Fatal SARS-CoV-2 Infection among Children, Japan, January-September 20221.

To determine the characteristics of pediatric patients 0-19 years of age who died after onset of SARS-CoV-2 infection in Japan during January 1-September 30, 2022, we reviewed multiple sources. We identified 62 cases, collected detailed information from medical records and death certificates, and conducted interviews, resulting in 53 patients with detailed information for our study. Among 46 patients with internal causes of death (i.e., not external causes such as trauma), 15% were <1 year of age, 59% had no underlying disease, and 88% eligible for vaccination were unvaccinated. Nonrespiratory symptoms were more common than respiratory symptoms. Out-of-hospital cardiac arrest affected 46% of patients, and time from symptom onset to death was <7 days for 77%. Main suspected causes of death were central nervous system abnormalities (35%) and cardiac abnormalities (20%). We recommend careful follow-up of pediatric patients after SARS-CoV-2 infection during the first week after symptom onset, regardless of underlying diseases.

Genetics of the rabbit haemorrhagic disease virus strains responsible for rabbit haemorrhagic disease outbreaks in Japan between 2000 and 2020.

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease in rabbits caused by the rabbit haemorrhagic disease virus (RHDV), which includes two genotypes, RHDV-GI.1 and RHDV2-GI.2. RHDVs tend to recombine among different strains, resulting in significant genetic evolution. This study evaluated the genetics of Japanese RHDV strains associated with six outbreaks between 2000 and 2020 using whole-genome sequencing, genomic recombination and phylogenetic analyses. Genomic recombination analysis using near-complete genomic sequences revealed that two Japanese strains detected in 2000 and 2002 were non-recombinant GI.1 (variant RHDVa-GI.1a) strains of different origins, most closely related to strains identified in PR China in 1997 and the USA in 2001, respectively. In contrast, four recent Japanese GI.2 strains detected between 2019 and 2020 were recombinant viruses harbouring structural protein (SP) genes from GI.2 strains and non-SP (NSP) genes from a benign rabbit calicivirus (RCV) strain of genotype RCV-E1-GI.3 (GI.3P-GI.2) or an RHDV G1-GI.1b variant (GI.1bP-GI.2). Phylogenetic analysis based on SP and NSP regions revealed that the GI.1bP-GI.2 recombinant virus detected in Ehime prefecture and the GI.3P-GI.2 recombinant viruses detected in Ibaraki, Tochigi and Chiba prefectures were most closely related to recombinant viruses identified in Australia in 2017 and Germany in 2017, respectively. These results suggested that past RHD outbreaks in Japan did not result from the evolution of domestic RHDVs but rather represented incursions of foreign RHDV strains, implying that Japan is constantly at risk of RHDV incursion from other countries.

[Safety and Efficacy of Robot-Assisted Surgery for Early Uterine Cancer in Our Institution over Five Years Since Its Introduction].

Lacking experience of laparoscopic surgery against gynecologic malignancies, we started performing robot-assisted surgery for uterine cancer in September 2017. Here we compared the safety and efficacy of robot-assisted surgery with those of open surgery in early-stage uterine cancer patients. The surgical time was significantly longer and the blood loss and hospital stay were significantly shorter for robot-assisted versus open surgery. No recurrence was observed after robot-assisted surgery. Robot-assisted surgery can be safely performed even in general hospitals and is an effective treatment option for early-stage uterine cancer patients.

Systemic and intestinal porcine epidemic diarrhea virus-specific antibody response and distribution of antibody-secreting cells in experimentally infected conventional pigs.

Porcine epidemic diarrhea (PED) is a coronavirus disease characterized by the rapid spread of severe diarrhea among pigs. PED virus (PEDV) infects and replicates mainly in the epithelial cells of the duodenum, jejunum, ileum and colon. Serum or mucosal IgA antibody levels have been used to predict both vaccine efficacy and the level of protective immunity to enteric infectious diseases in individuals or herds. Details of the B-cell immune response upon PEDV infection, such as the systemic and mucosal PEDV IgA antibody response, the distribution of IgA antibody-secreting cells (ASCs), and their role in virus clearance are not yet clear. In this experimental infection study, we observed similar fluctuations in PEDV IgA antibody levels in serum and intestinal contents of the upper and lower jejunum and ileum, but not fecal samples, over the 4-week experimental course. ASCs that actively secrete PEDV IgA antibody without in vitro stimulation were distributed mainly in the upper jejunum, whereas memory B cells that showed enhanced PEDV IgA antibody production upon in vitro stimulation were observed in mesenteric lymph nodes and the ileum. Our findings will contribute to the development of effective vaccines and diagnostic methods for PEDV.

