Incidence of Garlic common latent virus in Argentina, and phylogenetic and recombination analyses of isolates

The objective of this work was to estimate the incidence and prevalence of Garlic common latent virus (GarCLV) in the main production regions of garlic (Allium sativum) in Argentina, and to perform phylogenetic and recombination analyses in isolates from these regions. Leaf samples (3,050) were taken from four garlic commercial types, in 13 departments of the four main garlic-producing provinces of Argentina, in a 1,175-ha sampling area. Virus infection was evaluated with DAS-Elisa test using specific antiserum, and the phylogenetic and recombination analyses were done with capsid protein (CP) nucleotide sequence of seven GarCLV isolates from the provinces. The incidence of GarCLV in the evaluated provinces varied between 6.7 and 22% of the samples, whereas the prevalence varied between 52.6 and 70%. In the analysis of garlic commercial types, Morado showed the highest incidence of the virus, in the province of San Juan, whereas Rosado Paraguayo had the lowest incidence, in the province of Cordoba. Nucleotide identity in the CP sequences ranged between 80.3 and 97.6%. The phylogenetic analysis shows the presence of two main groups of GarCLV and of a possible third group that would include only a German isolate. The recombination analysis between isolates from different parts of the world evidences the presence of recombinant isolates from Poland and Australia.

Distribution, Abundance and Molecular Analysis of Genus Barbadocladius Cranston & Krosch (Diptera, Chironomidae) in Tropical, High Altitude Andean Streams and Rivers

The distribution of the genus Barbadocladius Cranston & Krosch (Diptera: Chironomidae), previously reported from Chile to Bolivia, has extended northwards. Larvae, pupae and pupal exuviae of this genus have been found in the high mountain tropical streams of Peru to 9°22′56″, but are restricted to very high altitude streams (altitudes over 3,278 m asl) compared to the lower altitude streams (below 1,100 m asl) in which the genus is reported in Chile and Argentina. Based on morphological studies, both described species in the genus, Barbadocladius andinus Cranston & Krosch and Barbadocladius limay Cranston & Krosch, have been found in Peru as pupae or pupal exuviae. Morphological analysis of the larvae and pupae revealed no differences between the two described species from Patagonia and Peru, which are of similar size and with a similar armament of hooklets and spines in pupal tergites and sternites. However, molecular analysis of larvae and pupae revealed that in Peru, there are at least two different evolutionary lines, one distributed widely and another restricted to one site. Phylogenetic analysis (using cox1 mitochondrial sequences) of all available sequences of Barbadocladius shows that the Chilean and Argentinean material differs from that of Peru. Therefore, a total of four molecular segregates are identified, although morphologically, neither larvae nor the pupae may be differentiated.

Analysis of the nucleotide sequence of the coat protein and 3'-untranslated region of two Brazilian Potato virus Y isolates

Two Brazilian Potato virus Y (PVY) isolates were biologically characterized as necrotic (PVY-NBR) and common (PVY-OBR) based upon symptoms on test plants. Additional characterization was performed by sequencing a cDNA corresponding to the 3' terminal region of the viral genome. The sequence consisted of 195 nucleotides (nt) coding part of the nuclear inclusion body b (NIb) gene, 804 nt of the coat protein (CP) gene, and 328 nt (PVY-OBR) or 326 nt (PVY-NBR) of the 3'-untranslated region (UTR). Translation of the sequence resulted in one single open reading frame with part of the NIb and a CP of 267 amino acids. The two isolates shared 95.1% similarity in the CP amino acid sequence. The CP and the 3'-UTR sequence of the Brazilian isolates were compared to those of other PVY isolates previously reported and unrooted phylogenetic trees were constructed. The trees revealed a separation of two distinct clusters, one comprising most of the common strains and the other comprising the necrotic strains. PVY-OBR was clustered in the common group and PVY-NBR in the necrotic one.

