Epidemiology and molecular evolution of GI.1 sapovirus in the recent era.

Sapovirus (SaV) infection is increasing worldwide. Herein, we provided evidence of a significant increase in SaV infection in Japan during 2010-2022, primarily due to the considerable (p = 0.0003) rise of the GI.1 genotype. Furthermore, we found that all major and minor SaV outbreaks in Japan, including the largest SaV outbreak in 2021-2022, were caused by the GI.1 genotype. Therefore, to get insight into the underlying molecular mechanism behind this rising trend of the SaV GI.1 type, we selected 15 SaV GI.1 outbreak strains for complete genome analysis through next-generation sequencing. Phylogenetically, our strains remained clustered in different branches in lineages I and II among the GI.1 genotype. We showed all amino acid (aa) substitutions in different open reading frames (ORFs) in these strains. Importantly, we have demonstrated that the strains involved in the largest SaV outbreak in Japan in 2021-2022 belonged to lineage II and possessed the third ORF. We have identified some unique aa mutations in these major outbreak strains in the NS1 and NS6-NS7 regions that are thought to be associated with viral pathogenicity, cell tropism, and epidemiological competence. Thus, in addition to enriching the database of SaV's complete sequences, this study provides insights into its important mutations.

Comparison of adverse events following the second/third dose of BNT162b2 in a medical institute in Japan.

Background: Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for ending the pandemic of coronavirus disease 2019 (COVID-19). Currently, the cumulative effect of booster shots of mRNA vaccines on adverse events is not sufficiently characterized. Methods: A survey-based study on vaccine adverse events was conducted in a Japanese medical institute after the third dose of Pfizer BNT162b2. Adverse events were grouped using network analysis, and a heteroscedastic probit model was built to analyse adverse events. Results: There were two main clusters of adverse events, systemic and local injection site-associated events. Subject background and the experience of previous vaccine-related adverse events were variably associated with the occurrence and intensity of adverse events following the third dose. Among adverse events, only lymphadenopathy increased prominently following the third dose, while the largest increase in other systemic adverse events occurred generally following the second dose. Conclusions: The effect of repeated booster vaccines on the frequency and intensity of adverse events differs depending on the kind of adverse event.

Precautionary findings on the utilization of FilmArray® to detect human astroviruses in fecal and sewage samples.

The FilmArray® Gastrointestinal (GI) Panel is a modern, sensitive, and comprehensive stool testing technique for identifying common gastrointestinal pathogens, including viruses, bacteria, and parasites. Its increasing demand is due to ease of operation and automation. Pathogens, particularly viruses, undergo constant genetic evolution. For instance, human astrovirus (HAstV), which causes gastroenteritis in children, the elderly, and immune-compromised individuals, can be identified by the GI Panel. HAstV has evolved into several clades, including the classic (HAstV1-8), novel Melbourne (MLB1-3), and Virginia (VA1-5) clades. This study investigated whether the GI Panel accurately detects all HAstV clades. A total of 12 stool and three sewage water (SW) samples were selected post-confirmation of distinct HAstV strains using conventional RT-PCR and sequence-based genotyping for reassessment by the GI Panel. The GI Panel accurately detected the classic HAstV in stool and SW samples. However, our results confirm the GI Panel's inability to detect the novel MLB (MLB1-3) and VA (VA2) clades in fecal samples, raising the possibility of false-negative results in HAstV testing. Although the GI Panel is useful for identifying a variety of gastrointestinal pathogens in stool and SW samples in a single test, our findings highlight the need to exercise caution when interpreting HAstV results from the GI Panel.

SARS-CoV-2 detection in pediatric dental clinic wastewater reflects the number of local COVID-19 cases in children under 10 years old.

This was the first longitudinal study to analyze dental clinic wastewater to estimate asymptomatic SARS-CoV-2 infection trends in children. We monitored wastewater over a 14-month period, spanning three major COVID-19 waves driven by the Alpha, Delta, and Omicron variants. Each Saturday, wastewater was sampled at the Pediatric Dental Clinic of the only dental hospital in Japan's Saitama Prefecture. The relationship between the weekly number of cases in Saitama Prefecture among residents aged < 10 years (exposure) and wastewater SARS-CoV-2 RNA detection (outcome) was examined. The number of cases was significantly associated with wastewater SARS-CoV-2 RNA positivity (risk ratio, 5.36; 95% confidence interval, 1.72-16.67; Fisher's exact test, p = 0.0005). A sample from Week 8 of 2022 harbored the Omicron variant. Compared to sporadic individual testing, this approach allows continuous population-level surveillance, which is less affected by healthcare seeking and test availability. Since wastewater from pediatric dental clinics originates from the oral cavities of asymptomatic children, such testing can provide important information regarding asymptomatic COVID-19 in children, complementing clinical pediatric data.

