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.

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.

Genome Characteristics of the Cyclophragma Undans Nucleopolyhedrovirus: A Distinct Species in Group I of Alphabaculovirus.

The Cyclophragma undans nucleopolyhedrovirus (CyunNPV), a potential pest control agent, was isolated from Cyclophragma undans (Lepidoptera: Lasiocampidae), an important forest pest. In the present study, we performed detailed genome analysis of CyunNPV and compared its genome to those of other Group I alphabaculoviruses. Sequencing of the CyunNPV genome using the Roche 454 sequencing system generated 142,900 bp with a G + C content of 45%. Genome analysis predicted a total of 147 hypothetical open reading frames comprising 38 baculoviral core genes, 24 lepidopteran baculovirus conserved genes, nine Group I Alphabaculovirus conserved genes, 71 common genes, and five genes that are unique to CyunNPV. In addition, the genome contains 13 homologous repeated sequences (hrs). Phylogenetic analysis groups CyunNPV under a distinct branch within clade "a" of Group I in the genus Alphabaculovirus. Unlike other members of Group I, CyunNPV harbors only nine of the 11 genes previously determined to be specific to Group I viruses. Furthermore, the CyunNPV lacks the tyrosine phosphatase gene and the ac30 gene. The CyunNPV F-like protein contains two insertions of continuous polar amino acids, one at the conventional fusion peptide and a second insertion at the pre-transmembrane domain. The insertions are likely to affect the fusion function and suggest an evolutionary process that led to inactivation of the F-like protein. The above findings imply that CyunNPV is a distinct species under Group I Alphabaculovirus.

Lacanobia oleracea nucleopolyhedrovirus (LaolNPV): A new European species of alphabaculovirus with a narrow host range.

During an insect sampling program in alfalfa crops near Montpellier, France in 2011, Lacanobia oleracea larvae were collected that died due to nucleopolyhedrovirus infection (LaolNPV). This virus was subjected to molecular and biological characterization. The virus was a multiple nucleocapsid NPV that showed similar restriction profiles to Mamestra configurata NPV-A (MacoNPV-A) but with significant differences. Polypeptide analysis demonstrated similar proteins in occlusion bodies and occlusion derived virions, to those observed in NPVs from Mamestra spp. Terminal sequencing revealed that the genome organization shared similarity with that of MacoNPV-A. The most homologous virus was MacoNPV-A 90/2 isolate (95.63% identity and 96.47% similarity), followed by MacoNPV-A 90/4 strain (95.37% and 96.26%), MacoNPV-B (89.21% and 93.53%) and M. brassicae MNPV (89.42% and 93.74%). Phylogenetic analysis performed with lef-8, lef-9, polh and a concatenated set of genes showed that LaolNPV and the Mamestra spp. NPVs clustered together with HaMNPV, but with a closer genetic distance to MacoNPV-A strains. The Kimura 2-parameter (K-2-P) distances of the complete genes were greater than 0.05 between LaolNPV and the MbMNPV/MacoNPV-B/HaMNPV complex, which indicates that LaolNPV is a distinct species. K-2-P distances were in the range 0.015-0.050 for comparisons of LaolNPV with MacoNPV-A strains, such that additional biological characteristics should be evaluated to determine species status. While MacoNPV-A was pathogenic to seven lepidopteran species tested, LaolNPV was only pathogenic to Chrysodeixis chalcites. Given these findings, Lacanobia oleracea nucleopolyhedrovirus should be considered as a new species in the Alphabaculovirus genus.

Analysis of codon usage bias of envelope glycoprotein genes in nuclear polyhedrosis virus (NPV) and its relation to evolution.

BACKGROUND: Analysis of codon usage bias is an extremely versatile method using in furthering understanding of the genetic and evolutionary paths of species. Codon usage bias of envelope glycoprotein genes in nuclear polyhedrosis virus (NPV) has remained largely unexplored at present. Hence, the codon usage bias of NPV envelope glycoprotein was analyzed here to reveal the genetic and evolutionary relationships between different viral species in baculovirus genus. RESULTS: A total of 9236 codons from 18 different species of NPV of the baculovirus genera were used to perform this analysis. Glycoprotein of NPV exhibits weaker codon usage bias. Neutrality plot analysis and correlation analysis of effective number of codons (ENC) values indicate that natural selection is the main factor influencing codon usage bias, and that the impact of mutation pressure is relatively smaller. Another cluster analysis shows that the kinship or evolutionary relationships of these viral species can be divided into two broad categories despite all of these 18 species are from the same baculovirus genus. CONCLUSIONS: There are many elements that can affect codon bias, such as the composition of amino acids, mutation pressure, natural selection, gene expression level, and etc. In the meantime, cluster analysis also illustrates that codon usage bias of virus envelope glycoprotein can serve as an effective means of evolutionary classification in baculovirus genus.

