Browsing by Author "Valenzuela, PDT"

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    Complete sequence of the genome of the human isolate of Andes virus CHI-7913
    (2003) Tischler, ND; Fernández, J; Müller, I; Martínez, R; Galeno, H; Villagra, E; Mora, J; Ramírez, E; Rosemblatt, M; Valenzuela, PDT
    We report here the complete genomic sequence of the Chilean human isolate of Andes virus CHI-7913. The S, M. and L genome segment sequences of this isolate are 1,802, 3,641 and 6,466 bases in length, with an overall GC content of 38.7%. These genome segments code for a nucleocapsid protein of 428 amino acids, a glycoprotein precursor protein of 1,138 amino acids and a RNA-dependent RNA polymerase of 2,152 amino acids. In addition, the genome also has other ORFs coding for putative proteins of 34 to 103 amino acids. The encoded proteins have greater than 98% overall similarity with the proteins of Andes virus isolates AH-1 and Chile R123. Among other sequenced Hantavirus, CHI-7913 is more closely related to Sin Nombre virus, with an overall protein similarity of 92%. The characteristics of the encoded proteins of this isolate, such as hydrophobic domains, glycosylation sites, and conserved amino acid motifs shared with other Hantavirus and other members of the Bunyaviridae family, are identified and discussed.
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    Hantavirus Gc glycoprotein: evidence for a class II fusion protein
    (2005) Tischler, ND; Gonzalez, A; Perez-Acle, T; Rosemblatt, M; Valenzuela, PDT
    Hantavirus cell entry is promoted by its envelope glycoproteins, Gin and Gc, through cell attachment and by fusion between viral and endosomal membranes at low pH. However, the role of Gn and Gc in receptor binding and cell fusion has not yet been defined. In this work, a sequence presenting characteristics similar to those of class II fusion peptides (FPs) of alphavirus Ell and flavivirus E proteins is identified within the hantavirus Gc glycoprotein. A three-dimensional comparative molecular model based on crystallographic data of tick-borne encephalitis virus E protein is proposed for the Andes virus (ANDV) Gc ectodomain, which supports a feasible class II fusion-protein fold. In vitro experimental evidence is provided for the binding activity of the ANDV FP candidate to artificial membranes, as demonstrated by fluorescence anisotropy assays. Taken together, these results support the hypothesis that the Gc glycoprotein of hantaviruses and of other members of the family Bunyaviridae directs the viral fusion activity and that it may be classified as a class II viral fusion protein.
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    Human and rodent humoral immune responses to Andes virus structural proteins
    (2005) Tischler, ND; Galeno, H; Rosemblatt, M; Valenzuela, PDT
    In the present work we identified B-cell epitopes recognized by sera of humans and rodents naturally infected with Andes virus, a hantavirus present in Chile and Argentina. Analysis of patient and rodent sera with overlapping peptides revealed 21 human and rodent epitopes on the three structural proteins. Whereas in the nucleoprotein the region comprising aa 248-260 was shown to be the key determinant of human sera, the major antigenic site of rodent antibody reactivity is located at aa 326-338. In G1, the main epitope recognized by human sera was mapped to aa 14-26, while rodent antibodies bound predominantly to aa 599-611. In contrast, humans and mice had strong responses to three regions in G2 (aa 691-703, aa 918-930, aa 955-967), of which the last two are associated with neutralization of Hantaan virus. This insight affords important information for the development of immunotherapies for the acute phase of hantavirus cardiopulmonary syndrome. (c) 2005 Elsevier Inc. All rights reserved.
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    Isolation and expression of the genes coding for the membrane bound transglycosylase B (MltB) and the transferrin binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis
    (2004) Wilhelm, V; Morales, C; Martínez, R; Rosemblatt, M; Burzio, LO; Valenzuela, PDT
    We have isolated and sequenced the genes encoding the membrane bound transglycosylase B (MltB) and the transferring binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis. The results of the sequence revealed two open reading frames that encode proteins with calculated molecular weights of 38,830 and 85,140. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from phylogenetically related microorganisms. Partial sequences coding the amino and carboxyl regions of MltB and a sequence of 761 base pairs encoding the amino region of TbpB have been expressed in E. coli. The strong Immoral response elicited by these proteins in mouse confirmed the immunogenic properties of the recombinant proteins. A similar response was elicited by both proteins when injected intraperitoneally in Atlantic salmon. The present data indicates that these proteins are good candidates to be used in formulations to study the protective immunity of salmon to infection by P. salmonis.
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    Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis
    (2005) Wilhelm, V; Soza, C; Martínez, R; Rosemblatt, M; Burzio, LO; Valenzuela, PDT
    We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.