Direct sequence determination of the influenza B HA-1 gene after PCR amplification of clinical specimens from an infected volunteer

Abstract

When clinical isolates of influenza A and B viruses are propagated in embryonated hens' eggs or tissue culture cells, different selective pressures in vitro result in specific amino acid substitutions in the haemagglutinin (HA) gene. A proportion of such viruses which lose a potential 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 infected volunteer were performed. The nucleotide sequences of the viral HA-1 from the nasopharynx of the infected volunteer were the same as that of the original infecting strain. Antigenic analysis of both the original infecting virus and the viruses isolated from sequential samples collected from the volunteer, all of which were cultivated on Madin-Darby Canine Kidney (MDCK) cells and in embryonated hens' eggs, revealed variation in the HA of viruses only after egg adaptation. In particular, we describe the use of direct nucleotide sequencing techniques without the use of cloning strategies in order to determine the sequence of the HA-1 gene after direct PCR amplification of clinical nasal wash material.