Assessment of Survival Kinetics for Emergent Highly Pathogenic Clade 2.3.4.4 H5Nx Avian Influenza Viruses

High pathogenicity avian influenza viruses (HPAIVs) cause high morbidity and mortality in poultry species. HPAIV prevalence means high numbers of infected wild birds could lead to spill over events for farmed poultry. How these pathogens survive in the environment is important for disease maintenance and potential dissemination. We evaluated the temperature-associated survival kinetics for five clade 2.3.4.4 H5Nx HPAIVs (UK field strains between 2014 and 2021) incubated at up to three temperatures for up to ten weeks. The selected temperatures represented northern European winter (4 °C) and summer (20 °C); and a southern European summer temperature (30 °C). For each clade 2.3.4.4 HPAIV, the time in days to reduce the viral infectivity by 90% at temperature T was established (DT), showing that a lower incubation temperature prolonged virus survival (stability), where DT ranged from days to weeks. The fastest loss of viral infectivity was observed at 30 °C. Extrapolation of the graphical DT plots to the x-axis intercept provided the corresponding time to extinction for viral decay. Statistical tests of the difference between the DT values and extinction times of each clade 2.3.4.4 strain at each temperature indicated that the majority displayed different survival kinetics from the other strains at 4 °C and 20 °C.

AIV survival in different environments is a critical component of understanding incursion risk to different poultry sectors [17].The survival of infectious AIVs has been demonstrated in water, soil, and faeces [18][19][20][21], and in different egg products [22].Multiple factors contribute to AIV survival metrics [23], hence comparisons in distilled water and saline [24,25], plus the effects of pH and temperature have featured in assessments of viral decay [26,27].In this study, we investigated the survival kinetics for five clade 2.3.4.4 H5Nx HPAIVs linked to UK incursions which were incubated at up to three temperatures.

Viruses and Safety Statement
Five H5Nx clade 2.3.4.4 HPAIV isolates derived from outbreak events in the United Kingdom (UK) were investigated in this study (Table 1) as representatives of viruses which caused disease events during five UK/European clade 2.3.4.4 epizootic seasons between 2014 and 2021.Viruses were propagated in 9-to 11-day-old specified pathogen-free embryonated fowls' eggs (SPF EFEs) [28].The H5Nx clade 2.3.4.4 HPAIVs are categorised in the UK by the Specified Animal Pathogens Order at level 4 and the Advisory Committee on Dangerous Pathogens at level 3, hence all laboratory work was carried out in licensed biosafety level 3 laboratories [29].

Preparation of Replicate Samples for Infectivity Assessment at Different Temperatures
Each virus stock was diluted in serum-free medium to prepare a working concentration at a 50% tissue culture infectious dose (TCID 50 ) of 1 × 10 6 TCID 50 /mL (Table 1).At time zero, three replicate samples (1.2 mL) were prepared, using sterile safe lock Eppendorf tubes (Anachem, Clonakilty, Ireland), for each investigated timepoint at a given temperature (detailed below).An additional eight to 10 aliquots of the working concentration of each virus were prepared and frozen promptly, to serve as time zero positive controls representative of no incubation treatment.

Virus Quantification after Incubation at Different Temperatures
The infectivity of each timepoint replicate was quantified as a 50% TCID 50 value of 96-well plates (Nunc, ThermoFisher, Waltham, MA, USA) which contained 80% confluent MDCK monolayers.Virus survival was determined as the time (days) for the viral infectivity to be reduced by 90% at the incubation temperature T (D T ), or a one log 10 reduction.Before titration, the 96-well MDCK plates were washed in 100 µL of serum-free EMEM and replaced with 100 µL of fresh media.Once each stored biological replicate had thawed, 100 µL of media was removed from all wells in the first column of a 96-well MDCK plate, with all remaining wells containing 100 µL of serum-free EMEM.Each biological replicate was divided between wells in column 1 and applied as eight 146 µL volumes (technical replicates).Forty-six µL volumes were removed from the first column and diluted into the second column with titration repeated sequentially from column 2 into column 3 and continued across to column 11 to produce a 0.5 log 10 dilution series.As an internal negative control, no virus was titrated into column 12.For the positive virus control (no incubation treatment), one time zero frozen aliquot was thawed, and applied for quantification in a separate 96-well plate of MDCK cells.After incubation at 37 • C (±2 • C) for 60 min, microtiter plates were washed using 100 µL of serum-free EMEM, before being overlaid with 100 µL of fresh serum-free EMEM.Each plate was incubated at 37 • C (±2 • C) and 5% CO 2 for up to four days, for the cytopathic effect to develop.
Following incubation, the medium was removed, and all wells were washed in 100 µL of serum-free EMEM and blotted before 50 µL of crystal violet solution (1% (w/v) in water: ethanol (1:5.3);Sigma-Aldrich, St. Louis, MI, USA) was added to each well.After 30 min incubation at room temperature, the crystal violet solution was discarded, the wells were washed using 100 µL of 0.1 M phosphate-buffered saline (pH 7.2), and the plates were blotted dry.All titrations of biological replicate samples were performed in triplicate and a TCID 50 /mL was determined for each from the mean of the eight technical replicates assessed [30].The limit of detection for virus infectivity was a virus titre of 1.625 TCID 50 /mL, below which a zero titre was recorded.

