ABSTRACT
INTRODUCTION: Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier. OBJECTIVE: The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method. MATERIALS AND METHODS: Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct. RESULTS: Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase. CONCLUSION: Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.
Subject(s)
Alphainfluenzavirus , Influenza A Virus, H1N1 Subtype , Orthomyxoviridae , Influenza A Virus, H1N1 Subtype/genetics , Orthomyxoviridae/genetics , Orthomyxoviridae/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Temperature , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolismABSTRACT
As an alternative to the classical method of erythrocyte hemagglutination, a latex agglutination assay based on the interaction of influenza viruses with the sialoglycoprotein fetuin immobilized on the surface of polystyrene microspheres has been developed. Twelve influenza A virus strains of different subtypes and two influenza B viruses of different lines were tested. Simultaneous titration of viruses using the classical hemagglutination test and the proposed latex agglutination assay showed similar sensitivity and a high degree of correlation (R = 0.94). The obtained microspheres can be used for titration of viruses that recognize and bind sialylated glycans as receptors. In particular, latex aggregation was also induced by the Newcastle disease virus.