Evidence of recent interspecies horizontal gene transfer regarding nucleopolyhedrovirus infection of Spodoptera frugiperda.

BACKGROUND: Baculoviruses are insect-associated viruses carrying large, circular double-stranded-DNA genomes with significant biotechnological applications such as biological pest control, recombinant protein production, gene delivery in mammals and as a model of DNA genome evolution. These pathogens infect insects from the orders Lepidoptera, Hymenoptera and Diptera, and have high species diversity which is expressed in their diverse biological properties including morphology, virulence or pathogenicity. Spodoptera frugiperda (Lepidoptera: Noctuidae), the fall armyworm, represents a significant pest for agriculture in America; it is a host for baculoviruses such as the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) (Colombia strain, genotype A) having been classified as a Group II alphabaculovirus making it a very attractive target for bioinsecticidal use. RESULTS: Genome analysis by pyrosequencing revealed that SfMNPV ColA has 145 ORFs, 2 of which were not present in the other sequenced genotypes of the virus (SfMNPV-NicB, SfMNPV-NicG, SfMNPV-19 and SfMNPV-3AP2). An in-depth bioinformatics study showed that ORF023 and ORF024 were acquired by a recent homologous recombination process between Spodoptera frugiperda and Spodoptera litura (the Oriental leafworm moth) nucleopolyhedroviruses. Auxiliary genes are numerous in the affected locus which has a homologous region (hr3), a repetitive sequence associated with genome replication which became lost in SfColA along with 1 ORF. Besides, the mRNAs associated with two acquired genes appeared in the virus' life-cycle during the larval stage. Predictive studies concerning the theoretical proteins identified that ORF023 protein would be a phosphatase involved in DNA repair and that the ORF024 protein would be a membrane polypeptide associated with cell transport. CONCLUSIONS: The SfColA genome was thus revealed to be a natural recombinant virus showing evidence of recent horizontal gene transfer between different baculovirus species occurring in nature. This feature could be the cause of its high insecticidal power and therefore SfColA becomes a great candidate for bioinsecticide formulations.

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±3.9 kbp. 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 4.37×10(3)OBs/ml occlusion bodies (OBs) per ml. The median survival time (ST50) was 181 h at a dose of 1×10(6)OBs/ml and 167 h at a dose of 1×10(8)OBs/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.

Genome sequence of a Bombyx mori nucleopolyhedrovirus strain with cubic occlusion bodies.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical species of Baculoviridae. The complete genome sequence of a BmNPV strain with cubic occlusion bodies is reported here. The genome of this strain consists of 127,465 nucleotides with a G+C content of 40.36% and is 97.3% and 97.5% identical to those of BmNPV strain T3 and Bombyx mandarina NPV S1, respectively. Despite the abnormal polyhedra it forms, the polyhedrin gene of the BmNPV cubic strain is 100% identical to those of the other two strains. Baculovirus repeated ORFs and homologous repeat regions cause the major differences in genome size of these BmNPV isolates.

AcMNPV enhances infection by ThorNPV in Sf21 cells and SeMNPV in Hi5 cells.