Statistical Analyses
Univariate linear regression analyses [24], were undertaken for each biological sample at each temperature treatment.Values for the viral log 10 TCID 50 /mL were plotted against time in days to determine viral decay kinetics at a chosen temperature.The slope of the corresponding line of best fit was used to calculate D T for a 90% reduction in viable viral population [31].Since three biological replicates per timepoint and temperature profile were prepared from the same working stock, each 50% TCID 50 calculation represented an independent test.To compare the best graphical fit for virus survival against time, the extra-sum-of-squares F test was used to determine how well these observed values fitted those expected from a regression analysis.Deviation of the slope from zero was considered statistically significant at the 5% level when p < 0.05.The R 2 values reflected the fit of these data to the regression line.Extrapolation of the straight line to the x-axis intercept (y = 1 log 10 TCID 50 /mL) gave a corresponding time to extrapolated extinction (days) for a given H5Nx HPAIV at the stated incubation temperature.The statistical significance of differences in the D T and extinction times between isolates was carried out using the analysis of covariance tool in Matlab v2019b (MathWorks, Natick, MA, USA), adjusted for multiple comparisons using a Tukey test.All other statistical analysis were performed using GraphPad Prism, version 8.4.2 (GraphPad Software, La Jolla, CA, USA).

Results and Discussion
The ability of AIV infectivity to survive in the environment is a major contributing factor to viral persistence and spread [17], and merited investigation because of the extensive nature of the most recent H5N1-2021 clade 2.3.4.4b epizootic [16].Experimental evidence has underlined the essential waterfowl-adapted tropism of H5Nx clade 2.3.4.4 HPAIVs, where the efficient acquisition and onward transmission of infection correlated with waterfowl interactions, within a strongly virus-contaminated environment [32][33][34][35][36].This sustained viral maintenance within wild anseriformes produces the infection pressure for associated poultry outbreaks [7], with viral environmental contamination also recorded at outbreak premises [37,38].We compared the temperature-associated virus stability (infectivity) of five UK-origin H5Nx clade 2.3.4.4 strains (Table 1) at three European outdoor temperatures, where 4 • C mirrored winter, while 20 • C and 30 • C corresponded to summer temperatures in northern and southern Europe, respectively.Viral stability was determined from the D T values at a given temperature [24], following incubation for up to 10 weeks.Extrapolation of the predicted straight line provided an intercept to also show the time to extinction of viral infectivity (Figures 1 and 2, Table 2).Each clade 2.3.4.4 isolate showed greatest stability at 4 • C, whereas increased incubation temperatures, namely 20 • C and 30 • C, resulted in a faster reduction in viral infectivity (Figures 1 and 2, Table 2), which was consistent with an inverse relationship for AIV survival kinetics and temperature [24,39].
using the analysis of covariance tool in Matlab v2019b (MathWorks, Natick, MA, USA), adjusted for multiple comparisons using a Tukey test.All other statistical analysis were performed using GraphPad Prism, version 8.4.2 (GraphPad Software, La Jolla, CA, USA).

