Isolation and characterization of a neprilysin-like protein from Venturia canescens virus-like particles

Maternal protein secretions from endoparasitoid wasps are evolutionary adaptations to regulate host physiology as part of an extended wasp phenotype. Virus-like particles (VLPs) produced in the calyx region of Venturia canescens wasps are involved in immune evasion of the developing parasitoid inside the host. In contrast to polydnaviruses (PDVs), VcVLPs are devoid of any nucleic acids. To understand the role of these particles in the regulation of host physiology and phylogenetic relationship between VLPs and PDVs, it is essential to identify particle proteins. In this paper, we describe the isolation and molecular cloning of a neprilysin-like gene (VcNEP) coding for a 94 kDa VcVLP protein and discuss its possible role in host regulation.

New status, species, distribution records and phylogeny for Australian mandibulate Chironomidae (Diptera)

All life-history stages of the Australian Podonominae (Chironomidae) genus Archaeochlus Brundin are revised, providing evidence for recognition of a separate clade, named here as Austrochlus Cranston. Based on molecular and morphological evidence, the clade contains two additional species: Austrochlus parabrundini Cranston, Edward and Cook sp. n. is described from Western Australia where its granite outcrop seepage habitat and geographical range is almost identical to that of the type species Austrochlus brundini Cranston, Edward and Colless (n. comb); Austrochlus centralaustralis Cranston, Edward and Cook sp. n. is described from ephemeral seepage/flows in the MacDonnell and James Ranges of central Australia. Molecular studies reported here confirm species distinctions, relationships to African taxa, and basal relationships within the Chironomidae. Modelled distributions provide evidence for range restriction by seasonal rainfall patterns.

A preliminary phylogeny of the scale insects (Hemiptera : Sternorrhyncha : Coccoidea) based on nuclear small-subunit ribosomal DNA

Scale insects (Hemiptera: Sternorrhyncha: Coccoidea) are a speciose and morphologically specialized group of plant-feeding bugs in which evolutionary relationships and thus higher classification are controversial. Sequences derived from nuclear small-subunit ribosomal DNA were used to generate a preliminary molecular phylogeny for the Coccoidea based on 39 species representing 14 putative families. Monophyly of the archaeococcoids (comprising Ortheziidae, Margarodidae sensu lato, and Phenacoleachia) was equivocal, whereas monophyly of the neococcoids was supported. Putoidae, represented by Puto yuccae, was found to be outside the remainder of the neococcoid clade. These data are consistent with a single origin (in the ancestor of the neococcoid clade) of a chromosome system involving paternal genome elimination in males. Pseudococcidae (mealybugs) appear to be sister to the rest of the neococcoids and there are indications that Coccidae (soft scales) and Kerriidae (lac scales) are sister taxa. The Eriococcidae (felt scales) was not recovered as a monophyletic group and the eriococcid genus Eriococcus sensu lato was polyphyletic. (C) 2002 Elsevier Science (USA). All rights reserved.

Molecular evolution and phylogeny of elapid snake venom three-finger toxins

Animal venom components are of considerable interest to researchers across a wide variety of disciplines, including molecular biology, biochemistry, medicine, and evolutionary genetics. The three-finger family of snake venom peptides is a particularly interesting and biochemically complex group of venom peptides, because they are encoded by a large multigene family and display a diverse array of functional activities. In addition, understanding how this complex and highly varied multigene family evolved is an interesting question to researchers investigating the biochemical diversity of these peptides and their impact on human health. Therefore, the purpose of our study was to investigate the long-term evolutionary patterns exhibited by these snake venom toxins to understand the mechanisms by which they diversified into a large, biochemically diverse, multigene family. Our results show a much greater diversity of family members than was previously known, including a number of subfamilies that did not fall within any previously identified groups with characterized activities. In addition, we found that the long-term evolutionary processes that gave rise to the diversity of three-finger toxins are consistent with the birth-and-death model of multigene family evolution. It is anticipated that this three-finger toxin toolkit will prove to be useful in providing a clearer picture of the diversity of investigational ligands or potential therapeutics available within this important family.

