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Baculoviruses infect insects, producing two distinct phenotypes during the viral life cycle: the budded virus (BV) and the occlusion-derived virus (ODV) for intra- and inter-host spread, respectively. Since the 1980s, several countries have been using Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent against the velvet bean caterpillar, A. gemmatalis. The genome of AgMNPV isolate 2D (AgMNPV-2D) carries at least 152 potential genes, with 24 that possibly code for structural proteins. Proteomic studies have been carried out on a few baculoviruses, with six ODV and two BV proteomes completed so far. Moreover, there are limited data on virion proteins carried by AgMNPV-2D. Therefore, structural proteins of AgMNPV-2D were analysed by MALDI- quadrupole-TOF and liquid chromatography MS/MS. A total of 44 proteins were associated with the ODV and 33 with the BV of AgMNPV-2D. Although 38 structural proteins were already known, we found six new proteins in the ODV and seven new proteins carried by the AgMNPV-2D BV. Eleven cellular proteins that were found on several other enveloped viruses were also identified, which are possibly carried with the virion. These findings may provide novel insights into baculovirus biology and their host interaction. Moreover, our data may be helpful in subsequent applied studies aiming to improve AgMNPV use as a biopesticide and a biotechnology tool for gene expression or delivery.

A bunyavirus surveillance was performed in 2,600 pools consisting of 45,728 mosquitoes collected in north-central Florida from May 2006 to April 2007. Fifteen mosquito pools were found to be virus-positive from the total 2,600 mosquito pools tested (0.6% infection rate), which resulted in a minimum infection rate of 0.33 per 1,000 mosquitoes. Sequence data identified the virus to be Tensaw virus, a member of the Bunyaviridae family. All the virus-positive samples were obtained from pools collected from May to October 2006, in 3 of the 4 major locations studied, revealing the presence of Tensaw virus in north-central Florida mosquito populations in 2006.

A certain level of genetic diversity and connectivity between populations is necessary to allow small populations to persist over time. In this study, we investigate the last two populations of the highly endangered Miami blue butterfly, one of which was extirpated in 2010. We investigate whether an erosion of genetic diversity was the cause for the extirpation of the Bahia Honda State Park (BHSP) population. As the level of existing molecular diversity within and gene flow between the last two populations of the endangered Miami blue butterfly was unknown, we present data from twelve polymorphic microsatellite loci as an assessment. We show that greater genetic diversity (allelic richness and observed heterozygosity) than originally predicted existed in the BHSP population prior to its extirpation and values were even greater in the Key West National Wildlife Refuge population. Allelic frequencies show short-range gene flow between neighboring habitat patches and underline the importance of habitat connectivity. It does not appear that loss of genetic diversity was the cause for the loss of the BHSP population. Greater consideration of habitat connectivity should be considered for short and long-term conservation and management of this taxon.

The genome of the Neodiprion sertifer nucleopolyhedrovirus (NeseNPV), which infects the European pine sawfly, N. sertifer (Hymenoptera: Diprionidae), was sequenced and analyzed. The genome was 86,462 bp in size. The C+G content of 34% was lower than that of the majority of baculoviruses. A total of 90 methionine-initiated open reading frames (ORFs) with more than 50 amino acids and minimal overlapping were found. From those, 43 ORFs were homologous to other baculovirus ORFs, and 29 of these were from the 30 conserved core genes among all baculoviruses. A NeseNPV homolog to the ld130 gene, which is present in all other baculovirus genomes sequenced to date, could not be identified. Six NeseNPV ORFs were similar to non-baculovirus-related genes, one of which was a trypsin-like gene. Only one iap gene, containing a single BIR motif and a RING finger, was found in NeseNPV. Two NeseNPV ORFs (nese18 and nese19) were duplicates transcribed in opposite orientations from each other. NeseNPV did not have an AcMNPV ORF 2 homolog characterized as the baculovirus repeat ORF ( bro ). Six homologous regions ( hr s) were located within the NeseNPV genome, each containing small palindromes embedded within direct repeats. A phylogenetic analysis was done to root the tree based upon the sequences of DNA polymerase genes of NeseNPV, 23 other baculoviruses, and other phyla. Baculovirus phylogeny was then constructed with 29 conserved genes from 24 baculovirus genomes. Culex nigripalpus nucleopolyhedrovirus (CuniNPV) was the most distantly related baculovirus, branching to the hymenopteran NeseNPV and the lepidopteran nucleopolyhedroviruses and granuloviruses.

La deteccion de Escherichia coli O157:H7 en las lecherias es importante para mejorar la seguridad de los productos lacteos, y se ha llevado a cabo principalmente mediante el aislamiento de las bacterias a partir de las muestras de estiercol. Sin embargo, los componentes biliares presentes en el estiercol complica la identificacion genetica utilizando la tecnica del PCR, y el aislamiento microbiologico se dificulta por la presencia de bacterias competidoras que comparten caracteristicas microbiologicas similares. El aislamiento de E. coli O157:H7 a partir de la mosca domestica evita las dificultades asociadas con el estiercol del ganado. El aislamiento de patogenos a partir de las moscas domesticas proporciona informacion adicional sobre el potencial impacto epidemiologico de la dispersion de la mosca domestica en la distribucion de patogenos, ya que las moscas domesticas se dispersan desde las lecherias donde la E. coli O157:H7 existe en forma endemica en el ganado. En este estudio, se encontro que las moscas domesticas son 2,6 veces mas sensibles para la deteccion de E. coli O157:H7 en las lecherias. Las moscas son mas faciles de capturar y manejar que el estiercol, y deberian ser utilizadas en cualquier ensayo para detectar E. coli O157:H7 en las lecherias y otros establecimientos.

