Hemagglutinin and neuraminidase of an H7N7 non-pathogenic avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens

Hemagglutinin and neuraminidase of an H7N7 non-pathogenic avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hemagglutinin and neuraminidase of an H7N7 non-pathogenic avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens Takaya Ichikawa, Takahiro Hiono, Masatoshi Okamatsu, Junki Maruyama, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4161114/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Sep, 2024 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named Vac2sub-P0, which is non-pathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs and a virus with high intravenous pathogenicity, namely, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 is only partially lethal in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the passaged virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages are responsible for increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with the parental virus in vitro; the virus growth at 40°C was higher, the binding affinity to a sialic acid receptor was lower, and the release activity by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study critically demonstrated that additional adaptations were required for the highly pathogenic virus via intravenous administration in chickens for increased pathogenicity via intranasal administration. High pathogenicity avian influenza virus Intranasal pathogenicity Chicken Hemagglutinin Neuraminidase Figures Figure 1 Figure 2 Figure 3 Introduction The natural hosts of influenza A viruses are wild waterbirds, particularly migratory ducks [ 9 , 15 , 37 ]. Previous studies revealed that duck influenza viruses transmit to chickens via domestic waterfowl or terrestrial birds [ 17 , 22 ]. With multiple repeated infections in a chicken population, high pathogenicity avian influenza viruses (HPAIVs) can only be selected from H5 or H7 viruses [ 1 , 14 , 25 ]. Hemagglutinin (HA) of the low pathogenicity avian influenza virus (LPAIV) is cleaved only by trypsin-like serine proteases expressed in the respiratory or intestinal epithelia, leading to mild or asymptomatic local infection. In contrast, the HA of HPAIV has polybasic amino acid residues at its cleavage site, which permit ubiquitous proteases such as furin and PC6 to cleave the precursor HA0 into HA1 and HA2 subunits, causing severe systemic infection in chickens [ 27 ]. The presence of polybasic amino acids is a prerequisite for the fatal pathogenicity in chickens. In contrast, previous investigations demonstrated that LPAIVs did not show high pathogenicity after the artificial introduction of a specific sequence at the HA cleavage site [ 3 , 18 , 30 , 33 , 36 ]. These results strongly suggested that additional viral factors are required for LPAIVs to acquire high pathogenicity in chickens. For example, a virus with three characteristics, filamentous form, high polymerase activity, and high stability of the matrix (M1) protein, was selected during the passage of a genetically modified H5N1 virus in chickens [ 40 ]. In our previous study, a genetically modified H7N7 duck influenza virus was designed by artificially introducing the basic amino acid residue KRRRRR at the HA cleavage site of an H7N7 LPAIV, A/duck/Hokkaido/Vac-2/2004 (Vac2), namely the Vac2sub-P0 strain [ 18 ]. After consecutive passages of Vac2sub-P0 in chicks, the passaged virus Vac2sub-P3 acquired high intravenous pathogenicity (Intravenous Pathogenicity Index: IVPI = 2.54). According to the experimental infection of mutant viruses using site-directed mutagenesis and reverse genetics, amino acid substitutions at two positions, E227G and I388T, in the HA of Vac2sub-P3 are essential for its high pathogenicity in chickens. In contrast to intravenous infection, intranasal inoculation with Vac2sub-P3 resulted in limited viral replication in organs and mortality in chickens. Discrepancies may arise in pathogenicity between the intranasal and intravenous inoculation routes. In the present study, Vac2sub-P3, which showed high pathogenicity via intravenous inoculation but partial pathogenicity via intranasal inoculation, was serially passaged in the lungs of chickens to acquire higher intranasal pathogenicity. We obtained a virus with comparable pathogenicity to field isolates and characterized the virus to demonstrate functional changes in viral proteins during the acquisition of intravenous and intranasal pathogenicity in chickens. Materials and methods Viruses The viruses used in this study were obtained after three passages in chick air sacs (Vac2sub-P3) with the A/duck/Hokkaido/Vac-2/2004 (H7N7) strain, which introduced the amino acid residue KRRRRR at the HA cleavage site (rgVac2sub-P0), as described previously [ 18 ]. All viruses were propagated in 10-day-old embryonated chicken eggs for 30–48 h at 35°C. Cells Madin-Darby canine kidney (MDCK) cells were maintained in minimum essential medium (MEM, Nissui, Tokyo, Japan) supplemented with 0.3 mg/mL L-glutamine (FUJIFILM Wako Pure Chemical, Osaka, Japan), 5% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin G (Meiji Seika Pharma, Tokyo, Japan), 0.1 g/mL streptomycin (Meiji Seika Pharma), and 8.0 g/mL gentamicin (MSD, Rahway, NJ, USA). Chicken embryonic fibroblasts (CEFs) were prepared from 10-day-old chicken embryos and cultured in MEM supplemented with l-glutamine, fetal bovine serum, and antibiotics. Plaque assay Ten-fold dilutions of viruses were inoculated onto confluent monolayers of MDCK cells and incubated at 35°C for 1 h. Unbound viruses were removed by washing cells with MEM. Cells were then overlaid with MEM containing 0.7% Bacto-agar (Becton Dickinson, Franklin Lakes, NJ, USA) in the presence of 5 µg/mL trypsin acetylated (Sigma-Aldrich, St. Louis, MO, USA). After 48 h of incubation at 35°C, cells were stained with 0.005% neutral red (Sigma-Aldrich). Consecutive passages of Vac2sub-P3 in chickens Four-week-old chickens (Boris Brown) free from antibodies against the H7 influenza virus were obtained from Hokkaido Chuo Shukeijou, Hokkaido, Japan. Three chickens were intranasally inoculated with 100 µL each of Vac2sub-P3. Three days post-inoculation (dpi), the chickens were sacrificed and their lungs were collected. A pooled 10% tissue suspension of the infected organs was serially passaged in three 4-week-old chickens via the intranasal route. After four passages, the isolate named Vac2sub-P3L4 (“L” indicates lungs) was obtained. Passaged viruses were propagated in the allantoic cavities of 10-day-old embryonated eggs for 48 h at 35°C. Experimental infection of chickens with mutant viruses Four-week-old chickens were used to test the pathogenicity of the viruses used in this study. Six chickens were intranasally inoculated with 100 µL of allantoic fluid containing each virus at 10 5.0 plaque forming units (PFU) and observed for 14 days. Specific antibodies against homologous viruses were detected in the serum at 14 dpi using the hemagglutination inhibition (HI) test. To study viral replication, three more chickens were inoculated with each virus, as described above and euthanized at 3 dpi, after which tissue and blood were collected aseptically. To prepare a 10% suspension in MEM, tissue samples were homogenized using a multi-bead shocker (Yasui Kikai, Osaka, Japan). Virus titers of these suspensions were calculated using a plaque assay and expressed as PFU/g of tissue or mL of blood. All experiments were performed in self-containing isolator units (Tokiwa Kagaku, Tokyo, Japan) in a biosafety level 3 (BSL3) facility at the Faculty of Veterinary Medicine, Hokkaido University, Japan. Reverse genetics Mutations were introduced into specific regions of the plasmid-encoded PA, HA, NA, M, and NS genes of Vac2sub-P3 using a QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA), as described previously [ 30 ]. The mutant viruses listed in Fig. 1 A were rescued employing reverse genetics [ 12 ], and all eight segments of the genome were sequenced to confirm that the desired mutations were induced and the undesired ones were eliminated. Co-cultures of human embryonic kidney 293T and MDCK cells were transfected with plasmids using 2 mg/mL polyethylenimine (Polysciences, Warrington, PA, USA). At 48 h post transfection, the culture supernatant was collected and propagated in 10-day-old embryonated chicken eggs. Three-dimensional (3D) structural-based mapping of the HA protein Mutations were mapped to the H7 HA protein (Protein Data Bank accession 1TI8 [ 24 ]) using the UCSF Chimera X version 1.2 (University of California, San Francisco, CA, USA). Monomeric HA is depicted as a soft-surface model and ribbon diagram. Virus growth in cell culture Viruses at a multiplicity of infection (M.O.I.) of 0.01 were inoculated onto confluent monolayers of CEF cells and incubated at 35°C for 1 h. Unbound viruses were removed by washing the cells with MEM, and the cells were overlaid with MEM in the presence of trypsin accetylated (5 µg/mL). The plates were incubated at 35 or 40°C under 5% CO 2 . At various times post-infection, the viral titers in the cell culture supernatant were determined using a plaque assay with MDCK cells. Solid-phase direct binding assay Receptor binding affinity of the viruses was assessed using a solid-phase direct binding assay with sialylglycopolymers, 3′-Sialyllactose-PAA (3′-SL) (Cosmo Bio, Tokyo, Japan), as described previously [ 28 ]. 3′-SL was added to each well of a Universal-BIND™, 96 well polystyrene stripwell microplate (Corning, Corning, NY, USA). After the wells were blocked with 2% bovine serum albumin, a solution containing influenza viruses (32 HA unit) was added and incubated at 4°C for 16 h. Mouse anti-H7 HA monoclonal antibody 253/1 [ 26 ] and goat anti-mouse IgG-HRP conjugate (Bio-Rad, Hercules, CA, USA) were used to detect the viruses. 