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• W4367333919 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Influenza A viruses in animal reservoirs repeatedly cross species barriers to infect humans. Dogs are the closest companion animals to humans, but the role of dogs in the ecology of influenza viruses is unclear. H3N2 avian influenza viruses were transmitted to dogs around 2006 and have formed stable lineages. The long-term epidemic of avian-origin H3N2 virus in canines offers the best models to investigate the effect of dogs on the evolution of influenza viruses. Here, we carried out a systematic and comparative identification of the biological characteristics of H3N2 canine influenza viruses (CIVs) isolated worldwide over 10 years. We found that, during adaptation in dogs, H3N2 CIVs became able to recognize the human-like SAα2,6-Gal receptor, showed gradually increased hemagglutination (HA) acid stability and replication ability in human airway epithelial cells, and acquired a 100% transmission rate via respiratory droplets in a ferret model. We also found that human populations lack immunity to H3N2 CIVs, and even preexisting immunity derived from the present human seasonal influenza viruses cannot provide protection against H3N2 CIVs. Our results showed that canines may serve as intermediates for the adaptation of avian influenza viruses to humans. Continuous surveillance coordinated with risk assessment for CIVs is necessary. Editor's evaluation This paper focuses on the avian H3N2 influenza virus that has recently started infecting and spreading between dogs. Using exhaustive and impressive experimental approaches, the authors demonstrate how this virus is adapting to dogs over time, gaining more and more properties consistent with robust infection of mammals. This paper is destined to become part of the canon on emerging viruses. https://doi.org/10.7554/eLife.83470.sa0 Decision letter Reviews on Sciety eLife's review process Introduction In the 21st century, newly emerging viruses, such as influenza A H7N9, Ebola virus, Zika virus, and the SARS-CoV-2 virus, are posing serious challenges to healthcare systems (Jacob et al., 2020; Imai et al., 2017; Yakob and Walker, 2016; Thakur and Ratho, 2022). These challenges are constant reminders to the scientific community to pay attention to emerging animal-borne zoonotic diseases. Influenza A viruses have a relatively broad host range (Long et al., 2019). When animal-borne viruses with different antigenicities acquire human-human aerosol transmission abilities, they become epidemic in the population. The four human pandemic viruses in recorded history underwent avian or swine influenza virus gene reassortment with human influenza virus or acquired human adaptive mutations (Vijaykrishna et al., 2010). Animal-borne virus adaption to a mammalian intermediate host is an important way that they are able to establish infections in humans (Parrish et al., 2015). Swine are considered a typical intermediate host; for example, the Eurasian avian-origin lineage of H1 subtype swine influenza viruses that originated in European swine in the 1970s gradually accumulated amino-acid mutations related to human adaptation and gained increased infectivity in humans (Mena et al., 2016; Brown, 2013). However, the role of other mammals in viral ecology is still unclear. Similar to that of pigs, the canine respiratory tract contains both types of sialic acid receptors used by influenza viruses (α2,3- and α2,6-linked) (Wasik et al., 2017; Ning et al., 2012). Dogs are susceptible to natural influenza virus infections caused by transmission from avian (H3N2 and H5N1), equine (H3N8), or human (pdmH1N1 and H3N2) virus reservoirs (Lin et al., 2012a; Crawford et al., 2005; Song et al., 2008). Several reassortment events in influenza viruses have occurred from different host sources in dogs (Lee et al., 2016b; Voorhees et al., 2017), such as reassortment between canine H3N2 and human H1N1 viruses (Song et al., 2012; Moon et al., 2015) and canine H3N2 and swine influenza viruses (Eichorst et al., 2018). Dogs are important companion animals, and once a new zoonotic disease appears in dogs, there is a high chance to infect humans. However, whether dogs can act as intermediate hosts to produce