Dense populations of extracellular bacteria were detected in midgut crypts of the southern chinch bug, Blissus insularis Barber (Hemiptera: Blissidae). Examination by epifluorescent and transmission electron microscopy revealed that the bacteria covered the luminal surface of the crypts and filled the entire lumen. Attempts to culture the extracellular endosymbionts in various media failed. Sequencing and phylogenetic analyses of 16S rRNA gene clones obtained from insects of five Florida populations showed high nucleotide homology to either betaproteobacterial Burkholderia spp. (243 clones from five populations) or gammaproteobacterial Pseudomonas spp. (58 clones from one population). Using Burkholderia-specific primers, bacteria were detected in the egg, nymph, and adult stages. Fluorescent in situ hybridization with genus-specific oligonucleotide probes confirmed the localization of Burkholderia in the crypts. Quantitative real-time PCR showed that antibiotic treatments of nymphs significantly reduced the amount of Burkholderia 16S rRNA gene copies in chinch bugs sampled 11 days after the treatment. Furthermore, these treatments resulted in retarded development and high mortality of B. insularis, indicating a beneficial impact of Burkholderia on its host.

The chilli thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) is an emerging pest of many economically important vegetable and ornamental crops grown in the United States. Accurate identification of this pest is a fundamental requirement in development of effective quarantine and management strategies. Using scanning electron microscopy, high resolution images of important taxonomic traits of this pest were produced, which will aid research, regulatory and extension personnel to identify this pest. High resolution images were obtained for identifying characters of S. dorsalis including tergites with antecostal ridges; head with 3 pairs of ocellar setae, metanotum presenting longitudinal striations with medially located pair of setae; veins of forewing presenting widely spaced setae; segment VIII with complete posteromarginal comb of microtrichia; and sternites lacking discal setae but covered with rows of microtrichia except in the antero-medial region. Further, a preliminary comparison of morphological traits of S. dorsalis populations from different geographical regions was conducted, which can help in understanding the phenotype of this pest. Specimens of S. dorsalis were obtained from 5 distinct geographical regions: New Delhi, India; Shizouka, Japan; Negev, Israel; St. Vincent and Florida in the United States. Fourteen morphological characters of each population of S. dorsalis were measured and compared among the 5 populations. No significant differences were observed between the body lengths of the various S. dorsalis populations, which ranged from 0.85 mm (Negev) to 0.98 mm (Florida). When comparing 12 morphological characters, we found no significant differences among New Delhi, St. Vincent, Negev and Florida populations. However, when S. dorsalis populations of these 4 regions were compared with Shizouka, significant differences were detected for either 2 or 5 morphological characters depending on the population, suggesting the Japan population is more robust i.e., longer and wider mesothorax and metathorax, and wider abdomens. Also, the mean lengths of body size among different populations did not vary directly or inversely with latitude.

A species-specific multiplex polymerase chain reaction targeting the cytochrome b gene of cattle, horses, humans, and dogs was developed to determine the blood meal sources of stable flies, Stomoxys calcitrans (L.), collected from Florida equine facilities. Of 595 presumptive blood-fed stable flies analyzed, successful host amplification was obtained in 350, for a field host-detection efficiency of 58.8%. The majority of analyzed stable flies had fed on cattle (64.6%), followed by horses (24.3%), humans (9.5%), and dogs (1.6%). A survey of animal-enclosed pastures occurring within 3 km of stable fly collection sites revealed that the nearest cattle were between 0.8 and 1.5 km from the four horse farm sampling sites. Cattle-feeding frequencies were greater on farms where cattle were located at distances of 0.8 km, suggesting that between farm differences in host-feeding frequency is related to the number of and distance from a particular host type. Time course evaluations of previously laboratory-fed stable flies demonstrated that host-detection efficiency with this system was 100, 50, and 0% when flies were evaluated at 16, 24, and 48 h postblood feeding, respectively. The results of this study suggest short-term stable fly dispersal of up to 1.5 km in a 48-h time period. The implications of these findings are discussed.

The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Autographa californica nuclear polyhedrosis virus) vector and shown by SDS-PAGE and Western blot analysis to be about 57 kDa. Transmission electron microscopy revealed fully assembled nucleocapsid-like particles (NLPs) exhibiting the typical helical herringbone morphology. These NLPs were approximately 20-22 nm in diameter and varied in length from 50 to 100 nm. Purified DMV-N protein was used as antigen in an indirect ELISA (iELISA) and shown to react with rabbit and human antisera to measles virus (MV) and dog sera with antibodies to canine distemper virus (CDV). The iELISA was used for the demonstration of morbillivirus antibodies in the serum of cetaceans and manatees, showing potential as a serological tool for the mass screening of morbillivirus antibodies in marine mammals.

Bacteria carried by wild house flies (Musca domestica L.) collected near the rear entrances and dumpsters of 4 restaurants in north central Florida were identified. Live house flies were collected and individually transferred to blood agar plates for 1 h. After removing the flies, the plates were incubated overnight at 37C. Bacterial colonies that were morphologically distinct were isolated from other colonies by streaking onto new plates. The bacteria were identified by fatty acid analysis and sequence of their 16S rRNA gene. The bacterial isolates included 5 new bacterial records for house flies: Acinetobacter baumanni, Bacillus pumilus, Cronobacter sakazakii, Methylobacterium persicinum, and Staphylococcus sciuri. Other bacteria identified have been associated previously with house flies, including Bacillus cereus, B. thuringiensis, Escherichia coli 0157:H7, Shigella dysenteriae, Staphylococcus saprophyticus, and Staphylococcus xylosus. Most of the organisms recovered from the house fly are se...

Mosquitoes from different species endemic to North Florida were collected over a 12-month period, and blood meal analyses were conducted on engorged females to determine the range of vertebrate hosts each species fed on. Thirty-one mosquito blood meals from 6 mosquito species were identified, and blood meal hosts identified included horse, cow, armadillo, deer, raccoon, rabbit, and owl. Several mosquitoes captured in the study should be considered potential bridge vectors for encephalitis viruses considering their ubiquity, their preference for mammalian blood meals, and their competence as viral vectors. Translation provided by the authors.

