Application of the heteroduplex mobility assay (HMA) for the investigation of the genomic diversity among noroviruses in environmental samples.
Recent studies have demonstrated the widespread contamination of river and seawater with noroviruses (NV), often with more than one strain. The heteroduplex mobility assay (HMA) in which amplicons from study samples are hybridised (by denaturing and reannealing) to amplicons from reference strains and resolved by electrophoresis, has the potential to provide a simple and rapid means to identify samples containing multiple NV strains and to establish the diversity of strains within that sample. PCR amplicons from environmental samples that were tested directly in the HMA assay were shown to contain more than one strain. In order to evaluate HMA for investigations of NV diversity in environmental samples, amplicons from three representative samples were cloned and, for each, 20 amplicons derived from individual clones were analysed by HMA. Between two and six different HMA profiles were demonstrated among clones from a single sample indicating the extent of NV diversity in the sample. Sequence analysis confirmed the relationship of HMA profile and NV 'genotype'. Far greater diversity was seen among Genogroup (G) II (Ni/E3) amplicons than Genogroup (G) I (Ando/E3) amplicons (generated from the RNA dependent RNA polymerase region of the ORF1 of noroviruses), which often contained only a single strain, which is reflective of the greater prevalence of GII NVs over GI NVs. Overall, four GII and four GI strains were identified in these environmental water/sewage samples.
Green
Jonathan
J
Enteric, Respiratory and Neurological Virus Laboratory, Specialist and Reference Microbiology Division, Health Protection Agency, Colindale, London, UK.
Gallimore
Chris I
CI
Shore
Jane
J
Sellwood
Jane
J
Brown
David W G
DW
D001483:Base Sequence; D016172:DNA Fingerprinting; D018076:DNA, Complementary / Q000032:analysis / Q000302:isolation & purification; D004783:Environmental Microbiology; D014644:Genetic Variation; D005838:Genotype; D020180:Heteroduplex Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D029322:Norovirus / Q000235:genetics* / Q000302:isolation & purification*; D010802:Phylogeny; D012367:RNA, Viral / Q000302:isolation & purification / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012623:Seawater / Q000821:virology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012722:Sewage / Q000821:virology; D014764:Viral Proteins / Q000235:genetics
D018076:DNA, Complementary; D012367:RNA, Viral; D012722:Sewage; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase