Diversity and Reassortment Rate of Influenza A Viruses in Wild Ducks and Gulls

Influenza A viruses (IAVs) evolve via point mutations and reassortment of viral gene segments. The patterns of reassortment in different host species differ considerably. We investigated the genetic diversity of IAVs in wild ducks and compared it with the viral diversity in gulls. The complete genomes of 38 IAVs of H1N1, H1N2, H3N1, H3N2, H3N6, H3N8, H4N6, H5N3, H6N2, H11N6, and H11N9 subtypes isolated from wild mallard ducks and gulls resting in a city pond in Moscow, Russia were sequenced. The analysis of phylogenetic trees showed that stable viral genotypes do not persist from year to year in ducks owing to frequent gene reassortment. For comparison, similar analyses were carried out using sequences of IAVs isolated in the same period from ducks and gulls in The Netherlands. Our results revealed a significant difference in diversity and rates of reassortment of IAVs in ducks and gulls.


Introduction
Wild aquatic birds of the orders Anseriformes (ducks, geese, and swans) and Charadriiformes (gulls, terns, waders, and plovers) are natural hosts of IAVs [1]. In these birds, 16 antigenic subtypes of hemagglutinin (HA) and 9 antigenic subtypes of neuraminidase (NA) in various combinations were found [2]. IAVs of H1-H12, H14, and H15 HA subtypes mainly circulate in ducks; these IAVs replicate in the intestine, cause asymptomatic infections, and transmit by the fecal-oral route through the water [1]. IAVs of H13 and H16 HA subtypes mainly circulate in gulls, causing epidemics among chicks in densely populated breeding colonies. These gull IAVs replicate in the intestine and the respiratory tract, and can transmit both by the fecal-oral route and through the air [3][4][5]. Thus, the patterns of replication and transmission differ between IAVs of ducks and gulls.
IAVs in domestic poultry evolve from IAVs of wild aquatic birds. IAVs of poultry can occasionally transmit and initiate host-specific evolutionary lineages in pigs, humans, and other mammals [1,2]. Long-term adaptation in ducks enables efficient replication and release of the virus into the environment without significant pathogenic effects to the host [2]. In gulls, the virus also replicates asymptomatically, while in poultry it may evolve The aim of our work was to compare the IAV reassortment rate in wild ducks and gulls. From 2006 to 2019, we isolated IAVs from wild birds resting in a city pond in Moscow, Russia during their autumn migration. Thirty-seven duck IAVs and one duckorigin spillover H6N2 gull isolate were fully sequenced. We did not find stable IAV genotypes perpetuated from year to year. For comparison, a similar study on genome constellations was carried using sequences of duck and gull IAVs isolated in the same years in The Netherlands.

Viruses
Fresh feces of mallard ducks and gulls were collected in 2006-2019 on the shore of a city pond in Moscow. Feces were suspended in 1 mL of phosphate-buffered saline (PBS) supplemented with 0.4 mg/mL gentamicin, 0.1 mg/mL kanamycin, 0.01 mg/mL nystatin, and 2% MycoKill AB solution (PAA Laboratories GmbH, Pasching, Austria). The suspension was centrifuged for 10 min at 4000 rpm, and 0.2 mL of the supernatant were inoculated into 10-day-old chicken embryos. Allantoic fluid was collected after 48 h and tested by hemagglutination assay with chicken red blood cells. Positive samples were taken for further passaging. All isolated IAV strains are stored in the virus repository of the Chumakov Federal Scientific Center (Moscow, Russia). Full names, designations of the viruses, and GenBank accession numbers are given in Table S1 of Supplementary Materials.

Sequencing
Viral RNA was isolated from the allantoic fluid using QIAamp Viral RNA mini kit (Qiagen, # 52904) according to the instructions of the manufacturer. Reverse transcription was carried out at 42 • C for 1 h in a 25 µL reaction mixture containing 8 µL RNA, 1 µL uni12 primer with a concentration of 50 ng/µL (13.5 pmol/µL), 10 µL water, 1 µL 10 mM dNTP, 5 µL 5× buffer and 100 units of MMLV (Alpha-Ferment Ltd., Moscow, Russia). The resulting cDNA was used in PCR with specific terminal primers to synthesize full-length genome segments. The amplified fragments were separated by electrophoresis in 1-1.3% agarose gel in the presence of ethidium bromide and were eluted from the gel with a Diatom DNA Elution kit (Isogen Laboratory Ltd., Russia, Moscow # D1031). Sequencing reactions were performed with terminal or internal primers [44] using the BrightDye™ Terminator Cycle Sequencing Kit v3.1 (Nimagen, The Netherlands), followed by analysis on an ABI PRISM 3100-Avant Genetic analyzer (Applied Biosystems 3100-Avant Genetic Analyzer, Foster City, CA, USA,). For assembly and analysis of nucleotide sequences, the Lasergene software package (DNASTAR, Inc. Madison, WI, USA) was used.