Tensaw virus (TSV) belongs to the genus Orthobunyavirus within the Bunyaviridae family. Although TSV does not cause hemorrhagic fever as some other members of its family, serological studies have shown that serum from Florida residents react against TSV indicating viral infection in humans. In this study, the three RNA genome segments of a TSV isolated from Anopheles crucians mosquitoes collected in North Central Florida in 2006 and a TSV isolate obtained from the CDC, Fort Collins, were sequenced and compared to other Bunyaviridae. The placement of the TSVs within the Bunyamwera serogroup was confirmed by phylogenetic analysis of the inferred amino acid (aa) sequence of proteins coded by each of the RNA segments separately as well as by the combined tree of the same three inferred proteins. The N terminal glycoprotein (Gn) encoded by the M segment contained the 18 conserved Cysteines present in Bunyamwera and California serogroups, the two glycosylation sites, and residues considered potential proteolytic cleavage sites conserved in other Bunyaviridae. The TSV L protein displayed all the strictly conserved amino acids in the four conserved regions known to be catalytically active for the RNA dependent RNA polymerase transcriptase and replicase activities. The amino acid conservation between the two TSV viral isolates was 100, 99.4, and 99.6% for the S, M, and L segments, respectively.

TheMusca domesticasalivary gland hypertrophy virus (MdSGHV) is a large dsDNA virus that infects and sterilizes adult houseflies. The transcriptome of this newly described virus was analysed by rapid amplification of cDNA 3′-ends (3′-RACE) and RT-PCR. Direct sequencing of 3′-RACE products revealed 78 poly(A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs not amplified by 3′-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either 3′-RACE or RT-PCR. A series of 5′-RACE reactions were conducted on selected ORFs that were identified by 3′-RACE to be transcribed in tandem (tandem transcripts). In the majority of cases, the downstream ORFs were detected as single transcripts as well as components of the tandem transcripts, whereas the upstream ORFs were found only in tandem transcripts. The only exception was the upstream ORF MdSGHV084, which was differentially transcribed as a single transcript at 1 and 2 days post-infection (days p.i.) and as a tandem transcript (MdSGHV084/085) at 2 days p.i. Transcriptome analysis of MdSGHV detected splicing in the 3′ untranslated region (3′-UTR) and extensive heterogeneity in the polyadenylation signals and cleavage sites. In addition, 23 overlapping antisense transcripts were found. In conclusion, sequencing the 3′-RACE products without cloning served as an alternative approach to detect both 3′-UTRs and transcript variants of this large DNA virus.

The genome of the virus that causes salivary gland hypertrophy in Musca domestica (MdSGHV) was sequenced. This non-classified, enveloped, double stranded, circular DNA virus had a 124,279bp genome. The G + C content was 43.5% with 108 putative methionine-initiated open reading frames (ORFs). Thirty ORFs had homology to database proteins: eleven to proteins coded by both baculoviruses and nudiviruses (p74, pif-1, pif-2, pif-3, odv-e66, rr1, rr2, iap, dUTPase, MMP, and Ac81-like), seven to nudiviruses (mcp, dhfr, ts, tk and three unknown proteins), one to baculovirus (Ac150-like), one to herpesvirus (dna pol), and ten to cellular proteins. Mass spectrum analysis of the viral particles' protein components identified 29 structural ORFs, with only p74 and odv-e66 previously characterized as baculovirus structural proteins. Although most of the homology observed was to nudiviruses, phylogenetic analysis showed that MdSGHV was not closely related to them or to the baculoviruses.

A simple method was developed to analyze the two morphologically indistinguishable host-associated strains of the fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith). Total DNA extracted from the FAW corn and rice strains, as well as from the S. frugiperda cell line (SF9) was used to PCR amplify a 569 base pairs region of the mitochondrial gene cytochrome oxidase subunit I (COI). The amplified DNA from the Spodoptera corn strain and the SF9 cell line contained a restriction fragment length polymorphism (RFLP) marker, the MspI recognition site that was not present in the rice strain. This PCR-RFLP method does not require purification of mitochondrial DNA (mtDNA) or the use of radioactive isotopes, and differs from previous methods in that only a few nanograms of total DNA are needed to yield clear and accurate strain identification of individual insects.

Seventeen plaque purified isolates of two viral preparations of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), were analyzed in terms of the genomic changes after digestion of their DNAs with HindIII and PstI restriction enzymes. The 1979 AgMNPV wild type preparation (AgMNPV-'79) resulted in six different variants and the 1985 viral commercial preparation (AgMNPV-'85), in eleven. The genomic variation of all the isolates was mapped showing that those from 1985 presented more heterogeneity with changes mapped in additional sites in comparison to the AgMNPV-'79 variants. Their virulence was compared by infecting two Lepidopteran cell lines, Spodoptera frugiperda (IPLB-SF-21AE) and Anticarsia gemmatalis (UFL-AG-286). The results indicated that there was some difference in virulence within the AgMNPV-'85 variants. This commercial preparation had been applied in soybean fields in Brazil over several years to control the velvetbean caterpillar defoliation.

Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (Ag M NPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0.25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the Ag M NPV DNA preparations were diluted 10- or 100-fold. Ag M NPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.

Sequences of the 16SrRNA gene of three microsporidia pathogenic to imported fire ants, Solenopsis invicta and Solenopsis richteri, were determined and compared to each other and 15 other species of microsporidia. The sequences of 2 Thelohania species are nearly identical (99.2% identity), supporting light-microscopic and ultrastructural evidence that Thelohania solenopsae and Thelohania sp. are closely related but probably not conspecific. Sequence comparisons further revealed that Vairimorpha sp. has a sequence identity of about 73% with the two Thelohania species and Vairimorpha necatrix, the type species of the genus Vairimorpha. This, together with information on spore morphology, suggests that Vairimorpha sp. represents a genus distinct from that of the fire ant Thelohania. Its placement in the genus Vairimorpha must also be reevaluated. Two new sister taxa, one containing T. solenopsae and Thelohania sp. and one containing Vairimorpha sp., were found to have diverged early in the microsporidian lineage.

