The mechanism to maintain homeostasis of the gut microbiota remains largely unknown despite its critical role in the body defense. In the intestines of mice with deficiency of activation-induced cytidine deaminase (AID), the absence of hypermutated IgA is partially compensated for by the presence of large amounts of unmutated IgM and normal expression levels of defensins and angiogenins. We show here a predominant and persistent expansion of segmented filamentous bacteria throughout the small intestine of AID(-/-) mice. Reconstitution of lamina propria IgA production in AID(-/-) mice recovered the normal composition of gut flora and abolished the local and systemic activation of the immune system. The results indicate that secretions of IgAs rather than innate defense peptides are critical to regulation of commensal bacterial flora and that the segmented filamentous bacteria antigens are strong stimuli of the mucosal immune system.
In the context of the coronavirus disease 2019 (COVID-19) pandemic, environmental surveillance for the detection of SARS-CoV-2 has become increasingly important. Studies have demonstrated that the SARS-CoV-2 RNA is present in the feces of infected individuals; further, its presence in wastewater has been reported. However, an optimized method for its detection in sewage has not yet been adequately investigated. Therefore, in this study, the efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater was investigated using two quantitative PCR assays. In particular, wastewater samples were collected from a manhole located in the commercial district of a metropolitan region in Japan, where COVID-19 is highly prevalent, and two wastewater treatment plants (WWTPs). The samples were concentrated using four separate methods, namely, electronegative membrane adsorption, polyethylene glycol precipitation, ultrafiltration, and solid precipitation. Each method revealed a significant concentration of pepper mild mottle virus (PMMoV) RNA, which is an indicator virus for wastewater. As expected, non-enveloped PMMoV RNA was enriched in the supernatant fraction such that relatively low concentrations were detected in the solid fraction of the wastewater samples. In contrast, higher SARS-CoV-2 RNA concentrations were consistently detected in the solid fractions compared with the supernatant fractions based on the other methods that were investigated in this study. Spearman's correlation tests showed that the SARS-CoV-2 RNA concentrations in wastewater samples from the WWTP were significantly correlated with the number of COVID-19 cases recorded during the data collection period. These results demonstrate that viral recovery from the solid fraction is an effective method for SARS-CoV-2 RNA surveillance in an aqueous environment.
Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1-/- mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm3 in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4+ or CD8+ T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifnγ, Tnfα, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1-/- mice and that macrophages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1-/- mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1-/- mice and may have relevance to relapsing hepatitis A in humans.
Activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is indispensable for somatic hypermutation (SHM), class switch recombination, and gene conversion of immunoglobulin genes, which indicates a common molecular mechanism for these phenomena. Here we show that ectopic expression of AID alone can induce hypermutation in an artificial GFP substrate in NIH 3T3 murine fibroblast cells. The frequency of mutations was closely correlated with the level of transcription of the target gene, and the distribution of mutations in NIH 3T3 cells was similar to those of SHM in B lymphocytes. These results indicate that AID is sufficient for the generation of SHM in an actively transcribed gene in fibroblasts, as well as B cells, and that any of the required cofactors must be present in these fibroblasts.
Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) class switch recombination and somatic hypermutation, and has also been implicated in translocations between Ig switch regions and c-Myc in plasma cell tumors in mice. We asked if AID is required for accelerated tumor development in pristane-treated Bcl-xL transgenic BALB/c mice deficient in AID (pBxAicda−/−). pBxAicda−/− mice developed tumors with a lower frequency (24 vs. 62%) and a longer mean latency (108 vs. 36 d) than AID-sufficient mice. The tumors appeared in oil granuloma tissue and did not form ascites. By interphase fluorescence in situ hybridization, six out of nine pBxAicda−/− primary tumors had T(12;15) and one had T(6;15) chromosomal translocations. Two tumors were transplantable and established as stable cell lines. Molecular and cytogenetic analyses showed that one had an unusual unbalanced T(12;15) translocation, with IgH Cμ and Pvt-1 oriented head to tail at the breakpoint, resulting in an elevated expression of c-Myc. In contrast, the second was T(12;15) negative, but had an elevated N-Myc expression caused by a paracentric inversion of chromosome 12. Thus, novel mechanisms juxtapose Ig and Myc-family genes in AID-deficient plasma cell tumors.
