Wojciech Rozek

Genetic typing of equine influenza virus isolated in Poland in 2005 and 2006.

Two equine influenza virus strains were isolated from horses during the local respiratory disease outbreaks in Poland in 2005 and 2006. The H3 equine influenza viral RNA was amplified directly from the clinical specimens with RT-PCR and HA1 fragments were sequenced. The highest homology of HA1 nucleotide sequences of A/eq/Pulawy/05 with A/eq/Aboyne/1/05 and A/eq/Pulawy/06 with A/eq/Essex/2/05 was found. The phylogenetic analysis based on amino acid sequences of HA1 fragments of 84 equine influenza virus strains isolated in Europe during the period of 1976-2007 was conducted to determine the evolutionary relationship of the two Polish and the other European isolates. The resulting phylogenetic tree clearly clustered A/eq/Pulawy/05 with the strains belonging to the European lineage of the equine influenza virus. On the other hand A/eq/Pulawy/06 was placed in the Florida sub-lineage of the American type strains. The presence of the same amino acids: methionine, asparagine and threonine at the positions 48, 159 and 163 respectively, in both Polish isolates, despite the fact that the strains are grouped in two different lineages may indicate the existence of the common ancestor. It is possible that A/eq/Pulawy/06 evolved locally rather than was introduced.

Mass spectrometry identification of granins and other proteins secreted by neuroblastoma cells

We used mass spectrometry-based protein identification to determine the presence of granins and other proteins in the mouse neuroblastoma secretome. We detected polypeptides derived from four members of the granin family: chromogranin A, chromogranin B, secretogranin III, and VGF. Many of them are derived from previously described biologically active regions; however, for VGF and CgB, we detected peptides not related to known bioactivities. Along with granins, we identified 115 other proteins secreted by mouse neuroblastoma cells, belonging to different functional categories. Fifty-six out of 119 detected proteins possess the signal fragments required for translocation into endoplasmic reticulum. Sequences of remaining 63 proteins were analyzed using SecretomeP algorithm to determine probability of nonclassical secretion. Identified proteins are involved in the regulation of cell cycle, proliferation, apoptosis, angiogenesis, proteolysis, and cell adhesion.

Chorioallantoic membranes of embryonated chicken eggs as an alternative system for isolation of equine influenza virus

BackgroundInfluenza virus isolation in embryonated chicken eggs (ECEs) is not applicable for rapid diagnosis, however it allows the recovery and propagation of the viable virus. A low number of infectious virus particles in the swabs, poor quality of samples or individual strain properties can lead to difficulties during the virus isolation process. We propose to utilize chorioallantoic membranes (CAM) of ECEs with the assistance of real-time RT PCR to facilitate equine influenza virus isolation.MethodsReal-time RT PCR was used to detect influenza virus genetic material in amniotic/allantoic fluids (AF) and CAM of ECEs. Haemagglutination assay was used for AF. We used highly diluted virus as a substitute of clinical specimen for ECEs inoculation.ResultsOur study demonstrated that real-time RT PCR testing of CAM homogenates was more useful than testing of AF for EIV detection in ECEs. Positive results from CAM allowed to select the embryos from those with haemagglutination assay (HA) - and real-time RT PCR-negative AF for further passages. Using homogenates of CAM for subsequent passages, we finally obtained HA-positive AF, which confirmed virus replication.ConclusionWe postulate that real-time RT PCR testing of CAM homogenates and their subsequent passages may facilitate the isolation of equine influenza viruses.