Highly Pathogenic H5N1 Influenza Viruses Carry Virulence Determinants beyond the Polybasic Hemagglutinin Cleavage Site

Highly pathogenic avian influenza viruses (HPAIV) originate from avirulent precursors but differ from all other influenza viruses by the presence of a polybasic cleavage site in their hemagglutinins (HA) of subtype H5 or H7. In this study, we investigated the ability of a low-pathogenic avian H5N1 strain to transform into an HPAIV. Using reverse genetics, we replaced the monobasic HA cleavage site of the low-pathogenic strain A/Teal/Germany/Wv632/2005 (H5N1) (TG05) by a polybasic motif from an HPAIV (TG05poly). To elucidate the virulence potential of all viral genes of HPAIV, we generated two reassortants carrying the HA from the HPAIV A/Swan/Germany/R65/06 (H5N1) (R65) plus the remaining genes from TG05 (TG05-HAR65) or in reversed composition the mutated TG05 HA plus the R65 genes (R65-HATG05poly). In vitro, TG05poly and both reassortants were able to replicate without the addition of trypsin, which is characteristic for HPAIV. Moreover, in contrast to avirulent TG05, the variants TG05poly, TG05-HAR65, and R65-HATG05poly are pathogenic in chicken to an increasing degree. Whereas the HA cleavage site mutant TG05poly led to temporary non-lethal disease in all animals, the reassortant TG05-HAR65 caused death in 3 of 10 animals. Furthermore, the reassortant R65-HATG05poly displayed the highest lethality as 8 of 10 chickens died, resembling “natural” HPAIV strains. Taken together, acquisition of a polybasic HA cleavage site is only one necessary step for evolution of low-pathogenic H5N1 strains into HPAIV. However, these low-pathogenic strains may already have cryptic virulence potential. Moreover, besides the polybasic cleavage site, the additional virulence determinants of H5N1 HPAIV are located within the HA itself and in other viral proteins

Warm or cold ischemia in animal models of lung ischemia-reperfusion injury: Is there a difference?

Objective: Experiments were designed to compare large animal models for lung ischemia-reperfusion injury using either 90 minutes' warm or 24 hours' cold ischemia. Methods: In 6 pigs, the left lung was perfused in situ with cold LPD solution. Reperfusion was started after 90 min of warm ischemia. The left lung of 6 donor pigs was perfused with LPD solution and the lung transplanted after 24 hours of cold storage into 6 recipient pigs. In both groups the right pulmonary artery and main bronchus were clamped after reperfusion of the left lung. Bronchoalveolar lavage (BAL) was obtained before ischemia and after 2 h of reperfusion. Hemodynamic and respiratory parameters were monitored. Surfactant activity was determined from the BAL in a pulsating bubble surfactometer. Results: Moderate lung injury was evident after reperfusion, but without any significant differences between groups. Surfactant composition and function were mildly impaired after reperfusion in both groups. Conclusions: Our results suggest that short periods of warm ischemia might be accepted as an accelerated model for ischemia-reperfusion injury of the lung after cold storage