Effect of acute aerobic exercise on arterial stiffness and thyroid-stimulating hormone in subclinical hypothyroidism.

Acute exercise has been reported to increase thyroid hormone levels and decrease arterial stiffness in healthy young subjects. However, the effect of acute aerobic exercise on circulating thyroid hormone levels and arterial stiffness in patients with subclinical hypothyroidism remains unclear. The aim of this study was to investigate the effects of acute aerobic exercise on arterial stiffness and thyroid hormone levels, and any relationship between these endpoints, in patients with subclinical hypothyroidism. We studied patients with untreated subclinical hypothyroidism (n = 53, 65 ± 12 years old) compared with euthyroid subjects (n = 55, 64 ± 10 years old). Exercise analysis was performed with a ramp cycle ergometer test. Arterial stiffness (cardio-ankle vascular index, CAVI) was measured at baseline and 5 min after exercise. We collected participant blood samples for serum thyroid-stimulating hormone (TSH) and free thyroxine (FT4) measurements before and 5 min after exercise. The CAVI and serum TSH levels significantly decreased after exercise in the subclinical hypothyroidism group (CAVI; 8.1 ± 1.6 vs. 8.5 ± 1.5, p < 0.001, TSH; 6.7 ± 1.4 vs. 7.6 ± 1.2 µIU/ml, p < 0.001) and euthyroid group (CAVI; 7.6 ± 1.0 vs. 8.3 ± 0.9, p < 0.001, TSH; 2.2 ± 1.1 vs. 2.4 ± 1.2 µIU/ml, p = 0.005). The changes in CAVI from baseline compared with after exercise were lower, in absolute values, in the subclinical hypothyroidism group than in the euthyroid group (subclinical hypothyroidism group vs euthyroid group; ΔCAVI: - 0.4 ± 0.6 vs. - 0.7 ± 0.7, p = 0.012). The changes in serum TSH from baseline to after exercise were higher, in absolute values, in the subclinical hypothyroidism group than in the euthyroid group (subclinical hypothyroidism group vs euthyroid group; Δ serum TSH: - 1.3 ± 1.4 vs. - 0.3 ± 0.5, p < 0.001). The changes in CAVI from baseline to after exercise were negatively correlated with changes in TSH (r = - 0.32, p = 0.038) in the subclinical hypothyroidism group. In conclusion, acute aerobic exercise decreased both arterial stiffness and serum TSH levels in patients with subclinical hypothyroidism and euthyroid subjects. While the absolute change in arterial stiffness decreased, the absolute change in serum TSH levels increased in patients with subclinical hypothyroidism compared with euthyroid subjects. These data suggest that subclinical hypothyroidism reduces CAVI during acute aerobic exercise. Further changes in absolute levels of serum TSH in subclinical hypothyroidism may result in reduced CAVI improvement by acute aerobic exercise.

Pig epidemic diarrhoea virus S gene variant with a large deletion non-lethal to colostrum-deprived newborn piglets.

We previously identified a third porcine epidemic diarrhoea virus (PEDV) S variant with a large deletion of 582 nucleotides in the 5' terminal region of the S gene, in addition to the North American type and the S INDELs type. To investigate the pathogenicity of this variant, TTR-2/JPN/2014, we performed experimental infection using colostrum-deprived piglets and compared the results with those from the North American type PEDV, OKN-1/JPN/2013. Fifteen newborn piglets were divided into two groups of 7-8 piglets each and inoculated orally with one of PEDV isolates maintained at the eighth passage in Vero cell culture. Although all PEDV-inoculated piglets showed acute watery diarrhoea, lethality clearly differed between both PEDV-inoculated groups. Moreover, there were differences in virus distribution and lesions on the intestines between the two PEDV-inoculated groups. Therefore, our data suggest that the OKN-1/JPN/2013 PEDV isolate is virulent, whereas the TTR-2/JPN/2014 PEDV isolate is avirulent.

