Simultaneous detection and genotyping of "Norwalk-like viruses" by oligonucleotide array in a reverse line blot hybridization format

Abstract

"Norwalk-like viruses" (NLVs) are the most common cause of outbreaks of nonbacterial gastroenteritis worldwide. To date, the method most widely used for typing of NLV strains is sequencing and subsequent phylogenetic analysis of reverse transcription (RT)-PCR products, which has revealed the existence of stable distinct lineages (genotypes). This typing method is rather costly, not routinely used in clinical laboratories, and not very suitable for the analysis of large numbers of samples. Therefore, we have developed a rapid and simple method for genotyping of NLVs. The method, designated reverse line blot hybridization, is based on the nucleotide divergence of a region of the gene for RNA polymerase which can be used to classify NLVs into genotypes. NLV RNA was amplified by RT-PCR and then hybridized to 18 different membrane-bound oligonucleotides that were able to discriminate among 13 NLV genotypes. Application of the method to a panel of 132 positive stool samples from 34 outbreaks and 20 sporadic cases of gastroenteritis collected in a 6-year period (1994 to 1999) resulted in successful genotyping of 124 samples (94%), as confirmed by phylogenetic analysis. The nucleotide sequences of the remaning eight strains (6%) from three outbreaks did not cluster with the known NLV genotypes. Phylogenetic analysis of the complete and partial open reading frame 2 (capsid gene) sequences of these strains revealed the existence of one novel genotype (Alphatron) and one potentially novel genotype (Amsterdam). This novel method, which allows simultaneous detection and genotyping of NLVs, is useful in the diagnosis and typing of NLVs obtained from outbreaks and in large-scale epidemiological studies.

FIG. 1

Detection and simultaneous typing of NLV strains from OBs (20 strains in panels B and D) and sporadic cases (6 strains in panel C) of gastroenteritis in The Netherlands. The probes used to detect GGIa and GGIb strains, GGII strains, 13 different genotypes (Table 1, genotypes 1 to 13), and strains Wortley and Rotterdam are indicated. For reference, the hybridization patterns of these 15 strains are shown in panel A. In panel B, strains belonging to the same OB have identical hybridization patterns (lanes 1 to 3, 4 to 6, 7 to 9, and 10 to 12), whereas panel D shows the hybridization patterns of two OBs in which two different strains were detected (lanes 1 to 4 and 5 to 8). Hybridization patterns of six different sporadic cases are shown in panel C.

FIG. 2

Phylogenetic relationships based on a 145-bp region of the RNA polymerase-encoding gene showing the relationships among representative strains of NLV genotypes from GGI (Norwalk/68 [NV], Southampton/91 [SOV], Desert Shield/90 [DSV], Queens Arms/89 [QA], Sindlesham/95 [SHV], Musgrove/89 [MGV], and Winchester/94 [WCV]) and GGII (Lordsdale/93 [LDV], Mexico/89 [MXV], Melksham/94 [MEV], Hawaii/72 [HV], Hillingdon/94 [HDV], Leeds/90 [LEV], viruses in the Rotterdam cluster [RDV], and Wortley/90 [WLV]), as well as the 26 strains previously typed by the RLB assay shown in Fig. 1 (in bold). Designations of RLB-typed strains refer to lanes in Fig. 1. Bootstrap values of the internal nodes are indicated.

FIG. 3

Phylogenetic relationships based on a 145-bp region of the RNA polymerase-encoding gene showing the relationships among NLV genotypes from GGI (Norwalk/68 [NV], Southampton/91 [SOV], and Desert Shield/90 [DSV]), GGII (Lordsdale/93 [LDV], Mexico/89 [MXV], Melksham/93 [MEV], Hawaii/72 [HV], Hillingdon/94 [HDV], and Leeds/90 [LEV]), and eight strains previously nontypeable by RLB (in bold). Bootstrap values of the internal nodes are indicated.