Analysis of Suspected Measles Cases with Discrepant Measles-Specific IgM and rRT-PCR Test Results, Japan.

We investigated clinically suspected measles cases that had discrepant real-time reverse transcription PCR (rRT-PCR) and measles-specific IgM test results to determine diagnoses. We performed rRT-PCR and measles-specific IgM testing on samples from 541 suspected measles cases. Of the 24 IgM-positive and rRT-PCR--negative cases, 20 were among children who received a measles-containing vaccine within the previous 6 months; most had low IgG relative avidity indexes (RAIs). The other 4 cases were among adults who had an unknown previous measles history, unknown vaccination status, and high RAIs. We detected viral nucleic acid for viruses other than measles in 15 (62.5%) of the 24 cases with discrepant rRT-PCR and IgM test results. Measles vaccination, measles history, and contact history should be considered in suspected measles cases with discrepant rRT-PCR and IgM test results. If in doubt, measles IgG avidity and PCR testing for other febrile exanthematous viruses can help confirm or refute the diagnosis.

Nationwide and long-term molecular epidemiologic studies of mumps viruses that circulated in Japan between 1986 and 2017.

In Japan, major mumps outbreaks still occur every 4-5 years because of low mumps vaccine coverage (30-40%) owing to the voluntary immunization program. Herein, to prepare for a regular immunization program, we aimed to reveal the nationwide and long-term molecular epidemiological trends of the mumps virus (MuV) in Japan. Additionally, we performed whole-genome sequencing (WGS) using next-generation sequencing to assess results from conventional genotyping using MuV sequences of the small-hydrophobic (SH) gene. We analyzed 1,064 SH gene sequences from mumps clinical samples and MuV isolates collected from 25 prefectures from 1986 to 2017. The results showed that six genotypes, namely B (110), F (1), G (900), H (3), J (41), and L (9) were identified, and the dominant genotypes changed every decade in Japan since the 1980s. Genotype G has been exclusively circulating since the early 2000s. Seven clades were identified for genotype G using SH sequence-based classification. To verify the results, we performed WGS on 77 representative isolates of genotype G using NGS and phylogenetically analyzed them. Five clades were identified with high bootstrap values and designated as Japanese clade (JPC)-1, -2, -3, -4, -5. JPC-1 and -3 accounted for over 80% of the total genotype G isolates (68.3 and 13.8%, respectively). Of these, JPC-2 and -5, were newly identified clades in Japan through this study. This is the first report describing the nationwide and long-term molecular epidemiology of MuV in Japan. The results provide information about Japanese domestic genotypes, which is essential for evaluating the mumps elimination progress in Japan after the forthcoming introduction of the mumps vaccine into Japan's regular immunization program. Furthermore, the study shows that WGS analysis using NGS is more accurate than results obtained from conventional SH sequence-based classification and is a powerful tool for accurate molecular epidemiology studies.

Severe Fever with Thrombocytopenia Syndrome Virus RNA in Ticks from Wild Mongooses in Okinawa Prefecture, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic tick-borne disease caused by SFTS virus (SFTSV). SFTSV has a wide spectrum of animal hosts and is considered to circulate in an enzootic tick-vertebrate-tick cycle. A previous seroepidemiological study showed the presence of anti-SFTSV antibodies in wild mongooses (Herpestes auropunctatus) and indicated that outdoor activity was associated with an increased risk of tick bites among Okinawa residents. However, the association of SFTSV with wild mongooses and ticks remains unclear. To understand the association between ticks and mongooses with respect to the SFTSV enzootic cycle, we investigated the presence of SFTSV RNA in ticks collected from wild mongooses on the Okinawa Island. A total of 638 ticks belonging to 2 genera and 3 species (Haemaphysalis hystricis, Haemaphysalis formosensis, and Ixodes granulatus) were collected from 22 wild mongooses from 2016 to 2021. SFTSV RNA was detected in two pools of H. hystricis larvae collected from a wild mongoose in the central area of the main island of Okinawa in 2017. Although the prevalence of SFTSV in ticks from wild mongooses is low, endemic circulation of the virus in Okinawa should be carefully monitored to prevent future infections.