Remarkable increase in the detection and molecular characterization of adenovirus F41 in children with acute gastroenteritis in Japan, 2017-2023.

Human adenovirus (HAdV) is one of the causative viruses of acute gastroenteritis (AGE) in children worldwide. Species F is known to be enteric adenovirus (genotypes 40 and 41) detected in stool samples. In Japan, we conducted an epidemiological study and molecular characterization of HAdV before and after the COVID-19 pandemic from 2017 to 2023. Among 821 patients, HAdV was detected in 118 AGE cases (14.4%). During a period of 6 years, the HAdV detection rates for each year were relatively low at 3.7% and 0%, in 2017-2018, and 2020-2021, respectively. However, the detection rate increased to remarkably high rates, ranging from 13.3% to 27.3% in the other 4-year periods. Of these HAdV-positive strains, 83.1% were F41 genotypes and 16.9% were other genotypes (A31, B3, C1, C2/C6, and C5). Phylogenetic analyses of the nucleotide and deduced amino acid sequences of the full-length hexon gene demonstrated that HAdV-F41 strains were comprised of three clades, and each clade was distributed across the study period from 2017 to 2023. Analysis of deduced amino acid sequences of the hexon gene of the representative HAdV-F41 strains from each clade revealed numerous amino acid substitutions across hypervariable regions (HVRs) from HVR-1 to HVR-7, two insertions in HVR-1 and HVR-7, and two deletions in HVR-1 and HVR-2 of the hexon gene compared to those of the prototype strain, particularly, those of clade 3 HAdV-F41 strains. The findings suggested that the HAdV-F41 of each clade was stable, conserved, and co-circulated for over two decades in Japan.

Guideline for gynecological practice in Japan: Japan Society of Obstetrics and Gynecology and Japan Association of Obstetricians and Gynecologists 2023 edition.

Twelve years after the first edition of The Guideline for Gynecological Practice, which was jointly edited by The Japan Society of Obstetrics and Gynecology and The Japan Association of Obstetricians and Gynecologists, the 5th Revised Edition was published in 2023. The 2023 Guidelines includes 5 additional clinical questions (CQs), which brings the total to 103 CQ (12 on infectious disease, 30 on oncology and benign tumors, 29 on endocrinology and infertility and 32 on healthcare for women). Currently, a consensus has been reached on the Guidelines, and therefore, the objective of this report is to present the general policies regarding diagnostic and treatment methods used in standard gynecological outpatient care that are considered appropriate. At the end of each answer, the corresponding Recommendation Level (A, B, C) is indicated.

Molecular Evolution of GII.P31/GII.4_Sydney_2012 Norovirus over a Decade in a Clinic in Japan.

Norovirus (NoV) genogroup II, polymerase type P31, capsid genotype 4, Sydney_2012 variant (GII.P31/GII.4_Sydney_2012) has been circulating at high levels for over a decade, raising the question of whether this strain is undergoing molecular alterations without demonstrating a substantial phylogenetic difference. Here, we applied next-generation sequencing to learn more about the genetic diversity of 14 GII.P31/GII.4_Sydney_2012 strains that caused epidemics in a specific region of Japan, with 12 from Kyoto and 2 from Shizuoka, between 2012 and 2022, with an emphasis on amino acid (aa) differences in all three ORFs. We found numerous notable aa alterations in antigenic locations in the capsid region (ORF2) as well as in other ORFs. In all three ORFs, earlier strains (2013-2016) remained phylogenetically distinct from later strains (2019-2022). This research is expected to shed light on the evolutionary properties of dominating GII.P31/GII.4_Sydney_2012 strains, which could provide useful information for viral diarrhea prevention and treatment.

Evaluation of a novel triplex immunochromatographic test for rapid simultaneous detection of norovirus, rotavirus, and adenovirus on a single strip test.

