(this information is current as of March 22, 2010): The following resources related to this article are available online at www.sciencemag.org seals. Although the two harbor seals with prov-en influenza B virus infection displayed respi-ratory symptoms during their rehabilitation pe-riod, this occurred at a time when many of the admitted juvenile seals suffered from lung-worm (Otostrongylus circumlitus and Parafi-laroides gymnurus) infections (17). Associa-tion of lungworm infections in pigs with influ-enza A virus pathogenesis and transmission has been described (25), but the evidence was con-sidered weak (26). The combined serological and virological data obtained from seal 99-012 indicate that shedding of influenza B virus in seals was prolonged as compared to shedding in hu-mans (27, 28) and that IgG antibody respons-es to NP and HA/NA were delayed. Possible explanations for this apparent suboptimal im-mune response upon infection may be asso-ciated with xenobiotic-related immunosup-pression (11) or the therapeutic use of corti-costeroids to combat the lungworm infections (17). Prolonged virus shedding in addition to the limited spreading of influenza B virus among seals (as shown in the SRRC and indicated by the limited seroprevalence of specific antibodies in the wild) may explain why little or no genetic and antigenic drift of influenza B virus is observed in seals. Our data not only highlight the fact that influenza B virus infections can emerge in seal populations but also show that seals may constitute an animal reservoir from which humans may be exposed to influenza B vi-ruses that have circulated in the past. 18. RNA was isolated by means of a high pure RNA isolation kit (Boehringer-Mannheim). RNA was used for RT-PCR analysis to amplify a 240 – base pair frag-ment of the influenza B virus NS gene segment using primers 5Ј-ATG GCC ATC GGA TCC TCA AC-3Ј and 5Ј-TGT CAG CTA T TA TGG AGC TG-3Ј, and AMV reverse transcriptase, Amplitaq DNA polymerase, re-combinant ribonuclease inhibitor (Promega) in the presence of 50 mM tris-HCl, 50 mM NaCl, 2 mM dithiothreitol, 7 mM MgCl 2 , 1 mM dNTP, and 400 nM each of primer. Cycling parameters were 30 min at 42°C, 4 min at 95°C, 1 min at 45°C, and 3 min at 72°C once; and then 1 min at 95°C, 1 min at 45°C, and 3 min at 72°C, repeated 39 times. PCR fragments were sequenced with a DYEnamic ET terminator cy-cle sequencing premix kit (Amersham) on an ABI-373A apparatus (Perkin Elmer). 19. Seal kidney cells were plated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, penicillin, and streptomycin at 1 ϫ 10 5 cells per well in 24-well plates. Cells were inoculated with 1 ϫ 10 5 TCID 50 of influenza virus B/Seal/Netherlands/1/99 in DMEM supplemented with 4% bovine serum albumin, 1% L-glutamine, penicillin, and streptomycin. Influenza B virus infection was detected by immunofluorescence with influenza B NP-specific antibodies, which were labeled with fluorescein isothiocyanate (IMAGEN In-fluenza AϩB, DAKO Diagnostics) after 24 hours. Cy-topathic changes and HA activity (titer ϭ 32) were detected in the culture cell after 48 hours. 20. J. T. Voeten et al., J. Clin. Microbiol. 36, 3527 (1998). 21. Antibody titers were determined with a recombinant fusion protein between maltose-binding protein (MBP) and NP or with HA/NA proteins purified from virions (both proteins were derived from B/Harbin/7/ 94). IgG antibody levels to HA/NA and NP proteins were determined by an indirect ELISA with antigen-coated plates and peroxidase-labeled protein A for detection. IgM antibody levels to NP were deter-mined by means of an antibody-capture ELISA with goat anti-dog IgM–coated plates and peroxidase-labeled MBP-NP antigen for detection. The goat anti-dog IgM antibody preparation specifically captures seal IgM, as was shown in routine serological tests for PDV and PHV.
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