Partial characterization of DNA from five entomopoxviruses

Extensive genomic heterogeneity was detected in the restriction endonuclease cleavage patterns of DNA from five entomopoxvirus isolates and vaccinia virus, strain WR. An 8.2 kilobase pair extra-chromosomal element was detected in Amsacta moorei entomopoxvirus and a 22 kilobase pair extra-chromosomal DNA element was isolated from Choristoneura biennis EPV. The extent of DNA base sequence homology was determined by Southern hybridization of HindIII and BamHI DNA restriction fragments of C. biennis EPV DNA and A. moorei EPV DNA with (α³²P)-labeledA. moorei EPV DNA. Methylation of 5′-C^mCGG-3′ sequences was not detected in the DNA of A. moorei, C. biennis, E. auxiliaris, M. sanguinipes, and A. conspersa entomopoxviruses after cleavage of the viral DNAs with MspI and HpaII restriction endonucleases. Based upon the DNA base sequence homology data presented here, the five entomopoxviruses used in this study appear to be unrelated.     

Characterization of the DNA and structural proteins of entomopoxviruses from Melanoplus sanguinipes,Arphia conspirsa, and Phoetaliotes nebrascensis (Orthoptera)

Entomopoxvirus (EPV) occlusion bodies were isolated from virus infected nymphs of the grasshoppers Melanoplus sanguinipes, Arphia conspirsa, and Phoetaliotes nebrascensis. Separation of the viral structural proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave unique protein patterns for each of the three viruses. An occlusion body protein of approximately 100,000 MW was isolated from each virus. Cleavage of viral DNA with HinddIII and BamHI restriction endonucleases and separation of the fragments by agarose gel electrophoresis gave different DNA fragment patterns for each of the three entomopoxviruses. Molecular weight estimates of 120 × 10⁶ for M. sanguinipes EPV DNA, 129 × 10⁶ for A. conspirsa EPV DNA, and 125 × 10⁶ for P. nebrascensis EPV DNA were calculated from the sizes of the viral DNA fragments. Approximately 55% base sequence homology was detected by Southern hybridization of α-³²P-labeledM. sanguinipes EPV DNA with P. nebrascensis DNA. No base sequence homology was detected by Southern hybridization of labeled M. sanguinipes EPV DNA to Othnonius batesi EPV DNA (Coleoptera), Amsacta moorei EPV DNA (Lepidoptera), Euxoa auxiliaris EPV DNA (Lepidoptera), and vaccinia virus DNA fragments.     

Structural proteins of Amsacta moorei,Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses

The structural proteins of Amsacta moorei, Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses (EPVs) were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. More than 35 structural proteins were detected in each virus. Based on the distribution and the variation in the molecular weights of the virus structural proteins little homology was detected between the EPVs and vaccinia virus. The molecular weight of Amsacta EPV occlusion body matrix protein (110,000) was determined by SDS-acrylamide gel electrophoresis. The occlusion body matrix protein of Amsacta EPV occluded virus isolated from infected E. acrea larvae was rapidly degraded at pH 10.6 to peptides of approximately 94,000 and 60,000 daltons. After 2 hr incubation at alkaline pH, Amsacta EPV occlusion body protein was degraded to approximately 56,000 daltons. Proteolysis of occlusion body protein was inhibited by SDS. No proteolytic degradation was detected in occlusion body matrix protein isolated from Amsacta EPV infected BTI-EAA cells. Amino acid analysis indicates that entomopoxvirus occlusion body matrix protein consists of approximately 20% acidic amino acids and 9% of the sulfur-containing amino acids cysteine and methionine.     

Molecular weight and base composition of DNA isolated from Melanoplus sanguinipes entomopoxvirus

The DNA genome of the orthopteran entomopoxvirus (EPV) isolated from Melanoplus sanguinipes was released from the virus by treatment with proteinase K and sodium dodecyl sulfate (SDS). The average length of the virus DNA molecule was determined by electron microscopy to be 62.8 μm, corresponding to a molecular weight of 124.3 × 10⁶ daltons (80 kb). The buoyant density of Melanoplus EPV DNA in cesium chloride was calculated to be 1.678 g/cm³, which corresponds to a base ratio of 18.6 mole% guanine + cytosine.     