An expression cassette containing the DsRed2 gene, which encodes the red fluorescent protein (RFP), was inserted into the wide-host-range Autographa californica M nucleopolyhedrovirus (AcMNPV) at the polyhedrin locus (vAcDsRed2). An expression cassette containing the enhanced green fluorescent protein (EGFP) gene was inserted at the gp37 locus of the narrow-host-range Thysanoplusia orichalcea MNPV (ThorMNPV) and the p10 locus of Spodoptera exigua MNPV (SeMNPV) to produce vThGFP and vSeGFP, respectively. vThGFP and vSeGFP are poor at infecting Sf21 and Hi5 cells, respectively, whereas vAcDsRed2 is highly infectious to both cell lines. During co-infection, vAcDsRed2 enhanced vThGFP infection in Sf21 cells by approximately 20-fold, and it enhanced vSeGFP infection in Hi5 cells by more than 300-fold, as detected by fluorescence measurements. In contrast, vThGFP reduced vAcDsRed2 infection by 5.4-fold in Sf21 cells, while vSeGFP reduced vAcDsRed2 by 3.2-fold in Hi5 cells. Plaque assay data did not suggest viral recombination, but vThGFP plaques surrounded by vAcDsRed2 plaques were observed. A viral DNA replication assay performed by real-time quantitative PCR suggested that the detected fluorescence correlated with virus replication. Sf21 cells infected with vAcDsRed2 were resistant to superinfection by viruses of the same type expressing EGFP (vAcGFP). These results demonstrated that AcMNPV could enhance replication of ThorMNPV and SeMNPV in non-permissive cells without recombination.

Identification and sequence analysis of the Condylorrhiza vestigialis MNPV p74 gene.

The baculovirus Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV), isolated from C. vestigialis infected larvae in Paraná (Brazil), was identified in our laboratory. A full-length clone was obtained from the CoveMNPV genome, of the gene that encodes the homolog to baculoviral p74, essential for oral infectivity which was then sequenced and characterized. The CoveMNPV p74 gene (GenBank accession number EU919397) contains an ORF of 1935 bp that encodes a deduced protein of 73.61 kDa. The phylogenetic affiliations of the CoveMNPV gene were determined by a heuristic search of 40 aligned baculovirus p74 nucleotide sequences using maximum parsimony (PAUP 4.0b4a). The phylogenetic analysis placed CoveMNPV within lepidopteran nucleopolyhedrovirus (NPV) Group I, Clade A, as being the closest to Choristoneura fumiferana defective NPV.

Establishment of an insect cell clone that harbours a partial baculoviral genome and is resistant to homologous virus infection.

After serially undiluted passage of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), persistently infected Se301 cells were established. A cell strain, in which no polyhedra or viral particles were observed, was cloned and designated P8-Se301-C1. The P8-Se301-C1 cells are morphologically similar to but grow slower than Se301 cells and they can homologously interfere with SeMNPV. PCR analysis showed that SeMNPV ie-0 and polyhedrin genes were present but DNA polymerase and orf67 genes were absent in P8-Se301-C1, suggesting that the cells harbour incomplete SeMNPV genomes. Dot-blot analysis demonstrated that 0.32+/-0.16 ng SeMNPV DNA was present in 1.25 x 10(5) P8-Se301-C1 cells. A quantitative real-time PCR assay showed that there were 13.2+/-4.3 copies of the SeMNPV polyhedrin gene in each cell. Nested RT-PCR demonstrated the presence of SeMNPV polyhedrin transcripts in P8-Se301-C1 cells. The fact that P8-Se301-C1 cells carry low levels of partial viral genome but do not produce viral progeny suggests a latent-like viral infection in the cells.

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus by loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid method for amplification of nucleic acids under isothermal conditions. In this report, a LAMP method was developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV), known previously as monodon baculovirus (MBV), using a set of six primers designed to specifically recognize the PemoNPV polyhedrin gene. The optimized time and temperature conditions for the LAMP assay were 60 min at 63 degrees C. The sensitivity of LAMP for PemoNPV detection was approximately 50 viral copies ng(-1) genomic DNA (equivalent to 150 viral copies per reaction). Using a DNA template extracted from PemoNPV-infected shrimp by a viral nucleic acid kit, the detection limit of LAMP was 0.7 fg while that of nested PCR was 70 fg; therefore, the LAMP assay was 100 times more sensitive than nested PCR. The LAMP method did not amplify a product using nucleic acid extracted from shrimp infected with other viruses including yellow head virus (YHV), Taura syndrome virus (TSV), white spot syndrome virus (WSSV), Penaeus stylirostris densovirus (PstDNV) known previously as infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Penaeus monodon densovirus (PmDNV) known previously as hepatopancreatic parvovirus (HPV).

A new nucleopolyhedrovirus strain (LdMNPV-like virus) with a defective fp25 gene from Lymantria xylina (Lepidoptera: Lymantriidae) in Taiwan.