Porcine aminopeptidase N is not a cellular receptor of porcine epidemic diarrhea virus, but promotes its infectivity via aminopeptidase activity.

Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhoea, has recently caused significant economic damage worldwide. Porcine aminopeptidase N (pAPN) has been reported to be the receptor for PEDV, although robust evidence is lacking. In the present study, we explored whether pAPN functions as a receptor for PEDV. Human HeLa cells expressing pAPN and pAPN-positive porcine CPK cells failed to support PEDV infection, but were susceptible to infection by transmissible gastroenteritis virus (TGEV), which utilizes pAPN as a functional receptor. In contrast to TGEV, PEDV did not bind soluble porcine aminopeptidases (pAPs) and infection was not inhibited by the soluble form of pAPs. However, overexpression of pAPN in porcine CPK cells (CPK-pAPN cells) slightly increased the production of PEDV, and the increased replication in CPK-pAPN cells was inhibited by bestatin, an inhibitor of the protease activity of aminopeptidase N. These results suggest that pAPN is not a functional receptor for PEDV, but promotes the infection of PEDV through its protease activity.

Integrin α6ß4 and TRPV1 channel coordinately regulate directional keratinocyte migration.

The directional migration of epithelial cells is crucial for wound healing. Among integrins, a family of cell adhesion receptors, integrin ß4 has been assumed to be a promigratory factor, in addition to its role in stable adhesion. In turn, Ca(2+) signaling is also a key coordinator of migration. Keratinocytes reportedly express transient receptor potential vanilloid channels (TRPV1); however, the function of these channels as a regulator of intracellular Ca(2+) level in cell migration has remained uncharacterized. In the present study, we investigated the role of TRPV1 in directional migration related to integrin ß4 using a scratch wound assay on a confluent monolayer sheet of murine keratinocytes (Pam212 cells). Double immunofluorescence staining revealed the de novo expression of integrin ß4 and TRPV1 in migrating cells at the wound edge in response to scratch wounding, and both expression levels were almost matched. Epidermal growth factor (EGF) not only promoted keratinocyte migration, but also caused the further up-regulation of both integrin ß4 and TRPV1. In addition, the knockdown of the integrin ß4 or TRPV1 gene significantly impeded wound closure. The TRPV1 agonist capsaicin significantly promoted migration, while a selective TRPV1 antagonist inhibited it. The gene knockdown of TRPV1 inhibited the expression of the integrin ß4 gene and that of ß4 protein in migrating cells. These findings suggest that TRPV1 may stimulate directional migration directly by eliciting a Ca(2+) signal or indirectly via integrin ß4 expression.

New porcine epidemic diarrhoea virus variant with a large deletion in the spike gene identified in domestic pigs.

From October 2013 to date, approximately 1,000 outbreaks of porcine epidemic diarrhoea virus (PEDV) have occurred in Japan. Porcine epidemic diarrhoea with non-lethal effects in piglets was identified in Tottori prefecture in October 2014. Complete genome analysis revealed that the causative pathogen, Tottori2, is a new PEDV variant with a large (582 nt) deletion in the spike gene. Phylogenetic analysis indicated that the Tottori2 PEDV strain might have been derived from the current PEDV strains circulating in domestic pigs. Moreover, the Tottori2 PEDV strain was successfully isolated in Vero cells by serial passage.

A "catch-and-release" protocol for alkyne-tagged molecules based on a resin-bound cobalt complex for peptide enrichment in aqueous media.

