Genotypic variation and presence of rare genotypes among Douglas-fir tussock moth multicapsid nucleopolyhedrovirus (OpMNPV) isolates in British Columbia

The Douglas-fir tussock moth (Orgyia pseudotsugata) multicapsid nucleopolyhedrovirus (OpMNPV) is periodically applied to suppress Douglas-fir tussock moth populations in British Columbia and in the western United States. The strain of OpMNPV in the product currently used for suppression is not genetically distinct from naturally occurring OpMNPV. To separate the mortality caused by the applied virus from that caused by the naturally occurring virus, a rare and genetically distinct strain of OpMNPV must be applied. To learn more about the genotypic diversity of OpMNPV populations in BC and to identify rare strains in this region, viral DNA was extracted from larvae reared from 208 field-collected egg masses found in five geographic regions of British Columbia and subjected to REN analysis. Nine, 12, and 9 different genotypes were detected using PstI, SalI, and HindIII, respectively. When the PstI, SalI, and HindIII profiles for each pure (single strain) isolate were grouped and considered as a combined PstI-SalI-HindIII genotype, 23 different genotypes were identified among 185 isolates. Nine rare OpMNPV genotypes were selected as ideal candidates for use as a potential 'marker strain' to accurately determine the efficacy of the treatment.     

Phylogenetic analysis of <Emphasis Type="Italic">Orgyia pseudotsugata</Emphasis> single-nucleocapsid nucleopolyhedrovirus

The Douglas-fir tussock moth Orgyia pseudotsugata (Lepidoptera: Lymantriidae) is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus (SNPV) isolated from O. pseudotsugata larvae in Canada (OpSNPV) was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group II NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for multiple, concatenated gene phylogenies to classify baculoviruses. Foundation item: Supported by a scholarship from the European Union (Functional Biodiversity and Crop Protection), contract n^o HPMT-CT-2000-00199.     

Characterization of a nucleopolyhedrovirus from the vapourer moth, Orgyia antiqua (Lepidoptera Lymantriidae).

The first characterization of a nucleopolyhedrovirus (NPV Baculoviridae) isolated from the vapourer moth, Orgyia antiqua (Lepidoptera Lymatriidae), in the United Kingdom is presented. Transmission electron microscopy revealed that the virus nucleocapsid rods were singly enveloped in the polyhedron inclusion body (PIB) so that the virus is assigned to the SNPV subgenus of the Baculoviridae. Restriction endonuclease analyses of viral DNA indicated a genomic size of approximately 148 kb. Restriction profiles of O. antiqua SNPV closely resembled those of heterologous NPVs isolated from four different Orgyia species and was most similar to a North American SNPV isolate from the Douglas-fir tussock moth, O. pseudotsugata. In host range tests, O. antiqua SNPV was not infectious to 23 lepidopteran species representing four families. Heterologous Orgyia NPV isolates were permissive in O. antiqua larvae. Using a diet plug bioassay method, the median lethal dose response (LD(50)) for this virus in second and third instar O. antiqua larvae were estimated at 52 and 539 PIBs per larva, respectively.     

Tests of the role of a nuclear polyhedrosis virus in the population dynamics of its host,douglas-fir tussock moth,Orgyia pseudotsugata (Lepidoptera: Lymantriidae)

Summary Outbreaks of the Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough), have recurred periodically, at 7- to 10-year intervals, since the first recorded observation in 1916 in Chase, British Columbia, Canada. Anderson and May (1981) hypothesized that microparasites are responsible for the periodic population fluctuations of some defoliating insects. We chose the association between the Douglas-fir tussock moth and a viral disease, caused by a nuclear polyhedrosis virus (NPV), to test whether their model, and variants thereof, can predict the observed population cycles. Density-dependent mortality, vertical transmission of the virus and an incubation period were added to the free-living stages model of Anderson and May (1981). Parameter values for the models were derived from published data and from an experiment.Sensitivity analyses conducted for each model showed that none of the models generated the behavior of the Douglas-fir tussock moth as observed in the field. Thus, the periodicity of the outbreaks in field populations of tussock moths cannot be explained solely by the dynamics of the viral disease as described by Anderson and May's class of models; the virus is too short-lived and the growth rate of the insect population too high. Dynamics of other system components such as predators, parasites or food of the tussock moth probably play a significant role in the insect's population dynamics.     

Development and Evaluation of Methods To Detect Nucleopolyhedroviruses in Larvae of the Douglas-Fir Tussock Moth, Orgyia pseudotsugata (McDunnough)

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r² = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.     

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC₅₀s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.     

Molecular characterization of Agrotis segetum nucleopolyhedrovirus from Poland

The turnip moth, Agrotis segetum (Lepidoptera, Noctuidae), is an important pest insect in Europe, Asia, and Africa. We have genetically characterized and classified a nucleopolyhedrovirus isolated from A. segetum larvae in Poland (AgseNPV-P). The restriction pattern of AgseNPV-P was distinct from an isolate from England/France (AgseNPV-UK and AgseNPV-F). Sequence analysis of three conserved baculovirus genes, polyhedrin, lef-8 and pif-2, revealed that AgseNPV-P differs substantially from the already described NPVs isolated from A. segetum and possibly represents a new NPV species. Phylogenetic analysis placed AgseNPV-P among group II NPVs and showed the closest relationship to Agrotis ipsilon (Agip) NPV and Spodoptera exigua (Se) MNPV.     

Serological relationships among plusiinae baculoviruses

Antisera were produced against nucleocapsids, NP-40 detergent soluble proteins, or polyhedral protein of the multiply embedded nuclear polyhedrosis virus (MNPV) of Autographa californica, nucleocapsids of Trichoplusia ni singly embedded virus (SNPV), and polyhedral protein of Lymantria dispar MNPV. Antigens consisting of nucleocapsids, NP-40 soluble proteins, and polyhedral protein were prepared from A. californica MNPV, T. ni MNPV, L. dispar MNPV, Rachiplusia ou MNPV, T. ni SNPV, and Pseudoplusia includens SNPV. Radial immunodiffusion patterns formed with Plusiinae nucleocapsid antigens and antiserum to nucleocapsids of A. californica MNPV or T. ni SNPV revealed a distinction between multiply and singly embedded viruses. The same alignment of Plusiinae viruses was observed in reactions between A. californica NP-40 soluble protein antiserum and the NP-40 soluble protein fractions from the Plusiinae NPVs. There were no reactions between the Plusiinae SNPV nucleocapsid antigens and the A. californica MNPV nucleocapsid antiserum. However, there were faint precipitin bands between MNPV nucleocapsid antigens and T. ni SNPV nucleocapsid antiserum. Each of the polyhedral protein fractions from the Plusiinae formed a single precipitin band with the antiserum to polyhedral protein of either A. californica or L. dispar. The precipitin bands formed with the A. californica antiserum by polyhedral proteins of T. ni SNPV, P. includens SNPV, and R. ou MNPV were confluent, and shared partial identity with those formed by A. californica MNPV and T. ni MNPV. All precipitin bands formed by Plusiinae polyhedral proteins against antiserum to L. dispar polyhedral protein were confluent, and shared partial identity with that formed by L. dispar polyhedral protein.     

European Leucoma salicis NPV is closely related to North American Orgyia pseudotsugata MNPV

The satin moth Leucoma salicis L. (Lepidoptera, Lymantriidae) is a frequent defoliator of poplar trees (Populus spp.) in Europe and Asia (China, Japan). Around 1920 the insect was introduced into the USA and Canada. In this paper, a multicapsid nucleopolyhedrovirus isolated from L. salicis larvae in Poland (LesaNPV) was characterized and appeared to be a variant of Orgyia pseudotsugata (Op) MNPV. O. pseudotsugata, the Douglas fir tussock moth (Lepidoptera, Lymantriidae), occurs exclusively in North America. Sequences of three conserved baculovirus genes, polyhedrin, lef-8, and pif-2, were amplified in polymerase chain reactions using degenerate primer sets, and revealed a high degree of homology to OpMNPV. Restriction enzyme analysis confirmed the close relationship between LesaNPV and OpMNPV, although a number of restriction fragment length polymorphisms were observed. The lef-7 gene, encoding late expression factor 7, and the ctl-2 gene, encoding a conotoxin-like protein, were chosen as putative molecular determinants of the respective viruses. The ctl-2 region appeared suitable for unequivocal identification of either virus as LesaNPV lacked a dUTPase gene in this region. Our observations may suggest that LesaNPV, along with L. salicis, was introduced into O. pseudotsugata after introduction of the former insect into North America in the 1920s.     

