Nationwide epidemiologic and genetic surveillance of hepatitis E in Japan, 2014-2021.

Hepatitis E virus (HEV) is an emerging causative agent of acute hepatitis. To clarify the epidemiology of HEV and characterize the genetic diversity of the virus in Japan, nationwide enhanced surveillance and molecular characterization studies of HEV in Japan were undertaken from 2014 to 2021. In total, 2770 hepatitis E cases were reported, of which 88% were domestic cases, while only 4.1% represented cases following infection abroad. In addition, 57% of domestic infections occurred in males aged in their 40s-70s. For domestic cases, infection via pork meat consumption continued to be the most reported route. Analysis of the 324 sequences detected between 2016 and 2021 showed that the majority of domestic HEV strains belong to Genotype 3a (G3a) and G3b. In contrast, six of eight cases of G1 HEV reflected infection abroad. Our results suggest that HEV is circulating widely in Japan, with genotypes G3a and G3b being most prevalent. Continued surveillance is necessary to monitor future trends and changes in the epidemiology of HEV in Japan.

Distribution of Human Sapovirus Strain Genotypes over the Last Four Decades in Japan: a Global Perspective.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages, both sporadically and as outbreaks. However, only a limited amount of SaV sequence information, especially whole-genome sequences for all the SaV genotypes, is publicly available. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001 to 2015 seasons in 13 prefectures across Japan. The genogroup GI was predominant (67%, n = 92), followed by genogroups GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified: GI.1 (n = 44), GI.2 (n = 40), GI.3 (n = 7), and GI.5 (n = 1). We then compared these Japanese SaV sequences with 3,119 publicly available human SaV sequences collected from 49 countries over the last 46 years. The results indicated that GI.1, and GI.2 have been the predominant genotypes in Japan, as well as in other countries, over at least four decades. The 138 newly determined Japanese SaV sequences together with the currently available SaV sequences, could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

Isolation of OXA-48-like carbapenemase-producing Escherichia coli susceptible to piperacillin/tazobactam in a Japanese patient without a history of travel abroad.

Oxacillinase (OXA)-48-like ß-lactamases are the most common carbapenemases in Enterobacterales in certain regions of the world and are being introduced on a regular basis into regions of non-endemicity. Japan has been characterized by low rates of carbapenemase-producing Enterobacterales, and among them, OXA-48-like carbapenemase-producing isolates are extremely rare. Here we describe a Japanese medical worker, without a history of travel abroad, who was diagnosed as having a community-acquired urinary tract infection, and whose urine sample was found to be positive for OXA-48-like carbapenemase-producing Escherichia coli. None of her close contacts had a history of foreign travel, and the same drug-resistant organism was not observed in other patients who had been hospitalized and undergone environmental culture tests in the same medical institution. This isolate was resistant to penicillins, narrow-spectrum cephalosporins, fluoroquinolones, and cefmetazole, but was susceptible to broad-spectrum cephalosporins, piperacillin/tazobactam, and meropenem and displayed reduced susceptibility to imipenem. The modified carbapenem inactivation test supported carbapenemase production, but inhibitor-based synergistic tests yielded negative results of carbapenemase production. Multiplex polymerase chain reaction revealed the presence of the carbapenemase gene (blaOXA-48) blaTEM and AmpC ß-lactamase gene (blaDHA). Singleplex polymerase chain reaction targeting the blaOXA-48 region amplified a product sequencing to nearly the full length (722 bp) and matching 100% with OXA-48. The present case highlights a new concern regarding OXA-48-like carbapenemase-producing Enterobacterales, which remain challenging to detect for clinical laboratories in regions of non-endemicity, and may already be latent in Japan.

Detection of Jingmenviruses in Japan with Evidence of Vertical Transmission in Ticks.

Jingmen tick virus (JMTV) and the related jingmenvirus-termed Alongshan virus are recognized as globally emerging human pathogenic tick-borne viruses. These viruses have been detected in various mammals and invertebrates, although their natural transmission cycles remain unknown. JMTV and a novel jingmenvirus, tentatively named Takachi virus (TAKV), have now been identified during a surveillance of tick-borne viruses in Japan. JMTV was shown to be distributed across extensive areas of Japan and has been detected repeatedly at the same collection sites over several years, suggesting viral circulation in natural transmission cycles in these areas. Interestingly, these jingmenviruses may exist in a host tick species-specific manner. Vertical transmission of the virus in host ticks in nature was also indicated by the presence of JMTV in unfed host-questing Amblyomma testudinarium larvae. Further epidemiological surveillance and etiological studies are necessary to assess the status and risk of jingmenvirus infection in Japan.

Increased Transmissibility of the SARS-CoV-2 Alpha Variant in a Japanese Population.

