Prevalence of Influenza A Viruses in Ducks Sampled in Northwestern Minnesota and Evidence for Predominance of H3N8 and H4N6 Subtypes in Mallards, 2007–2016

SUMMARY.

Long-term comprehensive studies of avian influenza virus subtypes in ducks not only contribute to understanding variations and patterns of subtype diversity, but also can be important in defining seasonal and temporal risks associated with transmission of potentially highly pathogenic H5 and H7 subtypes to domestic poultry. We analyzed influenza A virus (IAV) surveillance data from dabbling ducks collected at an important migratory stopover site in northwestern Minnesota from 2007–2016 and identified prevalence and subtype diversity throughout this period. In total, 13,228 cloacal and oropharyngeal swabs from waterfowl were tested over the 10-year period; the majority of these waterfowl were mallards sampled from late August through late September (n = 9133). From these, 1768 IAVs were isolated (19.4% mean annual prevalence, ranging from 11.0% in 2007 to 32.8% in 2011), and both hemagglutinin (HA) and neuraminidase were identified for 1588. Although subtype diversity and prevalence varied by year, H3 and H4 HA subtypes predominated in all years, accounting for 65.7% of the observed HA subtype diversity. The mechanisms driving this consistent pattern of subtype diversity and predominance are not understood but may include factors at the host, population, and virus level.

The extensive subtype diversity observed with influenza A viruses (IAVs) in waterfowl populations is well described, but patterns related to predominant and overrepresented subtypes as well as the seasonal and temporal variation in subtype diversity have received limited attention. In North American waterfowl, the occurrence of certain hemagglutinin (HA) and neuraminidase (NA) combinations such as H1N1, H2N3, H3N8, H4N6, H5N2, H6N1, H7N3, H8N4, H10N7, and H11N9 are overrepresented within a given HA subtype (3,9,17,18). Of these, two subtypes seem to predominate (H3N8 and H4N6) all other HA and NA combinations (18). Likewise, consistent seasonal shifts in subtype diversity have been reported in North American blue-winged teal populations, with H3 and H4 subtypes most commonly isolated during fall migration and H7 and H10 subtypes more common during spring migration (14). The mechanisms responsible for these patterns and their significance to the epidemiology of IAVs are not currently understood. This study builds upon previous short-term (2-year) studies of IAVs in staging and migrating waterfowl populations in northwestern Minnesota (3,18) by examining a 10-year data set (2007–2016) from this same area. The objectives of the study were the following: 1) to further describe potential variation in annual prevalence and subtype richness over the 10-year period and 2) to determine if H3N8 and H4N6 subtypes consistently predominate in ducks in northwestern Minnesota annually.

MATERIALS AND METHODS

Sample collection.

Samples were collected from nine species of dabbling ducks in summer and fall from 2007 to 2016 from three locations in northwestern Minnesota previously described by Wilcox, et al. (18): Roseau River Wildlife Management Area (WMA; Roseau County), Thief Lake WMA (Marshall County), and Agassiz National Wildlife Refuge (NWR; Marshall County). Due to their relative abundance, mallards (Anas platyrhynchos) were the most frequently sampled species (77.7% of all samples) and were the only species sampled from 2013–2016. Other dabbling duck species sampled prior to 2013 included American black duck (Anas rubripes; 0.3% of all samples), American green-winged teal (Anas carolinensis; 4.3%), American wigeon (Anas americana; 0.9%), blue-winged teal (Spatula discors; 11.8%), gadwall (Anas strepera; 1.4%), northern pintail (Anas. acuta; 1.2%), northern shoveler (Spatula clypeata; 2.1%), and mallard–unidentified species hybrid (0.2%).

Birds were captured by night-lighting with handheld nets (2007–2008) and rocket nets (2007–2016). From 2007–2011, cloacal (CL) swabs were collected as previously described (18); from 2012–2016, sampling included paired oropharyngeal and CL swabs. Samples were placed in viral transport media and stored at —80 C until testing, as previously described (18).

Virus isolation and subtyping.

