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Objectives: In Japan, population-based epidemiological data on respiratory syncytial virus (RSV) infections are limited. To elucidate the epidemiology of RSV before the introduction of new prophylactic drugs, we conducted a population-based study during and after the SARS-CoV-2 pandemic. Methods: This study was performed in four hospitals in Chiba City and three hospitals in Ichihara City. Clinical information and residual samples from RSV rapid antigen tests of infants under one year old were collected. Samples from patients with lower respiratory tract infections (LRTI) were analyzed using the FilmArray Respiratory 2.1 panels. Results: A total of 1200 infants underwent the RSV rapid antigen test, with 497 diagnosed with LRTI. Although five samples could not be stored, 252 out of 492 (51.2%) were positive for RSV. Among the RSV PCR-positive infants, 63 (25.0%) had underlying diseases, compared to 100 out of 240 (41.7%) RSV PCR-negative infants (P < 0.05). In Chiba City, the annual incidence of hospitalization per 1000 children was 12.7 in 2021, 4.4 in 2022, and 9.2 in 2023. Conclusions: During and after the SARS-CoV-2 pandemic, most hospitalized infants with RSV-LRTI did not have underlying diseases. Widespread use of prophylaxis in infants without underlying disease is desirable.

Molecular interactions between respiratory syncytial virus (RSV) fusion protein (F protein) and the cellular receptor Toll-like receptor 4 (TLR4) and myeloid differentiation factor-2 (MD-2) protein complex are unknown. Thus, to reveal the detailed molecular interactions between them, in silico analyses were performed using various bioinformatics techniques. The present simulation data showed that the neutralizing antibody (NT-Ab) binding sites in both prefusion and postfusion proteins at sites II and IV were involved in the interactions between them and the TLR4 molecule. Moreover, the binding affinity between postfusion proteins and the TLR4/MD-2 complex was higher than that between prefusion proteins and the TLR4/MD-2 complex. This increased binding affinity due to conformational changes in the F protein may be able to form syncytium in RSV-infected cells. These results may contribute to better understand the infectivity and pathogenicity (syncytium formation) of RSV.

In Japan’s Kanto region, the number of Salmonella enterica serovar Chester infections increased temporarily between 2014 and 2016. Concurrently with this temporal increase in the Kanto region, S. Chester isolates belonging to one clonal group were causing repetitive outbreaks in Europe. A recent study reported that the European outbreaks were associated with travelers who had been exposed to contaminated food in Morocco, possibly seafood. Because Japan imports a large amount of seafood from Morocco, we aimed to establish whether the temporal increase in S. Chester infections in the Kanto region was associated with imported Moroccan seafood. Short sequence reads from the whole-genome sequencing of 47 S. Chester isolates from people in the Kanto region (2014–2016), and the additional genome sequences from 58 isolates from the European outbreaks, were analyzed. The reads were compared with the complete genome sequence from a S. Chester reference strain, and 347 single nucleotide polymorphisms (SNPs) were identified. These SNPs were used in this study. Cluster and Bayesian cluster analyses showed that the Japanese and European isolates fell into two different clusters. Therefore, ΦPT and IAS values were calculated to evaluate genetic differences between these clusters. The results revealed that the Japanese and European isolates were genetically distinct populations. Our root-to-tip analysis showed that the Japanese isolates originating from one clone had accumulated mutations, suggesting that an emergence of this organism occurred. A minimum spanning tree analysis demonstrated no correlation between genetic and geographical distances in the Japanese isolates, suggesting that the emergence of the serovar in the Kanto region did not involve person-to-person contact; rather, it occurred through food consumption. The dN/dS ratio indicated that the Japanese strain has evolved under positive selection pressure. Generally, a population of bacterial clones in a reservoir faces negative selection pressure. Therefore, the Japanese strain must have existed outside of any reservoir during its emergence. In conclusion, S. Chester isolates originating from one clone probably emerged in the Kanto region via the consumption of contaminated foods other than imported Moroccan seafood. The emerging strain may have not established a reservoir for survival in the food supply chain resulting in its disappearance after 2017.

