New species and new records of Miroculis Edmunds, 1963 (Ephemeroptera: Leptophlebiidae) from Chacoan Domain in northeastern Brazil.

The genus Miroculis Edmunds, 1963 (Ephemeroptera: Leptophlebiidae) is endemic to South American with 27 described species. In Brazil, 21 of these are known to occur. The present paper describes three new species of Miroculis Edmunds, 1963. Miroculis (Miroculis) botafora sp. nov. is described based on a male imago collected in a transition area between the Cerrado and Caatinga biomes in Piauí State, Brazil. Two species are described based on nymphs and adults: Miroculis (Miroculis) azevedoi sp. nov. and Miroculis (Miroculis) quilombola sp. nov.; both were collected in a Cerrado region of Maranhão State, Brazil. Additionally, new distribution records of Miroculis (Miroculis) fittkaui Savage & Peters, 1983 are provided.

An updated distribution list of Leptophlebiidae Banks, 1900 (Ephemeroptera: Insecta) for Piauí state / Uma lista de distribuição atualizada de Leptophlebiidae Banks, 1900 (Ephemeroptera: Insecta) para o estado do Piauí

Abstract Leptophlebiidae is the second most diverse family within Ephemeroptera, with species distributed across various States in Brazil, but with gaps in distribution records in others. Currently, nine species of Leptophlebiidae are recorded for the state of Piauí. Based on this information gap, the objective of this study was to present an updated species list of the family Leptophlebiidae new occurrence records and distributional sites for the state of Piauí, Brazil. By analyzing 447 specimens, we have significantly expanded our knowledge about the distribution of Leptophlebiidae species in the state of Piauí, increasing the recorded species from nine to 17. We have also added new occurrence records for six species and, for the first time, documented the presence of four genera. It is important to highlight that there is still an extensive area within the Cerrado and Brazilian semiarid regions where the occurrence of Ephemeroptera is unknown, confirming that the diversity in this area is underestimated and that knowledge of Ephemeroptera species and their distributions can expand with increased sampling efforts in the coming years, reducing the Linnean and Wallacean shortfall regarding this group. Our results also demonstrate the urgent need for inventories in the southern part of the state of Piauí, particularly in the sub-basins of the middle and upper Parnaíba river, which are considered suitable for monoculture expansion in Brazil.

Resumo Leptophlebiidae é a segunda família mais diversa de Ephemeroptera, com espécies distribuídas em vários estados do Brasil, mas com lacunas no registro de distribuição em outros. Por exemplo, no estado do Piauí tem registrado apenas nove espécies de Leptophlebiidae. Baseada nessa lacuna de informação, o objetivo deste estudo foi apresentar uma lista atualizada de espécies da família Leptophlebiidae, novos registros de ocorrência e sítios de distribuição para o estado do Piauí, Brasil. Ao analisar 447 exemplares, ampliamos significativamente nosso conhecimento sobre a distribuição das espécies de Leptophlebiidae no estado do Piauí, aumentando o número de espécies registradas de nove para 17. Também adicionamos novos registros de ocorrência para seis espécies e, pela primeira vez, documentamos a presença de quatro gêneros. Destacamos que ainda existe uma extensa área do Cerrado e semiárido brasileiro que se desconhece a ocorrência de Ephemeroptera, confirmando que a diversidade nessa área é subestimada e que o conhecimento sobre as espécies de Ephemeroptera e suas distribuições podem se expandir com o aumento do esforço amostral nos próximos anos, diminuindo as lacunas Lineana e Wallaceana sobre esse grupo. Nossos resultados também demonstram a necessidade emergencial de inventários no sul do Estado do Piauí, principalmente nas sub-bacias do médio e alto rio Parnaíba que é considerado adequado para a expansão da monocultura no Brasil.

New species and records of Caenidae Newman, 1853 (Insecta: Ephemeroptera) from Northeastern Brazil.

New distributional records for four species and the description of a new species of Caenis Stephens from Parnaba River Basin, in a semiarid region of the state of Piau, Northeastern Brazil are given. Characters and illustrations to distinguish Caenis marataoan sp. nov. from all other species in Caenis are provided. The new species can be distinguished by the following combination of characters: 1) body length of male 1.52.0 mm, female 2.52.8 mm; 2) base of antennal flagellum not dilated; 3) forceps apically rounded, covered with trichomes and with 23 minute spines at apex; 4) styliger plate short with posterior margin convex; 5) penis fused and not laterally projected; 6) eggs oval-shaped; micropyle linear with narrow and long micropylar canal, preceded by an oval sperm guide.

