Genetic Inactivation of Dace, Leuciscus Leuciscus (L.), Gametes Using UV Irradiation
The results of genetic inactivation of dace, Leuciscus leuciscus (L.), gametes using UV irradiation are presented. Dace sperm was genetically inactivated with UV irradiation doses ranging from 384 to 3840 J m-2. The most effective dose, expressed as a high percentage of hatched haploid larvae, was 1920 J m-2. The UV treatment did not kill the spermatozoa, and following irradiation the activated spermatozoa exhibited a high percentage of motility. The percentage of hatched larvae in all the experimental groups, in which genetically inactivated dace eggs were fertilized with spermatozoa from the yellow form of ide, Leuciscus idus (L.), was much lower than in the control groups. All haploid larvae showed morphological abnormalities known as "haploid syndrome" that included stunted bodies and poorly formed retinas. The optimal doses of UV irradiation ranged from 3456 to 4608 J m-2, as it was within this range that almost 100% haploid larvae were produced at a hatching rate of over 15%. Lower UV doses led to abnormal embryonic development.
The immunoreactivity of haemagglutinin (HA) polypeptides of equine influenza virus was compared among the strains isolated in Poland, using H3 monoclonal antibody. A stronger signal in immunoblot reaction was observed for A/equi/Pulawy/2008 HA polypeptides compared to A/equi/Pulawy/2006, despite the fact that both strains are phylogenetically closely related and belong to Florida clade 2 of American lineage. The strongest signal, observed in the case of A/equi/Pulawy/2008, seemed to be connected with the presence of G135, I213, E379, and/or V530 instead of R135, M213, G379, and I530 present in A/equi/Pulawy/2006 HA sequence. This implies that point mutations within amino acid sequences of HA polypeptides of equine influenza virus may change their immunoreactivity even when they are not located within five basic antigenic sites.
The purpose of the experiment was to compare apoptosis induced by equine influenza virus (EIV A1 and EIV A2) infection in MDCK, RK13, and NEURO-2A cell lines. Flow cytometry was used to observe two symptoms of apoptosis: phosphatidylserine translocation in plasmalemma (annexin V assay) and the fragmentation of DNA generated by endonuclease activity (TUNEL assayterminal deoxynucleotidyl transferase biotin-dUTP nick end labelling). The differences in the onset of apoptosis in the studied cells was observed. In MDCK cells infected with EIV A1 and A2, a weak signal of the phosphatidylserine translocation was observed but more cells showed the DNA fragmentation. An opposite effect was observed in case of RK 13 cells. NEURO-2A cells displayed a similar number of annexin V and TUNEL positive cells after the infection with EIV A2, while in case of EIV A1 infection, only the early symptoms of apoptosis were noted. Differences between both viral serotypes could originate from functioning of viral proteins responsible for induction or inhibition of apoptosis. The differences between cell types may result from the activation of cellular pro or anti-apoptotic mechanisms.
Introduction: Traditionally, evolutionary analysis of equine influenza virus (EIV) is based on the HA gene. However, the specificity of the influenza virus enables the classification of viral strains into different phylogenetic groups, depending on the gene being analysed. The aim of the study was to analyse phylogenetic paths of EIV based on M gene with reference to the HA gene.
Material and Methods: M gene of Polish isolates has been sequenced and analysed along with all M sequences of EIV available in GenBank database. Phylogenetic analysis was performed using BioEdit, ClustalW, and MEGA7 softwares.
Results: The clustering of the strains isolated not only from Asia but also from Europe into one common Asian-like group of EIV was observed. Twelve nucleotide substitutions in the M gene of strains from the Asian-like group were crucial for the evolutionary analysis. We also observed homology in the M gene of the Asian-like and H7N7 strains.
Conclusions: M gene specific for the Asian-like group is present in strains recently isolated in Europe and Asia, which were classified previously in the Florida 2 clade based on HA. Therefore, Asian-like group does not seem to be assigned to a specific geographical region. Traces of H7N7 strains in more conservative genes like M of some contemporary EIV strains may indicate the link between the old phylogenetic group and recent H3N8 strains. Analysis of conservative genes may be more useful in tracking the direction of virus evolution than in the genes where the high variability rate may blur the original relationships.