BACKGROUND: Acute gastroenteritis is one of the major causes of morbidity and mortality in young children worldwide. Among these, rotavirus, norovirus, and adenovirus have been reported as the primary viral pathogens associated with the disease. Rapid diagnosis of viral pathogens is crucial when diarrhea outbreaks occur to ensure the timely administration of appropriate treatment and control measures. METHODS: We evaluated three immunochromatographic test kits designed for the detection of norovirus, rotavirus, and adenovirus in 71 stool specimens collected from children with diarrhea who visited clinics in Japan. The first kit is a triplex immunochromatographic test kit designed for simultaneous detections of norovirus, rotavirus, and adenovirus on a single strip (this kit was referred to as IC-A). The other two immunochromatographic test kits are a dual detection kit for rotavirus and adenovirus, and a single detection kit for norovirus (IC-B). The RT-PCR/PCR was used as the gold standard method. RESULTS: The results revealed that both IC-A and IC-B kits exhibited the same level of sensitivity of detection for rotavirus (72.7%) and adenovirus (22.7%), although the detection rate was lower than that of the RT-PCR/PCR method. However, there was a slight difference in the sensitivity of detection for norovirus between IC-A and IC-B, at 86.7% and 93.3%, respectively. The sensitivity of detection for adenovirus of both kits was relatively lower than those of RT-PCR method. This could be due to low viral load of adenovirus in clinical specimens below the detection limit of IC-A and IC-B kits. However, both immunochromatographic test kits (IC-A and IC-B) exhibited 100% specificity for norovirus, rotavirus, and adenovirus. CONCLUSIONS: The triplex immunochromatographic test kit (IC-A) designed for simultaneous detection of norovirus, rotavirus, and adenovirus has been proved to be more practical and convenient than the use of single or dual detection kits with more or less the same sensitivity and specificity of detections.

The Emergence and Widespread Circulation of Enteric Viruses Throughout the COVID-19 Pandemic: A Wastewater-Based Evidence.

Growing evidence shed light on the importance of wastewater-based epidemiology (WBE) during the pandemic, when the patients rarely visited the clinics despite the fact that the infections were still prevalent in the community as before. The abundance of infections in the community poses a constant threat of the emergence of new epidemic strains. Herein, we investigated enteric viruses in raw sewage water (SW) from Japan's Tohoku region and compared them to those from the Kansai region to better understand the circulating strains and their distribution across communities during the COVID-19 pandemic. Raw SW was collected between 2019 and 2022, concentrated by polyethylene-glycol-precipitation method, and investigated for major AGE viruses by RT-PCR. Sequence-based analyses were used to assess genotypes and evolutionary relationships. The most commonly detected enteric virus was rotavirus A (RVA) at 63.8%, followed by astrovirus (AstV) at 61.1%, norovirus (NoV) GII and adenovirus (AdV) at 33.3%, sapovirus (SV) at 25.0%, enterovirus (EV) at 19.4%, and NoV GI at 13.9%. The highest prevalence (46.0%) was found in the spring. Importantly, enteric viruses did not decline during the pandemic. Rather, several strains like NoV GII.2, DS-1-like human G3 (equine) RVA, MLB1 AstV, and different F41 HAdV emerged throughout the pandemic and spread widely over the Tohoku and Kansai regions. Tohoku's detection rate remained lower than that of the Kansai area (36 vs 58%). This study provides evidence for the emergence and spread of enteric viruses during the pandemic.

Genetic diversity and declining norovirus prevalence in infants and children during Japan's COVID-19 pandemic: a three-year molecular surveillance.

Noroviruses (NoVs) are a global concern, causing widespread outbreaks and sporadic acute gastroenteritis (AGE) cases across all age groups. Recent research has shed light on the emergence of novel recombinant strains of NoV in various countries. To delve deeper into this phenomenon, we extensively analyzed 1,175 stool samples collected from Japanese infants and children with AGE from six different prefectures in Japan over three years, from July 2018 to June 2021. Our investigation aimed to determine the prevalence and genetic characteristics of NoV associated with sporadic AGE while exploring the possibility of detecting NoV recombination events. Among the analyzed samples, we identified 355 cases positive for NoV, 11 cases attributed to GI genotypes, and 344 associated with GII genotypes. Notably, we discovered four distinct GI genotypes (GI.2, GI.3, GI.4, and GI.6) and seven diverse GII genotypes (GII.2, GII.3, GII.4, GII.6, GII.7, GII.14, and GII.17). The predominant genotypes were GII.4 (56.4%; 194 out of 344), followed by GII.2 and GII.3. Through dual genotyping based on sequencing of the ORF1/ORF2 junction region, we identified a total of 14 different RdRp/capsid genotypes. Of particular interest were the prevalent recombinant genotypes GII.4[P31] and GII.2[P16]. Notably, our study revealed a decrease in the number of children infected with NoV during and after the COVID-19 pandemic. These findings underscore the importance of continuous NoV surveillance efforts.

