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.

Roles of TGF-ß1 in Viral Infection during Pregnancy: Research Update and Perspectives.

Transforming growth factor-beta 1 (TGF-ß1) is a pleiotropic growth factor playing various roles in the human body including cell growth and development. More functions of TGF-ß1 have been discovered, especially its roles in viral infection. TGF-ß1 is abundant at the maternal-fetal interface during pregnancy and plays an important function in immune tolerance, an essential key factor for pregnancy success. It plays some critical roles in viral infection in pregnancy, such as its effects on the infection and replication of human cytomegalovirus in syncytiotrophoblasts. Interestingly, its role in the enhancement of Zika virus (ZIKV) infection and replication in first-trimester trophoblasts has recently been reported. The above up-to-date findings have opened one of the promising approaches to studying the mechanisms of viral infection during pregnancy with links to corresponding congenital syndromes. In this article, we review our current and recent advances in understanding the roles of TGF-ß1 in viral infection. Our discussion focuses on viral infection during pregnancy, especially in the first trimester. We highlight the mutual roles of viral infection and TGF-ß1 in specific contexts and possible functions of the Smad pathway in viral infection, with a special note on ZIKV infection. In addition, we discuss promising approaches to performing further studies on this topic.

Adverse events after BNT162b2 mRNA COVID-19 vaccination in health care workers and medical students in Japan.

To control the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the promotion of vaccination is important. However, adverse reactions following vaccination remain a concern. To investigate adverse events in the vaccinated Japanese population, we conducted a survey-based study among health care workers, including medical doctors and nurses; other medical staff; and medical university faculty, staff, and students in a single medical school and affiliated hospital in Japan. In addition, we analyzed the association of different adverse events with individual factors (e.g., age, sex) by performing network analysis. While young age and female sex are often considered risk factors for more severe adverse events, the regression models showed neither age nor sex was associated with local injection-site reactions after the second dose in this study. In contrast to local reactions, systemic adverse events were associated with young age and female sex. However, myalgia was unique in that it was not associated with younger age even though the network analysis showed that myalgia was consistently related to arthralgia and belonged to the group of systemic events after both the first and second vaccine doses. Further study is needed to ensure safe and effective vaccination to aid in controlling the COVID-19 pandemic.

Emerging norovirus GII.4 Sydney[P31] causing acute gastroenteritis outbreak in children in Japan, during COVID-19, 2021.

INTRODUCTION: Norovirus (NoV) is the most common agent causing outbreaks and sporadic cases of acute gastroenteritis among all ages, especially children under 5 years old. During the coronavirus disease 2019 (COVID-19) pandemic, NoV infection has decreased drastically in Japan due to school closures and no outbreak related to NoV infection had been reported. METHOD: In mid-September 2021, NoV outbreak occurred in kindergarten and nursery schools in Maizuru, Kyoto prefecture, Japan. Twenty-six stool samples collected from patients who were diagnosed of NoV gastroenteritis from the outbreak by an immunochromatographic (IC) kit at a pediatric outpatient clinic in Maizuru city during 3 weeks from September 13 to October 8, 2021 were examined for the presence of NoV GII by reverse transcriptase-polymerase chain reaction (RT-PCR), genome sequencing, and phylogenetic analysis. RESULT: All 26 samples were confirmed positive to NoV GII and their genotypes were identified as GII.4 Sydney[P31]. The amino acid substitutions in open reading frame1 (ORF1) and ORF2 genes were found when compared with previously detected sporadic NoV GII.4 Sydney[P31] strains isolated in Japan. The clinical characterization of infected children was described. Most of the children were mild cases and vomiting was the most frequent clinical symptom. CONCLUSION: This study reported a recent emergence of NoV GII.4 Sydney[P31] causing acute gastroenteritis outbreak in children in Japan during the COVID-19 pandemic and suggests a need for further monitoring of NoV GII.4 variants.

The association of three doses of the BNT162b2 mRNA vaccine with abnormal bleeding and an irregular menstrual cycle among premenopausal females: A single institute observation study.

BACKGROUND: The COVID-19 vaccine is effective in preventing severe cases of COVID-19. For women, gynecological adverse events, such as menstrual irregularities and irregular bleeding, could be a concern after COVID-19 vaccination. In this study, we investigated gynecological adverse events in the vaccinated Japanese female population. METHODS: We conducted a survey-based study with health-care workers, including medical doctors and nurses, medical coworkers, and medical university faculty, staff, and students, at a single medical school and affiliated hospital in Japan. We used McNemar's test and network analysis. RESULTS: Overall, we obtained 819 responses, and 424 were from females. After the exclusion of contradictory answers, 309 surveys were finally considered appropriate for the analysis. The frequencies of abnormal bleeding were 0.6%, 1.0%, and 3.0% for the first, second, and third doses, respectively. An irregular menstrual cycle was more common than abnormal bleeding: 1.9%, 4.9%, and 6.6% for the first, second, and third doses, respectively. Network analysis revealed that abnormal bleeding and an irregular menstrual cycle were not associated with other adverse reactions. CONCLUSION: The present study showed that the effects of COVID-19 vaccination on menstruation seem limited.

