A North American H7N3 Influenza Virus Supports Reassortment with 2009 Pandemic H1N1 and Induces Disease in Mice without Prior Adaptation

Abstract

Reassortment between H5 or H9 subtype avian and mammalian influenza A viruses (IAV) can generate a novel virus that causes disease and transmits between mammals. Such information is currently not available for H7 subtype viruses. We evaluated the ability of a low-pathogenicity North American avian H7N3 virus (A/shorebird/Delaware/22/2006) to reassort with mammalian or avian viruses using a plasmid-based competition assay. In addition to genome segments derived from an avian H7N9 virus, the H7N3 virus reassorted efficiently with the PB2, NA, and M segments from the 2009 pandemic H1N1 (PH1N1) virus.In vitro and in vivo evaluation of the H7N3:PH1N1 (7 + 1) reassortant viruses revealed that the PB2, NA, or M segments from PH1N1 largely do not attenuate the H7N3 virus, whereas the PB1, PA, NP, or NS genome segments from PH1N1 do. Additionally, we assessed the functionality of the H7N3:PH1N1 7 + 1 reassortant viruses by measuring the inflammatory response in vivo We found that infection with wild-type H7N3 resulted in increased inflammatory cytokine production relative to that seen with the PH1N1 strain and that the increase was further exacerbated by substitution of PH1N1 PB2 but not NA or M. Finally, we assessed if any adaptations occurred in the individually substituted segments after in vivo inoculation and found no mutations, suggesting that PH1N1 PB2, NA, and M are genetically stable in the background of this H7N3 virus. Taking the data together, we demonstrate that a North American avian H7N3 IAV is genetically and functionally compatible with multiple gene segments from the 2009 pandemic influenza virus strain without prior adaptation.

Importance: The 2009 pandemic H1N1 virus continues to circulate and reassort with other influenza viruses, creating novel viruses with increased replication and transmission potential in humans. Previous studies have found that this virus can also reassort with H5N1 and H9N2 avian influenza viruses. We now show that several genome segments of the 2009 H1N1 virus are also highly compatible with a low-pathogenicity avian H7N3 virus and that these reassortant viruses are stable and not attenuated in an animal model. These results highlight the potential for reassortment of H1N1 viruses with avian influenza virus and emphasize the need for continued surveillance of influenza viruses in areas of cocirculation between avian, human, and swine viruses.

FIG 1

A North American H7N3 virus preferentially selects avian and 2009 pandemic H1N1 genome segments following competitive transfection. (A) Seven genome segments derived from strain A/shorebird/Delaware/22/2006 (H7N3) were cotransfected with four (NA only) or five variants of the eighth genome segment. These variants included the homologous gene segment from A/shorebird/Delaware/22/2006 (H7N3) as well as those of A/mallard/Alberta/177/2004 (H7N9), A/Memphis/03/2008 (_SH1N1), A/California/04/2009 (_PH1N1), and A/Puerto Rico/08/1934 (_PR8H1N1) virus. Following transfection, individual virus particles were cultured in MDCK cells using a limiting dilution assay. Virus-positive wells were identified by HA assay, and the remaining supernatant was used to purify viral RNA. cDNA was generated using a universal influenza primer, and the segment of interest was amplified using segment-specific PCR primers and analyzed by restriction length fragment polymorphism (RFLP). RT-PCR, reverse transcription-PCR. (B) Pie charts depicting the relative distribution of four or five gene segments that were identified among the competitive reverse genetics-derived H7 viruses. The results represent the cumulative distribution of results from two or more independently repeated experiments; 334 viruses in total were sampled. The NA gene of A/Memphis/03/2008 (NA-inhibitor insensitive) was excluded from these analyses. Phylogenetic trees for each corresponding segment illustrate the genetic relationships between the assayed segments (bootstrapped 1,000×). The line beneath each phylogenetic tree represents 1 nucleotide substitution per 100 nucleotides.

FIG 2

Several H7 single-gene reassortant viruses possessing a _PH1N1 segment replicate efficiently in mammalian tissue culture. (A and B) MDCK cells were inoculated with 50 TCID₅₀ of the indicated virus, and the virus titer (TCID₅₀ per milliliter) in the supernatant was determined at 24 h (A) or 48 h (B) postinoculation by virus titration assay performed on MDCK cells. (C and D) A549 cells were inoculated with 10⁵ TCID₅₀ of the indicated virus, and the virus titer (TCID₅₀ per milliliter) in the supernatant was determined at 24 h (C) or 48 h (D) postinoculation by virus titration assay on MDCK cells. The limit of detection was 100 TCID₅₀ (dotted line). The data represent geometric means + standard errors of the means of results determined with four samples derived from two or three independent experiments performed in duplicate (*, P < 0.05; ***, P < 0.005 [compared to H7N3 values]).

FIG 3

PA protein from pandemic H1N1 virus increases the polymerase activity of H7N3 in human 293T cells. (A) A reporter assay was used to measure the relative levels of polymerase activity of different polymerase protein complex combinations between H7N3 and _PH1N1 viruses, including _PH1N1 PA P₂₉₅L, as described in Materials and Methods. The relative polymerase activity data were first normalized to the Renilla activity and compared to that of a full set of H7N3 polymerase proteins. Blue boxes represent H7N3 proteins, white boxes represent pH1N1 proteins, and spotted white boxes represent pH1N1 PA-P₂₉₅L. Data represent the average normalized values from three or five experiments performed in duplicate (**, P < 0.01; ***, P < 0.005 [compared to H7N3 values]).

FIG 5

Reassortant H7 viruses containing _PH1N1 PB2, NA, or M induce considerable morbidity in mice. (A to C) Groups of male C57BL/6J mice were inoculated intranasally with 10⁴ TCID₅₀ of the indicated viruses, and weight loss was measured for 7 days. Morbidity is displayed as percentages of original body weight remaining for mice inoculated with the indicated virus that showed weight loss that was significantly greater than (A), equal to (B), or significantly less than (C) that seen with the parental H7N3 virus. (D) Male C57BL/6J mice were inoculated intranasally with 10³ TCID₅₀ of H7N3, _PH1N1, or H7N3 containing the PB2 segment of _PH1N1 (H7N3_PB2), and weight loss was measured for 14 days. The data represent the averages and standard errors of the means of results determined for at least six animals from two or more independently performed experiments per virus. (*, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001 [compared to H7N3 values]; ##, P < 0.01; ###, P < 0.005; ####, P < 0.001 [compared to _PH1N1 values].)

FIG 5

H7N3 replicates extensively in the lungs of C57BL/6J mice, and _PH1N1 PB2, NA, or M does not attenuate H7N3 replication. Male C57BL/6J mice were inoculated with 10⁴ TCID₅₀ of H7N3, _PH1N1, or the indicated reassortant virus, and lung viral titers were quantified 3 and 7 days postinoculation. Virus titer present in whole-lung homogenate at day 3 (A) or day 7 (B) was determined by virus titration assay performed on MDCK cells and expressed as TCID₅₀ per milliliter. Viral titers were derived from results determined for five or more mice from two or more independently performed experiments. The limit of detection was 100 TCID₅₀ (dotted line). (*, P < 0.05; **, P < 0.01; ***, P < 0.005 [compared to H7N3 values].)