Influenza A virus environmental persistence is driven by the hemagglutinin and the neuraminidase

The transmission routes of Influenza A viruses (IAVs) submit virus particles to a wide range of environmental conditions that affect their transmission. In water, temperature, salinity and pH are important factors modulating viral persistence in a strain-dependant manner, and the viral factors driving IAV persistence remained to be described. We used an innovative method based on a real-time cell system analysis to quantify viral decay in an environmental model. Thus, we identified the viral hemagglutinin (HA) and neuraminidase (NA) as the main proteins driving the environmental persistence by comparing the inactivation slopes of several reassortant viruses. We also introduced synonymous and non-synonymous mutations in the HA or in the NA that modulated IAV persistence. Our results demonstrate that HA stability and expression level, as well as calcium-binding sites of the NA protein are molecular determinants of viral persistence. Finally, IAV particles could not trigger membrane fusion after environmental exposure, stressing the importance of the HA and the NA for environmental persistence.

Finally, IAV particles could not trigger membrane fusion after environmental exposure, 27 stressing the importance of the HA and the NA for environmental persistence. 28 29 Introduction 30 Influenza A viruses (IAVs) has a wide host range, which allows them to spread almost 31 everywhere on the planet. In nature, the H1N1 subtype infects several hosts such as 32 domestic and aquatic birds, humans, swine or dogs and thus spreads through a wide 33 range of environmental conditions. In humans, a pandemic H1N1 virus emerged in 2009 34 after the reassortment between a swine H1N1 virus from the Eurasian lineage and a swine 35 H2N1 virus descended from a triple reassortment between a swine H1N1 virus from the 36 North American lineage, an avian virus and the human H3N2 virus. In aquatic birds, which 37 are the main reservoir of these viruses, IAVs spread mainly by a fecal-oral route through 38 water. In poultry such as chicken and turkeys, or in mammalian species, IAVs mainly have 39 a respiratory tropism, and the virus spreads by contact between infected and susceptible 40 hosts or by contaminated fomites, as well as through aerosol or respiratory droplets. In 41 any case, the transmission routes of IAVs submit virus particles to a wide range of 42 environmental conditions, which more or less rapidly affect them. In water, the time for 43 IAV inactivation depends on widely studied abiotic factors such as temperature (Brown 44  In order to identify viral genetic drivers of the persistence phenotype, we generated 67 reassortants from H1N1 viruses, which do not have the same persistence in an 68 environmental model, and compared their inactivation slopes. As a model, we used saline 69 (35 g.L -1 NaCl) water at 35°C, because it is the average salt concentration in the ocean 70 and this temperature allows observing differences of viral persistence more rapidly. Using 71 fluorescence imaging microscopy, we also wanted to understand how our environmental 72 F453Y and Q299K in the HA of HAopti-NA/1999 virus greatly decreased the persistence of 164 the virus, with a mean inactivation slope of 9.9, 8.0, 9.8 and 9.6 CIT50.day -1 , respectively. 165 The S53K substitution however had less impact on the persistence, with a mean 166 inactivation slope of 7.6 CIT50.day -1 . Altogether, these results demonstrate that 167 synonymous mutations or a single amino acid change in the HA is sufficient to negatively 168 affect the viral persistence outside the host. 169 Viral particles cannot trigger membrane fusion after being exposed to saline water 170 at 35°C 171 Since the loss of hemagglutination titre of viral particles is slower than their loss of 172 infectivity (Chu 1948) (Fig. 3A and Fig. S1), we decided to evaluate whether viruses were 173 still able to bind their cellular receptor. For this purpose, we immuno-labelled the viral 174 nucleoprotein (NP), which encapsidates the viral genome to form the ribonucleoprotein, 175 and used confocal microscopy for its detection in infected MDCK cells. In cells infected 176 with non-exposed HA-NA/2009 or HA-NA/1999 viruses, the NP protein was concentrated 177 within the nucleus 2 hours after infection (Fig. 3B). On the contrary, in MDCK cells infected 178 with exposed HA-NA/2009 or HA-NA/1999 viruses for 5 days to saline water at 35°C, we 179 detected the NP protein close to the cell membrane but not in the nucleus after 2 hours of 180 infection. We confirmed that NP localization of exposed viruses was similar to that of 181 non-exposed viruses after 20 min of infection, when virus entry is not completely achieved 182 (Fig. S2). We thus assessed the HA-triggered fusion of the viral membrane in infected 183 MDCK cells using R18-labelled viruses at self-quenched concentration, a widely used 184 technique to detect fusion of the viral membrane with the endosomal membrane (Pohl,185 Edinger, and Stertz 2014; Schelker et al. 2016). Fusion events were detected 20 min after 186 infection by confocal microscopy in cells infected with viruses exposed 5 days to saline 187 water at 35°C or non-exposed viruses (Fig. 3C). We detected almost no R18 dequenching 188 signal in cells infected with HA-NA/2009 or HA-NA/1999 exposed viruses, whereas we 189 observed numerous fusion events in cells infected with the non-exposed viruses. This 190 result demonstrates that the loss of infectivity of H1N1 viruses in saline water at 35°C is a 191 consequence of the HA inability to trigger membrane fusion. results demonstrated that amino-acids at the calcium binding sites in the NA are important 231 molecular determinants of virus environmental persistence. Viruses with a high 232 environmental persistence also had a stable NA, as shown in Fig. 5C and Fig. 5D, 233 measuring the NA activity of viruses exposed or not to saline water at 35°C. Viruses with 234 an important environmental persistence also presented a high NA activity (Fig. 5E). 235 Unfortunately, the NA activity for the HA-NA (341)/1999 virus was below our detection 236 threshold and could not be measured. 237 Similarly, the less stable HA∆K147 and HA/Y454F mutant viruses (Fig. 3B) have a high 280 sensitivity to low pH and lower HA expression level in infected cells compared to the other 281 mutants ( Fig. 4A and 4B). On the other hand, the whole/2009 and the Pol/1933 viruses 282 had the same persistence but significant differences of HA expression levels (Fig. 4C). inactivation is probably progressive in the environment as suggested by our results on the 294 loss hemagglutination activity (Fig. 3A). 295 We provided evidences that the NA protein is also a driver of IAVs environmental

