Alejandra Garcia-Maruniak

Sequence Analysis of the Genome of the Neodiprion sertifer Nucleopolyhedrovirus

ABSTRACT 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 (hrs) 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.

STRAIN IDENTIFICATION OF SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) INSECTS AND CELL LINE: PCR-RFLP OF CYTOCHROME OXIDASE C SUBUNIT I GENE

Abstract 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.

A variable region of Anticarsia gemmatalis nuclear polyhedrosis virus contains tandemly repeated DNA sequences

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.

Two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica are related and form a distinct phylogenetic clade.

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.

WILD FLORIDA HOUSE FLIES (MUSCA DOMESTICA) AS CARRIERS OF PATHOGENIC BACTERIA

ABSTRACT 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 serious pathogens, known to produce diseases such as meningitis, food poisoning, diarrhea, abscesses, bloodstream infections, and hemorrhagic colitis. The possible exception is Bacillus thuringiensis, a known pathogen for insects that only occasionally produces allergic reactions in humans. If these organisms are not prevented from entering the food preparation and consumption areas, they could become a serious risk in the transmission of diseases.

Physical maps and virulence of Anticarsia gemmatalis nucleopolyhedrovirus genomic variants.

Summary. 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.

Proteomic analyses of baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus budded and occluded virus.

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.

Impact of House Fly Salivary Gland Hypertrophy Virus (MdSGHV) on a Heterologous Host, Stomoxys calcitrans

ABSTRACT 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. Virusinjected 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 ≈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 ≈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.

Expression from baculovirus and serological reactivity of the nucleocapsid protein of dolphin morbillivirus.

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.

Tensaw virus genome sequence and its relation to other Bunyaviridae.

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.