of the HA conformational change, thermo-stability of HA protein, and virus sensitivity to drugs preventing the endosomal acidifica- tion of mutated virus, correspond (although not to the full extent) to those of the human influenza isolates. Moreover, by applying the FMA technique, we could reveal that the conformational change of the original, but not the mutated HA, occurs already at pH 5.8. Interestingly, the introduction of the 58KRI substitution impaired the viral growth of the pH stable mutant in HBE cells. A similar finding was made for the HA2 G23RC mutant of A/ Nederlands/219/03 (H7N2) virus with a decreased pH of HA activation showing a reduced replication in MDCK cells [23]. Thus, decreasing the pH value of HA activation to the level of human viruses might not be beneficial for growth in tissue culture. These data are consistent with the previous observation showing that the adaptation of human influenza viruses to high growth in MDCK cells was accompanied with the appearance of mutations that elevate the pH of HA membrane fusion [28]. In contrast to the tissue culture results, the intranasal infection of mice revealed that the pH stable variant was more infectious in the mouse respiratory tract, including nasal mucosa, trachea, and lungs, as demonstrated by immunohistochemistry. Such a difference could not be explained by the superior replication capacity of the mutant virus, since NS1 protein deletion abrogated the multicycle replication. [20,29]. For an analogous pair of viruses comprising a functional NS1, the HA2 modification diminished the MID50 25 times compared to the non-mutated strain. These results are in agreement with the data of Ilyushina et al., showing that equivalent modifications of the HA of an H7N7 virus decreased the mouse lethal dose 50 by 3 log10 units [23]. Reed et al. demonstrated, for a highly pathogenic H5N1 strain, that the reduction of the pH of HA activation increased the viral shedding from the trachea but not from the cloaca of infected birds, indicating that the threshold pH of HA conformational change could be important for host and/or tissue tropism [19]. Consistently for better viral uptake, the VN1203DNS1-K58I virus appeared to be more immunogenic in mice, inducing significantly higher HAI and neutralizing antibody titers as well as mucosal IgA antibodies. We observed a similar regularity for seasonal influenza viruses in ferrets where the stable virus induced an enhanced antibody response when compared to the mutant having HA destabilizing mutation [30]. The acidification of the mucosal surfaces is one of the oldest primary innate defense mechanisms in mammals against a variety of pathogens. The degree of acidification depends on the distribution of the submucosal glands responsible for the liquid secretion and, therefore, might differ in various animal species [31]. There is evidence that mouse epithelium cell composition and ion transport traits are similar to that of the human conducting airways [32,33]. The nasal epithelia of humans and mice are abundantly supplied with qualitatively similar glands [34]. The pH of the human nasal mucosa in healthy adults is known to be slightly acidic [12–15]. Moreover, the human nasal airway epithelium starts to release acid in reaction to organic dust or inflammation [22,34]. In combination with the receptor specificity factor, the human nasal compartment might be suboptimal for an efficient infection by HPAI viruses, hence requiring a low respiratory tract delivery by aerosols (reviewed in [26]). In this regard, mutations decreasing the pH of HA conformational change and, therefore, stabilizing the HA of H5 HPAIVs might be a prerequisite for their efficient spread in humans via a large droplet mechanism infecting the upper respiratory tract (nose). A relatively high pH of HA activation (ranging from 5.6 to 5.9) was described as being essential for Increased Infectivity of H5N1 Influenza Virus infectivity, shedding, and transmission in wild ducks for HPAIVs [19]. However, for the efficient infection of mice and probably other mammals including humans, we found that a lower pH optimum of the HA activation might be required. Interestingly, the mutation HA2 K58RI was first described as stabilizing the HA towards a low pH and thereby conferring resistance to the antiviral substance amantadine in a variant of the HPAI virus Rostock (H7N1) [35]. Amantadine, besides for inhibiting virus uncoating during the entry step, also prevents the release of infectious influenza virus particles comprising an HA with a polybasic cleavage site [36]. As our results indicate that the mutation HA2 K58RI increases the infectivity of H5 viruses for mice, the use of amantadine in clinics, or especially in farming, could support the appearance of H5 HPAIVs more infectious for mammals [37]. It is noteworthy that LPAIVs (H5N1) are known to have a pH range of HA activation similar to that of human influenza viruses and, therefore, are extremely stable at a low pH [18]. This property is essential for the efficient replication of viruses in the low-pH intestinal tract, which is the main site of replication for these viruses. In addition, LPAIVs circulate in wild birds and transmit through the fecal-oral route, requiring virus stability in fresh water, usually with a pH of around 6.0 [38]. In contrast, HPAIVs circulate mostly in domestic poultry and are excreted mainly from the trachea (upper respiratory tract), rather than the cloaca, and they do not persist very long in water [39]. Theoretically, stable LPAIVs might be potentially more infectious for the human upper respiratory tract. However, other factors such as the incompatibility of the avian virus polymerase complex with mammalian host factors or the temperature optimum of the polymerase activity were shown to restrict their efficient replication in mammals [40–45]. In this regard, it seems logical that a live influenza H5N1vaccine reassortant virus comprising the HA of the LPAIV A/Potsdam/1402-6/86 (H5N3) in combination with the polymerase complex of a human cold-adapted influenza virus was shown to replicate in the upper respiratory tract of humans [11], whereas analogous vaccine candidates comprising the HAs of VN1203 or A/Hong Kong/213/03 did not. Intranasal vaccination with live attenuated influenza vaccines implies the infection of the nasal mucosa with the large droplet mode. Our findings suggest that an efficient intranasal vaccination may require a certain level of pH and the temperature stability of HA in order to achieve an optimal uptake of the attenuated virus by the nasal epithelial cells. In this context, any mutation destabilizing the HA appearing during vaccine construction or production can severely affect vaccine immunogenicity. In addition, since the nasal mucosa serves as the first target for large droplet settling, we believe that the HA mutations involved in modulating the pH and temperature stability, might be also important for the virus transmissibility besides the mutations regulating receptor binding specificity. Materials and Methods Cell culture A Vero cell line was obtained from the European Collection of Cell Cultures and was adapted and further cultivated at 37uC and 5% CO2 in a serum-free cultivation medium (SFM; Opti-pro medium supplemented with 4 mM L-glutamine; Invitrogen). Madin-Darby canine kidney (MDCK; ATCC CCL-34) cells were cultivated at 37uC and 5% CO2 in DMEM medium (Invitrogen) comprising 2% Fetal Bovine Serum (FBS, Invitrogen) and 2 mM L-glutamine. Human bronchial epithelial 16HBE14o– (HBE) cells (obtained from J. Seipelt, Austria) were grown in MEM PLoS ONE | www.plosone.org 9 April 2011 | Volume 6 | Issue 4 | e18577