The development of new and mild protocols for the specific enrichment of biomolecules is of significant interest from the perspective of chemical biology. A cobalt-phosphine complex immobilised on a solid-phase resin has been found to selectively bind to a propargyl carbamate tag, that is, "catch", under dilute aqueous conditions (pH 7) at 4 °C. Upon acidic treatment of the resulting resin-bound alkyne-cobalt complex, the Nicholas reaction was induced to "release" the alkyne-tagged molecule from the resin as a free amine. Model studies revealed that selective enrichment of the alkyne-tagged molecule could be achieved with high efficiency at 4 °C. The proof-of-concept was applied to an alkyne-tagged amino acid and dipeptide. Studies using an alkyne-tagged dipeptide proved that this protocol is compatible with various amino acids bearing a range of functionalities in the side-chain. In addition, selective enrichment and detection of an amine derived from the "catch and release" of an alkyne-tagged dipeptide in the presence of various peptides has been accomplished under highly dilute conditions, as determined by mass spectrometry.

Trifluoromethylation reactions for the synthesis of ß-trifluoromethylamines.

A multitalented system: N-migratory oxytrifluoromethylation and one-pot three-component reactions of allylamines as well as the aminotrifluoromethylation of alkenyl amines all proceeded efficiently in the presence of the Togni reagent (1) and CuI to afford a variety of ß-trifluoromethylamine derivatives (see scheme).

Analysis of the excretion dynamics and genotypic characteristics of rotavirus A during the lives of pigs raised on farms for meat production.

To determine the excretion dynamics and genotypic characteristics of rotavirus A (RVA), a longitudinal observational study was performed in 10 pigs from 3 litters at a farrow-to-finish farm. A total of 400 fecal samples were directly collected from the rectums of individual pigs (aged 7 to 217 days) at 3- to 14-day intervals. Seventy-one samples (17.5%) were positive for RVA by reverse transcription-PCR designed to detect the VP7 and VP4 genes. At least 13 combinations of 5 G (G2, G4, G5, G9, and G11) and 6 P (P[6], P[7], P[13], P[23], P[27], and P[34]) genotypes were identified by direct sequencing of the PCR products. We were able to detect RVA VP7 sequences from each pig 4 to 6 times with intervals of 7 to 52 days (from 7 to 119 days of age). Each pig harbored RVAs with at least 3 to 6 different combinations of G and P genotypes, while repeated excretions of RVAs carrying the same combinations of G and P genotypes were also observed. Virus shedding and changes in G and P genotypes appeared to be associated with movement of the pigs into weaning, growing, and finishing barns. These results indicated that, over their lifetimes, pigs raised for meat frequently and intermittently excrete genetically diverse RVAs.

Characterization of epidemic diarrhea outbreaks associated with bovine torovirus in adult cows.

Bovine torovirus (BToV) is recognized as an enteric pathogen of calves, but its etiological role in diarrhea and epidemiological characterization in adult cows remain unclear. In 2007-2008, three outbreaks of epidemic diarrhea occurred in adult cows at three dairy farms in Niigata Prefecture, Japan. BToV was the only enteric pathogen detected in these outbreaks, as determined by electron microscopy, reverse transcription-PCR, bacteria and parasite tests of fecal samples, and antibody tests with paired sera. The epidemiological features of the three outbreaks were similar to those of bovine coronavirus infection, except for the absence of bloody diarrhea, with diarrhea spreading among most adult cows, but not in calves, within several days and diarrhea lasting for 3-5 days with anorexia. Decreased milk production and mild respiratory symptoms were also observed in two of the outbreaks. Nucleotide sequence analysis of the BToV nucleocapsid, spike, and hemagglutinin-esterase (HE) genes revealed a close relatedness among the detected BToV strains from each outbreak and those of Japanese BToV strain Aichi/2004. Furthermore, we isolated a BToV strain, designated Niigata (TC), from a fecal sample using a human rectal tumor cell line. Sequence analysis of this isolate and Aichi/2004 indicated that both strains have truncated HE genes with deletions in the 3' region that occurred through cell culture-adaptation. The short projections that are believed to be formed by the HE protein on virus particles were not observed in these cultured strains by electron microscopy. Taken together, these results suggest that BToV causes epidemic diarrhea in adult cows and should be included in the differential diagnosis of diarrhea in adult cows. In addition, our findings indicate that the HE protein of BToV may not be necessary for viral replication.

Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle.