Comparative sequence analysis of the P-, M- and L-coding region of the measles virus CAM-70 live attenuated vaccine strain

Measles virus is a highly contagious agent which causes a major health problem in developing countries. The viral genomic RNA is single-stranded, nonsegmented and of negative polarity. Many live attenuated vaccines for measles virus have been developed using either the prototype Edmonston strain or other locally isolated measles strains. Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, there was remarkable sequence similarity of H, F and N genes among all vaccine strains. CAM-70 is a Japanese measles attenuated vaccine strain widely used in Brazilian children and produced by Bio-Manguinhos since 1982. Previous studies have characterized this vaccine biologically and genomically. Nevertheless, only the F, H and N genes have been sequenced. In the present study we have sequenced the remaining P, M and L genes (approximately 1.6, 1.4 and 6.5 kb, respectively) to complete the genomic characterization of CAM-70 and to assess the extent of genetic relationship between CAM-70 and other current vaccines. These genes were amplified using long-range or standard RT-PCR techniques, and the cDNA was cloned and automatically sequenced using the dideoxy chain-termination method. The sequence analysis comparing previously sequenced genotype A strains with the CAM-70 Bio-Manguinhos strain showed a low divergence among them. However, the CAM-70 strains (CAM-70 Bio-Manguinhos and a recently sequenced CAM-70 submaster seed strain) were assigned to a specific group by phylogenetic analysis using the neighbor-joining method. Information about our product at the genomic level is important for monitoring vaccination campaigns and for future studies of measles virus attenuation.

Molecular analysis of the bovine coronavirus S1 gene by direct sequencing of diarrheic fecal specimens

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8% identity for nucleotides and 96.8% identity for amino acids) and more similar to the BCoV-ENT strain (98.7% for nucleotides and 98.7% for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.

Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ß turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

Cloning, bioinformatics analysis, and expression of the dust mite allergen Der f 5 of Dermatophagoides farinae

Crude extracts of house dust mites are used clinically for diagnosis and immunotherapy of allergic diseases, including bronchial asthma, perennial rhinitis, and atopic dermatitis. However, crude extracts are complexes with non-allergenic antigens and lack effective concentrations of important allergens, resulting in several side effects. Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) is one of the predominant sources of dust mite allergens, which has more than 30 groups of allergen. The cDNA coding for the group 5 allergen of D. farinae from China was cloned, sequenced and expressed. According to alignment using the VECTOR NTI 9.0 software, there were eight mismatched nucleotides in five cDNA clones resulting in seven incompatible amino acid residues, suggesting that the Der f 5 allergen might have sequence polymorphism. Bioinformatics analysis revealed that the matured Der f 5 allergen has a molecular mass of 13604.03 Da, a theoretical pI of 5.43 and is probably hydrophobic and cytoplasmic. Similarities in amino acid sequences between Der f 5 and allergens of other domestic mite species, viz. Der p 5, Blo t 5, Sui m 5, and Lep d 5, were 79, 48, 53, and 37%, respectively. Phylogenetic analysis indicated that Der f 5 and Der p 5 clustered together. Blo t 5 and Ale o 5 also clustered together, although Blomia tropicalis and Aleuroglyphus ovatus belong to different mite families, viz. Echimyopodidae and Acaridae, respectively.

Phylogenetic relationships withinPelargonium sect. Peristera (Geraniaceae) inferred from nrDNA and cpDNA sequence comparisons

Phylogenetic analysis of nrDNA ITS and trnL (UAA) 5′ exon-trnF (GAA) chloroplast DNA sequences from 17 species ofPelargonium sect.Peristera, together with nine putative outgroups, suggests paraphyly for the section and a close relationship between the highly disjunct South African and Australian species of sect.Peristera. Representatives fromPelargonium sectt.Reniformia, Ligularia s. l. andIsopetalum (the St. Helena endemicP. cotyledonis) appear to be nested within thePeristera clade. The close relationship between the South African and AustralianPeristera is interpreted as being caused by long-range dispersal to Australia, probably as recent as the late Pliocene.

