The early response of Atlantic salmon (Salmo salar) macrophages exposed in vitro to Aeromonas salmonicida cultured in broth and in fish.

Aeromonas salmonicida is a fish pathogen that causes furunculosis. Virulent strains of this bacterium are able to infect salmonid macrophages and survive within them, although mechanisms favouring intracellular survival are not completely understood. It is known that A. salmonicida cultured in vivo in the peritoneal cavity of the host undergoes changes in gene expression and surface architecture compared with cultures grown in vitro in broth. Therefore, in this study, the early macrophage responses to A. salmonicida grown in vivo and in vitro were compared. Macrophage-enriched cell preparations from head kidney of Atlantic salmon (Salmo salar) were infected in vitro in 96-well microtitre dishes and changes in gene expression during the infection process were monitored using a custom Atlantic salmon cDNA microarray. A. salmonicida cultures grown in tryptic soy broth and in peritoneal implants were used to infect the macrophages. The macrophages were harvested at 0.5, 1.0 and 2.0h after addition of the bacteria to the medium. Significant changes in gene expression were evident by microarray analysis at 2.0h post-infection in macrophages infected with broth-grown and implant-grown bacteria; however, qPCR analysis revealed earlier up-regulation of JunB and TNF-alpha in macrophages exposed to the implant-grown bacteria. Up-regulation of those genes and others is consistent with the effects of extracellular products of aeromonad bacteria on macrophages and also suggests initiation of the innate immune response.

Alphavirus replicon particles encoding the fusion or attachment glycoproteins of respiratory syncytial virus elicit protective immune responses in BALB/c mice and functional serum antibodies in rhesus macaques.

Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract disease in humans. Towards development of a prophylactic vaccine, we genetically engineered Venezuelan equine encephalitis virus (VEEV) replicons encoding the fusion (Fa) or attachment (Ga or Gb) proteins of the A or B subgroups of RSV. Intramuscular immunization with a formulation composed of equal amounts of each replicon particle (3vRSV replicon vaccine) generated serum neutralizing antibodies against A and B strains of RSV in BALB/c mice and rhesus macaques. When contrasted with purified natural protein or formalin-inactivated RSV formulated with alum, the 3vRSV replicon vaccine induced balanced Th1/Th2 T cell responses in mice. This was evident in the increased number of RSV-specific IFN-gamma(+) splenocytes following F or G peptide stimulation, diminished quantity of eosinophils and type 2 T cell cytokines in the lungs after challenge, and increased in vivo lysis of RSV peptide-loaded target cells. The immune responses in mice were also protective against intranasal challenge with RSV. Thus, the replicon-based platform represents a promising new strategy for vaccines against RSV.

An efficient helper-virus-free method for rescue of recombinant paramyxoviruses and rhadoviruses from a cell line suitable for vaccine development.

Recovery of recombinant, negative-strand, nonsegmented RNA viruses from a genomic cDNA clone requires a rescue system that promotes de novo assembly of a functional ribonucleoprotein (RNP) complex in the cell cytoplasm. This is accomplished typically by cotransfecting permissive cells with multiple plasmids that encode the positive-sense genomic RNA, the nucleocapsid protein (N or NP), and the two subunits of the viral RNA-dependent RNA polymerase (L and P). The transfected plasmids are transcribed in the cell cytoplasm by phage T7 RNA polymerase (T7 RNAP), which usually is supplied by infection with a recombinant vaccinia virus or through use of a stable cell line that expresses the polymerase. Although both methods of providing T7 RNAP are effective neither is ideal for viral vaccine development for a number of reasons. Therefore, it was necessary to modify existing technology to make it possible to routinely rescue a variety of recombinant viruses when T7 RNAP was provided by a cotransfected expression plasmid. Development of a broadly applicable procedure required optimization of the helper-virus-free methodology, which resulted in several modifications that improved rescue efficiency such as inclusion of plasmids encoding viral glycoproteins and matrix protein, heat shock treatment, and use of electroporation. The combined effect of these enhancements produced several important benefits including: (1) a helper-virus-free methodology capable of rescuing a diverse variety of paramyxoviruses and recombinant vesicular stomatitis virus (rVSV); (2) methodology that functioned effectively when using Vero cells, a suitable substrate for vaccine production; and (3) a method that enabled rescue of highly attenuated recombinant viruses, which had proven refractory to rescue using published procedures.

Identification and characterisation of the genomic segment 7 of the infectious salmon anaemia virus genome.

The isolation and characterisation of a gene encoding the putative matrix proteins of infectious salmon anaemia virus (ISAV) is reported. Following identification of an ISAV-specific sequence from a cDNA library, RACE-PCR was used to identify a mRNA transcript of approximately 1.2 kb containing the ISAV consensus sequence GCTAAGA at the 5' end. Although the cDNA transcript and its putative protein product did not possess high homology with other orthomyxoviral sequences, similarity to a paramyxovirus fusion glycoprotein and viral cell surface proteins was identified. The size of this transcript suggested that it was derived from segment 7 of the ISAV genome and encoded the matrix proteins. Like syntenic segments of other orthomyxoviruses, this segment was shown to encode at least two matrix proteins, M1 and M2. The existence of M1 and M2 ISAV mRNA was demonstrated by RT-PCR and sequencing, with the M1 transcript being more abundant than the M2 in infected cell cultures, as is found in other orthomyxoviruses. Nucleotide sequence comparison of segment 7 of the ISAV genome from isolates of different geographic origin indicated it to be the one of the most variable of the ISAV genes characterised to date.