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A recombinant virus lacking the sf32 gene (Sf32null), unique to the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), was generated by homologous recombination from a bacmid comprising the complete viral genome (Sfbac). Transcriptional analysis revealed that sf32 is an early gene. Occlusion bodies (OBs) of Sf32null contained 62% more genomic DNA than viruses containing the sf32 gene, Sfbac and Sf32null-repair, although Sf32null DNA was three-fold less infective when injected in vivo. Sf32null OBs were 18% larger in diameter and contained 17% more nucleocapsids within ODVs than those of Sfbac. No significant differences were detected in OB pathogenicity (50% lethal concentration), speed-of-kill or budded virus production in vivo. In contrast, the production of OBs/larva was reduced by 39% in insects infected by Sf32null compared to those infected by Sfbac. The SF32 predicted protein sequence showed homology (25% identity, 44% similarity) to two adhesion proteins from Streptococcus pyogenes and a single N-mirystoylation site was predicted. We conclude that SF32 is a non-essential protein that could be involved in nucleocapsid organization during ODV assembly and occlusion, resulting in increased numbers of nucleocapsids within ODVs. Source

Palma L.,Institute Agrobiotecnologia | Munoz D.,Public University of Navarra | Berry C.,University of Cardiff | Murillo J.,Public University of Navarra | And 2 more authors.
Toxins | Year: 2014

Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities. © 2014 by the authors; licensee MDPI, Basel, Switzerland. Source

De Miguel M.J.,CSIC - Centro de Investigacion y Tecnologia Agroalimentaria | Marin C.M.,CSIC - Centro de Investigacion y Tecnologia Agroalimentaria | Munoz P.M.,CSIC - Centro de Investigacion y Tecnologia Agroalimentaria | Dieste L.,CSIC - Centro de Investigacion y Tecnologia Agroalimentaria | And 2 more authors.
Journal of Clinical Microbiology | Year: 2011

Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA's performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis. Copyright © 2011, American Society for Microbiology. Source

Barrera G.,Institute Agrobiotecnologia | Simon O.,Institute Agrobiotecnologia | Villamizar L.,Corporacion Colombiana de Investigacion Agropecuaria CORPOICA | Williams T.,Institute Ecologia AC | And 2 more authors.
Biological Control | Year: 2011

Thirty-eight isolates of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), collected from infected larvae on pastures, maize, and sorghum plants in three different geographical regions of Colombia, were subjected to molecular characterization and were compared with a previously characterized Nicaraguan isolate (SfNIC). Restriction endonuclease analysis (REN) using six different enzymes showed two different patterns among Colombian isolates, one profile was particularly frequent (92%) and was named SfCOL. The physical map of SfCOL was constructed and the genome was estimated to be 133.9. kb, with few differences in terms of number and position of restriction sites between the genomes of SfNIC and SfCOL. The PstI-K and PstI-M fragments were characteristic of SfCOL. These fragments were sequenced to reveal the presence of seven complete and two partial ORFs. This region was collinear with SfMNPV sf20-sf27. However, two ORFs (4 and 5) had no homologies with SfMNPV ORFs, but were homologous with Spodoptera exigua MNPV (se21 and se22/. se23) and Spodoptera litura NPV (splt20 and splt21). Biological characterization was performed against two different colonies of S. frugiperda, one originating from Colombia and one from Mexico. Occlusion bodies (OBs) of the SfCOL isolate were as potent (in terms of concentration-mortality metrics) as SfNIC OBs towards the Mexican insect colony. However, SfCOL OBs were 12 times more potent for the Colombian colony than SfNIC OBs and three times more potent for the Colombian colony than for the Mexican colony. SfCOL and SfNIC showed a slower speed of kill (by ~50. h) in insects from the Colombian colony compared to the Mexican colony, which was correlated with a higher production of OBs/larvae. SfCOL is a new strain of SfMNPV that presents pathogenic characteristics that favor its development as the basis for a biopesticide product in Colombia. © 2011 Elsevier Inc. Source

Simon O.,Institute Agrobiotecnologia | Williams T.,Institute Ecologia AC | Lopez-Ferber M.,Ecole des Mines dAles | Caballero P.,Institute Agrobiotecnologia | Caballero P.,Public University of Navarra
Journal of Invertebrate Pathology | Year: 2012

A Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, includes fast-killing genotypes with deletions in the egt region. Four bacmid based recombinants were constructed to determine the role of egt in this phenotype. SfdelF bacmid encompassed the deletion found in the NIC-F genotype. Sfdel3AP2 bacmid was constructed using the deletion reported in SfMNPV-3AP2 (Missouri, fast-killing isolate), whereas Sfdelegt and Sfdel27 bacmids lacked the single genes egt and the adjacent sf27 gene, respectively. No significant differences were observed in occlusion body (OB) concentration-mortality metrics (LC50 values) among the viruses. Larvae infected by NIC-B (a natural genotype with the largest genome), Sfbac (a bacmid with NIC-B genome) and Sfdel27 survived significantly longer than insects infected by NIC-F, SfdelF, SfMNPV-3AP2, Sfdel3AP2 or Sfdelegt. Fast-killing viruses produced ~6-13-fold fewer OBs/larva compared to other viruses tested. We conclude that deletion/disruption of egt is responsible for the fast-killing phenotypes of naturally-occurring genotypes in SfMNPV populations from Missouri and Nicaragua. © 2012 Elsevier Inc. Source

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