A qPCR Assay for the Quantification of Selected Genotypic Variants of Spodoptera frugiperda Multiple Nucleopolyhedrovirus (Baculoviridae).

Alphabaculoviruses are lethal dsDNA viruses of Lepidoptera that have high genetic diversity and are transmitted in aggregates within proteinaceous occlusion bodies. This mode of transmission has implications for their efficacy as biological insecticides. A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV-NIC) comprising nine genotypic variants has been the subject of considerable study due to the influence of variant interactions on the insecticidal properties of mixed-variant occlusion bodies. As part of a systematic study on the replication and transmission of variant mixtures, a tool for the accurate quantification of a selection of genotypic variants was developed based on the quantitative PCR technique (qPCR). First, primer pairs were designed around a region of high variability in four variants named SfNic-A, SfNic-B, SfNic-C and SfNic-E to produce amplicons of 103-150 bp. Then, using cloned purified amplicons as standards, amplification was demonstrated over a dynamic range of 108-101 copies of each target. The assay was efficient (mean ± SD: 98.5 ± 0.8%), reproducible, as shown by low inter- and intra-assay coefficients of variation (<5%), and specific to the target variants (99.7-100% specificity across variants). The quantification method was validated on mixtures of genotype-specific amplicons and demonstrated accurate quantification. Finally, mixtures of the four variants were quantified based on mixtures of budded virions and mixtures of DNA extracted from occlusion-derived virions. In both cases, mixed-variant preparations compared favorably to total viral genome numbers by quantification of the polyhedrin (polh) gene that is present in all variants. This technique should prove invaluable in elucidating the influence of variant diversity on the transmission and insecticidal characteristics of this pathogen.

Genomic analyses of a new baculovirus isolated from the wheat armyworm, Mythimna sequax (Franclemont) (Lepidoptera: Noctuidae).

We report the genomic analysis of a novel alphabaculovirus, Mythimna sequax nucleopolyhedrovirus isolate CNPSo-98 (MyseNPV-CNPSo-98), obtained from cadavers of the winter crop pest, Mythimna sequax Franclemont (Lepidoptera: Noctuidae). The insects were collected from rice fields in Southern Brazil in the 1980's and belongs to the 'EMBRAPA-Soja' Virus Collection. High-throughput sequencing reads of DNA from MyseNPV occlusion bodies and assembly of the data yielded an AT-rich circular genome contig of 148,403 bp in length with 163 annotated opening reading frames (ORFs) and four homologous regions (hrs). Phylogenetic inference based on baculovirus core protein sequence alignments indicated that MyseNPV-CNPSo-98 is a member of Alphabaculovirus genus that clustered with other group II noctuid-infecting baculoviruses, including viruses isolated from Helicoverpa armigera and Mamestra spp. The genomes of the clade share strict collinearity and high pairwise nucleotide identity, with a common set of 149 genes, evolving under negative selection, except a bro gene. Branch lengths and Kimura-2-parameter pairwise nucleotide distances indicated that MyseNPV-CNPSo-98 represents a distinct lineage that may not be classified in any of the currently listed species in the genus.

Haplotype determination of the Bombyx mori nucleopolyhedrovirus by Nanopore sequencing and linkage of single nucleotide variants.

Naturally occurring isolates of baculoviruses, such as the Bombyx mori nucleopolyhedrovirus (BmNPV), usually consist of numerous genetically different haplotypes. Deciphering the different haplotypes of such isolates is hampered by the large size of the dsDNA genome, as well as the short read length of next generation sequencing (NGS) techniques that are widely applied for baculovirus isolate characterization. In this study, we addressed this challenge by combining the accuracy of NGS to determine single nucleotide variants (SNVs) as genetic markers with the long read length of Nanopore sequencing technique. This hybrid approach allowed the comprehensive analysis of genetically homogeneous and heterogeneous isolates of BmNPV. Specifically, this allowed the identification of two putative major haplotypes in the heterogeneous isolate BmNPV-Ja by SNV position linkage. SNV positions, which were determined based on NGS data, were linked by the long Nanopore reads in a Position Weight Matrix. Using a modified Expectation-Maximization algorithm, the Nanopore reads were assigned according to the occurrence of variable SNV positions by machine learning. The cohorts of reads were de novo assembled, which led to the identification of BmNPV haplotypes. The method demonstrated the strength of the combined approach of short- and long-read sequencing techniques to decipher the genetic diversity of baculovirus isolates.

