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.
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 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.
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.
The Musca domestica salivary 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 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.
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 65x550 nm and contains a 124 279 bp genome (approximately 44 mol% G+C content) that codes for 108 putative open reading frames (ORFs). GpSGHV, measuring 50x1000 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.
The Miami blue butterfly (Cyclargus thomasi bethunebakeri) is a state-endangered taxon in Florida and a candidate for federal listing. Here we report 12 polymorphic microsatellite loci appropriate for use in population and conservation studies. We genotyped 114 individuals sampled from a metapopulation in the lower Florida Keys over a 2-year period (2005-2006). These results show 4-14 alleles per locus, and ranges of observed and expected heterozygosities are 0.02679-0.79630 and 0.06154-0.69565, respectively. Large deviations from Hardy-Weinberg equilibrium (HWE) are observed across the whole sample set. When a single breeding population is analysed alone, seven of the loci are in HWE.
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.
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