Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 20, Number 5—May 2014

Serologic Evidence of Influenza A(H1N1)pdm09 Virus Infection in Northern Sea Otters

Cite This Article

To the Editor: Sporadic epizootics of pneumonia among marine mammals have been associated with multiple animal-origin influenza A virus subtypes (16); seals are the only known nonhuman host for influenza B viruses (7). Recently, we reported serologic evidence of influenza A virus infection in free-ranging northern sea otters (Enhydra lutris kenyoni) captured off the coast of Washington, USA, in August 2011 (8). To investigate further which influenza A virus subtype infected these otters, we tested serum samples from these otters by ELISA for antibody-binding activity against 12 recombinant hemagglutinins (rHAs) from 7 influenza A hemagglutinin (HA) subtypes and 2 lineages of influenza B virus (Technical Appendix Table 1). Estimated ages for the otters were 2–19 years (Technical Appendix Table 2); we also tested archived serum samples from sea otters of similar ages collected from a study conducted during 2001–2002 along the Washington coast (9).


Thumbnail of Results of ELISA and hemagglutinin inhibition (HI) testing for influenza viruses in serum samples from northern sea otters captured off the coast of Washington, USA, during studies conducted in August 2011 (n = 30) and 2001–2002 (n = 21). A) IgG for influenza A(H1N1)pdm09 strain A/Texas/05/2009 detected by using standard indirect ELISA techniques with HRP-Protein A (Sigma, St. Louis, MO, USA). The ELISA titer was read as the reciprocal of the highest dilution of serum with an OD450n

Figure. Results of ELISA and hemagglutination inhibition (HI) testing for influenza viruses in serum samples from northern sea otters captured off the coast of Washington, USA, during studies conducted in August 2011...

Of the 30 sea otter serum samples collected during 2011, a total of 21 (70%) had detectable IgG (>200) for rHA of influenza A(H1N1)pdm09 virus (pH1N1) strain A/Texas/05/2009. Four of 7 serum samples that showed IgG ≥6,400 against pH1N1 rHA also showed low cross-reactivity (IgG 200) against rHA of A/Brisbane/59/2007, a previous seasonal influenza A(H1N1) virus (Figure, panel A; Technical Appendix Table 1). No IgG was detected in any samples for any of the other 11 rHAs tested (IgG ≤100), and the sea otter serum samples collected during 2001–2002 did not react with any of the rHAs tested, including pH1N1 (IgG ≤100; Figure, panel A).

Next, we tested serum samples by using a hemagglutination inhibition (HI) assay with whole influenza virus to detect strain-specific antibodies that inhibit receptor binding. Of the 30 samples collected during 2011, a total of 22 (73%) showed HI antibody titers of ≥40 against pH1N1 virus. Titers against all other human and avian viruses tested were ≤10 for all samples by HI assay using turkey red blood cells (RBCs) (Figure, panel B; Technical Appendix Table 3). No influenza A or B virus–specific HI antibodies were detected in the samples collected during 2001–2002 (data not shown). Although nasal swab specimens were collected from sea otters in the 2011 study, all specimens were negative for influenza virus by testing in embryonated eggs and by real-time PCR for detection of influenza A viral RNA (data not shown). These results suggest that sea otters were infected with influenza A virus sometime before the August 2011 sample collection date.

Although none of the 2011 samples showed HI titers to influenza A/duck/New York/96 (H1N1) virus (dk/NY/96) by testing using turkey RBCs (Technical Appendix Table 2), titers against this strain were detected when using horse RBCs, which is a more sensitive means for the detection of mammalian antibodies against some avian influenza subtypes (10). Of the 22 samples that had HI titers >40 to pH1N1 virus, 16 also had HI titers >40 against dk/NY/96 by horse RBC HI assay (Technical Appendix Table 2). However, titers against this strain were on average ≈4–8-fold lower than those for the pH1N1 virus strain, which suggests that that the titers against dk/NY/96 were the result of serologic cross-reactivity with avian- and swine-origin pH1N1 viruses.

To further test for cross-reactivity, 4 sea otter serum samples were adsorbed with purified pH1N1 and dk/NY/96 virions. Adsorption with pH1N1, but not dk/NY/96, removed HI antibodies to pH1N1, whereas adsorption with either virus removed HI antibodies against dk/NY/96 (Technical Appendix Table 4). A comparison of amino acid sequences comprising the known HA antigenic sites on the pH1N1 structure confirmed high sequence identity and structural similarity with dk/NY/96 HA in Sa (12/13 aa residues) and Sb (8/12 aa residues) antigenic sites (data not shown). These results indicate that HI antibodies detected in sea otters are the result of pH1N1 virus infection but cross-react with the avian influenza A(H1N1) virus.

Although we cannot exclude the possibility that sea otters were infected with classical swine influenza A(H1N1) virus, which shares high HA genetic and antigenic similarity with pH1N1 virus, our serologic evidence is consistent with isolation of pH1N1 virus from northern elephant seals (1). Therefore, we conclude that these sea otters were infected with pH1N1 virus. The origin and transmission route of pH1N1 virus infection in sea otters remain unknown. Potential contact between northern elephant seals and sea otters is one possibility; elephant seals’ summer feeding ranges and breeding areas along the Northeast Pacific coast overlap with areas where the Washington sea otter population is distributed (1).