3,3′-tetramethylbenzidine (TMB) and 0.04% H 2 O 2 were added as substrates and the absorbance was measured at 450/630 using a Model 680 Microplate Reader (Bio-Rad). The affinity constant for each virus was calculated as described previously [ 23 ]. Blank-corrected data were plotted and fitted to an established binding model using R version 3.0.1 (The R Foundation for Statistical Computing, Vienna, Austria). The formula for the model is as follows: $$\text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}=\frac{{A}_{max}\times C}{Kd+C}$$ where C indicates the concentration of 3′-SL; Amax indicates the maximum of binding absorbance; and Kd is the dissociation constant. The affinity constant (Ka) determines the reciprocal of Kd. Erythrocyte elution assay The elution ability of NA from chicken erythrocytes was determined as previously described [ 5 ]. Briefly, viruses containing 128 HA units were serially diluted in calcium saline buffer (6.8 mM CaCl 2 − 154 mM NaCl in 20 mM borate buffer, pH7.2) and incubated with 50 µL of 0.5% chicken erythrocytes in microtiter plates at 4°C for 1 h. The plates were then stored at 37°C, and the reduction of HA titers was monitored periodically for 10 h. NA enzymatic activity NA enzymatic activity was measured using 2′-(4-Methylumbelliferyl)–α–D– N -acetylneuraminic acid (MUNANA; Sigma-Aldrich) as a soluble substrate, as described previously [ 6 ]. Briefly, the viruses were mixed with serially diluted MUNANA in 96-well black-bottomed plates. The mixture was exerted at 360 nm at 1-min intervals, and the emission signal was periodically monitored for 30 min at 37°C using POWER SCAN 4 (Agilent Technologies). The initial reaction rate under each condition was calculated and plotted to obtain the Michaelis constant, Km, using R software. Ethics statement Animal experiments were approved by the Institutional Animal Care and Use Committee of the Faculty of Veterinary Medicine, Hokkaido University (approval numbers: 13–0108, 13–0162, 18–0037, 18–0040). All experiments involving genetically modified organisms, including mutant viruses generated by reverse genetics, were authorized by the Safety Committee on Genetic Recombination Experiments of Hokkaido University (approval numbers: 2015-019, 2020-009), and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (30–963, 2-654). This study was approved by the Biosafety Management Committee on Pathogens and Other Hazardous Agents of the Faculty of Veterinary Medicine at Hokkaido University (approval numbers: 2017-1-61, 2018-1-59, and 2019-1-62). Results Amino acid changes of the viruses during consecutive passages of Vac2sub-P3 in chickens The Vac2sub-P3 virus was obtained from a previous study and used for additional consecutive passages. Each isolate obtained from lungs was tested for pathogenicity. After four passages, the resultant virus Vac2sub-P3L4 showed high intranasal pathogenicity in chickens; all six chickens intranasally inoculated with the virus died within 5 days. The sequences of the passaged viruses were compared to identify amino acid substitutions (Table 1 ). Seven amino acid substitutions (S409I in PA, T32I and R65K in HA, L10S in NA, R101K in M1, D44G in M2, and E229K in NS1) and a deletion (at positions 40–73 in NA) were observed between Vac2sub-P3 and Vac2sub-P3L4. Among the six substitutions (K123E in PB2, N16D in PB1, E227G and I388T in HA, G228R in M1, and L46P in M2) between Vac2sub-P0 and Vac2sub-P3, G228R in M1 and L46P in M2 were reverted during additional passages to Vac2sub-P3L4. Table 1 Amino acid changes during consecutive passages of Vac2sub-P0 Virus PB2 PB1 PA HA NA M1 M2 NS1 123 a 16 409 32 65 227 388 10 40–73 101 228 44 46 229 Vac2sub- P0 K N S T R E I L ∗ b R G D L E P3 E D ཥ c ཥ ཥ G T ཥ ∗ ཥ R ཥ P ཥ P3L1 E D ཥ ཥ ཥ G T S Δ d ཥ ཥ D/G e ཥ ཥ P3L2 E D ཥ ཥ ཥ G T S Δ ཥ ཥ G ཥ ཥ P3L3 E D ཥ T/I ཥ G T S Δ ཥ ཥ G ཥ ཥ P3L4 E D I I K G T S Δ K ཥ G ཥ K a Methionine encoded by the AUG start codon is defined as position 1 b Asterisk indicates amino acids sequence GPKQKENLTCTTINQNNTTVVENTYVNNTTIITK c Periods indicate same amino acids as the parent virus d Delta (Δ) indicates the deletion of 40 to 73 amino acid residues in NA e Amino acid quasispecies were observed Amino acid diffenrence between Vac2sub-P0 and Vac2sub-P3 were already published by Maruyama et al., [ 18 ] Intranasal pathogenicity of rgVac2sub-P0, rgVac2sub-P3, rgVac2sub-P3L4, and mutant viruses in chickens To investigate the contribution of each mutation to the high intranasal pathogenicity of the passaged virus, we generated recombinant viruses using reverse genetics and compared their pathogenicity with that of Vac2sub-P0, Vac2sub-P3, and Vac2sub-P3L4 (Fig. 1 B). Consistent with a previous report [ 17 ], all the chickens intranasally inoculated with rgVac2sub-P0 lived. Two of the six chickens inoculated with rgVac2sub-P3 died during the observation period. All chickens inoculated with rgVac2sub-P3L4 died within 4 days. A previous study demonstrated that two mutations in HA, E227G and I388T, are critical for intravenous pathogenicity acquisition by Vac2sub-P3 [ 18 ]. Accordingly, we first focused on the contribution of these mutations to the HA segments. The virus rgP3L4/P0-HA, which has a Vac2sub-P0 HA segment with a Vac2sub-P3L4 backbone, did not kill any of the inoculated chickens. These results further highlight the importance of the four HA mutations for acquiring pathogenicity throughout consecutive passages. Moreover, infection with rgP3L4-P3HA showed significantly delayed mortality compared to infection with rgVac2sub-P3L4, suggesting that two additional mutations, T32I and R65K, in HA also contribute to the acquisition of intranasal pathogenicity. The functional balance between HA and NA is important for viral replication in cultured cells and pathogenesis in animals [ 2 , 20 , 29 , 38 ]. Therefore, we focused on mutations observed in NA. For this purpose, rgP3L4/P0-NA, harboring the NA of Vac2sub-P0 with a Vac2sub-P3L4 backbone, was generated. Similar to rgP3L4/P3-HA, infection with rgP3L4/P0-NA showed significantly delayed mortality compared with infection with rgVac2sub-P3L4. However, infection with rgP0/P3L4-HA,NA, which possesses HA and NA derived from Vac2sub-P3L4 with the Vac2sub-P0 backbone, resulted in a 2/3 mortality rate in infected chickens, indicating a significant contribution of mutations in other gene segments. To evaluate viral growth in chickens, we euthanized the infected chickens at 3 dpi and collected organ and blood samples to determine the virus titer (Table 2 ). No viruses were detected in chickens inoculated with rgVac2sub-P0 or rgP3L4/P0-HA, whereas rgVac2sub-P3L4 was systemically recovered from chickens at high titers. Viral titers recovered from chickens inoculated with either rgP3L4/P3-HA or rgP3L4/P0NA were lower than those recovered from chickens inoculated with rgVac2sub-P3L4. No virus was recovered from one of three chickens inoculated with rgP0/P3L4-HA or NA, which was consistent with their mortality rates and delayed mortality in chickens (Fig. 1 B). Table 2 Virus recovery from the chickens intranasally inoculated with each virus on 3 dpi Virus Chicken ID Virus recovery (log PFU/g) Brain Trachea Lung Liver Kidney Colon Blood a rgVac2sub-P0 1 - b - - - - - - 2 - - - - - - - 3 - - - - - - - rgVac2sub-P3 4 - - - - - - - 5 - - - - - - - 6 - - - - - - - rgVac2sub-P3L4 7 6.7 5.0 4.7 2.8 5.3 4.3 3.0 8 7.7 6.3 6.7 4.3 7.2 5.4 4.2 9 7.7 5.6 5.2 3.2 6.6 4.3 3.5 rgP3L4/P0-HA 10 - - - - - - - 11 - - - - - - - 12 - - - - - - - rgP3L4/P3-HA 13 4.6 4.3 4.1 2.9 4.1 3.5 3.1 14 6.3 3.4 3,0 2.3 4.4 - 3.0 15 4.6 3.8 3.2 2.3 3.7 2.3 - rgP3L4/P0-NA 16 3.4 3.8 3.0 - 3.9 2.5 - 17 6.0 4.3 4.3 - 5.6 2.3 - 18 6.8 4.2 4.6 2.3 6.0 3.0 - rgP0/P3L4-HA,NA 19 6.6 5.3 4.5 3.3 5.1 3.7 3.0 20 - - - - - - - 21 5.3 4.4 4.1 3.9 5.5 4.5 3.0 a log PFU/ml b < 2.0 (< 1.3 for blood samples) Phenotypical analyses of the HA of passaged viruses Th e in vivo analyses suggested that functional changes in HA and NA are crucial for acquiring intranasal pathogenicity of passaged viruses in chickens. First, we visualized the mutations observed in the 3D structure of HA (Fig. 2 A). Amino acid position 227 is located in the peripheral region of the receptor binding site. Position 388 is located in the alpha helix of the HA2 subunit. Position 32 was located in the N-terminal region of the HA1 subunit and near the HA1/2 cleavage site in the 3D structure. Position 65 is located in the hinge domain between the globular head and stem of the HA1 subunit. Notably, mutations at positions 227 and 388 were observed during the acquisition of intravenous pathogenicity in chickens in our previous study [ 18 ]. Position 227 faces opposite to the receptor-binding pocket. Accordingly, we hypothesized that this mutation affects receptor-binding avidity. Then, binding analyses of rgVac2sub-P0, rgVac2sub-P3L4, and rgP0/HA-227G with 3′-SL were conducted to calculate the affinity constant (Ka) of each virus with the receptor analog. 