To assess the relative transmissibility of the SARS-CoV-2 Alpha variant compared to the pre-existing SARS-CoV-2 in Japan, we performed a cross-sectional study to determine the secondary attack rate of COVID-19 in household contacts before and after the Alpha variant became dominant in Osaka. We accessed 290 household contacts whose index cases were diagnosed between 1 and 20 December 2020 (the third epidemic group), at a time when Osaka was free of the Alpha variant. We also accessed 398 household contacts whose index cases were diagnosed between 20 April and 3 May 2021 (the fourth epidemic group), by which time the Alpha variant had become dominant. We identified 124 household contacts whose index case was determined positive for the Alpha variant (Alpha group) in this fourth group. The secondary attack rates in the fourth group (34.7%) and the Alpha group (38.7%) were significantly higher than that in the third group (19.3%, p < 0.001). Multivariable Poisson regression analysis with a robust error variance showed a significant excess risk in the fourth group (1.90, 95% CI = 1.47-2.48) and the Alpha group (2.34, 95% CI = 1.71-3.21). This finding indicates that the SARS-CoV-2 Alpha variant has an approximately 1.9-2.3-fold higher transmissibility than the pre-existing virus in the Japanese population.

Subtype Screening of blaIMP Genes Using Bipartite Primers for DNA Sequencing.

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.

Isolation and plasmid characterisation of Salmonella enterica serovar Albany harbouring mcr-5 from retail chicken meat in Japan.

The emergence of plasmid-mediated colistin resistance genes (mcr), which is occurring in numerous countries, is a worldwide concern, primarily because colistin is a last-resort antibiotic. Compared to E. coli, prevalence of mcr genes in Salmonella is unclear in Japan. Here we screened for mcr-1-5 genes in our collection of Salmonella strains isolated from retail meat products collected in Japan from 2012 through 2016. We found that Salmonella Albany strain 27A-368 encodes mcr-5 and that mcr genes were undetectable among the remaining 202 isolates. The resistance plasmid p27A-368 was transferred by conjugation to S. Infantis and was stably retained as a transconjugant. Whole-genome sequencing revealed that mcr-5 resided on a 115 kb plasmid (p27A-368). The plasmid backbone of p27A-368 is more similar to that of pCOV27, an ESBL-encoding plasmid recovered from avian pathogenic E. coli, rather than pSE13-SA01718 of S. Paratyphi B that encodes mcr-5. Further, mcr-5 is located on a transposon, and its sequence is similar to that of pSE13-SA01718. A phylogenetic tree based on single nucleotide variants implies a relationship between 27A-368 and S. Albany isolated in Southeast Asian countries.

Single-Tube Multiplex Polymerase Chain Reaction for the Detection of Genes Encoding Enterobacteriaceae Carbapenemase.

A multiplex PCR assay in a single tube was developed for the detection of the carbapenemase genes of Enterobacteriaceae. Primers were designed to amplify the following six carbapenemase genes: blaKPC, blaIMP, blaNDM, blaVIM, blaOXA-48-like, and blaGES. Of 70 blaIMP variants, 67 subtypes were simulated to be PCR-positive based on in silico simulation and the primer-design strategy. After determining the optimal PCR conditions and performing in vitro assays, the performance of the PCR assay was evaluated using 51 and 91 clinical isolates with and without carbapenemase genes, respectively. In conclusion, the combination of multiplex PCR primers and QIAGEN Multiplex PCR Plus Kit was used to determine the best performance for the rapid and efficient screening of carbapenemase genes in Enterobacteriaceae. The assay had an overall sensitivity and specificity of 100%. This PCR assay compensates for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, in clinical and public health settings.

Characterization of the Ca2+-coordination structures of L- and T-plastins in combination with their synthetic peptide analogs by FTIR spectroscopy.

FTIR spectroscopy was employed to characterize the coordination structures of divalent cations (M2+ = Ca2+ or Mg2+) bound by L- and T-plastins, which contain two EF-hand motifs. We focused on the N-terminal headpieces in the L- and T-plastins to analyze the regions of COO- stretching and amide-I in solution. The spectral profiles indicated that these headpieces have EF-hand calcium-binding sites because bands at 1551 cm-1 and 1555 cm-1 were observed for the bidentate coordination mode of Glu at the 12th position of the Ca2+-binding site of Ca2+-loaded L-plastin and T-plastin, respectively. The amide-I profile of the Mg2+-loaded L-plastin headpiece was identical with that of the apo L-plastin headpiece, meaning that L-plastin has a lower affinity for Mg2+. The amide-I profiles for apo, Mg2+-loaded and Ca2+-loaded T-plastin suggested that aggregation was generated in protein solution at a concentration of 1 mM. The implications of the FTIR spectral data for these plastin headpieces are discussed on the basis of data obtained for synthetic peptide analogs corresponding to the Ca2+-binding site.