A one-step multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection of five viruses causing diarrhea in adult cattle: bovine group A rotavirus (GAR), bovine group B rotavirus (GBR), bovine group C rotavirus (GCR), bovine coronavirus (BCV), and bovine torovirus (BToV). The detection limit of the one-step multiplex RT-PCR for GAR, GCR, BCV, and BToV was 10(2), 10(0), 10(1), and 10(2) TCID(50)/ml, respectively, and that for GBR was 10(6) copies/ml. The one-step multiplex RT-PCR with newly designed primers to detect GAR had higher sensitivity than a single RT-PCR with conventional primers, with no false-positive reactions observed for ten other kinds of bovine RNA viruses To assess its field applicability, 59 of 60 fecal samples containing one of these five viruses from all 25 epidemic diarrhea outbreaks in adult cattle were positive in the one-step multiplex RT-PCR assay. Furthermore, using four additional fecal samples containing two viruses (GBR and BCV or BToV), two amplified products of the expected sizes were obtained simultaneously. In contrast, all 80 fecal samples lacking the five target viruses from normal adult cattle were negative in the multiplex assay. Taken together, our results indicate that the one-step multiplex RT-PCR developed here for the detection of GAR, GBR, GCR, BCV, and BToV can be expected to be a useful tool for the rapid and cost-effective diagnosis and surveillance of viral diarrhea in adult cattle.

Whole-genome sequencing of live attenuated bovine adenovirus type 7 vaccine strain TS-GT suggests biomarkers for virulence attenuation.

Bovine adenovirus type 7 (BAdV-7) is one of the most important respiratory and enteric pathogens in the cattle industry. Although live attenuated vaccines are used to control the virus in Japan, limited information is available on the genomic regions that determine viral pathogenicity. We determined the complete genome sequence of the attenuated BAdV-7 strain TS-GT. The genome is 30,052 bp long and contains 45-bp inverted terminal repeats and 30 predicted genes. A genome sequence comparison showed that 99.9% of the TS-GT genome is identical to the prototypic and pathogenic BAdV-7 strain Fukuroi; however, the TS-GT genome contains a novel mutation and four indels. We describe here potential relationships between these genomic changes and the biological characteristics of BAdV-7.

Isolation and immortalization of macrophages derived from fetal porcine small intestine and their susceptibility to porcine viral pathogen infections.

Macrophages are a heterogeneous population of cells that are present in all vertebrate tissues. They play a key role in the innate immune system, and thus, in vitro cultures of macrophages provide a valuable model for exploring their tissue-specific functions and interactions with pathogens. Porcine macrophage cultures are often used for the identification and characterization of porcine viral pathogens. Recently, we have developed a simple and efficient method for isolating primary macrophages from the kidneys and livers of swine. Here, we applied this protocol to fetal porcine intestinal tissues and demonstrated that porcine intestinal macrophages (PIM) can be isolated from mixed primary cultures of porcine small intestine-derived cells. Since the proliferative capacity of primary PIM is limited, we attempted to immortalize them by transferring the SV40 large T antigen and porcine telomerase reverse transcriptase genes using lentiviral vectors. Consequently, immortalized PIM (IPIM) were successfully generated and confirmed to retain various features of primary PIM. We further revealed that IPIM are susceptible to infection by the African swine fever virus and the porcine reproductive and respiratory syndrome virus and support their replication. These findings suggest that the IPIM cell line is a useful tool for developing in vitro models that mimic the intestinal mucosal microenvironments of swine, and for studying the interactions between porcine pathogens and host immune cells.

Iliac artery-enteric fistula developed during bevacizumab-containing chemotherapy for recurrent cervical cancer: A case report and literature review.

An arterioenteric fistula is a devastating and life-threatening condition. As patients often present in extremis from hemorrhage shock, an early diagnosis and prompt life-saving interventions have to be performed. In this report, we describe a case of a 38-year-old Japanese woman who presented with hematochezia that rapidly progressed to hemorrhagic shock secondary to an iliac artery-enteric fistula that developed during bevacizumab-containing chemotherapy for recurrent cervical cancer. The patient underwent successful endovascular treatment with a covered stent-graft as a bridge to definitive open surgery.

Escherichia coli Nissle 1917 Enhances Efficacy of Oral Attenuated Human Rotavirus Vaccine in a Gnotobiotic Piglet Model.