3′-SL is a sialylglycopolymer terminating with sialic acid α2,3 galactose and a model of the receptor for avian influenza viruses (Fig. 2 B). The Ka values of rgVac2sub-P3L4 and rgP0/HA-227G were significantly lower than those of rgVac2sub-P0 (Fig. 2 C). This result indicated that receptor-binding avidity was attenuated by the HA-E227G mutation. Mutations in the helix of HA2 stem affect the thermostability of HA molecule [ 13 ]. The growth of these viruses at different temperatures was assessed using CEFs. Although few differences were observed between the viruses at 35°C (Fig. 2 D), the virus titer of rgVac2sub-P3L4 and rgP0/HA-388T were significantly higher than that of rgVac2sub-P0 and rgP0/HA-227G at 40°C (Fig. 2 E). The present results indicate that viruses with HA-388T have a higher capacity to grow at high temperatures, such as in the chicken body. Phenotypical analyses of the NA of passaged viruses HA and NA functionally coevolve during viral adaptation to novel host species. The deletion of the NA stalk domain is among the most well-characterized changes in molecular coevolution. To examine the effects of NA stalk deletion on its function, the elution abilities from chicken erythrocytes of rgVac2sub-P0, rgVac2sub-P3L4 and rgP0/NAΔ40-73 (the mutant virus with the 34 amino acid truncation at the NA stalk with Vac2sub-P0 backbone) were assessed using an erythrocyte elution assay (Fig. 3 A). The rgVac2sub-P0 virus was eluted over time, whereas rgVac2sub-P3L4 and rgP0/NAΔ40-73 with shortened NA stalks, showed little or no elution. In addition, the sialidase activities of rgVac2sub-P0 and rgVac2sub-P3L4 were tested in a standard assay using MUNANA as a soluble substrate. As expected, no significant difference was observed in sialidase activity between these viruses (Fig. 3 B), indicating that the NA stalk deletion did not affect the removal of sialic acid from a soluble substrate [ 7 ]. Discussion The sequence of polybasic amino acids at the HA cleavage site permits ubiquitous proteases to cleave the HA, which is not sufficient but necessary for inducing severe systemic infection [ 8 , 14 , 30 ]. Some viral factors are also required for pathogenicity in chickens [ 10 , 34 , 36 , 40 ]. In addition, this study critically demonstrated that additional adaptations were required for the virus with high pathogenicity in chickens via intravenous administration for acquiring high pathogenicity via intranasal administration. In the present study, we investigated the characteristics of passaged H7N7 avian influenza viruses in a laboratory setting to determine the key factors responsible for their high intranasal pathogenicity. Receptor binding analysis demonstrated that viruses with HA-E227G showed lower glycan-binding avidity. Our previous study showed that this mutation, located near the receptor-binding domain, contributes critically to the acquisition of intravenous pathogenicity in chickens [ 18 ]. Soon after the additional passage of rgVac2sub-P3 into the respiratory tract of chickens, a 34 amino acid deletion in the NA stalk was observed between Vac2sub-P3 and Vac2sub-P3L1. Such deletions are often detected in avian influenza viruses isolated from terrestrial poultry, such as chickens and quails, but not in aquatic birds [ 1 , 16 , 19 , 32 ]. NA stalk deletion facilitates the adaptation of avian influenza viruses to terrestrial poultry [ 4 , 10 , 39 ] and increases their pathogenicity in the hosts [ 21 , 41 ]. Given that the virus used in this study was derived from the duck influenza virus [a reassortant strain between A/duck/Mongolia/736/2002 (H7N7) and A/duck/Hokkaido/49/1998 (H9N2)] [ 31 ], the NA stalk deletion following the present passages is consistent with these previous findings. Viruses with shortened NA stalks, such as rgVac2sub-P3L4 and rgP0/NAΔ40-73, showed little or no elution from chicken erythrocytes, in contrast to rgVac2sub-P0, indicating that the release activity from infected cells of the passaged virus was lower than that from the parent virus. Several studies have suggested that the balance between HA and NA functions is important for viral replication in cultured cells and experimental animals [ 2 , 20 , 29 , 38 ]. Accordingly, this selection might occur to adjust its function to optimize a balance with the HA function, which decreases the receptor-binding affinity during previous passages by Vac2sub-P3. However, it remains unclear how the shortened NA stalk region influences the adaptation of aquatic birds to terrestrial poultry. We hypothesized that the difference in tissue tropism between water birds and terrestrial poultry could be important; viruses infect and replicate in the colon of water birds and in the respiratory tract of terrestrial poultry. The basis of this hypothesis is that no NA stalk deletion was observed during other consecutive passages of Vac2sub-P3, in which the brain was used as a pooled sample in parallel (data not shown) and that viruses with shortened NA stalks replicated efficiently in the respiratory tract of chickens [ 21 , 32 ]. Viruses with low binding affinity and release activity may have some advantages in replicating the ciliated epithelial cells of the respiratory tract. The ancestral strain of H5 HPAIVs, A/goose/Guangdong/1/1996 (H5N1) (Gs/GD), had no deletion in its NA stalk domain, whereas the N1 NA of subsequent Gs/GD-like strains had 19 or 20 amino acid deletions in the stalk domain after the outbreak in Hong Kong in 1997. Later, descendants of the Gs/GD strain belonging to the HA genetic clade 2.3.4.4, underwent reassortment with viruses with various NA subtypes, resulting in the generation of H5Nx viruses. Among these, the N6 NA of H5N6 viruses has an 11 amino acid deletion in its stalk region, whereas no deletion has been observed in the N8 NA of H5N8 viruses. Furthermore, contemporary H5N1 viruses belonging to the HA genetic clade 2.3.4.4b obtained N1 NA without deletion in the NA-stalk domain by reassortment with non-pathogenic avian influenza viruses circulating among wild birds. Interestingly, these viruses show lethal pathogenicity to chickens in field settings, although there are strain-by-strain differences in their pathogenicity [ 35 ]. This suggests that HPAIVs are highly pathogenic in field settings without deletions at the NA stalk. In contrast, H5Nx viruses show altered receptor-binding specificity; these viruses recognize fucosylated receptors in addition to non-modified receptors [ 11 ]. The changes in the receptor-binding specificity of these viruses may be related to their potential to accept various NA with or without stalk deletion. Viruses harboring HA-I388T (rgVac2sub-P3L4 and rgP0/HA-388T) showed significantly higher growth and thermostability in CEFs. Mutations in the stem region of HA are often observed during experimental adaptation to influenza viruses, and these mutations are associated with a prior mutation in the (vicinity of) receptor-binding domain. The most well-known mutation is the T318I substitution in HA; this mutation was observed during the selection of airborne transmissible HPAIV in ferrets and increased the acid stability of HA [ 13 ]. It remains unclear whether these variants are selected to compensate for the instability of HA caused by the mutation in the receptor-binding domain or the phenotypic changes caused by the HA stem mutation are essential for adaptation. In addition, amino acid T32 comprises an N -glycosylation sequon, and the mutation T32I, which was observed after P3L3, abrogated the glycosylation at N30. Nevertheless, we did not obtain critical evidence to support glycosylation at N30 in P0-HA or non-glycosylation in P3L4-HA (data not shown). Perhaps, minor changes in glycan occupancy at this site may have contributed to the further adaptation of Vac2sub-P3L4 from Vac2sub-P3. In conclusion, the present study revealed that HA and NA underwent functional coevolution during the acquisition of high intranasal pathogenicity in chickens. Phenotypic changes in these proteins, along with polybasic amino acid residues at the HA cleavage site, are critical for this process. We installed polybasic amino acid residues at the HA cleavage sites to accelerate the adaptation process in a laboratory, and consecutive passages of the virus enhanced its growth capacity in chickens. The processes are vice versa; in the natural setting, the virus first acquires the ability to grow and be transmitted efficiently in chickens, and then the virus acquires the polybasic amino acid residues to the HA cleavage sites during the multiple transmission events in chicken flocks. Thus, further studies are needed to elucidate the definitive factors responsible for the difference between high pathogenicity of HPAIVs in chickens caused by intravenous and intranasal inoculations. Declarations Acknowledgements We thank Ms. Yuka Sato and Ms. Mayumi Endo for their technical assistance. Funding This work was supported by the Japan Initiative for World-Leading Vaccine Research and Development Centers [Grant No. JP233fa627005]. This study was partially funded by the Japan International Cooperation Agency within the framework of the Science and Technology Research Partnership for Sustainable Development [Grant No. JP23jm0110019]. This study was partially supported by the Japanese Initiative for the Progress of Research on Infectious Diseases for Global Epidemics (J-PRIDE) (Grant No. JP18fm0208026), and Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) (Grant No. JP18fm0108008) of the Japan Agency for Medical Research and Development (AMED). This study was partially supported by the Japan Science and Technology Agency (Grant No. JPMJSP2119); the World-Leading Innovative and Smart Education Program (1801) of the Ministry of Education, Culture, Sports, Science, and Technology, Japan; and the Japan Society for the Promotion of Science KAKENHI (Grant No. JP23KJ0059). Competing interests The authors declare that they have no competing interests. Author contributions Conceptualization: TI, MO, TH, HK, and YS; methodology: TI, MO, and TH; formal analysis: TI, MO, TH, and KM; investigation: TI, MO, TH, JM, DK, and YS; data curation: MO, TH, and KM; and writing – original draft preparation: TI and MO. TH: writing –review and editing, all authors; supervision, MO, HK, and YS; funding acquisition, MO and HK. All the authors have read and agreed to the published version of the manuscript. Data availability statement All data are available within the manuscript. Ethics approval Animal experiments were approved by the Institutional Animal Care and Use Committee of the Faculty of Veterinary Medicine, Hokkaido University (approval numbers: 13-0108, 13-0162, 18-0037, 18-0040). All experiments involving genetically modified organisms, including mutant viruses generated by reverse genetics, were authorized by the Safety Committee on Genetic Recombination Experiments of Hokkaido University (approval numbers: 2015-019, 2020-009), and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (30-963, 2-654). This study was approved by the Biosafety Management Committee on Pathogens and Other Hazardous Agents of the Faculty of Veterinary Medicine at Hokkaido University (approval numbers: 2017-1-61, 2018-1-59, and 2019-1-62). 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Vaccine 26:2127–2134. 10.1016/j.vaccine.2008.02.001 Senne D, Panigrahy B, Kawaoka Y, Pearson J, Suss J, Lipkind M, Kida H, Webster R (1996) Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: Amino acid sequence at the HA cleavage site as a marker of pathogenicity potential. Avian Dis 40:425–437. 10.2307/1592241 Shichinohe S, Okamatsu M, Sakoda Y, Kida H (2013) Selection of H3 avian influenza viruses with SAα2,6Gal receptor specificity in pigs. Virology 444:404–408. 10.1016/j.virol.2013.07.007 Shtyrya Y, Mochalova L, Voznova G, Rudneva I, Shilov A, Kaverin N, Bovin N (2009) Adjustment of receptor-binding and neuraminidase substrate specificties in avian–human reassortant influenza viruses. Glycoconj J 26:99–109. 10.1007/s10719-008-9169-x Soda K, Asakura S, Okamatsu M, Sakoda Y, kida H (2011) H9N2 influenza virus acquires intravenous pathogenicity on the introduction of a pair of di-basic amino acid residues at the cleavage site of the hemagglutinin and consecutive passages in chickens. Virol J 8:64. 10.1186/1743-422X-8-64 Soda K, Sakoda Y, Isoda N, Kajihara M, Haraguchi Y, Shibuya H, Yoshida H, Sasaki T, Sakamoto R, Saijo K, Hagiwara J, Kida H (2008) Development of vaccine strains of H5 and H7 influenza viruses. Jpn J Vet Res 55:93–98 Sorrell EM, Song H, Pena L, Perez DR (2010) A 27-amino-acid deletion in the neuraminidase stalk supports replication of an avian H2N2 influenza A virus in the respiratory tract of chickens. J Virol 84:11831–11840. 10.1128/jvi.01460-10 Stech O, Veits J, Weber S, Deckers D, Schröer, Vahlenkamp TW, Breithaupt A, Teifke J, Mettenleiter TC, Stech J (2009) Acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus Is not sufficient for immediate transformation into a highly pathogenic strain. J Virol 83:5864–5868. 10.1128/jvi.02649-08 Tada T, Suzuki K, Sakurai Y, Kubo M, Okada H, Ito T, Tsukamoto K (2011) NP body domain and PB2 contribute to increased virulence of H5N1 highly pathogenic avian influenza viruses in chickens. J Virol 85:1834–1846. 10.1128/jvi.01648-10 Takadate Y, Tsunekuni R, Kumagai A, Mine J, Kikutani Y, Sakuma S, Miyazawa K, Uchida Y (2023) Different Infectivity and Transmissibility of H5N8 and H5N1 High Pathogenicity Avian Influenza Viruses Isolated from Chickens in Japan in the 2021/2022 Season. Viruses 15. 10.3390/v15020265 Veits J, Weber S, Stech O, Breithaupt A, Gräber M, Gohrbandt S, Bogs J, Hundt J, Teifke JP, Mettenleiter TC, Stech J (2012) Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype. Proceedings of the National Academy of Sciences 109:2579–2584. 10.1073/pnas.1109397109 Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiol Rev 56:152–179. 10.1128/mr.56.1.152-179.1992 Xu R, Zhu X, Ryan M, Nycholat CM, Yu W, Paulson JC, Wilson IA (2012) Functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the 2009 H1N1 influenza pandemic. J Virol 86:9221–9232. 10.1128/jvi.00697-12 Yamada S, Shinya K, Takada A, Ito T, Suzuki T, Suzuki Y, Le QM, Ebina M, Kasai N, Kida H, Horimoto T, Rivailler P, Chen LM, Donis RO, Kawaoka Y (2012) Adaptation of a Duck Influenza A Virus in Quail. J Virol 86:1411–1420. 10.1128/JVI.06100-11 Yamamoto N, Soda K, Sakoda Y, Okamatsu M, Kida H (2013) Proteins of duck influenza virus responsible for acquisition of pathogenicity in chickens. Virus Res 173:294–298. 10.1016/j.virusres.2013.02.001 Zhou H, Yu Z, Hu Y, Tu J, Zou W, Peng Y, Zhu J, Li Y, Zhang A, Yu Z, Ye Z, Chen H, Jin M (2009) The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus. PLoS ONE 4:e6277. 10.1371/journal.pone.0006277 Cite Share Download PDF Status: Published Journal Publication published 22 Sep, 2024 Read the published version in Archives of Virology → Version 1 posted Editorial decision: Minor Revision 18 Jun, 2024 Reviewers agreed at journal 31 Mar, 2024 Reviewers invited by journal 29 Mar, 2024 Editor assigned by journal 26 Mar, 2024 First submitted to journal 24 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4161114","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285504762,"identity":"c802ff85-a6a0-4b5b-aed6-43edd8e798cb","order_by":0,"name":"Takaya Ichikawa","email":"","orcid":"","institution":"Hokkaido University: Hokkaido Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Takaya","middleName":"","lastName":"Ichikawa","suffix":""},{"id":285504763,"identity":"862c1440-80ff-48b1-9854-1817dc4363e4","order_by":1,"name":"Takahiro Hiono","email":"","orcid":"","institution":"Hokkaido University: Hokkaido Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Takahiro","middleName":"","lastName":"Hiono","suffix":""},{"id":285504764,"identity":"42532202-cf22-4bfd-9064-2497e0ca9ffb","order_by":2,"name":"Masatoshi Okamatsu","email":"","orcid":"","institution":"Hokkaido University: Hokkaido Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Masatoshi","middleName":"","lastName":"Okamatsu","suffix":""},{"id":285504765,"identity":"729f35ef-1f33-40cb-8e43-8e9fababe03f","order_by":3,"name":"Junki Maruyama","email":"","orcid":"","institution":"The University of Texas Medical Branch at Galveston","correspondingAuthor":false,"prefix":"","firstName":"Junki","middleName":"","lastName":"Maruyama","suffix":""},{"id":285504766,"identity":"79f39717-7cdd-41e3-be98-a5f1bcef48e4","order_by":4,"name":"Daiki Kobayashi","email":"","orcid":"","institution":"Hokkaido University: Hokkaido Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Daiki","middleName":"","lastName":"Kobayashi","suffix":""},{"id":285504767,"identity":"2716781f-809d-4b8d-90be-c9f2c2e3e823","order_by":5,"name":"Keita Matsuno","email":"","orcid":"","institution":"Hokkaido University: Hokkaido Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Keita","middleName":"","lastName":"Matsuno","suffix":""},{"id":285504768,"identity":"78c3decf-bb39-4a41-a80d-c567a0135655","order_by":6,"name":"Hiroshi Kida","email":"","orcid":"","institution":"Hokkaido University: Hokkaido Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Kida","suffix":""},{"id":285504769,"identity":"b9eaaff8-3ed5-4bb9-a2ec-201f3b19c60f","order_by":7,"name":"Yoshihiro Sakoda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABG0lEQVRIiWNgGAWjYFACNgYGHgYbCJuH4QCDBAODAUMCmHsAn5Y0ME2SlsNoWvAB+fZjiQ/e1JxP7JfvBjIY7shJzkje+OEBg508A+NZrNYYnEk7bDjn2O3EmW28mw3nMDwzlpZIK5ZIYEg2bGA4l4BVC0N6mzQP221jg2O826SBLkycJ5FjANTCDFR+BqsL5fufA7X8O2dsf4x3+2+oFuMfCQz1OLUw3Eg7Js3bdkDOgI13GzNIy2yJHDOgLYdxajG48SzZcG5fspzEsdzNknMMDhtL9jwrs0gwOG7YhsMv8v1phg/efLPj4W8+u/HDm4rDchLHkzff/FFRLc8vgT3EMEIDwWCTOEOEDlTA30OyllEwCkbBKBiWAACFSGE1CQYPTgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-7021-1688","institution":"Hokkaido University","correspondingAuthor":true,"prefix":"","firstName":"Yoshihiro","middleName":"","lastName":"Sakoda","suffix":""}],"badges":[],"createdAt":"2024-03-25 06:31:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4161114/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4161114/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-024-06118-z","type":"published","date":"2024-09-22T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54010095,"identity":"4c55971f-8ce5-4cde-9854-0e6d191000e0","added_by":"auto","created_at":"2024-04-03 10:34:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24892,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of chickens intranasally inoculated with each virus.\u003c/p\u003e\n\u003cp\u003e(A) rgVac2sub-P0, rgVac2sub-P3, rgVac2sub-P3L4, and hemagglutinin/neuraminidase (HA/NA) reassortant viruses were generated using reverse genetics. Open boxes indicate gene segments derived from rgVac2sub-P0, shaded boxes indicate those from rgVac2sub-P3, and closed boxes indicate those from rgVac2sub-P3L4. (B) Six 4-week-old chickens were intranasally inoculated with 100 µL of each virus at 10\u003csup\u003e5.0\u003c/sup\u003e plague forming units (PFU) and observed for 14 days. ** indicates \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, * indicates \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 in the comparison of rgVac2sub-P3L4 using a log-rank test.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-4161114/v1/fac3a36f5bdb78ad7a1110d2.png"},{"id":54010097,"identity":"a4217102-7b95-4d1e-9967-e2907fd23f5a","added_by":"auto","created_at":"2024-04-03 10:34:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":151853,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the NA function of each virus.\u003c/p\u003e\n\u003cp\u003e(A) The positions of four substitutions were mapped on the 3D structure of the H7 HA (Protein Data Bank accession 1TI8). The HA was depicted in the soft surface model (left panel) and ribbon diagram (right panel). HA1 was colored blue, and HA2 was colored red in the surface model, whereas the protein Cα-chain was colored dim gray in the ribbon diagram. Mutations were colored lime in both models. (B), (C) The binding of rgVac2sub-P0, rgVac2sub-P3L4, and rgP0/HA-227G to the receptor was investigated using a solid-phase direct binding assay to a sialylglycopolymer containing 3′-Sialyllactose-PAA (3′-SL), SAcα2-3Galβ1-4Glc (B). The binding affinity constant (Ka) of each virus to 3′-SL was calculated and represented as the mean ±SEM of triplicate experiments. * indicates \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 in the comparison of rgVac2sub-P0 (C). (D), (E) Chicken embryonic fibroblasts (CEFs) were inoculated with rgVac2sub-P0, rgVac2sub-P3L4, rgP0/HA-227G, and rgP0/HA-338T at M.O.I of 0.01 at 35 °C (D) or 40 °C (E). The data represented as the mean ± SEM of triplicate experiments. a: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 between rgVac2sub-P0 and rgVac2sub-P3L4, b: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 between rgVac2sub-P0 and rgP0/HA-388T.