Emerging Microbes & Infections 2023, VOL. 12, 2218945 (10 pages) https://doi.org/10.1080/22221751.2023.2218945 Diverse infectivity, transmissibility, and pathobiology of clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses in chickens Jung-Hoon Kwona,b*, Kateri Bertrana,c,d*, Dong-Hun Leee, Miria Ferreira Criadoa,f, Lindsay Killmastera, Mary J. Pantin-Jackwooda and David E. Swaynea** a Agricultural Research Service, U.S. Department of Agriculture, U.S. National Poultry Research Center, Athens, GA, USA; bCollege of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea; cUnitat mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Spain; dIRTA. Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, Spain; eCollege of Veterinary Medicine, Konkuk University, Seoul, Republic of Korea; fDepartment of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL, USA ABSTRACT Clade 2.3.4.4 Eurasian lineage H5Nx highly pathogenic avian influenza virus (HPAIV) has become the globally dominant clade and caused global outbreaks since 2014. The clade 2.3.4.4 viruses have evolved into eight hemagglutinin subgroups (2.3.4.4a-h). In this study, we evaluated the infectivity, pathobiology, and transmissibility of seven clade 2.3.4.4 viruses (two 2.3.4.4a, two 2.3.4.4b, one 2.3.4.4c and two 2.3.4.4e) in chickens. The two clade 2.3.4.4e viruses caused 100% mortality and transmissibility in chickens. However, clade 2.3.4.4a and c viruses showed 80–90% mortality and 67% transmissibility. Clade 2.3.4.4b viruses showed 100% mortality, but no transmission to co-housed chickens was observed based on lack of seroconversion. All the infected chickens died showing systemic infection, irrespective of subgroup. The results highlight that all the clade 2.3.4.4 HPAIVs used in this study caused high mortality in infected chickens, but the transmissibility of the viruses in chickens was variable in contrast to that of previous Eurasian-lineage H5N1 HPAIVs. Changes in the pathogenicity and transmissibility of clade 2.3.4.4 HPAIVs warrant careful monitoring of the viruses to establish effective control strategies. ARTICLE HISTORY Received 13 February 2023; Revised 17 May 2023; Accepted 23 May 2023 KEYWORDS Avian influenza virus; highly pathogenic avian influenza virus; clade 2.3.4.4; infectivity; pathogenicity; transmissibility; chicken Introduction domestic ducks and wild birds [3–7]. Clade 2.3.4.4 H5Nx HPAIVs have caused infections in multiple The Gs/GD lineage H5 highly pathogenic avian species, including poultry, wild birds, several mamma- influenza virus (HPAIV) was first detected in geese lian species, and humans; thus, the virus represents a from Guangdong, China, in 1996 (A/goose/Guang- serious threat to veterinary and public health and dong/1/1996 [H5N1], Gs/GD lineage). Since then, it the poultry industries [4,6–8]. has caused continuous outbreaks in enzootic countries Since 2014, clade 2.3.4.4 H5Nx HPAIVs have been and has evolved into multiple hemagglutinin (HA) detected in wild waterfowl and have spread world- genetic clades [1,2]. H5N1 was the dominant HA wide, including Europe, Africa, and the Americas and neuraminidase (NA) subtypes of the Gs/GD line- [4,9,10]. During 2014–2016, clade 2.3.4.4c H5N8 age; however, since 2008, novel NA subtypes of Gs/GD HPAIVs caused multiple outbreaks in various regions lineage HPAIVs belonging to the third order clade worldwide, including the first Gs/GD lineage HPAIV 2.3.4, such as H5N2, H5N5, H5N6, and H5N8, have outbreak in North America [10,11]. In May–June been identified in China and subsequently the HA 2016, a novel reassortant clade 2.3.4.4b H5N8 evolved into additional subclades, including fourth HPAIV was detected in wild birds in Qinghai Lake, order clade 2.3.4.4 [3–5]. In addition, clade 2.3.4.4 China, and Uvs-Nuur Lake in Siberia. Subsequently, H5Nx has evolved into eight fifth order HA genetic the virus spread to various regions in Asia, Africa, subgroups (a-h) and multiple genotypes, bearing vari- and Europe [4,12–14]. The related clade 2.3.4.4b able gene segment constellations by reassortment with H5Nx HPAIVs have circulated in the wild bird popu- low pathogenicity avian influenza viruses (LPAIVs) in lation, and multiple genotypes bearing gene segments CONTACT Dong-hun Lee donghunlee@konkuk.ac.kr College of Veterinary Medicine, Konkuk University, Seoul 05029, Republic of Korea; David E. Swayne birdfluvet@gmail.com Agricultural Research Service, U.S. Department of Agriculture, U.S. National Poultry Research Center, Athens, GA 30605, USA *These authors contributed equally to this work ** Current Address: David E Swayne, Birdflu Veterinarian LLC, Watkinsville, GA 30677, USA Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2023.2218945 This work was authored as part of the Contributor’s official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law. This is an Open Access article that has been identified as being free of known restrictions under copyright law, including all related and neighboring rights (https://creativecommons.org/publicdomain/mark/1.0/). You can copy, modify, distribute and per- form the work, even for commercial purposes, all without asking permission 2 J.