zoonotic influenza viruses is not established. Although dogs have been found infected with multiple influenza viruses, only equine-origin H3N8 and avian-origin H3N2 viruses have established lineages in dogs (Hayward et al., 2010; Anderson et al., 2012; Zhu et al., 2015; Lyu et al., 2019). Compared with H3N8 CIV, H3N2 CIV has a broader host range, infecting multiple mammalian animals, including ferrets, guinea pigs, mice, and cats (Lee et al., 2013, Lyoo et al., 2015; Jeoung et al., 2013; Song et al., 2011). H3N2 CIV was first isolated in 2006 from Guangdong Province in China, and was found to be genetically most closely related to the H3N2 avian influenza viruses prevalent in aquatic birds in South Korea for all eight gene segments (Li et al., 2010; Su et al., 2012). Since then, H3N2 CIV has been prevalent in China (Sun et al., 2013; Yang et al., 2014; Lin et al., 2012b; Wu et al., 2021) and South Korea (Lee et al., 2016a), and has circulated in the United States since 2015 (Voorhees et al., 2017; Voorhees et al., 2018; Martinez-Sobrido et al., 2020; Dalziel et al., 2014). The long-term epidemic of avian-origin H3N2 virus in canines offers the best opportunities to investigate the potential role of dogs in the ecology of influenza A viruses. In the present study, we thus systematically investigated the evolution of genetic and biological properties of this avian-origin virus during its circulation in dogs. We found that during the adaptation of H3N2 CIVs to dogs, H3N2 CIVs became able to recognize the human-like SAα2, 6Gal receptor, showed gradually increased HA acid stability and replication ability in human airway epithelial cells, and had a 100% transmission rate via respiratory droplets in a ferret mode. Our results revealed that dogs might serve as potential intermediate hosts for animal influenza viruses’ adaption to humans. Results Continued genetic evolution of avian-origin H3N2 CIVs in dogs From 2012 to 2019, we collected tracheal swab samples from 4174 dogs with signs of respiratory disease from animal hospitals and kennels in nine provinces or municipalities of China (Figure 1—figure supplement 1). A total of 235 samples (5.63%) were positive for H3N2 infection. The mean positive rates for each year increased from 1.98% in 2012 to 10.85% in 2019 (Figure 1—figure supplement 2), with a sharp increase after 2016. According to isolation time and location, 117 representative viruses were selected for full genome sequencing, including 51 strains isolated from 2012–2017 that were previously uploaded to the GenBank database by our laboratory (Lyu et al., 2019). The whole genomes of these viruses were analyzed along with all complete H3N2 CIV genomes publicly available in GenBank and the GISAID database (https://www.gisaid.org/) (n=229), and we constructed the maximum-likelihood phylogenic trees of eight viral gene segments (Figure 1—figure supplements 3–10). The inferred trees for all genomic segments exhibited a similar topology; thus, we grouped the viruses into six clades (clades 0–5) according to HA phylogeny, with several viruses consistently clustering together with high posterior probability values (bootstrap values ≥70). Clade 0 contains viruses isolated in China from 2006 to 2007, and clade 1 contains isolates exclusively from South Korea from 2007 and 2012. Clade 2 represents viruses isolated in China from 2009 to 2016, Thailand in 2012, and the United States in 2017; and clade 3 contains viruses collected in South Korea from 2012 and 2013. Clade 4 contains strains isolated in the United States from 2015 to 2017 and South Korea in 2015, and clade 5 encompasses isolates from China from 2016 to 2019 and the United States from 2017 to 2018. Most H3N2 CIVs after 2019 isolated in China have formed a further subclade 5.1 (Figure 1A). Comparing sequences of H3N2 CIVs with human influenza viruses and ancestral avian influenza viruses showed that, compared with ancestral avian influenza viruses, H3N2 CIVs that were initially introduced to dogs possessed several substitutions identical to human influenza viruses with high frequencies (>90%). A noteworthy observation was the number of human-like amino-acid substitutions that had gradually accumulated during the evolution of H3N2 CIVs in dogs and increased significantly after 