Genogroup-specific PCR primers for detection of Norwalk-like viruses

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Abstract

Norwalk-like viruses (NLV) are a major causative agent of nonbacterial gastroenteritis. There are still many NLV strains that are refractory to gene amplification by ordinary reverse transcription-polymerase chain reaction. This is due mainly to the genetic diversity among NLV, especially mismatches in the primer sequences, which limits this technique in clinical utility. In this study, improved primer sets based on the capsid region, to detect both genogroup I and II NLV by genogroup-specific manner, were developed. When stool specimens from gastroenteritis patients, that were positive for NLV by electron microscopy, were tested by this new primer set, all specimens were positive by RT-PCR. Primers described previously for RdRp and capsid protein were capable of amplifying the specimens by 31 and 77%, respectively. Therefore, new primer sets are extremely useful for the amplification and rapid diagnosis of nonbacterial gastroenteritis due to NLV as well as for epidemiological studies.

Introduction

Norwalk-like viruses (NLV), in the family Caliciviridae (Green et al., 2000), are the major causative agent of non-bacterial acute gastroenteritis that frequently occurs in the winter season (Xi et al., 1990). NLV are a small icosahedral virus with a single-stranded positive-sense RNA genome. Complete nucleotide sequences have been determined for several strains, including Norwalk virus (Jiang et al., 1993), Southampton virus (Lambden et al., 1993), Lordsdale virus (Dingle et al., 1995), Camberwell virus (Seah et al., 1999a), Hawaii virus (Lew et al., 1994a), Chiba virus (Someya et al., 2000) and the BS5 strain (Schreier et al., 2000). The genome of NLV contains three open reading frames (ORFs). ORF1 encodes a large polypeptide containing amino acid sequence motifs observed in many RNA viruses, such as RNA helicase, 3C-like protease and RNA-dependent RNA polymerase (Dingle et al., 1995). ORF2 encodes the capsid protein with an apparent molecular mass of 58 kDa. The capsid protein has several domains, including the N domain facing the interior of the capsid, the S domain involving the formation of the icosahedral shell and the P domain forming the prominent protrusion emanating from the shell (Prasad et al., 1999). ORF3 encodes a small protein abundant in basic amino acids (Seah et al., 1999b). Although the precise role of the ORF3 protein is unknown, it is likely that it is a minor structural protein that interacts with the genome RNA when the virion formation occurs (Glass et al., 2000). Phylogenetic analyses, based on the entire genome, revealed that the NLV could be segregated into two genetic groups, genogroup I (GI) and genogroup II (GII) (Green et al., 1994, Wang et al., 1994). The nucleotide sequence similarity between GI and GII NLV is <60% and that within each genogroup is ≈75% (Green et al., 1993). Thus, NLV comprises many genetically heterogeneous viruses.

Although there is neither cell culture systems nor small animal models for propagating the virus, detection of the virus in stool specimens from patients with acute gastroenteritis has been accomplished by electron microscopy (EM) or immune EM. Ever since complete nucleotide sequences of Norwalk virus, the prototype strain of NLV (Jiang et al., 1993) and Southampton virus (Lambden et al., 1993) were determined in 1993, reverse transcription-polymerase chain reaction (RT-PCR) followed by either hybridization or nucleotide sequence analysis has became a common tool to identify NLV.

Sensitive RT-PCR-based assay relies heavily on the design of the appropriate primers for the PCR. The RNA-dependent RNA polymerase (RdRp) region has been considered to be functionally the most conserved domain of the NLV genome (Green et al., 1993, Ando et al., 1995). Although many primers designed in the RdRp region amplified a broad range of NLV (Wang et al., 1994, Ando et al., 1995), the genetic diversity of NLV has made it difficult to detect all NLV with these primers.

Kobayashi et al. (2000b) used a set of primers, designated to amplify the N terminal domain and a part of the shell domain of the capsid protein (capsid N/S domain) of NLV. Success in amplifying many strains with this primer set led us to use them for the detection of NLV. However, when 35 EM-positive stool specimens were examined by RT-PCR, this primer set was not capable of amplifying eight strains.

In this study, genogroup-specific primers improved further, were investigated by using sequences from published databases and eight additional NLV sequences.

Section snippets

Stool specimens

Thirty-five stool specimens were collected from 19 gastroenteritis outbreaks in the Saitama Prefecture, Japan, between 1997 and 1999. All stool specimens were positive for NLV by EM and stored at −80 °C.

Extraction of RNA

Viral RNA was extracted from 140 l of 10% water suspensions of the stool specimens with the QIAamp viral RNA extraction kit (Qiagen), according to manufacturer's instructions. RNA was eluted with 60 l of diethyl pyrocarbonate-treated water and stored at −80 °C until use in RT-PCR assays.

Primers and RT-PCR

The primer

Determination and optimization of primers for efficient amplification

During routine examination of NLV, the detection rate by RT-PCR of the EM-positive stool specimens from gastroenteritis patients was found to be relatively low when primer sets C was employed. For example, only 11 out of 35 EM-positive stool specimens (31%) were positive in the first RT-PCR (data not shown) and 28 (80%) become positive when the nested PCR was carried out. In contrast, primer set A, which amplified the capsid N/S region, was capable of amplifying 27 specimens (77%) by the first

Discussion

Since NLV cannot be propagated in either small experimental animals or tissue culture systems, detection of NLV has relied mainly on a classical EM method. Although many RT-PCR has been explored as a means for detecting NLV (Green et al., 1993, Jiang et al., 1999), the application of this method has been limited by the lack of suitable primers.

Previous studies have used the RdRp region for the amplification because this region was believed to contain the most conserved nucleotide sequence in

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