Hagenulopsis Ulmer (Ephemeroptera: Leptophlebiidae): re-description, morphological notes and a new species from South America, Brazil.

Hagenulopsis diptera Ulmer, type species of the genus Hagenulopsis, was originally described based on imagos from Santa Catarina State, Southern Brazil. Misconceptions of H. diptera circumscription led to erroneous attribution of material from Minas Gerais and Esprito Santo, Southeastern Brazil, to H. diptera. Despite the increase in the number of species attributed to Hagenulopsis, little attention has been given to the type species. After comparative examination of photographs of the holotype and fresh material of H. diptera from Southeastern Brazil, we conclude that many specimens previously assigned to H. diptera represent a new species. Thus, we redescribe H. diptera and describe a new species Hagenulopsis perere sp. nov. based on eggs, nymphs and imagos. Diagnostic features of Hagenulopsis perere sp. nov. include cross veins between C and RP1 strongly clouded with brown and outer surface of mid femur with a brown spot at midlength. Finally, comments and new records are presented for Hagenulopsis minuta Spieth.

Interactions and nuclear import of the N and P proteins of sonchus yellow net virus, a plant nucleorhabdovirus.

We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization.

Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus.

We have recently used a green fluorescent protein (GFP) fusion to the gammab protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The gammab protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAbeta but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, betab (TGB1), betac (TGB3), and betad (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.

Retention of a small replicase gene segment in tomato bushy stunt virus defective RNAs inhibits their helper-mediated trans-accumulation.

Tomato bushy stunt virus (TBSV) and other tombusviruses are notorious for their propensity to accumulate defective interfering RNAs (DIs) upon serial passage through experimental Nicotiana species. Hallmarks of this occurrence include reduced levels of helper RNA and protein accumulation and amelioration of the lethal necrosis induced upon infection of the host with the helper viruses alone. The objective of this study was to determine whether the prolific trans-accumulation of defective RNAs typically occurs for all replicase-deficient TBSV mutants, or if this process is influenced by internal cis-acting elements that have been excised from DIs. For this purpose, various replicase-deficient TBSV cDNA constructs were generated and their transcripts were tested for trans-accumulation competence in the presence of helper virus. The results revealed that a region of ca. 150 nucleotides near the center of the replicase gene, with a predicted high degree of secondary structure, was a potent inhibitor of trans-rescue (ITR) by TBSV. Relocation of the ITR into efficiently trans-replicating DIs inhibited their accumulation drastically, but only when inserted in the reverse orientation and with an intact 5' ITR-specific predicted hairpin structure. Insertion of the ITR element in the positive orientation yielded DI transcripts that were able to replicate, but failed to interfere noticeably with either accumulation of the helper RNA or the onset of the lethal necrosis phenotype in N. benthamiana. In conclusion, the ITR has an intrinsic capacity to inhibit trans-accumulation of defective RNAs, but its stringency and biological effects are strongly influenced by the overall sequence context.

Requirements for cell-to-cell movement of Barley stripe mosaic virus in monocot and dicot hosts.

Summary The Barley stripe mosaic virus (BSMV) RNAss genome contains a series of overlapping open reading frames termed the triple gene block. The three most abundant proteins, betab, betac and betad, have been shown to have essential roles in infectivity, but their function in cell-to-cell movement has not previously been unambiguously defined, nor has the role of a minor translational read-through protein, betad' been characterized. We have now examined the direct involvement of each of these proteins in cell-to-cell movement in planta by engineering fusions of the green fluorescent protein (GFP) to a cysteine-rich regulatory protein designated gammab. Microscopic examination of inoculated and systemically infected barley and oat leaves revealed high levels of fluorescence that moved rapidly through the compact striate vascular tissue without infecting epidermal cells. In contrast, a radial pattern of fluorescence spread through a large number of epidermal and mesophyll cells before entry into the reticulate vascular tissue of the dicot hosts Nicotiania benthamiana and Chenopodium amaranticolor. Mutational analyses indicated that the betab, betac and betad proteins are each essential for cell-to-cell movement in local lesion and systemic hosts, whereas the betad' protein is dispensable. Collectively, these results demonstrate conclusively that the three major triple gene block-encoded proteins act in concert to mediate cell-to-cell movement of BSMV.