Seven foodborne norovirus outbreaks attributable to the GII.P17-GII.17 strain were reported across Japan in 2017, causing illness in a total of 2,094 persons. Nori (dried shredded seaweed) was implicated in all outbreaks and tested positive for norovirus. Our data highlight the stability of norovirus in dehydrated food products.

During the 2016–17 winter season in Japan, human norovirus GII.P16-GII.2 strains (2016 strains) caused large outbreaks of acute gastroenteritis. Phylogenetic analyses suggested that the 2016 strains derived from the GII.2 strains detected during 2010–12. Immunochromatography between 2016 strains and the pre-2016 GII.2 strains showed similar reactivity.

To elucidate the evolution of human respirovirus 3 (HRV3), we performed detailed genetic analyses of the F gene (full-length) detected from hundreds of HRV3 strains obtained from various geographic regions. First, we performed time-scaled evolutionary analyses using the Bayesian Markov chain Monte Carlo method. Then, we performed analyses of phylodynamics, similarity, phylogenetic distance, selective pressure, and conformational B-cell epitope with the F-protein structural analyses. Time-scaled phylogenetic tree showed that the common ancestor of HRV3 and bovine respirovirus 3 diverged over 300 years ago and subdivided it into three major clusters and four subclusters during the most recent 100 years. The overall evolutionary rate was approximately 10–3 substitutions/site/year. Indigenous similarity was seen in the present strains, and the mean phylogenetic distance were 0.033. Many negative selection sites were seen in the ectodomain. The conformational epitopes did not correspond to the neutralizing antibody binding sites. These results suggest that the HRV3 F gene is relatively conserved and restricted in this diversity to preserve the protein function, although these strains form many branches on the phylogenetic tree. Furthermore, HRV3 reinfection may be responsible for discordances between the conformational epitopes and the neutralizing antibody binding sites of the F protein. These findings contribute to a better understanding of HRV3 virology.

Noroviruses are a major cause of viral epidemic gastroenteritis in humans worldwide. The protease (Pro) encoded in open reading frame 1 (ORF1) is an essential enzyme for proteolysis of the viral polyprotein. Although there are some reports regarding the evolutionary analysis of norovirus GII-encoding genes, there are few reports focused on the Pro region. We analyzed the molecular evolution of the Pro region of norovirus GII using bioinformatics approaches. A time-scaled phylogenetic tree of the Pro region constructed using a Bayesian Markov chain Monte Carlo method indicated that the common ancestor of GII diverged from GIV around 1680 CE [95% highest posterior density (HPD), 1607–1749]. The GII Pro region emerged around 1752 CE (95%HPD, 1707–1794), forming three further lineages. The evolutionary rate of GII Pro region was estimated at more than 10–3 substitutions/site/year. The distribution of the phylogenetic distances of each genotype differed, and showed genetic diversity. Mapping of the negative selection and substitution sites of the Pro structure showed that the substitution sites in the Pro protein were mostly produced under neutral selection in positions structurally adjacent to the active sites for proteolysis, whereas negative selection was observed in residues distant from the active sites. The phylodynamics of GII.P4, GII.P7, GII.P16, GII.P21, and GII.P31 indicated that their effective population sizes increased during the period from 2005 to 2016 and the increase in population size was almost consistent with the collection year of these genotypes. These results suggest that the Pro region of the norovirus GII evolved rapidly, but under no positive selection, with a high genetic divergence, similar to that of the RNA-dependent RNA polymerase (RdRp) region and the VP1 region of noroviruses.

Human norovirus (HuNoV) GII.P17-GII.17 (Kawasaki2014 variant) reportedly emerged in 2014 and caused gastroenteritis outbreaks worldwide. To clarify the evolution of both VP1 and RNA-dependent RNA polymerase (RdRp) regions of GII.P17-GII.17, we analyzed both global and novel Japanese strains detected during 2013–2017. Time-scaled phylogenetic trees revealed that the ancestral GII.17 VP1 region diverged around 1949, while the ancestral GII.P17 RdRp region diverged around 2010. The evolutionary rates of the VP1 and RdRp regions were estimated at ~2.7 × 10−3 and ~2.3 × 10−3 substitutions/site/year, respectively. The phylogenetic distances of the VP1 region exhibited no overlaps between intra-cluster and inter-cluster peaks in the GII.17 strains, whereas those of the RdRp region exhibited a unimodal distribution in the GII.P17 strains. Conformational epitope positions in the VP1 protein of the GII.P17-GII.17 strains were similar, although some substitutions, insertions and deletions had occurred. Strains belonging to the same cluster also harbored substitutions around the binding sites for the histo-blood group antigens of the VP1 protein. Moreover, some amino acid substitutions were estimated to be near the interface between monomers and the active site of the RdRp protein. These results suggest that the GII.P17-GII.17 virus has produced variants with the potential to alter viral antigenicity, host-binding capability, and replication property over the past 10 years.