Poly I:C induces collective migration of HaCaT keratinocytes via IL-8.

BACKGROUND: Delayed wound healing reduces the quality of life (QOL) of patients. Thus, understanding the mechanism of wound healing is indispensable for better management. However, the role of innate immunity in wound healing is thus far unknown. Recently the involvement of TLR3 in wound healing has been evaluated. The systemic administration of polyriboinosinic-polyribocytidylic acid (poly I:C ; a substitute for viral dsRNA and a ligand of toll-like receptor 3), enhances wound healing in vivo. The aim of this study is to improve our understanding of the link between innate immunity and human wound healing, particularly in re-epithelialization. RESULTS: The present study showed that poly I:C significantly accelerated collective HaCaT cell migration in a scratch assay. Poly I:C also increased IL-8 and bFGF production, and anti-IL-8 antibodies significantly inhibited the migration caused by poly I:C. Human recombinant IL-8 also accelerated collective HaCaT cell migration. An immunofluorescence assay and enzyme-linked immunosorbent assay (ELISA) also revealed that poly I:C decreased E-cadherin protein levels and increased vimentin protein levels, and anti-IL-8 antibody reversed this effect. In contrast, nucleic/cytosolic protein ratios of Snail 1 were unchanged in all tested conditions. CONCLUSION: Our findings demonstrated that poly I:C accelerated collective HaCaT cell migration via autocrine/paracrine secretions of IL-8 and the subsequent incomplete epithelial-mesenchymal transition (EMT). Our findings provide a new strategy for wound healing by regulating innate immune systems in re-epithelialization.

Prevalence and Antimicrobial Resistance of Campylobacterjejuni and Campylobacter coli from Adult Hospitalized Patients with Diarrhea in Thailand.

BACKGROUND: Infection with Campylobacter jejuni and C. coli are recognized as the major cause of bacterial gastroenteritis in humans. METHODS: A total of 310 fecal samples collected from Thai adult patients with diarrhea in 2008 were screened for the presence of Campylobacter by PCR. Resistance to fluoroquinolone and macrolides of the detected Campylobacter strains were analyzed by studying the mutations in the gyrA and 23S rRNA genes, respectively. RESULTS: Campylobacter species were detected in 4/310 (1.3%) of diarrheal patients, and C. jejuni was found in 3 of the 4 cases (75%). Fluoroquinolone resistance was noted in 2 cases (50%); however, no resistance to macrolides was observed. CONCLUSIONS: Campylobacter was detected in a low prevalence in adult Thai patients hospitalized with diarrhea, and the resistance to fluoroquinolones is still a matter of concern in case antibiotic therapy is required.

Seroprevalence of Chlamydophila pneumoniae in HIV-infected children in Vietnam.

A total of 89 blood samples collected from HIV-infected infants and children from provinces of southern Vietnam who were hospitalized at Children's Hospital 1, Ho Chi Minh City, during the 1-year period from October 2004 to September 2005 were submitted to serological screening for IgG, IgA, and IgM antibodies against Chlamydophila pneumoniae (C. pneumoniae). The presence of this microorganism was also evaluated by PCR. The results showed that 64 % of the samples were positive for anti-C. pneumoniae IgG, 31.5 % were positive for IgA, and 3.4 % were positive for IgM. The highest prevalences of IgG and IgA positivity, 75 % and 66.7 %, respectively, were noted in the 1- to 2-year-old age group. However, all the samples were negative for C. pneumoniae by PCR. The study revealed a high seroprevalence of C. pneumoniae in Vietnamese infants and children with HIV/AIDS.

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.

Recent Research in Cell Stress and Microbial Infection.

Microbial infection, including bacterial, viral, fungal, and parasitic, is a common human disease leading to various cell stresses [...].

Low Susceptibility of Rubella Virus in First-Trimester Trophoblast Cell Lines.

