Although the influenza D virus has not been isolated from humans, a cross-sectional serological study was performed on human serum samples from 35 cattle-exposed and 11 non-cattle-exposed adults to detect IDV antibodies using Hemagglutination Inhibition (HI) and Microneutralization (MN) assays. Overall, 94% of the cattle-exposed persons had a positive HI and MN titer. Characteristics of seropositive cattle-exposed persons included: 41% worked with sick cattle, 4% worked with swine diagnosed with respiratory illness, and 41% self-reported febrile illness in the previous year. When testing the people living close to cattle farms (with potential risk for infection), 8 of 741 serum samples collected were positive for HI antibodies (titers ≥ 40) [7].
Song et al. have analyzed the biological characteristics of the major surface glycoproteins in both IAV and IDV [14]. It was found that IAV can specifically bind to the receptors of N-Acetylneuraminyl-(2-3)-Galactose (originating in poultry) or N-Acetylneuraminyl-(2-6)-Galactose (human), and some subtypes of IAV can bind to both receptors. The bovine-derived IDV receptors are N-Acetylneuraminyl-(2-3)- Galactose and N-Acetylneuraminyl-(2-6)-Galactose, while the swine-derived IDV receptor is N-Acetylneuraminyl-(2-6)-Galactose, but not N-Acetylneuraminyl-(2-3)- Galactose. These findings suggest that the bovine-derived IDV may be transmitted between species just like IAV and even with more widespread cell tropism [15]. At the same time, the saccharide microarray experiment proves that IDV and ICV are similar in structure. The three-dimensional crystal structure of the HEF protein receptor complex of IDV shows the segments close to the top of HEF1, together with the 230-helix, 270-ring, 170-ring. In addition, unlike in ICV, three amino acids (T239, K235, D269) form a shallow cavity with an open channel, which may be one of the reasons why IDV gets a broader host spectrum [16–17]. Although deadly infections have not been reported in humans, IDV significance in public health should not be neglected.
We can predict the key sites that may influence interspecies transmission by building a random mutation library of the IDV PB2 protein and screening the unique mutation sites. This study used amino acid point mutations at positions 627 and 701 of influenza D viruses. There are two positions in the PB2 protein that affect the growth of influenza A viruses in mammalian cells: the amino acid at position 627 (PB2-627) and the amino acid at position 701 (PB2-701) [10]. Several credible studies have found that there is always an E627K mutation of PB2 in clinical strains isolated from people who had died or were severely infected with specific subtypes of influenza viruses. Furthermore, this phenomenon occurred not only in one influenza subtype, but the PB2-E627K mutation can also be found in multiple isolates from the patients who were seriously infected or died from influenza, including H5N1 H7N7, H7N9, H9N2, and H10N8 subtypes. Hence, the PB2-E627K mutation may be closely related to the high pathogenicity to humans in these influenza subtypes [18–21]. Mutation D701N in the PB2 protein has been found in human influenza virus subtype H3N2, highly pathogenic avian influenza virus subtype H5N1, and highly pathogenic avian influenza virus subtype H7N7, which has also been verified to enhance the pathogenicity of influenza virus in mice [22–24]. Such mutation can enhance the virus replication capacity in mammals by increasing the viral polymerase activity [25]. We decided to focus on amino acids at positions 627 and 701 in influenza D viruses based on these previous studies. We found that in IDV PB2, mutations of the position 627E and 701P are not the key sites that could affect the activities of influenza D virus polymerase.
Based on the results of the amino acid positions at 627 and 701 of IDV PB2, we determined some other amino acids in the IDV PB2 protein and analyzed their effects on polymerase activity. The results have been classified into three sections. First, mutations at specific amino acid sites lead to a reduction in viral polymerase activity or even a loss of the expression capacity of the polymerase protein. Secondly, mutations at some specific amino acid sites show no effect on viral polymerase activity. Thirdly, mutations at specific amino acid sites contribute to increasing the activities of the viral polymerase. The statistical analysis found three amino acid sites (M532, D533, and L534) that had enormous implications for IDV polymerase activities. Mutations M532I, D533S, and L534I increased the polymerase activities by 3.26 times, 9.92 times, and 2.66 times, respectively, indicating that these positions are critical. In addition, mutations at the positions 515-519, such as N515G, D516E, and E519Y, increased the polymerase activities by 4.73 times, 2.26 times, and 2.33 times, respectively. Those positions should be considered and focused on the surveillance efforts of influenza D viruses. Moreover, the screening and characterization of other amino acid sites are still under investigation.
Through the study of PB2 protein structure elucidation, residues 256-689 of the PB2 subunit (the PB2-CTD), which comprise the mid, cap-binding, and 627 domains, are highly flexible and could not be resolved in either the apo or promoter-bound states. The two key sites are respectively located in the linker domains and 627 domains of the PB2 protein (Fig. 4), which are too flexible to be elucidated [26]. It makes us think more about the existence of critical sites in the domains that we have not yet determined could affect the activity of influenza D virus polymerase, which may affect the characteristics of interspecies transmission.
As a consequence, our approach represents a strategy to evaluate the capacity of IDV replication and pathogenicity based on the polymerase activity. Accordingly, the results of this research contribute to understanding, assessing, and monitoring the risk of a potential IDV pandemic that may occur in the future. That is a way to identify which of those mutations under observation during the viral surveillance is more likely to cause a significant increase in virus replication, pathogenicity, and even transmission. There are, definitely, some possible additional factors, including environmental and epidemiological factors, etc., not captured in this study. The data on amino acid mutations in this study can be combined with other data revealing evolution to explore the risks of IDV cross-species transmission.