Seroepidemiological study of severe fever with thrombocytopenia syndrome in animals and humans in Okinawa, Japan.

In Okinawa prefecture, Japan, the first case of severe fever with thrombocytopenia syndrome (SFTS) was confirmed in August 2016, and this case remains to be the only reported case of SFTS in Okinawa. The epidemiological investigation indicated that the patient had been infected on the main island of Okinawa, but source and route of infection were unknown. Therefore, to understand the possible source and route of SFTS virus (SFTSV) infection in Okinawa, we performed a seroepidemiological study of SFTSV among animals and dwellers in Okinawa and conducted a questionnaire survey to investigate risk factors for tick bites in Okinawa. Among the 1,035 serum samples from four different animal species, anti-SFTSV antibodies were detected in only 4.2% wild mongoose (Herpestes auropunctatus) serum samples. To our knowledge, this is the first study to report the detection of anti-SFTSV antibodies in wild mongooses. Meanwhile, all 1,104 human inhabitants tested negative for anti-SFTSV antibodies, suggesting that the frequency of SFTSV exposure is low in Okinawa. Logistic regression analysis of the questionnaire results showed that outdoor activity was associated with an increased risk of tick bite among Okinawa residents. Despite the current low frequency of SFTSV infection in animals and humans, endemic circulation of the virus in Okinawa should be carefully monitored in the area for preventing future infections.

Characteristics of Household Transmission of COVID-19 during Its Outbreak in Okinawa, Japan from February to May 2020.

The Okinawa prefecture confirmed 142 cases of coronavirus disease from February 14 to May 2020. Among them, 78 were the first cases of a household with 174 household contacts. Of the 174 contacts, 21 contracted the disease, indicating a secondary attack rate of 12.1% (95% confidence interval [CI] 7.6-17.9%). No significant differences were observed in the demographics and quantitative reverse transcription-polymerase chain reaction test results between the first cases that became the sources of infection to the household members. The secondary attack rates with respect to the various characteristics of the household members were significantly different: aged >69 years (40.9% [95% CI 20.7-63.6%]) and those with underlying diseases (36.0% [95% CI 18.0-57.5%]). When the period from the onset to isolation of the first household case was within 3 days, the secondary attack rate was low (4.5% [95% CI 0.1-22.8%]). Among the 21 secondary cases, 11 (52.4%) developed within 5 days of symptom onset in the first case within the same household. This indicates that secondary infection within the household occurred immediately after symptom onset in the first case. Therefore, isolation of a suspected patient can help reduce secondary household infections.

Molecular and serological epidemiology of Leptospira infection in cats in Okinawa Island, Japan.

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Cats have been reported to be infected with Leptospira spp. and shed the bacteria in the urine. However, the importance of cats as an infection source for humans remains unclear. In this study, Leptospira infection in cats in Okinawa Prefecture, Japan, where leptospirosis is endemic, was investigated by leptospiral antibody and DNA detection using microscopic agglutination test and nested PCR, respectively. Moreover, multilocus sequence typing (MLST) and whole genome sequencing (WGS) were conducted on the Leptospira borgpetersenii serogroup Javanica isolated from cats, black rats, a mongoose, and humans. Anti-Leptospira antibodies were detected in 16.6% (40/241) of the cats tested, and the predominant reactive serogroup was Javanica. The leptospiral flaB gene was detected in 7.1% (3/42) of cat urine samples, and their sequences were identical and identified as L. borgpetersenii. MLST and WGS revealed the genetic relatedness of L. borgpetersenii serogroup Javanica isolates. This study indicated that most seropositive cats had antibodies against the serogroup Javanica and that cats excreted L. borgpetersenii in the urine after infection. Further, genetic relatedness between cat and human isolates suggests that cats may be a maintenance host for L. borgpetersenii serogroup Javanica and a source for human infection.