A new multiple nucleopolyhedrovirus strain was isolated from casuarina moth, Lymantria xylina Swinhoe, (Lepidoptera: Lymantriidae) in Taiwan. This Lymantria-derived virus can be propagated in IPLB-LD-652Y and NTU-LY cell lines and showed a few polyhedra (occlusion bodies) CPE in the infected cells. The restriction fragment length polymorphism (RFLP) profiles of whole genome indicated that this virus is distinct from LyxyMNPV and the virus genome size was approximately 139 kbps, which was smaller than that of LyxyMNPV. The molecular phylogenetic analyses of three important genes (polyhedrin, lef-8 and lef-9) were performed. Polyhedrin, LEF-8 and LEF-9 putative amino acid analyses of this virus revealed that this virus belongs to Group II NPV and closely related to LdMNPV than to LyxyMNPV. The phylogenetic distance analysis was further clarified the relationship to LdMNPV and this virus provisionally named LdMNPV-like virus. A significant deletion of a 44bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. In ultrastructural observations, the nuclei of the infected LD host cells contained large occlusion bodies (OBs), and few OBs, which presented as one or two OBs in a nucleus that was otherwise filled with free nuclocapsids and virions. We concluded that this LdMNPV-like virus is a new LdMNPV strain from L. xylina.

Genomic and host range studies of Maruca vitrata nucleopolyhedrovirus.

The complete genome of the Maruca vitrata nucleopolyhedrovirus (MaviNPV) isolated from the legume pod borer, Maruca vitrata (Lepidoptera: Pyralidae), was sequenced. It was found to be 111 953 bp in length, with an overall 39 % G+C content, and contained 126 open reading frames (ORFs) encoding predicted proteins of over 50 aa. The gene content and gene order of MaviNPV have the highest similarity to those of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and their shared homologous genes are 100 % collinear. In fact, MaviNPV seems to be a mini-AcMNPV that is native to Taiwan and possesses a smaller genome with fewer auxiliary genes than the AcMNPV type species. Except for one ORF (Mv74), all of the MaviNPV ORFs have homologues in the AcMNPV genome. MaviNPV is the first lepidopteran-specific baculovirus to lack homologues of vfgf and odv-e66. In addition, MaviNPV lacks the baculovirus repeat ORF (bro) gene that corresponds to AcMNPV ORF2. Five homologous regions (hrs) were located within the MaviNPV genome, and these contained a total of 44 imperfect palindromes. Phylogenetic analysis of the whole genome revealed that MaviNPV was separated from the common ancestor of AcMNPV and Bombyx mori nucleopolyhedrovirus before these two viral species diverged from each other. Moreover, replication of MaviNPV in several cell lines and an egfp-MaviNPV infection assay revealed that IPLB-LD-652Y cells are only partially permissive to MaviNPV, which supports our conclusion that MaviNPV is a distinct species of the group I lepidopteran NPVs.

Structural and phylogenetic relationship of ORF 31 from the Anticarsia gemmatalis MNPV to poly (ADP-ribose) polymerases (PARP).

ORF 31 is a unique baculovirus gene in the genome of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D). It encodes a putative polypeptide of 369 aa homologous to poly (ADP-ribose) polymerase (PARP) found in the genomes of several organisms. Moreover, we found a phylogenetic association with Group I PARP proteins and a 3D homology model of its conserved PARP C-terminal catalytic domain indicating that had almost an exact spatial superimposition of <1 A with other PARP available structures. The 5' end of ORF 31 mRNA was located at the first nucleotide of a CATT motif at position -27. Using real-time PCR we detected transcripts at 3 h post-infection (p.i.) increasing until 24 h p.i., which coincides with the onset of DNA replication, suggestive of a possible role in DNA metabolism.

Characterization of a nucleopolyhedrovirus with a deletion of the baculovirus core gene Bm67.

Open reading frame (ORF) 67 (Bm67) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that is found in all completely sequenced baculoviruses; its function is unknown. In the present study, a Bm67-knockout virus was generated for studying the role of Bm67 in the BmNPV infection cycle. Furthermore, a Bm67-repair bacmid was constructed by transposing the Bm67 native promoter-promoted Bm67 ORF into the polyhedrin locus of the Bm67-knockout bacmid. After these recombinant bacmids were transfected into BmN cells, the Bm67-knockout bacmid caused defects in the production of infectious budded viruses. However, the Bm67-repair bacmid could rescue the defect, and budded virus titres reached wild-type levels. Quantitative real-time PCR analysis indicated that Bm67 is required for normal levels of DNA synthesis or for the stability of nascent viral DNA at the early stage. Electron microscopic analysis revealed that the formation of normal-appearing nucleocapsids is reduced in Bm67-knockout bacmid-transfected cells, and nucleocapsids are rarely found in the cytoplasm. The presence of 'enveloped' nucleocapsids at the nucleoplasm bilayer indicated that they are enveloped abnormally. These results indicated that Bm67 is required for the production of infectious budded viruses and for assembly of envelope and nucleocapsids.

