Research Article: Adaptive Heterosubtypic Immunity to Low Pathogenic Avian Influenza Viruses in Experimentally Infected Mallards

Date Published: January 20, 2017

Publisher: Public Library of Science

Author(s): Karen M. Segovia, David E. Stallknecht, Darrell R. Kapczynski, Lisa Stabler, Roy D. Berghaus, Alinde Fotjik, Neus Latorre-Margalef, Monique S. França, Charles J. Russell.


Mallards are widely recognized as reservoirs for Influenza A viruses (IAV); however, host factors that might prompt seasonality and trends in subtype diversity of IAV such as adaptive heterosubtypic immunity (HSI) are not well understood. To investigate this, we inoculated mallards with a prevailing H3N8 low pathogenic avian influenza virus (LPAIV) subtype in waterfowl to determine if prior infection with this virus would be protective against heterosubtypic infections with the H4N6, H10N7 and H14N5 LPAIV subtypes after one, two and three months, respectively. Also, we investigated the effect of cumulative immunity after sequential inoculation of mallards with these viruses in one-month intervals. Humoral immunity was assessed by microneutralization assays using a subset of representative LPAIV subtypes as antigens. Our results indicate that prior inoculation with the H3N8 virus confers partial protective immunity against subsequent heterosubtypic infections with the robustness of HSI related to the phylogenetic similarity of the HA protein of the strains used. Furthermore, induced HSI was boosted and followed by repeated exposure to more than one LPAIV subtype. Our findings provide further information on the contributions of HSI and its role in the dynamics of IAV subtype diversity in mallards.

Partial Text

Wild aquatic birds from the order Anseriformes and Charadriiformes are the major reservoir of Influenza A viruses (IAV) [1, 2]. Mallard (Anas platyrhynchos) is the most common dabbling duck species in North America and Europe and is an important species in the ecology of avian influenza [3, 4]. Seasonal patterns of IAV infections in waterfowl have been described in the Northern Hemisphere (North America, Europe, and Asia) [1, 5–7] where IAV prevalence increases at the end of the summer and peaks in early fall as a result of the congregation of adult and immunologically naïve young birds in breeding grounds prior to Southern migration [2, 3]. Detection rates drop during winters with slight increases during spring migrations [5, 8]. Prevalence of IAV is higher among juvenile ducks as compared to adults, which is probably a result of immunity induced by previous IAV infections in adult birds [5, 9, 10]. In North America, the more common HA subtypes of IAV reported in ducks are H3 and H4, followed by H1, H2, H6, H7, H10, and H11, while the H14 subtype has rarely been detected in surveillance studies [3, 5, 11, 12]. Although the H4 and H6 subtypes were also frequent in surveillance studies in Europe, recurrent detection of other subtypes was not significantly different [5, 13]. Seasonal patterns of IAV prevalence among wild birds have been described; however, factors and mechanisms that drive diversity and prevalence of IAV subtypes such as the effects of homo- and heterosubtypic immunity remain unclear [14, 15]. Previous studies have demonstrated the induction of homosubtypic and partial heterosubtypic immunity in mallards [16–20], and this has been supported further by field observations [21]. At the same time, additional studies are needed to understand better the effect of reinfections with common and less frequently detected subtypes of LPAIV on the ecology of influenza in the wild bird reservoir.

Overt clinical signs were not observed in any of the experimental groups, and all OP and CL samples collected immediately before each inoculation tested negative for IAV by virus isolation and qRT-PCR. Serum samples from all ducks collected before IAV inoculation tested negative by both ELISA and MN. Back titration of the inocula determined a titer of 106.2 EID50/0.1 ml for H3N8, 106.0 EID50/0.1 ml for H4N6, 106.3 EID50/0.1 ml for H10N7 and 105.7 EID50/0.1 ml for H14N5. Also, subtyping by RT-PCR of the viruses isolated after each infection confirmed that birds were excreting only the subtype of virus inoculated at each time point (data not shown).

Previous infection with the H3N8 did not prevent reinfection with the H4N6, H10N7, and H14N5 LPAIVs after one, two and three months, respectively; but induced different levels of protective immunity as evidenced by a decrease in the duration and amount of viral shedding after heterosubtypic infections. The induced protective immunity was boosted following exposure to more than one virus subtype, with complete abrogation of viral shedding upon inoculation with H14N5 in birds previously exposed to three different subtypes (H3N8xH4N6xH10N7). Moreover, three out of five ducks primed with H3N8 were protected against secondary infection with the HA and NA clade-related H14N5 virus three months later, as none of them had detectable levels of viral excretion by virus isolation or RT-PCR after 1 dpi. Shedding of H10N7 was delayed in some birds primed with H3N8 or H3N8xH4N6, and the viral shedding was lower in ducks previously exposed to H3N8xH4N6 viruses.