Virus isolation was attempted in specific-pathogen-free eggs (11,17,18). Harvested amnio-allantoic fluid was tested by hemagglutination assay (6), and all assay-positive samples were screened for IAVs by matrix real-time reverse transcriptase PCR (RRT-PCR) (2). All RRT-PCR positive samples were further screened with H5- and H7-specific RRT-PCR (16), and any positive samples were submitted to the National Veterinary Services Laboratories (Ames, Iowa) for confirmation of pathogenicity and final subtype determination. All other subtypes were identified by serotyping, sequencing, or with subtype-specific RT-PCR, depending on the year of recovery (1,7,12).

Data analysis.

Variability in sampling efforts existed both temporally and species-wise throughout the sample period. Because prevalence and subtype diversity are temporally and host-species dependent (18), analyses focused on the most ubiquitously sampled species (mallards) and a consistent sampling period in order to standardize data for comparisons of prevalence and subtype diversity across all years. Analyses were restricted to data collected within five contiguous weeks from August 23 to September 26. At this site, this period is reported to correspond with mallard staging and migration and also coincides with the highest IAV subtype diversity (18).

The frequency of H3N8 and H4N6 viruses compared with other subtypes was evaluated using chi-square tests comparing the observed frequency with an expected frequency based on the assumption that all subtypes had an equal probability of detection.

RESULTS AND DISCUSSION

All waterfowl species, all time periods, 2007–2016.

Of the 13,228 wild waterfowl samples collected from 2007–2016, 2329 IAV isolates were confirmed by IAV matrix RRT-PCR (17.4% mean prevalence). Complete HA and NA subtypes were identified for 2106 (90.4%) of these isolates, most of which were identified as H3 or H4 HA subtypes (31.4% and 34.3% of total isolates, respectively). Similar patterns were identified in both mallards and nonmallard species. Of the 2954 samples collected from nonmallard species from 2007–2012, 250 isolates were recovered. Of these, 74.4% were identified as H3 or H4 HA subtypes, mostly represented by the H3N8 and H4N6 subtype combinations. Prevalence estimates for IAVs in samples from these species included American black duck, 18.6% (n = 43); American green-winged teal, 8.2% (n = 573); American wigeon, 6.8% (n = 117); blue-winged teal, 7.2% (n = 1566); gadwall, 9.0% (n = 188); northern pintail, 11.5% (n = 165); northern shoveler, 12.7% (n = 275); and mallard–unidentified species hybrid, 14.8% (n = 27).

Mallards collected August 23–September 26, 2007–2016.

The majority of samples collected over the 10-year period were obtained from mallards during late August through September (n = 9133). Of these, 1768 (19.4%) IAVs were isolated; annual prevalence ranged from 11.0% in 2007 to 32.8% in 2011 (Fig. 1). The majority of the tested samples were from juvenile birds (n = 6352; 69.5% of samples), and overall mean IAV prevalence was higher in juvenile (23.3%) compared to adult (8.0%) mallards. This relationship between age and IAV prevalence has been repeatedly observed in ducks (5,13) and was consistent among years of this study.

Fig. 1.

Prevalence of all influenza A viruses (points connected by dotted line) and those of H3 (gray bars) and H4 (black bars) hemagglutinin subtypes in mallard samples from northwestern Minnesota, August 23 to September 26, 2007–2016. Error bars represent 95% confidence intervals.

Subtypes were determined for 1588 (89.8%) IAVs isolated from mallards from August 23 to September 26, 2007–2016 (Table 1), and 67 unique HA and NA combinations were identified (Table 2). HA subtypes 1–12 and NA subtypes 1–9 were detected at least once through the 10-year period, with an average of 7.8 HA subtypes detected per year (Table 2). Although subtype diversity in mallards varied by year, H3 and H4 were the most prevalent HA subtypes over the 10-year period (Table 1). Overall, the mean annual prevalence (based on number of mallards sampled) observed for viruses of H3 and H4 subtypes were 5.3% and 6.5%, respectively, and these subtypes accounted for 65.1% of the total subtype diversity observed. Furthermore, H3 and H4 were both the most predominant HA subtypes each year in both adults and juvenile mallards from 2008–2016 (Table 1). An exception occurred in 2007 that was characterized by a low overall prevalence in both adults and juvenile birds (11.0%; Fig. 1). In that year, H3 and H4 subtypes represented 23.6% and 10.6% of total subtype diversity but other subtypes were well represented (15.5% H1, 12.4% H7, and 10.6% H10). However, during 2007, a large number of positive samples were collected prior to the 5-week sample period selected for this study; during that early period (July 11–August 9) and for the total 2007 sample, H3N8 and H4N6 subtypes did predominate (18).