We recently published an article about myelin oligodendrocyte glycoprotein-independent rubella infection of keratinocytes in vitro, in which first-trimester trophoblast cells were shown as rubella virus (RuV)-resistant. Given an incident rate as high as 90% of congenital rubella syndrome in the first eight weeks of pregnancy, the RuV infection of first-trimester trophoblasts is considered key to opening the gate to transplacental transmission mechanisms. Therefore, with this study, we aimed to verify the susceptibility/resistance of first-trimester trophoblast cell lines, HTR-8/SVneo and Swan.71, against RuV. Cells cultured on multi-well plates were challenged with a RuV clinical strain at a multiplicity of infection from 5 to 10 for 3 h. The infectivity was investigated by immunofluorescence (IF) assay and flow cytometry (FCM) analysis. Supernatants collected during the post-infection period were used to determine virus-progeny production. The scattered signaling of RuV infection of these cells was noted by IF assay, and the FCM analysis showed an average of 4-5% of gated cells infected with RuV. In addition, a small but significant production of virus progeny was also observed. In conclusion, by employing appropriate approaches, we determined the low infectivity of RuV in first-trimester trophoblast cell lines but not resistance as in our previous report.

Parenchymal calcification is associated with the neurological prognosis in patients with congenital rubella syndrome.

Congenital rubella syndrome (CRS) results from maternal rubella virus infection in early pregnancy. Abnormal neuroimaging findings have been analyzed in a small number of CRS patients in the past; however, their clinical significance has been poorly addressed. Therefore, we have investigated the neuroimaging findings of 31 patients with CRS from previous studies. The most common finding was parenchymal calcification, which was observed in 18 of 31 patients (58.1%). A multivariable logistic regression model showed that it was associated with psychomotor or mental retardation (p = 0.018), suggesting that parenchymal calcification in CRS could be a prognostic factor.

TGF-ß1 Promotes Zika Virus Infection in Immortalized Human First-Trimester Trophoblasts via the Smad Pathway.

The Zika virus (ZIKV) is well known for causing congenital Zika syndrome if the infection occurs during pregnancy; however, the mechanism by which the virus infects and crosses the placenta barrier has not been completely understood. In pregnancy, TGF-ß1 is abundant at the maternal-fetal interface. TGF-ß1 has been reported to enhance rubella virus binding and infection in human lung epithelial cells. Therefore, in this study, we investigate the role of TGF-ß1 in ZIKV infection in the immortalized human first-trimester trophoblasts, i.e., Swan.71. The cells were treated with TGF-ß1 (10 ng/mL) for two days before being inoculated with the virus (American strain PRVABC59) at a multiplicity of infection of five. The results showed an enhancement of ZIKV infection, as demonstrated by the immunofluorescent assay and flow cytometry analysis. Such enhanced infection effects were abolished using SB431542 or SB525334, inhibitors of the TGF-ß/Smad signaling pathway. An approximately 2-fold increase in the virus binding to the studied trophoblasts was found. In the presence of the Smad inhibitors, virus replication was significantly suppressed. An enhancement in Tyro3 and AXL (receptors for ZIKV) expression induced by TGF-ß1 was also noted. The results suggest that TGF-ß1 promotes the virus infection via the Smad pathway. Further studies should be carried out to clarify the underlying mechanisms of these findings.

Inhibition of Epithelial-Mesenchymal Transition Maintains Stemness in Human Amniotic Epithelial Cells.

Human amniotic epithelial cells (hAECs), which are a type of placental stem cell, express stem cell marker genes and are capable of differentiating into all three germ layers under appropriate culture conditions. hAECs are known to undergo TGF-ß-dependent epithelial-mesenchymal transition (EMT); however, the impact of EMT on the stemness or differentiation of hAECs has not yet been determined. Here, we first confirmed that hAECs undergo EMT immediately after starting primary culture. Comprehensive transcriptome analysis using RNA-seq revealed that inhibition of TGF-ß-dependent EMT maintained the expression of stemness-related genes, including NANOG and POU5F1, in hAECs. Moreover, the maintenance of stemness did not affect the nontumorigenic characteristics of hAECs. We showed for the first time that TGF-ß-dependent EMT negatively affected the stemness of hAECs, providing novel insight into cellular processes of placental stem cells.

Enhancement of Rubella Virus Infection in Immortalized Human First-Trimester Trophoblasts Under Low-Glucose Stress Conditions.