Classification of Gene Variants and Detection of Gene Reassortants
All lineages and subordinate lineages were classified according to the topology of the phylogenetic trees using the approach described in [47], but with more detail. The major clades on each gene tree were defined by strong bootstrap support (>95%) and numbered. The minor clades within the major clade were designated by corresponding number and a letter.

Results
During the autumn periods of 2006-2019, feces of wild waterfowl were collected on the bank of a pond in Moscow city, and IAVs were isolated. Over 14 years, about 4000 samples were collected, and 38 strains of influenza A viruses of subtypes H1N1, H1N2, H3N1, H3N2, H3N6, H3N8, H4N6, H5N3, H6N2, H11N6, and H11N9 were isolated and completely sequenced ( Table 1 and Table S1 of Supplementary Materials). All isolates replicated in chick embryos to high titers, were infectious and immunogenic in mice, although these animals were generally not killed by the viruses. Five viruses tested were non-pathogenic in chickens [48,49].
The subtypes of isolates differed in different years. Until 2013, IAVs with hemagglutinins H3 and H4 dominated. These subtypes were no longer isolated after 2014 and were substituted by IAVs of the subtypes H1N2, H1N1, and H11N6. This change in the virus isolation pattern correlated with data from databases on the isolation of IAVs from ducks in Europe.

Evolutionary Relationships of Gene Segments
To study evolutionary relationships of gene segments, we expanded the set of IAVs isolated in Moscow by including the 191 mallard IAVs isolated in the Netherlands in the same period of time (2006-2019) [39]. We built phylogenetic trees for internal gene segments; clades/subclades on each tree were identified and numbered (Figures S1-S6 of Supplementary Materials).
The A/gull/Moscow/3100/2006 (H6N2) was not fundamentally separated from mallard IAVs. Thus, for all gene segments of the gull isolate, closely related variants can be found among the duck viruses ( Table 1 and Table S2 (Table 1).
Importantly, although segments of the same clade/subclade were found in duck IAVs isolated in different years, these viruses never preserved their full genome constellations and always contained a unique mixture of segments that belonged to different clades/subclades. Although four pairs of IAVs with identical constellations were detected among the Moscow viruses (Table 1), the viruses of each pair were isolated almost simultaneously and in the same place. These results support previous conclusion of Dugan and colleagues [34] that IAVs circulate in wild birds as a pool of gene segments, which reassort extensively and appear in a new combination each year.

Diversity of Gene Segment Constellations of IAVs in Ducks and Gulls
To compare the rate of natural reassortment of IAVs in ducks and gulls, we analyzed viruses isolated from ducks and viruses isolated from gulls in the Netherlands in the same period of time (2006-2019) [39]. The gull viruses were almost exclusively isolated from black headed gulls and were represented by 252 viruses of the H13 subtype, 94 viruses of the H16 subtype, and 11 mixed isolates.
The evolutionary trees for the gene segments were generated, and the clades and subclades were numbered (Figures S7-S12 of Supplementary Materials). The data on genome constellations of 228 duck IAVs viruses and 357 gull IAVs are presented in Tables S2-S5 of the Supplementary Materials; selected data are shown in Figure 1 and Table 3. Among 228 duck IAVs, we identified 187 distinctive genotypes. Pairs of isolates that matched in all gene segments, as a rule, were isolated on the same day.
One can conclude that two H13N3 isolates originated from H16N3 IAVs, in which HA and a number of other genes were replaced by gene segments of H13 IAVs via reassortment. Apparently, such reassortants are not viable enough; they were found in only two isolates despite the fact that there were seven mixed isolates in the same set (Table S4).
Some of the H16N3 IAVs represent reassortants containing internal gene segments of the H13 viruses. Thus, among 94 H16N3 viruses analyzed, we recognized 17 nonpersistent reassortants, with 13 of them carrying gene segments of H13 viruses (Table S4 of Supplementary Materials). Among 252 H13 viruses studied, we found 14 non-persistent reassortants, nine of which acquired at least one gene segment from H16 viruses. About 10% of the gull viruses isolated within one season were reassortants, this number being of the same order as the number of mixed isolates (3%) (see Table S3 of Supplementary  Materials).
Thus, although partial reassortment of gene segments can be observed over years among H13 and H16 IAVs, constellations of several segments remained stable for prolonged periods of time. The observed pattern is very different from what we observed in duck viruses of H1-H6 and H11 subtypes. No stable gene constellations could be detected in the sets of duck IAVs studied.

