Increased Acid Stability of the Hemagglutinin Protein Enhances H5N1 Influenza Virus Growth in the Upper Respiratory Tract but Is Insufficient for Transmission in Ferrets

Abstract

bInfluenza virus entry is mediated by the acidic-pH-induced activation of hemagglutinin (HA) protein. Here, we investigated how a decrease in the HA activation pH (an increase in acid stability) influences the properties of highly pathogenic H5N1 influenza virus in mammalian hosts. We generated isogenic A/Vietnam/1203/2004 (H5N1) (VN1203) viruses containing either wild-type HA protein (activation pH 6.0) or an HA2-K58I point mutation (K to I at position 58) (activation pH 5.5). The VN1203-HA2-K58I virus had replication kinetics similar to those of wild-type VN1203 in MDCK and normal human bronchial epithelial cells and yet had reduced growth in human alveolar A549 cells, which were found to have a higher endosomal pH than MDCK cells. Wild-type and HA2-K58I viruses promoted similar levels of morbidity and mortality in C57BL/6J mice and ferrets, and neither virus transmitted efficiently to naive contact cage-mate ferrets. The acid-stabilizing HA2-K58I mutation, which diminishes H5N1 replication and transmission in ducks, increased the virus load in the ferret nasal cavity early during infection while simultaneously reducing the virus load in the lungs. Overall, a single, acid-stabilizing mutation was found to enhance the growth of an H5N1 influenza virus in the mammalian upper respiratory tract, and yet it was insufficient to enable contact transmission in ferrets in the absence of additional mutations that confer ␣(2,6) receptor binding specificity and remove a critical N-linked glycosylation site. The information provided here on the contribution of HA acid stability to H5N1 influenza virus fitness and transmissibility in mammals in the background of a non-laboratory-adapted virus provides essential information for the surveillance and assessment of the pandemic potential of currently circulating H5N1 viruses.

Stochastic Model of Acidification, Activation of Hemagglutinin and Escape of Influenza Viruses from an Endosome

“…We use data from endosomal acidification in MDCK cells where the pH inside endosomes decreases very quickly within the first 10-15 min (Figure 2) to reach a steady-state pH around 5.5 after 20 min, in agreement with [22]. The steady-state pH is pH early ∞ = 6.0 and pH late ∞ = 5.5 for early and late endosomes respectively [23].…”

Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein

“…Similarly, two independent studies showed that adaptation of H5 viruses to the upper respiratory tracts of ferrets and the acquisition of airborne transmissibility required a mutation that lowered the HA activation pH from 5.6 to 5.2-5.4 (7,8). In addition, α-2,6 sialic acid receptor specificity and efficient polymerase activity at 33°C (the temperature of mammalian upper airways) were also required (8,23,25). In contrast to the upper respiratory tract, in the lungs, a lower HA activation pH has been associated with reduced virus growth and reduced or delayed pathogenicity for H5N1 viruses (23,26).…”

“…In avian-like H5N1 viruses, relatively high HA activation pH values (5.6-6.0) were associated with greater growth and pathogenicity in chickens (20,21) and greater growth and transmission in mallard ducks (22). A stabilizing mutation that reduced H5N1 HA activation pH from 5.9 to 5.4 attenuated growth and eliminated transmission in ducks but enhanced growth in the upper (but not lower) respiratory tracts of mice and ferrets (22)(23)(24). Here, stabilizing mutations that reduced pH1N1 HA activation pH from 6.0 to 5.3 enhanced upper respiratory growth and airborne transmissibility in ferrets.…”

“…In addition, α-2,6 sialic acid receptor specificity and efficient polymerase activity at 33°C (the temperature of mammalian upper airways) were also required (8,23,25). In contrast to the upper respiratory tract, in the lungs, a lower HA activation pH has been associated with reduced virus growth and reduced or delayed pathogenicity for H5N1 viruses (23,26). An opposite effect is reported here for pH1N1, for which a lower HA activation pH has been linked to increased growth in the lungs and increased pathogenicity.…”

“…A higher HA activation pH (5.6-6.0) enhances replication of HPAI viruses in the enteric and respiratory tracts of ducks and chickens by facilitating membrane fusion (20,22). Such facile activation of the HA protein leads to virion inactivation in mildly acidic pH environments, such as the mammalian upper respiratory tract (7,8,23,25). The airways of mammals are mildly acidic (pH 5.5-6.9) (27) and become more acidic (pH 5.2) during influenza virus infection (28).…”