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0343502030
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unpublished data
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A. D. Osterhaus, G. F. Rimmelzwaan, B. E. Martina, T. M. Bestebroer, R. A. Fouchier, unpublished data.
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Osterhaus, A.D.1
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18
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0343937882
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note
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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 cycle sequencing premix kit (Amersham) on an ABI-373A apparatus (Perkin Elmer).
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19
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0342631836
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note
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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 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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21
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0342631837
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note
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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 antidog IgM antibody preparation specifically captures seal IgM, as was shown in routine serological tests for PDV and PHV.
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22
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0004050245
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Blackwell Scientific, Oxford, ed. 8
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I. M. Roitt, Essential Immunology (Blackwell Scientific, Oxford, ed. 8, 1994).
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Roitt, I.M.1
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23
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0342631528
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The sequences of influenza B virus HA and NS genes are available at www.flu.lanl.gov/.
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24
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0343502028
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note
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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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28
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0018641820
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C. B. Hall, R. G. Douglas, J. M. Geiman, M. P. Meagher, J. Infect. Dis. 140, 610 (1979).
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Geiman, J.M.3
Meagher, M.P.4
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29
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0343502023
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note
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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).
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30
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0343502024
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note
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We are grateful to L. van der Kemp, C. 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.
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