Isolation and characterization of the equine influenza virus causing the 2006 outbreak in Chile.

The equine influenza virus is the causal agent of influenza in horses. In July 2006, horses from various regions of Chile presented fever, serious nasal discharge, dry cough, anorexia and depression. Here we describe the isolation and characterization of the virus responsible for this outbreak. The virus was identified as equine influenza virus H3N8, since haemagglutination was inhibited by an anti-A/equi/1/H3N8 serum, but not by an anti-A/equi/1/H7N7 serum. The isolate was named A/equi/2/Lonquén/06 (H3N8). In addition, we describe the isolation and sequencing of the haemagglutinin, neuraminidase and nucleoprotein genes of this new isolate. Sequence alignments show important differences with the Santiago/85 isolate and a closer relation to North American isolates, especially with the Florida lineage, and to Argentina isolates from 1990s.

Isolation, sequencing and phylogenetic analysis of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 and H3N8.

We report here on the isolation and sequencing of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 (A/equi-1/Santiago/77, Sa77) and H3N8 (A/equi-2/Santiago/85, Sa85). The sequences obtained allowed a variability analysis, which indicated significant differences when compared with other isolates. We found that Chilean isolates are more similar to the North American variety than to European isolates. Isolate Sa77 is a good candidate for inclusion in a vaccine as it is the latest isolate of the subtype H7N7 and is probably better-adapted to the equine host. Isolate Sa85, of subtype H3N8, also appears to be a good candidate since it has no significant differences in the main antigenic sites with recent isolates.

Isolation, sequencing and phylogenetic analysis of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 and H3N8

We report here on the isolation and sequencing of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 (A⁄equi-1⁄Santiago⁄77, Sa77) and H3N8 (A⁄equi-2⁄Santiago⁄85, Sa85) . The sequences obtained allowed a variability analysis, which indicated significant differences when compared with other isolates. We found that Chilean isolates are more similar to the North American variety than to European isolates. Isolate Sa77 is a good candidate for inclusion in a vaccine as it is the latest isolate of the subtype H7N7 and is probably better-adapted to the equine host. Isolate Sa85, of subtype H3N8, also appears to be a good candidate since it has no significant differences in the main antigenic sites with recent isolates.

Immunoresponse of Coho salmon immunized with a gene expression library from Piscirickettsia salmonis.

We have used the expression library immunization technology to study the protection of Coho salmon Oncorhynchus kisutch to the infection with Piscirickettsia salmonis. Purified DNA from this bacterium was sonicated and the fragments were cloned in the expression vector pCMV-Bios. Two libraries were obtained containing 22,000 and 28,000 colonies and corresponding to approximately 8 and 10 times the genome of the pathogen, respectively. On average, the size of the inserts ranged between 300 and 1,000 bp. The plasmid DNA isolated from one of these libraries was purified and 20 micrograms were injected intramuscularly into 60 fish followed by a second dose of 10 micrograms applied 40 days later. As control, fish were injected with the same amount of DNA of the vector pCMV-Bios without insert. The titer of IgM anti-P. salmonis of vaccinated fish, evaluated 60 days post-injection, was significantly higher than that of the control group injected with the vector alone. Moreover, this response was specific against P. salmonis antigens, since no cross reaction was detected with Renibacterium salmoninarum and Yersinia ruckeri. The vaccinated and control fish were challenged 60 days after the second dose of DNA with 2.5 x 10(7) P. salmonis corresponding to 7.5 times the LD50. At 30 days post-challenge, 100% mortality was obtained with the control fish while 20% of the vaccinated animals survived. All surviving fish exhibited a lower bacterial load in the kidney than control fish. The expression library was also tested in Balb/c mice and it was found that the humoral immune response was specific to P. salmonis and it was dependent on the amount of DNA injected.

Complete sequence of the genome of the human isolate of Andes virus CHI-7913: comparative sequence and protein structure analysis.

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.

Complete sequence of the genome of the human isolate of Andes virus CHI-7913: comparative sequence and protein structure analysis

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 percent. 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 percent 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 percent. 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

Cloning and comparison of ten gene sequences of a Chilean H. pylori strain with other H. pylori strains revealed higher variability for VacA and CagA virulence factors.

