Highly pathogenic H5N1 influenza viruses carry virulence determinants beyond the polybasic hemagglutinin cleavage site

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 (TG05(poly)). 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-HA(R65)) or in reversed composition the mutated TG05 HA plus the R65 genes (R65-HA(TG05poly)). In vitro, TG05(poly) 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 TG05(poly), TG05-HA(R65), and R65-HA(TG05poly) are pathogenic in chicken to an increasing degree. Whereas the HA cleavage site mutant TG05(poly) led to temporary non-lethal disease in all animals, the reassortant TG05-HA(R65) caused death in 3 of 10 animals. Furthermore, the reassortant R65-HA(TG05poly) 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.

Figure 1. Multicycle replication in-vitro.

Plaque assays of TG05 in comparison with TG05_poly, TG05-HA_R65, and R65-HA_TG05poly on MDCK cells in the presence and in the absence of trypsin.

Figure 2. Proteolytic HA activation.

Western blots of DF-1 cell lysates infected with TG05, TG05_poly, TG05-HA_R65 or R65-HA_TG05poly at multiplicity of infection of 0.1 incubated in the presence (T) and in the absence (-) of trypsin.

Figure 3. Growth kinetics.

DF-1 cells were inoculated with TG05 (blue), TG05_poly (magenta), TG05-HA_R65 (yellow), and R65-HA_TG05poly (red) at multiplicity of infection 10⁻³ in the presence (diamonds) and in the absence (circles) of trypsin.

Figure 4. Virulence in chicken.

Daily clinical score after oculonasal inoculation with 10⁵ pfu of TG05 (blue), TG05_poly (magenta), TG05-HA_R65 (yellow) or R65-HA_TG05poly (red). The birds were observed for 10 days for clinical signs and classified as healthy (0), ill (1), severely ill (2), or dead (3); the daily clinical score was calculated from the sum of individual clinical scores from all birds divided by the number of animals per group (10 chickens).

Figure 5. Viral organ tropism in chicken.

Immunohistochemical detection of influenza A virus nucleoprotein (brown) in lung and cerebellum from chickens sacrificed on day 4 post inoculation with 10⁵ pfu of TG05 or R65-HA_TG05poly, and from moribund chickens sacrificed on day 2 post inoculation with 10⁶ TCID₅₀ R65.