Results and Discussion
The ability of AIV infectivity to survive in the environment is a major contributing factor to viral persistence and spread [17], and merited investigation because of the extensive nature of the most recent H5N1-2021 clade 2.3.4.4b epizootic [16].Experimental evidence has underlined the essential waterfowl-adapted tropism of H5Nx clade 2.3.4.4 HPAIVs, where the efficient acquisition and onward transmission of infection correlated with waterfowl interactions, within a strongly virus-contaminated environment [32][33][34][35][36].This sustained viral maintenance within wild anseriformes produces the infection pressure for associated poultry outbreaks [7], with viral environmental contamination also recorded at outbreak premises [37,38].We compared the temperature-associated virus stability (infectivity) of five UK-origin H5Nx clade 2.3.4.4 strains (Table 1) at three European outdoor temperatures, where 4 °C mirrored winter, while 20 °C and 30 °C corresponded to summer temperatures in northern and southern Europe, respectively.Viral stability was determined from the DT values at a given temperature [24], following incubation for up to 10 weeks.Extrapolation of the predicted straight line provided an intercept to also show the time to extinction of viral infectivity (Figures 1 and 2, Table 2).Each clade 2.3.4.4 isolate showed greatest stability at 4 °C, whereas increased incubation temperatures, namely 20 °C and 30 °C, resulted in a faster reduction in viral infectivity (Figures 1 and 2, Table 2), which was consistent with an inverse relationship for AIV survival kinetics and temperature [24,39].considered statistically significant at the 5% level when p < 0.05.The R 2 values reflected the fit of these data to the regression line.Extrapolation of the straight line to the x-axis intercept (y = 1 log10 TCID50/mL) gave a corresponding time to extrapolated extinction (days) for a given H5Nx HPAIV at the stated incubation temperature.The statistical significance of differences in the DT and extinction times between isolates was carried out using the analysis of covariance tool in Matlab v2019b (MathWorks, Natick, MA, USA), adjusted for multiple comparisons using a Tukey test.All other statistical analysis were performed using GraphPad Prism, version 8.4.2 (GraphPad Software, La Jolla, CA, USA).

Results and Discussion
The ability of AIV infectivity to survive in the environment is a major contributing factor to viral persistence and spread [17], and merited investigation because of the extensive nature of the most recent H5N1-2021 clade 2.3.4.4b epizootic [16].Experimental evidence has underlined the essential waterfowl-adapted tropism of H5Nx clade 2.3.4.4 HPAIVs, where the efficient acquisition and onward transmission of infection correlated with waterfowl interactions, within a strongly virus-contaminated environment [32][33][34][35][36].This sustained viral maintenance within wild anseriformes produces the infection pressure for associated poultry outbreaks [7], with viral environmental contamination also recorded at outbreak premises [37,38].We compared the temperature-associated virus stability (infectivity) of five UK-origin H5Nx clade 2.3.4.4 strains (Table 1) at three European outdoor temperatures, where 4 °C mirrored winter, while 20 °C and 30 °C corresponded to summer temperatures in northern and southern Europe, respectively.Viral stability was determined from the DT values at a given temperature [24], following incubation for up to 10 weeks.Extrapolation of the predicted straight line provided an intercept to also show the time to extinction of viral infectivity (Figures 1 and 2, Table 2).Each clade 2.3.4.4 isolate showed greatest stability at 4 °C, whereas increased incubation temperatures, namely 20 °C and 30 °C, resulted in a faster reduction in viral infectivity (Figures 1 and 2, Table 2), which was consistent with an inverse relationship for AIV survival kinetics and temperature [24,39].  1 Extinction (days) is the x axis intercept (y = 1 log10 TCID50/mL).
For the five viruses assessed at 4 °C and 20 °C, pairwise comparisons of their DT values and extrapolated extinction times revealed that, in the majority of cases, the virus survival (stability) of each virus was significantly different from the others (p < 0.05; Tables 3  and 4).The only exceptions among the 4 °C DT comparisons were those for H5N8-2014 versus H5N1-2021 (p = 0.33), and H5N8-2016 versus H5N6-2017 (p = 0.06), with comparison of H5N1-2021 versus H5N6-2017 being non-significant for both their DT values (p = 0.22) and extinction times (p = 0.58; Table 3).Exceptions among the 20 °C comparisons were noted for the DT values for H5N1-2021 versus H5N8-2014 (p = 0.55), for the extinction times for H5N1-2021 versus H5N6-2017 (p = 0.13), with comparison of H5N6-2017 versus H5N8-2014 being non-significant for both their DT values (p = 0.35) and extinction times (p = 0.97; Table 4).However, comparisons of both parameters obtained at 30 °C showed that only H5N8-2016 had a significantly lower extinction time than H5N8-2020 and H5N1-2021 (p < 0.001), with no significant difference between the extinction times of H5N8-2020 and H5N1-2021 (p = 0.33).There were no significant differences between the DT values among any of the three isolates at 30 °C (p > 0.28).up to 10 weeks.Extrapolation of the predicted straight line provided an intercept to also show the time to extinction of viral infectivity (Figures 1 and 2, Table 2).Each clade 2.3.4.4 isolate showed greatest stability at 4 °C, whereas increased incubation temperatures, namely 20 °C and 30 °C, resulted in a faster reduction in viral infectivity (Figures 1 and 2, Table 2), which was consistent with an inverse relationship for AIV survival kinetics and temperature [24,39].Overall, for a given H5Nx clade 2.3.4.4 isolate, these data showed the reduction in survival time at 20 • C was at least 2.5-fold faster than the D T values observed at the low incubation temperature (4 • C).Wild waterfowl cases, during the clade 2.3.4.4 epizootic waves, peaked in the European winter months when these heightened infection pressures would result in accompanying incursions in farmed poultry [12,16].The two least stable viruses, at least when ranked by their extinction times, were H5N8-2014 and H5N6-2017 (Table 2), and their corresponding significance tests at 20 • C (Table 4) showed this virus pair was significantly different from the other viruses.Interestingly, the European winter incursions of H5N8-2014 and H5N6-2017 were the smallest of the five H5Nx clade 2.3.4.4 epizootics, which in the case of H5N6-2017 were essentially limited to wild birds with no accompanying commercial poultry outbreaks during winter 2017-2018 [12,40].The relative instability of H5N8-2014 and H5N6-2017, reflected in low D T and extinction values, may have contributed to the limited scale of these incursions.
In field settings, there is the confounder of matrix effects upon virus deposited in the environment, which may unpredictably affect the complexity of virus survival.Therefore, it is important to apply uniform and consistent assessment of temperature-associated survival of any viral pathogen which is excreted into the environment, hence all five H5Nx HPAIVs were each isolated and propagated by using SPF EFEs.Progeny AIVs are produced during the orthomyxovirus replication cycle [8,41], during which they acquire an external lipid envelope of plasma membrane origin from the host [42].Therefore, lipid envelope consistency was ensured by focusing the temperature stability investigations on viruses propagated in EFEs.This approach ensured the variation in infectivity survival was determined by mainly differences among the viral envelope proteins, namely the HA and NA [23,43].However, potential contributions to viral stability by other internal structural proteins could not be entirely excluded [23,44], and may also extend to the stability of the encapsulated viral polymerase enzymes, so these could have contributed to the stability differences observed in our study for the three H5N8 clade 2.3.4.4 subtypes (Table 2).
Temperature-associated viral stability reflected in environmental survival is but one factor which may influence the scale and extent of epizootics.The recent H5N1 clade 2.3.4.4bHPAIV epizootic has included an expansion of the avian host range to include seabirds with many cases reported on a global scale, thereby enhancing viral spread and associated infection pressure [10,15,45].However, the dynamics of spread within waterfowl may be modulated by host responses, including innate and/or H5-specific humoral immunity acquired during previous AIV incursions [46,47].Other species-specific differences have been noted, where environmental contamination may not have such a prominent role in H5Nx clade 2.3.4.4 HPAIV spread among chickens [48,49].

