Multiple reassortment between pandemic (H1N1) 2009 and endemic influenza viruses in pigs, United States

Abstract

As a result of human-to-pig transmission, pandemic influenza A (H1N1) 2009 virus was detected in pigs soon after it emerged in humans. In the United States, this transmission was quickly followed by multiple reassortment between the pandemic virus and endemic swine viruses. Nine reassortant viruses representing 7 genotypes were detected in commercial pig farms in the United States. Field observations suggested that the newly described reassortant viruses did not differ substantially from pandemic (H1N1) 2009 or endemic strains in their ability to cause disease. Comparable growth properties of reassortant and endemic viruses in vitro supported these observations; similarly, a representative reassortant virus replicated in ferrets to the same extent as did pandemic (H1N1) 2009 and endemic swine virus. These novel reassortant viruses highlight the increasing complexity of influenza viruses within pig populations and the frequency at which viral diversification occurs in this ecologically important viral reservoir.

Figure 1

Lineages of North American reassortant swine influenza viruses identified through phylogenetic analyses. Pandemic and endemic gene segments are represented in black and white, respectively. *Denotes partial sequences. Isolates sw/NC/226124/10 and sw/NC/226125/10 (not shown) have the same genotype as sw/NC/226126/10. PB2, polymerase basic 2; PB1, polymerase basic 1; PA, polymerase acidic; HA, hemagglutinin; NP, nucleoprotein; NA, neuraminidase; M, matrix; NS, nonstructural.

Figure 2

Phylogenetic trees of pandemic reassortant swine influenza viruses compared with currently circulating swine influenza strains: A) hemagglutinin (H); B) neuraminidase (N); C) matrix genes. The trees were constructed by using the neighbor-joining method (Kimura 2-parameter) with 1,000 bootstrap replicates. Only bootstrap values >74 are shown. Swine reassortant strains characterized in this study are indicated with a closed circle. Boldface indicates pandemic segments. Greek letters indicate virus genogroups; α represents classical swine influenza virus and δ seasonal human-like swine influenza virus. TR indicates swine triple reassortant influenza virus. Scale bars indicate nucleotide substitutions per site.

Figure 3

Replication of North American reassortant and endemic swine influenza viruses on swine testicle (ST) cells and in ferrets. A) The growth of 6 endemic swine viruses and 3 reassortant viruses (sw/MN/239105/09, sw/MN/239106/10, and sw/NC/239108/10) from 2009–2010 were analyzed in vitro. One curve corresponds to 1 isolate. Black lines and symbols indicate swine pandemic influenza reassortant viruses; red lines and symbols indicate swine triple reassortant (TR) influenza viruses. The progeny viruses released from infected ST cultures were collected at the indicated time points and titrated in ST cells by performing a 50% tissue culture infectious dose (TCID₅₀) assay. Negative TCID₅₀ titers were given the value 0.5; log₁₀ TCID₅₀ >8.5 were given the value 8.5. B) Virus titers in the upper respiratory tract (nasal washes) of ferrets infected with 10⁶ 50% egg infectious dose (EID₅₀) pandemic (H1N1) 2009 TN/560/09 (35); 10⁶ TCID₅₀ swine TR influenza virus sw/TX/4199/98; or 10⁶ EID₅₀ swine pandemic reassortant virus sw/MN/239105/09. Values are the mean ± SD for 4, 3, and 5 ferrets for TN/560/09, sw/TX/4199/98, and sw/MN/239105/09, respectively.