Table 1.

Summary of influenza A viruses (IAVs) recovered from mallards in northwestern Minnesota, August 23 to September 26, 2007–2016.

	Year
	2007	2008	2009	2010	2011	2012	2013	2014	2015	2016	Total
Samples tested (n)	1466	983	1102	892	1215	967	638	532	586	752	9133
IAV isolates (%)	161 (11.0)A	237 (24.1)	159 (14.4)	183 (20.5)	399 (32.8)	160 (16.5)	96 (15.0)	168 (31.6)	87 (14.8)	118 (15.7)	1768 (19.6)
IAV identified to HAB subtype	147	237	156	180	371	129	93	165	83	114	1675
IAV identified to NAC subtype	142	231	157	179	344	122	88	165	84	118	1630
IAV identified to HA and NA subtype	131	230	155	176	331	122	85	163	81	114	1588
No. HA types	10	8	8	7	9	8	6	6	9	9	12
No. NA types	9	8	8	9	9	9	6	7	7	7	9
H3NxD isolates (%)	38 (23.6)	74 (31.2)	45 (28.3)	99 (54.1)	92 (23.1)	41 (25.6)	30 (31.3)	34 (20.3)	9 (10.3)	25 (21.2)	487 (27.5)
H4Nx isolates (%)	17 (10.6)	76 (32.1)	50 (31.4)	38 (20.8)	146 (36.6)	30 (18.6)	55 (57.3)	98 (58.3)	44 (50.6)	42 (35.6)	596 (33.7)
H3N8E	19 (11.8)**	32 (13.5)**	31 (19.4)**	56 (30.6)**	67 (16.8)**	26 (16.3)**	15 (15.6)**	19 (11.3)**	7 (8.0)*	20 (16.9)**	292 (16.5)**
H4N6 E	16 (9.9)**	47 (19.8)**	18 (11.3)**	23 (12.6)**	111 (27.8)**	24 (15.0)**	44 (45.8)**	76 (45.2)**	29 (33.3)**	25 (21.2)**	413 (23.4)**

^ANumbcr of IAVs recovered (prevalence as % of total annual collection).

^bHA = hemagglutinin.

^CNA = neuraminidase.

^DIncludes any NA type, including mixed and Nx (unknown).

^E*Observed frequency of H3N8 and H4N6 exceeds expected frequency (P ≤ 0.05)

^**Observed frequency of H3N8 and H4N6 exceeds expected frequency (P ≤ 0.001).

Table 2.

Number of influenza A virus hemagglutinin (HA) and neuraminidase (NA) subtypes detected in mallards, northwestern Minnesota, August 23 to September 26, 2007–2016.^AB

	N1	N2	N3	N4	N5	N6	N7	N8	N9	Total
H1	48**	2	2	3	3	2	—	3	3	66
H2	3	14	61**	2	12	5	1	21	5	124
H3	21	34*	9	1	5	93**	1	292**	13	469**
H4	8	42**	3	1	5	413**	—	90**	3	565**
H5	11	25	1	1	1	—	—	1	2	42
H6	88**	45**	2	1	9	4	—	18	1	168
H7	—	—	18	—	—	—	—	1	1	20
H8	—	—	—	1	—	—	—	—	—	1
H9	—	—	—	—	—	—	—	—	1	1
H10	1	3	1	—	—	2	34*	—	1	42
H11	3	8	3	—	—	1	—	—	60**	75
H12	—	—	—	—	14	—	—	—	1	15
Total	183	173	100	10	49	520	36	426	91	1588

^A*Observed frequency of HA/NA subtype or HA (Total column) exceeds expected frequency (P ≤ 0.01)

^**Observed frequency of HA/NA subtype or HA (Total column) exceeds expected frequency (P ≤ 0.001).

