Michiyo Shinohara

Broadly Reactive and Highly Sensitive Assay for Norwalk-Like Viruses Based on Real-Time Quantitative Reverse Transcription-PCR

ABSTRACT We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.

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

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.

Coexistence of Multiple Genotypes, Including Newly Identified Genotypes, in Outbreaks of Gastroenteritis Due to Norovirus in Japan

ABSTRACT Norovirus (NV) (formerly called Norwalk-like virus) is the most common cause of acute nonbacterial gastroenteritis in humans. Recently, we reported an NV genotyping scheme based on variability in the capsid N-terminal/shell (N/S) domain gene (Katayama et al., Virology 299:225-239, 2002). We found 19 genotypes, including nine of genogroup I and 10 of genogroup II. In the present study, we investigated the molecular epidemiology of NV from 66 outbreaks that occurred in Saitama Prefecture, Japan, from 1997 to 2002. We screened 416 stool specimens by a real-time reverse transcription (RT)-PCR method (Kageyama et al., J. Clin. Microbiol. 41:1548-1557, 2003) and detected 156 NV-positive specimens, from which we amplified the capsid N/S domain gene by RT-PCR and then cloned the PCR products. After sequencing these clones, we obtained 368 sequence variants (strains). By applying our classification scheme to the strains from Saitama and other published strains, we identified a total of 31 genotypes, including an additional five genotypes for genogroup I and seven for genogroup II. Of the 31 genotypes, 26 were present in the Saitama area during that time period. These results provide additional evidence for the great diversity of human NV genotypes. Specimens from all shellfish-related infections contained multiple genotypes, including several new genotypes. On the other hand, single genotypes were observed mostly in outbreaks that originated in semiclosed communities. Thus, the number of NV genotypes in each outbreak depended on the route of transmission.

Detection, Quantitation, and Phylogenetic Analysis of Noroviruses in Japanese Oysters

ABSTRACT Noroviruses (NVs) cause many cases of oyster- or clam-associated gastroenteritis in various countries. We collected 191 samples from Japanese oysters intended for raw consumption that had been harvested from the sea in two different areas between December 2001 and February 2002. To detect, quantitate, and phylogenetically analyze the NV genome in purified concentrates from the stomachs and digestive diverticula of these oysters, we amplified the NV capsid gene by reverse transcription-PCR. Phylogenetic analysis was performed by using the neighbor-joining method. We detected the NV genome in 17 of 191 oysters (9%). Phylogenetic analysis indicated genogroup I (Norwalk virus type) in 3 of the 17 oysters and genogroup II (Snow Mountain virus type) in the other 14. Both genogroups showed wide genetic diversity. To quantify the NV capsid gene in these oysters, we performed real-time PCR using genogroup-specific probes. More than 102 copies of the NV genome were detected in 11 of 17 oysters. The results suggested that about 10% of Japanese oysters intended for raw consumption harbored NVs, and more than 50% of those oysters in which NVs were detected had a large amount.

Genetic analyses of GII.17 norovirus strains in diarrheal disease outbreaks from December 2014 to March 2015 in Japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region

A novel GII.P17-GII.17 variant norovirus emerged as a major cause of norovirus outbreaks from December 2014 to March 2015 in Japan. Named Hu/GII/JP/2014/GII.P17-GII.17, this variant has a newly identified GII.P17 type RNA-dependent RNA polymerase, while the capsid sequence displays amino acid substitutions around histo-blood group antigen (HBGA) binding sites. Several variants caused by mutations in the capsid region have previously been observed in the GII.4 genotype. Monitoring the GII.17 variants geographical spread and evolution is important.

Characterization of Mumps Virus Isolated in Saitama Prefecture, Japan, by Sequence Analysis of the SH Gene

Mumps virus (MuV) strains isolated from cerebrospinal fluid and throat swabs from patients in Saitama Prefecture and Tokyo, Japan, from 1997 to 2000 were examined by analyzing the SH gene nucleotide sequence (316‐nt). Eighteen of the 20 strains studied were divided into three genotypes, recognized as B, G, and H in previous reports. Two genotypes (G and H) are believed to be new in Japan. Two of the 20 strains belonged to none of the previously reported genotypes (A–I), but were closely related to two known strains, MP94‐H and Lougl/UK97. We propose that the two strains identified in this study together with the previously reported strains, MP94‐H and Lougl/UK97, form a new genotype, designated J, based on the divergence of the SH gene nucleotide sequences between these four strains and other strains reported (genotypes A–I). Our results also suggest that more than two genotypes circulated in Saitama Prefecture from 1997 to 1999, but only one, genotype G, was in evidence in 2000. Genotype B was earlier reported as the predominant strain in Japan, but it became undetectable by the year 2000. These results provide important epidemiological data on mumps in Japan.

Characterization of the F Gene of Contemporary Mumps Virus Strains Isolated in Japan

Mumps virus (MuV) strains isolated in Saitama Prefecture, Japan, from 1997 to 2001, were examined by analyzing the SH and the F gene nucleotide sequences. The results of the SH gene analysis showed that only genotype G was found in 2001 as well as in 2000, and that genotype J, which we proposed as a new genotype in a previous study, was from a different lineage than the genotype J described by Tecle et al. (J. Gen. Virol. 82, 2675–2680). We therefore, propose to rename the genotype as K to avoid confusion. Then, the F gene of genotypes G, H, and K strains were analyzed together with previously reported strains in this study. The results of phylogenetic analysis of the F gene nucleotide sequences showed that these strains formed a cluster as described by the SH gene analysis. Alignment of the F amino acid sequences showed that the F protein was well conserved among strains of different genotypes with a few amino acid differences. These results provide better information for the characterization of contemporary MuV strains in Japan.

Novel concentration method for the detection of norovirus and sapovirus from water using minute particles of amorphous calcium phosphate

A novel concentration method using minute particles of amorphous calcium phosphate (ACP) was developed for the detection of caliciviruses including norovirus and sapovirus, agents of human gastroenteritis, from water. In seeding experiments with feline calicivirus (FCV), ACP particles were able to adsorb efficiently the viruses in water, and the FCV-concentrated solution was obtained by dissolution of the virus-adsorbing ACP particles with citric acid after centrifugation. By quantitative real-time RT-PCR, the recovery efficiencies from 300 ml ultrapure water seeded with 10³, 10⁴ and >10⁵ copies of FCV were 48, 68 and >100 %, respectively. A comparative study showed that in the addition of viruses at <10⁵ copies, the recovery efficiency of our method was significantly higher (P<0.05) than that of the similar calcium flocculation-citrate dissolution method. Using our newly developed method, we successfully detected 2.1 x 10⁴ copies l⁻¹ of norovirus (each of genogroups I and II) and 5.4 x 10³ copies l⁻¹ of sapovirus (genogroups I, II, IV and V) from river water. The data suggest that our new viral concentration is a rapid, simple, cost efficient and high virus recovery method, and it can be used for routine monitoring of norovirus and sapovirus in water, especially environmental water.

Molecular Evolution of the RNA-Dependent RNA Polymerase and Capsid Genes of Human Norovirus Genotype GII.2 in Japan during 2004–2015

The RNA-dependent RNA polymerase (RdRp) and capsid (VP1) genes of 51 GII.2 human norovirus (HuNoV) strains collected during the period of 2004–2015 in Japan were analyzed. Full-length analyses of the genes were performed using next-generation sequencing. Based on the gene sequences, we constructed the time-scale evolutionary trees by Bayesian Markov chain Monte Carlo methods. Time-scale phylogenies showed that the RdRp and VP1 genes evolved uniquely and independently. Four genotypes of GII.2 (major types: GII.P2-GII.2 and GII.P16-GII.2) were detected. A common ancestor of the GII.2 VP1 gene existed until about 1956. The evolutionary rates of the genes were high (over 10−3 substitutions/site/year). Moreover, the VP1 gene evolution may depend on the RdRp gene. Based on these results, we hypothesized that transfer of the RdRp gene accelerated the VP1 gene evolution of HuNoV genotype GII.2. Consequently, recombination between ORF1 (polymerase) and ORF2 (capsid) might promote changes of GII.2 antigenicity.

Application of a simple method using minute particles of amorphous calcium phosphate for recovery of norovirus from cabbage, lettuce, and ham

In this study, the amorphous calcium phosphate (ACP) method developed previously for calicivirus concentration from water was applied for norovirus detection from food. The viral recovery from cabbage, lettuce, or ham (10g of each) was firstly examined in seeding experiments with feline caliciviruses (FCVs). The viruses were concentrated by viral adsorption to ACP particles (0.3g) in the eluent solution (40ml) from foods, collection of the particles by centrifugation, followed by dissolution of the particles with 3.3M citric acid (3ml). In ham, FCV recovery was improved by addition of ascorbic acids into the eluent solution before ACP-particle adsorption. Quantitative real-time reverse transcription-PCR (qRT-PCR) revealed that FCV recoveries were 32-33%, 50-55%, and 37-46% from cabbage, lettuce, and ham, respectively, when seeded with 10(3)-10(4) viruses, and detection limits were estimated ∼10(3) genomic copies in all 3 foods. Subsequently, the ACP-concentration method was evaluated for norovirus (NoV) detection from these 3 foods. The recoveries and detection limit of NoVs determined by qRT-PCR were 12-41% and 10(3) (genomic copies) from cabbage, 30-57% and 10(3) from lettuce, and 20-26% and 10(4) from ham, when seeded with 10(3)-10(5) viruses. This simple method may be suitable for NoV detection from these foods.