Desenvolvimento de metodologia para quantificação de vírus Influenza por RT-qPCR

Abstract

Influenza A viruses A H1N1, A H3N2 and type B (Victoria lineage), are part of the current trivalent Influenza vaccine composition produced by the Instituto Butantan. The WHO recommendation is based on data about viruses circulation around the globe. Due to this constant change of strains, the production time, from the announcement of the strains to be used until the start of the annual campaign, is just over 7 months. Therefore, the quest for vaccine producers to streamline any and all procedures, without compromising quality, is constant. In this context, obtaining a good dose/egg yield is a crucial point, being directly proportional to the success of the strain replication and, therefore, to the production yield. Vaccine production begins with master seed lots and working seed lots production, subsequently used for bulk batches production. The precise, accurate and quicly quantification can improve yield. In this study, the standardization of an RT-qPCR methodology for the quantification of viral nucleic acid copies number was performed following a statistical correlation between virus data determined by EID50/mL. To achieve the objectives, a standard curve of synthetic DNA was prepared for each Influenza virus (A H1N1, A H3N2 and B (Victoria)), which allowed the quantification of copies number by the standardized methodology. The methodologies were applied to the viral strains and validated by the parameters of linearity, limits of detection and quantification, precision and specificity, reaching the acceptance criteria. A good correlation was established between the number of copies and EDI50/mL, with r of ~0.99 for each of the 3 standardized methods, demonstrating the possibility of using the methodologies in the steps that need the most exact quantification of viruses, generating data for viral replication monitoring and optimizing production processes