**1. Introduction**

In addition to pandemic COVID-19 virus infections, influenza virus remains a major threat to public health and causes significant morbidity and mortality worldwide from annual seasonal epidemics and periodic pandemics [1,2]. According to WHO, seasonal influenza affects 5–10% of the global population annually and results in 3-5 million hospitalizations and about half a million deaths per year. Pandemic influenza A virus (IAV) represents an even greater concern. Due to the rapid mutation rate of human IAVs, antigenically novel viruses are emerging periodically, which are difficult to predict, prevent, or treat [3]. Zoonotic influenza viruses of H5N1, H9N2 and recently, H7N9 and H10N8 subtypes have been identified as potentially pandemic [4,5].

Influenza viruses belong to the *Orthomyxoviridae* family and comprise negative-sense, single stranded, segmented RNA genome. The RNA genome segments are loosely encapsidated by the nucleoprotein into virus particle. There are four types of influenza virus—types A, B, C, and D. Influenza A viruses (IAV) and type B viruses are clinically relevant for humans.

IAV are further sub-divided based on the antigenic properties of surface glycoproteins into 18 hemagglutinin (HA) and 11 neuraminidase (NA) subtypes. Only a few IAV subtypes have been known to infect humans, while the majority of them are harbored in their natural hosts such as waterfowl, shorebirds, and other species [6]. Cases of H7N9 human infections caused by an avian-origin H7N9 virus emerged in eastern China in March 2013 [7,8]. This novel virus has immediately raised pandemic concerns as historically, pandemics were caused by the introduction of new subtypes into immunologically naïve human populations [9]. Phylogenetic results indicate that novel H7N9 virus was a triple reassortant derived from avian influenza viruses [7]. Since 2013, surveillance of live poultry markets routinely detected H7N9 virus [10]. Human infections with H7N9 virus were associated mainly with the exposure to infected poultry [11] and were identified in many cities in China [12]. Both low pathogenicity avian influenza (LPAI) and high pathogenicity avian influenza (HPAI) H7N9 viruses have been recorded. The first wave of H7N9 was associated with LPAI virus and lasted from March until September 2013. The following four waves occurred annually until 2017 (Figure 1). During the fifth wave in the 2016/17 season, the emergence of HPAI H7N9 viruses was detected. After no reported human cases of HPAI H7N9 for over a year, another HPAI H7N9 case with severe disease was reported in mainland China in late March 2019, indicating the continuing public health threat from the H7N9 subtype [13]. HPAI subtype H5 and H7 proteins contain multiple basic amino acid cleavage sites between HA1 and HA2 domains within HA proteins, which can be cleaved by furin-like proteases [14] in many host cells and organs that can lead to the efficient spread of the virus and severe disease in humans. In contrast, HA of LPAI viruses does not have the furin cleavage site.

**Figure 1.** Phylogenetic tree of hemagglutinin (HA) sequences derived from human H7N9 viruses [15]. The evolutionary history was inferred using the neighbor-joining method with Kimura distances. Five major clusters are shown as a collapsed branch. A/Netherlands/219/2003 is defined as an outgroup. The Yangtze River Delta and Pearl River Delta lineages are circulating in China. HPAI H7N9 viruses, which harbor multiple basic amino acids in the HA cleave site, are included in the Yangtze River Delta lineage. Permission: Viruses https://doi.org/10.3390/v11020149.

A fatality rate of up to 38% was reported for H7N9 viruses [16], which highlights the need for a safe and effective vaccine [17]. Several candidate H7N9 vaccine viruses have been prepared and listed by WHO (Table 1). These candidate vaccine viruses are available to vaccine developers for the preparation of H7N9 vaccine in the case of a pandemic. The majority of current vaccine manufacturers prepare vaccines either as split subvirions or live-attenuated viruses, and they are mostly dependent on fertilized chicken eggs as production "bioreactors". This technology is unlikely to meet the vaccine production demand during the rapid pandemic spread [18]. Scalability issues (one vaccine dose/egg), the relatively long 6-month time period from strain isolation to final dose formulation and the requirement of biosecurity facilities for HPAI are the major obstacles for egg-based production [19]. In addition, IAV can acquire adaptive mutations when grown in eggs, which can interfere with the vaccine performance and efficacy. According to the action plan published by WHO in 2006, more than 2.34 billion monovalent vaccine doses will be needed in the case of a global pandemic, which justifies the development of novel technologies capable of supporting surge demand for pandemic influenza vaccine within a short period of time [20].


**Table 1.** WHO-recommended vaccine strains for H7N9 virus (adapted from [21]).
