Lef-9 Genes Research Articles

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

The complete genome of a European pine sawfly Neodiprion sertifer nucleopolyhedrovirus (NeseNPV-TR) was sequenced and characterized from next-generation sequencing data of N. sertifer larva from Türkiye. This genome was analyzed and compared to previously reported genomes of baculoviruses. The baculovirus phylogeny was reconstructed and the species identity of the NeseNPV-TR was delineated using K2P distance. The length of the genome was 82,052bp, with a G + C content of 33.28%. It contained 83 putative ORFs, including 38 baculovirus core genes, three lepidopteran baculovirus core genes, and three non-conserved genes. It had five hrs with 20.6% overall mean distance on average. The pairwise K2P distances of lef-8, lef-9, and polh genes and combinations of three genes and 38 genes between NeseNPV-TR and NeseNPV were slightly higher than the specified threshold values for species demarcation. The most variable genes were lef-2, helicase, p40, desmoplakin, pif7, 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 genus Gammabaculovirus. Taking into account the phylogenetic position, K2P distances, and NJ tree, the NeseNPV-TR can be classified in the same species (Gammabaculovirus nesertiferis) 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.

Molecular characterization of Spodoptera litura Nucleopolyhedrovirus (SpltNPV) from Bogor using late expression factor-8 gene

Spodoptera litura Nucleopolyhedrovirus (SpltNPV) is a biological agent successfully developed as a bio insecticide to control S. litura. NPV is host-specific. Therefore, it only infects particular insects. NPV identification is needed so its use can be right on target. Molecular Identification by PCR has been used to characterise Baculovirus. Ac17, lef-2, polh, p35, and 16s RNA, are genes that are often used to identify NPVs. However, the lef-8 gene is the most conserved gene for NPV identification. We conducted this research to study SpltNPV molecular characterisation through nucleotide and amino acid using the lef-8 gene. The NPV isolate was propagated in the Insect Pathology Laboratory. We use the CTAB method to purify the NPV DNA. Forward TCCAAACGTCGATTACG and reverse GACAGGCAGGTTTCTAA primers were used to amplify the lef-8 gene of SpltNPV. The molecular character was used to describe the relationship of SpltNPV isolate with the other NPV reported in GenBank. The lef-8 gene can be used to identify SpltNPV. Based on nucleotides and amino acids sequence of the lef-8 gene, the homology of SpltNPV Bogor has the highest similarity to SpltNPV of German isolates of 99% and 94%. Based on the left-8 gene sequence phylogeny, the Bogor SpltNPV isolate is a member of the same group as the NPV on Spodoptera and Amsacta.

Influence of delayed density and ultraviolet radiation on caterpillar baculovirus infection and mortality.

Infectious disease is an important potential driver of population cycles but must occur through delayed density-dependent infection and resulting fitness effects. Delayed density-dependent infection by baculoviruses can be caused by environmental persistence of viral occlusion bodies (OBs), which can be influenced by environmental factors. Specifically, ultraviolet radiation is potentially important in reducing the environmental persistence of viruses by inactivating OBs. Delayed density-dependent viral infection has rarely been observed empirically at the population level although theory predicts that it is necessary for pathogens to drive population cycles. Similarly, field studies have not examined the effects of ultraviolet radiation on viral infection rates in natural animal populations. We tested if viral infection is delayed density-dependent with the potential to drive cyclic dynamics and if ultraviolet radiation influences viral infection levels. We censused 18 Ranchman's tiger moth (Arctia virginalis) populations across 9° of latitude over 2 years and quantified the effects of direct and delayed density and ultraviolet radiation on proportion infected by baculovirus, infection severity and survival to adulthood. Caterpillars were collected from field populations and reared in the laboratory. Baculovirus has not previously been described infecting A. virginalis, and we used genetic methods to confirm the identity of the virus. We found that proportion infected, infection severity and survival to adulthood exhibited delayed density dependence. Ultraviolet radiation in the previous summer decreased infection severity, which increased caterpillar survival probability. Structural equation modelling indicated that the effect of lagged density on caterpillar survival was mediated through proportion infected and infection severity and was 2.5-fold stronger than the indirect effect of ultraviolet. We successfully amplified polh, lef-8 and lef-9 viral genes from caterpillars, and BLAST results confirmed that the virus was a nucleopolyhedrovirus. Our findings provide clear evidence that delayed density-dependent mortality can arise through viral infection rate and severity in insects, which supports the role of viral disease as a mechanism, among others, that may drive insect population cycles. Furthermore, our findings support predictions that ultraviolet radiation can modify viral disease dynamics in insect populations, most likely through attenuating viral persistence in the environment.

CRISPR/Cas9-Mediated Disruption of the lef8 and lef9 to Inhibit Nucleopolyhedrovirus Replication in Silkworms.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV ie-1 and me53 genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target lef8 and lef9, which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV lef8 and lef9 genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.

Identification of a new nucleopolyhedrovirus isolated from the olive leaf moth, Palpita vitrealis, from two locations in Egypt

The olive leaf moth (jasmine moth), Palpita vitrealis (Lepidoptera: Crambidae), is an important insect pest of olives in several Mediterranean countries. A new alphabaculovirus was isolated from diseased larvae of P. vitrealis in Egypt, first in Giza in spring 2005 and again in Marsa Matrouh in 2019.The larvae exhibited typical symptoms of a baculovirus infection. Light and scanning electron microscopy studies revealed polyhedral occlusion bodies. Transmission electron microscopy of ultrathin sections of purified OBs revealed virions with multiple embedded nucleocapsids. The identity of the two virus isolates was confirmed by sequencing the partial polyhedrin and lef-8 genes, and sequence comparison suggested a relationship to group I alphabaculoviruses. Therefore, this virus was termed Palpita vitrealis nucleopolyhedrovirus (PaviNPV). Whole genome sequencing of the PaviNPV isolate from Giza (Gz05) revealed a genome of 117,533 bp, 131 open reading frames (ORFs) and four homologous repeat (hr) regions. Phylogenetic reconstruction and genetic distance analyses using 38 core genes indicated that PaviNPV should be considered to belong to a novel species within the genus Alphabaculovirus. In bioassays, PaviNPV was highly virulent against second-instar larvae of P. vitrealis. The study reports a novel baculovirus that might have potential as a biological control agent of the olive leaf moth.

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.

Characterization of alphabaculovirus: HearNPV-IIPR05 isolate infecting Helicoverpa armigera (Hubner) larvae

BackgroundThe alphabaculoviruses are lethal pathogens of lepidopteran caterpillars including a polyphagous and globally recognized pest, Helicoverpa armigera (Hubner) infesting economically important agriculture crops worldwide. The biological and molecular characterizations of indigenous nucleopolyhedrovirus of the genus Alphabaculovirus isolated from H. armigera in chickpea fields are described.ResultsThe virulence of virus isolate was tested in 3rd instar H. armigera larvae, and LC50 (median lethal concentration) was estimated to be 2.69 × 104 OBs ml−1. The ST50 (median survival time) was 4 days post-inoculation, when the 3rd instar H. armigera larvae were inoculated by OB (occlusion body) concentration equivalent to LC90. An average incubation period of the virus isolate in 3rd instar ranged between 4 and 6 days post-inoculation. The OBs of a virus isolate appeared irregular in shape and variable in size with diameter ranging from 0.57 to 1.46 μm on the longest edge and average of 1.071 ± 0.068 μm (mean ± SE). On the basis of phylogenetic analysis of polh, pif-1, and lef-8 genes, the isolate was found to be a member of the genus Alphabaculovirus. The isolate showed a genetic affinity with species of group II Alphabaculoviruses and appeared to be a group II NPV.ConclusionsOn the basis of molecular phylogeny and associated host insect, this indigenous isolate was designated as HearNPV-IIPR05 isolate, which could be a potential candidate for the biological control of H. armigera infesting legumes and other commercial crops.

Characterization of a novel baculovirus isolate from Malacosoma neustria (Linnaeus, 1758) (Lepidoptera: Lasiocampidae) in Samsun and its pathogenicity in different hosts

Malacosoma neustria (Linnaeus, 1758) (Lepidoptera: Lasiocampidae) causes economic losses in apple, pear, plum, willow, oak and other economically important trees. In this study, an alphabaculovirus was isolated from the larval population of M. neustria from Samsun in the central Black Sea Region of Turkey between 2015 and 2016. Electron microscope analysis of occlusion bodies (OBs) obtained from dead larvae showed that the nucleocapsids of a new isolate (ManeNPV-T4) are multiply enveloped. The Kimura two-parameter analysis and the phylogenetic tree were performed based on concatenated nucleotide and amino acid sequences of the partial lef-8, lef-9 and polh genes from ManeNPV-T4 isolate compared to those of other 51 baculoviruses. Insecticidal activity tests against third instar M. neustria larvae produced 48 to 100% mortalities. The LC 50 of ManeNPV-T4 was 0.78 x 10 3 OBs/ml in M. neustria . Additionally, the isolate caused mortalities lower than 50% in Spodoptera exigua (Hübner, 1808) (Lepidoptera: Noctuidae) , Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae) , Hyphantria cunea (Drury, 1773) (Lepidoptera: Arctiidae) and Helicoverpa armigera (Hübner, 1805) (Lepidoptera: Noctuidae) larvae. Consequently, the new nucleopolyhedrovirus is infectious on M. neustria larvae and other lepidopterans.

Genetic Identification and Biological Characterization of Baculovirus Isolated from Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil

Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) larvae are polyphagous, aggressive, and have been found in many of Brazil's agricultural areas. Biological control with baculoviruses is part of an integrated pest management (IPM) strategy to manage this insect. Three isolates of nucleopolyhedrovirus (NPV) were collected from Brazilian populations of H. armigera, and compared genetically and biologically to Gemstar® (an imported nucleopolyhedrovirus), and to Nucleopolyedrovirus spodoptera (SPNPV) that was passed serially through H. armigera. Genetic sequencing of lef-8 and lef-9 genes revealed that the Brazilian isolates were closely related to nucleopolyhedrovirus species from Australia, South Africa, China, and India. The isolates caused high mortality rates in third instar H. armigera larvae. The mean lethal dose (LD50) and lethal time (LT50) differed between isolates, but was highest for HearNPV-BR2. This is the first report of HearNPV in Brazil, and the insecticidal properties of the BR2 isolate support its potential use in the production of biopesticides to manage H. armigera in Brazil.

Identification of an Argentinean isolate of Spodoptera frugiperda granulovirus

The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is an important maize pest. Due to the environmental impact and emergence of resistance caused by chemical pesticides and transgenic events, the use of baculoviruses becomes a safe and useful alternative for its control in integrated pest management strategies. Here we report the identification of a novel isolate of a granulovirus of S. frugiperda native to the central region of Argentina, named SfGV ARG. We observed that larvae infected with SfGV ARG showed a yellowish coloration, swollen body and, in some cases, severe lesions in the last abdominal segments. We confirmed the identity of the isolate by sequencing fragments of the lef-8, lef-9 and granulin genes and by calculating evolutionary distances using the Kimura-2-Parameter model. SfGV ARG DNA restriction pattern allowed to estimate a genome of at least 135 kb.

First Report ofChrysodeixis includensNucleopolyhedrovirus (ChinNPV) InfectingChrysodeixis includens(Lepidoptera: Noctuidae) in Argentina

Typical baculovirus infection symptoms were observed in Chrysodeixis includens Walker (Lepidoptera: Noctuidae) larvae from a laboratory rearing in Tucuman, Argentina. Scanning electron microscopy revealed the occurrence of polyhedral occlusion bodies displaying a heterogeneous morphology. Amplification, sequencing, and phylogenetic analysis of conserved polyhedrin, lef-8 and lef-9 genes identified the pathogen, for the first time in Argentina, as a variant of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV). The biocontrol potential of this new isolate is worthy of future research.

Natural Enemies of the Sugar Beet Army Worm, Spodoptera exigua (Lepidoptera: Noctuidae) in Northeast Iran

During 2014-2015, a survey was carried out to determine the natural enemies of the sugar beet armyworm, Spodoptera exigua (Hübner) in the Mashhad region of northeastern Iran. Through extensive sampling, different larval stages of S. exigua with possible signs or symptoms of infection were collected from beet fields, kept in laboratory conditions, fed with fresh beet leaves, and checked daily until the emergence of parasitoids. Herein, 12 species of parasitoids, a virus: Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), an entomopathogenic fungus: Beauveria varroae, and an entomophilic nematode, Pristionchus pacificus are reported. The parasitoids were Meteorus rubens, Microplitis rufiventris, Cotesia sp., Chelonus (Chelonus) annulipes (Braconidae), Euplectrus sp. (Eulophidae), Sinophorus nr. xanthostomus, Anomalon sp., Diadegma pulchripes, Temelucha sp. (Ichenumonidae), two species of Drino genus and Exorista sp. (Tachinidae). The body tissues of the virus-infected larvae were liquefied, swollen, and glossy with a very thin cuticle. SEM photographs of viral occlusion bodies (OBs) showed polyhedral and irregular shapes. A cross-section of an OB indicated that each virion contained multiple nucleocapsids arranged randomly within the occlusion matrix. Genetic analysis based on lef-8 and Polyhedrin gene sequences confirmed the identity of the Iranian strain as SeMNPV. The virus was transmitted vertically and horizontally. Another pathogen was Beauveria varroae, isolated from a lepidopteran insect for the first time; S. exigua was indicated as a new host for this fungus. Both pathogens are herein introduced as new records for Iran microflora. The results of the current work highlight the diversity of natural enemies on the sugar beet armyworm in Iran.

Development and application of a duplex PCR assay for detection of Crangon crangon bacilliform virus in populations of European brown shrimp (Crangon crangon).

Crangon crangon bacilliform virus (CcBV) was first discovered in 2004 in European brown shrimp (Crangon crangon) caught along the English coast. This study describes a duplex PCR assay developed for the detection of CcBV, based on amplification of the lef-8 gene (211 bp) of CcBV and the E75 gene (105 bp) of C. crangon as an internal amplification control. The lef-8 and E75 primer pairs were designed based on preliminary genome sequencing information of the virus and transcriptomic data available for C. crangon, respectively. Sequencing of the resulting amplicons confirmed the specificity of this PCR assay and sequence analysis of the lef-8 fragment revealed amino acid identity percentages ranging between 31 and 42% with members of the Nudiviridae, proposing that CcBV may reside within this family. Finally, the duplex PCR assay was applied to samples of C. crangon hepatopancreas tissue collected along the Belgian coast to screen for the presence of CcBV. The prevalence of CcBV averaged 87%, which is comparable to previous reports of high prevalence, based upon histological analysis, in shrimp collected along the English coast. Development of a specific and sensitive PCR assay to detect CcBV will provide a useful tool for future aquaculture and research programs involving C. crangon.

Comparison of a South African and Canadian Isolate of the Nucleopolyhedrosis Virus Infecting the InsectTrichoplusia ni

The nucleotide and amino acid sequence of the ecdysteroid UDP-glucosyltransferase (egt), polyhedrin (pol), inhibitor of apoptosis (iap2 and iap3), late expression factor (lef) 9 and vp1054 genes of a South African isolate of Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) was compared to a Canadian isolate of TnSNPV. Nucleotide and amino acid sequence identity for the compared genes ranged from 89%to 98%and 95%to 99%, respectively. The deduced amino acid sequences of the TnSNPV egt, pol, iap3, lef-9 and vp1054 genes were used to infer phylogenetic trees and these were compared to the tree inferred from combined data sets consisting of the amino acid sequences of polyhedrin/granulin, lef-8 and lef-9 genes of 48 completely sequenced baculoviruses. The topologies of trees for the baculovirus core genes pol, lef-9 and vp1054 were better resolved than that of the auxiliary genes iap2, iap3 and egt when compared to the tree inferred from complete genome sequences. Sequence and phylogenetic analysis confirm...

Molecular identification and phylogenetic analyses of multiple nucleopolyhedrovirus isolated from Lymantria obfuscata (Lepidoptera: Lymantriidae) in India

The Lymantria obfuscata Walker (Lyob) multiple (M) nucleopolyhedrovirus (NPV) (LyobMNPV) has been isolated and successfully applied for the management of the Indian gypsy moth, L. obfuscata in Jammu and Kashmir (J&K), India. The present work aimed to investigate the variability of LyobMNPV isolates from six localities of J&K through molecular [amplification of the polyhedrin (polh), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes] and biological (bioassays) characterization. To identify the position of LyobMNPV in the phylogenetic tree of baculoviruses, partial sequences of the polh, lef-8 and lef-9 genes were determined by using the DNA sequences within their coding regions by optimizing the polymerase chain reaction with degenerate primers. The sequence alignment revealed that LyobMNPV isolates exhibited seven, five and eleven single nucleotide polymorphic sites in the case of polh, lef-8 and lef-9, respectively. The phylogenetic analyses supported placing LyobMNPV with the Lymantria dispar L. MNPV (LdMNPV) isolates from different countries, and showed that it was more closely related to LdMNPV than to Lymantria xylina Swinhoe NPV and Lymantria monacha L. NPV. The contaminated diet plug bioassays using 2nd instar larvae indicated that the median lethal dose (LD50) and median survival time (ST50) of different isolates of LyobMNPV against L. obfuscata were lower than those of LdMNPV against L. dispar. LyobMNPV was more closely related to LdMNPV but its LD50 and ST50 were lower than those of LdMNPV. The study provides novel information on the position of LyobMNPV in the phylogenetic tree of baculoviruses and about biological and genetic variation of Lymantria species’ NPV isolates from different parts of the world.

Karakter morfologi, biologi, dan molekuler tiga isolat Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) terhadap penggerek buah kapas Helicoverpa armigera Hubner (Lepidoptera: Noctuidae)

Helicoverpa armigera Hubner (Lepidoptera: Noctuidae) is one of the most important pests on cotton. Yield losses due to this pest can reach up to 70%. Pest control using the NPV has been proven to be effective in the field. However NPV isolates from different geographical locations could be showed variation in genetic and their biology. The purpose of this study was to compare morphology, biology, and molecular characteristics between three HearNPV isolates from cotton field in South Sulawesi, Central Java, and East Java. Scanning electron microscope (SEM) was used to look at morphological characters, whereas biological characters were observed by dipping shelled of young corn cob on each isolate suspension with a density of 2.5 x 103, 2.5 x 104, 2.5 x 105, 2.5 x 106, 2.5 x 107 POBs /ml and control then fed to the first instar larvae of H. armigera. Probit analysis was used to determine LC and LT. The molecular assay used polymerase chain reaction (PCR) with lef-8 gene. The results showed that the three isolates had the shape and size of polyhedra which was not significantly different with the mean diameter were 0.86 μm. The result showed that in 7 days post infection, HearNPV isolate from East Java was the most virulent isolates with the lowest LC50, which is 6.38 POBs/ml, but lowest LT50 value in 2.5 x 107 POBs/ml were showed both from East Java and South Sulawesi, which are 2.7 days post infection. Meanwhile, the DNA sequencing of the three isolates showed a high similarity (100% homology).

An alphabaculovirus isolated from dead Lymantria dispar larvae shows high genetic similarity to baculovirus previously isolated from Lymantria monacha – An example of adaptation to a new host

A new isolate of baculovirus, Lymantria dispar multiple nucleopolyhedrovirus-BNP (LdMNPV-BNP), was found in dead gypsy moth (L. dispar) caterpillars collected in the Biebrzanski National Park in Poland. Here, we examined its biological activity, structure, genetic content and phylogeny. Multiple nucleocapsids of LdMNPV-BNP are enveloped together in 2–26 virions embedded in occluded bodies (OBs) very similar to the OBs previously described in viruses infecting Lymantriinae. This isolate kills pest larvae in a relatively short time (LT50 of approximately 9days for a dose of 2×107OBs/ml), highlighting the possibility for its use as a biopesticide. Next-generation sequencing of LdMNPV-BNP revealed gene content (e.g. DNA photolyase) that is not present in any LdMNPV isolate sequenced to date. The genome is 157,270 base pairs long and has a notably lower G+C content in comparison to other LdMNPVs (50.3% G+C content compared to an average of 57.4% among other LdMNPVs). According to our phylogenetic analysis based on 37 core genes, LdMNPV-BNP is a member of group II alphabaculoviruses, which are closely related to LdMNPV and LyxyMNPV (Lymantria xylina multiple nucleopolyhedrovirus). Molecular evolution inference based on the partial sequence of lef-8, lef-9 and polh genes shows that LdMNPV-BNP and isolates of Lymantria monacha nucleopolyhedrovirus (LymoNPV) may share a very recent common ancestor or be isolates of the same virus species. LdMNPV-BNP, like other baculoviruses, could be beneficial as an active component of biopesticides that can be used during forest integrated pest management.

Lymantria mathura nucleopolyhedrovirus: Identification, occurrence and genetic diversity in Iwate Prefecture, Japan

A high prevalence of nuclear polyhedrosis has been observed in larval outbreaks of Lymantria mathura in Iwate Prefecture, Japan. However, the virus responsible has not been identified. Here the virus was designated Lymantria mathura nucleopolyhedrovirus (LymaNPV), based on partial sequence results of the lef-8, lef-9, and polh genes and transmission electron microscopic observations. Diagnosis by polymerase chain reaction targeting of the partial polh gene and a subsequent restriction fragment length polymorphism (RFLP) analysis indicated that LymaNPV was an exclusive causative agent for the nuclear polyhedrosis of the L. mathura larvae. LymaNPV was also detected from a very small fraction of L. dispar larvae that co-occurred with L. mathura larvae where the prevalence of LymaNPV-caused disease was high. A bioassay using LymaNPV against L. dispar larvae produced infection with the inoculated virus. This finding was consistent with the results of the field sample analysis. LymaNPV contained six genome types in the L. mathura populations sampled, as determined by RFLP of LymaNPV genomic DNA and southern blot hybridization analyses. None of the genome types was unique to any sampling site, indicating that some flow had occurred among sites. However, genome-type composition seemed to differ among sites. This study provides basic information about the interaction between L. mathura and LymaNPV.

Bombyx mori nucleopolyhedrovirus lef8 gene: effects of deletion and implications for gene transduction applications.

In this study, we have deleted the lef8 gene of the baculovirus BmNPV, which encodes one of the viral RNA polymerase subunits, in order to create a knockout bacmid, Δlef8, directing cytopathology-free single-cell infections for gene transduction and recombinant protein production. However, while removal of the complete lef8 ORF produced the expected phenotype, it also affected the function of the closely linked essential gene orf40, thus hampering the mutant bacmid rescue in cultured Bombyx cells expressing recombinant LEF8. Subsequently, we determined that several diverse sequences can substitute for the orf40 5'-upstream sequences that were removed by the deletion of the lef8 gene and also showed that neither a physical linkage nor expression of the two relevant genes under native promoter control is a prerequisite for a fully functional virus. Based on these findings, we generated a rescue-competent lef8-null vector, which contained a heterologous promoter-driven orf40. This lef8-deficient vector, which produces productive infections and progeny virus lacking lef8 in deficiency-complementing cells expressing LEF8, could be used as the basis for an alternative to current silkmoth transduction systems.

Occurrence and characterization of a tetrahedral nucleopolyhedrovirus from Spilarctia obliqua (Walker)

Spilarctia obliqua Walker (Lepidoptera: Arctiidae) is a polyphagous insect pest damaging pulses, oil seeds, cereals, vegetables and medicinal and aromatic plants in India. The pest also infests turmeric and ginger sporadically in Kerala. We observed an epizootic caused by a nucleopolyhedrovirus (NPV) in field populations of the insects in December 2013. The NPV was purified and characterized. The isolate was tetrahedral in shape and belonged to multicapsid NPV. The REN profile of the SpobNPV genome with Pst I, Xho I and HindIII enzymes showed a genome size of 99.1±3.9kbp. Partialpolh, lef-8 and lef-9 gene sequences of the isolate showed a close relationship with HycuNPV and SpphNPV. Phylogram and K-2-P distances between similar isolates suggested inclusion of the present SpobNPV isolate to group I NPV. The biological activity of the isolate was tested under laboratory conditions against third instar larvae of S. obliqua and the LC50 was 4.37×103OBs/ml occlusion bodies (OBs) per ml. The median survival time (ST50) was 181h at a dose of 1×106OBs/ml and 167h at a dose of 1×108OBs/ml. SpobNPV merits further field evaluation as a potential biological control agent of S. obliqua, a serious pest of many agriculturally important crops in the Oriental region.