We have cloned and sequenced ten Helicobacter pylori genes from a Chilean strain (CH-CTX1) including: a cytotoxin VacA fragment, a CagA fragment (A17), a species-specific protein (TsaA), urease subunits (UreA, UreB), a flagellin subunit (FlaB), heat shock proteins (HspA and HspB), adhesin (HpaA) and a lipoprotein (Lpp20). We compared their deduced amino acid sequences with the corresponding sequences from three unrelated H. pylori strains, including fully sequenced strains 26695(UK) and J99(USA), and found that eight of them (UreA, UreB, FlaB, HspA, HspB, Lpp20, TsaA and HpaA) presented more than 97.3% identity. In contrast, VacA partial sequence showed lower identity values (93.2-94.9%). Moreover, we found major differences in the A17 region respect to the number and arrangement of the internal repeated elements when sequences from different strains were aligned. The A17 regions from strains CH-CTX1 and 26695 are very similar (91.8% identity) but lacked 6 repeated elements when compared to the Australian strains ATCC 43526 and NCTC 11637. The CCUG 17874 A17 region showed the largest deletion involving 9 repeats. A17 size differences between strains CCUG 17874 and CH-CTX1 were verified by PCR and polypeptide size. Such differences may explain variations in virulence among H. pylori strains as well as diversity in serum immunoreactivity.

A putative RNA editing from U to C in a mouse mitochondrial transcript.

Recently, we isolated and characterized a new mouse mitochondrial RNA molecule containing the mitochondrial 16S RNA plus 121 nt joined to the 5' end of the RNA. This fragment arises from the L strand of the same gene and we have named this transcript chimeric RNA. At position 121 of the RNA there is a C, which, according to the sequence of the mitochondrial 16S RNA gene, should be a U. We hypothesized that this RNA is synthesized having a U at position 121, which is later substituted to a C by a putative editing reaction. Based on the presence of sites for the restriction endonucleases RsaI and Fnu4HI around position 121, both forms of the RNA were detected in mouse tissues. To confirm the presence of the non-edited and putative edited RNA, a fragment containing the first 154 nt of the RNA was amplified by RT-PCR and cloned. The substitution of U for C was demonstrated by sequencing these clones. In vitro transcription experiments demonstrated that the substitution of U for C is not due to artifact of amplification or cloning. Moreover, in mitochondria from testis only the non-edited form was found. This, together with other experimental evidence, demonstrated that the base substitution was not due to polymorphism of the mitochondrial 16S RNA gene. This is the first demonstration of a substitution reaction from U to C in a mammalian mitochondrial transcript.

Cloning and comparison of ten gene sequences of a Chilean H. pylori strain with other H. Pylori strains revealed higher variability for VacA and CagA virulence factors

We have cloned and sequenced ten Helicobacter pylori genes from a Chilean strain (CH-CTX1) including: a cytotoxin VacA fragment, a CagA fragment (A17), a species-specific protein (TsaA), urease subunits (UreA, UreB), a flagellin subunit (FlaB), heat shock proteins (HspA and HspB), adhesin (HpaA) and a lipoprotein (Lpp20). We compared their deduced amino acid sequences with the corresponding sequences from three unrelated H. pylori strains, including fully sequenced strains 26695(UK) and J99(USA), and found that eight of them (UreA, UreB, FlaB, HspA, HspB, Lpp20, TsaA and HpaA) presented more than 97.3% identity. In contrast, VacA partial sequence showed lower identity values (93.2-94.9%). Moreover, we found major differences in the A17 region respect to the number and arrangement of the internal repeated elements when sequences from different strains were aligned. The A17 regions from strains CH-CTX1 and 26695 are very similar (91.8% identity) but lacked 6 repeated elements when compared to the Australian strains ATCC 43526 and NCTC 11637. The CCUG 17874 A17 region showed the largest deletion involving 9 repeats. A17 size differences between strains CCUG 17874 and CH-CTX1 were verified by PCR and polypeptide size. Such differences may explain variations in virulence among H. pylori strains as well as diversity in serum immunoreactivity.