Leucoma salicis nucleopolyhedrovirus (LesaNPV) genome sequence shed new light on the origin of the Alphabaculovirus orpseudotsugatae species.

LesaNPV (Leucoma salicis nucleopolyhedrovirus) is an alphabaculovirus group Ib. Potentially, it can be an eco-friendly agent to control the white satin moth Leucoma salicis population. In this study, we have established the relationship between LesaNPV and other closely related alphabaculoviruses. Environmental samples of late instar of white satin moth collected in Poland infected with baculovirus have been homogenized, polyhedra were purified and subjected to scanning and transmission electron microscopy. Viral DNA was sequenced using the Illumina platform and the whole-genome sequence was established by de novo assembly of paired reads. Genome annotation and phylogenetic analyses were performed with the use of bioinformatics tools. The genome of LesaNPV is 132 549 bp long with 154 ORFs and 54.9% GC content. Whole-genome sequencing revealed deletion of dUTPase as well as ribonucleoside reductases small and large subunits region in LesaNPV genome compared to Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) and Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) where this region is complete. Phylogenetic analysis of Baculoviridae family members showed that LesaNPV is less divergent from a common ancestor than closely related species DapuNPV and OpMNPV. This is interesting because their hosts do not occur in the same area. The baculoviruses described in this manuscript are probably isolates of one species and could be assigned to recently denominated species Alphabaculovirus orpseudotsugatae, historically originating from OpMNPV. This finding could have significant implications for the classification and understanding of the phylogeographical spread of baculoviruses.

An electrochemical immunosensor based on hybrid self-assembled monolayers for rapid detection of Bombyx mori nucleopolyhedrovirus.

Bombyx mori nucleopolyhedrovirus (BmNPV) is highly contagious and poses a serious threat to sericulture production. Because there are currently no effective treatments for BmNPV, a rapid and simple detection method is urgently needed. This paper describes an electrochemical immunosensor for the detection of BmNPV. The immunosensor was fabricated by covalently immobilizing anti-BmNPV, a biorecognition element, onto the surface of the working gold electrode via 11-mercaptoundecanoic acid (MUA)/ß-mercaptoethanol (ME) hybrid self-assembled monolayers. Electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) were used to characterize the electrochemical performance and morphology of the immunosensor, respectively. Under optimum conditions, the developed immunosensor exhibited a linear response to BmNPV polyhedrin in the range of 1 × 102-1 × 108 fg/mL, with a low detection limit of 14.54 fg/mL. The immunosensor also exhibited remarkable repeatability, reproducibility, specificity, accuracy, and regeneration. Normal silkworm blood was mixed with BmNPV polyhedrin and analyzed quantitatively using this sensor, and the recovery was 92.31 %-100.61 %. Additionally, the sensor was used to analyze silkworm blood samples at different time points after BmNPV infection, and an obvious antigen signal was detected at 12 h post infection. Although this result agreed with that provided by the conventional polymerase chain reaction (PCR) method, the electroanalysis method established in this study was simpler, shorter in detection period, and lower in material cost. Furthermore, this innovative electrochemical immunosensor, developed for the ultra-sensitive and rapid detection of BmNPV, can be used for the early detection of virus-infected silkworms.

Patterns in Genotype Composition of Indian Isolates of the Bombyx mori Nucleopolyhedrovirus and Bombyx mori Bidensovirus.