Phylogeny, evolution and historical zoogeography of ticks: a review of recent progress

There has been much progress in our understanding of the phylogeny and evolution of ticks, particularly hard ticks, in the past 5 years. Indeed, a consensus about the phylogeny of the hard ticks has emerged. Our current working hypothesis for the phylogeny of ticks is quite different to the working hypothesis of 5 years ago. So that the classification reflects our knowledge of ticks, several changes to the nomenclature of ticks are imminent. One subfamily, the Hyalomminae, will probably be sunk, yet another, the Bothriocrotoninae n. subfamily, will be created. Bothriocrotoninae n. subfamily, and Bothriocroton n. genus, are being created to house an early-diverging ('basal') lineage of endemic Australian ticks that used to be in the genus Aponomma (ticks of reptiles). There has been progress in our understanding of the subfamily Rhipicephalinae. The genus Rhipicephalus is almost certainly paraphyletic with respect to the genus Boophilus. Thus, the genus Boophilus will probably become a subgenus of Rhipicephalus. This change to the nomenclature, unlike other options, will keep the name Boophilus in common usage. Rhipicephalus (Boophilus) microplus may still called B. microplus, and Rhipicephalus (Boophilus) annulatus may still be called B. annulatus, but the nomenclature will have been changed to reflect our knowledge of the phylogeny and evolution of these ticks. New insights into the historical zoogeography of ticks will also be presented.

Phylogeny and evolution of anomalous roots in Daviesia (Fabaceae : Mirbelieae)

The phylogeny of the Australian legume genus Daviesia was estimated using sequences of the internal transcribed spacers of nuclear ribosomal DNA. Partial congruence was found with previous analyses using morphology, including strong support for monophyly of the genus and for a sister group relationship between the clade D. pachyloma and the rest of the genus. A previously unplaced bird-pollinated species, anceps + D. D. epiphyllum, was well supported as sister to the only other bird-pollinated species in the genus, D. speciosa, indicating a single origin of bird pollination in their common ancestor. Other morphological groups within Daviesia were not supported and require reassessment. A strong and previously unreported sister clade of Daviesia consists of the two monotypic genera Erichsenia and Viminaria. These share phyllode-like leaves and indehiscent fruits. The evolutionary history of cord roots, which have anomalous secondary thickening, was explored using parsimony. Cord roots are limited to three separate clades but have a complex history involving a small number of gains (most likely 0-3) and losses (0-5). The anomalous structure of cord roots ( adventitious vascular strands embedded in a parenchymatous matrix) may facilitate nutrient storage, and the roots may be contractile. Both functions may be related to a postfire resprouting adaptation. Alternatively, cord roots may be an adaptation to the low-nutrient lateritic soils of Western Australia. However, tests for association between root type, soil type, and growth habit were equivocal, depending on whether the variables were treated as phylogenetically dependent (insignificant) or independent ( significant).

The higher-level phylogeny of Archosauria (Tetrapoda: Diapsida)

Crown group Archosauria, which includes birds, dinosaurs, crocodylomorphs, and several extinct Mesozoic groups, is a primary division of the vertebrate tree of life. However, the higher-level phylogenetic relationships within Archosauria are poorly resolved and controversial, despite years of study. The phylogeny of crocodile-line archosaurs (Crurotarsi) is particularly contentious, and has been plagued by problematic taxon and character sampling. Recent discoveries and renewed focus on archosaur anatomy enable the compilation of a new dataset, which assimilates and standardizes character data pertinent to higher-level archosaur phylogeny, and is scored across the largest group of taxa yet analysed. This dataset includes 47 new characters (25% of total) and eight taxa that have yet to be included in an analysis, and total taxonomic sampling is more than twice that of any previous study. This analysis produces a well-resolved phylogeny, which recovers mostly traditional relationships within Avemetatarsalia, places Phytosauria as a basal crurotarsan clade, finds a close relationship between Aetosauria and Crocodylomorpha, and recovers a monophyletic Rauisuchia comprised of two major subclades. Support values are low, suggesting rampant homoplasy and missing data within Archosauria, but the phylogeny is highly congruent with stratigraphy. Comparison with alternative analyses identifies numerous scoring differences, but indicates that character sampling is the main source of incongruence. The phylogeny implies major missing lineages in the Early Triassic and may support a Carnian-Norian extinction event.