Discussion
Comparison of duck and gull viruses showed a large difference in the detection rate of reassortment in the main duck subtypes (H1, H2, H3, H4, H5, H6, and H11) and the gull subtypes H13 and H16. In gull viruses, reassortment was a fairly frequent, wellrecorded phenomenon, while in duck viruses, gene mixing was so intense that it was almost impossible to find viruses with the same genome constellation. There may be several explanations for this difference.
A key difference between gulls and ducks is that gulls breed in dense colonies with much mixing of chicks, whereas ducks are dispersed during breeding. As virus spreads among the gull chicks in nesting colonies, a single newly introduced variant can infect many birds. Large numbers of nearly identical strains can be isolated at this time. In the separated gull colonies, an outbreak of another variant of the virus may occur. Thus, three clusters of closely related isolates from 2008 (one H16N3 cluster and two H13N8 clusters) represent independent epidemics in three different colonies [5]. Sometimes, the virus is carried from one colony to another, leading to emergence of mix-isolates and reassortants. However, these events are rather exceptions than the rule. There are even lower chances of mixed infections in gulls during their seasonal migration, as gulls do not form large flocks during migration.
Because ducks do not breed in colonies, transmission among young ducks is limited during breeding. On the other hand, during the moult and the fall flight, ducks from multiple breeding areas may mix, creating ideal conditions for mixed infections with distinctive influenza viruses. On the pond where the Moscow duck viruses were isolated, hundreds of mallards accumulate in the fall gathering along the edges of the pond, where children throw pieces of bread. Ducks arrive from the north of Europe [50,51]. Mallards spend about 2 months on this pond. The first birds arrive in mid-September, followed by constantly increasing numbers of birds. As shown by Wille and colleagues, ducks can be infected sequentially by several variants of IAVs, and excrete virus intensively in their feces. Therefore, each introduced virus multiplies and infects other ducks, thus promoting multiple infections and reassortments [52].
The second factor affecting the rate of reassortment is the pressure of natural selection, which sweeps unsuccessful combinations. In the secondary hosts, such as chickens and humans, this factor is probably the main reason for the persistence of certain optimal gene constellations over the years. For example, during the epidemics in humans, when two subtypes co-circulate, co-infection and even reassortment is quite possible [13][14][15][16], but reassortants are usually less viable than the parental variants and do not become fixed in the population [18,19].
Probably, the same reason explains the stability of the genomes of highly pathogenic influenza viruses. After moving from wild birds to poultry, the viruses adapt to a new host and a new route of virus transmission. The adaptation of IAVs to chickens is associated with an increase of the virus pathogenicity [53]. This effect can be explained, at least in part, by the IAV evolution towards efficient transmission in infected poultry owing to cannibalism (that is, pecking and eating of sick individuals). A characteristic feature of chicken influenza is the selection of IAVs with polybasic cleavage site in the HA, which enables the virus to infect endothelial cells and to cause generalized infection [54,55]. Acquisition of new properties requires the coordinated evolution of all genes. In each of the evolutionary branches of IAVs that adapt to chickens, unique concerted changes in the genes could take place, so that exchange of some segments by reassortment can destroy an interplay between the genes and/or their products and make the virus less viable. Naturally, such reassortants will be swept by natural selection, leading to the persistence of specific constellations of the gene segments.
In duck viruses, stable genome constellations are largely absent [34]. Gull viruses, by contrast, have semi-stable genome constellations, among them, stable combinations of HA and NA. The H16 HA is tightly associated with the N3 NA. The NAs of IAVs with H13 HA (N2, N6, and N8 NAs) separated in the course of evolution from the ancestral viruses of ducks and adapted to the viruses of gulls. The variants with H13 and N3 did not form stable evolutionary lineages, probably, they are not viable enough. The functional balance of HA and NA is an essential element of the viability of IAVs [56]. The receptor specificity of gull IAVs differs from that of duck IAVs. In contrast to duck IAVs, gull IAVs efficiently bind to fucosylated receptors. Unlike all other IAVs of aquatic birds, H16 IAVs recognize both 2-3 and 2-6 sialyl-galactose receptors, being in this respect more similar to swine viruses than to duck viruses [57]. Likely, the neuraminidase of H16N3 viruses is specifically adapted to H16 HA.
The internal gene segment of the H13 and H16N3 viruses, as well as the HA and NA segments, represent separate evolutionary branches adapted to gulls. The internal genes of the H13 and H16 viruses are still interchangeable, but tend to form relatively stable constellations in each subtype.

Conclusions
Duck AIVs represent a unique variant of the symbiosis of the virus with the host, where the virus does not persist as a specific genome, but as a pool of genes, from which new genomic combinations are constantly formed [34]. However, in non-duck species, including gulls, a different evolution scenario is common, when the virus evolves in the host in the form of a whole genome.  Table S1: Full names, designations and accession number of genes of IAV isolated in Moscow in 2006-2019., Table S2: Duck IAV isolated in Moscow and the Netherlands in 2006-2019. Viruses are grouped by hemagglutinin subtype. The cells contain clade and subclade numbers for the corresponding genes. The strain names of IAV isolates containing all gene segments of the same subclade highlighted in orange. If the date of collection coincides, the names of strains is written in black. If the date of collection differed within the same year, the names of strains are written in red. If the strains were collected on different years, the name is written in green. Table S3