The mulberry silkworm, Bombyx mori (L.), is a model organism of lepidopteran insects with high economic importance. The viral diseases of the silkworm caused by Bombyx mori nucleopolyhedrovirus (BmNPV) and Bombyx mori bidensovirus (BmBDV) inflict huge economic losses and significantly impact the sericulture industry of India and other countries. To understand the distribution of Indian isolates of the BmNPV and to investigate their genetic composition, an in-depth population structure analysis was conducted using comprehensive and newly developed genomic analysis methods. The seven new Indian BmNPV isolates from Anantapur, Dehradun, Ghumarwin, Jammu, Kashmir, Mysore and Salem grouped in the BmNPV clade, and are most closely related to Autographa californica multiple nucleopolyhedrovirus and Rachiplusia ou multiple nucleopolyhedrovirus on the basis of gene sequencing and phylogenetic analyses of the partial polh, lef-8 and lef-9 gene fragments. The whole genome sequencing of three Indian BmNPV isolates from Mysore (-My), Jammu (-Ja) and Dehradun (-De) was conducted, and intra-isolate genetic variability was analyzed on the basis of variable SNP positions and the frequencies of alternative nucleotides. The results revealed that the BmNPV-De and BmNPV-Ja isolates are highly similar in their genotypic composition, whereas the population structure of BmNPV-My appeared rather pure and homogenous, with almost no or few genetic variations. The BmNPV-De and BmNPV-Ja samples further contained a significant amount of BmBDV belonging to the Bidnaviridae family. We elucidated the genotype composition within Indian BmNPV and BmBDV isolates, and the results presented have broad implications for our understanding of the genetic diversity and evolution of BmNPV and co-occurring BmBDV isolates.

Genomic analyses of Biston suppressaria nucleopolyhedrovirus: a viral isolate obtained from the tea looper caterpillar, Biston suppressaria (Guenée, 1857).

We described the complete genome sequence of a novel baculovirus isolate of species Buzura suppressaria nucleopolyhedrovirus, called by isolate CNPSo-25. The occlusion bodies were found to be polyhedral in shape and to contain virions with singly embedded nucleocapsids. The size of the genome is 121,377 bp with a G+C content of 36.7%. We annotated 131 ORFs that cover 90.42% of the genome. Moreover, phylogenetic inference indicated that CNPSo-25 is a member of genus Alphabaculovirus that clustered together with two other Chinese isolates of the same species. We called the virus by Biston suppressaria nucleopolyhedrovirus isolate CNPSo-25 (BisuNPV-CNPSo-25), as Buzura was placed inside the lepidopteran genus Biston. As expected, we detected intra-population variability in the virus sample when the novel isolate was compared to the Chinese isolates: 292 single nucleotide variants were found in the genome, with 181 affecting the protein product. The closest representatives of other species to BisuNPV-CNPSo-25 was found to be Sucra jujuba nucleopolyhedrovirus and Hyposidra talaca nucleopolyhedrovirus, two other virus isolates of geometrid caterpillars. The study of baculovirus genomes is of importance for the development of tools for insect pest biological control and biotechnology.

Genome Analysis of an Alphabaculovirus Isolated from the Larch Looper, Erannis ankeraria.

The larch looper, Erannis ankeraria Staudinger (Lepidoptera: Geometridae), is one of the major insect pests of larch forests, widely distributed from southeastern Europe to East Asia. A naturally occurring baculovirus, Erannis ankeraria nucleopolyhedrovirus (EranNPV), was isolated from E. ankeraria larvae. This virus was characterized by electron microscopy and by sequencing the whole viral genome. The occlusion bodies (OBs) of EranNPV exhibited irregular polyhedral shapes containing multiple enveloped rod-shaped virions with a single nucleocapsid per virion. The EranNPV genome was 125,247 bp in length with a nucleotide distribution of 34.9% G+C. A total of 131 hypothetical open reading frames (ORFs) were identified, including the 38 baculovirus core genes and five multi-copy genes. Five homologous regions (hrs) were found in the EranNPV genome. Phylogeny and pairwise kimura 2-parameter analysis indicated that EranNPV was a novel group II alphabaculovirus and was most closely related to Apocheima cinerarium NPV (ApciNPV). Field trials showed that EranNPV was effective in controlling E. ankeraria in larch forests. The above results will be relevant to the functional research on EranNPV and promote the use of this virus as a biocontrol agent.

Identification of miRNAs encoded by Autographa californica nucleopolyhedrovirus.