Human rotavirus (HRV) infection is a major cause of viral gastroenteritis in young children worldwide. Current oral vaccines perform poorly in developing countries where efficacious vaccines are needed the most. Therefore, an alternative affordable strategy to enhance efficacy of the current RV vaccines is necessary. This study evaluated the effects of colonization of neonatal gnotobiotic (Gn) pigs with Escherichia coli Nissle (EcN) 1917 and Lacticaseibacillus rhamnosus GG (LGG) probiotics on immunogenicity and protective efficacy of oral attenuated (Att) HRV vaccine. EcN-colonized pigs had reduced virulent HRV (VirHRV) shedding and decreased diarrhea severity compared with the LGG-colonized group. They also had enhanced HRV-specific IgA antibody titers in serum and antibody secreting cell numbers in tissues pre/post VirHRV challenge, HRV-specific IgA antibody titers in intestinal contents, and B-cell subpopulations in tissues post VirHRV challenge. EcN colonization also enhanced T-cell immune response, promoted dendritic cells and NK cell function, reduced production of proinflammatory cytokines/Toll like receptor (TLR), and increased production of immunoregulatory cytokines/TLR expression in various tissues pre/post VirHRV challenge. Thus, EcN probiotic adjuvant with AttHRV vaccine enhances the immunogenicity and protective efficacy of AttHRV to a greater extent than LGG and it can be used as a safe and economical oral vaccine adjuvant.

Genetic divergence and classification of non-structural protein 1 among porcine rotaviruses of species B.

Porcine rotavirus B (RVB) has frequently been detected in diarrhoea of suckling and weaned pigs. Moreover, epidemiological studies using ELISA have demonstrated high antibody prevalence in sera from sows, indicating that RVB infections are widespread. Because it is difficult to propagate RVBs serially in cell culture, genetic analysis of RNA segments of porcine RVBs other than those encoding VP7 and NSP2 has been scarcely performed. We conducted sequence and phylogenetic analyses focusing on non-structural protein 1 (NSP1), using 15 porcine RVB strains isolated from diarrhoeic faeces collected around Japan. Sequence analysis showed that the porcine NSP1 gene contains two overlapping ORFs. Especially, peptide 2 of NSP1 retains highly conserved cysteine and histidine residues among RVBs. Comparison of NSP1 nucleotide and deduced amino acid sequences from porcine RVB strains demonstrated low identities to those from other RVB strains. Phylogenetic analysis of RVB NSP1 revealed the presence of murine, human, ovine, bovine and porcine clusters. Furthermore, the NSP1 genes of porcine RVBs were divided into three genotypes, suggesting the possibility that porcine species might be an original host of RVB infection. Of nine strains common to those used in our previous study, only one strain was classified into a different genotype from the others in the analysis of VP7, in contrast to the analysis of NSP1, where all belonged to the same cluster. This fact suggests the occurrence of gene reassortment among porcine RVBs. These findings should provide more beneficent information to understand the evolution and functions of RVBs.

Genetic diversity of group A rotaviruses associated with repeated outbreaks of diarrhea in a farrow-to-finish farm: identification of a porcine rotavirus strain bearing a novel VP7 genotype, G26.

Group A rotaviruses (GARs) are one of the most common causes of diarrhea in suckling pigs. Although a number of G and P genotypes have been identified in porcine GARs, few attempts have been made to study the molecular epidemiology of these viruses associated with diarrhea outbreaks within a farm over an extended period of time. Here, we investigated the molecular characteristics of GARs that caused four outbreaks of diarrhea among suckling pigs in a farrow-to-finish farm over the course of a year. G and P genotyping of GARs detected at each outbreak demonstrated genetic diversity in this farm as follows: G9P[23] was detected at the first outbreak, G9P[13]/[22] and G9P[23] at the second, G3P[7] at the third, and G9P[23], G5P[13]/[22], and P[7] combined with an untypeable G genotype at the fourth. Sequence analysis of the detected GARs revealed that such genetic diversity could have resulted not only from the introduction of new GAR strains, but also from gene reassortment between GAR strains within the farm. Further, the GAR strain carrying the untypeable G genotype was shown to be a novel porcine GAR bearing a new G26 genotype, as confirmed by the Rotavirus Classification Working Group.