The detection of species and strains of baculoviruses in natural field insect populations facilitates ecological studies, risk assessment evaluation and quality control programs where wild type and/or recombinant baculoviruses are used. The polymerase chain reaction (PCR) was evaluated as a detection tool for determining viral persistence and spread in Anticarsia gemmatalis Hubner and predatory hemipteran populations in fields of soybean, Glycine max (L.) Merril. Two isolates of the A. gemmatalis nucleopolyhedrovirus (AgMNPV-2D and AgMNPV-D7) were applied individually and mixed in replicated field plots. The insect populations were sampled 1, 10, 15, 30, and 45 d after viral application and taken to the laboratory for PCR analysis. PCR analysis targeted the coding region of the viral polyhedrin gene and the highly variable homologous region 4 (hr4 region). The size of the expected products was 575 base pairs (bp) for the polyhedrin-PCR, and 1,726 bp (AgMNPV-2D) and 1,345 bp (AgMNPV-D7) for the hr4 region. The virus was detected in A. gemmatalis larvae and predators collected on all sample dates by PCR amplification of the polyhedrin region. AgMNPV was detected in the host and predator populations 1–45 d after application in the field. Virus movement into the control plots indicated that AgMNPV dispersed at a rate of 2–3 m per day. PCR amplification of the hr4 region was effective in distinguishing between AgMNPV-2D and AgMNPV-D7 isolates. The polymerase chain reaction proved to be a useful tool in ecological studies of this baculovirus.

A technique using the polymerase chain reaction (PCR) and restriction analysis was developed for the simultaneous detection of eight baculoviruses. The baculoviruses detected by this technique were Autographa californica multiple-embedded nuclear polyhedrosis virus (MNPV), Anticarsia gemmatalis MNPV, Bombyx mori MNPV, Orgyia pseudot-sugata MNPV, Spodoptera frugiperda MNPV, S. exigua MNPV, Anagrapha falcifera MNPV, Heliothis zea single-embedded nuclear polyhedrosis virus (SNPV). A highly conserved DNA sequence within the coding region of the polyhedrin gene was targeted for amplification. One pair of degenerate primers was designed, and PCR conditions were optimized to produce 575 base pair fragments for all eight baculoviruses. Restriction analysis of the PCR products resulted in distinct profiles for each virus. This technique would be useful in monitoring the release of wild type as well as genetically engineered baculoviruses.

Primary powders of Bacillus sphaericus strain S2 isolated from soil samples in Brazil, and strain 2362 were produced in a 14 liter fermentor. Growth patterns and sporulation observed in three trials with strains S2 and 2362 in the fermentor were similar. Second-instar larvae of Culex quinquefasciatus, Anopheles albimanus, Anopheles quadrimaculatus, and Aedes aegypti exposed for 48 hr to strain S2 responded with LC50 values of 0.25, 5.95, 12.28 and 140.0 ppb of lyophilized primary powder, respectively. Under the same conditions, strain 2362 resulted in LC50 values of 0.39, 7.16, 16.93 and 307.0 ppb of lyophilized primary powder, respectively, in those mosquito larvae. Statistical analysis of the bioassay data did not show significant differences among LC50 values observed in B. sphaericus strains S2 and 2362, at the 0.05 level. Toxins of strains S2 and 2362 were extracted at pH 12 with NaOH. Electrophoresis of the extracts in polyacrylamide gel under denaturing conditions revealed the 51 and 42 kDa toxins in both S2 and 2362 B. sphaericus strains. The presence of the 42 kDa peptide in the extracts was confirmed by Western blot and Elisa, with anti-42 kDa IgG previously prepared from strain 2362.

The Spodoptera frugiperda multiple nucleocapsid nuclear polyhedrosis virus (SfMNPV) gp41 structural protein gene was located in the 1.9 kbp EcoRI-S fragment and sequenced. An open reading frame (ORF) of 999 nucleotides was detected that encoded a protein of 332 amino acids. The gp41 gene transcript was detected after 12 h post-infection (p.i.) and remained detectable at 48 h p.i. Two major mRNAs, about 1.6 and 2.8 kb in length, were determined by Northern blot analysis. Primer extension analysis demonstrated that the gp41 gene promoter region contains three transcription start sites. Two of the gp41 gene transcription start sites were located at -42 and -41 nucleotides from the ATG translation start codon within a consensus late transcription start site (TAAG) and another transcription start site was located at -140 nucleotides from the ATG translation start codon for which no consensus motif has been determined. Comparison of SfMNPV gp41 nucleotide and amino acid sequences with the gp41 genes from Autographa californica, Bombyx mori, and Helicoverpa zea NPVs showed 60% homology of nucleotide sequences and 70% similarity of amino acid sequences.

The effect of Musca domestica salivary gland hypertrophy virus (MdSGHV) on selected fitness parameters of stable flies, Stomoxys calcitrans (L.), was examined in the laboratory. Virus-injected stable flies of both genders suffered substantially higher mortality than control flies. By day 9, female mortality was 59.3 +/- 10.1% in the virus group compared with 23.7 +/- 3.7% in the controls; mortality in virus-injected males was 78.1 +/- 3.1% compared with 33.3 +/- 9.3% for controls. Fecundity of control flies on days 6-9 was 49-54 eggs deposited per live female per day (total, 8,996 eggs deposited), whereas virus-injected flies produced four to five eggs per female on days 6-7 and less then one egg per female per day thereafter (total, 251 eggs). Fecal spot deposition by virus-injected flies was comparable to controls initially but decreased to approximately 50% of control levels by day 4 after injection; infected flies produced only 26% as many fecal spots as healthy flies on days 6 and 7. None of the virus-injected stable flies developed symptoms of salivary gland hypertrophy. Quantitative real-time polymerase chain reaction demonstrated virus replication in injected stable flies, with increasing titers of virus genome copies from one to four days after injection. MdSGHV in stable flies displayed tissue tropism similar to that observed in house fly hosts, with higher viral copy numbers in fat body and salivary glands compared with ovaries. Virus titers were approximately 2 orders of magnitude higher in house fly than in stable fly hosts, and this difference was probably due to the absence of salivary gland hypertrophy in the latter species.

A strain of Spodoptera frugiperda nuclear polyhedrosis virus (SfNPV) was selected in previous research for an increased rate of vertical transmission by isolating it from pupae that had been infected through vertical transmission. Differences were detected between the wild (PW) and the selected (PS) isolates of SfNPV when the viral DNA was digested with the restriction enzymes Eco RI, Hind III, and Bam HI. The restriction profiles indicated a possible DNA insertion in the PS isolate. Probes with 32 P-labeled SfNPV DNA and S. frugiperda cellular DNA indicated that there was only viral DNA, not S. frugiperda cellular DNA, in the genome of the PS isolate. Therefore, the differences between the PW and the PS isolates were due to rearrangement of DNA within the viral genome. Cluster analysis based on numbers of DNA fragments shared among SfNPV isolates from the Eco RI, Hind III, and Bam HI enzyme digests indicated that the PS isolate had one of the more unique genomes when compared with the PW isolate and 22 other SfNPV isolates analyzed in previous research. However, the PS DNA enzyme digests did not fall outside the range of patterns of digests observed in the natural isolates.

