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Marine sponges contain diverse brominated compounds as secondary metabolites and the sponge habitat appears to enrich for a population of anaerobic dehalogenating bacteria. Hence, there is interest in understanding how these natural and anthropogenic compounds are degraded in the marine environment. Compound specific isotope analysis (CSIA) is a useful tool to monitor and to quantify the degradation and fate of aquatic pollutants. The objective of this study was to evaluate whether reductive dehalogenation of brominated phenols by sponge-associated bacteria, including Desulfoluna spongiiphila, can be monitored by CSIA. Debromination of 2,6-dibromophenol to phenol by sponge-associated cultures resulted in measurable stable carbon isotope fractionation. All sponge-associated cultures showed similar isotopic enrichment factors (��). The �� values for two independent sponge-derived dehalogenating cultures were ���3.1 �� 1.5���, and ���3.0 �� 0.3���, and that of sponge associated sediment cultures ���2.0 �� 0.3���. Thus, we demonstrate that reductive debromination of 2,6-dibromophenol resulted in measurable carbon isotope fractionation and that CSIA can be used to assess reductive debromination and to monitor and estimate in vivo dehalogenation in a sponge animal., [Display omitted], ���Compound specific isotope analysis (CSIA) was evaluated for monitoring reductive dehalogenation.���Reductive debromination of 2,6-dibromophenol to phenol results in carbon isotope fractionation.���CSIA can assess reductive debromination and estimate in vivo organohalide flux in a sponge animal.

Anaerobic microorganisms associated with marine sponges can reductively dehalogenate organohalide compounds. Desulfoluna spongiiphila strain AA1 isolated from the marine sponge Aplysina aerophoba is an anaerobic bacterium capable of growth by respiratory dehalogenation. To investigate the presence of D. spongiiphila within other marine sponges and the potential ability for in situ dehalogenation, specimens of Pseudaxinella lunaecharta, Chondrilla nucula and Clathria prolifera were examined. Sponges amended with 2,6-dibromophenol (at 100 ��M) demonstrated dehalogenation activity at a rate of over 1.5 mM day-1 kg sponge-1 for P. lunaecharta and C. prolifera. Desulfoluna spongiiphila-like bacteria were also found within these sponge tissues. To determine the genes associated with debromination, the genome of Desulfoluna spongiiphila strain AA1 was sequenced, and three putative reductive dehalogenase genes were identified. Initially, the reductive dehalogenase enzyme was found to be a corrinoid dependent enzyme through propyl iodide reversible inhibition. Then, a search of corrinoid binding motifs in open reading frames yielded a number of putative reductive dehalogenase genes each containing the two conserved motifs found in most reductive dehalogenases: e.g., a tat signal sequence and iron-sulfur clusters. Each of these putative dehalogenase genes was associated with a downstream gene encoding for the potential membrane anchoring protein and a gene for a transcriptional regulator. A transcription assay with D. spongiiphila strain AA1 was attempted to verify involvement of the putative genes in debromination. In summary, marine sponges have the potential for dehalogenation in the environment and D. spongiiphila-like bacteria may be involved in this process in different sponge species. The putative gene(s) encoding the reductive dehalogenase genes of Desulfoluna spongiiphila strain AA1 have been uncovered.

Desulfoluna spongiiphila strain AA1 is an organohalide respiring bacterium, isolated from the marine sponge Aplysina aerophoba, that can use brominated and iodinated phenols, in addition to sulfate and thiosulfate as terminal electron acceptors. The genome of Desulfoluna spongiiphila strain AA1 is approximately 6.5 Mb. Three putative reductive dehalogenase (rdhA) genes involved in respiratory metabolism of organohalides were identified within the sequence. Conserved motifs found in respiratory reductive dehalogenases (a twin arginine translocation signal sequence and two iron-sulfur clusters) were present in all three putative AA1 rdhA genes. Transcription of one of the three rdhA genes was significantly upregulated during respiration of 2,6-dibromophenol and sponge extracts. Strain AA1 appears to have the ability to synthesize cobalamin, the key cofactor of most characterized reductive dehalogenase enzymes. The genome contains genes involved in cobalamin synthesis and uptake and can grow without cobalamin supplementation. Identification of this target gene associated with debromination lays the foundation for understanding how dehalogenating bacteria control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle. In the sponge environment, D. spongiiphila strain AA1 may thus take advantage of both brominated compounds and sulfate as electron acceptors for respiration.

Tesis de Maestría

Tacaribe virus (TACV) is an arenavirus that is genetically and antigenically closely related to Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever (AHF). It is well established that TACV protects experimental animals fully against an otherwise lethal challenge with JUNV. To gain information on the nature of the antigens involved in cross-protection, recombinant vaccinia viruses were constructed that express the glycoprotein precursor (VV-GTac) or the nucleocapsid protein (VV-N) of TACV. TACV proteins expressed by vaccinia virus were indistinguishable from authentic virus proteins by gel electrophoresis. Guinea pigs inoculated with VV-GTac or VV-N elicited antibodies that immunoprecipitated authentic TACV proteins. Antibodies generated by VV-GTac neutralized TACV infectivity. Levels of antibodies after priming and boosting with recombinant vaccinia virus were comparable to those elicited in TACV infection. To evaluate the ability of recombinant vaccinia virus to protect against experimental AHF, guinea pigs were challenged with lethal doses of JUNV. Fifty per cent of the animals immunized with VV-GTac survived, whereas all animals inoculated with VV-N or vaccinia virus died. Having established that the heterologous glycoprotein protects against JUNV challenge, a recombinant vaccinia virus was constructed that expresses JUNV glycoprotein precursor (VV-GJun). The size and reactivity to monoclonal antibodies of the vaccinia virus-expressed and authentic JUNV glycoproteins were indistinguishable. Seventy-two per cent of the animals inoculated with two doses of VV-GJun survived lethal JUNV challenge. Protection with either VV-GJun or VV-GTac occurred in the presence of low or undetectable levels of neutralizing antibodies to JUNV. Fil: Lopez, Nora Mabel. Ministerio de Produccion y Trabajo. Secretaria de Gobierno de Agroindustria. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Centro de Virologia Animal. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Centro de Virologia Animal.; Argentina Fil: Scolaro, Luis Alberto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rossi, Carlos. Ministerio de Produccion y Trabajo. Secretaria de Gobierno de Agroindustria. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Centro de Virologia Animal. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Centro de Virologia Animal.; Argentina Fil: Jacamo, Rodrigo. Ministerio de Produccion y Trabajo. Secretaria de Gobierno de Agroindustria. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Centro de Virologia Animal. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Centro de Virologia Animal.; Argentina Fil: Candurra, Nélida Alicia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Pujol, Carlos Alberto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Damonte, Elsa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Damonte, Elsa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Franze Fernández, María T.. Ministerio de Produccion y Trabajo. Secretaria de Gobierno de Agroindustria. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Centro de Virologia Animal. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Centro de Virologia Animal.; Argentina

In March 1995 the first case of a familiar outbreak of Hantavirus Pulmonary Syndrome (HPS) was notified in El Bolson, in the South of Argentina. Until December 15, 1996, a total of 77 cases of HPS had been notified with 48% mortality, distributed in three geographical areas of the country, South, North and Center. During 1996, of the 19 cases from El Bolsón, North and Center. During 1996, of the 19 cases from El Bolsón, three were local physicians, one of whom --during the prodrome of her illness-- travelled to Buenos Aires to be attended. In the hospital, two of the physicians who assisted her, developed HPS 27 and 28 days after the first contact. These data indicate for the first time the possibility of interhuman transmission of the Hantavirus responsible for the pulmonary syndrome. En marzo de 1995 se notificó el primer caso de un brote familiar del síndrome pulmonar por Hantavirus (HPS) en El Bolsón en el sur de la Argentina. Hasta el 15 de diciembre de 1996 se habían notificado un total de 77 casos de HPS con un 48% de mortalidad, distribuidos en tres áreas geográficas del país, sur, norte y centro. Durante 1996, de los 19 casos en El Bolsón, tres fueron médicos de la zona, uno de los cuales en los prodromos de su enfermedad viajó a Buenos Aires para ser atendida. Entre los médicos en contacto con ella en el hospital, dos desarrollaron HPS a los 27 y 28 días del primer contacto. De esta manera, por primera vez se presentan evidencias de contagio interhumano por el Hantavirus que induce el síndrome pulmonar. Fil: Enria, Delia. ANLIS Dr.C.G.Malbrán; Argentina. Fil: Padula, Paula. ANLIS Dr.C.G.Malbrán; Argentina. Fil: Segura, Elsa L. ANLIS Dr.C.G.Malbrán; Argentina. Fil: Pini, Noemi. ANLIS Dr.C.G.Malbrán; Argentina. Fil: Lopez, Nora. ANLIS Dr.C.G.Malbrán; Argentina. Fil: Riva Posse, Clara. ANLIS Dr.C.G.Malbrán; Argentina. Fil: Weissenbacher, Mercedes C. ANLIS Dr.C.G.Malbrán; Argentina.

Tacaribe virus (TCRV) belongs to the Arenaviridae family. Its bisegmented negative-stranded RNA genome encodes the nucleoprotein (N), the precursor of the envelope glycoproteins, the polymerase (L), and a RING finger matrix (Z) protein. The 570-amino-acid N protein binds to viral RNA, forming nucleocapsids, which are the template for transcription and replication by the viral polymerase. We have previously shown that the interaction between N and Z is required for assembly of infectious virus-like particles (VLPs) (J. C. Casabona et al., J. Virol. 83:7029-7039, 2009). Here, we examine the functional organization of TCRV N protein. A series of deletions and point mutations were introduced into the N-coding sequence, and the ability of the mutants to sustain heterotypic (N-Z) or homotypic (N-N) interactions was analyzed. We found that N protein displays two functional domains. By using coimmunoprecipitation studies, VLP incorporation assays, and double immunofluorescence staining, the carboxy-terminal region of N was found to be required for N-Z interaction and also necessary for incorporation of N protein into VLPs. Moreover, further analysis of this region showed that the integrity of a putative zinc-finger motif, as well as its amino-flanking sequence (residues 461 to 489), are critical for Z binding and N incorporation into VLPs. In addition, we provide evidence of an essential role of the amino-terminal region of N protein for N-N interaction. In this regard, using reciprocal coimmunoprecipitation analysis, we identified a 28-residue region predicted to form a coiled-coil domain (residues 92 to 119) as a newly recognized molecular determinant of N homotypic interactions.