Molecular and phylogenetic analysis of haemagglutinin and neuraminidase sequences from recent human influenza type A (H3N2) viral isolates in Southern Greece.

Eighteen haemagglutinin (HA1) gene segments and eleven neuraminidase (NA) genes of human influenza type A (H3N2) viruses isolated from non-vaccinated individuals presenting severe influenza-like illness at peak influenza activity in Southern Greece during the surveillance period 1996-1999, were subjected to sequence and phylogenetic analyses following propagation in embryonated hen's eggs. The HA1 gene segment of the clinical isolates differed from the recent reference influenza type A (H3N2) vaccine strains in an Ile at residue 186, a Val at residue 194 and a Val at residue 226 for one, two and thirteen isolates of the 1996-1997 and 1996-1999 periods, respectively. The analogous differences in the NA gene were confined in an Asp to Asn substitution at residue 198 in one A/Wuhan/359/95 (H3N2)-like isolate of the 1996-1997 period, primarily. In addition, phylogenetic analysis revealed that an isolate of the 1997-1998 period was a recombinant with its HA1 gene segment being closely related to that of A/Wuhan/359/95-like viruses and its NA to viruses of the A/Sydney/5/97 (H3N2) lineage. These findings confirmed the profound genetic instability of influenza type A (H3N2) viruses and underscored the importance for periodic molecular surveys of HA and NA in the effective prevention and management of viral outbreaks. Most importantly, however, they contributed the first complete epidemiological material for influenza in Southern Greece, the archival nature of which constitutes valuable reference for future surveys.

High sequence divergence in the 5' non-coding region of reference Coxsackie B and ECHO viral strains and clinical isolates revealed by restriction fragment length polymorphism analysis.

We report the restriction fragment length polymorphism (RFLP) patterns of a 440-bp-long 5' non-coding region (5' NCR) amplification target of all 34 reference Coxsackie B and ECHO (enteric cytopathic human orphan) enterovirus strains and a total of 42 serotypically pre-assigned clinical isolates, in order to afford meaningful comparisons among these patterns and those of polioviruses. The RFLP patterns of reference Coxsackie B strains differed from one another and from those of polio and ECHO reference enteroviruses except from Coxsackie B1 and B2, which, although they differed from one another, had identical RFLP patterns with ECHO 17 and 13, respectively. The 28 ECHO reference strains formed a more variable viral group including strains with RFLP patterns distinct from one another and from those of polio and Coxsackie B enteroviruses, and others with RFLP pattern identities common to other ECHO viruses and Coxsackie B1 and B2 but not polioviruses. The RFLP patterns of the clinical isolates and their corresponding serotypically assigned reference Coxsackie B and ECHO strains presented the most notable variations. The observed differences between serotype and genotype-dependent assignments within the 440-bp long 5' NCR target sequence of Coxsackie B and ECHO enteroviruses were in sharp contrast to the analogous situation with polioviruses. These findings support the specificity of the described method for clinical diagnostic genotyping of polioviruses and demonstrate that the 440-bp-long target sequence follows a different evolutionary process in polio and non-polio enteroviruses that is particularly prominent between reference non-polio strains and their serotypically assigned clinical isolates.