Sapovirus infections in Japan before and after the emergence of the COVID-19 pandemic: An alarming update.

An increasing trend of sapovirus (SaV) infections in Japanese children during 2009-2019, particularly after the introduction of the voluntary rotavirus (RV)-vaccination program has been observed. Herein, we investigated the epidemiological situation of SaV infections from 2019 to 2022 when people adopted a precautionary lifestyle due to the emergence of the COVID-19 pandemic, and RV vaccines had been implemented as routine vaccines. Stool samples were collected from children who attended outpatient clinics with acute gastroenteritis and analyzed by reverse transcriptase-polymerase chain reaction to determine viral etiology. Among 961 stool samples, 80 (8.3%) were positive for SaV: 2019-2020 (6.5%), 2020-2021 (0%), and 2021-2022 (12.8%). The trend of SaV infection in Japanese children yet remained upward with statistical significance (p = 0.000). The major genotype was GI.1 (75%) which caused a large outbreak in Kyoto between December 2021 and February 2022. Phylogenetic, gene sequence and deduced amino acid sequence analyses suggested that these GI.1 strains detected in the outbreak and other places during 2021-2022 or 2019-2020 remained genetically identical and widely spread. This study reveals that SaV infection is increasing among Japanese children which is a grave concern and demands immediate attention to be paid before SaV attains a serious public health problem.

A New Method to Detect Variants of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Bioluminescent Assay in Real Time (RT-LAMP-BART).

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), of which there are several variants. The three major variants (Alpha, Delta, and Omicron) carry the N501Y, L452R, and Q493R/Q498R mutations, respectively, in the S gene. Control of COVID-19 requires rapid and reliable detection of not only SARS-CoV-2 but also its variants. We previously developed a reverse transcription loop-mediated isothermal amplification assay combined with a bioluminescent assay in real time (RT-LAMP-BART) to detect the L452R mutation in the SARS-CoV-2 spike protein. In this study, we established LAMP primers and peptide nucleic acid probes to detect N501Y and Q493R/Q498R. The LAMP primer sets and PNA probes were designed for the N501Y and Q493R/Q498R mutations on the S gene of SARS-CoV-2. The specificities of RT-LAMP-BART assays were evaluated using five viral and four bacterial reference strains. The sensitivities of RT-LAMP-BART assays were evaluated using synthetic RNAs that included the target sequences, together with RNA-spiked clinical nasopharyngeal and salivary specimens. The results were compared with those of conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. The method correctly identified N501Y and Q493R/Q498R. Within 30 min, the RT-LAMP-BART assays detected up to 100-200 copies of the target genes; conventional real-time RT-PCR required 130 min and detected up to 500-3000 copies. Surprisingly, the real-time RT-PCR for N501Y did not detect the BA.1 and BA.2 variants (Omicron) that exhibited the N501Y mutation. The novel RT-LAMP-BART assay is highly specific and more sensitive than conventional real-time RT-PCR. The new assay is simple, inexpensive, and rapid; thus, it can be useful in efforts to identify SARS-CoV-2 variants of concern.

Whole genome sequencing and genomic characterization of a DS-1-like G2P[4] group A rotavirus in Japan.

After rotavirus was discovered in 1973, it became the leading pathogen in causing acute gastroenteritis in humans worldwide. In this study, we performed whole genome sequencing and genomic characterization of a DS-1-like G2P[4] group A rotavirus in feces of a Japanese child with acute gastroenteritis who was fully Rotarix® vaccinated. The genomic investigation determined a genomic constellation G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 of this rotavirus strain. Its antigenic epitopes of the VP7 and VP4 proteins had significant mismatches compared with the vaccine strains. Our study is the latest attempt to investigate the evolution of the VP7 and VP4 genes of emerging G2P[4] rotavirus in Japan.

Outbreak of human astroviruses 1 and Melbourne 2 in acute gastroenteritis pediatric patients in Japan during the COVID-19 pandemic, 2021.