2016 (Figure 1B). These results indicated that H3N2 CIVs may have increased their adaptability to humans during their evolution in dogs. Figure 1 with 10 supplements see all Download asset Open asset Genetic and antigenic characterization of H3N2 CIVs. (A) Maximum-likelihood phylogenetic tree of hemagglutinin (HA) genomic segment of H3N2 CIVs. The phylogenetic tree of the HA gene was estimated using genetic distances calculated by maximum likelihood under the GTRGAMMA +I model. Viruses with full names in the tree in (A) were selected for animal experiments. Black, red, dark purple, and aqua blue indicate H3N2 CIVs from China, the United States, Thailand, and South Korea, respectively. A, B, C, D, E, F, and G represent different antigen groups of H3N2 CIVs, respectively. A full detailed HA gene tree with a consistent topology is shown in Figure 1—figure supplement 3 (scale bar is in units of nucleotide substitutions per site). (B) Prevalence of mammalian adaption markers among H3N2 CIVs. The sequences of H3N2 CIVs available in NCBI have compared with avian and human influenza A viruses. Color indicates the frequency of indicated substitutions in H3N2 CIVs for each indicated time period. (C) Antigenic map based on the HI assay data. Open squares and filled circles represent the positions of antisera and viruses, respectively. A k-means clustering algorithm identified clusters. Strains belonging to the same antigenic cluster are encircled with an oval. The vertical and horizontal axes both represent antigenic distance. The spacing between grid lines is 1 unit of antigenic distance, corresponding to a twofold dilution of antiserum in the HI assay. Details of the hemagglutinin inhibition (HI) assay data are shown in Supplementary file 1. Humans lack immunity to H3N2 CIVs We performed an antigenicity test for representative H3N2 CIVs from different clades. Supplementary file 1 and Figure 1C show that H3N2 CIVs continuously occurred antigenic changes worldwide. The cross-reactive titers between different antigenic groups were greater than or equal to fourfold lower than those of homologous reactions. The reaction patterns of clade 0 and clade 1 were similar and belonged to antigenic group A. Some clade 2 viruses were in antigenic group B, while other clade 2 viruses belonged to antigenic group C. The antigenicity of clade 3 and clade 4, which belong to antigenic group D, is different from other clades. Clade 5 included viruses belonging to antigenic groups E, F, or G. The co-circulation of different antigenic group viruses in recent years increased the difficulty of preventing and controlling canine influenza viruses. Additionally, we found that no H3N2 CIV was recognized by antisera to H3N2 human seasonal influenza virus in the hemagglutinin inhibition (HI) and neuraminidase inhibition (NI) assays (Supplementary file 1 and Supplementary file 2). To further investigate whether humans have existing immune protection against H3N2 CIVs, we tested sera collected from children (≤10 year old, n=100), adults (25–53 year old, n=100), and elderly adults (≥60 year old, n=100) against four viruses (BJ/1230/16, human seasonal H3N2 influenza; Cn/BJ/38/16, group C; Cn/FJ/1109/18, group D and Cn/GZ/011/19, group E) for HI, NI, and microneutralization (MNT) antibodies, as described previously (Potter and Oxford, 1979; Rowe et al., 1999; Sandbulte et al., 2009). We found that 15.0%, 1.0%, 1.0%, and 2.0% of children; 8.0%, 0.0%, 1.0%; 1.0% of adults; and 5.0%, 0.0%, 0.0%, and 1.0% of elderly adults had HI antibody titers of ≥40 to BJ/1230/16, Cn/BJ/38/16, Cn/FJ/1109/18, and Cn/GZ/011/19, respectively (Table 1). In addition, 26.0%, 2.0%, 2.0%, and 3.0% of children had NI antibody titers of ≥10, and 12.0%, 1.0%, 2.0%, and 1.0% of adults and 12.0%, 1.0%, 1.0%, and 2.0% of elderly adults had NI antibody titers of ≥10 to BJ/1230/16, Cn/BJ/38/16, Cn/FJ/1109/18, and Cn/GZ/011/19, respectively. Furthermore, 14.0%, 1.0%, 1.0%, and 2.0% of children had MNT antibody titers of ≥40; 6.0%, 0%, 0%, and 0% of adults; and 4.0%, 0%, 0%, and 0% of elderly adults had MNT antibody titers of ≥80 to BJ/1230/16, Cn/BJ/38/16, Cn/FJ/1109/18, and Cn/GZ/011/19, respectively. Table 1 Cross-reactive antibody responses against influenza A (H3N2) viruses of human sera collected in China. Age group (year)Antigen%HI titer ≥40 (95% CI*)%NI titer ≥10 (95% CI*)%NT titer ≥40 for children or ≥80 for adults (95% CI*)≤10, n=100BJ/1230/16 (human)15.0 (7.9–22.1)26.0 (17.4–34.6)14.0 (7.2–20.8)Cn/BJ/38/161.0 (0–2.9)2.0 (0–4.7)1.0 (0–2.9)Cn/FJ/1109/181.0 (0–2.9)2.0 (0–4.7)1.0 (0–2.9)Cn/GZ/011/192.0 (0–4.7)3.0 (0–6.3)2.0 (0–4.7)25–53, n=100BJ/1230/16 (human)8.0 (2.7–13.3)12.0 (5.6–18.4)6.0 (1.4–10.6)Cn/BJ/38/1601.0 (0–2.9)0Cn/FJ/1109/181.0 (0–2.9)2.0 (0–4.7)0Cn/GZ/011/191.0 (0–2.9)1.0 (0–2.9)0≥60, n=100BJ/1230/16 (human)5.0 (0.7–9.3)12.0 (5.6–18.4)4.0 (0.2–7.8)Cn/BJ/38/1601.0 (0–2.9)0Cn/FJ/1109/181.0 (0–2.9)1.0 (0–2.9)0Cn/GZ/011/191.0 (0–2.9)2.0 (0–4.7)0 * Confidence interval. These results indicated that human populations lack immunity to H3N2 CIV, and even preexisting immunity derived from the present human seasonal influenza viruses cannot provide protection against H3N2 CIVs. H3N2 CIVs obtained human-type receptor-binding properties and their acid stability increased stepwise Our genetic analysis found that humanized adaptive mutations increased significantly along with the prevalence of H3N2 CIVs in dogs, while humans lacked preexisting immunity to the H3N2 CIVs, indicating that H3N2 CIVs might spread in populations once they are adapted to humans. Therefore, we further evaluated the potential threat of H3N2 CIVs to public health. The binding preference of HA for the host Saα2 6 Gal receptor and low activation pH are critical determinants for cross-species transmission of influenza virus to humans (Connor et al., 1994; Matrosovich et al., 2000). Therefore, we examined the receptor-binding preference of 11 H3N2 canine influenza viruses isolated from 2006–2019 (Figure 2—figure supplement 1). We noticed that, compared with the H3N2 avian influenza virus Dk/KR/JS53/04 and H3N2 CIVs from clades 0, 1, 2, and 3, which only recognized α–2,3-linked sialosides (Figure 2A), H3N2 CIVs belonging to clades 4, 5, and 5.1, represented by Cn/US/M17/17, Cn/SH/159/17, and Cn/HaiN/079/19, showed dual binding specificity to both α–2,3- and α–2,6-linked sialosides. The HA acid stability of clade 5 viruses (activation pH 5.3) was higher than that of clade 0, 1, 2, 3, and 4 viruses (activation pH 5.4) (Figure 2B and Figure 2—figure supplement 2). Compared with the H3N2 CIVs that were circulating before 2019 (activation pH 5.3), the viruses belonging to clade 5.1 circulating after 2019 had higher HA acid stability (activation pH 5.2) and identical HA fusion pH to that of the human H3N2 virus. Figure 2 with 2 supplements see all Download asset Open asset Binding specificities toward α–2, 3-, or α–2, 6-linked sialic acid receptors and hemagglutinin (HA) acid stability. (A) Characterization of receptor-binding properties of H3N2 CIVs. Direct binding of the virus to sialylglycopolymers containing either 2,3-linked (blue) or 2,6-linked (red) sialic acids was tested (n=3 biological replicates and n=3 technical replicates). Values are expressed as means ± standard deviations (SD). (B) HA activation pH measured by syncytia assay. Representative fields of cells infected with the indicated viruses and exposed to pH 5.2, 5.3, 5.4, 5.5, or 5.6 are shown. Scale bar, 100μm. The experiments were repeated three times, with similar results. The replication efficiency of CIVs exhibiting different receptor-binding specificity and HA acid stability were evaluated in vitro. In A549 cells, the titers of clade 5 and 5.1 viruses were significantly higher (up to nearly 100-fold higher) than those of other clades over 12 to 72 hr (p<0.01) (Figure 3A), and clade 5.1 viruses showed comparable virus outputs with the human seasonal H3N2 virus at each time point. Infection of NHBE cells with H3N2 CIVs produced similar progeny results. The titers of clade 5 and 5.1 viruses in NHBE cells were significantly (up to nearly 100-fold higher) higher than those of viruses from other clades between 24 hpi and 72 hpi (p<0.01), and clade 5.1 viruses and human seasonal H3N2 virus had also replicated to similar levels at each time point (Figure 3B). Collectively, H3N2 CIVs obtained human-type receptor binding properties, and their HA acid stability and replication ability in human cells increased stepwise during their circulation in the dog population. Figure 3 Download asset Open asset Viral growth properties in A549 (A) and NHBE (B) cells. Cells were infected with indicated viruses at MOI of 0.01 and incubated at 37 °C or 33 °C. Supernatants were harvested at the indicated time points, and the virus titers were determined in MDCK cells. Values are expressed as means ± standard deviations (SD) (n=3 biological replicates and n=3 technical replicates). **p<0.01,statistical significance was assessed using two-way ANOVA, the titers of BJ/1230/16 (human seasonal H3N2 virus) and clade 5 or 5.1 viruses were significantly higher than other viruses. The dashed black lines indicate the lower limit of detection. Improved replication and transmissibility of H3N2 CIVs in dogs To evaluate the infectivity and transmission ability of H3N2 CIVs in dogs, we inoculated intranasally six dogs with 106 TCID50 of each virus strain (Figure 4—figure supplement 1). Twenty-four hours later, three dogs inoculated with each virus strain were individually paired and cohoused with a direct-contact dog. At 4 dpi, nasal turbinates, tracheas, lungs, and tonsils were collected from another three inoculated dogs in each infection group for virus titration. We found that the H3N2 avian influenza virus could not be detected in dogs at 4 dpi, while H3N2 CIVs from clades 5 and 5.1 replicated efficiently in both the upper (nasal turbinate and trachea) and lower (lung) respiratory tracts and tonsils of dogs, where they were present in significantly higher amounts than the other clade viruses (p<0.01) (Figure 4A). Additionally, we monitored the clinical signs of the three inoculated dogs used for the transmission experiment for 14 days, and we found that H3N2 avian influenza virus (Dk/KR/JS53/04) and H3N2 CIVs (from clades 0, 1, 2, 3, and 4) caused only mild clinical signs with mean clinical scores ranging from 0.5 to 2.5 and a mean body temperature ranging from 37.7 to 39.7°C (Figure 4—figure supplement 1 and Figure 4B and C). However, infection with H3N2 CIVs from clades 5 and 5.1 resulted in more severe clinical symptoms such as pyrexia, sneezing, wheezing, and coughing, with a higher mean clinical score ranging from 2.8 to 3.3 and a higher mean body temperature ranging from 39.9 to 40.2°C. Furthermore, the virus identification of nasal swabs showed that H3N2 CIVs were efficiently transmitted to all naive dogs by direct contact (Figure 4D and Figure 4—figure supplement 1). In contrast, the H3N2 avian influenza virus was not transmitted between dogs. H3N2 CIVs (from clade 0, 1, 2, 3, and 4) were detected in two of the three naïve animals and all contact animals at 4 dpi and 6 dpi, respectively, and seroconversion was detected in 2/3 contacts (1:80 to 1:160) or all contacts (1:160 to 1:320) (Supplementary file 3). Noteworthy, clade 5 and 5.1 viruses, represented by Cn/BJ/38/16, Cn/SH/159/17, Cn/CA/BRW003/18, Cn/HaiN/079/19, and Cn/GZ/1180/19 were transmitted to all three contact animals at 2 dpi with seroconversion in all contacts (1:320 to 1:640), which was earlier than clade 0, 1, 2, 3, and 4 viral transmissions to naïve animals. Furthermore, clade 5 and 5.1 viruses also replicated more efficiently in all donors than other clade viruses (p<0.01). Thus, the replication and transmissibility of H3N2 CIVs gradually increased in dogs. Figure 4 with 1 supplement see all Download asset Open asset Infectivity and transmissibility of H3N2 CIVs in dogs. (A) Virus replication in the indicated organ. Three dogs were infected intranasally with 106 EID50 of each virus and euthanized at 4 dpi for virus titration. Each color bar represents the virus titer of an individual animal. (B) Clinical symptoms score of dogs infected with H3N2 CIVs. (C) Body temperatures of dogs infected with H3N2 CIVs. The results are shown as the means ± standard deviations (n = 3) (*p<0.05; **p<0.01). (D) Direct contact transmission of H3N2 CIVs in dogs. Each virus was tested with a total of three donors that were in direct contact with each group. Dogs housed in the same cage are denoted by the same color. The dashed black lines indicate the lower limit of detection. Statistical significance of clade 5 or clade 5.1 viruses relative to other viruses in the inoculated animals was assessed using two-way ANOVA (*p<0.05; **p<0.01). H3N2 CIVs acquired efficient aerosol transmissibility