Predicting Directions of Changes in Genotype Proportions Between Norovirus Seasons in Japan.

The norovirus forecasting system (NOROCAST) has been developed for predicting directions of changes in genotype proportions between human norovirus (HuNoV) seasons in Japan through modeling herd immunity to structural protein 1 (VP1). Here 404 nearly complete genomic sequences of HuNoV were analyzed to examine whether the performance of NOROCAST could be improved by modeling herd immunity to VP2 and non-structural proteins (NS) in addition to VP1. It was found that the applicability of NOROCAST may be extended by compensating for unavailable sequence data and observed genotype proportions of 0 in each season. Incorporation of herd immunity to VP2 and NS did not appear to improve the performance of NOROCAST, suggesting that VP1 may be a suitable target of vaccines.

Seroprevalence of severe fever with thrombocytopenia syndrome (SFTS) virus antibodies in humans and animals in Ehime prefecture, Japan, an endemic region of SFTS.

Severe fever with thrombocytopenia syndrome (SFTS) was first identified as an emerging tick-borne infectious disease caused by the SFTS virus (SFTSV) in China and has also been found to be endemic to Japan and South Korea, indicating that SFTS is of great concern in East Asia. The aim of the present study was to determine the seroprevalence of SFTSV antibodies in humans and animals in SFTS-endemic regions of Japan. One of 694 (0.14%) healthy persons over 50 years of age and 20 of 107 (18.7%) wild and domestic animals in Ehime prefecture of western Japan were determined to be seropositive for SFTSV antibodies by virus neutralization test and ELISA, respectively. The seropositive person, a healthy 74-year-old woman, was a resident of the southwest part of Ehime prefecture engaged in citriculture and field work. This woman's sample exhibited neutralizing activity against SFTSV although she had neither a clear experience with tick bites nor SFTS-like clinical illness. These findings indicate that most people living in the endemic regions are not infected with SFTSV and suggest that most of the SFTS patients reported so far do not reflect the tip of an iceberg of people infected with SFTSV, but at the same time, that SFTSV infection does not always induce severe SFTS-associated symptoms. These findings also suggested that SFTSV has been maintained in nature within animal species and ticks.

Evaluation of a surface plasmon resonance imaging-based multiplex O-antigen serogrouping for Escherichia coli using eleven major serotypes of Shiga -toxin-producing E. coli.

The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 106 and 17.6 × 106 CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed.

Characterization of a novel thogotovirus isolated from Amblyomma testudinarium ticks in Ehime, Japan: A significant phylogenetic relationship to Bourbon virus.

The genus Thogotovirus, as represented by Thogoto virus and Dhori virus, comprises a group of arthropod-borne viruses, most members of which are transmitted by ticks. Here we report the genetic and biological characterization of a new thogotovirus, designated Oz virus (OZV), isolated from the hard tick Amblyomma testudinarium in Ehime, Japan. OZV efficiently replicated and induced a cytopathic effect in Vero cells, from which enveloped pleomorphic virus particles were formed by budding. OZV could also replicate in BHK-21 and DH82 cells and caused high mortality in suckling mice after intracerebral inoculation. Phylogenetic analyses of six viral proteins indicated that OZV is clustered with Dhori and related viruses, and is most closely related in glycoprotein (GP) and matrix protein (M) sequences to Bourbon virus, a human-pathogenic thogotovirus discovered recently in the United States. Our findings emphasize the need for understanding the geographic distribution and ecology of OZV and related viruses and for reevaluation of the medical and public health importance of thogotoviruses.

Genetic Analysis of Human Norovirus Strains in Japan in 2016-2017.

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.

Phylogeny and Immunoreactivity of Norovirus GII.P16-GII.2, Japan, 2016-17.

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.

A patient with severe fever with thrombocytopenia syndrome and hemophagocytic lymphohistiocytosis-associated involvement of the central nervous system.

