Influenza A and B viruses circulate among humans causing epidemics almost annually. While various hosts for influenza A viruses exist, influenza B viruses have been detected only in humans and seals. However, recurrent infections of seals in Dutch coastal waters with influenza B viruses that are antigenetically distinct from influenza B viruses circulating among humans suggest that influenza B viruses have been introduced into this seal population by another, non-human, host. Harbour porpoises (Phocoena phocoena) are sympatric with seals in these waters and are also occasion

Bildt, M.W.G. (Marco) van de

Bodewes, R. (Rogier)

Bunskoek, P.E. (Paulien)

Elk, C.E. (Niels) van

Kuiken, T. (Thijs)

Osterhaus, A.D.M.E. (Albert)

Smits, S.L. (Saskia)

Vijver, D.A.M.C. (David) van de

Influenza A and B viruses circulate among humans causing epidemics almost annually. While various hosts for influenza A viruses exist, influenza B viruses have been detected only in humans and seals. However, recurrent infections of seals in Dutch coastal waters with influenza B viruses that are antigenetically distinct from influenza B viruses circulating among humans suggest that influenza B viruses have been introduced into this seal population by another, non-human, host. Harbour porpoises (Phocoena phocoena) are sympatric with seals in these waters and are also occasionally in close contact with humans after stranding and subsequent rehabilitation. In addition, virus attachment studies demonstrated that influenza B viruses can bind to cells of the respiratory tract of these animals. Therefore, we hypothesized that harbour porpoises might be a reservoir of influenza B viruses. In the present study, an unique set of serum samples from 79 harbour porpoises, stranded alive on the Dutch coast between 2003 and 2013, was tested for the presence of antibodies against influenza B viruses by use of the hemagglutination inhibition test and for antibodies against influenza A viruses by use of a competitive influenza A nucleoprotein ELISA. No antibodies were detected against either virus, suggesting that influenza A and B virus infections of harbour porpoises in Dutch coastal waters are not common, which was supported by statistical analysis of the dataset