A double-stranded DNA virus was isolated from hyperplasic salivary glands of male and female houseflies, Musca domestica L. (Diptera: Muscidae), collected from a dairy in Alachua County, Florida, U.S.A. Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) of this housefly salivary gland hyperplasia (SGH) virus revealed the presence of two major and eight minor structural polypeptides. Restriction endonuclease analysis indicated that the c. 137 kilobase pair DNA was double-stranded. Weekly sweep-net sampling of the fly population throughout the season (May-October, 1991) showed that 1.5-18.5% of the dissected flies possessed hyperplasic salivary glands. The virus replicated within the nuclei of the salivary gland cells and was transmitted per os to newly-emerged healthy adult flies.

The cDNA sequence encoding mature human C9 protein and its signal peptide was cloned into three expression vectors for expression in COS-7 (mammalian), Spodoptera frugiperda IPLB-SF-21AE (insect), and Saccharomyces cerevisiae (yeast) cells. In addition, C9 cDNA encoding only the mature protein was fused to the yeast invertase leader sequence (SUC2) and cloned for expression in yeast. Under optimal conditions COS-7 and IPLB-SF-21AE cells secreted recombinant C9 (rC9) at concentrations of about 111 and 700 ng C9/ml culture supernatant, respectively. By comparison S. cerevisiae, whether transformed with C9 cDNA containing its native or yeast invertase leader sequence, secreted only very small amounts of rC9 (5-10 ng/ml). However, upon lysis concentrations of up to 500 ng/mg dry wt were found in yeast cells transformed with C9 cDNA. SDS-PAGE followed by Western blot analysis revealed COS-7 cell and S. cerevisiae expressed rC9 to have a MW similar to that of native C9 purified from human serum, while rC9 from IPLB-SF-21AE cells was about 4 kDa smaller. No hemolytic activity of S. cerevisiae secreted rC9 could be detected and the specific hemolytic activity of S. cerevisiae intracellular rC9 was also very low. However, the specific hemolytic activities of COS-7 and IPLB-SF-21AE secreted rC9 were indistinguishable from that of purified native human C9. Thus, for future studies on the structure and function of C9 where the production of large quantities of mutant protein would be desirable, the baculovirus-insect cell expression system appears to offer considerable advantages.

The genomic region of the Anticarsia gemmatalis multiple nucleocapsid nuclear polyhedrosis virus (AgMNPV) strain 2D encoding the polyhedrin gene was cloned and mapped, and a 2085 bp SphI-PstI fragment containing the gene was sequenced. The polyhedrin polypeptide of the parental isolate AgMNPV was manually sequenced, and the amino acid sequence obtained agreed with that deduced from the DNA coding region sequence. AgMNPV and Orgyia pseudotsugata MNPV (OpMNPV) are similar in terms of promoter structure and polyhedrin primary sequence, and the polyhedrin gene of both viruses is transcribed in the anti-clockwise direction in relation to their physical maps. The region upstream from the polyhedrin gene of AgMNPV, OpMNPV, Bombyx mori NPV and Autographa californica MNPV (AcMNPV) was compared and this showed that the open reading frame (ORF) common to all four viruses (ORF 5) has sequence homology with the AcMNPV 25K gene. The sequences between ORF 5 and the polyhedrin gene were found to be variable among the polyhedrin gene loci compared. Additionally, conserved elements in the promoters of the very late genes encoding polyhedrin and granulin, and those encoding two p10 proteins were found to share sequence homology and positional similarity with consensus regions in the conserved boxes A and C, responsible for binding transcription factors to eukaryotic 5S ribosomal RNA genes, and to box C of tRNA genes.

The objective of this study was to examine the extrinsic risk factors of West Nile virus (WNV) clinical disease in Florida horses as established from confirmed and negative horses tested within the state from 2001 to 2003. An Arboviral Case Information Form (ACF) was submitted by a referring veterinarian at the time of testing to the Florida Department of Agriculture and Consumer Services on every horse suspected of a viral encephalitis in Florida. A follow-up survey that focused on arbovirus prevention and farm ecology was created and mailed to the owner of each tested horse. Data from the follow-up survey indicated peak WNV prevalence in the late summer months in Florida. Quarter horses were the most commonly affected breed. The WNV vaccine was highly protective and natural water on the property also had a protective association. Factors that increased the risk of WNV to horses were the use of fans and a stable construction of solid wood or cement. Some risk indicators were dead birds on the property and other ill animals on the property. Data from this retrospective study have helped identify factors associated with WNV transmission in equines in Florida. Horses that have not been vaccinated and show clinical signs of arboviral infection from June to November should be tested for WNV. Horses that have been vaccinated and show clinical signs should be tested when the vaccination was administered within 1 month or greater than 6 months prior to the onset of clinical symptoms associated with WN infection.

Glossina pallidipes and Musca domestica salivary gland hypertrophy viruses (GpSGHV and MdSGHV) replicate in the nucleus of salivary gland cells causing distinct tissue hypertrophy and reduction of host fertility. They share general characteristics with the non-occluded insect nudiviruses, such as being insect-pathogenic, having enveloped, rod-shaped virions, and large circular double-stranded DNA genomes. MdSGHV measures 65×550 nm and contains a 124 279 bp genome (∼44 mol% G+C content) that codes for 108 putative open reading frames (ORFs). GpSGHV, measuring 50×1000 nm, contains a 190 032 bp genome (28 mol% G+C content) with 160 putative ORFs. Comparative genomic analysis demonstrates that 37 MdSGHV ORFs have homology to 42 GpSGHV ORFs, as some MdSGHV ORFs have homology to two different GpSGHV ORFs. Nine genes with known functions (dnapol, ts, pif-1, pif-2, pif-3, mmp, p74, odv-e66 and helicase-2), a homologue of the conserved baculovirus gene Ac81 and at least 13 virion proteins are present in both SGHVs. The amino acid identity ranged from 19 to 39 % among ORFs. An (A/T/G)TAAG motif, similar to the baculovirus late promoter motif, was enriched 100 bp upstream of the ORF transcription initiation sites of both viruses. Six and seven putative microRNA sequences were found in MdSGHV and GpSGHV genomes, respectively. There was genome. Collinearity between the two SGHVs, but not between the SGHVs and the nudiviruses. Phylogenetic analysis of conserved genes clustered both SGHVs in a single clade separated from the nudiviruses and baculoviruses. Although MdSGHV and GpSGHV are different viruses, their pathology, host range and genome composition indicate that they are related.