-H. KWON ET AL. of wild bird-origin LPAIVs have been detected mainly clade 2.3.4.4 H5Nx HPAIVs have showed varying in Eurasia [12–14]. Since 2021, novel reassortment and relatively lower mortality rates in experimentally clade 2.3.4.4b H5N1 HPAIVs have caused worldwide inoculated or contact exposed chickens [24–26]. In epizootics in Europe, Africa, Asia, and the Americas addition, clade 2.3.4.4 virus-infected duck species var- [15–18]. Unlike the previous viruses, the clade ied from asymptomatic to high mortality but shed 2.3.4.4b virus has been maintained in wild birds for high viral titers [25,27,28]. Most of the clade 2.3.4.4 a long time, causing changes in the epidemiology of viruses exhibited low mortality in duck species; how- HPAIVs [19,20]. ever, an exceptional high mortality rate was observed The clades 2.3.4.4d-h of the H5N6 subtype have when mallards were experimentally infected with the been predominant in China since 2014 and their geo- A/Tufted-duck/Denmark/11470/LWPL/2016 H5N8 graphic distribution has been limited to East Asia. Par- clade 2.3.4.4b virus [27,28]. While diverse subgroups ticularly, the clade 2.3.4.4e H5N6 was detected in wild and genotypes of clade 2.3.4.4 HPAIVs have been birds in 2016 and subsequently caused large outbreaks reported, their pathogenicity in avian species have in South Korea and Japan during 2016–2017 [6,21,22], been examined for only a few strains. Furthermore, with multiple reassortants being detected in both the pathogenicity and transmissibility of clade 2.3.4.4 domestic poultry and wild birds [22,23]. Clade viruses in chickens have been evaluated in different 2.3.4.4a H5N6 virus has only been detected in Asian laboratories under different experimental conditions. enzootic countries since 2013, and distant geographi- To determine whether the biological characteristics cal spread to other regions has not been reported [6,7]. are different for these genetically divergent clade Unlike previous H5N1 HPAIVs of the Gs/GD line- 2.3.4.4 viruses, we evaluated the pathogenicity and age that caused almost 100% mortality in chickens, transmissibility of seven H5Nx HPAIV strains repre- senting clade 2.3.4.4 subgroups a, b, c, and e in specific Table 1. Mortality, virus shedding, and seroconversion of pathogen free (SPF) White Leghorn chickens. chickens inoculated with and contact of clade 2.3.4.4 highly pathogenic avian influenza viruses. Inoculum dose for intrachoanal inoculation included in first column. Materials and methods Challenge Mortality Viral viruses (dead/ sheddingc Animals (clade) total) (positive/ Seroconversiond (titera) Group (MDTb) total) (positive/total) One hundred one SPF White Leghorn chickens A/Ck/VNM/ Inoculated 9/10 (3.3) 9/10 0/1 obtained from the U.S. National Poultry Research 14 Contact 2/3 (4.5) 2/3 0/1 (2.3.4.4a) Center (USNPRC) in-house flocks were used. All (105.7) birds were transferred at 6 weeks of age to the animal A/Dk/VNM/ Inoculated 9/10 (3.2) 9/10 0/1 15 Contact 2/3 (4.5) 2/3 0/1 biosafety level 3 enhanced facilities and housed in (2.3.4.4a) negative-pressure HEPA-filtered isolators for the (105.9) HPAIV challenge. The birds were provided ad libitum A/TD/DNK/ Inoculated 10/10 (2.1) 10/10 N/A 16 Contact 0/3 0/3 0/3 access to feed and water throughout the experiment. (2.3.4.4b) All procedures were performed according to protocols (106.1) A/Tk/HUG/ Inoculated 10/10 (2.1) 10/10 N/A approved by the Institutional Laboratory Animal Care 16 Contact 0/3 0/3 0/3 and Use Committee of the USNPRC. (2.3.4.4b) (106.5) A/Tk/MN/ Inoculated 8/10 (2.1) 8/10 0/2 15 Contact 2/3 (3.5) 2/3 0/1 Viruses (2.3.4.4c) (105.9) The following seven clade 2.3.4.4 H5Nx HPAIVs were A/Ck/Hok/ Inoculated 10/10 (2) 10/10 N/A 16 Contact 3/3 (3) 3/3 N/A used as challenge viruses and obtained from the (2.3.4.4e) Southeast Poultry Research Laboratory, USNPRC (106.3) A/MD/KOR/ Inoculated 10/10 (2) 10/10 N/A HPAIV repository: A/chicken/Vietnam/NCVD14- 16 Contact 3/3 (2) 3/3 N/A A324/2014 (A/Ck/VNM/14; H5N6, 2.3.4.4a)(original (2.3.4.4e) (106.5) source, Nguyen Van Long, Department of Animal Sham (PBS) Negative 0/10 0/10 0/10 Health, Vietnam), A/duck/Vietnam/NCVD-15A74/ control 2015 (A/Dk/VNM/15; H5N6, 2.3.4.4a)(original Note: N/A: not applicable. a The viruses were inoculated via the intra-choanal route with approxi- source, Nguyen Van Long, Department of Animal mately 106 EID50 in 0.1 mL of each isolate, and the inoculum was Health, Vietnam), A/tufted-duck/Denmark/11740/ back tittered. b MDT, Mean death time. 2016 (A/TD/DNK/16; H5N8, 2.3.4.4b)(original c Number of birds shedding detectable viruses by the oral and/or cloacal source, Lars Erik Larsen, University of Copenhagen, route in at least one time point / total number of birds. The viral shed- Denmark), A/turkey/Hungary/53433/2016 (A/Tk/ ding was evaluated and quantified by H5 gene-specific qRT-PCR. d Determined in 14 dpi serum samples from surviving chickens by HI assay HUG/16; H5N8, 2.3.4.4b)(original source, Ian using homologous antigen. Brown, Animal and Plant Health Agency, United EMERGING MICROBES & INFECTIONS 3 Kingdom), A/turkey/Minnesota/12582/2015 (A/Tk/ hemagglutination inhibition (HI) assay using homolo- MN/15; H5N2, 2.3.4.4c)(original source, Mia Torch- gous antigens [31]. All the surviving birds were huma- etti, National Veterinary Services Laboratories, nely euthanized by ketamine/xylazine anesthesia USA), A/chicken/Hokkaido/1/2016 (A/Ck/Hok/16; followed by cervical dislocation at 14 dpi. H5N6, 2.3.4.4e)(original source, Hiroshi Kida, Hok- kaido University, Japan), and A/mandarin_duck/ Virus quantification Korea/K16-187-3/2016 (A/MD/KOR/16; H5N6, 2.3.4.4e)(original source, Chang-Seon Song, Konkuk Total viral RNA was extracted from 50 μL of the swab University, South Korea). The viruses were propa- material using the MagMAX-96 AI/ND Viral RNA gated and titrated using 9-to-11-day-old SPF embryo- Isolation Kit (Thermo Fisher Scientific, Carlsbad, nating chicken eggs in biosecurity level-3 enhanced CA, USA) following the manufacturer’s instructions. facilities in the USNPRC [29]. Tissues were homogenized in BHI broth to a 10% sol- ution, and total RNA was extracted from the 250 μL supernatant of tissue homogenates by RNA precipi- Experimental design and sample collection tation using the Trizol LS reagent (Invitrogen/Thermo The birds were divided into seven groups of 13 birds Fisher Scientific, Grand Island, NY, USA) and the each for virus challenge and one group of 10 birds RNeasy Mini Kit (Qiagen Corp, Valencia, CA, USA). as a negative control. At 6 weeks of age, ten birds Total RNA extracted from the tissue samples was from each group were challenged via the intrachoanal quantified using an Epoch spectrophotometer (Biotek, route with approximately 106 50% egg infectious doses Winooski, VT, USA) and diluted in PBS to obtain (EID50) in 0.1 mL of each isolate, and the inoculum 50 ng/µL for normalization. was back titered on the day of inoculation (Table 1). Because of the low compatibility of the previous To evaluate contact transmissibility, the remaining influenza virus matrix gene-targeting real-time poly- three birds per group were transferred into the isola- merase chain reaction (PCR) system [32] for clade tors with the inoculated birds on 1-day post-inocu- 2.3.4.4 viruses, the amount of influenza virus RNA lation (dpi). Negative control birds were sham- was quantified by customized H5 gene-specific quan- inoculated with phosphate-buffered saline (PBS). titative reverse transcription-PCR (qRT-PCR). Two All chickens were monitored daily for clinical signs primers, H5-380F (5′ -AAACACCTRTTGAGCA- and mortality until 14 dpi. Chickens showing severe GAATAAATCATT-3′ ) and H5-525R (5′ -AGCCA- clinical signs, which might include severe ataxia, TACCACATTTCTGAA-3′ ), and a probe, H5-421P severe lethargy, or inability to eat or drink, were eutha- (5′ -FAM-GATCATCCCCARGAGTTCTTGG- nized and counted as dead the next day in the mean TAMRA-3′ ), were used for qRT-PCR. Quantitative death time (MDT) calculations. Oropharyngeal (OP) real-time RT–PCR was performed using the AgPath- swabs and cloacal (CL) swabs were collected at 1, 2, ID one-step RT–PCR kit (Ambion/Thermo Scientific, 4, 7, 10 and 14 dpi using synthetic swab on plastic Grand Island, NY, USA) according to the manufac- stem and 1.5 mL brain heart infusion (BHI) medium turer’s instructions. (Becton, Dickinson and Company, Sparks, MD, To extrapolate the cycle threshold (Ct) values to USA) with antibiotics and antifungal (penicillin/strep- infectious unit equivalents, the known titer of the chal- tomycin/amphotericin B; Hyclone, Logan, UT, USA) lenge virus stocks was subjected to 10-fold serial and stored at −80C. dilutions. Viral RNA was extracted from these Three dead or euthanized chickens from each chal- dilutions and run in duplicate using qRT-PCR to pro- lenge group were necropsied at 2 dpi to examine for duce a standard curve for each challenge virus, as gross lesions and to collect tissues for histological described above. Previous AIV challenge studies in evaluation. The nasal turbinates, trachea, lungs, chickens have shown a high correlation between the heart, brain, intestine, pancreas, liver, spleen, thymus, quantity of RNA as determined using qRT-PCR and cloacal bursa, kidneys, and skeletal muscle were col- the infectious titer [33]. The detection limit was 102.5 lected in 10% neutral buffered formalin for histo- EID50/mL for all viruses. pathological examination by hematoxylin–eosin (HE) staining and influenza nucleoprotein viral anti- Statistical analysis gen detection by immunohistochemical (IHC) stain- ing [30]. Other sections of the brain, heart, lungs, Statistical analyses were performed using the Prism 5 and spleen were collected for viral RNA detection software (GraphPad Software, San Diego, CA, USA). and stored at −80C. Statistical differences in mean viral titers and antibody Serum samples were collected on the day of the levels among groups were analysed using one-way challenge (0 dpi) from two birds in each group, and ANOVA with Tukey’s post hoc test. Fisher’s exact at 14 dpi from all surviving birds. Seroconversion test was used to analyse the statistical significance of against the challenge virus was evaluated by a the number of birds shedding detectable virus titers. 4 J.-H. KWON ET AL. Figure 1. Egg infectious dose-equivalent titers from the oropharyngeal (OP) and cloacal (CL) swabs collected from chickens inocu- lated with or in contact with chickens shedding clade 2.3.4.4 highly pathogenic avian influenza viruses. Chickens in contact group were transferred into the isolators with the inoculated birds on 1 dpi. All samples were negative at 7, 10, and 14 dpi and are not shown. The lower detection limit was 102.5 50% egg infectious dose (EID50)/mL. Negative RT-PCR samples were treated as 102.4 EID50/mL for statistical purposes. During virus shedding analysis, the samples not three chickens in the clade 2.3.4.4a and 2.3.4.4c virus detected using qRT-PCR were treated as 102.4 EID50/ inoculated groups died at 4–6 days post contact. mL for statistical purposes. Viral shedding and seroconversion Results Most dead birds had high titers of virus in the oro- pharynx (mean: 106.3 EID50/mL) and cloaca (mean: Mortality and transmissibility 105.9 EID50/mL) at 2 dpi (Figure 1). At 1 dpi, higher The back titration results indicated that birds were titers of virus were detected in OP swab samples inoculated with 105.7–106.5 EID50 of each clade (mean: 104.3 EID50/mL) than in CL swab samples 2.3.4.4 virus, and these viruses caused different mor- (mean: 102.9 EID50/mL) in all groups, but statistically tality in the inoculated chickens (Table 1). All chickens significant (p < 0.05) differences between OP and CL inoculated with clade 2.3.4.4b viruses (A/TD/DNK/16 viral titers were only detected in clades 2.3.4.4b and and A/Tk/HUG/16) and clade 2.3.4.4e viruses (A/Ck/ 2.3.4.4e virus-inoculated groups. At 2 dpi, however, Hok/16 and A/MD/KOR/16) died within 3 dpi. Con- similar virus titers were detected in both OP and CL versely, two of the 10 chickens in the clade 2.3.4.4c swabs. virus (A/Tk/MN/15) inoculated group and one of In clade 2.3.4.4b, 2.3.4.4c, and 2.3.4.4e virus-inocu- the 10 chickens in the clade 2.3.4.4a virus (A/Ck/ lated groups, viral shedding was detected until 2 dpi, VNM/14 and A/Dk/VNM/15) inoculated groups sur- because all the infected birds were dead before 3 dpi. vived and showed no clinical signs through the end of However, one bird in the A/Ck/VNM/14 virus-inocu- the experiment on 14 dpi. All chickens in the sham lated group and three birds in the A/Dk/VNM/15- group survived and showed no clinical signs through inoculated group were positive for virus shedding at 14 dpi. The shortest MDT (Table 1) was detected in 4 dpi and died within 6 dpi. The peak viral shedding the two clade 2.3.4.4e (2 days), A/Tk/HUG/16 titers and the number of chickens shedding detectable (2.3.4.4b) (2.1 days) and A/Tk/MN/15 (2.3.4.4c) (2.1 virus did not show statistically significant differences days) virus inoculated groups, and a relatively long among groups. MDT was detected in the two clade 2.3.4.4a virus At 2 and 4 dpi, low viral titers (less than 103.0 inoculated groups (3.2 and 3.3 days). EID50/mL) were detected in the OP and CL swabs of Different transmissibility rates for clade 2.3.4.4 some birds that survived until the end of experiment, viruses were detected in co-housed chickens (Table probably due to mechanical virus exposure from con- 1). All three chickens co-housed with clade 2.3.4.4e taminated food and water during co-housing with the inoculated groups died at 3–4 days post contact. How- infected birds, rather than due to active infection. ever, all three chickens co-housed with clade 2.3.4.4b- From 7 dpi onwards, viral shedding was not detected inoculated birds survived through 14 dpi. Two of the from any surviving birds. To assess the possibility of EMERGING MICROBES & INFECTIONS 5 Table 2. Microscopic lesions and viral antigen distribution in chickens inoculated with clade 2.3.4.4 viruses, 2 days post- inoculation. A/Ck/VNM/14 A/Dk/VNM/15 A/TD/DNK/16 A/Tk/HUG/16 A/Tk/MN/15 A/Ck/Hok/16 A/MD/KOR/16 (2.3.4.4a) (2.3.4.4a)a (2.3.4.4b) (2.3.4.4b) (2.3.4.4c) (2.3.4.4e) (2.3.4.4e) Tissue