Abstract

Interspecies transmission of influenza A is an important factor in the evolution and ecology of influenza viruses. Marine mammals are in contact with a number of influenza reservoirs, including aquatic birds and humans, and this may facilitate transmission among avian and mammalian hosts.

Virus isolation, whole genome sequencing, and hemaglutination inhibition assay confirmed that exposure to pandemic H1N1 influenza virus occurred among free-ranging Northern Elephant Seals (Mirounga angustirostris) in 2010. Nasal swabs were collected from 42 adult female seals in April 2010, just after the animals had returned to the central California coast from their short post-breeding migration in the northeast Pacific. Swabs from two seals tested positive by RT-PCR for the matrix gene, and virus was isolated from each by inoculation into embryonic chicken eggs. Whole genome sequencing revealed greater than 99% homology with A/California/04/2009 (H1N1) that emerged in humans from swine in 2009. Analysis of more than 300 serum samples from free-ranging adults and rehabilitating pups showed that samples collected in early 2010 (n = 100) were negative and by April 2010, seals began to test positive for antibodies against the pH1N1 virus (HI titer of ≥ 1:40), supporting the molecular findings. In vitro characterization studies revealed that viral replication was indistinguishable from that of reference strains of pH1N1 in canine kidney cells, but replication was inefficient in human epithelial respiratory cells, indicating these isolates may be elephant seal-adapted viruses.

In summary, molecular analysis, virus isolation, and serologic assays confirmed that exposure to pandemic H1N1 that was circulating in people in 2009 occurred among free-ranging Northern Elephant Seals in 2010 off the central California coast. This is the first report of pH1N1 (A/Elephant seal/California/1/2010) in any marine mammal and provides evidence for cross species transmission of influenza viruses in free-ranging wildlife and for movement of influenza viruses between humans and wildlife.

Acknowledgements

Funding for this research was provided by CRIP (Center for Research on Influenza Pathogenesis), under the Centers of Excellence for Influenza Research and Surveillance (CEIRS) program supported by the National Institute of Allergy and Infectious Disease (contract HHSN266200700009C), the Tagging of Pacific Pelagics program with grants from the National Ocean Partnership Program (N00014-02-1-1012), the Office of Naval Research (N00014-00-1-0880, N00014-03-1-0651 and N00014-08-1-1195), International Association of Oil and Gas Producers contract JIP2207-23, the Moore, Packard, and Sloan Foundations, and benefitted from intellectual developments from the PREDICT project of the United States Agency for International Development (USAID) Emerging Pandemic Threats Program, CONICYT Insertion of Human Capital to the Academy grant (79100014) and by the Program Iniciativa Científica Milenio from the Chilean Ministry of Economy, Development and Tourism. We thank the Año Nuevo State Reserve and Rangers for logistical support, laboratory personnel at the University of California Davis and Mount Sinai School of Medicine, especially Brett Smith, Magdalena Plancarte, LeAnn Lindsay and Richard Cadagan, for processing of samples and virus isolates and technical assistance; students from the Costa Lab at UC Santa Cruz for collection of samples during field procedures especially Sarah Peterson and Chandra Goetsch; and the staff and volunteers of The Marine Mammal Center for performing the sample collection. This work was completed under National Marine Fisheries Service Marine Mammal permits # 836, 87-1743 and 932-1489-00. All procedures on free-ranging elephant seals were approved by the UCSC IACUC Committee.

* Presenting author