Rubella virus (RuV) infections in pregnant women, especially first-trimester infections, can lead to congenital rubella syndrome (CRS). However, the mechanisms of fetal RuV infection are not completely understood, and it is not observed in every pregnant woman infected with RuV. As gestational diabetes mellitus is a risk factor for congenital viral infections, we investigated the possible roles of hypoglycemia-related endoplasmic reticulum (ER) stress as a key factor for vertical RuV infection using immortalized human first-trimester trophoblasts. Low-glucose stress was induced prior to RuV infection by culturing HTR-8/SVneo and Swan.71 cells in low-glucose (LG) medium for 24 h or high-glucose medium for 6 h and then LG medium for an additional 18 h. Clinically isolated RuV was inoculated at a multiplicity of infection of 5 to 10. The intracellular localization of the RuV capsid protein was investigated 24 to 48 h post-infection (pi) with flow cytometry (FCM) analysis and fluorescence microscopy. Viral progeny production was monitored by FCM analysis. Increases in RuV infection in LG-induced ER-stressed trophoblasts were observed. No significant increase in apoptosis of RuV-infected cells was noted at days 2 and 5 pi, and substantial viral progeny production was observed until day 5 pi. An approximate fivefold increase in viral binding was noted for the LG-stressed cells. Although the detailed mechanisms underlying viral entry into LG-stressed cells are not known and require further investigation, these findings suggest that a certain degree of LG stress in early pregnancy may facilitate infection and cause CRS.

Restriction of SARS-CoV-2 replication in the human placenta.

Although SARS-CoV-2 can infect human placental tissue, vertical transmission is rare. Therefore, the placenta may function as a barrier to inhibit viral transmission to the foetus, though the mechanisms remain unclear. In this study, we confirmed the presence of the SARS-CoV-2 genome in human placental tissue by in situ hybridization with antisense probes targeting the spike protein; tissue staining was much lower when using sense probes for the spike protein. To the best of our knowledge, this is the first evidence directly indicating inefficient viral replication in the SARS-CoV-2-infected placenta. Additional studies are required to reveal the detailed mechanisms.

Human parechovirus infection in children hospitalized with acute gastroenteritis in Sri Lanka.

Of 362 fecal specimens collected from infants and children hospitalized with acute gastroenteritis in Sri Lanka from September 2005 to August 2006, 30 (8.3%) were positive for human parechovirus (HPeV). Six different HPeV genotypes, including HPeV1, -3, -4, -5, -10, and -11, were identified, of these, HPeV11 was reported for the first time.

Human bocavirus infection in children with acute gastroenteritis in Japan and Thailand.

A total of 329 fecal specimens, which had been known to be negative for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus, and which were collected from infants and children with acute gastroenteritis in Japan and Thailand during 2005-2008 were screened for human bocavirus (HBoV). HBoV was detected by PCR with a primer pair that amplified the NP1 region of its genome and was genotyped by sequencing of the VP1/VP2 region. Of the 329 samples tested, 6 (1.8%) were positive for HBoV. Of these, five samples were collected from Japan and one sample was from Thailand, and the detection rates of HBoV in each country were 2% and 1.2%, respectively. For the detected HBoV, the capsid VP1/VP2 gene of all HBoV strains was successfully sequenced. Four Japanese HBoV strains studied were clustered into group 1, while the remaining Japanese strain and a unique Thai strain belonged to group 2. No severe acute gastroenteritis associated with HBoV was noted. This study provides better understanding on the epidemiology of HBoV infections in children with acute gastroenteritis in Japan and Thailand.

The Epithelial-to-Mesenchymal Transition-Like Process Induced by TGF-ß1 Enhances Rubella Virus Binding and Infection in A549 Cells via the Smad Pathway.

Virus-host cell interactions in rubella virus (RuV) are of great interest in current research in the field, as their mechanism is not yet well understood. By hypothesizing that the epithelial-to-mesenchymal transition (EMT) may play a role in RuV infection, this study aimed to investigate the influence of TGF-ß1-induced EMT of human lung epithelial A549 cells on the infectivity of RuV. A549 cells were cultured and treated with TGF-ß1 for 1 to 2 days prior to virus infection (with a clinical strain). Viral infectivity was determined by flow cytometry analysis of cells harvested at 24 and 48 h post-infection (hpi) and by titration of supernatants collected at 48 hpi. The results showed that the percentages of the TGF-ß1-treated A549 cells that were positive for RuV were at least twofold higher than those of the control, and the viral progeny titers in the supernatants collected at 48 hpi were significantly higher in the treatment group than in the control group. In addition, the virus binding assay showed a strong increase (more than threefold) in the percentages of RuV-positive cells, as determined by flow cytometry analysis and further confirmed by real-time PCR. Such an enhancement effect on RuV infectivity was abolished using LY364947 or SB431542, inhibitors of the TGF-ß/Smad signaling pathway. The findings suggest that the TGF-ß1-induced EMT-like process enhances RuV binding and infection in A549 cells via the Smad pathway. Further studies are necessary to identify possible proteins that facilitate viral binding and entry into treated cells.