Molecular detection and epidemiology of Sapporo-like viruses

Abstract

Sapporo-like viruses (SLVs) are associated with acute gastroenteritis in humans. Due to a limited supply of available reagents for diagnosis, little is known about the incidence and pathogenicity of these viruses. We have developed a first-generation generic reverse transcriptase (RT) PCR assay based on a single primer pair targeting the RNA polymerase gene. With this assay, 55 (93%) of the 59 stool specimens collected in a 10-year period of time (1988 to 1998) and containing typical caliciviruses by electron microscopy tested positive and could be confirmed by Southern hybridization. By phylogenetic analysis, most SLV strains could be classified into one of the three recently described genotypes. However, three samples clustered separately, forming a potential new genotype. We sequenced the complete capsid gene of one of the strains in this cluster: Hu/SLV/Stockholm/97/SE. Alignment of the capsid sequences showed 40 to 74% amino acid identity among strains of the different clusters. Phylogenetic analysis of the aligned sequences confirmed the placing of Hu/SLV/Stockholm/97/SE into a new distinct genetic cluster. This is the first report on the development of a broadly reactive RT-PCR assay for the detection of SLVs.

FIG. 1

Electron micrograph of typical calicivirus particles in a stool specimen. Bar, 50 nm.

FIG. 2

Phylogenetic analysis of published SLV sequences and sequences from a selection of strains detected in the United Kingdom and Sweden (strain numbers correspond to those in Table 1) and analyzed in this study. Multiple alignments were created based on a 147-nucleotide region of the RNA-dependent RNA polymerase gene (A) and deduced amino acid sequences (B). Distance matrixes were calculated using the Jukes and Cantor correction for evolutionary rate (26). One hundred bootstrapped data sets were analyzed, and evolutionary trees were drawn using the UPGMA clustering method. GenBank accession numbers for the prototype SLVs are as follows: X86560 (Manchester), S77903 (Sapporo), U73124 (Parkville), U43287 (VanderBijlpark), U67858 (London), U67856 (Houston/86), and U67859 (Houston/90).

FIG. 3

Results of ethidium bromide staining (A and C) and corresponding Southern hybridization (B and D) of products of generic SLV RT-PCR (320-bp product) (A and B) and NLV RT-PCR (326-bp product) (C and D). The templates were RNA from strains representing the four different SLV genotypes (Sapporo [SAP], Parkville [PAR], London [LON], and Stockholm [STO]) and RNA representing strains from NLV genogroup I (Norwalk [NV] and Southampton [SOV]) and genogroup II (Lordsdale [LDV] and Mexico [MXV]). M, DNA molecular weight marker V.