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-4161114/v1/9cfa1e00ad8bbb5e9447f603.png"},{"id":54010563,"identity":"397fb920-4f55-4fa0-94a1-04d4287e6d33","added_by":"auto","created_at":"2024-04-03 10:42:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17534,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the NA function of each virus.\u003c/p\u003e\n\u003cp\u003e(A) The abilities of the NAs of rgVac2sub-P0 with long NA stalk and those of rgVac2sub-P3L4 and rgP0/NAΔ40–73 with short NA stalk were assessed using the erythrocyte elution assay. Viruses containing HA titers of 1:128 were incubated with chicken erythrocytes at 4 °C for 1 h and then incubated at 37 °C. The HA titers were monitored periodically for 10 h. (B) Sialidase activity of rgVac2sub-P0 (open circles) and rgVac2sub-P3L4 (closed circles) to MUNANA was measured. The initial reaction rate at each condition was calculated and plotted to obtain the Michaelis constant, Km. The data were represented as the mean ± SEM of three independent experiments.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-4161114/v1/eb364188c263c93f9a886a8e.png"},{"id":65103991,"identity":"0a9b87df-a82d-4088-b77b-f9a5048b3690","added_by":"auto","created_at":"2024-09-23 16:10:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1019190,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4161114/v1/f8e68e23-733c-4ff9-87db-47fd7c0e98a2.pdf"}],"financialInterests":"","formattedTitle":"Hemagglutinin and neuraminidase of an H7N7 non-pathogenic avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe natural hosts of influenza A viruses are wild waterbirds, particularly migratory ducks [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Previous studies revealed that duck influenza viruses transmit to chickens via domestic waterfowl or terrestrial birds [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. With multiple repeated infections in a chicken population, high pathogenicity avian influenza viruses (HPAIVs) can only be selected from H5 or H7 viruses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Hemagglutinin (HA) of the low pathogenicity avian influenza virus (LPAIV) is cleaved only by trypsin-like serine proteases expressed in the respiratory or intestinal epithelia, leading to mild or asymptomatic local infection. In contrast, the HA of HPAIV has polybasic amino acid residues at its cleavage site, which permit ubiquitous proteases such as furin and PC6 to cleave the precursor HA0 into HA1 and HA2 subunits, causing severe systemic infection in chickens [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The presence of polybasic amino acids is a prerequisite for the fatal pathogenicity in chickens. In contrast, previous investigations demonstrated that LPAIVs did not show high pathogenicity after the artificial introduction of a specific sequence at the HA cleavage site [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These results strongly suggested that additional viral factors are required for LPAIVs to acquire high pathogenicity in chickens. For example, a virus with three characteristics, filamentous form, high polymerase activity, and high stability of the matrix (M1) protein, was selected during the passage of a genetically modified H5N1 virus in chickens [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our previous study, a genetically modified H7N7 duck influenza virus was designed by artificially introducing the basic amino acid residue KRRRRR at the HA cleavage site of an H7N7 LPAIV, A/duck/Hokkaido/Vac-2/2004 (Vac2), namely the Vac2sub-P0 strain [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. After consecutive passages of Vac2sub-P0 in chicks, the passaged virus Vac2sub-P3 acquired high intravenous pathogenicity (Intravenous Pathogenicity Index: IVPI\u0026thinsp;=\u0026thinsp;2.54). According to the experimental infection of mutant viruses using site-directed mutagenesis and reverse genetics, amino acid substitutions at two positions, E227G and I388T, in the HA of Vac2sub-P3 are essential for its high pathogenicity in chickens. In contrast to intravenous infection, intranasal inoculation with Vac2sub-P3 resulted in limited viral replication in organs and mortality in chickens. Discrepancies may arise in pathogenicity between the intranasal and intravenous inoculation routes. In the present study, Vac2sub-P3, which showed high pathogenicity via intravenous inoculation but partial pathogenicity via intranasal inoculation, was serially passaged in the lungs of chickens to acquire higher intranasal pathogenicity. We obtained a virus with comparable pathogenicity to field isolates and characterized the virus to demonstrate functional changes in viral proteins during the acquisition of intravenous and intranasal pathogenicity in chickens.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eViruses\u003c/h2\u003e \u003cp\u003eThe viruses used in this study were obtained after three passages in chick air sacs (Vac2sub-P3) with the A/duck/Hokkaido/Vac-2/2004 (H7N7) strain, which introduced the amino acid residue KRRRRR at the HA cleavage site (rgVac2sub-P0), as described previously [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. All viruses were propagated in 10-day-old embryonated chicken eggs for 30\u0026ndash;48 h at 35\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCells\u003c/h2\u003e \u003cp\u003eMadin-Darby canine kidney (MDCK) cells were maintained in minimum essential medium (MEM, Nissui, Tokyo, Japan) supplemented with 0.3 mg/mL L-glutamine (FUJIFILM Wako Pure Chemical, Osaka, Japan), 5% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin G (Meiji Seika Pharma, Tokyo, Japan), 0.1 g/mL streptomycin (Meiji Seika Pharma), and 8.0 g/mL gentamicin (MSD, Rahway, NJ, USA). Chicken embryonic fibroblasts (CEFs) were prepared from 10-day-old chicken embryos and cultured in MEM supplemented with l-glutamine, fetal bovine serum, and antibiotics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePlaque assay\u003c/h2\u003e \u003cp\u003eTen-fold dilutions of viruses were inoculated onto confluent monolayers of MDCK cells and incubated at 35\u0026deg;C for 1 h. Unbound viruses were removed by washing cells with MEM. Cells were then overlaid with MEM containing 0.7% Bacto-agar (Becton Dickinson, Franklin Lakes, NJ, USA) in the presence of 5 \u0026micro;g/mL trypsin acetylated (Sigma-Aldrich, St. Louis, MO, USA). After 48 h of incubation at 35\u0026deg;C, cells were stained with 0.005% neutral red (Sigma-Aldrich).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eConsecutive passages of Vac2sub-P3 in chickens\u003c/h2\u003e \u003cp\u003eFour-week-old chickens (Boris Brown) free from antibodies against the H7 influenza virus were obtained from Hokkaido Chuo Shukeijou, Hokkaido, Japan. Three chickens were intranasally inoculated with 100 \u0026micro;L each of Vac2sub-P3. Three days post-inoculation (dpi), the chickens were sacrificed and their lungs were collected. A pooled 10% tissue suspension of the infected organs was serially passaged in three 4-week-old chickens via the intranasal route. After four passages, the isolate named Vac2sub-P3L4 (\u0026ldquo;L\u0026rdquo; indicates lungs) was obtained. Passaged viruses were propagated in the allantoic cavities of 10-day-old embryonated eggs for 48 h at 35\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExperimental infection of chickens with mutant viruses\u003c/h2\u003e \u003cp\u003eFour-week-old chickens were used to test the pathogenicity of the viruses used in this study. Six chickens were intranasally inoculated with 100 \u0026micro;L of allantoic fluid containing each virus at 10\u003csup\u003e5.0\u003c/sup\u003e plaque forming units (PFU) and observed for 14 days. Specific antibodies against homologous viruses were detected in the serum at 14 dpi using the hemagglutination inhibition (HI) test. To study viral replication, three more chickens were inoculated with each virus, as described above and euthanized at 3 dpi, after which tissue and blood were collected aseptically. To prepare a 10% suspension in MEM, tissue samples were homogenized using a multi-bead shocker (Yasui Kikai, Osaka, Japan). Virus titers of these suspensions were calculated using a plaque assay and expressed as PFU/g of tissue or mL of blood. All experiments were performed in self-containing isolator units (Tokiwa Kagaku, Tokyo, Japan) in a biosafety level 3 (BSL3) facility at the Faculty of Veterinary Medicine, Hokkaido University, Japan.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReverse genetics\u003c/h2\u003e \u003cp\u003eMutations were introduced into specific regions of the plasmid-encoded PA, HA, NA, M, and NS genes of Vac2sub-P3 using a QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA), as described previously [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The mutant viruses listed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA were rescued employing reverse genetics [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and all eight segments of the genome were sequenced to confirm that the desired mutations were induced and the undesired ones were eliminated. Co-cultures of human embryonic kidney 293T and MDCK cells were transfected with plasmids using 2 mg/mL polyethylenimine (Polysciences, Warrington, PA, USA). At 48 h post transfection, the culture supernatant was collected and propagated in 10-day-old embryonated chicken eggs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eThree-dimensional (3D) structural-based mapping of the HA protein\u003c/h2\u003e \u003cp\u003eMutations were mapped to the H7 HA protein (Protein Data Bank accession 1TI8 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]) using the UCSF Chimera X version 1.2 (University of California, San Francisco, CA, USA). Monomeric HA is depicted as a soft-surface model and ribbon diagram.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eVirus growth in cell culture\u003c/h2\u003e \u003cp\u003eViruses at a multiplicity of infection (M.O.I.) of 0.01 were inoculated onto confluent monolayers of CEF cells and incubated at 35\u0026deg;C for 1 h. Unbound viruses were removed by washing the cells with MEM, and the cells were overlaid with MEM in the presence of trypsin accetylated (5 \u0026micro;g/mL). The plates were incubated at 35 or 40\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. At various times post-infection, the viral titers in the cell culture supernatant were determined using a plaque assay with MDCK cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSolid-phase direct binding assay\u003c/h2\u003e \u003cp\u003eReceptor binding affinity of the viruses was assessed using a solid-phase direct binding assay with sialylglycopolymers, 3\u0026prime;-Sialyllactose-PAA (3\u0026prime;-SL) (Cosmo Bio, Tokyo, Japan), as described previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 3\u0026prime;-SL was added to each well of a Universal-BIND\u0026trade;, 96 well polystyrene stripwell microplate (Corning, Corning, NY, USA). After the wells were blocked with 2% bovine serum albumin, a solution containing influenza viruses (32 HA unit) was added and incubated at 4\u0026deg;C for 16 h. Mouse anti-H7 HA monoclonal antibody 253/1 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and goat anti-mouse IgG-HRP conjugate (Bio-Rad, Hercules, CA, USA) were used to detect the viruses. 3,3\u0026prime;-tetramethylbenzidine (TMB) and 0.04% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were added as substrates and the absorbance was measured at 450/630 using a Model 680 Microplate Reader (Bio-Rad).\u003c/p\u003e \u003cp\u003eThe affinity constant for each virus was calculated as described previously [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Blank-corrected data were plotted and fitted to an established binding model using R version 3.0.1 (The R Foundation for Statistical Computing, Vienna, Austria). The formula for the model is as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{a}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}=\\frac{{A}_{max}\\times C}{Kd+C}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere C indicates the concentration of 3\u0026prime;-SL; Amax indicates the maximum of binding absorbance; and Kd is the dissociation constant. The affinity constant (Ka) determines the reciprocal of Kd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eErythrocyte elution assay\u003c/h2\u003e \u003cp\u003eThe elution ability of NA from chicken erythrocytes was determined as previously described [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Briefly, viruses containing 128 HA units were serially diluted in calcium saline buffer (6.8 mM CaCl\u003csub\u003e2\u003c/sub\u003e \u0026minus;\u0026thinsp;154 mM NaCl in 20 mM borate buffer, pH7.2) and incubated with 50 \u0026micro;L of 0.5% chicken erythrocytes in microtiter plates at 4\u0026deg;C for 1 h. The plates were then stored at 37\u0026deg;C, and the reduction of HA titers was monitored periodically for 10 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNA enzymatic activity\u003c/h2\u003e \u003cp\u003eNA enzymatic activity was measured using 2\u0026prime;-(4-Methylumbelliferyl)\u0026ndash;α\u0026ndash;D\u0026ndash;\u003cem\u003eN\u003c/em\u003e-acetylneuraminic acid (MUNANA; Sigma-Aldrich) as a soluble substrate, as described previously [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Briefly, the viruses were mixed with serially diluted MUNANA in 96-well black-bottomed plates. The mixture was exerted at 360 nm at 1-min intervals, and the emission signal was periodically monitored for 30 min at 37\u0026deg;C using POWER SCAN 4 (Agilent Technologies). The initial reaction rate under each condition was calculated and plotted to obtain the Michaelis constant, Km, using R software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e Animal experiments were approved by the Institutional Animal Care and Use Committee of the Faculty of Veterinary Medicine, Hokkaido University (approval numbers: 13\u0026ndash;0108, 13\u0026ndash;0162, 18\u0026ndash;0037, 18\u0026ndash;0040). All experiments involving genetically modified organisms, including mutant viruses generated by reverse genetics, were authorized by the Safety Committee on Genetic Recombination Experiments of Hokkaido University (approval numbers: 2015-019, 2020-009), and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (30\u0026ndash;963, 2-654). This study was approved by the Biosafety Management Committee on Pathogens and Other Hazardous Agents of the Faculty of Veterinary Medicine at Hokkaido University (approval numbers: 2017-1-61, 2018-1-59, and 2019-1-62).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAmino acid changes of the viruses during consecutive passages of Vac2sub-P3 in chickens\u003c/h2\u003e \u003cp\u003eThe Vac2sub-P3 virus was obtained from a previous study and used for additional consecutive passages. Each isolate obtained from lungs was tested for pathogenicity. After four passages, the resultant virus Vac2sub-P3L4 showed high intranasal pathogenicity in chickens; all six chickens intranasally inoculated with the virus died within 5 days. The sequences of the passaged viruses were compared to identify amino acid substitutions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Seven amino acid substitutions (S409I in PA, T32I and R65K in HA, L10S in NA, R101K in M1, D44G in M2, and E229K in NS1) and a deletion (at positions 40\u0026ndash;73 in NA) were observed between Vac2sub-P3 and Vac2sub-P3L4. Among the six substitutions (K123E in PB2, N16D in PB1, E227G and I388T in HA, G228R in M1, and L46P in M2) between Vac2sub-P0 and Vac2sub-P3, G228R in M1 and L46P in M2 were reverted during additional passages to Vac2sub-P3L4.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAmino acid changes during consecutive passages of Vac2sub-P0\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"23\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c18\" colnum=\"18\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c19\" colnum=\"19\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c20\" colnum=\"20\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c21\" colnum=\"21\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c22\" colnum=\"22\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c23\" colnum=\"23\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eVirus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePB2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePB1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c12\" namest=\"c9\"\u003e \u003cp\u003eHA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003eM1\u003c/p\u003e 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\u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e227\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e388\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003e40\u0026ndash;73\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c17\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c18\"\u003e \u003cp\u003e228\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c20\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c21\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c22\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c23\"\u003e \u003cp\u003e229\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVac2sub-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lowast;\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eཥ\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lowast;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP3L1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eΔ\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eD/G\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP3L2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eΔ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP3L3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eT/I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eΔ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP3L4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eΔ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003eཥ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"23\"\u003e\u003csup\u003ea\u003c/sup\u003e Methionine encoded by the AUG start codon is defined as position 1\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"23\"\u003e\u003csup\u003eb\u003c/sup\u003e Asterisk indicates amino acids sequence GPKQKENLTCTTINQNNTTVVENTYVNNTTIITK\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"23\"\u003e\u003csup\u003ec\u003c/sup\u003e Periods indicate same amino acids as the parent virus\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"23\"\u003e\u003csup\u003ed\u003c/sup\u003e Delta (Δ) indicates the deletion of 40 to 73 amino acid residues in NA\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"23\"\u003e\u003csup\u003ee\u003c/sup\u003e Amino acid quasispecies were observed\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"23\"\u003eAmino acid diffenrence between Vac2sub-P0 and Vac2sub-P3 were already published by Maruyama et al., [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIntranasal pathogenicity of rgVac2sub-P0, rgVac2sub-P3, rgVac2sub-P3L4, and mutant viruses in chickens\u003c/h2\u003e \u003cp\u003eTo investigate the contribution of each mutation to the high intranasal pathogenicity of the passaged virus, we generated recombinant viruses using reverse genetics and compared their pathogenicity with that of Vac2sub-P0, Vac2sub-P3, and Vac2sub-P3L4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistent with a previous report [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], all the chickens intranasally inoculated with rgVac2sub-P0 lived. Two of the six chickens inoculated with rgVac2sub-P3 died during the observation period. All chickens inoculated with rgVac2sub-P3L4 died within 4 days. A previous study demonstrated that two mutations in HA, E227G and I388T, are critical for intravenous pathogenicity acquisition by Vac2sub-P3 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Accordingly, we first focused on the contribution of these mutations to the HA segments. The virus rgP3L4/P0-HA, which has a Vac2sub-P0 HA segment with a Vac2sub-P3L4 backbone, did not kill any of the inoculated chickens. These results further highlight the importance of the four HA mutations for acquiring pathogenicity throughout consecutive passages. Moreover, infection with rgP3L4-P3HA showed significantly delayed mortality compared to infection with rgVac2sub-P3L4, suggesting that two additional mutations, T32I and R65K, in HA also contribute to the acquisition of intranasal pathogenicity.\u003c/p\u003e \u003cp\u003eThe functional balance between HA and NA is important for viral replication in cultured cells and pathogenesis in animals [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, we focused on mutations observed in NA. For this purpose, rgP3L4/P0-NA, harboring the NA of Vac2sub-P0 with a Vac2sub-P3L4 backbone, was generated. Similar to rgP3L4/P3-HA, infection with rgP3L4/P0-NA showed significantly delayed mortality compared with infection with rgVac2sub-P3L4. However, infection with rgP0/P3L4-HA,NA, which possesses HA and NA derived from Vac2sub-P3L4 with the Vac2sub-P0 backbone, resulted in a 2/3 mortality rate in infected chickens, indicating a significant contribution of mutations in other gene segments.\u003c/p\u003e \u003cp\u003eTo evaluate viral growth in chickens, we euthanized the infected chickens at 3 dpi and collected organ and blood samples to determine the virus titer (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). No viruses were detected in chickens inoculated with rgVac2sub-P0 or rgP3L4/P0-HA, whereas rgVac2sub-P3L4 was systemically recovered from chickens at high titers. Viral titers recovered from chickens inoculated with either rgP3L4/P3-HA or rgP3L4/P0NA were lower than those recovered from chickens inoculated with rgVac2sub-P3L4. No virus was recovered from one of three chickens inoculated with rgP0/P3L4-HA or NA, which was consistent with their mortality rates and delayed mortality in chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVirus recovery from the chickens intranasally inoculated with each virus on 3 dpi\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVirus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChicken ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c9\" namest=\"c3\"\u003e \u003cp\u003eVirus recovery (log PFU/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrachea\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLung\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eKidney\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eColon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eBlood\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ergVac2sub-P0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ergVac2sub-P3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ergVac2sub-P3L4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ergP3L4/P0-HA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ergP3L4/P3-HA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ergP3L4/P0-NA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ergP0/P3L4-HA,NA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003ea\u003c/sup\u003e log PFU/ml\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003csup\u003eb\u003c/sup\u003e \u0026lt; 2.0 (\u0026lt;\u0026thinsp;1.3 for blood samples)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypical analyses of the HA of passaged viruses\u003c/h2\u003e \u003cp\u003eTh\u003cem\u003ee in vivo\u003c/em\u003e analyses suggested that functional changes in HA and NA are crucial for acquiring intranasal pathogenicity of passaged viruses in chickens. First, we visualized the mutations observed in the 3D structure of HA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Amino acid position 227 is located in the peripheral region of the receptor binding site. Position 388 is located in the alpha helix of the HA2 subunit. Position 32 was located in the N-terminal region of the HA1 subunit and near the HA1/2 cleavage site in the 3D structure. Position 65 is located in the hinge domain between the globular head and stem of the HA1 subunit.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, mutations at positions 227 and 388 were observed during the acquisition of intravenous pathogenicity in chickens in our previous study [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Position 227 faces opposite to the receptor-binding pocket. Accordingly, we hypothesized that this mutation affects receptor-binding avidity. Then, binding analyses of rgVac2sub-P0, rgVac2sub-P3L4, and rgP0/HA-227G with 3\u0026prime;-SL were conducted to calculate the affinity constant (Ka) of each virus with the receptor analog. 3\u0026prime;-SL is a sialylglycopolymer terminating with sialic acid α2,3 galactose and a model of the receptor for avian influenza viruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The Ka values of rgVac2sub-P3L4 and rgP0/HA-227G were significantly lower than those of rgVac2sub-P0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This result indicated that receptor-binding avidity was attenuated by the HA-E227G mutation.\u003c/p\u003e \u003cp\u003eMutations in the helix of HA2 stem affect the thermostability of HA molecule [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The growth of these viruses at different temperatures was assessed using CEFs. Although few differences were observed between the viruses at 35\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), the virus titer of rgVac2sub-P3L4 and rgP0/HA-388T were significantly higher than that of rgVac2sub-P0 and rgP0/HA-227G at 40\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The present results indicate that viruses with HA-388T have a higher capacity to grow at high temperatures, such as in the chicken body.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypical analyses of the NA of passaged viruses\u003c/h2\u003e \u003cp\u003eHA and NA functionally coevolve during viral adaptation to novel host species. The deletion of the NA stalk domain is among the most well-characterized changes in molecular coevolution. To examine the effects of NA stalk deletion on its function, the elution abilities from chicken erythrocytes of rgVac2sub-P0, rgVac2sub-P3L4 and rgP0/NAΔ40-73 (the mutant virus with the 34 amino acid truncation at the NA stalk with Vac2sub-P0 backbone) were assessed using an erythrocyte elution assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The rgVac2sub-P0 virus was eluted over time, whereas rgVac2sub-P3L4 and rgP0/NAΔ40-73 with shortened NA stalks, showed little or no elution. In addition, the sialidase activities of rgVac2sub-P0 and rgVac2sub-P3L4 were tested in a standard assay using MUNANA as a soluble substrate. As expected, no significant difference was observed in sialidase activity between these viruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating that the NA stalk deletion did not affect the removal of sialic acid from a soluble substrate [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe sequence of polybasic amino acids at the HA cleavage site permits ubiquitous proteases to cleave the HA, which is not sufficient but necessary for inducing severe systemic infection [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Some viral factors are also required for pathogenicity in chickens [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition, this study critically demonstrated that additional adaptations were required for the virus with high pathogenicity in chickens via intravenous administration for acquiring high pathogenicity via intranasal administration. In the present study, we investigated the characteristics of passaged H7N7 avian influenza viruses in a laboratory setting to determine the key factors responsible for their high intranasal pathogenicity.\u003c/p\u003e \u003cp\u003eReceptor binding analysis demonstrated that viruses with HA-E227G showed lower glycan-binding avidity. Our previous study showed that this mutation, located near the receptor-binding domain, contributes critically to the acquisition of intravenous pathogenicity in chickens [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Soon after the additional passage of rgVac2sub-P3 into the respiratory tract of chickens, a 34 amino acid deletion in the NA stalk was observed between Vac2sub-P3 and Vac2sub-P3L1. Such deletions are often detected in avian influenza viruses isolated from terrestrial poultry, such as chickens and quails, but not in aquatic birds [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. NA stalk deletion facilitates the adaptation of avian influenza viruses to terrestrial poultry [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and increases their pathogenicity in the hosts [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Given that the virus used in this study was derived from the duck influenza virus [a reassortant strain between A/duck/Mongolia/736/2002 (H7N7) and A/duck/Hokkaido/49/1998 (H9N2)] [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], the NA stalk deletion following the present passages is consistent with these previous findings. Viruses with shortened NA stalks, such as rgVac2sub-P3L4 and rgP0/NAΔ40-73, showed little or no elution from chicken erythrocytes, in contrast to rgVac2sub-P0, indicating that the release activity from infected cells of the passaged virus was lower than that from the parent virus. Several studies have suggested that the balance between HA and NA functions is important for viral replication in cultured cells and experimental animals [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccordingly, this selection might occur to adjust its function to optimize a balance with the HA function, which decreases the receptor-binding affinity during previous passages by Vac2sub-P3. However, it remains unclear how the shortened NA stalk region influences the adaptation of aquatic birds to terrestrial poultry. We hypothesized that the difference in tissue tropism between water birds and terrestrial poultry could be important; viruses infect and replicate in the colon of water birds and in the respiratory tract of terrestrial poultry. The basis of this hypothesis is that no NA stalk deletion was observed during other consecutive passages of Vac2sub-P3, in which the brain was used as a pooled sample in parallel (data not shown) and that viruses with shortened NA stalks replicated efficiently in the respiratory tract of chickens [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Viruses with low binding affinity and release activity may have some advantages in replicating the ciliated epithelial cells of the respiratory tract.\u003c/p\u003e \u003cp\u003eThe ancestral strain of H5 HPAIVs, A/goose/Guangdong/1/1996 (H5N1) (Gs/GD), had no deletion in its NA stalk domain, whereas the N1 NA of subsequent Gs/GD-like strains had 19 or 20 amino acid deletions in the stalk domain after the outbreak in Hong Kong in 1997. Later, descendants of the Gs/GD strain belonging to the HA genetic clade 2.3.4.4, underwent reassortment with viruses with various NA subtypes, resulting in the generation of H5Nx viruses. Among these, the N6 NA of H5N6 viruses has an 11 amino acid deletion in its stalk region, whereas no deletion has been observed in the N8 NA of H5N8 viruses. Furthermore, contemporary H5N1 viruses belonging to the HA genetic clade 2.3.4.4b obtained N1 NA without deletion in the NA-stalk domain by reassortment with non-pathogenic avian influenza viruses circulating among wild birds. Interestingly, these viruses show lethal pathogenicity to chickens in field settings, although there are strain-by-strain differences in their pathogenicity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This suggests that HPAIVs are highly pathogenic in field settings without deletions at the NA stalk. In contrast, H5Nx viruses show altered receptor-binding specificity; these viruses recognize fucosylated receptors in addition to non-modified receptors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The changes in the receptor-binding specificity of these viruses may be related to their potential to accept various NA with or without stalk deletion.\u003c/p\u003e \u003cp\u003eViruses harboring HA-I388T (rgVac2sub-P3L4 and rgP0/HA-388T) showed significantly higher growth and thermostability in CEFs. Mutations in the stem region of HA are often observed during experimental adaptation to influenza viruses, and these mutations are associated with a prior mutation in the (vicinity of) receptor-binding domain. The most well-known mutation is the T318I substitution in HA; this mutation was observed during the selection of airborne transmissible HPAIV in ferrets and increased the acid stability of HA [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It remains unclear whether these variants are selected to compensate for the instability of HA caused by the mutation in the receptor-binding domain or the phenotypic changes caused by the HA stem mutation are essential for adaptation. In addition, amino acid T32 comprises an \u003cem\u003eN\u003c/em\u003e-glycosylation sequon, and the mutation T32I, which was observed after P3L3, abrogated the glycosylation at N30. Nevertheless, we did not obtain critical evidence to support glycosylation at N30 in P0-HA or non-glycosylation in P3L4-HA (data not shown). Perhaps, minor changes in glycan occupancy at this site may have contributed to the further adaptation of Vac2sub-P3L4 from Vac2sub-P3.\u003c/p\u003e \u003cp\u003eIn conclusion, the present study revealed that HA and NA underwent functional coevolution during the acquisition of high intranasal pathogenicity in chickens. Phenotypic changes in these proteins, along with polybasic amino acid residues at the HA cleavage site, are critical for this process. We installed polybasic amino acid residues at the HA cleavage sites to accelerate the adaptation process in a laboratory, and consecutive passages of the virus enhanced its growth capacity in chickens. The processes are vice versa; in the natural setting, the virus first acquires the ability to grow and be transmitted efficiently in chickens, and then the virus acquires the polybasic amino acid residues to the HA cleavage sites during the multiple transmission events in chicken flocks. Thus, further studies are needed to elucidate the definitive factors responsible for the difference between high pathogenicity of HPAIVs in chickens caused by intravenous and intranasal inoculations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Ms. Yuka Sato and Ms. Mayumi Endo for their technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Japan Initiative for World-Leading Vaccine Research and Development Centers [Grant No. JP233fa627005]. This study was partially funded by the Japan International Cooperation Agency within the framework of the Science and Technology Research Partnership for Sustainable Development [Grant No. JP23jm0110019]. This study was partially supported by the Japanese Initiative for the Progress of Research on Infectious Diseases for Global Epidemics (J-PRIDE) (Grant No. JP18fm0208026), and Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) (Grant No. JP18fm0108008) of the Japan Agency for Medical Research and Development (AMED). This study was partially supported by the Japan Science and Technology Agency (Grant No. JPMJSP2119); the World-Leading Innovative and Smart Education Program (1801) of the Ministry of Education, Culture, Sports, Science, and Technology, Japan; and the Japan Society for the Promotion of Science KAKENHI (Grant No. JP23KJ0059).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: TI, MO, TH, HK, and YS; methodology: TI, MO, and TH; formal analysis: TI, MO, TH, and KM; investigation: TI, MO, TH, JM, DK, and YS; data curation: MO, TH, and KM; and writing \u0026ndash; original draft preparation: TI and MO. TH: writing \u0026ndash;review and editing, all authors; supervision, MO, HK, and YS; funding acquisition, MO and HK. All the authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were approved by the Institutional Animal Care and Use Committee of the Faculty of Veterinary Medicine, Hokkaido University (approval numbers: 13-0108, 13-0162, 18-0037, 18-0040). All experiments involving genetically modified organisms, including mutant viruses generated by reverse genetics, were authorized by the Safety Committee on Genetic Recombination Experiments of Hokkaido University (approval numbers: 2015-019, 2020-009), and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (30-963, 2-654). This study was approved by the Biosafety Management Committee on Pathogens and Other Hazardous Agents of the Faculty of Veterinary Medicine at Hokkaido University (approval numbers: 2017-1-61, 2018-1-59, and 2019-1-62).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBanks J, Speidel ES, Moore E, Plowright L, Piccirillo A, Capua I, Cordioli P, Fioretti A, Alexander DJ (2001) Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1 avian influenza viruses in Italy. 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PLoS ONE 4:e6277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0006277\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0006277\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"High pathogenicity avian influenza virus, Intranasal pathogenicity, Chicken, Hemagglutinin, Neuraminidase","lastPublishedDoi":"10.21203/rs.3.rs-4161114/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4161114/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named Vac2sub-P0, which is non-pathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs and a virus with high intravenous pathogenicity, namely, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 is only partially lethal in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the passaged virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages are responsible for increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with the parental virus in vitro; the virus growth at 40\u0026deg;C was higher, the binding affinity to a sialic acid receptor was lower, and the release activity by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study critically demonstrated that additional adaptations were required for the highly pathogenic virus via intravenous administration in chickens for increased pathogenicity via intranasal administration.\u003c/p\u003e","manuscriptTitle":"Hemagglutinin and neuraminidase of an H7N7 non-pathogenic avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-03 10:33:58","doi":"10.21203/rs.3.rs-4161114/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2024-06-18T17:36:29+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-01T00:06:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-29T21:48:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-26T07:36:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2024-03-25T02:31:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bca6064c-2b09-4e3c-9529-516753797f24","owner":[],"postedDate":"April 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:02:06+00:00","versionOfRecord":{"articleIdentity":"rs-4161114","link":"https://doi.org/10.1007/s00705-024-06118-z","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2024-09-22 15:57:35","publishedOnDateReadable":"September 22nd, 2024"},"versionCreatedAt":"2024-04-03 10:33:58","video":"","vorDoi":"10.1007/s00705-024-06118-z","vorDoiUrl":"https://doi.org/10.1007/s00705-024-06118-z","workflowStages":[]},"version":"v1","identity":"rs-4161114","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4161114","identity":"rs-4161114","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}