Virus persistence in saline water at 35°C 386
Virus persistence in saline water at 35°C was studied as follow: viral suspensions were 387 diluted 10 times in saline distilled water (35 g.L -1 NaCl) and placed in a humidified 388 incubator (5% CO2, 35°C) for 1 hour, 24 hours or 48 hours. The pH of the saline water 389 did not vary between experiments. In order to quantify their residual infectivity, MDCK 390 cells were seeded on a 16-well microtiter plate (30000 cells per well) coated with 391 microelectrode sensors in the xCELLigence® Real-Time Cell Analysis (RTCA) DP 392 instrument (ACEA Bioscience, Inc.) and grown for 24 hours (5% CO2, 37°C). Cells were 393 then infected by exposed viruses and cell impedance, expressed as an arbitrary unit called 394 the Cell Index (CI), was measured through the electrodes every 15 min. Cell Index 395 decrease due to virus-induced cytopathogenic effect was quantified with the CIT50 value, 396 which is the necessary time in hours to measure a 50% decrease from the initial CI, always 397 set as the CI value 5 hours after infection. CIT50 values are linearly correlated to the TCID50 398 titres of the viral suspensions (47) (Fig. S4). Thus, the increase of the CIT50 values over 399 time reflects the loss of infectivity. For each exposed viral suspension, CIT50 values were 400 obtained at different exposure times and plotted to calculate a linear regression slope 401 referred to as the inactivation slope. All data shown on viral persistence have been 402 obtained at different periods and with different viral stock productions. Each replicate value 403 of the inactivation slopes refers to a replicate virus suspension tested for persistence. 404

Virus concentration for functional analyses 405
For the experiments described below, harvested supernatants of stock viruses were 406 clarified and concentrated using a vivaspin 20 centrifugal concentrator (1 000 000 MWCO, 407 Sartorius), to reduce the initial volume by 10 times. Concentrated supernatants were then 408 diluted in saline distilled water (35g.L -1 NaCl) at a ratio of 1:10 and placed either at 4°C (0 409 day exposure) or at 35°C for 5 days and then kept at 4°C until used in further analysis. 410

Hemagglutination assay 411
Exposed viruses were diluted in two-fold dilution steps with PBS in a 96-wells plate and 412 mixed with an equal volume of a 0.75% suspension of fresh guinea pig erythrocytes. The 413 mixture was incubated for 1 hour at room temperature before observing erythrocytes 414 aggregation. 415

Neuraminidase activity assay 416
Neuraminidase activity of exposed viruses was measured using a NA-Fluor kit (Applied    (A) Viral particles were diluted in saline water (35 g.L -1 NaCl) and exposed to a 687 temperature of 35°C for 0, 1 or 2 days. MDCK cells were infected by exposed viruses and 688 impedance was monitored continuously and plotted as "Cell Index" (CI). (B) CI decrease 689 due to virus-induced cytopathogenic effect was quantified with the CIT50 value, which is 690 the necessary time in hours to measure a 50% decrease from the initial CI, always set as