Molecular phylogeny of the freshwater prawn genus Macrobrachium (Decapoda, Palaemonidae), with emphasis on the relationships among selected American species

The genus Macrobrachium Bate, 1868 is one of the best examples of widespread crustacean genera distributed globally throughout tropical and subtropical waters. Previous investigators have noted the systematic complexity of the group, and have suggested rearrangements within the family Palaemonidae. Our phylogenetic analysis of new mitochondrial DNA sequences of 58 species of Macrobrachium distributed mainly in America support the hypothesis of monophyly of this genus, if Cryphiops Dana, 1852 is accepted as a generic synonym. We concluded that the independent evolution of different types of life cycle (abbreviated larval development-ALD and extended larval development-ELD) must have occurred more than once in the history of the group. Similarly, we also concluded that the current type species of the genus, Macrobrachium americanum Bate, 1868, should not be considered valid, as previously proposed. The synonymy of two members of the `olfersi` species complex (M. birai Lobao, Melo&Fernandes, 1986 and M. holthuisi Genofre&Lobao, 1978) with M. olfersi (Wiegmann, 1836) was confirmed. Similar results were found in comparing M. petronioi Melo, Lobao&Fernandes, 1986 and M. potiuna (Muller, 1880), in which the genetic divergence placed M. petronioi within the level of intraspecific variation of M. potiuna. The taxonomic status of the genus Cryphiops, as well as theories on the origin of Macrobrachium, is also called into question.

Phylogeny of Grumichellini Morse, 1981 (Trichoptera: Leptoceridae) with the description of a new genus from southeastern Peru

Osflintia manu, new genus, new species, of long-horned caddisfly (Leptoceridae: Triplectidinae: Grumichellini) is described and illustrated from southeastern Peru. The phylogeny of Grumichellini Morse (Leptoceridae: Triplectidinae) is revisited and hypotheses of homology of some morphological characters are reconsidered. The monophyly of the tribe is corroborated and the phylogenetic relationships of its included genera are inferred to be (Triplexa (Gracilipsodes ((Grumichella, Amazonatolica) (Atanatolica, Osflintia, n. gen.)))) from adult and larval characters. Diagnostic characters of the new genus include the following: reduced tibial spur formula (2, 2, 2), loss of forewing crossvein sc-r1, hind wing discoidal cell closed, hind wing fork IV present, pair of long setae on tergum IX of the male genitalia, and pair of processes on the apex of segment X.

Phylogeny of the Limnophilinae (Limoniidae) and early evolution of the Tipulomorpha (Diptera)

Tipulomorpha (craneflies) comprise one of the largest subgroups of Diptera, but its phylogeny at different levels has been poorly explored. This study presents the most comprehensive cladistic analysis of the group ever made, with emphasis on the genera and subgenera of the subfamily Limnophilinae (Limoniidae), assumed to include some of the earliest lineages of Tipulomorpha sensu stricto and therefore important for the understanding of the early patterns in the evolution of the craneflies. Eighty-eight characters of the male imago were scored for 104 exemplar species. The most parsimonious trees were searched using implied weighting, in the framework of a sensitivity analysis with different values of k (2 to 6). The dataset based on the characters of adult male morphology showed high levels of homoplasy and yielded very incongruent and unstable phylogenetic results, which are very sensitive to changes in analytical parameters. In the preferred and most parsimonious phylogenetic hypothesis, the Pediciidae is the sister-group of all other Tipulomorpha sensu stricto. The results indicate the paraphyly of the Limoniidae with respect to the Cylindrotomidae and Tipulidae, which are considered sister-groups. The Limoniidae subfamilies Limnophilinae, Limoniinae and Chioneinae are considered non-monophyletic. The study allowed a reconstruction of the possible ground plan condition of selected features of the adult male morphology of craneflies. The genera/subgenera Epiphragma (Epiphragma), Acantholimnophila, Shannonomyia, Limnophila (Arctolimnophila), Eloeophila, Conosia, Polymera, Polymera (Polymerodes), Prionolabis, Eutonia, Phylidorea (Phylidorea), Metalimnophila, Gynoplistia (Cerozodia), Gynoplistia (Dirhipis), Nothophila, Pseudolimnophila (Pseudolimnophila), Pilaria and Ulomorpha are considered monophyletic, but in general are defined by combinations of very homoplastic character states. Two Temperate Gondwanan clades, (Tonnoirella + (Edwardsomyia + (Tinemyia + (Rhamphophila + (Nothophila))))) and ((Notholimnophila + Bergrothomyia) + (Mesolimnophila + (Chilelimnophila + Ctenolimnophila))) are recovered. The genera Limnophila, Neolimnomyia, Gynoplistia (sensu lato) and Hexatoma (sensu lato) are considered non-monophyletic. The systematic position and some morphological characters of `problematic` taxa, such as Dactylolabis, Elephantomyia, Helius and Atarba are discussed on the light of the proposed phylogeny and the analysis of the characters. Character states are richly illustrated. A detailed study of the morphology of the male genitalia is made, and several genera and species have the morphology of the male genitalia illustrated for the first time.

Varicella zoster virus in bell`s palsy: a prospective study

Although Bell`s palsy is the major cause of acute peripheral facial palsy, its pathogenesis remains unknown. Reactivation of the varicella zoster virus has been implicated as one of the main causes of Bell`s palsy, however, studies which investigate the varicella zoster virus reactivation in Bell`s palsy patients are mostly Japanese and, therefore, personal and geographic characteristics are quite different from our population. Aims: To determine varicella zoster virus frequency in saliva samples from patients with Bell`s palsy, using PCR. Material and Method: One hundred seventy one patients with acute peripheral facial palsy were prospectively enrolled in this study. One hundred twenty were clinically diagnosed with Bell`s palsy, within one week of onset of the disease and no previous anti-viral therapy. We had 20 healthy adults as controls. Three saliva samples were collected from patients and controls at initial examination and at one and two weeks later. The detection of the varicella zoster virus DNA was performed using PCR. Results: Varicella zoster virus was detected in two patients (1.7%). The virus was not identified in saliva samples from the controls. Conclusions: Varicella zoster virus was detected in 1.7% of saliva samples from patients with Bell`s palsy, using PCR.

Improved method for counting virus and virus like particles

An improved method for counting virus and virus like particles by electron microscopy (EM) was developed. The procedure involves the determination of the absolute concentration of pure or semi-pure particles once deposited evenly on EM grids using either centrifugation or antibody capture techniques. The counting of particles was done with a Microfiche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low magnification (2500 x) by EM. Initially, latex particles of a known concentration were counted using this approach, to prove the accuracy of the technique. The latex particles were deposited evenly on an EM grid using centrifugation (Modified Beckmen EM-90 Airfuge technique). Subsequently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a hovel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventional method, showed good agreement. The method is simple, accurate, rapid, and reproducible when used with either pure particles or with particles from crude cell culture extracts.

CD4+T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes

P>Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4+ and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

Human immunodeficiency virus test-seeking blood donors in a large blood bank in Sao Paulo, Brazil

BACKGROUND: Persons with human immunodeficiency virus (HIV) risk behaviors are excluded from donation to reduce the risk of transfusion-transmitted infection. Persons donating to be tested for HIV may therefore deny risk behaviors. STUDY DESIGN AND METHODS: A random sample of donors completed a survey on motivations, knowledge, and attitudes on the screening process. Donors were considered test seekers if they agreed with two statements ""I think that blood donation is a good, fast, and anonymous way to get my blood tested"" and ""I donate to get my test results."" This study was conducted from June to November 2006 at the largest blood bank in Sao Paulo, Brazil. RESULTS: Of 3061 participants, 208 (7%) were test seekers. They tended to be male and had a lower educational level. They were more likely to have incorrect knowledge about blood safety (e.g., not knowing that a unit can test antibody negative and still transmit infection, 60% vs. 42%, p = 0.02), express dissatisfaction with screening questions (e.g., feeling that important questions were not asked, 14% vs. 5%, p < 0.01), and concur that donors do not answer questions truthfully (e.g., donors have more sexual partners than they admit, 29% vs. 18%, p < 0.01). Test seekers were more likely to believe that it is acceptable to donate blood to get tested for HIV (41% vs. 10%, p < 0.01). CONCLUSIONS: Test-seeking motivation, coupled with low knowledge of window period risk, is counter to improving blood safety and to donor prevention needs. Donor education needs to be improved along with availability of appropriate HIV counseling and testing.