Genotyping of GII.4 and GIIb norovirus RT-PCR amplicons by RFLP analysis.
GII.4 and GIIb/Hilversum norovirus (NoV) strains appear to have a prominent epidemiological role in outbreaks or sporadic cases of human gastroenteritis. Sequence analysis, although laborious, is the reference method used for characterization of noroviruses. In this study a screening test is proposed to characterize GIIb and GII.4 NoVs based on restriction fragment length polymorphism (RFLP) analysis of amplicons obtained from the RNA-dependent RNA polymerase (RdRp) region. Virtual analysis of 793 RdRp sequences of GGI and GGII NoVs, retrieved from GenBank, and representative of global geographical origins on a long-time period, permitted the selection of four restriction enzymes, XmnI, AhdI, BstXI, and AcuI, suitable for correct identification of GIIb and GII.4 NoV genotypes. Experimental analysis by the RT-PCR RFLP analysis of 41 NoV strains detected in Palermo during the years 2002-2005 allowed to recognize all the Italian strains as belonging to GIIb/Hilversum or GII.4, and sequence analysis confirmed these results. The PCR-RFLP protocol developed in this study proved to be a simple and reliable proxy for sequence-based classification of the GIIb/Hilversum and GII.4 NoV variants displaying high specificity (100%) and sensitivity (94%).
Ramirez
Stefania
S
Department of Hygiene and Microbiology, University of Palermo, via del Vespro 133, Palermo, Italy.
Giammanco
Giovanni M
GM
De Grazia
Simona
S
Colomba
Claudia
C
Martella
Vito
V
Arista
Serenella
S
D017250:Caliciviridae Infections / Q000821:virology*; D002675:Child, Preschool; D005759:Gastroenteritis / Q000821:virology; D005838:Genotype; D006801:Humans; D007223:Infant; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D012150:Polymorphism, Restriction Fragment Length; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012367:RNA, Viral