Salivary gland hypertrophy viruses (SGHVs) have been identified from different dipteran species, such as the tsetse fly Glossina pallidipes (GpSGHV), the housefly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). These viruses share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 500–1,000 nm in length and 50–100 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120 to 190 kbp and having G + C ratios ranging from 28 to 44%; (iii) they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility. The available information on the complete genome sequence of GpSGHV and MdSGHV indicates significant co-linearity between the two viral genomes, whereas no co-linearity was observed with baculoviruses, ascoviruses, entomopoxviruses, iridoviruses and nudiviruses, other large invertebrate DNA viruses. The DNA polymerases encoded by the SGHVs are of the type B and closely related, but they are phylogenetically distant from DNA polymerases encoded by other large dsDNA viruses. The great majority of SGHV ORFs could not be assigned by sequence comparison. Phylogenetic analysis of conserved genes clustered both SGHVs, but distantly from the nudiviruses and baculoviruses. On the basis of the available morphological, (patho)biological, genomic and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This proposed family currently comprises two unassigned species, G. pallidipes salivary gland hypertrophy virus and M. domestica salivary gland hypertrophy virus, and a tentative unassigned species, M. equestris salivary gland hypertrophy virus. Here, we present the characteristics and the justification for establishing this new virus family.

1. 1. The sensitivity of insect cells to whole ricin and its catalytic subunit has been examined. Evidence is presented that although cells from Trichoplusia ni and Spodoptera frugiperda are resistant to ricin, cell-free protein synthesis extracts are inhibited by ricin A chain and that the isolated insect ribosomes are sensitive to the N-glycosidase depurization activity of the A chain. 2. 2. These results are consistent with the model that, as with other sensitive eukaryotic ribosomes, insect ribosomes appear to have a consensus sequence in the ribosomal RNA on the surface of the ribosome which is sensitive to this N-glycosidase.

Baculovirus-insect cell system (BICS) is considered one of the most efficient eukaryotic gene expression systems. This system has also been used for producing different recombinant baculoviruses with increased insect toxicity as potential biopesticides. Establishing a universal gene silencing (UGS) system is very important due to the increasing number of studies using RNA interference (RNAi) with BICS. In this work, the enhanced green fluorescent protein (EGFP) was used as the RNAi consistent target sequence located downstream of a depressant insect-neurotoxin gene, LqqIT2 used as a model of the gene of interest. Small interfering RNA (siRNA) and inverted repeats of EGFP gene (IR-EG) were examined in targeting the EGFP-LqqIT2 (EL)-fusion mRNA or LqqIT2-EGFP (LE)-bicistronic mRNA for degradation. Suppression efficiencies using these inducers were examined transiently and stably in uninfected and infected insect Sf9 cells. Moreover, RNAi showed persistence for 4 and 8 days in baculovirus-infected as well as uninfected Sf9 cells, respectively. Bicistronic RNA seems an efficient way to lower cost and effort of the gene silencing approach while maintaining the biological activity of the protein of interest when the RNAi is not in use.

Outbreaks of West Nile virus on a Florida alligator farm prompted an investigation of which species of mosquitoes were feeding on the animals at the farm. Mosquitoes were collected on 4 separate overnight trips in September and October 2003 by using CO2-baited Centers for Disease Control light traps and wooden resting boxes that were placed inside or near the alligator housing pens. Mosquitoes were identified to species, bloodfed individuals were separated, and their abdomens were removed for DNA extraction. The DNA was tested to determine the vertebrate origin of the blood meal by polymerase chain reaction (PCR) amplification by using 4 primer sets specific to crocodilians, alligators, mammals, and birds. PCR products were sequenced to identify hosts. Of the 37 mosquito blood meals tested, 13 blood meals were positively identified to species, and 7 blood meals of those 13 were from Alligator mississippiensis, the American alligator. Alligator blood was found in Culex erraticus, Mansonia dyari, and Ma. titillans, and to our knowledge, this represents the first report of these mosquito species feeding on American alligators.

The genome of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D), which is the most extensively used virus pesticide in the world, was completely sequenced and shown to have 132 239 bp (G+C content 44.5 mol%) and to be capable of encoding 152 non-overlapping open reading frames (ORFs). Three ORFs were unique to AgMNPV-2D, one of which (ag31) had similarity to eukaryotic poly(ADP-ribose) polymerases. The lack of chiA and v-cath may explain some of the success and growth of the AgMNPV biological control programme, as it may explain the high recovery of polyhedra sequestered inside dead larvae in the field, which are collected and used for further application as biological pesticides in soybean fields. The genome organization was similar to that of the Choristoneura fumiferana defective MNPV (CfDefNPV). Most of the variation between the two genomes took place near highly repetitive regions, which were also closely associated with bro-coding regions. The separation of the NPVs into groups I and II was supported by: (i) a phenogram of the complete genomes of 28 baculovirus and Heliothis zea virus 1, (ii) the most parsimonious reconstruction of gene content along the phenograms and (iii) comparisons of genomic features. Moreover, these data also reinforced the notion that group I of the NPVs can be split further into the AgMNPV lineage (AgMNPV, CfDefNPV, Epiphyas postvittana NPV, Orgyia pseudotsugata MNPV and C. fumiferana MNPV), sharing eight defining genes, and the Autographa californica MNPV (AcMNPV) lineage (AcMNPV, Rachiplusia ou NPV and Bombyx mori NPV), sharing nine defining genes.