Novel genotypes of Leptospira interrogans serogroup Sejroe isolated from human patients in Okinawa Prefecture, Japan.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of Leptospira species. It is a public health issue in the tropics, including Okinawa, the southernmost prefecture of Japan. This study reports the first isolation of L. interrogans serogroup Sejroe from two human patients in Japan, and describes its molecular characterization using multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). MLST on the two isolates, 168036 and 178129, showed that pfkB in 178129 is a novel allele, and that both isolates constitute novel sequence types (STs); ST286 for 168036 and ST287 for 178129. A minimum spanning tree based on seven alleles of L. interrogans indicates that both isolates are genetically close, but are distinct from known L. interrogans serogroup Sejroe strains. MLVA using 11 loci demonstrated that seven of the 11 loci were identical between the two isolates, whereas the identity between the isolates and the seven reference strains of L. interrogans serogroup Sejroe was zero to three loci. These results indicate that the isolates investigated in this study have novel genotypes, and are genetically closest to each other among the known L. interrogans serogroup Sejroe strains.

Emergent measles-containing vaccination recommendation for aged 6-11 months and detection of vaccine-associated measles during a large measles outbreak in Okinawa, Japan, in 2018.

Okinawa Prefecture, Japan, experienced a large measles outbreak from March to May 2018. During this outbreak, there were 99 laboratory-confirmed cases and 14 vaccine-associated measles cases. In addition to the reinforcement of routine immunization, Okinawa prefectural government introduced emergent measles-containing vaccination recommendations for infants aged 6-11 months as part of the outbreak response. Increased concern exists in Okinawa about measles in infants following a previous outbreak from 1998 to 2001, when nine children including four infants died. Of 8062 infants aged 6-11 months who received measles-containing vaccine (MCV), six developed vaccine-associated measles; incidence was 0.74 per 1000 doses (95%CI 0.27-1.62). This was similar to that of first dose routine immunization recipients at one year of age (IR 0.60, 95%CI 0.20-1.78). Among 14 vaccine-associated measles cases, throat swab samples showed the highest positive rate (92.9%) by real-time reverse transcription polymerase chain reaction (RT-qPCR), followed by urine (25.0%) and whole blood (7.7%) samples. Furthermore, one throat swab sample classified as equivocal by RT-qPCR was positive by conventional RT-PCR (RT-PCR). During an outbreak, it is critical to distinguish between cases with measles-like symptoms caused by wild circulating virus and those caused by vaccine-derived virus as accurately and urgently as possible because the public health response will be quite different. No infant deaths were observed during this outbreak, and no severe adverse events following immunization were seen among infants 6-11 months old who were given MCV as a public health response. Thus, we conclude that introduction of emergent MCV was effective and describing the characteristics of vaccine-associated measles cases during a measles outbreak will be helpful for future outbreak response efforts.

Purification, cDNA cloning, and characterization of plant chitinase with a novel domain combination from lycophyte Selaginella doederleinii.