Distribution of mosquitoes in the South East of Argentina and first report on the analysis based on 18S rDNA and COI sequences

Although Mar del Plata is the most important city on the Atlantic coast of Argentina, mosquitoes inhabiting such area are almost uncharacterized. To increase our knowledge in their distribution, we sampled specimens of natural populations. After the morphological identification based on taxonomic keys, sequences of DNA from small ribosomal subunit (18S rDNA) and cytochrome c oxidase I (COI) genes were obtained from native species and the phylogenetic analysis of these sequences were done. Fourteen species from the genera Uranotaenia, Culex, Ochlerotatus and Psorophora were found and identified. Our 18S rDNA and COI-based analysis indicates the relationships among groups at the supra-species level in concordance with mosquito taxonomy. The introduction and spread of vectors and diseases carried by them are not known in Mar del Plata, but some of the species found in this study were reported as pathogen vectors.

Phylogenetic relationships, diversification and biogeography in Neotropical Brotogeris parakeets

Aim We present a molecular phylogenetic analysis of Brotogeris (Psittacidae) using several distinct and complementary approaches: we test the monophyly of the genus, delineate the basal taxa within it, uncover their phylogenetic relationships, and finally, based on these results, we perform temporal and spatial comparative analyses to help elucidate the historical biogeography of the Neotropical region. Location Neotropical lowlands, including dry and humid forests. Methods Phylogenetic relationships within Brotogeris were investigated using the complete sequences of the mitochondrial genes cyt b and ND2, and partial sequences of the nuclear intron 7 of the gene for Beta Fibrinogen for all eight species and 12 of the 17 taxa recognized within the genus (total of 63 individuals). In order to delinetae the basal taxa within the genus we used both molecular and plumage variation, the latter being based on the examination of 597 skin specimens. Dates of divergence and confidence intervals were estimated using penalized likelihood. Spatial and temporal comparative analyses were performed including several closely related parrot genera. Results Brotogeris was found to be a monophyletic genus, sister to Myiopsitta. The phylogenetic analyses recovered eight well-supported clades representing the recognized biological species. Although some described subspecies are diagnosably distinct based on morphology, there was generally little intraspecific mtDNA variation. The Amazonian species had different phylogenetic affinities and did not group in a monophyletic clade. Brotogeris diversification took place during the last 6 Myr, the same time-frame as previously found for Pionus and Pyrilia. Main conclusions The biogeographical history of Brotogeris implies a dynamic history for South American biomes since the Pliocene. It corroborates the idea that the geological evolution of Amazonia has been important in shaping its biodiversity, argues against the idea that the region has been environmentally stable during the Quaternary, and suggests dynamic interactions between wet and dry forest habitats in South America, with representatives of the Amazonian biota having several independent close relationships with taxa endemic to other biomes.

Morphology and phylogenetic relationships of a remarkable new genus and two new species of Neotropical freshwater stingrays from the Amazon basin (Chondrichthyes: Potamotrygonidae)

The morphology and phylogenetic relationships of a new genus and two new species of Neotropical freshwater stingrays, family Potamotrygonidae, are investigated and described in detail. The new genus, Heliotrygon, n. gen., and its two new species, Heliotrygon gomesi, n. sp. (type-species) and Heliotrygon rosai, n. sp., are compared to all genera and species of potamotrygonids, based on revisions in progress. Some of the derived features of Heliotrygon include its unique disc proportions (disc highly circular, convex anteriorly at snout region, its width and length very similar), extreme subdivision of suborbital canal (forming a complex honeycomb-like pattern anterolaterally on disc), stout and triangular pelvic girdle, extremely reduced caudal sting, basibranchial copula with very slender and acute anterior extension, and precerebral and frontoparietal fontanellae of about equal width, tapering very little posteriorly. Both new species can be distinguished by their unique color patterns: Heliotrygon gomesi is uniform gray to light tan or brownish dorsally, without distinct patterns, whereas Heliotrygon rosai is characterized by numerous white to creamy-white vermiculate markings over a light brown, tan or gray background color. Additional proportional characters that may further distinguish both species are also discussed. Morphological descriptions are provided for dermal denticles, ventral lateral-line canals, skeleton, and cranial, hyoid and mandibular muscles of Heliotrygon, which clearly corroborate it as the sister group of Paratrygon. Both genera share numerous derived features of the ventral lateral-line canals, neurocranium, scapulocoracoid, pectoral basals, clasper morphology, and specific patterns of the adductor mandibulae and spiracularis medialis muscles. Potamotrygon and Plesiotrygon are demonstrated to share derived characters of their ventral lateral-line canals, in addition to the presence of angular cartilages. Our morphological phylogeny is further corroborated by a molecular phylogenetic analysis of cytochrome b based on four sequences (637 base pairs in length), representing two distinct haplotypes for Heliotrygon gomesi. Parsimony analysis produced a single most parsimonious tree revealing Heliotrygon and Paratrygon as sister taxa (boot-strap proportion of 70%), which together are the sister group to a clade including Plesiotrygon and species of Potamotrygon. These unusual stingrays highlight that potamotrygonid diversity, both in terms of species composition and undetected morphological and molecular patterns, is still poorly known.