HEb IHCc HE IHC HE IHC HE IHC HE IHC HE IHC HE IHC Nasal turbinates ++ +++ ++ +++ ++ +++ − + ++ +++ ++ +++ ++ +++ − + ++ +++ ++ ++ +++ +++ ++ ++ ++ +++ ++ ++ ++ +++ +++ +++ +++ +++ ++ + ++ +++ ++ +++ Trachea − + − − nt nt − + − ++ − + − + + ++ − − ++ ++ − − − − ++ +++ − + − − ++ ++ − − − + − − ++ ++ Lung + ++ +++ +++ ++ ++ +++ +++ +++ ++ − − + ++ +++ +++ +++ +++ ++ +++ +++ +++ ++ ++ − − + ++ +++ +++ +++ +++ +++ +++ +++ +++ − ++ ++ +++ Heart +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ − − − − +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ Brain ++ ++ +++ +++ ++ +++ − ++ + ++ ++ ++ ++ ++ +++ +++ ++ +++ − + + ++ + + ++ +++ ++ ++ ++ +++ ++ +++ − ++ + + ++ ++ + + Duodenum − + − + − + − ++ nt nt − ++ + +++ − +++ − ++ − ++ − ++ − + + +++ − ++ − +++ + +++ − − − − + +++ ++ +++ Ileum nt nt − + ++ +++ − ++ nt nt − − − − − ++ − − ++ ++ − − − + − ++ − +++ − ++ + + − − nt nt − ++ nt nt Cecal tonsil nt nt − + nt nt − + nt nt − ++ − +++ − ++ − − nt nt − ++ − ++ − +++ +++ +++ − ++ nt nt − ++ − − − − − ++ Pancreas − + − ++ ++ ++ + ++ − − − ++ − + − ++ − − − − − ++ − + − ++ − − + ++ ++ + − − − − + ++ − + Liver − − − + − + − + − + + +++ − ++ − +++ − − − + − − − − + ++ − ++ + +++ − ++ − − − − + +++ − ++ Spleen ++ ++ ++ +++ +++ − +++ +++ ++ +++ nt nt − ++ ++ ++ ++ +++ ++ − +++ +++ ++ ++ +++ +++ +++ +++ ++ +++ +++ +++ +++ ++ ++ ++ − ++ +++ +++ Thymus + ++ +++ +++ − + +++ +++ +++ +++ − − − − +++ +++ +++ +++ +++ +++ +++ +++ − + ++ +++ − + +++ +++ +++ +++ +++ +++ + ++ + − − + Cloacal bursa + − ++ +++ +++ − +++ + + + ++ ++ − − +++ +++ ++ ++ +++ − +++ − + + − + +++ ++ +++ ++ +++ + − − − − − + − + Kidney − − − ++ + + ++ + − − + ++ − − − ++ − + − + − + − − − − − + ++ +++ ++ ++ − + − − − − − − Skeletal muscle − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − Note: nt: no tissue. a One bird from the A/Dk/VNM/15-inoculated group showed no microscopic lesions or IHC staining in any tissue. Because this bird had no virus shedding or PCR-positive tissues, it was considered non-infected and was excluded from the histopathological evaluation. b Histopathology score of lesions in hematoxylin-eosin (HE) staining: −: no lesions; +: mild; ++: moderate; +++: severe. c Immunohistochemical (IHC) staining: −: no antigen staining; +: infrequent; ++: common; +++: widespread. subclinical infection of clade 2.3.4.4 HPAIVs in the viruses and tissues was necrosis of parenchymal cells, surviving chickens, we evaluated the HA antibody often accompanied by lymphoplasmacytic or hetero- responses to challenge viruses using the HI test. HI philic inflammatory infiltrates (Table 3). Viral antigen antibodies were not detected in any of the pre-chal- was consistently detected in parenchymal cells and lenge serum samples. Serum samples collected at vascular endothelial cells of most tissues, regardless 14 dpi were all negative, both from sham-inoculated of the challenge virus, in nasal tissues, lung, heart, chickens and from surviving HPAIV-inoculated brain, spleen, and thymus, which were the most chickens supporting the latter lacked active infection. affected tissues. Marked lesion heterogeneity in immune tissues (spleen, cloacal bursa, and thymus) was observed among birds. In these tissues, lymphoid Microscopic lesions and viral antigen depletion due to necrosis was common, although in distribution some cases, viral antigen was widespread in distri- Collectively, similar types of histological lesions and bution, indicating active replication at the time of types of cells expressing virus antigen were observed necropsy. In other cases, no viral staining could be in infected chickens with all the viruses tested (Tables observed, suggesting active replication prior to 2 and 3, Figure 2). The most prominent lesion across necropsy. 6 J.-H. KWON ET AL. Table 3. Microscopic lesions and viral antigen distribution in chickens inoculated with clade 2.3.4.4a-e highly pathogenic avian influenza viruses, 2 days post-inoculation. Tissue Lesions Cell types expressing virus antigen Nasal Epithelial cell necrosis and desquamation, rhinitis, sinusitis, Vascular endothelial cells, vascular endothelial cells of mucosal cavity mononuclear cell infiltrates, heterophilic rhinitis glands, nasal epithelial cells, respiratory epithelial cells, nasal gland epithelium, mononuclear cells Trachea Focal necrosis, lymphoplasmacytic infiltrates Pseudostratified epithelial cells, vascular endothelial cells, sternotrachealis muscle Lung Interstitial pneumonia, edema, congestion, necrosis, Vascular endothelial cells, pneumocytes, mononuclear cells lymphoplasmacytic infiltrates Heart Focal necrosis of myocardiocytes Myocardiocytes, vascular endothelial cells Brain Neuronal necrosis, gliosis, chromatolysis of Purkinje cell layer Neurons, Purkinje cells, ependymal cells, glial cells, vascular endothelial cells Intestine Focal necrosis, lymphoplasmacytic