Severe fever with thrombocytopenia syndrome (SFTS), a severe infectious disease caused by novel bunyavirus, SFTS virus (SFTSV), is endemic to China, Korea, and Japan. Most SFTS patients show abnormalities in consciousness. Pathological findings in the central nervous system (CNS) of SFTS patients are not reported. A 53-year-old Japanese man was admitted to Uwajima City Hospital with an 8-day history of fever and diarrhea. Laboratory tests revealed leukopenia, thrombocytopenia, and liver enzyme elevation. He was diagnosed as having severe fever with thrombocytopenia syndrome (SFTS) following detection of the SFTSV genome in his blood. Bone marrow aspiration revealed hemophagocytic lymphohistiocytosis. He suffered progressive CNS disturbance and died on day 13 from onset of first symptoms. The SFTSV genome load in blood and levels of certain cytokines increased over the disease course. Necrotizing lymphadenitis with systemic lymphoid tissues positive for nucleocapsid protein (NP) of SFTSV was revealed by immunohistochemical (IHC) analysis. SFTSV-NP-positive immunoblasts were detected in all organs examined, including the CNS, and in the vascular lumina of each organ. Parenchymal cells of all organs examined were negative for SFTSV-NP on IHC analysis. Microscopic examination of the pons showed focal neuronal cell degeneration with hemosiderin-laden macrophages around extended microvessels with perivascular inflammatory cell infiltration and intravascular fibrin deposition. Autopsy confirmed this patient with SFTS was positive for systemic hemophagocytic lymphohistiocytosis including in the CNS. This patient's neurological abnormalities may have been caused by both functional and organic abnormalities. These novel findings provide important insights into the pathophysiology of SFTS.

Unusual presentation of a severely ill patient having severe fever with thrombocytopenia syndrome: a case report.

BACKGROUND: Severe fever with thrombocytopenia syndrome is an emerging infectious disease caused by a novel phlebovirus belonging to the family Bunyaviridate. Emergence of encephalitis/encephalopathy during severe fever with thrombocytopenia syndrome progression has been identified as a major risk factor associated with a poor prognosis. Here we report the case of a severely ill patient with severe fever with thrombocytopenia syndrome virus-associated encephalitis/encephalopathy characterized by a lesion of the splenium, which resolved later. CASE PRESENTATION: A 56-year-old Japanese man presented with fever and diarrhea, followed by dysarthria. Diffusion-weighted magnetic resonance imaging demonstrated high signal intensity in the splenium of the corpus callosum. The severe fever with thrombocytopenia syndrome virus genome was detected in our patient's serum, and the clinical course was characterized by convulsion, stupor, and hemorrhagic manifestations, with disseminated intravascular coagulation and hemophagocytic lymphohistiocytosis. Supportive therapy not including administration of corticosteroids led to gradual improvement of the clinical and laboratory findings, and magnetic resonance imaging demonstrated resolution of the splenial lesion. The serum severe fever with thrombocytopenia syndrome viral copy number, which was determined with the quantitative reverse-transcription polymerase chain reaction, rapidly decreased despite the severe clinical course. Our patient's overall condition improved, allowing him to be eventually discharged. CONCLUSIONS: Patients with encephalitis/encephalopathy due to severe fever with thrombocytopenia syndrome virus infection may have a favorable outcome, even if they exhibit splenial lesions and a severe clinical course; monitoring the serum viral load may be of value for prediction of outcome and potentially enables the avoidance of corticosteroids to intentionally cause opportunistic infection.

[Genetic Investigation of bla(CTX-M)-typing in Extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae from Clinical Specimens and Commercially Available Chicken Liver].

A genetic investigation consisting of the bla(CTX-M) typing was performed using 40 extended spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates from chicken liver and 43 ESBL-producing E. coli and 42 ESBL-producing Klebsiella pneumoniae isolates from patients. The types were determined using a sequence analysis. In 31 isolates in the bla(CTX-M-1) group, there were 13 with the bla(CTX-M-1) and all were from chicken liver. Nine E. coli isolates from chicken liver and one E. coli isolate from patients were found to be bla(CTX-M-55). In the bla(CTX-M-15), there were 6 E. coli isolates and one K. pneumoniae isolate from patients. All 39 isolates in the bla(CTX-M-2) group had the blac(CTX-M-2). Fifty-five isolates were found in the bla(CTX-M-9) group, the highest detection frequency, with 36 possessing bla(CTX-M-14) : 20 E. coli and 13 K. pneumoniae from patients and 3 E. coli from chicken liver. There were 17 bla(CTX-M-27) isolates, including 10 E. coli and 7 K. pneumoniae from patients. One bla(CTX-M-90) K. pneumoniae isolate and one bla(CTX-M-9) E. coli isolate were also obtained from patients. These results indicate that the E. coli isolates from chicken liver consist of bla(CTX-M-1), bla(CTX-M-2) and bla(CTX-M-55) ; the E. coli isolates from patients consist of bla(CTX-M-14) and bla(CTX-M-27) ; and the K. pneumoniae isolates from patients consist of bla(CTX-M-2), bla(CTX-M-14) and bla(CTX-M-27). Therefore, the bla(CTX-M) type differs in isolates from chicken liver and those from humans. These results suggest that it is unlikely that ESBL-producing E. coli from chicken liver are firmly established in the human intestinal tract.

Molecular evolution of the capsid gene in human norovirus genogroup II.

Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10(-3) substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>10(2)) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans.