Abstract
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.
Highlights
Pathogenic avian influenza viruses (HPAIV) are the causative agents of fowl plague [1,2] which causes devastating economic losses in gallinaceous poultry
Generation of Recombinant Viruses As parental strains we used a recent H5N1 lowpathogenic avian influenza A viruses (LPAIV) isolated in Germany in 2005, A/Teal/Germany/Wv632/2005 (H5N1) [43] as well as the first Highly pathogenic avian influenza viruses (HPAIV) H5N1 isolate derived from the outbreak in wild swans on the island of Rugen in February 2006, A/Swan/ Germany/R65/06 (H5N1) [44]
Influenza virus virulence is a polygenic trait which had been established by generating reassortants neurovirulent for mice from two apathogenic strains, and reassortants apathogenic in chicken from two virulent strains [46,47]
Summary
Pathogenic avian influenza viruses (HPAIV) are the causative agents of fowl plague [1,2] which causes devastating economic losses in gallinaceous poultry. HPAIV possess a polybasic HA cleavage site cleavable by furin [4,5], which is ubiquitous and supports systemic viral replication This polybasic HA cleavage site is the prime virulence determinant of HPAIV [6,7,8] which originate from LPAIV precursors [4,9,10,11,12,13,14,15,16]. We demonstrated that the acquisition of a polybasic cleavage site by an LPAIV H3N8 strain is not sufficient for immediate transformation into an HPAIV, and that additional virulence determinants other than the polybasic HA cleavage site are required [42]. To elucidate the virulence potential of all viral genes of H5N1 HPAIV in chicken further, we generated two H5N1 reassortants carrying an HPAIV HA plus the remaining LPAIV genes, or, in reversed composition, the LPAIV HA with engineered polybasic cleavage site plus the HPAIV genes
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