Group I but not group II NPV induces antiviral effects in mammalian cells.

Nucleopolyhedrovirus (NPV) is divided into Group I and Group II based on the phylogenetic analysis. It has been reported that Group I NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group II NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group II had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.

Sequencing and characterisation of p74 gene in two isolates of Anticarsia gemmatalis MNPV.

P74 is a protein encoded in the genome of baculoviruses, associated with the envelopes of occluded virus. Its presence proved to be essential for per os infection. In first place, in this work we designed two universal primers to amplify a sequence region of the p74 ORF in baculoviruses from different classification groups. Then, by the use of these amplicons we obtained the complete sequence of the p74 locus from two isolates of AgMNPV, 2D (Brazil) and SF (Argentina). In the flanking regions we determined the complete sequence of p10 gene and a portion of p26 gene. Comparing both p74 sequence data (ORFs of 1935 bp) we found fifteen nucleotide changes that result in six amino acid changes. Comparisons of AgMNPV p74s with other baculovirus homologous genes indicate a close relationship with other group I Nucleopolyhedrovirus, in particular CfDEFNPV. These results were based on ORF sequence, amino acid sequence and gene order. The predictive studies about secondary structure and hydrophobic index point at six regions potentially associated to its function or native conformation. Finally, the detection of p74 mRNA after virus DNA replication confirms a late expression pattern.

Characterization and polyhedrin gene cloning of Lymantria xylina multiple nucleopolyhedrovirus.

A baculovirus has been isolated from infected larvae of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae) in Taiwan. Ultrastructural observation revealed that this virus is a multiple nucleopolyhedrovirus (MNPV), and the name L. xylina MNPV (LyxyMNPV) was proposed. Restriction endonuclease (BamHI, EcoRI, and EcoRV) profiles of LyxyMNPV genome differed from those of other known NPVs. The size of the LyxyMNPV genome was estimated to be 154+/-1.26 kbp (mean+/-SE). The polyhedrin gene is located in the BamHI-D, EcoRI-C, and EcoRV-K fragments of LyxyMNPV genome. The gene organization of the LyxyMNPV EcoRV-K fragment and the phylogenetic analysis based on the polyhedrin gene sequences showed that LyxyMNPV is closely related to the L. dispar MNPV (LdMNPV). Furthermore, a rapid assay method was developed to distinguish LyxyMNPV from LdMNPV based on PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis of polyhedrin genes.

Nucleotide sequence and phylogenetic analyses of the DNA polymerase gene of Anticarsia gemmatalis nucleopolyhedrovirus.

The DNA polymerase from Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) was identified and sequenced, and its amino acid sequence was compared with other viral DNA polymerases to identify conserved regions and to reconstruct a phylogenetic tree. The sequence analysis of the AgMNPV DNA polymerase gene revealed the presence of a 2976 nucleotides open reading frame (ORF) encoding a polypeptide of 991 amino acid residues with a predicted molecular mass of 114.7 kDa. Among the baculovirus DNA polymerase genes identified to date, the AgMNPV DNA polymerase gene shared maximum amino acid sequence identity with the DNA polymerase gene of Choristoneura fumiferana nucleopolyhedrovirus defective strain (CfDEFNPV) (94%). The alignment of 140 virus sequences, 23 of them from baculovirus, showed that, of the 10 conserved regions identified, 5 are exclusive to baculoviruses (R1, R5, R9, R6 and R10), only 2 of them (R6 and R10) previously described as such in the literature. Our analysis, based on their positions in the AgMNPV DNA polymerase model, suggests that R9 and R10 could interact with DNA. Phylogenetic analysis of DNA polymerase sequences places the enzyme from AgMNPV within the cluster containing the polymerases of Group I Nucleopolyhedrovirus and suggests that the AgMNPV DNA polymerase is more closely related to that of CfDEFNPV than to those of other baculoviruses.