Conclusions
These quantified virus survival data provide evidence to refine disease prevention strategies for poultry units and inform future veterinary risk assessments for outbreak management, particularly in view of the continuing H5N1 clade 2.3.4.4 HPAIV global epizootic [50,51].Importantly, these virus survival outcomes contribute to the understanding of AIV persistence in the environment, thereby informing protection of poultry health and commercial production systems.These data highlight HPAIV persistence at low temperatures, so presenting a greater infection risk to avian species during the cooler months in temperate latitudes, especially in the case of clade 2.3.4.4 H5Nx HPAIVs to waterfowl with subsequent incursion risks for farmed poultry.Our statistical data, using pairwise comparisons of virus D T values and extrapolated extinction times at 4 • C and 20 • C, (p < 0.05; Tables 3 and 4), revealed that, in many instances, the virus survival (stability) of each isolate was significantly different from the others.The relevance of these experimental findings has been underlined by the detection of H5N1 HPAIV in the immediate farm environment during UK clade 2.3.4.4 outbreaks in 2023 [38], affirming observations during earlier H5N1 GsGd HPAIV outbreaks [37].These experimental and field-based studies are now identifying the consequences of environmental contamination, which have arisen from AIV incursions (either wild birds or poultry) and may influence onward spread.The temperature stability of isolates can also inform the likelihood of continuing infectivity, particularly during the vulnerable period prior to statutory cleansing and on-farm disinfection interventions [52,53].Interestingly, heat treatment may provide an alternative to chemical disinfection, thereby giving additional importance to the outcomes of viral temperature stability investigations [54,55].

Table 3 .
Resulting p-values from significance tests of the difference between the D T values and extinction times at 4 • C for five different H5Nx HPAIV isolates.single p-value cell means that the same p-value applies to both the D T value and extinction time.-indicates that either (i) table diagonals: result not applicable as it is for the virus strain versus itself, or (ii) lower half of table: result available in the top half of the table. A

Table 4 .
Resulting p-values from significance tests of the difference between the D T values and extinction times at 20 • C for five different H5Nx HPAIV isolates.single p-value cell means that the same p-value applies to both the D T value and extinction time.-indicates that either (i) table diagonals: result not applicable as it is for the virus strain versus itself, or (ii) lower half of table: result available in the top-half of the table. A