Genomic comparison of Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) and Neodiprion lecontei nucleopolyhedrovirus (NeleNPV) showed that the hymenopteran baculoviruses had features in common and were distinct from other, fully sequenced lepidopteran and dipteran baculoviruses. Their genomes were small in size (86 462 and 81 755 bp, respectively), had low G+C contents (33.8 and 33.3 mol%, respectively) and contained fewer open reading frames (ORFs) (90 and 89, respectively) than other baculoviruses. They shared 69 ORFs (48.6 % mean amino acid identity overall), 43 of which were previously identified baculovirus homologues. The remaining shared ORFs could be common to other baculoviruses, but low amino acid identities precluded identifying them as such. Some may also be unique to hymenopteran baculoviruses. These included a trypsin-like protease, a zinc-finger protein, regulator of chromosome condensation proteins, a densovirus capsid-like protein and a phosphotransferase. Structural analysis, the presence of conserved domains and phylogenetic studies suggested that some of these ORFs may be functional and could have been transferred horizontally from an insect host. ORFs found only in NeseNPV and NeleNPV may play a role in host specificity and/or tissue tropism, as hymenopteran baculoviruses are restricted to the midgut. The genomes were basically collinear, but contained non-syntenic regions (NSRs) with large numbers of repeats between their polyhedrin and dbp genes. They differed from each other in the number of ORFs and the G+C content of their NSRs and the presence of homologous regions in the NeseNPV genome. NeleNPV also had a short inversion relative to NeseNPV. NeseNPV contained 21 ORFs not found in NeleNPV and NeleNPV had 20 ORFs not found in NeseNPV.

Novel poxviruses were identified in skin lesions of several species of cetaceans and pinnipeds using polymerase chain reaction targeting DNA polymerase and DNA topoisomerase I genes of members of the subfamily Chordopoxvirinae. With the exception of parapoxviruses, no molecular data of marine mammal poxviruses were available to infer genetic and evolutionary relatedness to terrestrial vertebrate poxviruses. Viruses were assigned to a cetacean poxvirus 1 (CPV-1) group based on nucleotide and amino acid identities of gene fragments amplified from skin lesions of Asian bottlenose (Tursiops aduncus), Atlantic bottlenose (Tursiops truncatus), rough-toothed (Steno bredanensis), and striped (Stenella coeruleoalba) dolphins. A different poxvirus was detected in skin lesions of a bowhead whale (Balaena mysticetus) and provisionally assigned to a CPV-2 group. These viruses showed highest identity to terrestrial poxviruses of the genera Orthopoxvirus and Suipoxvirus. A novel species-specific poxvirus was also identified in skin lesions of Steller sea lions (Eumetopias jubatus). None of these poxviruses were found to have amplifiable hemagglutinin gene sequences. Novel parapoxviruses were also identified in skin lesions of Steller sea lions and spotted seals (Phoca largha). A significant degree of divergence was observed in sequences of Steller sea lion parapoxviruses, while those of spotted seals and harbor seals (Phoca vitulina) were highly conserved.

Four clones encoding the insect depressant toxin LqqIT2 have been isolated from the Egyptian scorpion Leiurus quinquestriatus quinquestriatus using RT-PCR. The four clones have been sequenced and their deduced amino acid sequences have been compared with the original amino acid sequence determined from the purified LqqIT2 protein and polymorphisms have been shown. This study succeeded in isolating more than one copy of the LqqIT2 gene, although only one amino acid sequence has been identified from the purified LqqIT2 toxin.

A newly sequenced Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) gp41 gene was used to reconstruct the phylogeny for gp41 by comparison with Autographa californica MNPV, Bombyx mori MNPV, Helicoverpa zea single nucleopolyhedrovirus (SNPV), Lymantria dispar MNPV, Orgyia pseudotsugata MNPV and Spodoptera frugiperda MNPV. The 3.5 kb fragment of the AgMNPV gp41 region not only contained the gp41 gene but also three other open reading frames that had significant homology with the very late factor (vlf-1) of baculoviruses, AcMNPV ORF78, AcMNPV ORF79, and one partial open reading frame homologous to AcMNPV ORF81. The reconstructed phylogenetic tree of baculovirus gp41 genes compared with the polyhedrin gene tree produced similar topologies. Two other phylogenetic trees were reconstructed based on either combined gp41 and polyhedrin nucleotide sequences (total evidence) or combined evolutionary histories of both genes (strict consensus tree). The former had an identical tree topology as the gp41 gene tree alone, and the latter lost resolution in the branch of AcMNPV and BmMNPV. Mutation rate analysis showed the gp41 gene had a higher nucleotide substitution rate than the polyhedrin gene, implying that the polyhedrin gene may have a different selection constraint than the gp41 gene. Both genes have nonsynonymous/synonymous substitution values close to 0.1, similar to other DNA viruses.

A 16-kDa protein toxin was purified from Hirsutella thompsonii var thompsonii and named hirsutellin A (HtA). At 0.5 and 5.0 microM concentrations, HtA caused detectable cytopathic effects on Spodoptera frugiperda cells (Sf-9) within 2-4 hr and completely inhibited Sf-9 cell growth at 4 days posttreatment. Electron microscope data showed that the HtA treated Sf-9 cells became hypotrophied and internal organelles and cell membranes were disrupted. At the same concentration, HtA effectively inhibited Brome mosaic virus protein synthesis of both rabbit reticulocyte and wheat germ in vitro translation system. The ribosomal RNA extracted from HtA treated Sf-9 cells produced a smaller RNA (approximately 528 bases) than untreated Sf-9 cells. In summary, HtA is the first mycotoxin of a invertebrate mycopathogen determined to possess ribosomal inhibiting activity and appears to possess some specificity to invertebrate cells.