Infection of Locusta migratoria with entomopoxviruses from Arphia conspersa and Melanoplus sanguinipes grasshoppers

Entomopoxvirus (EPV) occlusion bodies isolated from Arphia conspersa and Melanoplus sanguinipes grasshoppers were fed to 3rd and 4th instar Locusta migratoria nymphs. Locus mortality induced by A. conspersa EPV was first detected 18 days after addition of virus to the diet, and reached a level of approximately 68% of the colony population by 60 days after virus inoculation. In a similar population of L. migratoria nymphs, mortality induced by M. sanguinipes virus reached 90% 60 days after virus inoculation. Entomopoxvirus was isolated from M. sanguinipes EPV infected locust nymphs and the viral DNA was cleaved with several restriction endonucleases. The DNA fragment patterns obtained after agarose gel electrophoresis were compared with the fragment patterns from the original sample of M. sanguinipes EPV DNA cleaved with the same restriction endonucleases. No differences in the cleavage patterns were detected between the two virus DNA samples. Virus structural proteins of M. sanguinipes EPV purified from infected locust nymphs were compared by polyacrylamide gel electrophoresis with virus proteins isolated from the original sample of M. sanguinipes EPV. A total of six different virus protein bands were detected between the two poxvirus preparations.     

Virus DNA replication and protein synthesis in Amsacta moorei entomopoxvirus-infected Estigmene acrea cells

Amsacta moorei entomopoxvirus DNA synthesis was detected in Estigmene acrea cells by [³H]thymidine incorporation 12 hr after virus inoculation. Hybridization of ³²P-labeled Amsacta entomopoxvirus DNA to the DNA from virus-infected cells indicated that viral-specific DNA synthesis was initiated between 6 and 12 hr after virus inoculation. A rapid increase in the rate of virus DNA synthesis was detected from 12 to 24 hr after virus inoculation. Amsacta entomopoxvirus protein biosynthesis in E. acrea cells was studied by [su35S]methionine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracellular virus and virus-containing occlusion bodies were first detected in virus-infected cell cultures 18 hr after virus inoculation. Thirty-seven virus structural proteins, ranging in molecular weight from 13,000 to 208,000 were detected in both occluded and nonoccluded forms of the virus. The biosynthesis of virus structural proteins increased rapidly from 18 to 34 hr after infection. A major viral-induced protein corresponding in molecular weight to viral occlusion body protein (110,000) was detected approximately 24 hr after virus inoculation.     

New Insights into the Evolution of Entomopoxvirinae from the Complete Genome Sequences of Four Entomopoxviruses Infecting Adoxophyes honmai,Choristoneura biennis,Choristoneura rosaceana,and Mythimna separata

Poxviruses are nucleocytoplasmic large DNA viruses encompassing two subfamilies, the Chordopoxvirinae and the Entomopoxvirinae, infecting vertebrates and insects, respectively. While chordopoxvirus genomics have been widely studied, only two entomopoxvirus (EPV) genomes have been entirely sequenced. We report the genome sequences of four EPVs of the Betaentomopoxvirus genus infecting the Lepidoptera: Adoxophyes honmai EPV (AHEV), Choristoneura biennis EPV (CBEV), Choristoneura rosaceana EPV (CREV), and Mythimna separata EPV (MySEV). The genomes are 80% AT rich, are 228 to 307 kbp long, and contain 247 to 334 open reading frames (ORFs). Most genes are homologous to those of Amsacta moorei entomopoxvirus and encode several protein families repeated in tandem in terminal regions. Some genomes also encode proteins of unknown functions with similarity to those of other insect viruses. Comparative genomic analyses highlight a high colinearity among the lepidopteran EPV genomes and little gene order conservation with other poxvirus genomes. As with previously sequenced EPVs, the genomes include a relatively conserved central region flanked by inverted terminal repeats. Protein clustering identified 104 core EPV genes. Among betaentomopoxviruses, 148 core genes were found in relatively high synteny, pointing to low genomic diversity. Whole-genome and spheroidin gene phylogenetic analyses showed that the lepidopteran EPVs group closely in a monophyletic lineage, corroborating their affiliation with the Betaentomopoxvirus genus as well as a clear division of the EPVs according to the orders of insect hosts (Lepidoptera, Coleoptera, and Orthoptera). This suggests an ancient coevolution of EPVs with their insect hosts and the need to revise the current EPV taxonomy to separate orthopteran EPVs from the lepidopteran-specific betaentomopoxviruses so as to form a new genus.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Production and partial characterization of monoclonal antibodies for detection of entomopoxvirus from Melanoplus sanguinipes