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No Serological Evidence that Harbour Porpoises AreAdditional Hosts of Influenza B VirusesRogier Bodewes1*, Marco W. G. van de Bildt1, Cornelis E. van Elk1,2,3, Paulien E. Bunskoek2,3,David A. M. C. van de Vijver1, Saskia L. Smits1,4, Albert D. M. E. Osterhaus1,4, Thijs Kuiken11Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands, 2 SOS Dolphin Foundation, Harderwijk, the Netherlands, 3Dolfinarium Harderwijk,Harderwijk, the Netherlands, 4Viroclinics Biosciences B.V., Rotterdam, the NetherlandsAbstractInfluenza A and B viruses circulate among humans causing epidemics almost annually. While various hosts for influenza Aviruses exist, influenza B viruses have been detected only in humans and seals. However, recurrent infections of seals inDutch coastal waters with influenza B viruses that are antigenetically distinct from influenza B viruses circulating amonghumans suggest that influenza B viruses have been introduced into this seal population by another, non-human, host.Harbour porpoises (Phocoena phocoena) are sympatric with seals in these waters and are also occasionally in close contactwith humans after stranding and subsequent rehabilitation. In addition, virus attachment studies demonstrated thatinfluenza B viruses can bind to cells of the respiratory tract of these animals. Therefore, we hypothesized that harbourporpoises might be a reservoir of influenza B viruses. In the present study, an unique set of serum samples from 79 harbourporpoises, stranded alive on the Dutch coast between 2003 and 2013, was tested for the presence of antibodies againstinfluenza B viruses by use of the hemagglutination inhibition test and for antibodies against influenza A viruses by use of acompetitive influenza A nucleoprotein ELISA. No antibodies were detected against either virus, suggesting that influenza Aand B virus infections of harbour porpoises in Dutch coastal waters are not common, which was supported by statisticalanalysis of the dataset.Citation: Bodewes R, van de Bildt MWG, van Elk CE, Bunskoek PE, van de Vijver DAMC, et al. (2014) No Serological Evidence that Harbour Porpoises Are AdditionalHosts of Influenza B Viruses. PLoS ONE 9(2): e89058. doi:10.1371/journal.pone.0089058Editor: Leo L. M. Poon, University of Hong Kong, Hong KongReceived December 2, 2013; Accepted January 13, 2014; Published February 13, 2014Copyright:  2014 Bodewes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) underthe project ‘‘European Management Platform for Emerging and Re-emerging Infectious disease Entities’’ (EMPERIE) EC grant agreement number 223498 and theVIRGO Consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: Two authors of the manuscript have interests to declare: Prof. Dr. A.D.M.E. Osterhaus and Dr. S.L. Smits are part time chief scientific officerand senior scientist respectively of Viroclinics Biosciences B.V. There are no patents, products in development or marketed products to declare. These interests donot alter our adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors* E-mail: r.bodewes@erasmusmc.nlIntroductionInfluenza A and B viruses circulate among humans causingepidemics almost annually. While various hosts were identified forinfluenza A viruses (for review see [1,2]), influenza B viruses wereuntil 1999 only detected in humans. In 1999, an influenza B viruswas isolated from harbor seals (Phoca vitulina) which was differentfrom influenza B viruses circulating among humans at that time[3]. In addition, during a sero-survey of seals of the Dutch coastalwaters using serum samples collected from 2002 to 2012,antibodies were detected against influenza B virus in only 9 sealsin 2010 (43% of the tested serum samples of this year) and one in2011 (0.7% of the tested serum samples of this year; both harborseals and grey seals [Halichoerus grypus]) [4]. Additional analysissuggested that these seals were probably infected with an influenzaB virus that was similar to influenza B/Yamanashi/166/1998 andB/Jiangsu/010/2003, and was different from both the influenza Bviruses that were isolated from seals in 1999 and influenza Bviruses circulating in humans in 2010–2011 [3,5]. These findingsmight be explained by introduction of influenza B viruses into theseal population of the Dutch coastal waters both in 1999 and 2010by another, non-human, host. Since this non-human host mightpotentially be a reservoir of influenza B viruses that areantigenetically distinct from influenza B viruses that currentlycirculate among humans, it is of interest from a public healthperspective to identify this/these novel host(s).Based on results of our previous studies [3,4], a currentlyunknown host