BACKGROUND: Human astrovirus (HAstV) infection is one of the leading causes of acute gastroenteritis in young children. The present study reports the outbreak of HAstV in children with acute gastroenteritis in Kyoto, Japan, during the COVID-19 pandemic, 2021. METHODS: A total of 61 stool samples were collected from children with acute gastroenteritis who visited a pediatric outpatient clinic in Maizuru city, Kyoto, Japan from July to October, 2021. HAstV was screened by RT-PCR, and the genotypes were identified by nucleotide sequence analysis. RESULTS: Of 61 cases of acute gastroenteritis, 20 were mono-infected with HAstV alone. In addition, mixed infection of HAstV and NoV, and HAstV and RVA were also detected in 15 and 1 cases, respectively. Of 36 HAstV strains detected in this outbreak, 29 and 7 were HAstV1 and MLB2 genotypes, respectively. All HAstV1 strains were closely related to the HAstV1 reported from Thailand and Japan in 2021 and all of them belonged to subgenotype HAstV1a. Among MLB2, they were most closely related to the MLB2 strains reported from China in 2016 and 2018. CONCLUSIONS: After the kindergartens and schools were re-opened at the middle of 2021 in Japan, an outbreak of HAstV was reported. Control measures against the COVID-19 pandemics might affect the spread of diarrheal virus infection. Here we report the outbreak of HAstV1 and MLB2 in Kyoto, Japan, during COVID-19 pandemic in 2021.

A Comparative Study of Acute Gastroenteritis Symptoms in Single- versus Multiple-Virus Infections.

Many different enteric viruses can cause acute gastroenteritis in humans worldwide. While a single virus can indeed cause disease, multiple-virus infections are commonly reported. However, data regarding a comparison between single- and multiple-virus infections upon clinical manifestations of acute gastroenteritis are relatively limited. In this study, a total of 2383 fecal specimens were collected from children with acute gastroenteritis during June 2014-July 2017 in a pediatric clinic in Japan and tested for 11 viruses by multiplex RT-PCR. At least 1 virus was found in 1706 (71.6%) specimens and norovirus GII was the most frequent agent, followed by rotavirus A and other viruses. Multiple-virus infections were identified in 565 cases (33.1%). While major clinical symptoms were found to be significantly different in some single- vs. multiple-virus infections, the disease severity was statistically non-significant. Our study highlights the burden of multiple-virus infections for acute gastroenteritis and the clinical features of patients with multiple-virus infections.

Molecular Epidemiology of Classic, MLB, and VA Astroviruses in Children with Acute Gastroenteritis, 2014-2021: Emergence of MLB3 Strain in Japan.

Human astroviruses (HAstVs) are important causative pathogens of acute gastroenteritis (AGE) in children worldwide. MLB and VA HAstVs, which are genetically distinct from the previously known classic HAstVs, have been detected since 2008. To investigate the role of HAstVs in AGE, we conducted molecular detection and characterization of HAstVs circulating in children with AGE in Japan from 2014 to 2021. Out of 2,841 stool samples, HAstVs were detected in 130 (4.6%). MLB1 was the predominant genotype detected (45.4%), followed by HAstV1 (39.2%), MLB2 (7.4%), VA2 (3.1%), HAstV3 (2.3%), HAstV4, HAstV5, and MLB3 (0.8% each). The results demonstrated that HAstV infection in pediatric patients in Japan was dominated by the two major genotypes MLB1 and HAstV1, with a small proportion of other genotypes. The overall infection rates of MLB and VA HAstVs were higher than those of classic HAstVs. The HAstV1 strains detected in this study belonged solely to lineage 1a. The rare MLB3 genotype was detected for the first time in Japan. All three HAstV3 strains belonged to lineage 3c based on the ORF2 nucleotide sequence and were shown to be recombinant strains. IMPORTANCE HAstVs are one of the pathogens of viral AGE and are considered the third most common viral agents of AGE after rotavirus and norovirus. HAstVs are also suspected to be the causative agents of encephalitis or meningitis in immunocompromised patients and elderly persons. However, little is known about the epidemiology of HAstVs in Japan, especially that of MLBs and VA HAstVs. This study demonstrated epidemiological features and molecular characterization of human astroviruses encompassing a 7-year study period in Japan. This study highlights the genetic diversity of HAstV circulating in pediatric patients with acute AGE in Japan.

