Maria F. Caeiro

Vaccinia virus mutants with alanine substitutions in the conserved G5R gene fail to initiate morphogenesis at the nonpermissive temperature.

ABSTRACT The initial characterization of the product of the vaccinia virus G5R gene, which is conserved in all poxviruses sequenced to date, is described. The G5 protein was detected in the core fraction of purified virions, and transcription and translation of the G5R open reading frame occurred early in infection, independently of DNA replication. Attempts to delete the G5R gene and isolate a replication-competent virus were unsuccessful, suggesting that G5R encodes an essential function. We engineered vaccinia virus mutants with clusters of charged amino acids changed to alanines and determined that several were unable to replicate at 40°C but grew well at 37°C. At the nonpermissive temperature, viral gene expression and DNA replication and processing were unperturbed. However, tyrosine phosphorylation and proteolytic cleavage of the A17 membrane protein and proteolytic cleavage of core proteins were inhibited at 40°C, suggesting an assembly defect. The cytoplasm of cells that had been infected at the nonpermissive temperature contained large granular areas devoid of cellular organelles or virus structures except for occasional short crescent-shaped membranes and electron-dense lacy structures. The temperature-sensitive phenotype of the G5R mutants closely resembled the phenotypes of vaccinia virus mutants carrying conditionally lethal F10R protein kinase and H5R mutations. F10, although required for phosphorylation of A17 and viral membrane formation, was synthesized by the G5R mutants under nonpermissive conditions. An intriguing possibility is that G5 participates in the formation of viral membranes, a poorly understood event in poxvirus assembly.

Phylogeny and Differentiation of Reptilian and Amphibian Ranaviruses Detected in Europe

Ranaviruses in amphibians and fish are considered emerging pathogens and several isolates have been extensively characterized in different studies. Ranaviruses have also been detected in reptiles with increasing frequency, but the role of reptilian hosts is still unclear and only limited sequence data has been provided. In this study, we characterized a number of ranaviruses detected in wild and captive animals in Europe based on sequence data from six genomic regions (major capsid protein (MCP), DNA polymerase (DNApol), ribonucleoside diphosphate reductase alpha and beta subunit-like proteins (RNR-α and -β), viral homolog of the alpha subunit of eukaryotic initiation factor 2, eIF-2α (vIF-2α) genes and microsatellite region). A total of ten different isolates from reptiles (tortoises, lizards, and a snake) and four ranaviruses from amphibians (anurans, urodeles) were included in the study. Furthermore, the complete genome sequences of three reptilian isolates were determined and a new PCR for rapid classification of the different variants of the genomic arrangement was developed. All ranaviruses showed slight variations on the partial nucleotide sequences from the different genomic regions (92.6–100%). Some very similar isolates could be distinguished by the size of the band from the microsatellite region. Three of the lizard isolates had a truncated vIF-2α gene; the other ranaviruses had full-length genes. In the phylogenetic analyses of concatenated sequences from different genes (3223 nt/10287 aa), the reptilian ranaviruses were often more closely related to amphibian ranaviruses than to each other, and most clustered together with previously detected ranaviruses from the same geographic region of origin. Comparative analyses show that among the closely related amphibian-like ranaviruses (ALRVs) described to date, three recently split and independently evolving distinct genetic groups can be distinguished. These findings underline the wide host range of ranaviruses and the emergence of pathogen pollution via animal trade of ectothermic vertebrates.

New viruses from Lacerta monticola (Serra da Estrela, Portugal): further evidence for a new group of nucleo-cytoplasmic large deoxyriboviruses.

Lizard erythrocytic viruses (LEVs) have previously been described in Lacerta monticola from Serra da Estrela, Portugal. Like other known erythrocytic viruses of heterothermic vertebrates, these viruses have never been adapted to cell cultures and remain uncharacterized at the molecular level. In this study, we made attempts to adapt the virus to cell cultures that resulted instead in the isolation of a previously undetected Ranavirus closely related to FV3. The Ranavirus was subsequently detected by polymerase chain reaction (PCR) in the blood of infected lizards using primers for a conserved portion of the Ranavirus major capsid protein gene. Electron microscopic study of the new Ranavirus disclosed, among other features, the presence of intranuclear viruses that may be related to an unrecognized intranuclear morphogenetic process. Attempts to detect by PCR a portion of the DNA polymerase gene of the LEV in infected lizard blood were successful. The recovered sequence had 65.2/69.4% nt/aa% homology with a previously detected sequence from a snake erythrocytic virus from Florida, which is ultrastructurally different from the studied LEV. These results further support the hypothesis that erythrocytic viruses are related to one another and may represent a new group of nucleo-cytoplasmic large deoxyriboviruses.

Adaptation of Ranaviruses from Peneda-Gerês National Park (Portugal) to Cell Cultures and their Characterization

Ranaviruses are known to produce iseases in fish, amphibians and reptiles, being involved in high mortality episodes in aquaculture farms and in the decline of amphibian populations worldwide. Mass mortality episodes were previously detected in Triturus marmoratus from two lagoons of the Peneda-Geres National Park (PGNP) (NW Portugal). Ranavirus-like particles were identified in tissues of diseased newts T. marmoratus and T. boscai . Here we report on the molecular and ultrastructural characterization of these viruses.

Gymnodinium catenatum Graham isolated from the Portuguese coast: Toxin content and genetic characterization

The bloom forming marine dinoflagellate Gymnodinium catenatum Graham has been linked to paralytic shellfish poisoning (PSP) outbreaks in humans. Along the Portuguese coast (NE Atlantic), G. catenatum shows a complex bloom pattern, raising questions about the origin and affinities of each bloom population. In this work, the variability within six cultured strains of G. catenatum isolated from Portuguese coastal waters (S coast, W coast and NW coast), between 1999 and 2011, was investigated. The strains were analyzed for toxin profiling and intra-specific genetic diversity. Regarding the toxin profile, differences recorded between strains could not be assigned to the time of isolation or geographical origin. The parameter that most influenced the toxin profile was the life-cycle stage that originated the culture: vegetative cell versus hypnozygote (resting cyst). At the genetic level, all strains showed similar sequences for the D1-D2 region of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA) and shared complete identity with strains from Spain, Algeria, China and Australia. Conversely, we did not find a total identity match for the ITS-5.8S nuclear rDNA fragment. After sequence analysis, two guanine/adenine (R) single nucleotide polymorphisms (SNP 1 and 2) were detected for all strains, in the ITS1 region. This species has been reported to present very conservative LSU and ITS-5.8S rDNA regions, though with few SNP, including SNP1 of this study, already attributed to strains from certain locations. The SNP here described characterize G. catenatum populations from Portuguese waters and may represent valuable genetic markers for studies on the phylogeography of this species.

Use of drift substrates to characterize marine fungal communities from the west coast of Portugal

This survey reports the occurrence, diversity and similarity of marine fungi associated with five categories of drift substrates (Arundo donax, Phragmites australis, Spartina maritima, “other stems” and driftwood) collected on four sandy beaches of the western coast of Portugal. “Other stems” and driftwood are composite samples with a variety of identified and unidentified pieces of non-woody and woody substrates respectively. Fifty-six taxa were identified, including 38 Ascomycota and 18 anamorphic fungi. Twenty-six taxa were generalists; however several cases of “substrate recurrence” were identified. The very frequent fungi differed among the categories of studied substrates, with the exception of Corollospora maritima, very frequent on four categories. Except for S. maritima, P. australis and driftwood, cases of multiple fungal colonization were rare. S. maritima was the single substrate with five different marine fungi on one sample, as well as with the highest number of very frequent fungi, highest percentage of colonization and average number of fungi per sample. Driftwood presented the highest value of fungal richness (37 taxa) and A. donax the lowest (22 taxa). ANOSIM analysis of similarity showed that all substrates supported different fungal communities with the exception of the pair P. australis/”other stems”. The effect of sample size on estimated fungal richness was tested, and the results let us conclude that, although most of the sporadic fungi (<1% occurrence) will be detected only in a very large number of samples, 60 samples of A. donax and “other stems” and 70 samples of all the other substrates may suffice to assess their respective representative marine mycota.

BIODIVERSITY OF FILAMENTOUS FUNGI ON SOILS AND SANDS

The diversity of soil organisms is more extensive than in any other existing environment as far as the living forms are considered. The soil biota contains representatives of all groups of microorganisms (fungi, bacteria, algae and viruses), as well as microfauna, such as protozoa and nematodes (Campbell and Puri, 2002).

Lulworthia atlantica: a new species supported by molecular phylogeny and morphological analysis

ABSTRACT This study presents morphological and molecular evidence for the establishment of a new species of Lulworthia based on the evaluation of 11 isolates of Lulworthiales collected in two marinas on the west coast of Portugal initially classified as Lulworthia sp. Maximum likelihood and Bayesian methods were applied to obtain phylogenetic trees for 18S-28S and internal transcribed spacer (ITS1-5.8S-ITS2 = internal transcribed spacer [ITS]) nuc rDNA. The isolates consistently grouped together in a distinct and highly supported clade; all other Lulworthiales analyzed, including the available sequences of Lulworthia spp., segregated separately. These results were supported by common and distinctive morphological characters and enabled recognition and description of the new species Lulworthia atlantica. Two Portuguese isolates were confirmed as Lulworthia cf. purpurea, and two were confirmed as asexual morphs of Zalerion maritima.

Discovery of New Nucleo Cytoplasmic Large Deoxiriboviruses by Transmission Electron Microscopy

1. Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Quinta da Granja, Monte de Caparica, 2829-511 Caparica, Portugal. 2. Centro de Estudos do Ambiente e do Mar (CESAM/FCUL), Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal 3. Laboklin GmbH & Co. KG, Bad Kissingen, Germany. 4. Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences of the Hebrew University of Jerusalem, Rehovot 76100, Israel.

Portuguese marine fungi and the contribution of differential interference contrast (DIC) microscopy for their morphological identification

Marine fungi occur either in Open Ocean or in the intertidal zone of sandy beaches, salt marshes and mangroves, where their hosts and substrates are found. The development of morphological adaptations like appendages and sheaths of the spores are vital to the settlement and attachment to substrate surfaces, floatation and dispersion on seawater. The morphological features of these appendages and sheaths of spores also have an important role in the identification of marine fungi. Differential interference contrast (DIC) microscopy is an essential tool for the observation of these mucilaginous structures in marine fungi spores and was therefore applied to marine mycota from surveys along the Portuguese coast.