Comparative pathogenic potential of avian influenza H7N3 viruses isolated from wild birds in Egypt and their sensitivity to commercial antiviral drugs

Archives of Virology (2023) 168:82 https://doi.org/10.1007/s00705-022-05646-w ORIGINAL ARTICLE Comparative pathogenic potential of avian influenza H7N3 viruses isolated from wild birds in Egypt and their sensitivity to commercial antiviral drugs Ahmed E. Kayed1 · Omnia Kutkat1 · Ahmed Kandeil1 · Yassmin Moatasim1 · Ahmed El Taweel1 · Mohamed El Sayes1 · Rabeh El‑Shesheny1 · Basma Emad Aboulhoda2 · Nourtan F. Abdeltawab3 · Ghazi Kayali4 · Mohamed A. Ali1 · Mohammed A. Ramadan3 Received: 28 June 2022 / Accepted: 3 November 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2023 Abstract Active surveillance and studying the virological features of avian-origin influenza viruses are essential for early warning and preparedness for the next potential pandemic. During our active surveillance of avian influenza viruses in wild birds in Egypt in the period 2014-2017, multiple reassortant low-pathogenic avian influenza H7N3 viruses were isolated. In this study, we investigated and compared the infectivity, pathogenicity, and transmission of four different constellation forms of Egyptian H7N3 viruses in chickens and mice and assessed the sensitivity of these viruses to different commercial antiviral drugs in vitro. Considerable variation in virus pathogenicity was observed in mice infected with different H7N3 viruses. The mortality rate ranged from 20 to 100% in infected mice. Infected chickens showed only ocular clinical signs at three days postinfection as well as systemic viral infection in different organs. Efficient virus replication and transmission in chickens was observed within each group, indicating that these subtypes can spread easily from wild birds to poultry without prior adaptation. Mutations in the viral proteins associated with antiviral drug resistance were not detected, and all strains were sensitive to the antiviral drugs tested. In conclusion, all of the viruses studied had the ability to infect mice and chickens. H7N3 viruses circulating among wild birds in Egypt could threaten poultry production and public health. Introduction Handling Editor: Sheela Ramamoorthy. * Ghazi Kayali * Mohamed A. Ali * Mohammed A. Ramadan 1 Environmental Research Division, Centre of Scientific Excellence for Influenza Viruses, National Research Centre, Giza 12622, Egypt 2 Department of Anatomy and Embryology, Faculty of Medicine, Cairo University, Cairo 12613, Egypt 3 Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo 12613, Egypt 4 Human Link DMCC, Dubai, United Arab Emirates Each year, avian influenza viruses (AIVs) cause regional outbreaks in birds in various parts of the world, and they sometimes give rise to influenza viruses that cause human influenza pandemics [27]. These pandemics usually occur due to antigenic shifts caused by an exchange of viral genes among human-, swine-, and wild- and/or domestic-birdadapted strains [25]. Wild birds are frequently exposed to AIV, and wild aquatic birds are considered the primary virus reservoir [38]. Influenza viruses with more than 80 different combinations of HA and NA subtypes have been isolated from wild birds [28]. Asymptomatic transmission of AIVs among wild birds in particular increases the potential of AIVs to spread between countries or even continents during their annual migration [17]. Anseriforms (waterfowl) carry a distinct pool of influenza viruses [19, 45], and H7 AIV is one of the frequent subtypes identified in migratory waterfowl [2, 11]. Lowpathogenic avian influenza virus (LPAIV) subtype H7N3 was one of the strains most frequently detected during our 13 Vol.:(0123456789) 82 Page 2 of 15 previous surveillance study of AIVs in wild birds in Egypt [16]. Infection with LPAIV H7 and H5 subtypes in waterfowl is asymptomatic, and the birds can transmit the virus over long distances during their annual migration and spill the infection over into domestic poultry [21]. During infection of domestic galliforms with LPAIV H5 and H7 subtypes, the virus can potentially evolve into a highly pathogenic avian influenza virus (HPAIV) by insertion or substitution of basic amino acids at the HA0 cleavage site [3, 32, 46]. Alternatively, coinfection with more than one AIV can result in recombination of the HA gene with other viral genes of non-homologous viruses, giving rise to a more pathogenic virus that causes severe systemic disease and mortality [39]. H7 viruses occasionally spill over from wild birds into domestic poultry, causing economic losses in poultry production and sporadic infections in humans [18]. The first isolation of H7N3 viruses was reported in Britain in 1963, during an outbreak on a turkey farm, and in 1994, Australia and Pakistan were affected by HPAI variants [1]. Other outbreaks of H7N3 HPAIV in poultry were reported in China in 2002 [33], Canada in 2004 [14] and 2007 [6], and Chile in 2002 [39]. In 2020, an outbreak of H7N3 LPAIV occurred on a turkey farm in the USA, and the virus was found to be closely related to wild-bird isolates of North American lineage [44]. Genetic analysis of Egyptian H7N3 viruses isolated during our active surveillance study of wild birds during the period 2014-2016 indicated the presence of four different H7N3 viruses with different genome constellations [16]. Here, we investigated the replication and pathogenicity of these viruses in avian and mammalian hosts as well as their sensitivity in vitro to different commercial antivirals, including oseltamivir and zanamivir (neuraminidase inhibitors) [22], amantadine (an M2 blocker) [41], and favipiravir (an RdRp inhibitor) [13]. Materials and methods Viruses The LPAIVs used in this study were isolated through routine AIV surveillance of wild birds [16]. All four viruses – A/teal/Egypt/MB.D125OP/2015, A/teal/Egypt/ MB.D128OP/2015, A/teal/Egypt/MB.D487OP/2016, and A/northern shoveler/Egypt/MB.D690OP/2016 (hereafter abbreviated as 125OP, 128OP, 487OP, and 690OP) – were isolated from Damietta governorate in the same migration season (at the end of 2015 and beginning of 2016). Full genome sequencing was conducted [10], and the sequences of the segments were subjected to BLAST analysis on the Global Initiative on Sharing All Influenza Data (GISAID) 13 A. E. Kayed et al. platform (Fig. 1). Sequences are available in the GenBank database under the accession numbers listed in Table 1. The viruses were purified by plaque assay [12] and then propagated in specific-pathogen-free (SPF) eggs. Aliquots were stored at -80 °C and titrated using egg infectious dose 50 (EID50) and plaque assays [15, 31]. Pathogenicity of the virus isolates in SPF chickens Twelve 12 30-day-old SPF chickens obtained from the SPF Egg Production Farm, Koum Osheim, El-Fayoum, Egypt, were infected with 1 06 EID50 of each virus in a volume of 200 µl via the intraocular and intranasal routes. Chickens were kept in class II isolator cages and monitored daily for m (...truncated)