Entomopoxviruses (EPV) are currently being considered as candidate grasshopper (Orthoptera: Acrididae) microbial control agents. Classical techniques for diagnosing infections in grasshoppers are laborious, time consuming, and sometimes inaccurate. Specific murine monoclonal antibodies were developed against an EPV from Melanoplus sanguinipes (Fab) for use in a nitrocellulose-based enzyme-linked immunoassay (dot-blot). An IgG_2b monoclonal antibody was used to diagnose infections in grasshoppers 13 days following injection with virions. Of 25 grasshoppers that had patent infections microscopically, 22 produced positive results on the dot-blot. In a second test, 39 patently infected grasshoppers all produced positive results. Seven additional grasshoppers in the first test and 2 in the second test gave positive reactions in the dot-blot method but virus was not detected upon microscopic examination. The monoclonal antibody did not cross-react with other commonly occurring grasshopper pathogens. The dot-blot method detected as few as 2.5×10⁶ purified EPV virions. The improvement over existing detection techniques should facilitate evaluation of EPV for field use.

---

Prodution et caractérisation partielle d'anticorps monoclonaux pour la détection d'entomopoxyvirus de Melanoplus sanguinipes

Résumé Les criquets sont très nuisibles aux pâturages de l'Ouest des USA et du Canada. Les méthodes classiques de protection sont basées sur les traitements chimiques lors des pullulations. Un entomopoxvirus (EPV) extrait de M. sanguinipes est généralement considéré comme un outil, pour le contrôle des populations sur une longue période, en vue de la suppression des criquets. Les méthodes actuelles d'isolement de EPV sont longues, pénibles et peu fiables. Les tests d'adsorption des antigènes sur l'anticorps fixé et le dosage per l'anticorps enzymatiquement marqué sont efficaces, sûrs et donnent à temps des résultats pour déceler des entomopathogènes. Nous avons produit des anticorps monoclonaux de souris contre EPV de M. sanguinipes, et les avons utilisés dans des dosages immunoenzymatiques d'extraits protéiques adsorbés sur nitrocellulose. Les EPV sont recherchés sur des criquets injectés de virions 13 jours avant. Sur 25 criquets qui présentaient des infections nettes lors d'examens microscopiques, 22 ont donné des résultats positifs par sérologie. Dans un second test, sur 38 criquets nettement contaminés, tous ont donné des résultats positifs; 7 criquets suppl'ementaires mentaires dans le premier test et 2 dans le second test ont donné des résultats positifs en sérologie alors que l'examen microsopique n]avait pas test ont donné des résultats positifs en sérologie alors que l'examen microsopique n'avait pas révélé de virus. Ceci montre que ce type de détection est plus sûr que la méthode ordinaire. Les anticorps monoclonaux ne donnent pas de réactions avec les autres pathogènes courants des criquets. La méthode sérologique a permis de détecter même une concentration de 2,5×10⁶ virions EPV.

     

Detection of Amsacta moorei entomopoxvirus and vaccinia virus proteins in cell cultures restrictive for poxvirus multiplication

The titer of Amsacta entomopoxvirus (EPV) protein detected in murine L-929 cells by enzyme-linked immunosorbent assay (ELISA) decreased to within preimmune serum levels by 24 hr after inoculation of the virus which indicates that Amsacta EPV structural protein biosynthesis does not occur in the vertebrate cell line. A viral-induced protein of approximately 100,000 M_r was detected by [³⁵S]methionine incorporation 4 hr after inoculation of Tn-368 cells with Amsacta EPV. Biosynthesis of protein which reacted with vaccina antiserum was detected in Estigmene acrea (BTI-EAA) cells by ELISA 10 hr after inoculation with 10 PFU of virus per cell. The amount of putative vaccinia structural protein detected in BTI-EAA cells increased approximately twofold by 70 hr after virus inoculation. No increase in vaccinia structural protein biosynthesis was detected in BTI-EAA cells inoculated with vaccinia virus previously inactivated by heat and UV light.     

Identification and expression of a conserved ubiquitin gene homologue of Spodoptera litura nucleopolyhedrovirus (SpltNPV-I)

An ORF having a potential to code for a polypeptide of 79 amino acids has been identified within 993 nt sequence of 2 kb EcoRI-W fragment of Spodoptera litura nucleopolyhedrovirus (SpltNPV-I). Nucleotide and deduced amino acid sequence analyses showed its identity with the ubiquitin homologue of eukaryotes (79–80%), Melanoplus sanguinipes entomopoxvirus (76%) and other baculoviruses (72–89%). The ORF is under baculovirus late promoter motif RTAAG but unlike other baculoviruses, three such motifs at –6, –10 and –27 position are present in SpltNPV. The ORF expresses as a 10 kDa protein in E. coli and the purified recombinant protein showed crossreactivity with the rabbit anti-ubiquitin antibodies.     