Coding behavioural data for cladistic analysis: using dynamic homology without parsimony

Many of the controversies around the concept of homology rest on the subjectivity inherent to primary homology propositions. Dynamic homology partially solves this problem, but there has been up to now scant application of it outside of the molecular domain. This is probably because morphological and behavioural characters are rich in properties, connections and qualities, so that there is less space for conflicting character delimitations. Here we present a new method for the direct optimization of behavioural data, a method that relies on the richness of this database to delimit the characters, and on dynamic procedures to establish character state identity. We use between-species congruence in the data matrix and topological stability to choose the best cladogram. We test the methodology using sequences of predatory behaviour in a group of spiders that evolved the highly modified predatory technique of spitting glue onto prey. The cladogram recovered is fully compatible with previous analyses in the literature, and thus the method seems consistent. Besides the advantage of enhanced objectivity in character proposition, the new procedure allows the use of complex, context-dependent behavioural characters in an evolutionary framework, an important step towards the practical integration of the evolutionary and ecological perspectives on diversity. (C) The Willi Hennig Society 2010.

Systematic revision and cladistic analysis of the Brazilian subfamily Sodreaninae (Opiliones: Gonyleptidae)

Sodreaninae is reviewed and all ten species are combined under its type genus, Sodreana Mello-Leitao, 1922, according to a cladistic analysis of morphological characters, which revealed a pectinate pattern of clades. The subfamily is endemic to the Brazilian Atlantic rainforest from Santa Catarina state to Rio de Janeiro state. Sodreana is herein considered a senior synonym of Stygnobates Mello-Leitao, 1927, Zortalia Mello-Leitao, 1936, Gertia B. Soares & H. Soares, 1946 and Annampheres H. Soares, 1979. The following new combinations are proposed: Sodreana barbiellinii (Mello-Leitao, 1927), Sodreana hatschbachi (B. Soares & H. Soares, 1946), Sodreana inscripta (Mello-Leitao, 1939), Sodreana leprevosti (B. Soares & H. Soares, 1947b), Sodreana bicalcarata (Mello-Leitao, 1936). Sodreana granulata (Mello-Leitao, 1937) is revalidated from the synonymy of Sodreana sodreana Mello-Leitao, 1922. Three new species are described: Sodreana glaucoi from Ilhabela and Boraceia, Sao Paulo state; S. curupira from Parque Nacional da Serra dos Orgaos, Rio de Janeiro state, and S. caipora from Ubatuba, Sao Paulo state. Sodreaninae species are restricted to forested areas and most occur in the southern part of the coastal Atlantic rainforest, one species occurs in interior Atlantic rainforest. The biogeographical analysis (Brooks Parsimony Analysis) resulted in a single and fully resolved most parsimonious tree with three main: components: northern (Bahia and Serra do Espinhaco), southern (Santa Catarina, Parana, Serra do Mar of Sao Paulo), and central (Espirito Santo, Serra da Bocaina, southern state of Rio de Janeiro, Serra dos Orgaos, Serra da Mantiqueira, Serra do Mar of Sao Paulo).