Development of a multiplex real-time PCR assay for detection of human enteric viruses other than norovirus using samples collected from gastroenteritis patients in Fukui Prefecture, Japan.

There are many varieties of gastroenteritis viruses, of which norovirus (NoV) accounts for over 90% of the viral food poisoning incidents in Japan. However, protocols for rapidly identifying other gastroenteritis viruses need to be established to investigate NoV-negative cases intensively. In this study, a multiplex real-time PCR assay targeting rotavirus A, rotavirus C, sapovirus, astrovirus, adenovirus, and enterovirus was developed using stool samples collected from gastroenteritis patients between 2010 and 2013 in Fukui Prefecture, Japan. Of the 126 samples collected sporadically from pediatric patients with suspected infectious gastroenteritis, 51 were positive for non-NoV target viruses, whereas 27 were positive for NoV, showing a high prevalence of non-NoV viruses in pediatric patients. In contrast, testing in 382 samples of 58 gastroenteritis outbreaks showed that non-NoV viruses were detected in 13 samples, with NoV in 267. Of the 267 NoV-positive patients, only two were co-infected with non-NoV target viruses, suggesting that testing for non-NoV gastroenteritis viruses in NoV-positive samples was mostly unnecessary in outbreak investigations. Given these results, multiplex real-time PCR testing for non-NoV gastroenteritis viruses, conducted separately from NoV testing, may be helpful to deal with two types of epidemiological investigations, regular surveillance of infectious gastroenteritis and urgent testing when gastroenteritis outbreaks occur.

Molecular evolution of the fusion protein (F) gene in human respiratory syncytial virus subgroup B.

In this study, we examined the molecular evolution of the fusion protein (F) gene in human respiratory syncytial virus subgroup B (HRSV-B). First, we performed time-scale evolution analyses using the Bayesian Markov chain Monte Carlo (MCMC) method. Next, we performed genetic distance, linear B-cell epitope prediction, N-glycosylation, positive/negative selection site, and Bayesian skyline plot analyses. We also constructed a structural model of the F protein and mapped the amino acid substitutions and the predicted B-cell epitopes. The MCMC-constructed phylogenetic tree indicated that the HRSV F gene diverged from the bovine respiratory syncytial virus gene approximately 580years ago and had a relatively low evolutionary rate (7.14×10-4substitutions/site/year). Furthermore, a common ancestor of HRSV-A and -B diverged approximately 290years ago, while HRSV-B diverged into three clusters for approximately 60years. The genetic similarity of the present strains was very high. Although a maximum of 11 amino acid substitutions were observed in the structural model of the F protein, only one strain possessed an amino acid substitution located within the palivizumab epitope. Four epitopes were predicted, although these did not correspond to the neutralization sites of the F protein including the palivizumab epitope. In addition, five N-glycosylation sites of the present HRSV-B strains were inferred. No positive selection sites were identified; however, many sites were found to be under negative selection. The effective population size of the gene has remained almost constant. On the basis of these results, it can be concluded that the HRSV-B F gene is highly conserved, as is the F protein of HRSV-A. Moreover, our prediction of B-cell epitopes does not show that the palivizumab reaction site may be recognized as an epitope during naturally occurring infections.

Molecular evolution of the hypervariable region of the attachment glycoprotein gene in human respiratory syncytial virus subgroup B genotypes BA9 and BA10.

We studied the molecular evolution of the C-terminal 3rd hypervariable region in the attachment glycoprotein gene of human respiratory syncytial virus subgroup B (HRSV-B) genotypes BA9 and BA10. We performed time-scaled phylogenetic analyses using Bayesian Markov chain Monte Carlo methods. We also performed a genetic distance analysis (p-distance analysis), positive and negative selection analyses, and a Bayesian skyline plot (BSP) analysis. We found that genotype BA9 diverged from the common ancestor of genotypes BA7, BA8, and BA10, while genotype BA10 diverged from the ancestor of genotypes BA7 and BA8. Strains of both genotypes were distributed worldwide. BA9 and BA10 diverged between 1999 and 2001. Both BA9 and BA10 evolved rapidly (about 4.8×10(-3)substitutions/site/year) and formed three distinct lineages in a 10-year period. BA10 strains belonging to lineage 3 had large genetic distances (p-distance>0.07). Thus, it may be possible to classify these strains as a new genotype, BA11. No positive selection site was detected in either genotype. Phylodynamic analyses showed that the effective population size of BA10 decreased gradually since 2010 and BA9 slightly decreased since 2009. The results suggested that the recently prevalent HRSV-B genotypes BA9 and BA10 evolved uniquely, leading to epidemics of HRSV-B worldwide over a 15-year period.

Molecular evolution of human respiratory syncytial virus attachment glycoprotein (G) gene of new genotype ON1 and ancestor NA1.

We conducted a comprehensive genetic analysis of the C-terminal 3rd hypervariable region of the attachment glycoprotein (G) gene in human respiratory syncytial virus subgroup A (HRSV-A) genotype ON1 (93 strains) and ancestor NA1 (125 strains). Genotype ON1 contains a unique mutation of a 72 nucleotide tandem repeat insertion (corresponding to 24 amino acids) in the hypervariable region. The Bayesian Markov chain Monte Carlo (MCMC) method was used to conduct phylogenetic analysis and a time scale for evolution. We also calculated pairwise distances (p-distances) and estimated the selective pressure. Phylogenetic analysis showed that the analyzed ON1 and NA1 strains formed 4 lineages. A strain belonging to lineage 4 of ON1 showed wide genetic divergence (p-distance, 0.072), which suggests that it might be a candidate new genotype, namely ON2. The emergence of genotype NA1 was estimated to have occurred in 2000 (95% of highest probability density, HPD; 1997-2002) and that of genotype ON1 in 2005 (95% HPD; 2000-2010) based on the time-scaled phylogenetic tree. The evolutionary rate of genotype ON1 was higher than that of ancestral genotype NA1 (6.03×10(-3) vs. 4.61×10(-3) substitutions/site/year, p<0.05). Some positive and many negative selection sites were found in both ON1 and NA1 strains. The results suggested that the new genotype ON1 is rapidly evolving with antigenic changes, leading to epidemics of HRSV infection in various countries.

Characteristics of human metapneumovirus infection prevailing in hospital wards housing patients with severe disabilities.

Epidemics of infectious diseases often occur at long-term inpatient facilities for patients with severe motor and intellectual disabilities. However, the pathogens causing these infections remain unknown in approximately half of such epidemics. Two epidemics of respiratory tract infection occurred in 2 wards in the National Hospital Organization Ehime Hospital (prevalence 1, 34 infected out of 59 inpatients in the A ward in September 2011; prevalence 2, 8 infected out of 58 inpatients in the B ward in June 2012). Human metapneumovirus (HMPV) was detected from the nasal (and some pharyngeal) swabs from 17 patients. Based on phylogenetic analysis of viral genomes, the virus was grouped in subgroup A2 (prevalence 1) and B2 (prevalence 2). We considered that the viruses had spread through the 2 wards. The average duration of high fever in the 42 patients was 6.8 days, with the majority of fevers exceeding 38℃ (79%) and being accompanied by a productive cough. Ten out of 17 patients (59%) in whom HMPV was detected had decreased lymphocyte and increased monocyte counts in the blood. Eleven cases (65%) had elevated-C reactive protein levels and fever protraction as well as images of bronchitis or pneumonia on chest radiographs approximately 1 week after onset. Anti-HMPV antibody in the blood was positive in 95% of patients (151 of 159 inpatients), indicating no relation between HMPV infection and antibody titer but revealing recurrent infections. In view of the fever protraction and frequent co-occurrence of bronchitis and pneumonia at long-term inpatient facilities for immunocompromised patients such as the ones in this study, the prevalence of HMPV must be carefully monitored, and preventive measures and early-stage treatments are required.

Molecular epidemiology of human metapneumovirus from 2005 to 2011 in Fukui, Japan.

To investigate the molecular epidemiology of human metapneumovirus (HMPV) infections in acute respiratory infections (ARI), we performed genetic analysis of the F gene in HMPV from patients with ARI in Fukui Prefecture from August 2005 to July 2011. HMPV was detected in 53 of 741 nasopharyngeal swabs (7.2%). Phylogenetic analysis helped us assign 31 strains to subgroup A2, 1 strain to subgroup B1, and 21 strains to subgroup B2. The prevalence of HMPV was peaked between January and June. A high degree of nucleotide identity was seen among subgroup A2 strains (95.6-100%) and subgroup B2 strains (97.5-100%). In addition, no positively selected sites (substitutions) were found in the F gene in these HMPV strains. The results suggest that the prevalent HMPV strains in Fukui were associated with various ARI in Japan during the investigation period.

Molecular epidemiology of the attachment glycoprotein (G) gene in respiratory syncytial virus in children with acute respiratory infection in Japan in 2009/2010.

This study performed a detailed genetic analysis of the glycoprotein (G) gene of respiratory syncytial virus (RSV) detected in 50 Japanese children with acute respiratory infection (ARI) in the 2009/2010 season. A phylogenetic tree constructed by the neighbour-joining method showed that 34 and 16 of the RSV strains could be classified into subgroups A and B, respectively. Strains belonging to subgroups A and B were further subdivided into GA2 and BA, respectively. The nucleotide and deduced amino acid sequence identities were relatively high among these strains (>90%). The deduced amino acid sequences implied that a relatively high frequency of amino acid substitutions occurred in the C-terminal 3rd hypervariable region of the G protein in these strains. In addition, some positively selected sites were estimated. The results suggest that RSV with genotypes GA2 and BA was associated with ARI in Japanese children in 2009/2010.

Surveillance of adenovirus D in patients with epidemic keratoconjunctivitis from Fukui Prefecture, Japan, 1995-2010.

Human adenoviruses species D (HAdV-D) are known to cause severe epidemic keratoconjunctivitis. However, the isolation rate of HAdV-D is not high, because HAdV-D is usually slow to propagate. Although new types of HAdV-D have been reported, accurate surveillance has not been performed because of difficulties in culturing the viruses and lack of a practical identification method. In this study, HAdV-Ds were detected and identified from patients with epidemic keratoconjunctivitis in the Fukui Prefecture during 1995-2010 by PCR, loop-mediated isothermal amplification (LAMP) of DNA, and conventional virus isolation and neutralization tests. All samples were subjected to culture and PCR and LAMP. A total of 124 strains of HAdV-D were detected from 157 patients with epidemic keratoconjunctivitis. The strains consisted of the following types: D8 (n = 8), D19 (n = 4), D37 (n = 40), D53 (n = 5), D54 (n = 66), and D56 (n = 1). Among these, D53, D54, and D56 are new types that have been reported recently. The results of this study demonstrated that new types of HAdV-D caused epidemic keratoconjunctivitis during 1995-2010, and included an outbreak of keratoconjunctivitis caused by HAdV-D54. The LAMP method was able to detect and identify HAdV-D53 and HAdV-D54 in 1 hr, and may therefore be applicable for use at the bedside.

PB-2, a polysaccharide fraction from lichen Flavoparmelia baltimorensis, peripherally promotes the induction of long-term potentiation in the rat dentate gyrus in vivo.

We have previously found that oral or intravenous administration of PC-2, a polysaccharide fraction purified from extracts of lichen Flavoparmelia caperata, facilitates the induction of long-term potentiation (LTP) in the dentate gyrus of anesthetized rats. PC-2 could be useful in the development of therapeutic drugs for senile dementia. However, it has been very difficult to obtain the material Flavoparmelia caperata because of its scarcity. In the present study, we therefore investigated whether PB-2, a polysaccharide fraction from another lichen Flavoparmelia baltimorensis, has similar biological effects. Oral administration of PB-2 (100-200 mg/kg) did not affect basal evoked potentials, but significantly promoted the induction of LTP following tetanic stimulation (30 pulses at 60 Hz) in the dentate gyrus of anesthetized rats. Intravenous injection of PB-2 (1-5 mg/kg) was also effective in promoting the induction of LTP, but intracerebroventricular injection of PB-2 (1-2 mg/brain) was ineffective. PB-2, as well as PC-2, should be valuable in identifying factors that promote synaptic plasticity in the hippocampus.