Two Autographa californica nucleopolyhedrovirus (AcMNPV) encoded miRNAs, AcMNPV-miR-1 and AcMNPV-miR-3, have been reported by us in 2013 and 2019, respectively. Here, we present an integrated investigation of AcMNPV-encoded miRNAs, which include the above two miRNAs and three additional newly identified miRNAs. Six candidate miRNAs were predicted through small RNA deep sequencing and bioinformatics, of which, five were validated. Three miRNAs are located opposite the coding sequences, the other two are located in the coding sequences of viral genes. Targets in both virus and host were predicted and subsequently tested using dual-luciferase reporter assays. The validated targets were found mainly in AcMNPV, except for the targets of AcMNPV-miR-4, which are all host genes. Based on reporter assays, the five miRNAs predominantly function by down-regulating their targets. The transcription start sites of these miRNAs were bioinformatic screened based on known baculovirus promoter motifs. Our study reveals that AcMNPV-encoded miRNAs function as fine modulators of the interactions between host and virus by regulating viral and/or host genes.

Proliferation of Bombyx mori nucleopolyhedrovirus strain H4 in BmN cells is enhanced by exchange of the F gene sequence with type strain T3.

The Bombyx mori nucleopolyhedrovirus (BmNPV)-based baculoviral expression vector system is among the most efficient expression vector systems for eukaryotic proteins especially when used in combination with silkworms as a host. We newly isolated a novel BmNPV strain (BmNPV H4) in Hokkaido, Japan that outperforms the type strain T3 in terms of both proliferation and expression of polyhedrin protein in silkworm larvae; however, it proliferates poorly in the BmN cell line. We inferred the gene responsible for the differences in proliferation between viral strains by quantifying amino acid similarity distances in protein functional domains and identifying highly divergent alleles between the H4 and T3 strains. Among proteins that differ markedly in functional domain sequence between H4 and T3, we identified the F gene, which encodes the F protein, as a putative cause of proliferative differences between the two strains. Using recombinant viruses with the F protein-coding sequence exchanged between H4 and T3, we determined that the T3 F protein increases H4 proliferation in BmN while the H4 F protein does not improve T3 proliferation in silkworm larvae. Our results suggest that the BmNPV F protein can strongly affect viral proliferation in a genetic background-specific manner and may be an important target for manipulating the proliferation characteristics of BmNPV-based expression vectors.

A new single nucleopolyhedrovirus isolate from Cabbage looper, Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae): Detection, characterization and virulence.

This study was focused on the detection, characterization and virulence of a new baculovirus isolate from the larvae of cabbage looper, Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae). T. ni is a polyphagous pest, and it has cosmopolitan distribution. An infected T. ni larvae was collected from a cabbage field in Turkey. Scanning electron microscopy studies showed the presence of typical occlusion bodies (OBs) with average size of 0.76 to 1.46 µm in the collected larvae. Since the virions have single nucleocapsid within the envelopes, the isolate was named as Trichoplusia ni single nucleopolyhedrovirus Turkey isolate (TrniSNPV- TR). The total genome of the TrniSNPV-TR was determined as 122.9 kb in size. Sequence analysis of the amplified late expression factor 8 (lef8), late expression factor 9 (lef9) and polyhedrin (polh) genes showed that the virus is a new isolate of nucleopolyhedroviruses and close to Trichoplusia ni SNPV isolates mentioned in the literature. However, this is the first study for the detection and characterization of a baculovirus from T. ni in Eurasian region. Insecticidal activities of the TrniSNPV-TR isolate (106 OBs/ml-1) against neonate, 3rd and 5th instar larvae of T. ni and Helicoverpa armigera showed 98%-91%, 91%-87% and 65%-60% mortalities, respectively, within 14 days. LC50 of TrniSNPV-TR was determined as 2×103-9×103, 3×104-7×104 and 1×105-2×105 OBs/ml on neonate, 3rd and 5th instar larvae, respectively. All these results showed that, TrniSNPV-TR has good potential to be utilized as a bio-pesticide against T. ni larvae in the future. Keywords: baculovirus; nucleopolyhedrovirus; Trichoplusia ni; TrniSNPV-TR; biological control.

Characterization of a novel alphabaculovirus isolated from the Southern armyworm, Spodoptera eridania (Cramer, 1782) (Lepidoptera: Noctuidae) and the evolution of odv-e66, a bacterium-acquired baculoviral chondroitinase gene.

The Southern armyworm Spodoptera eridania (Lepidoptera: Noctuidae) is native to the American tropics and a polyphagous pest of several crops. Here we characterized a novel alphabaculovirus isolated from S. eridania, isolate Spodoptera eridania nucleopolyhedrivurus CNPSo-165 (SperNPV-CNPSo-165). SperNPV-CNPSo-165 occlusion bodies were found to be polyhedral and to contain virions with multiple nucleocapsids. The virus was lethal to S. eridania and S. albula but not to S. frugiperda. The SperNPV-CNPSo-165 genome was 137.373 bp in size with a G + C content of 42.8%. We annotated 151 ORFs with 16 ORFs unique among baculoviruses. Phylogenetic inference indicated that this virus was closely related to the most recent common ancestor of other Spodoptera-isolated viruses.

Genome sequence analysis of a Helicoverpa armigera single nucleopolyhedrovirus (HearNPV-TR) isolated from Heliothis peltigera in Turkey.

The entire genome of Helicoverpa armigera single nucleopolyhedrovirus (HearNPV-TR) was sequenced, and compared to genomes of other existing isolates. HearNPV-TR genome is 130.691 base pairs with a 38.9% G+C content and has 137 open reading frames (ORFs) of ≥ 150 nucleotides. Five homologous repeated sequences (hrs) and two baculovirus repeated ORFs (bro-a and bro-b) were identified. Phylogenetic analysis showed that HearNPV-TR is closer to HaSNPV-C1, HaSNPV-G4, HaSNPV-AU and HasNPV. However, there are significant differences in hr3, hr5 regions and in bro-a gene. Pairwise Kimura-2 parameter analysis of 38 core genes sequences of HearNPV-TR and other Helicoverpa NPVs showed that the genetic distances for these sequences were below 0.015 substitutions/site. Genomic differences as revealed by restriction profiles indicated that hr3, hr5 regions and bro-a gene may play a role in the virulence of HearNPV-TR.

Genetic variants in Argentinean isolates of Spodoptera frugiperda Multiple Nucleopolyhedrovirus.

The fall armyworm, Spodoptera frugiperda (JE Smith) is a key pest in the Americas. Control strategies are mainly carried out by use of chemical insecticides and transgenic crops expressing Bacillus thuringiensis toxins. In the last years, resistance of S. frugiperda populations to transgenic corn was reported in different Latin American countries. The baculovirus Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) is a pathogenic agent for the fall armyworm and a potential alternative for its control in integrated pest management strategies. In this work, we analyze some characteristics of two baculovirus isolates collected from maize (SfMNPV-M) and cotton (SfMNPV-C) fields from Argentina. The isolates were compared by restriction enzymes patterns and the analysis reveals the presence of genotypic variants in the SfMNPV-M isolate. We confirmed a deletion by sequencing fragments encompassing egt gene and most part of its contiguous gene (orf A) in a SfMNVP-M genotypic variant. Additionally, we estimated the 50% lethal dose and median survival time of each isolate in bioassays with S. frugiperda larvae.

Quantitative label-free proteomic analysis reveals differentially expressed proteins in the digestive juice of resistant versus susceptible silkworm strains and their predicted impacts on BmNPV infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity. In this study, antiviral activity examination of different resistant strains showed that the digestive juice of the resistant strain (A35) had higher inhibition to virus than the susceptible strain (P50). Subsequently, the label-free quantitative proteomics was used to study the midgut digestive juice response to BmNPV infection in P50 and A35 strains. A total of 98 proteins were identified, of which 80 were differentially expressed proteins (DEPs) with 54 enzymes and 26 nonenzymatic proteins by comparing the proteomes of infected and non-infected P50 and A35 silkworms. These DEPs are mainly involved in metabolism, proteolysis, neuroactive ligand receptor interaction, starch and sucrose metabolism and glutathione metabolism. After removing the genetic background and individual immune stress response proteins, 9 DEPs were identified potentially involved in resistance to BmNPV. Further studies showed that a serine protease, an alkaline phosphatase and serine protease inhibitor 2 isoform X1 were differentially expressed in A35 compared to P50 or post BmNPV infection. Taken together, these results provide insights into the potential mechanisms for silkworm digestive juice to provide resistance to BmNPV infection. Signifcance: Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, which has a great impact on the sericulture. BmNPV entered the midgut lumen and exposed to digestive juices after oral infection. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity, however, current information on the digestive juice proteome of high resistant silkworm strain after BmNPV challenge compared to susceptible strain is incomprehensive. Here, we combined label-free quantification method, bioinformatics, RT-qPCR and western blot analysis and found that BmNPV infection causes some protein changes in the silkworm midgut digestive juice. The DEPs were identified in the digestive juices of different resistant strains following BmNPV infection, and screened out some proteins potentially related to resistance to BmNPV. Three important differentially expression proteins were validated by independent approaches. These findings uncover the potential role of silkworm digestive juice in providing resistance to BmNPV and supplemented the profile of the proteome of the digestive juices in B. mori.

Genome Analysis of a Novel Clade II.b Alphabaculovirus Obtained from Artaxa digramma.

Artaxa digramma is a lepidopteran pest distributed throughout southern China, Myanmar, Indonesia, and India. Artaxa digramma nucleopolyhedrovirus (ArdiNPV) is a specific viral pathogen of A. digramma and deemed as a promising biocontrol agent against the pest. In this study, the complete genome sequence of ArdiNPV was determined by deep sequencing. The genome of ArdiNPV contains a double-stranded DNA (dsDNA) of 161,734 bp in length and 39.1% G+C content. Further, 149 hypothetical open reading frames (ORFs) were predicted to encode proteins >50 amino acids in length, covering 83% of the whole genome. Among these ORFs, 38 were baculovirus core genes, 22 were lepidopteran baculovirus conserved genes, and seven were unique to ArdiNPV, respectively. No typical baculoviral homologous regions (hrs) were identified in the genome. ArdiNPV had five multi-copy genes including baculovirus repeated ORFs (bros), calcium/sodium antiporter B (chaB), DNA binding protein (dbp), inhibitor of apoptosis protein (iap), and p26. Interestingly, phylogenetic analyses showed that ArdiNPV belonged to Clade II.b of Group II Alphabaculoviruses, which all contain a second copy of dbp. The genome of ArdiNPV was the closest to Euproctis pseudoconspersa nucleopolyhedrovirus, with 57.4% whole-genome similarity. Therefore, these results suggest that ArdiNPV is a novel baculovirus belonging to a newly identified cluster of Clade II.b Alphabaculoviruses.

A Nymphalid-Infecting Group I Alphabaculovirus Isolated from the Major Passion Fruit Caterpillar Pest Dione juno juno (Lepidoptera: Nymphalidae).

Baculoviruses are capable of infecting a wide diversity of insect pests. In the 1990s, the Dione juno nucleopolyhedrovirus (DijuNPV) was isolated from larvae of the major passionfruit defoliator pest Dione juno juno (Nymphalidae) and described at ultrastructural and pathological levels. In this study, the complete genome sequence of DijuNPV was determined and analyzed. The circular genome presents 122,075 bp with a G + C content of 50.9%. DijuNPV is the first alphabaculovirus completely sequenced that was isolated from a nymphalid host and may represent a divergent species. It appeared closely related to Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and other Choristoneura-isolated group I alphabaculoviruses. We annotated 153 open reading frames (ORFs), including a set of 38 core genes, 26 ORFs identified as present in lepidopteran baculoviruses, 17 ORFs unique in baculovirus, and several auxiliary genes (e.g., bro, cathepsin, chitinase, iap-1, iap-2, and thymidylate kinase). The thymidylate kinase (tmk) gene was present fused to a dUTPase (dut) gene in other baculovirus genomes. DijuNPV likely lost the dut portion together with the iap-3 homolog. Overall, the genome sequencing of novel alphabaculoviruses enables a wide understanding of baculovirus evolution.