A variable region (PstI-T fragment) of two genotypic variants of the Anticarsia gemmatalis multiple nucleocapsid nuclear polyhedrosis virus (AgMNPV) was sequenced and compared. This region, which is known to have deletions and duplications in AgMNPV variants, was shown to be formed of units of a 127 bp tandemly repeated sequence containing two 30 bp imperfect palindromes. Southern blot experiments showed that the PstI-T fragment contained one of the four homologous regions mapped interspersed in the AgMNPV genome. The comparison of the nucleotide sequences of the two variants, AgMNPV-2D and AgMNPV-D7, showed that the difference between these two variants in one of these regions (hr4) was caused by the addition or elimination of 381 bp corresponding exactly to three of the 127 bp repeated sequences. An analysis of the sequence showed homology to the homologous regions (hr) of other baculoviruses. The sequence upstream of the repetitive region contained a sequence homologous to the N-terminal portion of the Autographa californica MNPV and Choristoneura fumiferana MNPV p74 gene.

Four insect cell lines were used to isolate two recombinant baculoviruses which had the beta-galactosidase (beta-gal) gene for colorimetric assay purposes. Plaque assays were performed using two Trichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and two Spodptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blue beta-gal+ recombinants) formed in the Trichoplusia cells was higher than in the Spodoptera cells. The appearance of Autographa californica NPV polyhedra was also faster in the T. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9 cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was also different.

The phylogenetic relationship of several baculovirus polyhedrin genes was investigated. Alignments of 18 polyhedrin gene amino acid sequences from which 12 were also available as DNA sequences were constructed based on positional homology and trees were calculated using character state and distance methods. Our results indicate that the Hymenopteran NsSNPV diverged from the Lepidopteran baculovirus before the separation of the NPV from the GV and subsequent radiation of these Lepidopteran groups. Moreover, this analysis indicates that lepidopteran NPV are clustered in at least two groups with distinct evolutionary rates. Group I includes the AcMNPV, BmMNPV, BmSNPV, OpMNPV, AgMNPV, and possibly the GmMNPV and AsMNPV. Group II includes the MbMNPV, PfMNPV, SfMNPV, SeMNPV, and possibly the OpSNPV. This grouping is also supported by a tree constructed from sequence data from the 5' untranslated leader region of the polyhedrin gene. Furthermore, the polyhedrin-based phylogeny was analyzed for its congruence to the genomic organization and other biological characteristics of the baculoviruses. This study shows that the SNPV and MNPV morphotypes have to be taken with caution as classificatory traits since they appear to be unpolarized. In addition, the comparison of the baculovirus phylogeny with the Lepidoptera phylogeny confirms the assumption that the evolutionary history of the baculoviruses is not entirely in agreement with the taxonomic organization of the Lepidoptera, implying that the common designation of baculoviruses based on the hosts may not be adequate for baculovirus classification since it does not reflect the genetic relatedness of the viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

A highly species-specific nuclear polyhedrosis virus isolated from velvetbean caterpillar larvae (VBC), Anticarsia gemmatalis, has been used widely as a biological control for VBC in Brazilian soybean. A single virus application of a similar Brazilian isolate significantly suppressed VBC populations for the remainder of the growing season in field trials in Florida. However, the virus had to be applied when larval populations were one half normal economic threshold densities to prevent yield loss under severe pressure. Studies were expanded to compare efficacy of the above-mentioned viruses with a less species-specific nuclear polyhedrosis virus isolated from Autographa californica larvae. All three viruses at two treatment rates provided significant suppression of the damaging middle and late instar VBC larvae. The impacts on nontarget pests and predators of each virus and a standard chemical insecticide treatment also were determined in this study. The chemical insecticide treatment did not affect populations of nontarget pests or predators, and tolerance of sub-economic VBC densities in the virus treatments did not enhance populations of predators. Consequently, there was little ecological advantage in our research for using baculoviruses for VBC control compared with the standard chemical insecticide. Baculoviruses may have greater commercial potential in the USA as biopesticides against other pests more difficult to control with chemical insecticides.

Salivary glands from male Caribbean fruit flies, Anastrepha suspensa, were isolated individually by dissection and cultured in Schneider's medium containing 10% Fetal Bovine Serum and antibiotics. A total of 103 salivary glands were successfully cultured. Two different procedures were used to evaluate viability. A trypan blue dye test revealed that no more than 10% of the cultured cells were non-viable, since they acquired blue coloration. In the second set of experiments, acid phosphatase activity of the cultured glands was measured colorimetrically from absorbance at 415 nm of liberated p-nitrophenol. This method indicated that the cultured glands stayed metabolically active, with enzyme activity equal to that of freshly excised glands on the fifth, eighth, and fifteenth days of the experiment. Scanning electron microscopy (SEM) showed no visual differences in tissue organization and size of individual cells when freshly excised salivary glands and cultured glands were compared. SEM showed that the cells of cultured glands were neither swollen nor shrunken, and close-up views showed no evidence of cell deterioration or lysing.

A non-occluded baculovirus was isolated from nymphs of the field cricket, Gryllus rubens. SDS-polyacrylamide gel electrophoresis revealed the presence of 6 major and 11 minor polypeptides in these particles. Restriction endonuclease analysis indicated that the genome, 87.0 +/- 1.8 kilobase pairs, was a closed circular DNA molecule. DNA-DNA hybridization in low strigency conditions revealed no homology with the genomes of Oryctes baculovirus or Autographa california NPV. The virus replicated in nuclei of fat body cells, and was transmitted per os to a small proportion of first instar G. rubens nymphs.

Regions of homology on the physical maps of Spodoptera exempta multiple-nucleocapsid nuclear polyhedrosis virus (SeMNPV-25), an Autographa californica MNPV genomic variant, and S. frugiperda (SfMNPV-2) baculovirus DNAs were identified by reciprocal DNA-DNA blot hybridization under conditions of an effective temperature of Tm -25 degrees C. In addition, cloned fragments of the viral genome which contained the homologous regions were used in hybridization experiments to confirm, refine and correlate the regions of the two physical maps. Five homologous regions conserved between the two physical maps were identified. When the stringency of the hybridization was increased (Tm -20 degrees C), only two of the original regions were identified by blot hybridization. One of the two regions contained the polyhedrin gene, and the other region was not associated with any known viral function. The five regions did not overlap with the intragenic homologous sequence (hr1 to hr5) regions on the SeMNPV-25 map or with restriction endonuclease variant (vI to vIV) regions on the SeMNPV-25 and SfMNPV-2 maps. The degree of similarity in the genomic organization of these two baculoviruses is discussed.