The Factors Influencing Pregnant Women's Selection of Media Sources to Obtain Information on COVID-19 in Japan in 2021.

Pregnant women presumably gather information about the coronavirus disease 2019 (COVID-19) from various sources. However, it is difficult for pregnant women who are not medical professionals to source the appropriate information because of the infodemic related to the COVID-19 pandemic. Therefore, the objective of our study was to investigate how pregnant women gathered information about COVID-19 and COVID-19 vaccination. To address this issue, we conducted an online questionnaire survey between 5 October and 22 November 2021, which was approved by the Ethics Committee of Nihon University School of Medicine. We received 4962 responses after excluding 1179 insufficient answers. Our study found that age, occupation, and infection-risk anxiety influenced the selection of media for obtaining information. Pregnant women who were older, medical professionals, public servants, or educators tended to rely on specialized medical websites, whereas housewives tended to use mass media, social media, and sources with uncertain scientific evidence. Additionally, the number of weeks of gestation and the method of conception (natural or assisted reproductive conception) affected the selection of media. The accessibility of COVID-19 information for pregnant women was determined by their social background and pregnancy status. We need to continue making efforts to ensure that appropriate information is readily available to pregnant women and their families.

Clinical and Microbiological Features of Fulminant Haemolysis Caused by Clostridium perfringens Bacteraemia: Unknown Pathogenesis.

Bacteraemia brought on by Clostridium perfringens has a very low incidence but is severe and fatal in fifty per cent of cases. C. perfringens is a commensal anaerobic bacterium found in the environment and in the intestinal tracts of animals; it is known to produce six major toxins: α-toxin, ß-toxin, ε-toxin, and others. C. perfringens is classified into seven types, A, B, C, D, E, F and G, according to its ability to produce α-toxin, enterotoxin, and necrotising enterotoxin. The bacterial isolates from humans include types A and F, which cause gas gangrene, hepatobiliary infection, and sepsis; massive intravascular haemolysis (MIH) occurs in 7-15% of C. perfringens bacteraemia cases, resulting in a rapid progression to death. We treated six patients with MIH at a single centre in Japan; however, unfortunately, they all passed away. From a clinical perspective, MIH patients tended to be younger and were more frequently male; however, there was no difference in the toxin type or genes of the bacterial isolates. In MIH cases, the level of θ-toxin in the culture supernatant of clinical isolates was proportional to the production of inflammatory cytokines in the peripheral blood, suggesting the occurrence of an intense cytokine storm. Severe and systemic haemolysis is considered an evolutionary maladaptation as it leads to the host's death before the bacterium obtains the benefit of iron utilisation from erythrocytes. The disease's extraordinarily quick progression and dismal prognosis necessitate a straightforward and expedient diagnosis and treatment. However, a reliable standard of diagnosis and treatment has yet to be put forward due to the lack of sufficient case analysis.

Ca2+ Dependent Formation/Collapse of Cylindrical Ca2+-ATPase Crystals in Scallop Sarcoplasmic Reticulum (SR) Vesicles: A Possible Dynamic Role of SR in Regulation of Muscle Contraction.

[Ca2+]-dependent crystallization of the Ca2+-ATPase molecules in sarcoplasmic reticulum (SR) vesicles isolated from scallop striated muscle elongated the vesicles in the absence of ATP, and ATP stabilized the crystals. Here, to determine the [Ca2+]-dependence of vesicle elongation in the presence of ATP, SR vesicles in various [Ca2+] environments were imaged using negative stain electron microscopy. The images obtained revealed the following phenomena. (i) Crystal-containing elongated vesicles appeared at ≤1.4 µM Ca2+ and almost disappeared at ≥18 µM Ca2+, where ATPase activity reaches its maximum. (ii) At ≥18 µM Ca2+, almost all SR vesicles were in the round form and covered by tightly clustered ATPase crystal patches. (iii) Round vesicles dried on electron microscopy grids occasionally had cracks, probably because surface tension crushed the solid three-dimensional spheres. (iv) [Ca2+]-dependent ATPase crystallization was rapid (<1 min) and reversible. These data prompt the hypothesis that SR vesicles autonomously elongate or contract with the help of a calcium-sensitive ATPase network/endoskeleton and that ATPase crystallization may modulate physical properties of the SR architecture, including the ryanodine receptors that control muscle contraction.