The complete genome of Rachiplusia nu nucleopolyhedrovirus (RanuNPV) and the identification of a baculoviral CPD-photolyase homolog.

We described a novel baculovirus isolated from the polyphagous insect pest Rachiplusia nu. The virus presented pyramidal-shaped occlusion bodies (OBs) with singly-embed nucleocapsids and a dose mortality response of 6.9 × 103 OBs/ml to third-instar larvae of R. nu. The virus genome is 128,587 bp long with a G + C content of 37.9% and 134 predicted ORFs. The virus is an alphabaculovirus closely related to Trichoplusia ni single nucleopolyhedrovirus, Chrysodeixis chalcites nucleopolyhedrovirus, and Chrysodeixis includens single nucleopolyhedrovirus and may constitute a new species. Surprisingly, we found co-evolution among the related viruses and their hosts at species level. Besides, auxiliary genes with homologs in other baculoviruses were found, e.g. a CPD-photolyase. The gene seemed to be result of a single event of horizontal transfer from lepidopterans to alphabaculovirus, followed by a transference from alpha to betabaculovirus. The predicted protein appears to be an active enzyme that ensures likely DNA protection from sunlight.

A Novel Alphabaculovirus from the Soybean Looper, Chrysodeixis includens, that Produces Tetrahedral Occlusion Bodies and Encodes Two Copies of he65.

Isolates of the alphabaculovirus species, Chrysodeixis includens nucleopolyhedrovirus, have been identified that produce polyhedral occlusion bodies and infect larvae of the soybean looper, Chrysodeixis includens. In this study, we report the discovery and characterization of a novel C. includens-infecting alphabaculovirus, Chrysodeixis includens nucleopolyhedrovirus #1 (ChinNPV#1), that produces tetrahedral occlusion bodies. In bioassays against C. includens larvae, ChinNPV #1 exhibited a degree of pathogenicity that was similar to that of other ChinNPV isolates, but killed larvae more slowly. The host range of ChinNPV#1 was found to be very narrow, with no indication of infection occurring in larvae of Trichoplusia ni and six other noctuid species. The ChinNPV#1 genome sequence was determined to be 130,540 bp, with 126 open reading frames (ORFs) annotated but containing no homologous repeat (hr) regions. Phylogenetic analysis placed ChinNPV#1 in a clade with other Group II alphabaculoviruses from hosts of lepidopteran subfamily Plusiinae, including Chrysodeixis chalcites nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus. A unique feature of the ChinNPV#1 genome was the presence of two full-length copies of the he65 ORF. The results indicate that ChinNPV#1 is related to, but distinct from, other ChinNPV isolates.

Genetic Variability of Chrysodeixis Includens Nucleopolyhedrovirus (ChinNPV) and the Insecticidal Characteristics of Selected Genotypic Variants.

Genetic variation in baculoviruses is recognized as a key factor, not only due to the influence of such variation on pathogen transmission and virulence traits, but also because genetic variants can form the basis for novel biological insecticides. In this study, we examined the genetic variability of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) present in field isolates obtained from virus-killed larvae. Different ChinNPV strains were identified by restriction endonuclease analysis, from which genetic variants were isolated by plaque assay. Biological characterization studies were based on pathogenicity, median time to death (MTD), and viral occlusion body (OB) production (OBs/larva). Nine different isolates were obtained from eleven virus-killed larvae collected from fields of soybean in Mexico. An equimolar mixture of these isolates, named ChinNPV-Mex1, showed good insecticidal properties and yielded 23 genetic variants by plaque assay, one of which (ChinNPV-R) caused the highest mortality in second instars of C. includens. Five of these variants were selected: ChinNPV-F, ChinNPV-J, ChinNPV-K, ChinNPV-R, and ChinNPV-V. No differences in median time to death were found between them, while ChinNPV-F, ChinNPV-K, ChinNPV-R and ChinNPV-V were more productive than ChinNPV-J and the original mixture of field isolates ChinNPV-Mex1. These results demonstrate the high variability present in natural populations of this virus and support the use of these new genetic variants as promising active substances for baculovirus-based bioinsecticides.