infiltrates in submucosa Villi enterocytes, vascular endothelial cells, mononuclear cells in lymphoid associated tissue, smooth muscle cells Pancreas Degeneration of individual pancreatic acinar cells, focal Acinar cells, duct cells, vascular endothelial cells lymphoplasmacytic infiltrates Liver Focal necrosis, lymphoplasmacytic infiltrates Kupffer cells, hepatocytes, vascular endothelial cells, macrophages Spleen Multifocal necrosis, hemorrhages, lymphoid depletion Mononuclear cells, vascular endothelial cells Thymus Focal to multifocal necrosis in medullary area (evident in Hassall’s Mononuclear cells, thymic epithelium in medullary area, vascular corpuscles), lymphocyte depletion, apoptotic lymphocytes in endothelial cells cortex area Cloacal Lymphocyte necrosis and apoptosis, lymphocyte depletion, Mononuclear cells, medullary support cells, vascular endothelial bursa phagocytic hyperplasia cells Kidney Focal necrosis of tubular epithelium with lymphoplasmacytic Tubular epithelial cells, glomerular cells, vascular endothelial cells infiltrates Skeletal No lesion No lesion muscle Some differences in the severity of lesions and detected, indicating lack of successful HPAIV intensity of viral staining were observed among the infection. viruses (Table 2). The intestine (duodenum, ileum, caecal tonsil) of clade 2.3.4.4a (A/Dk/VNM/15) and Discussion clade 2.3.4.4c infected chickens was unaffected or only showed mild viral antigen staining, whereas Clade 2.3.4.4 H5Nx viruses emerged by gradual clade 2.3.4.4a (A/Ck/VNM/14), 2.3.4.4b, and 2.3.4.4e mutations and multiple reassortment between H5N1 viruses replicated better in villi enterocytes and capil- HPAIVs and LPAIVs in ducks [3–6]. Previous studies laries, often causing focal areas of necrosis with lym- have shown that these viruses can efficiently replicate phoplasmacytic infiltrates. Viral staining in the liver in duck species without causing high mortality, indi- was mild and infrequent with clade 2.3.4.4a (A/Dk/ cating that clade 2.3.4.4 HPAIVs are highly adapted VNM/15), 2.3.4.4b, and 2.3.4.4c viruses but was to duck species [25,34,35]. Other studies have more widespread in clade 2.3.4.4e and 2.3.4.4a (A/ suggested that some clade 2.3.4.4 HPAIVs show rela- Ck/VNM/14) viruses. Although virus staining in vas- tively low infectivity and transmissibility in chickens cular endothelial cells was frequent for all viruses, compared to previous H5N1 viruses of the Gs/GD clade 2.3.4.4a and e viruses showed especially exten- lineage, which typically show 100% mortality in chick- sive vascular endothelial cell tropism. ens [24,26,36]. In this study, clades 2.3.4.4a and To evaluate the systemic replication of clade 2.3.4.4 2.3.4.4c HPAIVs showed 80–90% mortality in chick- viruses, viral titers in the brain, heart, lungs, and ens. All surviving chickens lacked seroconversion, spleen were determined (Figure 3). Clade 2.3.4.4 indicating no active infection despite co-housing virus-infected chickens showed high viral titers with infected birds that shed high viral titers. A pre- (>107.6 EID50/g) in all four tissues except one chicken vious infectivity study identified high 50% chicken in the A/Dk/VNM/15-challenged group. The highest infectious doses (105.7 and 104.4 EID50/mL) and lack viral titer was detected in the lungs of the clade of seroconversion in surviving chickens inoculated 2.3.4.4c virus challenge group and the heart of the with clade 2.3.4.4c H5N8 and H5N2 viruses, indicat- clade 2.3.4.4b challenge group. The clade 2.3.4.4e chal- ing lower infectivity of chickens to these clade lenge groups showed high viral titers in the brain and 2.3.4.4 viruses compared to previous Gs/GD HPAIVs heart. For clade 2.3.4.4a viruses, A/Ck/VNM/14-chal- [24]. In addition, less than 100% transmissibility was lenged chickens showed high titers in the lungs and identified in clade 2.3.4.4a-, b-, and c-exposed chick- spleen, but A/Dk/VNM/15-challenged chickens ens in the present study. The difference in the mor- showed high titers in the brain and heart. tality and transmissibility of viruses was not Viral antigen and RNA were not detected in any tis- statistically supported because of the low number of sue of one chicken of A/Dk/VNM/15-challenged birds, but less than 100% mortality and transmissibil- group. This chicken was euthanized for tissue sample ity are uncommon in other clades of Gs/GD lineage collection at 2 dpi, and viral shedding was not HPAIVs, including clade 2.3.2.1 and clade 2.2 EMERGING MICROBES & INFECTIONS 7 Figure 2. Histopathology and immunohistochemical staining for AIV antigen in tissues of chickens infected with clade 2.3.4.4 highly pathogenic avian influenza viruses, 2 days post-inoculation. Hematoxylin-eosin staining (A-E); immunohistochemistry (insets), virus staining of cells (nuclei – clearly stained circular bodies) in red. A. Lung, A/Ck/VNM/14 (clade 2.3.4.4a). Interstitial pneumonia with lymphoplasmacytic infiltrates. Inset: Lung, same area. Viral antigen in endothelial cells, pneumocytes, mono- nuclear cells. B. Pancreas, A/TD/DNK/16 (clade 2.3.4.4b). Necrotic pancreatic acinar cells with lymphoplasmacytic infiltrates. Inset: Pancreas, same area. Viral antigen in acinar cells, duct cells, endothelial cells. C. Thymus, A/TD/DNK/16 (clade 2.3.4.4b). Necrosis in medulla and lymphocyte depletion in cortex. Inset: Thymus, same area. Viral antigen in mononuclear cells, thymic epithelium in medullary area, endothelial cells. D. Duodenum, A/Ck/Hok/16 (clade 2.3.4.4e). Focal necrosis with lymphoplasma- cytic infiltrates in submucosa. Inset: Duodenum, same area. Viral antigen in enterocytes, endothelial cells, mononuclear cells in lymphoid associated tissue, smooth muscle cells. E. Cecal tonsil, A/MD/KOR/16 (clade 2.3.4.4e). Focal necrosis with lymphoplas- macytic infiltrates in submucosa. Inset: Cecal tonsil, same area. Viral antigen in enterocytes, endothelial cells, mononuclear cells in lymphoid associated tissue. [37,38]. All viruses used in this study caused systemic changes in molecular and biological characteristics of infections and lesions in the infected chickens. These this clade should be continuously monitored. Notably, results highlight that the mortality rate of clade in this study, the clade 2.3.4.4e virus isolated from a 2.3.4.4 viruses in chickens is still high, but the suscep- wild mandarin duck during the initial outbreak showed tibility of chickens to become infected depends on the high mortality and transmissibility in chickens without viral strain, the exposure dose and the virus’s degree of preadaptation. The poultry isolates of clade 2.3.4.4a adaptation to chickens. In 2014–2025, the 2.3.4.4c and 2.3.4.4c viruses showed less than 100% mortality clade viruses increased adaptation to chickens and tur- and transmissibility in this study. keys after they circulated on farms, decreasing the The viruses used in this study had H5 genes belong- dose of virus needed to produce infection and ing to different clade 2.3.4.4 subgroups of the Gs/GD increased the ability to spread between farms by lateral Eurasian lineage, and multiple gene constellations transmission [24,25]. with the other seven gene segments because of reas- The viruses belonging to the same subgroup showed sortment with LPAIVs [12,21,22,40]. For example, similar infectivity and transmissibility in this study. clade 2.3.4.4b H5N8 viruses have wild bird LPAIV ori- However, these biological characteristics can be altered gin gene segments, except for the HA, NA, and non- during viral evolution and adaptation. For example, structural (NS) genes [12]. Additionally, the clade during the 2014–2015 clade 2.3.4.4c H5N2 epizootic 2.3.4.4c H5N2 virus has North American wild bird in the USA, the viruses isolated from poultry during LPAIV origin polymerase basic 1 (PB1), nucleoprotein spring 2015 showed better adaption and higher infec- (NP), and NA gene segments [9]. Clade 2.3.4.4a and tivity in chickens, as evident by lower 50% chicken 2.3.4.4e H5N6 viruses also emerged by multiple reas- infectious dose, than the initial H5N2 virus isolated sortments with previous H5N1 HPAIVs and duck-ori- from a wild duck [25,39]. The clade 2.3.4.4b viruses gin LPAIVs [6]. Due to these multiple reassortments, used in this study were isolated from wild birds during the genetic mutations that contribute to the pathogen- the initial phase of the outbreak. Because the clade icity changes cannot be defined in this study. Although 2.3.4.4b viruses have caused ongoing worldwide out- the role of each gene segment in pathogenicity in breaks since 2016 until now, as of January 2023, the chickens or ducks has not yet been fully determined, 8 J.-H. KWON ET AL. Figure 3. Egg infectious dose-equivalent titers in brain (A), heart (B), lung (C) and spleen (D) collected from chickens inoculated with clade 2.3.4.4 highly pathogenic avian influenza viruses at 2 days post-inoculation (n = 3 chickens/virus). Viral titers sharing at least one superscript letter indicate no statistically significant differences (p ≥ 0.05). previous studies have shown that internal gene seg- and may limit early disease control. Sharing updated ments, including polymerase complex genes, can con- pathogenicity information with field veterinarians tribute to changes in virus replication and and poultry farmers will be crucial for the early detec- pathogenicity in chickens and ducks [39,41,42]. The tion and control of HPAIVs. clade 2.3.4.4 viruses are continuously evolving into During the 2016–2017 clade 2.3.4.4b HPAIV epi- multiple genotypes by reassortment with prevailing zootic in Europe, most poultry cases were detected LPAIVs in wild birds [6,12,13,15,16,43]. Although in domestic waterfowl, including ducks and