In conclusion, our results show that sea otters are susceptible to infection with influenza A virus and highlight the complex nature of interspecies transmission of influenza viruses in the marine environment. Further surveillance, especially in other sea otter populations, is required to determine virus origin, potential pathogenesis, and consequences for the marine ecosystem.



We thank Heather Tatum, Leilani Thomas, and Peter Browning for specimen management and Tina Egstad, Katy Griffin, Renee Long, and Zac Najacht for technical assistance.

Sample collection was done in collaboration with United States Geological Survey–Alaska Science Center, Monterey Bay Aquarium, and Seattle Aquarium. Recombinant HAs with Histidine Tag from A/Japan/305/57 (H2N2), FR-700; A/Netherlands/219/2003 (H7N7), FR-71; A/Hong Kong/1073/99 (H9N2), FR-88; A/shorebird/DE/68/2004 (H13N9), FR-73; globular head domain HA1 rHAs of B/Brisbane/60/2008, FR-836; and B/Wisconsin/1/2010 FR-843, were obtained through the Influenza Reagent Resource, Influenza Division, World Health Organization Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention.


Zhu-Nan Li, Hon S. Ip, Jessica F. Trost, C. LeAnn White, Michael J. Murray, Paul J. Carney, Xiang-Jie Sun, James Stevens, Min Z. Levine, and Jacqueline M. KatzComments to Author 
Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (Z.-N. Li, J.F. Trost, P.J. Carney, X.-J. Sun, J. Stevens, M.Z. Levine, J.M. Katz); United States Geological Survey National Wildlife Health Center, Madison, Wisconsin, USA (H.S. Ip, C.L. White); Monterey Bay Aquarium, Monterey, California, USA (M.J. Murray)



  1. Goldstein  T, Mena  I, Anthony  SJ, Medina  R, Robinson  PW, Greig  DJ, Pandemic H1N1 influenza isolated from free-ranging Northern Elephant Seals in 2010 off the central California coast. PLoS ONE. 2013;8:e62259 . DOIPubMedGoogle Scholar
  2. Hinshaw  VS, Bean  WJ, Geraci  J, Fiorelli  P, Early  G, Webster  RG. Characterization of two influenza A viruses from a pilot whale. J Virol. 1986;58:6556 .PubMedGoogle Scholar
  3. Hinshaw  VS, Bean  WJ, Webster  RG, Rehg  JE, Fiorelli  P, Early  G, Are seals frequently infected with avian influenza viruses? J Virol. 1984;51:8635 .PubMedGoogle Scholar
  4. Geraci  JR, St Aubin  DJ, Barker  IK, Webster  RG, Hinshaw  VS, Bean  WJ, Mass mortality of harbor seals: pneumonia associated with influenza A virus. Science. 1982;215:112931 . DOIPubMedGoogle Scholar
  5. Anthony  SJ, St Leger  JA, Pugliares  K, Ip  HS, Chan  JM, Carpenter  ZW, Emergence of fatal avian influenza in New England harbor seals. MBio. 2012;3:e0016612 . DOIPubMedGoogle Scholar
  6. Callan  RJ, Early  G, Kida  H, Hinshaw  VS. The appearance of H3 influenza viruses in seals. J Gen Virol. 1995;76:199203 . DOIPubMedGoogle Scholar
  7. Osterhaus  AD, Rimmelzwaan  GF, Martina  BE, Bestebroer  TM, Fouchier  RA. Influenza B virus in seals. Science. 2000;288:10513. DOIPubMedGoogle Scholar
  8. White  CL, Schuler  KL, Thomas  NJ, Webb  JL, Saliki  JT, Ip  HS, Pathogen exposure and blood chemistry in the Washington, USA population of northern sea otters (Enhydra lutris kenyoni). J Wildl Dis. 2013;49:88799 . DOIPubMedGoogle Scholar
  9. Brancato  MS, Milonas  L, Bowlby  CE, Jameson  R, Davis  JW. Chemical contaminants, pathogen exposure and general health status of live and beach-cast Washington sea otters (Enhydra lutris kenyoni). Marine Sanctuaries Conservation Series ONMS-09–01. Silver Spring (MD): US Department of Commerce, National Oceanic and Atmospheric Administration, Office of National Marine Sanctuaries; 2009. p. 181.
  10. Stephenson  I, Wood  JM, Nicholson  KG, Zambon  MC. Sialic acid receptor specificity on erythrocytes affects detection of antibody to avian influenza haemagglutinin. J Med Virol. 2003;70:3918. DOIPubMedGoogle Scholar




Cite This Article

DOI: 10.3201/eid2005.131890

Related Links


Table of Contents – Volume 20, Number 5—May 2014


Please use the form below to submit correspondence to the authors or contact them at the following address:

Jacqueline M. Katz, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, Mailstop G16, Atlanta, GA 30333, USA

Send To

10000 character(s) remaining.


Page created: April 17, 2014
Page updated: April 17, 2014
Page reviewed: April 17, 2014
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.