for influenza B viruses should meet a number ofconditions. First, this host species should be susceptible to infectionwith an influenza B virus and this virus should be able to spreadamong individuals of this host. Second, this host species shouldconsist of a population that is large enough to allow maintenanceof this acute virus infection. Third, this host species should sharethe habitat with seals of the Dutch coastal waters to facilitateoccasional spread of influenza B virus from this animal species toseals. Fourth, individuals of this host species should have been (atleast once) in close contact with humans to facilitate infection ofthis host with a human influenza B virus. However, animals shouldnot live in such close contact with humans that transmission occursregularly and similar influenza B viruses circulate among bothhumans and this species.Based on these conditions, there are a number of potentialadditional animal host reservoirs for influenza B viruses. Trans-mission of influenza A viruses from birds to marine mammals hasbeen reported [1], but there is no evidence that influenza B virusescirculate among birds. Influenza B viruses might also originatePLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e89058from migrating individuals of other seal species that inhabit Arcticwaters, since antibodies against influenza B viruses were detectedin various other species of otariids and phocids in other areas [6,7].Spread of viruses from these populations to seals of the North Sea,including Dutch coastal waters, has been suggested previously [8],but no serological surveys were performed to show the absence/presence of influenza B infections in these seal populations. Besidesseals, other species of marine mammals might be a source ofinfluenza B viruses that transmits to seals.Harbour porpoises (Phocoena phocoena), the most abundantmarine mammal species of the North Sea, are sympatric withseals in the Dutch coastal waters [9–11]. In March 2010, thepopulation of harbour porpoises in Dutch waters was approx-imately 66,000, but their number varies with the season [12]. Inaddition, harbour porpoises stranded alive on the Dutch coast arealso rehabilitated, indicating that a small proportion of the totalpopulation of these animals lives in close contact with humans (fora short period) and could be infected with a human influenza Bvirus during that stay. Furthermore, it has been demonstrated invirus attachment studies that influenza B viruses do bind to cells ofthe respiratory tract of harbour porpoises, although mainly to cellsof the lower respiratory tract [13]. Therefore, we hypothesizedthat harbour porpoises might be a host for influenza B viruses. Inthe present study, we evaluated an unique set of serum samplesfrom live stranded harbour porpoises for the presence ofantibodies against influenza A and B virus.Materials and MethodsEthics StatementAdmission and rehabilitation of live stranded wild harbourporpoises at the SOS Dolphin Foundation was authorized by thegovernment of the Netherlands (application number FF/75/2012/036). Blood samples used in the present study were collectedfrom harbour porpoises for routine diagnostics by qualified andexperienced veterinarians of the SOS Dolphin Foundation. TheSOS Dolphin Foundation provided permission to the Departmentof Viroscience, Erasmus Medical Centre to use the samples for thepresent study. No blood samples were collected from animals forresearch purposes.Sample CollectionSerum samples collected from 79 harbour porpoises that werestranded alive on different parts of the Dutch coast from 2003 to2013 were tested for the presence of antibodies against influenza Bviruses. In total, 42 serum samples were included from animalsthat were estimated to be less than 1 year of age (calves), 25 serumsamples from animals that were estimated to be between 1 and 3years of age (juveniles) and 12 serum samples from animals thatwere estimated to be older than 3 years of age (adults) (Figure 1).Age estimation was based on the length of the harbour porpoise incombination with the moment of stranding [14]. Of these 79animals, 40 were females and 39 were males. Blood samples werecentrifuged briefly and serum was harvested, aliquoted and storedat 220uC until further processing.SerologySamples were tested for the presence of antibodies againstinfluenza B virus strains B/Seal/1/1999, B/Jiangsu/010/2003,B/Yamanashi/166/1998 and B/Malaysia/2506/2004 using thehemagglutination inhibition assay as described previously [15].Influenza B virus strains were selected based on previous results inseals [3,4]. In brief, serum samples were pre-treated with cholerafiltrate for 16–18 hours at 37uC and two-fold serial