Influenza viruses are global zoonotic and human pathogens, and vaccination remains the main preventative measure against infection. The influenza virus is a member of the Orthomyxoviridae family. The genome is composed of eight negative single-sense RNA segments that determines the viral genus, alpha-, beta-, delta-, and gammainfluenzavirus that correspond to the species influenza A, B, D, and C viruses, respectively. Of the four influenza types, Types A and D are commonly isolated from animals, whereas influenza Types B and C are most commonly associated with human infection especially in children (1). The Type A influenza viruses are further classified into subtypes determined by the two major surface proteins, hemagglutinin (HA) and neuraminidase (NA). Currently, there are 18 HA subtypes and 11 NA subtypes that can be paired to create different influenza subtypes.

Influenza viruses are of international importance due to the widespread infection in different livestock, leading to vaccination being utilized across the veterinary field (2). Equine influenza viruses are important horse pathogens with policies in place that require horses be vaccinated for equine influenza viruses before participation in events or importation (3, 4). Furthermore, due to the transmission of influenza viruses from horses to dogs, as well as the endemic infection of influenza viruses in the canine population, canine vaccination is also recommended for dogs with high risk of exposure (5, 6). The swine industry uses primarily whole inactivated vaccines [WIVs—reviewed in reference (7)] that are developed using split-inactivated technologies (7, 8). The poultry industry utilizes the greatest variety of vaccine platforms, including split-inactivated virus, HA protein antigens, HA DNA vaccines, and recombinant technologies with other backbone viruses (9, 10).

However, during infection both HA and NA proteins are targets for neutralizing antibodies (11). The NA glycoprotein mediates viral egress and virion de-aggregation by cleaving sialic acids as well as contributing to motility through cleaving mucins in the upper respiratory tract (12, 13). Polyclonal NA-specific sera and NA inhibition (NAI) titers reduce, modulate, and protect against disease (14, 15). Further research has identified monoclonal NA-specific antibodies that neutralize viral growth (16). Although NA antibodies hinder viral replication, the induction of NAI antibody titers following vaccination is not as great as the induction of HAI titers, potentially due to either the split-inactivated vaccines lacking a standardized concentration of NA protein or the NA protein being destroyed during the split-inactivation process (15). Recently, research and vaccine development have focused on live-attenuated viruses that elicit NA antibodies, or protein vaccines that include the NA (17).

Currently, the enzyme-linked lectin assay (ELLA), MUNANA substrate, thiobarbituric acid (TBA) fluorescent-based assay, and NA-Star chemiluminescent assay are methods for measuring antibodies against the NA molecule (18–23). As the NA glycoprotein undergoes antigenic drift, the protein's ability to cleave sialic acid can be measured and quantified using these assays. All techniques assess the elicited antibody-specific inhibition of the NA after vaccination or infection. The ELLA measures the ability of the viral NA to cleave sialic acids from a large substrate (fetuin) similar to infection when sialic acids are expressed on the surface of the host cell, whereas the MUNANA and NA-Star techniques measure cleavage of small soluble chemical substrates (24). However, only the ELLA was proposed as the assay for measuring serum NA-inhibiting antibodies as a correlate for protection for humans (25).

Components in raw sera have non-specific inhibitory activity against NA activity (20). These initial findings were conducted with ferret sera that varied using different viruses from different influenza subtypes. However, treating sera with receptor-destroying enzyme (RDE) overnight and then heat-inactivating the sera for 8 h at 55°C mitigated the non-specific inhibition without loss of NA- or HA-specific inhibitory activity (20). The animal models used for influenza virus research are growing and now include more species. Not only is there a need to compare serological results between animal models that are used for human influenza viruses, but also endemic influenza virus infection in agricultural animal species requires a consistent method to quantify the NA-inhibiting antibodies as well. Therefore, it may be necessary to handle sera from different species differently when quantifying the NA inhibition responses, which may be key to determining overall vaccine effectiveness.

