Genome sequence analysis of H7N3 subtype avian influenza virus originated from wild birds and its potential infectivity in mice

In 2021, an H7N3 avian influenza virus (AIV) was isolated from a mallard in Tianhewan Yellow River National Wetland Park, Ningxia Hui Autonomous Region, China. Sequences analysis showed that this strain received its genes from H7, H6, H5, H3, and H1 AIVs of domestic poultry and wild birds in Asia and Europe. It was mild pathogenicity in mice. These results suggest the importance of continued surveillance of the H7N3 virus to better understand the ecology and evolution of the AIVs in poultry and wild birds and the potential threat to humans.


Introduction
Avian influenza viruses (AIVs) belong to the family Orthomyxoviridae, genus Alphainfluenzavirus. Based on the antigenic properties of the predominant viral surface glycoproteins, the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, AIVs are further classified into 16 HA and 9 NA subtypes. Aquatic birds are considered a natural reservoir for AIVs, in which all subtypes of avian influenza viruses have been detected. According to the pathogenicity of the virus in chickens, AIVs can be divided into high-pathogenicity (HP) viruses and low-pathogenicity (LP) viruses, and the LP viruses could be mutated into HP viruses. So far, only H5 and H7 subtypes of avian influenza viruses have the potential to mutate from LP to HP. For example, H7N9 LP viruses that arose in China in 2013 had been mutated into H7N9 HP viruses in 2017 [1].
The H7 subtype AIV was first reported in northern Italy in 1878 [2]. In recent years, the H7N3 subtype avian influenza was epidemic in America and Europe, such as the highly pathogenic H7N3 subtype avian influenza was epidemic in Italy in 2003 [3]. In 2004, an outbreak of H7N3 subtype avian influenza occurred in British Columbia, Canada, causing conjunctivitis and avian flu-like symptoms in 57 poultry farm workers in addition to 1.9 million chickens culling losses [4]. So far, many subtypes of H7 including H7N2, H7N3, H7N7, and H7N9 have caused human infection and death [5]. Nowadays, the H7N9 subtype avian influenza is one of the important poultry diseases that influence the poultry industry in China. However, the epidemiological and biological characteristics of other subtypes, such as H7N3, were not clear in China.
In order to investigate the biological characteristics of the H7N3 subtype AIV, in this study, an H7N3 AIV was isolated from mallard. The whole genome sequence of H7N3 AIV was analyzed, and the infectibility to mammals was preliminarily evaluated using mice as animal model.
In April 2021, during routine surveillance for avian influenza in Ningxia Hui Autonomous Region, China, we collected feces samples from mallards in Tianhewan Yellow River National Wetland Park. The samples were centrifuged at 10 000 g for 5 min at 4 °C, and the supernatants were inoculated into the allantoic cavities of 10-day-old specific-pathogen-free (SPF) chicken embryos. The embryos were incubated at 37 °C for 4 days and checked daily. The allantoic fluids were collected and tested by using the  TTC TAA CCG AGG TCG AAA C, andM229R, AAG CGT CTA  CGC TGC AGT CC). The M gene positive isolates were further amplified with primers for HA and NA genes and sequenced, and an H7N3 subtype AIV was isolated. It was named as A/mallard/Ningxia/Y37/2021(H7N3)(NX/Y37)(EpiFlu isolate ID: EPI_ISL_15773496). Subsequently, its entire genome was amplified with primers as described elsewhere by RT-PCR assay. RT-PCR products were purified with an agarose gel DNA extraction kit (Sangon, Shanghai, China) and sequenced using the ABI 3730xl DNA Analyzer [6].
To better understand the genetic relationship between the H7N3 strain in China and these in the world, the phylogenetic analyses were carried out with the software MEGA 6.05 of the ClustalW software package (www. megas oftwa re. net) using a neighbor-joining algorithm. Nucleotide substitutions were set under the Kimura 2-parameter model, and substitution rates among sites were set in gamma distribution. The gaps were handled by pairwise deletion. A bootstrap analysis was conducted using 1000 replicates.
Based on the deduced amino acid sequence of the hemagglutinin (HA), the NX/Y37 isolate has only one amino acid residue (PEIPKGR/GLF) at the cleavage site, which is typical for low-pathogenicity AIVs. The amino acid residues at the receptor binding site in the HA protein are G186, Q226, and G228 (H3 numbering), which indicates its avian-like receptor binding preference. The strain has five potential N-glycosylation sites of 22 NGT, 38 NAT, 240 NDT, 411 NWT, 483 NNT (H3 numbering) in the HA protein.