in a ferret model after the 2016 To further evaluate the potential risk of H3N2 CIVs to public health, we examined the replication and transmission of H3N2 viruses in a ferret model. A group of six ferrets was inoculated intranasally with 106 TCID50 of each virus strain. Twenty-four hours later, three inoculated ferrets for each virus strain were individually paired. An uninfected animal was housed in a wire-frame cage adjacent to the infected ferret to assess aerosol spread. Virus detection in nasal washes of the aerosol-spread animal showed that the H3N2 avian influenza virus (Dk/KR/JS53/04) and H3N2 CIVs (from clades 0, 1, 2, 3, and 4) did not transmit to ferrets through respiratory droplets (Figure 5). Clade 5 viruses, represented by Cn/BJ/38/16, Cn/SH/159/17, and Cn/CA/BRW003/18, transmitted to all naïve three ferrets via respiratory droplets by 6 dpi, and seroconversion was detected in all aerosols (1:160 to 1:320) (Supplementary file 4). More importantly, clade 5.1 viruses, represented by Cn/HaiN/079/19 and Cn/GZ/1180/19, were able to transmit to all three naïve ferrets as early as 4 dpi, with seroconversion in 3/3 aerosols (1:320 to 1:640). In addition, clade 5 and 5.1 viruses also replicated more efficiently than other clade viruses in all donors. Clade 5.1 viruses showed comparable virus outputs and transmissibility with the human seasonal H3N2 virus in the ferret model. Collectively, the evidence shows H3N2 CIVs obtained aerosol transmissibility during their evolution in dogs. Figure 5 Download asset Open asset Respiratory droplet transmission of H3N2 CIVs in ferrets. Groups of three ferrets were infected intranasally with 106 EID50 of indicated viruses and then housed separately in solid stainless-steel cages within an isolator. The next day, three uninfected respiratory droplet contact animals were individually housed in a wire-frame cage adjacent to the infected ferret. Nasal washes were collected every other day from all animals for virus-shedding detection from day 2 of the initial infection. Each color bar represents the virus titer of an individual animal. No data are displayed when no virus was detected in any of the groups. Dashed lines indicate the lower limit of virus detection. Statistical significance of the human influenza virus (BJ/1230/16) and clade 5.1 or clade 5 viruses relative to other viruses in the inoculated animals was assessed using two-way ANOVA (**p<0.01). Molecular determinants associated with efficient transmission reside in the HA and PB1 genes Since recent strains have increased replication and transmissibility in dogs and have gradually acquired 100% aerosol transmission ability in the ferret model, we further determined the molecular mechanisms responsible for the enhanced replication and transmission of H3N2 CIVs in mammals. We used Cn/BJ/256/15 (clade 2) as the backbone to generate reassortant viruses by individually replacing all genes from the Cn/HaiN/079/19 (clade 5.1) virus and testing the viral replication and transmissibility among dogs and ferrets. We found that PB1 and HA genes significantly enhanced the replication and transmission of single-gene reassortant viruses in both dogs and ferrets (Supplementary file 5 and Supplementary file 6), and seroconversion was detected in all contact dogs (1:160 to 1:320) and 2/3 aerosol ferrets (1:80 to 1:160). Next, we identified 15 conserved amino acid variations in the HA protein and PB1 protein of H3N2 CIVs that emerged in 2016–2019 (Figure 6—figure supplement 1). Among them, HA-146S, 188D, and PB1-154G were also highly enriched (>90%) among human H3N2 influenza A viruses isolated from 2006–2019 (Figure 6A). In addition, HA-16S was detected at a high frequency (>81%) among the H3N2 CIVs isolated after 2019, while all H3N2 CIVs before 2018 possessed HA-G16 (Figure 6A). Next, we evaluated the effect of these substitutions in receptor binding property assays and acid stability and thermal stability experiments. We found that the introduction of the HA-G146S enhanced binding to α–2,6-linked sialosides in Cn/BJ/256/15 (Figure 6B). The introduction of HA-G16S and HA-N188D increased the HA acid and temperature stability of Cn/BJ/256/15 (Figure 6C and D). Introduction of PB1-D154G increased the polymerase activity of Cn/BJ/256/15 (Figure 6E). Figure 6 with 1 supplement see all Download asset Open asset HA-G16S, G146S, N188D, and PB1-D154G mutations in H3N2 CIVs were the minimal molecular change required to facilitate the efficient aerosol transmissibility of the non-transmissible clade 2 virus. (A) Detection frequency of G/S at hemagglutinin (HA) residue 16 and 146, N/D at HA1 residue 188 (n= 437), and D/G at PB1 residue 154 (n=437) in avian, canine, and human influenza viruses. Amino acid residues are colored blue, red, and green, respectively. (B) Identification of mutations that confer binding to human-type receptors (n=3 biological replicates and n=3 technical replicates). Values are expressed as means ± standard deviations (SD). The wildtype data (Cn/BJ/256/15) were reproduced from Figure 2. (C) Representative fields of Vero cells expressing the indicated HAs and exposed to pH 5.2, 5.3, 5.4, or 5.5 are shown. Scale bar, 100μm. The experiments were repeated three times, with similar results. (D) HA protein stability as measured by the ability of viruses to agglutinate CRBCs after incubation at indicated temperatures for 40 min. Colors indicate the hemagglutination titers upon treatment at various temperatures for 40 min, as shown in the legend. The experiments were repeated three times with similar results. (E) The effects of PB1 from 19(Cn/HaiN/079/19) and amino-acid substitutions at PB1 residue 154 on the viral polymerase activity were determined using a minigenome assay in 293T cells. Values shown are the mean ± SD of the three independent experiments and are standardized to those of 15 (Cn/BJ/256/15) measured at 37℃ (100%) and 33℃ (100%). Statistical significance was assessed using two-way ANOVA (**p<0.01). (F) Aerosol transmissibility of the mutation viruses in ferrets. The wildtype data (Cn/BJ/256/15) were reproduced from Figure 5. Statistical significance of rgHA(G16S, G146S, N188D)PB1(D154G) viruses relative to other single substitution viruses were assessed using two-way ANOVA (*p<0.05). We then focused on these four mutations and evaluated the replication and respiratory droplet transmission ability of rgHA-(G16S), rgHA(G146S), rgHA(N188D), rgPB1(D154G), and rgHA(G16S, G146S, N188D)PB1(D154G) viruses in ferrets. We found that rgHA(G146S), rgHA(N188D), and rgPB1(D154G) viruses were transmitted to two of the three ferrets with seroconversion in 2/3 aerosols (1:160 to 1:320), and rgHA-(G16S), and rgHA(G16S, G146S, N188D)PB1(D154G) viruses were transmitted to all three ferrets with seroconversion in 3/3 aerosols (1:320 to 1:640) (Figure 6F). In addition, the viral titers in the nasal washes from the rgHA(G16S, G146S, N188D) PB1(D154G)-inoculated animals were higher than those of the other single substitution viruses (p<0.05) and were similar to that of the transmissible wild-type virus (Cn/HaiN/079/19). These results indicated that HA-G16S, G146S, N188D, and PB1-D154G are crucial for the replication and efficient transmissibility of H3N2 CIVs in ferrets. Discussion By evaluating the biological characteristics of avian-origin H3N2 CIVs isolated in dogs in different years, we found that effective infection and transmission in dogs were not intrinsic properties of H3N2 CIVs but appeared through stepwise adaptation. Of note, threats to human health might increase during H3N2 CIVs’ adaptation to dogs. Specifically, we observed changes in receptor binding specificity, from viruses recognizing only α–2,3-linked sialosides to those recognizing both α–2,3- and α–2,6-linked sialosides, as well as gradually increased HA acid stability and replication in human airway epithelial cells and ferrets and the ability to be transmitted by aerosol among ferrets. In addition, humans lack immunity to the H3N2 CIVs, and increased isolation of H3N2 CIVs in the do" @default.
• W4367333919 created "2023-04-30" @default.
• W4367333919 creator A5012500396 @default.
• W4367333919 date "2022-12-21" @default.
• W4367333919 modified "2023-09-28" @default.
• W4367333919 title "Editor's evaluation: Increased public health threat of avian-origin H3N2 influenza virus caused by its evolution in dogs" @default.
• W4367333919 doi "https://doi.org/10.7554/elife.83470.sa0" @default.
• W4367333919 hasPublicationYear "2022" @default.
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