Evidence of the simultaneous replications of active viruses in specimens positive for multiple respiratory viruses.

IMPORTANCE: Since the pandemic of coronavirus diseases 2019, the use of real-time PCR assay has become widespread among people who were not familiar with it in virus detection. As a result, whether a high real-time PCR value in one time test indicates virus transmissibly became a complicated social problem, regardless of the difference in assays and/or amplification conditions, the time and number of diagnostic test during the time course of infection. In addition, the multiple positives in the test of respiratory viruses further add to the confusion in the interpretation of the infection. To address this issue, we performed virus isolation using pediatric SARI (severe acute respiratory infections) specimens on air-liquid interface culture of human bronchial/tracheal epithelial cell culture. The result of this study can be a strong evidence that the specimens showing positivity for multiple agents in real-time PCR tests possibly contain infectious viruses.

Development of a duplex real-time RT-PCR assay for the detection and identification of two subgroups of human metapneumovirus in a single tube.

Human metapneumovirus (hMPV) is a common cause of respiratory infections in children. Many genetic diagnostic assays have been developed, but most detect hMPV regardless of the subgroup. In this study, we developed a real-time RT-PCR assay that can detect and identify the two major subgroups of hMPV (A and B) in one tube. Primers and probes were designed based on the sequences of recent clinical isolates in Japan. The assay showed comparable analytical sensitivity to a previously reported real-time RT-PCR assay and specific reactions to hMPV subgroups. The assay also showed no cross-reactivity to clinical isolates of 19 species of other respiratory viruses. In a validation assay using post-diagnosed clinical specimens, 98% (167/170) positivity was confirmed for the duplex assay, and the three specimens not detected were of low copy number. The duplex assay also successfully distinguished the two major subgroups for all 12 clinical specimens, for which the subgroup had already been determined by genomic sequencing analysis. The duplex assay described here will contribute to the rapid and accurate identification and surveillance of hMPV infections.

Single Amino Acid Substitution in the Receptor Binding Domain of Spike Protein Is Sufficient To Convert the Neutralization Profile between Ethiopian and Middle Eastern Isolates of Middle East Respiratory Coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes MERS, which is endemic in the Middle East. The absence of human cases in Africa despite the presence of MERS-CoV suggests virological differences between MERS-CoVs in Africa and the Middle East. In fact, in the laboratory, recombinant MERS-CoV carrying the spike (S) protein of Ethiopian isolates exhibits attenuated properties, being more easily neutralized and replicating slower than viruses carrying the S protein of Middle Eastern isolate, EMC. In this study, to identify the amino acids that define the different virological features between Ethiopian and Middle Eastern MERS-CoVs, neutralization titers and viral replication were evaluated using recombinant MERS-CoVs carrying amino acid substitution(s) in the S protein. A single amino acid difference introduced into the receptor binding domain was sufficient to reverse the difference in the neutralizing properties of the S protein between Ethiopian and Middle Eastern MERS-CoVs. Furthermore, amino acid mutations in the S1 and S2 regions of S protein were collectively involved in slow viral replication. Since even a single amino acid difference in S protein can reverse the viral properties of MERS-CoV, it should be noted that multiple mutations may induce a significant change. Careful monitoring of genetic alterations in MERS-CoVs in Africa is therefore required to detect the emergence of virulent strains generated by a few genetic differences. IMPORTANCE There have been no reported cases of human Middle East respiratory syndrome (MERS) in Africa, despite the presence of MERS coronavirus (MERS-CoV). Previous studies have shown that recombinant MERS-CoV carrying the S protein of an Ethiopian isolate replicated slower and was more easily neutralized relative to MERS-CoV carrying the S protein of a Middle Eastern isolate. In this study, we investigated the amino acid(s) in S protein associated with the different viral characteristics between Ethiopian and Middle Eastern MERS-CoVs. The results revealed that a single amino acid difference in the receptor binding domain was sufficient to reverse the neutralization profile. This implies that slight genetic changes can alter the predominant population of MERS-CoV, similar to the transition of variants of severe acute respiratory syndrome coronavirus-2. Careful genetic monitoring of isolates is important to detect the spread of possible virulent MERS-CoVs generated by mutation(s).

Essential role of TMPRSS2 in SARS-CoV-2 infection in murine airways.

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.

Hydrophobic Alpha-Helical Short Peptides in Overlapping Reading Frames of the Coronavirus Genome.

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

Nearly Complete Genome Sequences of 12 Types of Human Rhinoviruses Isolated from Pediatric Inpatients in Fukushima, Japan.

We reported nearly complete genomic sequences of 12 serotypes of human rhinoviruses (HRVs) isolated from pediatric inpatients in Fukushima, Japan using an air-liquid interface culture of human bronchial tracheal epithelial cells. We found that various serotypes of HRV circulated locally and simultaneously from 2018 to 2021.

Ten Nearly Complete Genome Sequences of Human Orthorubulavirus 4 Isolated from Pediatric Inpatients in Fukushima, Japan.

We report 10 nearly complete genomic sequences of human orthorubulavirus 4, also called human parainfluenza virus 4 (HPIV4), isolated from pediatric inpatients with respiratory infections in Fukushima, Japan, by using an air-liquid interface culture of human bronchial and tracheal epithelial cells.

Detection of the ORF1 Gene Is an Indicator of the Possible Isolation of Severe Acute Respiratory Syndrome Coronavirus 2.