Identification of Frankia Strains by Direct DNA Hybridization of Crushed Nodules

A hybridization procedure was developed to identify Frankia strains inside actinorhizae by direct probing of crushed root nodules. The probe consisted of an indigenous cryptic plasmid. This well-conserved, 8-kilobase plasmid was detected in Frankia isolates that were very close taxonomically (they possessed a very high DNA sequence homology). The probe did not hybridize to the DNA of Frankia isolates which did not carry the plasmid. Endophyte DNA was extracted by a modification of a technique originally developed for the detection of plasmids in Frankia isolates. The hybridization procedure applied to nodules collected in a stand of alder permitted determination of a distribution map of the plasmid-bearing Frankia strains.     

Nucleic Acid Homology of Murine Type-C Viral Genes

The nucleic acid sequence homology between various murine, endogenous type-C viruses (three host range classes of BALB/c virus, the AT-124 virus, and the CCL 52 virus) and two laboratory strains of murine leukemia virus (Rauscher and Kirsten) was determined by DNA:RNA hybridization. The viral sequences exhibit varying degrees of partial homology. DNA:DNA hybridizations were performed between [³H]DNA probes prepared from N- and X-tropic BALB/c endogenous viruses and cellular DNAs from BALB/c, NIH Swiss, and AKR inbred mouse strains as well as from California feral mice and the Asian mouse subspecies Mus musculus molossinus and M. musculus castaneus. All of these strains of mice are shown to possess multiple (six to seven per haploid genome), partially related copies of type-C virogenes in their DNAs. Thermal melting profiles of the DNA:RNA and DNA:DNA hybrids suggest that the partial homology of the viral nucleic acid sequences is the result of base alterations throughout the viral genomes, rather than the loss of discrete segments of viral sequences.     

Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M_r of its subunit was 77,000. The cells converted [¹⁴C]-l-phenylalanine into [¹⁴C]-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M_r 77, 137), a 22-bp 5′-noncoding region and a 207-bp 3′-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.     

Evidence for sub-families of actin genes in Dictyostelium as determined by comparisons of 3' end sequences

We have sequenced the 3′ end of five actin genomic clones and three actin complementary DNA clones from Dictyostelium. Comparison of the sequences shows that the protein coding regions are highly conserved, while the region corresponding to the 3′ untranslated regions are divergent. Additional analysis indicates regions of homology in the 3′ untranslated region between sets of actin genes. Southern DNA blot hybridization studies using labeled 3′ ends suggest that there are sub-families of actin genes that are related within the 3′ untranslated regions. No homology is found in the sequences outside the messenger RNA encoding regions. Analysis of the sequence data has shown that the difference in length between the ~1.25 × 10³ and ~1.35 × 10³ base actin messenger RNAs is in the lengths of the 3′ untranslated region.     

Specificity of Developmental Resistance in Gypsy Moth （Lymantria dispar） to two DNA-Insect Viruses

Gypsy moth (Lymantria dispar) larvae displayed marked developmental resistance within an instar to L. dispar M nucleopolyhedrovirus (LdMNPV) regardless of the route of infection (oral or intrahemocoelic) in a previous study, indicating that in gypsy moth, this resistance has a systemic component. In this study, gypsy moth larvae challenged with the Amsacta moorei entomopoxvirus (AMEV) showed developmental resistance within the fourth instar to oral, but not intrahemocoelic, inoculation. In general, gypsy moth is considered refractory to oral challenge with AMEV, but in this study, 43% mortality occurred in newly molted fourth instars fed a dose of 5×10⁶ large spheroids of AMEV; large spheroids were found to be more infectious than small spheroids when separated by a sucrose gradient. Developmental resistance within the fourth instar was reflected by a 2-fold reduction in mortality (18%–21%) with 5×10⁶ large spheroids in larvae orally challenged at 24, 48 or 72 h post-molt. Fourth instars were highly sensitive to intrahemocoelic challenge with AMEV; 1PFU produced approximately 80% mortality regardless of age within the instar. These results indicate that in gypsy moth, systemic developmental resistance may be specific to LdMNPV, reflecting a co-evolutionary relationship between the baculovirus and its host.