Koplik spots are considered a disease-specific sign for measles, although comprehensive virological studies have not been conducted to date. In Japan, a national survey of 3023 measles and measles-suspected cases was conducted between 2009 and 2014 using polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) to detect various rash/fever-associated viruses. Koplik spots were observed in 717 of 3023 cases (23.7%). Among these, the measles virus was detected in 202 cases (28.2%), while the rubella virus was detected in 125 cases (17.4%). Other viruses were detected in 51 cases having the spots (7.1%). In some of the cases with spots, two or three viruses, such as the rubella virus, parvovirus, and human herpesvirus type 6 were also detected. The sensitivity and specificity of Koplik spots as a diagnostic marker for measles were 48 and 80%, respectively. The results suggested that Koplik spots might appear not only in measles but also in other viral infections, such as rubella, as a clinical sign.

Noroviruses are the leading cause of viral gastroenteritis in humans across the world. RNA-dependent RNA polymerase (RdRp) plays a critical role in the replication of the viral genome. Although there have been some reports on a limited number of genotypes with respect to the norovirus evolution of the RdRp region, no comprehensive molecular evolution examination of the norovirus GII genotype has yet been undertaken. Therefore, we conducted an evolutionary analysis of the 25 genotypes of the norovirus GII RdRp region (full-length), collected globally using different bioinformatics technologies. The time-scaled phylogenetic tree, generated using the Bayesian Markov Chain Monte Carlo (MCMC) method, indicated that the common ancestor of GII diverged from GIV around 1443 CE [95% highest posterior density (HPD), 1336–1542]. The GII RdRp region emerged around 1731 CE (95% HPD, 1703–1757), forming three lineages. The evolutionary rate of the RdRp region of the norovirus GII strains was estimated at over 10−3 substitutions/site/year. The evolutionary rates were significantly distinct in each genotype. The composition of the phylogenetic distances differed among the strains for each genotype. Furthermore, we mapped the negative selection sites on the RdRp protein and many of these were predicted in the GII.P4 RdRp proteins. The phylodynamics of GII.P4, GII.P12, GII.P16, and GII.Pe showed that their effective population sizes increased during the period from 2003 to 2014. Our results cumulatively suggest that the RdRp region of the norovirus GII rapidly and uniquely evolved with a high divergence similar to that of the norovirus VP1 gene.

In the 2016/2017 winter season in Japan, HuNoV GII.P16-GII.2 strains (2016 strains) emerged and caused large outbreaks of acute gastroenteritis. To better understand the outbreaks, we examined the molecular evolution of the VP1 gene and RdRp region in 2016 strains from patients by studying their time-scale evolutionary phylogeny, positive/negative selection, conformational epitopes, and phylodynamics. The time-scale phylogeny suggested that the common ancestors of the 2016 strains VP1 gene and RdRp region diverged in 2006 and 1999, respectively, and that the 2016 strain was the progeny of a pre-2016 GII.2. The evolutionary rates of the VP1 gene and RdRp region were around 10-3 substitutions/site/year. Amino acid substitutions (position 341) in an epitope in the P2 domain of 2016 strains were not found in pre-2016 GII.2 strains. Bayesian skyline plot analyses showed that the effective population size of the VP1 gene in GII.2 strains was almost constant for those 50 years, although the number of patients with NoV GII.2 increased in 2016. The 2016 strain may be involved in future outbreaks in Japan and elsewhere.

Human norovirus (HuNoV) is a leading cause of viral gastroenteritis worldwide, of which GII.4 is the most predominant genotype. Unlike other genotypes, GII.4 has created various variants that escaped from previously acquired immunity of the host and caused repeated epidemics. However, the molecular evolutionary differences among all GII.4 variants, including recently discovered strains, have not been elucidated. Thus, we conducted a series of bioinformatic analyses using numerous, globally collected, full-length GII.4 major capsid (VP1) gene sequences (466 strains) to compare the evolutionary patterns among GII.4 variants. The time-scaled phylogenetic tree constructed using the Bayesian Markov chain Monte Carlo (MCMC) method showed that the common ancestor of the GII.4 VP1 gene diverged from GII.20 in 1840. The GII.4 genotype emerged in 1932, and then formed seven clusters including 14 known variants after 1980. The evolutionary rate of GII.4 strains was estimated to be 7.68 × 10−3 substitutions/site/year. The evolutionary rates probably differed among variants as well as domains [protruding 1 (P1), shell, and P2 domains]. The Osaka 2007 variant strains probably contained more nucleotide substitutions than any other variant. Few conformational epitopes were located in the shell and P1 domains, although most were contained in the P2 domain, which, as previously established, is associated with attachment to host factors and antigenicity. We found that positive selection sites for the whole GII.4 genotype existed in the shell and P1 domains, while Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants were under positive selection in the P2 domain. Amino acid substitutions overlapped with putative epitopes or were located around the epitopes in the P2 domain. The effective population sizes of the present strains increased stepwise for Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants. These results suggest that HuNoV GII.4 rapidly evolved in a few decades, created various variants, and altered its evolutionary rate and antigenicity.

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.

Two live attenuated oral rotavirus vaccines, Rotarix and RotaTeq, have been introduced as voluntary vaccination in Japan since 2011 and 2012, respectively. Effectiveness of the vaccines has been confirmed, whereas concerns such as shedding of the vaccine strains and gastroenteritis cases caused by vaccine strains are not well assessed. We aimed to identify the vaccine strains in children with acute gastroenteritis (AGE) to investigate the prevalence of AGE caused by vaccination or horizontal transmission of vaccine strains. A total of 1,824 stool samples were collected from children with AGE at six outpatient clinics in 2012-2015. Among all, 372 group A rotavirus (RVA) positive samples were screened for vaccine components by real-time RT-PCR which were designed to differentiate vaccine strains from rotavirus wild-type strains with high specificity. For samples possessing both vaccine and wild-type strains, analyses by next-generation sequencing (NGS) were conducted to characterize viruses existed in the intestine. As a result, Rotarix-derived strains were identified in 6 of 372 (1.6%) RVA positive samples whereas no RotaTeq strain was detected. Among six samples, four possessed Rotarix-derived strains while two possessed both Rotarix-derived strains and wild-type strains. In addition, other pathogens such as norovirus, enterovirus and E.coli were detected in four samples. The contribution of these vaccine strains to each patient's symptoms was unclear as all of the cases were vaccinated 2-14 days before sample collection. Proportion of average coverage for each segmented gene by NGS strongly suggested the concurrent infection of the vaccine-derived strain and the wild-type strain rather than reassortment of these two strains in one sample. This is the first study to report the prevalence of vaccine-derived strains in patients with RVA AGE in Japan as 1.6% without evidence of horizontal transmission. The results emphasized the importance of continuous monitoring on vaccine strains and their clinical impacts on children.

We obtain the discrete versions of integral inequalities of Bernoulli type obtained in Choi (2007) and give an application to study the boundedness of solutions of nonlinear Volterra difference equations.

We examine the various types of stability for the solutions of linear dynamic systems on time scales and give two examples.

We study the existence of almost periodic solutions for nonlinear discrete Volterra equations with unbounded delay, as a discrete analogue of the results for integro-differential equations by Y. Hamaya (1993).

We characterize the h-stability in variation and asymptotic equilibrium in variation for nonlinear difference systems via n∞-summable similarity and comparison principle. Furthermore we study the asymptotic equivalence between nonlinear difference systems and their variational difference systems by means of asymptotic equilibria of two systems.