diluted serumsamples were incubated with 4 hemagglutinating units of eachrespective influenza B virus antigen for 30 minutes at 37uC.Subsequently, 1% turkey erythrocytes were added to each well andpatterns were read after storage at 4uC for one hour. An antibodytiter of 20 was used as the cut-off value for a positive result. Pre-and post-infection sera of ferrets infected with the indicated viruseswere used as negative and positive controls, respectively. Inaddition to the influenza B serology, serum samples were alsotested for the presence of antibodies against the influenza A virusnucleoprotein using an influenza A virus nucleoprotein compet-itive ELISA according to the instructions of the manufacturer(IDEXX laboratories, Hoofddorp, the Netherlands).Statistical AnalysisSince the study size was dependent on the number of animalsthat were stranded, a formal power analysis before the studystarted was not possible. As a consequence, it cannot be excludedFigure 1. Overview of serum samples used in the presentstudy. Indicated are the number of serum samples from harbourporpoises stranded of each year used in the present study (light greyareas: calves; white areas: juveniles; dark grey area: adults).doi:10.1371/journal.pone.0089058.g001Figure 2. Probability that the actual unknown prevalence isgreater than 0% based on the 79 included animals of whomnone had antibodies. The calculations were performed using thebinomial distribution.doi:10.1371/journal.pone.0089058.g002No Flu A and B Antibodies in Harbour PorpoisesPLOS ONE | www.plosone.org 2 February 2014 | Volume 9 | Issue 2 | e89058that a particular prevalence of antibodies that was found in ourstudy was due to random variation and that the prevalence wouldhave been very different if a larger number of animals could havebeen included. Therefore, we used the number of includedanimals and the number of animals with antibodies to calculatethe probability that the actual prevalence was different from theprevalence we found. The probability was calculated in thestatistical package R, using the binomial distribution [16,17].Results and DiscussionNo antibodies were detected against any of the influenza B virusstrains in any of the tested serum samples of stranded harbourporpoises, while antibodies were detected in all positive controlferret sera. These results indicate that infections with influenza Bviruses are not common in harbour porpoises of the Dutch waters,especially since we tested samples from animals of different agesand that were stranded on different locations and on differentyears. However, since we only tested a relatively low number ofsamples compared to the total population of harbour porpoises inDutch waters, we may have missed incidental infections ofinfluenza B virus among harbour porpoises. Figure 2 shows theprobability that based on the 79 included animals of whom nonehad antibodies, the actual unknown prevalence was greater than0%. It can be seen that the probability that the actual prevalencewas 1%, 2% or 3% was, 0.45, 0.2, 0.09, respectively. Theprobability that the actual prevalence was 4% was already lessthan 0.05. It is unlikely that the actual prevalence exceeds 4% asthe probability that the actual prevalence is 5% is ,0.02 withstrongly reduced probability at higher actual prevalences.In addition to the absence of antibodies against influenza B virusin the tested serum samples, no antibodies were detected againstthe nucleoprotein of influenza A virus indicating that influenza Avirus infections are also not common in harbour porpoises of theDutch waters.Although the absence of antibodies against influenza A and Bviruses suggest that harbour porpoises of the Dutch coastal watersare not commonly infected with influenza A and B viruses, thisstudy has a few limitations. First, previous infections might beundetectable since the antibody response could be detected onlyfor a relative short time after infection in this animal species.Second, assays used in the present study might not be able todetect antibodies against influenza viruses in this animal species,although the assays have been used for various animal species andhumans [4,18–21]. Third, the use of serum samples collected fromstranded animals in the present study might have created a biassince these animals were weakened and more susceptible toinfection. Therefore, the results of this study do not providedefinite proof that influenza A and B viruses do not circulateamong harbour porpoises, but strongly suggest that influenza Aand B virus infections are not common.The results of the present study provided no answer whether,besides human beings, otariids and phocids, additional hosts existfor influenza B