Resumen de Characterization of pathogenicity factors of a lethal influenza A(H1N1)09 pandemic viral isolate
Influenza virus is responsible for seasonal outbreaks that result in more than 5 million hospitalization and 500 000 deaths every year. Analysis of two clinical AH1N1pdm09 pandemic isolates originated from a patient with mild (M strain) illness and a patient that deceased (F strain) from influenza related complication during the 2009 pandemic, highlighted three amino-acid changes PB2 A221T, PA D529N and HA S127L in the F isolate, that might be responsible for the increased virulence of this strain in vitro and in vivo. Furthermore, F isolate was able to accumulate less defective genomes (DGs) than M virus during the infection in cell culture as well as in animal model. Mutations found in F strain and their effects in F virus pathogenicity were studied by introducing its changes in A/California/04/09 (CAL) virus backbone. Although these mutations did not affect viral growth or polymerase activity in vitro, we found that PB2 A221T and HA S127L attenuated while PA D529N increased the already described pathogenicity of CAL virus in vivo. Besides 100 fold decreased LD50 , CAL-PA D529N virus showed high replication rates in lungs of infected animals and strong innate immune response cell influx (neutrophils, dendritic cells, monocytes, alveolar macrophages) and inflammation in early infection (2 day post-infection). Furthermore, CAL-PA D529N virus showed great potential of viral spread to extra-pulmonary organs. Animals infected with CAL-PA D529N virus had infectious viral particles in heart tissue for a prolonged period of time (up to 4 days of infection) and at higher frequency (80% of animals) than CAL infected animals. We demonstrated that beside high viral titre, viral mRNAs of NEP, only present in cells with ongoing infection, were present in heart tissue of animals infected with CAL- PA D529N virus. Analysis of electrocardiogram (ECG) monitorization of infected animals showed that those infected with CAL-PA D529N virus suffered from systolic bradycardia and conduct cardiac defects. Accordingly with the data we obtained from studying F strain, CAL-PA D529N virus have low accumulation of defective genomes in cell culture as well as low induction of antiviral genes (MxA, ISG56). Additionally, our work shows that PA D529N mutation is able to reduce the accumulation of DGs in viruses with high DG accumulation capacity.
In summary, PA D529N is responsible for increased pathogenicity of F strain. As a consequence of this mutation virus accumulates less DGs which partially delays timely antiviral response. This would allow virus to remain undetected by the infected cells for a relatively short but sufficient time period to enable viral replication. High replication rates in lungs of CAL-PA D529N virus and efficient viral spread into extra-pulmonary organs in animals increase its pathogenicity potential. These findings give us an insight into complex pathogenesis of influenza A viruses infections and the different ways it can affect final outcome of disease.
