Influenza B Virus in Seals

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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 fragment of the influenza B virus NS gene segment using primers 5′-ATG GCC ATC GGA TCC TCA AC-3′ and 5′-TGT CAG CTA TTA TGG AGC TG-3′, and AMV reverse transcriptase, Amplitaq DNA polymerase, recombinant ribonuclease inhibitor (Promega) in the presence of 50 mM tris-HCl, 50 mM NaCl, 2 mM dithiothreitol, 7 mM MgCl₂, 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 cycle sequencing premix kit (Amersham) on an ABI-373A apparatus (Perkin Elmer).

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 × 10⁵ cells per well in 24-well plates. Cells were inoculated with 1 × 10⁵ TCID₅₀ 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 Influenza A+B, DAKO Diagnostics) after 24 hours. Cytopathic changes and HA activity (titer = 32) were detected in the culture cell after 48 hours.

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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 determined 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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Analysis of the HA1 sequences from human influenza B virus strains that are available from the influenza sequence database showed that between 1991 and 1999, the proportion of strains with >98% nucleotide identity to B/Seal/Netherlands/1/99 was 0, 17, 27, 46, 62, 29, 0, 0, and 0% of all epidemic strains for each successive year, respectively.

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HA1 sequences were amplified with M13-tagged primers P1 (5′-29M13-GCA TTT TCT AAT ATC CAC AA-3′) and P4 (5′-21M13-TTT GGG AAG CCA CCA ATC TG-3′) or primers P3 (5′-29M13-CCT ATA ATG CAC GAC AGA AC-3′) and P6 (5′-21M13-AAA CCA GCA ATA GCT CCG AA-3′), followed by sequencing with primers 29M13 (5′-CAG GAA ACA GCT ATG ACC-3′) and 21M13 (5′-TGT AAA ACG ACG GCC AGT-3′) using a DYEnamic ET terminator cycle sequencing premix kit (Amersham) and an ABI-373A sequencing apparatus (Perkin Elmer).

We are grateful to L. van der Kemp, G. de Mutsert, and M. van der Bildt for technical assistance; J. Habova for electron microscopy; Solvay Pharmaceuticals, Weesp, the Netherlands, for providing HA/NA proteins from B/Harbin/7/94; and the SRRC staff for taking care of and samples from seals. R. F. is a fellow of the Royal Netherlands Academy of Arts and Sciences.