Characterisation of a nucleopolyhedrovirus and Spiroplasma sp. bacterium associated with outbreaking populations of the Antler moth Cerapteryx graminis

A broad survey was undertaken to characterise microbes associated with larval outbreaks of the Antler moth Cerapteryx graminis in Cumbria, United Kingdom. A nucleopolyhedrovirus present in all sampled populations at ?5% prevalence, was characterised via restriction fragment length polymorphism and partial sequencing the Polyhedrin, Lef-8 and Lef-9 genes; indicating a previously uncharacterised species most closely related to Agrotis ipsilon NPV. A survey of the host-associated bacterial community detected a species phylogenetically related to Spiroplasma sp., a male-killing phenotype previously isolated from Lepidoptera and Coleoptera, present at <63% prevalence in larvae. The implications of these associated microbes for host population dynamics are discussed.     

Baculovirus immediate-early promoter-mediated expression of the Zeocin™ resistance gene for use as a dominant selectable marker in Dipteran and Lepidopteran insect cell lines

The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 μg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 μg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were used to drive expression of the Zeocin resistance gene (ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3–4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by β-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.     

Phylogenetic Analysis of Orgyia pseudotsugata Single-nucleocapsid Nucleopolyhedrovirus

The Douglas-fir tussock moth Orgyia pseudotsugata (Lepidoptera: Lymantriidae) is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus (SNPV) isolated from O. pseudotsugata larvae in Canada (OpSNPV) was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group II NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for multiple, concatenated gene phylogenies to classify baculoviruses.     

Phylogenetic Analysis of Orgyia pseudotsugata Single-nucleocapsid Nucleopolyhedrovirus

The Douglas-fir tussock moth Orgyia pseudotsugata （Lepidoptera： Lymantriidae） is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus （SNPV） isolated from O. pseudotsugata larvae in Canada （OpSNPV） was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group Ⅱ NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for multiple, concatenated gene phylogenies to classify baculoviruses.     

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.     

Characterization of a baculovirus newly isolated from the tea slug moth, <Emphasis Type="Italic">Iragoidae fasciata</Emphasis>

The tea slug moth Iragoidae fasciata (Lepidoptera, Eucleidae) is one of the main insect pests that attack tea bushes. A new nucleopolyhedrovirus (NPV) called Iragoidae fasciata NPV (IrfaNPV) was recently isolated from diseased larvae. An 11,626 bp fragment of the viral genomic DNA containing the polyhedrin gene and other 12 genes was cloned and sequenced. Gene comparison and phylogenetic analysis showed that IrfaNPV is a member of the Group I NPVs. However, the genomic organization of IrfaNPV is highly distinct. In addition, electron microscopy analysis showed that IrfaNPV is a single nucleocapsid NPV (SNPV). An inoculation assay showed that IrfaNPV is semi-permissive in the Trichoplusia ni cell line Tn-5Bl-4. Bioassays on lethal concentration (LC₅₀) and lethal time (LT₅₀) were conducted to test the susceptibility of I. fasciata larvae to the virus.     

Comparative study on the susceptibility of cutworms (Lepidoptera: Noctuidae) to Agrotis segetum nucleopolyhedrovirus and Agrotis ipsilon nucleopolyhedrovirus

The common cutworm (Agrotis segetum) and the black cutworm (Agrotis ipsilon) are serious soil pests of many vegetable and field crops all over the world. We have demonstrated the cross-infectivity of two baculoviruses, A. segetum nucleopolyhedrovirus (AgseNPV) and A. ipsilon nucleopolyhedrovirus (AgipNPV) for these two insect pests. The susceptibility of A. segetum to AgipNPV was confirmed by DNA restriction endonuclease analyses of DNA isolated from virus harvested from infected A. segetum larvae. For an initial comparison of both viruses, partial polyhedrin sequences were amplified by PCR, cloned, and sequenced. Both viruses shared a very similar polyhedrin gene sequence resulting in only three amino acid substitutions. Phylogenetic analyses clearly demonstrated that both viruses belong to NPV group II and are most closely related to a clade consisting of Spodoptera exigua NPV, Spodoptera frugiperda NPV, and Spodoptera littoralis NPV. Since AgipNPV shows high virulence for both cutworm species, it appears to be a suitable candidate as a single biological control agent of A. segetum and A. ipsilon.     

Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10 % (v/v) Fetal Bovine Serum (FBS), 1 % (v/v) hemolymph extract, or 3 % (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID₅₀/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10 % (v/v) FBS and 1 % (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10 % (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication.     

Thermal inactivation characteristics of two strains of nucleopolyhedrosis virus (Baculovirus subgroup A) pathogenic for Orgyia pseudotsugata

Two nucleopolyhedrosis viruses of the Douglas-fir tussock moth, Orgyia pseudotsugata, one with a single nucleocapsid per envelope (SV) and one with multiple nucleocapsids per envelope (BV), are inactivated by a first-order reaction at 55° and 60°C. BV is the more thermostable of the two viruses: At both test temperatures, it has a lower inactivation rate than SV. BV is also the more virulent of the two viruses, with respect to acute course of the disease and severity of the histological lesions. The greater thermostability of BV and the acute course of the disease caused by this pathogen support the choice of BV as the virus most suitable for industrial production and field use.     

Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.) (Lepidoptera: Noctuidae)

A multicapsid nucleopolyhedrovirus (ThorMNPV) that was co-isolated with a single nucleocapid ThorSNPV from mixed infected larvae of Thysanoplusia orichalcea L. (Lepidoptea: Noctuidae) is characterized. Scanning electron microscopy of ThorMNPV showed a dodecahedral-shaped occlusion body (OB). The occluded virions contained one to as many as eight nucleocapsids/virion. Virion band profiles in gradient centrifugation were consistent in at least 10 rounds of centrifugation from different virion sample preparations. The ThorMNPV had high virulence to third instar Trichoplusia ni and Pseudoplusia includens with LD50 values of 17 and 242OBs per larva, respectively. However, ThorMNPV did not cause mortality in Spodoptera exigua, Spodoptera frugiperda, Spodoptera eridania, Anticarsia gemmatalis, and Helicoverpa zea. ThorMNPV replicates in cells of various tissues such as the fat body and tracheal epithelium cells. T. ni High 5 cells were permissive to ThorMNPV in terms of infection and viral DNA transfection, but SF-21 was less permissive and the infection process was slower. Production of OBs by ThorMNPV in the nuclei of SF-21 was not well pronounced. The genome size of ThorMNPV was estimated to be 136 kb. The polyhedrin gene open reading frame (ORF) was cloned and completely sequenced. The promoter sequence is identical to that of Autographa californica MNPV. Phylogenetic analyses using partial sequences of the polh, lef-8, and lef-9 revealed that ThorMNPV is a member of the Group I NPVs and is related but distinct from the AcMNPV/Rachiplusia ou NPV/Bombyx mori NPV cluster.     

Competition between wild-type and a marked recombinant baculovirus (Spodoptera exigua nucleopolyhedrovirus) with enhanced speed of action in insect larvae

Competition between virus genotypes in insect hosts is a key element of virus fitness, affecting their long-term persistence in agro-ecosystems. Little information is available on virus competition in insect hosts or during serial passages from one cohort of hosts to the next. Here we report on the competition between two genotypes of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), when serially passaged as mixtures in cohorts of 4th instar S. exigua larvae. One of the genotypes was a SeMNPV wild-type isolate, SeUS1, while the other was a SeMNPV recombinant (SeMNPV-XD1) having a greater speed of kill than SeUS1. SeXD1 lacks a suite of genes, including the ecdysteroid UDP-glucosyl transferase (egt) gene. SeXD1 expresses the green fluorescent protein (GFP) gene, enabling the identification of SeXD1 in cell culture and in insects. The relative proportion of SeUS1 and SeXD1 in successive passages of mixed infections in various ratios was determined by plaque assays of budded virus from infected larvae and by polymerase chain reactions and restriction enzyme analyses. The SeUS1 genotype outcompeted recombinant SeXD1 over successive passages. Depending on the initial virus genotype ratio, the recombinant SeXD1 was no longer detected after 6-12 passages. A mathematical model was developed to characterize the competition dynamics. Overall, the ratio SeUS1/XD1 increased by a factor 1.9 per passage. The findings suggest that under the experimental conditions recombinant SeXD1 is displaced by the wild-type strain SeUS1, but further studies are needed to ascertain that this is also the case when the same baculoviruses would be used in agro-ecosystems.