viruses. In addition to harbour porpoises, a fewother species (e.g. other members of the order of Cetacea) meet thecriteria indicated above but no serological studies have beenperformed yet to evaluate the presence of influenza B infections inthese animals. Additional studies using serum samples of potentialanimal reservoir species are necessary to elucidate whetheradditional hosts for influenza B viruses exist.Author ContributionsConceived and designed the experiments: RB TK. Performed theexperiments: RB MWGB. Analyzed the data: RB MWGB CEE PBDAMCV SLS ADMEO TK. Contributed reagents/materials/analysistools: CEE PB. Wrote the paper: RB DAMCV SLS TK.References1. Runstadler J, Hill N, Hussein IT, Puryear W, Keogh M (2013) Connecting thestudy of wild influenza with the potential for pandemic disease. Infect GenetEvol 17C: 162–187.2. Reperant LA, Rimmelzwaan GF, Kuiken T (2009) Avian influenza viruses inmammals. Rev Sci Tech 28: 137–159.3. Osterhaus AD, Rimmelzwaan GF, Martina BE, Bestebroer TM, Fouchier RA(2000) Influenza B virus in seals. Science 288: 1051–1053.4. Bodewes R, Morick D, de Mutsert G, Osinga N, Bestebroer T, et al. (2013)Recurring influenza B virus infections in seals. Emerg Infect Dis 19: 511–512.5. de Jong JC, Donker GA, Meijer A, van der Hoek W, Rimmelzwaan GF, et al.(2011) Het influenzaseizoen 2010/2011 in Nederland: het nieuwe A(H1N1)-virus van 2009 blijft actief. Ned Tijdschr Med Microbiol 19: 21–27.6. Blanc A, Ruchansky D, Clara M, Achaval F, Le Bas A, et al. (2009) Serologicevidence of influenza A and B viruses in South American fur seals(Arctocephalus australis). J Wildl Dis 45: 519–521.7. Ohishi K, Ninomiya A, Kida H, Park CH, Maruyama T, et al. (2002)Serological evidence of transmission of human influenza A and B viruses toCaspian seals (Phoca caspica). Microbiol Immunol 46: 639–644.8. Harkonen T, Dietz R, Reijnders P, Teilmann J, Harding K, et al. (2006) The1988 and 2002 phocine distemper virus epidemics in European harbour seals.Dis Aquat Organ 68: 115–130.9. Hammond PS, Berggren P, Benke H, Borchers DL, Collet A, et al. (2002)Abundance of harbour porpoise and other cetaceans in the North Sea andadjacent waters. J Appl Ecol 39: 361–376.10. ScansII Project Team (2008) Small Cetaceans in the European Atlantic andNorth Sea (SCANS II) Final Report to the European Commission under projectLIFE04NAT/GB/000245. Available from SMRU, G.M.L., University of StAndrews, St Andrews, Fife, KY16 8LB, UK, ed.11. Geelhoed SCV, Scheidat M, van Bemmelen RSA, Aarts G (2013) Abundance ofharbour porpoises (Phocoena phocoena) on the Dutch Continental Shelf, aerialsurveys in July 2010-March 2011. Lutra 56: 45–57.12. Scheidat M, Verdaat H, Aarts G (2012) Using aerial surveys to estimate densityand distribution of harbour porpoises in Dutch waters. Journal of Sea Research69: 1–7.13. Ramis AJ, van Riel D, van de Bildt MW, Osterhaus A, Kuiken T (2012)Influenza A and B virus attachment to respiratory tract in marine mammals.Emerg Infect Dis 18: 817–820.14. Read AJ (1998) Harbour Porpoise. In: Ridgway, S.H.; Harrison, R., editors.Handbook of Marine Mammals. London: Academic Press. p. 336.15. Palmer D, Dowle W, Coleman M, Schild G (1975) Haemagglutinationinhibition test. In: Advances laboratory techniques for influenza diagnosis:procedural guide Atlanta: US Department of Health, Education and Welfare: p.25–62.16. Armitage P, Berry G, Matthews JNS (2002) Statistical methods in medicalresearch. Oxford: Blackwell Science. p. 1–817.17. Bland M (2000) An introduction to medical statistics. Oxford: Oxford UniversityPress.18. de Boer GF, Back W, Osterhaus AD (1990) An ELISA for detection ofantibodies against influenza A nucleoprotein in humans and various animalspecies. Arch Virol 115: 47–61.19. Bodewes R, de Mutsert G, van der Klis FR, Ventresca M, Wilks S, et al. (2011)Prevalence of Antibodies against Seasonal Influenza A and B Viruses inChildren in Netherlands. Clin Vaccine Immunol 18: 469–476.20. Lebarbenchon C, Brown JD, Luttrell MP, Stallknecht DE (2012) Comparison oftwo commercial enzyme-linked immunosorbent assays for detection of InfluenzaA virus antibodies. J Vet Diagn Invest 24: 161–165.21. Nielsen O, Clavijo A, Boughen JA (2001) Serologic evidence of influenza Ainfection in marine mammals of arctic Canada. J Wildl Dis 37: 820–825.No Flu A and B Antibodies in Harbour PorpoisesPLOS ONE | www.plosone.org 3 February 2014 | Volume 9 | Issue 2 | e89058

No serological evidence that harbour porpoises are additional hosts of influenza B viruses

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No serological evidence that harbour porpoises are additional hosts of influenza B viruses

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