Chitinase-A from a lycophyte Selaginella doederleinii (SdChiA), having molecular mass of 53 kDa, was purified to homogeneity by column chromatography. The cDNA encoding SdChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1477 nucleotides and its open reading frame encoded a polypeptide of 467 amino acid residues. The deduced amino acid sequence indicated that SdChiA consisted of two N-terminal chitin-binding domains and a C-terminal plant class V chitinase catalytic domain, belonging to the carbohydrate-binding module family 18 (CBM18) and glycoside hydrolase family 18 (GH18), respectively. SdChiA had chitin-binding ability. The time-dependent cleavage pattern of (GlcNAc)4 by SdChiA showed that SdChiA specifically recognizes the ß-anomer in the + 2 subsite of the substrate (GlcNAc)4 and cleaves the glycoside bond at the center of the substrate. This is the first report of the occurrence of a family 18 chitinase containing CBM18 chitin-binding domains. ABBREVIATIONS: AtChiC: Arabidopsis thaliana class V chitinase; CBB: Coomassie brilliant blue R250; CBM: carbohydrate binding module family; CrChi-A: Cycas revolute chitinase-A; EaChiA: Equisetum arvense chitinase-A; GH: glycoside hydrolase family, GlxChi-B: gazyumaru latex chitinase-B; GlcNAc: N-acetylglucosamine; HPLC: high performance liquid chromatography; LysM; lysin motif; MtNFH1: Medicago truncatula ecotypes R108-1 chitinase; NCBI: national center for biotechnology information; NF: nodulation factor; NtChiV: Nicotiana tabacum class V chitinase; PCR: polymerase chain reaction; PrChi-A: Pteris ryukyuensis chitinase-A; RACE: rapid amplification of cDNA ends; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SdChiA: Selaginella doederleinii chitinase-A.

Molecular Epidemiological Study of Mumps Epidemics of 2015 in Okinawa, Japan.

Although major mumps epidemics occurred every 4-5 years in Okinawa Prefecture in Japan, no laboratory diagnoses were conducted. A mumps epidemic started in Okinawa in October 2014, and we collected clinical samples from 31 patients in 4 areas (Hokubu, Nanbu, Miyako, and Yaeyama) from July to December 2015, for virus isolation and RT-PCR, whose positive ratios were 52% and 87%, respectively. Phylogenetic analyses showed that all isolates were classified into genotype G, and with one exception, consisted of 2 subgenotypes, Ge (55.6%) and Gw (40.7%), which have been prominent in Japan recently. One isolate was classified in another lineage, which was detected in Japan for the first time, and was similar to a Hong Kong isolate from 2014. Remarkably, the geographic distributions of the 2 major lineages were separated. The Ge viruses were isolated from the main island of Okinawa and the Yaeyama Islands, whereas the Gw isolates were mainly detected from the Miyako Islands. These results suggest that the Ge and Gw mumps viruses mainly caused the mumps epidemics of 2015 in Okinawa, and that they spread independently in separate regions. This is the first report describing the molecular epidemiology of mumps epidemics in Okinawa Prefecture.

Molecular evolution of VP3, VP1, 3C(pro) and 3D(pol) coding regions in coxsackievirus group A type 24 variant isolates from acute hemorrhagic conjunctivitis in 2011 in Okinawa, Japan.

A large acute hemorrhagic conjunctivitis (AHC) outbreak occurred in 2011 in Okinawa Prefecture in Japan. Ten strains of coxsackievirus group A type 24 variant (CA24v) were isolated from patients with AHC and full sequence analysis of the VP3, VP1, 3C(pro) and 3D(pol) coding regions performed. To assess time-scale evolution, phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo method. In addition, similarity plots were constructed and pairwise distance (p-distance) and positive pressure analyses performed. A phylogenetic tree based on the VP1 coding region showed that the present strains belong to genotype 4 (G4). In addition, the present strains could have divided in about 2010 from the same lineages detected in other countries such as China, India and Australia. The mean rates of molecular evolution of four coding regions were estimated at about 6.15 to 7.86 × 10(-3) substitutions/site/year. Similarity plot analyses suggested that nucleotide similarities between the present strains and a prototype strain (EH24/70 strain) were 0.77-0.94. The p-distance of the present strains was relatively short (<0.01). Only one positive selected site (L25H) was identified in the VP1 protein. These findings suggest that the present CA24v strains causing AHC are genetically related to other AHC strains with rapid evolution and emerged in around 2010.

Comparative analyses of the two proliferating cell nuclear antigens from the hyperthermophilic archaeon, Thermococcus kodakarensis.

The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced, Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain.