093/sysbio/sys029\u003c/li\u003e\n\u003cli\u003eSeemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068\u0026ndash;2069. https://doi.org/10.1093/bioinformatics/btu153\u003c/li\u003e\n\u003cli\u003eSlack J, Arif BM (2007) The baculoviruses occlusion-derived virus: virion structure and function. Adv Virus Res 69:99\u0026ndash;165. https://doi.org/10.1016/S0065-3527(06)69003-9\u003c/li\u003e\n\u003cli\u003eSlack JM, Lawrence SD, Krell PJ, Arif BM (2008) Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection. J Gen Virol 89:2388\u0026ndash;2397. https://doi.org/10.1099/vir.0.2008/002543-0\u003c/li\u003e\n\u003cli\u003eSong J, Wang X, Hou D, Huang H, Liu X, Deng F, Wang H, Arif BM, Hu Z, Wang M (2016) The host specificities of baculovirus per os infectivity factors. PLoS One 11:e0159862. https://doi.org/10.1371/journal.pone.0159862\u003c/li\u003e\n\u003cli\u003eSu J, Lung O, Blissard GW (2011) The Autographa californica multiple nucleopolyhedrovirus lef-5 gene is required for productive infection. Virology 416:54\u0026ndash;64. https://doi.org/10.1016/j.virol.2011.04.019\u003c/li\u003e\n\u003cli\u003eSzewczyk B, Hoyos-Carvajal L, Paluszek M, Skrzecz I, Lobo de Souza M (2006) Baculoviruses \u0026mdash; re-emerging biopesticides. Biotechnol Adv 24:143\u0026ndash;160. https://doi.org/10.1016/j.biotechadv.2005.09.001\u003c/li\u003e\n\u003cli\u003eTamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725\u0026ndash;2729. https://doi.org/10.1093/molbev/mst197\u003c/li\u003e\n\u003cli\u003eThiem SM, Cheng X-W (2009) Baculovirus host-range. Virol Sin 24:436\u0026ndash;457. https://doi.org/10.1007/s12250-009-3058-8\u003c/li\u003e\n\u003cli\u003eTodd JW, Passarelli AL, Miller LK (1995) Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J Virol 69:968\u0026ndash;974. https://doi.org/10.1128/jvi.69.2.968-974.1995\u003c/li\u003e\n\u003cli\u003eVaidya G, Lohman DJ, Meier R (2011) SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27:171\u0026ndash;180. https://doi.org/10.1111/j.1096-0031.2010.00329.x\u003c/li\u003e\n\u003cli\u003evan Oers M, Vlak J (2007) Baculovirus genomics. Curr Drug Targets 8:1051\u0026ndash;1068. https://doi.org/10.2174/138945007782151333\u003c/li\u003e\n\u003cli\u003eVanarsdall AL, Okano K, Rohrmann GF (2006) Characterization of the role of very late expression factor 1 in baculovirus capsid structure and DNA processing. J Virol 80:1724\u0026ndash;1733. https://doi.org/10.1128/JVI.80.4.1724-1733.2006\u003c/li\u003e\n\u003cli\u003eVaux DL, Silke J (2005) IAPs, RINGs and ubiquitylation. Nat Rev Mol Cell Biol 6:287\u0026ndash;97. https://doi.org/10.1038/nrm1621\u003c/li\u003e\n\u003cli\u003eWang J, Hou D, Wang Q, Kuang W, Zhang L, Li J, Shen S, Deng F, Wang H, Hu Z, Wang M (2018) Genome analysis of a novel Group I alphabaculovirus obtained from Oxyplax ochracea. PLoS One 13:e0192279. https://doi.org/10.1371/journal.pone.0192279\u003c/li\u003e\n\u003cli\u003eWang M, Hu Z (2019) Advances in Molecular Biology of Baculoviruses. In: Insect Molecular Virology: Advances and Emerging Trends. Caister Academic Press\u003c/li\u003e\n\u003cli\u003eWang M, Tuladhar E, Shen S, Wang H, van Oers MM, Vlak JM, Westenberg M (2010) Specificity of baculovirus P6.9 basic DNA-binding proteins and critical role of the C terminus in virion formation. J Virol 84:8821\u0026ndash;8828. https://doi.org/10.1128/JVI.00072-10\u003c/li\u003e\n\u003cli\u003eWang X, Shang Y, Chen C, Liu S, Chang M, Zhang N, Hu H, Zhang F, Zhang T, Wang Z, Liu X, Lin Z, Deng F, Wang H, Zou Z, Vlak JM, Wang M, Hu Z (2019) Baculovirus per os infectivity factor complex: components and assembly. J Virol 93:e02053-18. https://doi.org/10.1128/JVI.02053-18\u003c/li\u003e\n\u003cli\u003eWennmann JT, Keilwagen J, Jehle JA (2018) Baculovirus Kimura two-parameter species demarcation criterion is confirmed by the distances of 38 core gene nucleotide sequences. J Gen Virol 99:1307\u0026ndash;1320. https://doi.org/10.1099/jgv.0.001100\u003c/li\u003e\n\u003cli\u003eWood DE, Lu J, Langmead B (2019) Improved metagenomic analysis with Kraken 2. Genome Biol 20:257. https://doi.org/10.1186/s13059-019-1891-0\u003c/li\u003e\n\u003cli\u003eWu X, Guarino LA (2003) Autographa californica nucleopolyhedrovirus orf69 encodes an RNA cap (nucleoside-2\u0026prime;- O)-methyltransferase. J Virol 77:3430\u0026ndash;3440. https://doi.org/10.1128/JVI.77.6.3430-3440.2003\u003c/li\u003e\n\u003cli\u003eZanotto PM, Kessing BD, Maruniak JE (1993) Phylogenetic interrelationships among baculoviruses: evolutionary rates and host associations. J Invertebr Pathol 62:147\u0026ndash;164. https://doi.org/10.1006/jipa.1993.1090\u003c/li\u003e\n\u003cli\u003eZhang J-H, Ohkawa T, Washburn JO, Volkman LE (2005) Effects of Ac150 on virulence and pathogenesis of \u003cem\u003eAutographa californica\u003c/em\u003e multiple nucleopolyhedrovirus in noctuid hosts. J Gen Virol 86:1619\u0026ndash;1627. https://doi.org/10.1099/vir.0.80930-0\u003c/li\u003e\n\u003cli\u003eZhao S, Kong X, Wu X (2021) RNAi-based immunity in insects against baculoviruses and the strategies of baculoviruses involved in siRNA and miRNA pathways to weaken the defense. Dev Comp Immunol 122:104116. https://doi.org/10.1016/j.dci.2021.104116\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"virus-genes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"viru","sideBox":"Learn more about [Virus Genes](http://link.springer.com/journal/11262)","snPcode":"11262","submissionUrl":"https://submission.nature.com/new-submission/11262/3","title":"Virus Genes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Baculoviruses, DNA viruses, european pine sawfly, gammabaculovirus, genetic divergence, nucleopolyhedrovirus","lastPublishedDoi":"10.21203/rs.3.rs-3466248/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3466248/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe complete genome of the European pine sawfly \u003cem\u003eNeodiprion sertifer\u003c/em\u003e nucleopolyhedrovirus was sequenced and characterized from next-generation sequencing data of the \u003cem\u003eN. sertifer\u003c/em\u003e larva from Turkiye. This genome was comparatively analysed by previously reported genomes baculoviruses. The baculovirus phylogeny was reconstructed and the species boundary of the NeseNPV-TR was delineated using K2P distance. The length of the genome was 82,052 bp, with a G\u0026thinsp;+\u0026thinsp;C content of 33.28%. It contained 82 putative ORFs, including 33 baculovirus core genes, three lepidopteran baculovirus core genes, three non-conserved genes. It had five \u003cem\u003ehrs\u003c/em\u003e with 20.6% overall mean distance on average. The pairwise K2P distances of \u003cem\u003elef-9\u003c/em\u003e and \u003cem\u003epolh\u003c/em\u003e genes were lower than the specified threshold value, while those of \u003cem\u003elef-8\u003c/em\u003e, combinations of three genes and 33 genes were slightly higher between NeseNPV-TR and NeseNPV. The most variable genes were \u003cem\u003elef-2\u003c/em\u003e, \u003cem\u003ehelicase\u003c/em\u003e, \u003cem\u003ep40\u003c/em\u003e, \u003cem\u003edesmoplakin\u003c/em\u003e, \u003cem\u003ep6.9\u003c/em\u003e, \u003cem\u003evp91\u003c/em\u003e and \u003cem\u003evp39\u003c/em\u003e, while the most conserved were \u003cem\u003elef-8\u003c/em\u003e, \u003cem\u003elef-9\u003c/em\u003e, \u003cem\u003eodv-e18\u003c/em\u003e, \u003cem\u003epif2\u003c/em\u003e and \u003cem\u003elef-5\u003c/em\u003e among baculoviruses. The genome of NeseNPV-TR is smaller and contains the fewest ORFs among baculoviruses. Some of unassigned ORFs had conserved domains and hence, we suggest further investigation to determine their structural and functional roles. Phylogenetic analyses confirmed its position within Gammabaculovirus. The NeseNPV-TR can be considered as the same species with NeseNPV. The different divergence rates in the baculovirus core genes may be related with different selection pressures acting on the genes. The lower genetic diversity of Group I alphabaculoviruses is most probably due to recent emergence.\u003c/p\u003e","manuscriptTitle":"Comparative genomics of the Neodiprion sertifer nucleopolyhedrovirus from Turkey with the fewest ORFs among baculoviruses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-23 23:35:30","doi":"10.21203/rs.3.rs-3466248/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2023-12-10T22:18:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-11-06T16:09:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6b110c3b-d977-40f6-944a-1350b208fa67","date":"2023-11-03T09:08:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a5e0350e-9c88-45ef-aadd-ae3e013af950","date":"2023-10-30T11:09:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-10-30T00:55:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-10-20T11:59:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-20T11:59:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virus Genes","date":"2023-10-19T10:40:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virus-genes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"viru","sideBox":"Learn more about [Virus Genes](http://link.springer.com/journal/11262)","snPcode":"11262","submissionUrl":"https://submission.nature.com/new-submission/11262/3","title":"Virus Genes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6f0282ce-b9b4-41a0-b6db-b9a80cc09af5","owner":[],"postedDate":"October 23rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-01-22T15:05:16+00:00","versionOfRecord":{"articleIdentity":"rs-3466248","link":"https://doi.org/10.1007/s11262-024-02050-1","journal":{"identity":"virus-genes","isVorOnly":false,"title":"Virus Genes"},"publishedOn":"2024-01-19 15:00:51","publishedOnDateReadable":"January 19th, 2024"},"versionCreatedAt":"2023-10-23 23:35:30","video":"","vorDoi":"10.1007/s11262-024-02050-1","vorDoiUrl":"https://doi.org/10.1007/s11262-024-02050-1","workflowStages":[]},"version":"v1","identity":"rs-3466248","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3466248","identity":"rs-3466248","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}