The research is dedicated to investigation of molecular-biological and physicochemical properties of iridovirus, isolated from mosquito Aedes flavescens in Kiev region. Our results showed that mosquito iridovirus A. flavescens was able to replicate in both large wax-moth larvae G. mellonella and continued cell culture A. aegypti Mоs 20A. But for virus propagation in high infectious titer it is more advisability to use larvae G. mellonella, where the virus titer reach up to 8,0-8,2 lg ІD50/ml. AFM and EM observations revealed hexagonal virions with a diameter of 200 nm. The purified iridovirus particles showed a dense central core and three-layered membrane including an external lipoprotein envelope, an inner protein capsid and a lipid-containing membrane. These virions were morphologically similar to those of the Iridoviridae. Iridovirus A. flavescens was sensitive to ethanol, chloroform but not sensitive to ether. Infectivity of virions was rapidly lost at pH 3.0 and 11.0 and by heating to 60 0C. Sensitivity was detected following treatment by UW radiation and proteinase K. No sensitivity was detected for electromagnetic radiation, 0,1 % SDS, 0,1 M EDTA and trypsin. Gradient SDS-PAGE of purified virions revealed the presence of 12 polypeptides ranging from 14,5 to 122 kDa. The molecular weight of a major capsid protein (MCP) was a 48 kDa. Interestingly that number of proteins of another mosquito iridovirus A. taeniorhynchus is 9 ranging from 15,5 to 98 kDa and molecular weight of MCP is approximately 55 kDa. Restriction enzyme analysis of viral DNA with the endonucleases EcoRI, BamHI, HindIII, XbaI, MspI, and HpaII has revealed that the restriction fragment patterns of iridovirus A. flavescens differ from those obtained from experiments with iridovirus A. taeniorhynchus and iridoviruses of lower vertebrates. DNA of iridovirus A. flavescens is degraded by both MspI and HpaII. These enzymes have the same cleavage site (CCGG), however, whereas MspI is able to cleave viral DNA in the presence of methylcytosine, HpaII is not. Thus, DNA must be unmethylated and exhibits the expected characteristics of invertebrate iridovirus DNA. Using nucleotide sequences of all iridoviruses a set of oligonucleotide primers was designed for ribonucleoside reductase small subunit, ATPase and thymidine kinase genes. After amplification of iridovirus A. flavescens genomic DNA, PCR products were visible on agarose gels. PCR product of ATPase target amplification of iridovirus A. flavescens don't show significant homologies to other iridoviruses, but demonstrate 75 % identity to ATP-binding protein of Pseudomonas fluorescens. Experimental infection indicate that mosquito iridovirus A. flavescens is pathogenic for carp, zebrafish and stone moroco. The cumulative mortality reached up to 60-80% within 20 days. Probably the mortality of experimental fish was caused by the toxicity of virus proteins, because UW inactivated virions also lead to fish death. It was shown that water soluble C60 fullerenes under visible light interact with virions of iridovirus A. flavescens and lead to their destruction. Specifically, the photodynamic inactivation of virus during 1 h reduced the infectious titer of virus on 4.5 lg ID50/ml. In addition among 8 new synthetic chemicals one was selected to have antiviral activity. This chemical reduced infectious titer of virus on 4.0 lg ID50/ml. Physicochemical and molecular approaches yielded results consistent with the suggestion that mosquito iridovirus A. flavescens was a member of the family Iridoviridae. Moreover RFLP analysis of viral DNA and molecular weight of MCP distinguished iridovirus A. flavescens from another mosquito iridovirus A. taeniorhynchus, the type species of the genus Chloriridovirus. Presently, a large number of mosquito iridoviruses, that have been reported in literature, are insufficiently studied and consequently they are not classified. Our observation may help to understand the relatedness of mosquito iridovirus A. flavescens within family Iridoviridae.
