DIRECT-SEQUENCE DETERMINATION OF THE INFLUENZA-B HA-1 GENE AFTER PCR AMPLIFICATION OF CLINICAL SPECIMENS FROM AN INFECTED VOLUNTEER

When clinical isolates of influenza A and B viruses are propagate in e mbryonated hens' eggs or tissue culture cells, different selective pre ssures in vitro result in specific amino acid substitutions in the hae magglutinin (HA) gene. A proportion of such viruses which lose a poten tial glycosylation site near the receptor binding region of the HA at amino acid positions 196-198 appear to have reduced virulence. Direct polymerase chain reaction (PCR) amplifications of cDNA and subsequent nucleotide sequence analysis of part of the HA-1 gene of the original infecting influenza B strain and the nasal wash material from an infec ted volunteer were performed. The nucleotide sequences of the viral HA -1 from the nasopharynx of the infected volunteer were the same as tha t of the original infecting strain. Antigenic analysis of both the ori ginal infecting virus and the viruses isolated from sequential samples collected from the volunteer, all of which were cultivated on Madin-D arby Canine Kidney (MDCK) cells and in embryonated hens' eggs, reveale d variation in the HA of viruses only after egg adaptation. In particu lar, we describe the use of direct nucleotide sequencing techniques wi thout the use of cloning strategies in order to determine the sequence of the HA-1 gene after direct PCR amplification of clinical nasal was h material.