Hand, foot, and mouth disease (HFMD) is a common febrile illness caused by enteroviruses in the Picornaviridae family. The major symptoms of HFMD are fever and a vesicular rash on the hand, foot, or oral mucosa. Acute meningitis and encephalitis are observed in rare cases. HFMD epidemics occur annually in Japan, usually in the summer season. Relatively large-scale outbreaks have occurred every two years since 2011. In this study, the epidemic patterns of HFMD in Japan are predicted four weeks in advance using a deep learning method. The time-series data were analyzed by a long short-term memory (LSTM) approach called a Recurrent Neural Network. The LSTM model was trained on the numbers of weekly HFMD cases in each prefecture. These data are reported in the Infectious Diseases Weekly Report, which compiles the national surveillance data from web sites at the National Institute of Infectious Diseases, Japan, under the Infectious Diseases Control Law. Consequently, our trained LSTM model distinguishes between relatively large-scale and small-scale epidemics. The trained model predicted the HFMD epidemics in 2018 and 2019, indicating that the LSTM approach can estimate the future epidemic patterns of HFMD in Japan.
Abstract Background Hepatitis B virus (HBV) infection is a global public health concern. Precise and sensitive detection of viral markers, including HBV DNA and HBs antigen (Ag), is essential to determine HBV infection. Methods The sensitivities and specificities of 5 HBV DNA and 14 HBsAg kits were evaluated using World Health Organization International Standards (WHO IS) and the Regional Reference Panel (RRP) consisting of 64 HBsAg-negative and 80 HBsAg-positive specimens. Results All 5 HBV DNA kits detected HBV DNA in the WHO IS at a concentration of 10 IU/mL. The sensitivity and specificity to the RRP were 98.8–100% and 96.9–100%, respectively. HBV DNA titers were well correlated among the 5 kits regardless of HBV genotype. However, discordance of the HBV DNA titer was found in 5 specimens measured by CAP/CTM HBV v2.0. Among 12 automated HBsAg kits, the minimum detectable concentrations in the WHO IS varied from 0.01 to 0.1 IU/mL. Two lateral flow assays were positive for WHO IS concentrations greater than or equal to 1.0 and 0.1 IU/mL, respectively. When analyzed by the RRP, 12 automated kits exhibited a sensitivity of 98.8–100%, and 2 lateral flow assays showed sensitivities of 93.8% and 100%. The specificities of HBsAg kits were 100%. In the quantification of HBsAg, some kits showed a poor correlation of measurements with each other and showed up to a 1.7-fold difference in the regression coefficient of HBsAg titers. There were variations in the correlations of measurements among HBsAg kits when analyzed by genotype. Conclusions Five HBV DNA kits showed sufficient sensitivity and specificity to determine HBV infection. HBV DNA titers were compatible with each other irrespective of HBV genotypes. HBsAg kits had enough sensitivity and specificity to screen for HBV infection. One of the lateral flow assays had a nearly equivalent sensitivity to that of the automated HBsAg kit. HBsAg titers quantified by the evaluated kits were not compatible across the kits. Genotype-dependent amino acid variations might affect the quantification of HBsAg titers.
Abstract The CreERT2 for conditional gene inactivation has become increasingly used in reverse mouse genetics, which enables temporal regulation of Cre activity using a mutant estrogen binding domain (ERT2) to keep Cre inactive until the administration of tamoxifen. In this study, we present the severe toxicity of ubiquitously expressed CreERT2 in adult mice and embryos. The toxicity of Cre recombinase or CreERT2 in vitro or in vivo organisms are still less sufficiently recognized considering the common use of Cre/loxP system, though the toxicity might compromise the phenotypic analysis of the gene of interest. We analyzed two independent lines in which CreERT2 is knocked-in into the Rosa26 locus (R26CreERT2 mice), and both lines showed thymus atrophy, severe anemia, and illegitimate chromosomal rearrangement in hematopoietic cells after the administration of tamoxifen, and demonstrated complete recovery of hematological toxicity in adult mice. In the hematopoietic tissues in R26CreERT2 mice, reduced proliferation and increased apoptosis was observed after the administration of tamoxifen. Flow cytometric analysis revealed that CreERT2 toxicity affected several hematopoietic lineages, and that immature cells in these lineages tend to be more sensitive to the toxicity. In vitro culturing of hematopoietic cells from these mice further demonstrated the direct toxicity of CreERT2 on growth and differentiation of hematopoietic cells. We further demonstrated the cleavage of the putative cryptic/pseudo loxP site in the genome after the activation of CreERT2 in vivo. We discussed how to avoid the misinterpretation of the experimental results from potential toxic effects due to the activated CreERT2.