Therefore, animal sera from different species were characterized for their inherent inhibition of the ELLA with a panel of influenza viruses. Sera were compared for their ability to non-specifically inhibit the NA proteins of many influenza viruses representing different viral subtypes. Sera were collected and tested from varying animal serum sources against H1 and H3 human- and swine-isolated influenza strains as well as avian-isolated viruses with N2 and N3 proteins. Overall, there are many different variables that contribute to the interpretation of the ELLA assay, and understanding the innate characteristics of the host origin of the sera is critical to conducting the assay and interpreting the results. Therefore, it is important to standardize methodologies that will allow for consistent and reproducible results to assess anti-NA antibodies.

Influenza vaccine formulations, including live-attenuated virus, whole-inactivated virus, and protein subunit minutes, use NA as a vaccine component to elicit NA-specific antibodies (28). However, components in raw sera have anti-NA properties that result in inhibition of NA activity. The ELLA is used to measure antibody-mediated NA inhibition for cleaving a large substrate, and has been used to assess the effectiveness of NA-containing vaccines and anti-NA antibodies (29–32).

In this study, seven raw animal sera were tested for inhibition of virus in the ELLA assay (Table 2). All sera, regardless of species, inhibited at least one influenza virus (50% inhibition) with a dilution of >1:10. Five of the seven samples inhibited 50% NAI activity at a titer of >1:100. Sera contain innate host influenza inhibitors, such as complement protein of the α-, β-, and γ-class serum inhibitors. In horse and pig sera, the α-2-macroglobulin (γ-class) is one of the major innate influenza virus-neutralizing factors (33, 34). The γ-class inhibitors express sialic acids that bind specifically to the HA protein on influenza viruses and may inhibit the NA through steric interactions. These γ-class inhibitors are inactivated through RDE treatment using Vibrio cholerae NA and are resistant to viral sialidase activity (34, 35). There appear to be minor innate factors that result in the ability of horse and pig sera to inhibit different viruses in the panel.

Not all sera inhibited NA activity of all viruses. There were distinct inhibition profiles against specific influenza viruses in the panel. Innate inhibitors interact with influenza viruses through competitive binding of sialic acids to the HA protein receptor-binding site (RBS) (α- and γ-class) and with mannose-binding lectins (β-class) (36, 37). Depending on the host origin of the virus, the HA RBS may have stronger affinity for α-2,3 or α-2,6 sialic acids. The glycosylation of HA proteins has been associated with mannose-binding lectins (37). Further research into the contributions of HA sialic acid binding specificity and the glycosylation of HA and NA surface proteins is needed to determine if it is significantly impacting the variation of NA inhibition observed here across the different viruses.

The innate NA inhibition of different species sera is useful for determining the appropriate treatment before conducting for ELLA assays. To account for the innate inhibitors observed here, sera may either be heat treated or RDE treated overnight at 37°C to cleave competing sialic acids from α- and γ-class inhibitors and heat inactivated at 56°C for a minimum of 30 min to inactivate the heat-labile β-class inhibitors and up to 8 h to fully inactivate the V. cholerae NA, when used with ferret sera (20). Immunoglobulins vary in their heat stability with IgG being more stable than IgA which is more stable than IgM (38). With researchers using different inactivation methods, it may be inappropriate to compare titers between sera heat inactivated for 30 min to RDE-treated sera that is heat inactivated for 8 h.

However, one of the major limitations of the study design was the inability to quantify within-species variability due to the limited sources of the sera. This variability can be further investigated to determine if age, sex, or husbandry practices, such as farm or laboratory origin animals, have any effect on the results. Furthermore, the serum inactivation procedure for conducting the ELLA may be different between species. To determine the appropriate method, positive control antiserum is necessary to confirm that no loss in NA-specific antibodies is observed during treatment. Given the wide panel of viruses and different animal models tested here, those samples were not available. Lastly, the wide variability in the NA activity titers observed between viruses (Table 1) may either be from increased enzymatic capacity, i.e., a virus' NA protein cleaves more sialic acid at a higher rate than another viral NA, or from having a higher NA content per PFU. Therefore, why different viruses had such variability in NA activity was undetermined.

In conclusion, with the increase in NA research, the RDE treatment, the inactivation time, and the temperature used to inactivate sialidases should be clearly described with the negative control data provided for each viral strain with serum species used for the assay in order to accurately interpret the results. This information will allow for comparison across species or if comparison of anti-NA serological results need to assessed within the same species.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by IACUC of University of Georgia.

AS: concept, writing, analysis, and statistics. TR: editing and writing. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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