Broad-spectrum protection against tombusviruses elicited by defective interfering RNAs in transgenic plants.

We have designed a DNA cassette to transcribe defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) and have investigated their potential to protect transgenic Nicotiana benthamiana plants from tombusvirus infections. To produce RNAs with authentic 5' and 3' termini identical to those of the native B10 DI RNA, the DI RNA sequences were flanked by ribozymes (RzDI). When RzDI RNAs transcribed in vitro were mixed with parental TBSV transcripts and inoculated into protoplasts or plants, they became amplified, reduced the accumulation of the parental RNA, and mediated attenuation of the lethal syndrome characteristic of TBSV infections. Analysis of F1 and F2 RzDI transformants indicated that uninfected plants expressed the DI RNAs in low abundance, but these RNAs were amplified to very high levels during TBSV infection. By two weeks postinoculation with TBSV, all untransformed N. benthamiana plants and transformed negative controls died. Although infection of transgenic RzDI plants initially induced moderate to severe symptoms, these plants subsequently recovered, flowered, and set seed. Plants from the same transgenic lines also exhibited broad-spectrum protection against related tombusviruses but remained susceptible to a distantly related tombus-like virus and to unrelated viruses.

Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene.

Nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (TBSV) failed to elicit effective virus resistance when inoculated with wildtype virus. Subsequently, R1 and R2 progeny from 13 transgenic lines were inoculated with a TBSV mutant containing a defective coat protein gene. Mild symptoms typical of those elicited in nontransformed plants infected with the TBSV mutant initially appeared. However, within 2 to 4 weeks, up to 20% of the transgenic plants sporadically began to develop the lethal syndrome characteristic of wild-type virus infections. RNA hybridization and immunoblot analyses of these plants and nontransformed N. benthamiana inoculated with virus from the transgenic lines indicated that wild-type virus had been regenerated by a double recombination event between the defective virus and the coat protein transgene. Similar results were obtained with a TBSV deletion mutant containing a nucleotide sequence marker, and with a chimeric cucumber necrosis virus (CNV) containing the defective TBSV coat protein gene. In both cases, purified virions contained wild-type TBSV RNA or CNV chimeric RNA derived by recombination with the transgenic coat protein mRNA. These results thus demonstrate that recombinant tombus-viruses can arise frequently from viral genes expressed in transgenic plants.

Acupuncture in the management of chronic pain.

This article reports the results of a survey of acupuncture practice in chronic pain clinics in the United Kingdom. The survey reveals that acupuncture is widely used in the treatment of chronic pain with 84% of those responding stating that is was available at their clinics. The majority of practitioners had attended a course at one of the 'acupuncture schools' but in about one fifth of the clinics the practitioner had not received any formal training.

Sonchus yellow net rhabdovirus nuclear viroplasms contain polymerase-associated proteins.

We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of beta-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.

Nucleotide sequence and infectivity of a full-length cDNA clone of panicum mosaic virus.

The sequence of an infectious cDNA clone of panicum mosaic virus (PMV) showed that the single-stranded RNA genome is 4326 nucleotides (nt) and a single highly abundant subgenomic (sg) RNA of 1475 nt was synthesized during PMV infection of pearl millet plants and protoplasts. Computer comparisons revealed strong similarities between the predicted amino acid sequences of the p48 and p112 open reading frames (ORFs) and replicase proteins of members of the Tombusviridae. The sgRNA has the potential to encode five proteins. Three small ORFs, p8, p8-FS, and/or p6.6 have similarity to ORFs of carmo-, necro-, and machlomoviruses thought to be involved in virus spread in plants. The sgRNA also has the potential to encode a 26-kDa capsid protein and a 15-kDa nested gene (p15) of unknown function. PMV transcripts also supported replication and movement of SPMV, the satellite virus. Genome organization, physicochemical properties, and biological features indicate that PMV is a member of the Tombusviridae family. However, PMV differs sufficiently from previously described members to warrant its placement in a new genus provisionally designated Panicovirus.