^BDash indicates that subtype was not detected.

Five subtype combinations represented the majority of subtype diversity: H3N6 (5.9% of isolates), H3N8 (18.5%), H4N6 (26.1%), H4N8 (5.7%), and H6N1 (5.6%). Only H2 and H3 viruses were detected in combination with all of the NA subtypes (N1–N9). A single H8N4 virus was isolated during the five focused weeks of this analysis, in 2014, but this rare subtype combination was identified in mallards 20 times in earlier weeks in four years (2007–2010).

It has been suggested that both influenza prevalence and HA prevalence follow a cyclic pattern (9,15). However, although overall IAV prevalence peaked every 3 years, with the highest prevalence occurring in 2008 (24.1%), 2011 (32.8%), and 2014 (31.6%), alternating shifts in HA predominance of H3 and H4 viruses were not observed (Fig. 1). The H3 viruses were most commonly isolated in 2007 (23.6% of all isolates), 2010 (54.1%), and 2012 (25.6%) and H4 in 2009 (31.4%), 2011 (36.6%), 2013 (57.3%), 2014 (58.3%), 2015 (50.6%), and 2016 (35.6%). In 2008, H3 and H4 viruses were codominant, representing 31.2% and 32.1% of the viruses isolated, respectively (Table 1).

Of the H3 and H4 subtypes observed in this study, the most frequently occurring HA and NA combinations were H3N8 and H4N6; these two subtypes were detected every year and represented 18.5% and 26.1% of the total subtype diversity observed over the 10-year period, respectively (Fig. 2). With two exceptions, they also represented the two predominant subtypes in individual years. In 2007, H1N1 and H3N8 were codominant, each representing 11.8% of all isolates, and in 2009, H6N1 and H3N8 predominated (19.5% and 11.3%, respectively) (Table 1). In all years, however, including 2007 and 2009, the frequencies of the H3N8 and H4N6 subtypes were higher than expected, assuming a random probability of occurrence (Table 1).

Fig. 2.

Diversity of H3 and H4 subtype combinations recovered from adult and juvenile mallards in northwestern Minnesota, August 23 to September 26, 2007–2016.

All nine NA subtypes were detected in combination with the H3 and H4 hemagglutinin genes except for N7, which was not detected in association with H4N6. Aside from H3N8 and H4N6, the H3N6 and H4N8 subtypes were also often detected (Table 2). The H3N6 and H4N8 subtypes were detected every year, and annual prevalence appeared proportional to that of H3N8 and H4N6 in both adult and juvenile birds (Fig. 2). It is probable that these combinations result from annual reassortment between the H3N8 and H4N6 subtypes.

The significance of the high proportion of H3N8 and H4N6 IAVs during periods of peak IAV prevalence in North American waterfowl is currently unclear. These infections with H3 and H4 viruses, however, could indirectly affect subsequent IAV prevalence and subtype diversity in these populations through resulting homo- and heterosubtypic immunity (10). Such immune pressure may serve to seasonally structure HA predominance, providing a means for H7 and H10 viruses to predominate during spring migration (14). These immune pressures may also have relevance to the potential for exotic IAVs such as the highly pathogenic A/Goose/ Guangdong/1/1996 lineage clade 2.3.4.4 H5N8 to successfully establish in North America (8). The mechanisms driving this consistent pattern of subtype diversity and predominance may include factors at the host, population, and virus level and warrant further investigation.

Abbreviations:

CL

cloacal

HA

hemagglutinin

IAV

influenza A virus

NA

neuraminidase

NWR

National Wildlife Refuge

RRT-PCR

real-time reverse transcriptase PCR

WMA

Wildlife Management Area