Molecular characterization of a new intergenotype Norovirus GII recombinant.

Human noroviruses (NoVs) of the Caliciviridae family are a major cause of epidemic gastroenteritis. The NoV genus is genetically diverse and recombination of viral RNA is known to depend upon various immunological and intracellular constraints that may allow the emergence of viable recombinants. In the present study, we report the development of a broadly reactive RT-PCR assay, which allowed the characterization of strain A6 at molecular level, established its genetic relationship at the sub-genogroup level and classified A6 strain at the sub-genotype level. The detection was carried out initially by enzyme-linked immunosorbent assay (ELISA) and the subsequent detection and molecular characterization of NoV strain was achieved by reverse transcription-PCR and sequencing. Based on the sequence analysis, A6 strain was revealed to belong to the GII genogroup of NoVs. Partial ORF1 gene sequencing analysis and complete ORF2 gene sequencing revealed that ORF1 and ORF2 belonged to two distinct genotypes GII/9 and GII/6, respectively, making obvious that A6 strain is a rare intergenotypic recombinant within the genogroup GII between GII.9 and GII.6 genotypes. A6 strain represents the first human NoV from Greece, whose genome has been partially (ORF1&ORF3) and completed (ORF2) sequenced. To our knowledge the recombination event GII.9/GII.6 in RdRp and capsid gene, respectively, that was revealed in the present study is reported for the first time.

Complete nucleotide sequence analysis of the VP1 genomic region of Echoviruses 6 isolated from sewage in Greece revealed 98% similarity with Echoviruses 6 that were characterized from an aseptic meningitis outbreak 1 year later.

The molecular characterization of two enterovirus strains (LR51A5 and LR61G3) isolated from the sewage treatment plant unit in Larissa, Greece, in May and June 2006 and the investigation of their relationship with enteroviruses of the same serotype isolated in Greece in 2001 and 2007 were performed by complete VP1 sequence analysis of the isolates. The close phylogenetic relationship and the high nucleotide similarity (98%) led to the conclusion that the virus isolated from sewage in 2006 was associated with that isolated from an aseptic meningitis outbreak 1 year later. Bootscan analysis of the VP1 genomic region revealed that intraserotypic multi-recombination events might have been involved in the evolutionary past history of the LR51A5 and LR61G3 isolates.

Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains.

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.