The enigma of pX: A host-dependent cis-acting element with variable effects on tombusvirus RNA accumulation.

Tomato bushy stunt virus (TBSV) is a small isometric virus that contains a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open reading frame (ORF) of 207 nucleotides, designated pX, which resides at the 3' end of the genome. Bioassays showed that deletions or additions of nucleotides at the 5' end of the pX gene that were designed to disrupt the ORF, or site-specific inactivation of its start codon, all gave rise to TBSV mutants which were unable to accumulate to detectable levels in cucumber or Nicotiana benthamiana protoplasts. Although these results suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect. However, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumulation. Therefore, we hypothesize that cis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of the cis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mutations that prohibited accumulation of TBSV RNA in cucumber or N. benthamiana, failed to interfere substantially with replication in Chenopodium quinoa protoplasts or plants. Irrespective of the host, the cis-acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replication in trans. In addition, the pX gene was not essential for in vitro translation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequences. However, it is possible that very early in the replication cycle of genomic RNA in vivo, the pX gene cis-acting element is essential for some other unidentified function which involves interaction with one or more host components whose composition varies slightly between different plants.

Genotypic and phenotypic variation of selected Saint Louis encephalitis viral strains isolated in California.

The mechanism for long-term maintenance of St. Louis encephalitis (SLE) virus in California is unknown. Two possibilities are 1) that the virus is maintained locally in discrete enzootic foci by one or more reservoir mechanisms, and/or 2) that the foci are ephemeral in nature and virus is reintroduced periodically from other enzootic areas by migratory birds or movement of vectors. We have investigated these epidemiologic alternatives by studies of genetic variation within a 277 nucleotide portion of the envelope-encoding region among 17 strains of SLE virus isolated since 1952 from different geographic locations in California. Three lineages of virus were detected. One lineage, Group A, consisted of four SLE virus strains isolated in California since 1972 from the Coachella, Sacramento, and San Joaquin Valleys. The group A strains were closely related to strain MSI-7 of SLE virus isolated in Mississippi in 1975. The 13 other strains formed the second and third lineages (Groups B1 and B2) that had geographically overlapping distributions. Group A (BFN 4585) and Group B2 (BFN 4820) appeared to be sympatric in the Sacramento Valley in 1972. Strains from the San Joaquin Valley isolated prior to 1989 (Groups B1 and B2) differed markedly from a 1989 isolate from the same location, Kern 373 (Group A). These results suggest that virus introduction(s) led to changes in genotype, or alternatively that the enzootic virus was subjected to selective pressure leading to rapid emergence of a new genotype. Nucleotide sequences of the envelope and 5' untranslated region of the viral genome of these virus strains did not correlate with virulence as measured by mortality in weanling mice, nor viremia levels and duration in chickens.

Characterization of the components and activity of Sonchus yellow net rhabdovirus polymerase.

Sonchus yellow net virus (SYNV) is the best-characterized member of a group of plant rhabdoviruses that replicate in the host cell nucleus. Using a recently developed method for partial purification of active SYNV polymerase by salt extraction of nuclei from infected plant tissue (J. D. O. Wagner et al, J. Virol. 70:468-477, 1996), we have identified the nucleocapsid (N), M2, and L proteins as polymerase complex components (based on copurification with the polymerase activity and by coimmunoprecipitation assays). Furthermore, the L protein was shown by antibody inhibition analysis to be a functional component of the polymerase. A second complex of M2 and L proteins, thought to be a precursor to the polymerase complex, was also identified. In addition, we conducted a detailed characterization of SYNV RNA synthesis in vitro. The results demonstrate that the RNAs are transcribed sequentially, beginning with the N mRNA and followed successively by the remaining five mRNAs in the order of their genome organization. Gene expression conforms to a cascade pattern, with synthesis of the 3'-proximal N mRNA occurring at the highest level, followed by consecutively lower levels of transcription from each subsequent gene. The reaction conditions favor transcription over minus-sense RNA replication, which, we posit, is inhibited near specific signal sequences located on the antigenomic template. The results support the concept that the mechanism of transcription is highly conserved among diverse rhabdoviruses and are compatible with a unified model for the regulation of genomic and antigenomic RNA synthesis.