geese, the clade 2.3.4.4e viruses showed higher mortality and outbreaks were focused in high duck density and transmissibility than other viruses in this study, areas such as France and Hungary [46]. In South the pathogenicity of a specific virus can be readily Korea, domestic ducks played a central role in the changed by further reassortment with other viruses. clade 2.3.4.4c H5N8 outbreak of 2014–2016; however, Therefore, the biological characteristics of newly clade 2.3.4.4e H5N6 outbreaks occurred mostly in emerging genotypes should be continuously moni- chickens [47,48]. Our results showed that clade tored to inform veterinary authorities of any increases 2.3.4.4e H5N6 viruses caused higher mortality and in adaptation to poultry and thus an increased risk of transmissibility than other clade 2.3.4.4 viruses in transmission. chickens, in line with field observations where clade Clade 2.3.4.4 H5Nx HPAIVs have become the 2.3.4.4e viruses predominated in chicken premises. dominant clade in wild birds since 2014, especially Understanding the pathogenicity and susceptibility the clade 2.3.4.4b HPAIVs which have caused global of poultry species to novel HPAIVs is a key to predict- outbreaks in Asia, Europe, Africa, and America ing disease outbreak patterns and establishing targeted [4,8,10,14–18,43–45]. The relatively low infectivity control strategies. and transmissibility of some clade 2.3.4.4 H5Nx We identified different levels of infectivity and viruses for chickens, especially broiler chickens, com- transmissibility rates, and pathobiology of clade pared to previous Gs/GD-lineage H5N1 HPAIVs may 2.3.4.4 H5Nx subgroups. In contrast to previous delay and hinder rapid, accurate diagnoses on farms H5N1 HPAIVs of the Gs/GD lineage, clade 2.3.4.4 EMERGING MICROBES & INFECTIONS 9 H5Nx HPAIVs continuously cause infection in wild have been detected in poultry and caused multiple birds and poultry, and evolve by reassortment with human infections in China. Emerg Microbes Infect. diverse LPAIVs in wild birds which can result in 2022 Dec;11(1):1174–1185. [9] Lee DH, Torchetti MK, Winker K, et al. changes in pathobiology of viruses [12,15,41]. The Intercontinental apread of Asian-origin H5N8 to evolution of HPAIVs and changes in their biological North America through Beringia by migratory birds. characteristics need to be continuously monitored J Virol. 2015 Jun;89(12):6521–6524. for the effective control of HPAIV outbreaks. [10] Global Consortium for H5N8 and Related Influenza Viruses. Role for migratory wild birds in the global spread of avian influenza H5N8. Science. 2016 Oct;354(6309):213–217. Acknowledgements [11] Lee DH, Torchetti MK, Killian ML, et al. Reoccurrence of avian influenza A(H5N2) virus clade 2.3.4.4 in wild The authors would like to thank Àlex Cobos for his assist- birds, Alaska, USA, 2016. Emerg Infect Dis. 2017 ance with the histopathology images. Feb;23(2):365–367. [12] Poen MJ, Venkatesh D, Bestebroer TM, et al. Co-cir- culation of genetically distinct highly pathogenic Disclosure statement avian influenza A clade 2.3.4.4 (H5N6) viruses in wild waterfowl and poultry in Europe and East Asia, No potential conflict of interest was reported by the 2017–18. Virus Evol. 2019 Jan;5(1):vez004. author(s). [13] Jeong S, Lee DH, Kwon JH, et al. Highly pathogenic avian influenza clade 2.3.4.4b subtype H5N8 virus iso- lated from mandarin duck in South Korea, 2020. Funding Viruses. 2020 Dec;12(12):1389. This study was supported by the U.S. Department of Agri- [14] King J, Schulze C, Engelhardt A, et al. Novel HPAIV culture, Agricultural Research Service Project 6040-32000- H5N8 reassortant (Clade 2.3.4.4b) detected in 081-00D. Jung-Hoon Kwon is supported by the Korea Insti- Germany. Viruses. 2020 Mar;12(3):281. tute of Planning and Evaluation for Technology in Food, [15] Bevins SN, Shriner SA, Cumbee JC, Jr., et al. Agriculture, Forestry (IPET) through Animal Disease Man- Intercontinental movement of highly pathogenic agement Technology Advancement Support Program, avian influenza A(H5N1) clade 2.3.4.4 virus to the funded by Ministry of Agriculture, Food and Rural Affairs United States, 2021. Emerg Infect Dis. 2022 May;28 (1220160-2). (5):1006–1011. [16] Nagy A, Cernikova L, Stara M. A new clade 2.3.4.4b H5N1 highly pathogenic avian influenza genotype detected in Europe in 2021. Arch Virol. 2022 References Jun;167(6):1455–1459. [1] Lee DH, Criado MF, Swayne DE. Pathobiological ori- [17] Sagong M, Lee YN, Song S, et al. Emergence of clade gins and evolutionary history of highly pathogenic 2.3.4.4b novel reassortant H5N1 high pathogenicity avian influenza viruses. Cold Spring Harbor Perspect avian influenza virus in South Korea during late 2021. Med. 2021 Feb;11(2):a038679. Transbound Emerg Dis. 2022 Sep;69(5):e3255–e3260. [2] Swayne DE. 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