Molecular surveillance of low pathogenic avian influenza viruses in wild birds across the United States: inferences from the hemagglutinin gene

Abstract

A United States interagency avian influenza surveillance plan was initiated in 2006 for early detection of highly pathogenic avian influenza viruses (HPAIV) in wild birds. The plan included a variety of wild bird sampling strategies including the testing of fecal samples from aquatic areas throughout the United States from April 2006 through December 2007. Although HPAIV was not detected through this surveillance effort we were able to obtain 759 fecal samples that were positive for low pathogenic avian influenza virus (LPAIV). We used 136 DNA sequences obtained from these samples along with samples from a public influenza sequence database for a phylogenetic assessment of hemagglutinin (HA) diversity in the United States. We analyzed sequences from all HA subtypes except H5, H7, H14 and H15 to examine genetic variation, exchange between Eurasia and North America, and geographic distribution of LPAIV in wild birds in the United States. This study confirms intercontinental exchange of some HA subtypes (including a newly documented H9 exchange event), as well as identifies subtypes that do not regularly experience intercontinental gene flow but have been circulating and evolving in North America for at least the past 20 years. These HA subtypes have high levels of genetic diversity with many lineages co-circulating within the wild birds of North America. The surveillance effort that provided these samples demonstrates that such efforts, albeit labor-intensive, provide important information about the ecology of LPAIV circulating in North America.

Figure 1. Geographic locations of avian fecal samples collected in the United States and HA sequences generated during USDA surveillance efforts from 2006 through 2007.

This map shows the total avian fecal samples collected across the United States and shows which of those samples were matrix positive and matrix negative. Of the matrix positive samples that we successfully sequenced we also show sampling localities and differentiate between sequences representing HA subtypes that have been previously documented in that state (HA sequences from the same subtype and state also found on NCBI Influenza Virus Resource during our searches) and those that were newly documented in that state through this study.

Figure 2. Numbers of sequences used in this study showing frequency of each subtype per continent and number surveillance sequences relative to sequences obtained from NCBI Influenza Virus Resource.

Figure 3. ML phylogram of H1 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H1. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 4. ML phylogram of H2 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H2. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 5. ML phylogram of H3 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H3. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 6. ML phylogram of H4 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H4. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 7. ML phylogram of H6 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H6. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support.

Figure 8. ML phylogram of H8 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H8. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 9. ML phylogram of H9 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H9. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 10. ML phylogram of H10 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H10. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 11. ML phylogram of H11 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H11. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 12. ML phylogram of H12 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H12. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 13. ML phylogram of H13 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H13. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.

Figure 14. ML phylogram of H16 sequences from North America and Eurasia including U.S. surveillance samples.

ML phylogram of influenza A subtype H16. Branch lengths represent genetic distance. Dots show nodes with significant bootstrap support. USDA surveillance sequences from avian fecal samples are shown in bold.