There were no amino acid substitutions associated with resistance to oseltamivir (E119G, Q136L, E276D, R292K, and R371K) in the viral neuraminidase protein (NA) [7]. The NX/Y37 virus does not have mutations associated with high pathogenicity of the virus in mammals (K526R, A588V, E627K, and D701N) identified in the viral PB2 protein [8][9][10]. A mutation of D622G occurred in the PB1 and H226R, N383D, and N409S in the PA protein which could enhance polymerase activity in mammalian cells [11,12]. Mutations of N30D and T215A occurred in the viral M protein and P42S and I106M in NS, which could increase the viral virulence in mice [13,14].
Sequences analysis showed that the HA and NA genes showed the highest sequence identities (98.3% and 97.8%) with those of South Korea strains A/wild duck/South Korea/#57/2020(H7N7) and A/mallard/South Korea/JB21-58/2019(H5N3), respectively. The internal genes showed high nucleotide identity with those of the viruses circulating in wild birds and domestic poultry, including H5N2, H5N3, H3N2, and H3N8 (Table 1).
Phylogenetic analysis showed that both HA and NA genes of NX/Y37 belonged to the Eurasian lineage. The HA gene was very closely related to H7N7 viruses circulating in Asia which was obviously different from H7N9 that appeared in China. The NA gene was very closely related to H1N3 and H5N3 viruses circulating in Asia. Phylogenetic analysis of internal genes showed that they were very closely related to H6, H5, H3, and H1 viruses circulating in Asia and Europe ( Figure S1A-H). According to the above-described analysis, NX/Y37 was a reassortant virus and received its genes maybe from several subtype viruses of poultry and wild birds in Asia and Europe.
To investigate the replication and virulence of the virus in a mammalian host, eight 6-week-old BALB/c mice were inoculated intranasally with 10 6 EID 50 of NX/Y37 isolate. On 3 days post-inoculation (dpi), three mice were euthanized to examine the virus activity in the organs, including nasal turbinate, lungs, spleen, brain, and liver; the other five mice were observed for a total of 14 days for body weight changes and death. On day 3 post-inoculation, high titers of viruses were detected in the nasal turbinate and lungs, and low titers of viruses in the brain and spleen were not detected in the kidney (Fig. 1A). The body weights of mice infected with NX/Y37 were slightly lost during 7 dpi, and subsequently slowly regained weight with a survival rate of 100% (Fig. 1B). These results suggested that the NX/Y37 virus was not capable of fatally infecting mice, but could effectively replicate in the nasal turbinate, and lung without preadaptation.
To investigate the antigenicity of the NX/Y37 and the current H7-Re4 vaccine used in China, we performed the hemagglutination inhibition (HI) assay. The NX/Y37 isolate reacted poorly with antiserum against H7-Re4, and the HI antibody titer was 16-fold lower than that against the H7-Re4 antigen. It indicated that the protection efficiency of H7-Re4 against NX/Y37 may be poor.
In conclusion, a novel H7N3 AIV was isolated from mallard in China, in 2021. This novel H7N3 AIV underwent wide reassortment between the viruses circulating in domestic poultry and wild birds in Asia and Europe. This reassortant H7N3 virus was found to have mild pathogenicity in mice. Wild birds play an important role in spreading of avian influenza viruses. For example, the global outbreaks were caused by H5N8 viruses and their further recombinants between these with other subtypes AIVs via wild bird migration [15,16]. Moreover, H7 viruses have the characteristic of mutating from low pathogenicity to high pathogenicity. These pose significant risks and threats to the poultry industry. Our results suggest the importance of continued surveillance of the H7N3 virus to better understand the ecology and evolution of the AIVs in poultry and wild birds.
Author contributions JW designed the study. YX, WT, and PC performed the experiments and analyzed the data together with LJ, LS, and HG. JW wrote the initial draft of the manuscript and LH revised the manuscript.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval All procedures performed in studies involving animals were following the ethical standards of China Animal Health and Epidemiology Center (No.2022-17). All applicable international, national, and institutional guidelines for the care and use of animals were followed. Fig. 1 The replication and virulence of A/mallard/Ningxia/ Y37/2021(H7N3) virus in mice. A Eight female BALB/c mice were intranasally inoculated with a 10 6 EID 50 virus. On 3 dpi, three mice were euthanatized after anesthetization, and the nasal turbinates, brains, lungs, livers, and spleens of the mice were pooled separately and homogenized in PBS containing antibiotics to make a 10% w/v tissue homogenate for virus titration in embryonated chicken eggs. Values are mean ± SD. The values labeled with a red star indicate that the virus was only detected in the organ(s). B Body weight changes of BALB/c mice were monitored daily for 14 days