In the ongoing coronavirus diseases 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), real-time RT-PCR based diagnostic assays have been used for the detection of infection, but the positive signal of real-time RT-PCR does not necessarily indicate the infectivity of the patient. Due to the unique replication system of the coronavirus, primer/probe sets targeted nucleocapsid (N) and spike (S) protein detect the abundantly synthesized subgenomic RNAs as well as the virus genome, possibly making the assay unsuitable for estimation of the infectivity of the specimen, although it has an advantage for the diagnostic tests. In this study, the primer/probe set targeting the open reading frame 1a (ORF1a) gene was developed to specifically detect viral genomic RNA. Then the relation between the ORF1a signal and infectivity of the clinical specimens was validated by virus isolation using VeroE6 cells, which constitutively express transmembrane protease, serine 2, (VeroE6/TMPRSS2). The analytical sensitivity of developed ORF1a set was similar to that of previously developed N and S sets. Nevertheless, in the assay of the clinical specimen, detection rate of the ORF1a gene was lower than that of the N and S genes. These data indicated that clinical specimens contain a significant amount of subgenomic RNAs. However, as expected, the isolation-succeeded specimen always showed an RT-PCR-positive signal for the ORF1a gene, suggesting ORF1a detection in combination with N and S sets could be a more rational indicator for the possible infectivity of the clinical specimens.

Thirteen Nearly Complete Genome Sequences of Human Bocavirus 1 Isolated from Pediatric Inpatients in Fukushima, Japan.

We report 13 genomic sequences of human bocavirus 1 isolated from pediatric inpatients in Fukushima, Japan, using an air-liquid interface culture of human bronchial tracheal epithelial cells. This work suggests the endemic circulation of a human bocavirus variant with a unique amino acid signature in Fukushima.

Anti-HBV drug entecavir ameliorates DSS-induced colitis through PD-L1 induction.

PD-L1-mediated signaling is one of the major processes that regulate local inflammatory responses in the gut. To date, protective effects against colitis through direct Fc-fused PD-L1 administration or indirect PD-L1 induction by probiotics have been reported. We have previously shown that the anti-HBV drug entecavir (ETV) induces PD-L1 expression in human hepatocytes. In the present study, we investigated whether ETV induces PD-L1 expression in intestinal cells and provides a protective effect against DSS-induced colitis. ETV induced PD-L1 expression in epithelial cells, rather than T and B cells, improving the symptoms of colitis. In the mechanistic analysis, Th17 cell differentiation was inhibited and B cell infiltration into the lamina propria was reduced. In addition, PD-L1 expression was positively correlated with Foxp3 or CSF1-R. In conclusion, ETV upregulated PD-L1 expression in epithelial cells and ameliorated inflammation in DSS-induced colitis. These results suggest that ETV may be a potential therapeutic agent as a PD-L1 enhancer for the treatment of human IBD.

Human hepatocyte-derived extracellular vesicles attenuate the carbon tetrachloride-induced acute liver injury in mice.

Acute liver injury (ALI) induced by chemicals or viruses can progress rapidly to acute liver failure (ALF), often resulting in death of patients without liver transplantation. Since liver transplantation is limited due to a paucity of donors, expensive surgical costs, and severe immune rejection, novel therapies are required to treat liver injury. Extracellular vesicles (EVs) are used for cellular communication, carrying RNAs, proteins, and lipids and delivering them intercellularly after being endocytosed by target cells. Recently, it was reported that EVs secreted from human hepatocytes have an ability to modulate the immune responses; however, these roles of EVs secreted from human hepatocytes were studied only with in vitro experiments. In the present study, we evidenced that EVs secreted from human hepatocytes attenuated the CCL4-induced ALI by inhibiting the recruitment of monocytes through downregulation of chemokine receptor in the bone marrow and recruitment of neutrophils through the reduction of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2 expression levels in the liver.

Extracellular vesicles secreted by HBV-infected cells modulate HBV persistence in hydrodynamic HBV transfection mouse model.

Hepatitis B, a viral infection that affects the liver, is thought to affect over 257 million people worldwide, and long-term infection can lead to life-threatening issues such as cirrhosis or liver cancer. Chronic hepatitis B develops by the interaction between hepatitis B virus (HBV) and host immune response. However, questions of how HBV-infected cells thwart immune system defenses remain unanswered. Extracellular vesicles (EVs) are used for cellular communication, carrying cargoes such as RNAs, proteins, and lipids and delivering them intracellularly after being endocytosed by target cells. HBV-infected liver cells secrete several types of EVs into body fluids such as complete and incomplete virions, and exosomes. We previously demonstrated that monocytes that incorporated EVs moved to immunoregulatory phenotypes via up-regulation of PD-L1, an immunocheckpoint molecule, and down-regulation of CD69, a leukocyte activation molecule. In this study, we transfected mice with HBV using hydrodynamic injection and studied the effects of EVs secreted by HBV-infected liver cells. EVs secreted from cells with HBV replication strongly suppressed the immune response, inhibiting the eradication of HBV-replicating cells in the mice transfected with HBV. EVs were systemically incorporated in multiple organs, including liver, bone marrow (BM), and intestine. Intriguingly, the BM cells that incorporated EVs acquired intestinal tropism and the dendritic cell populations in the intestine increased. These findings suggest that the EVs secreted by HBV-infected liver cells exert immunosuppressive functions, and that an association between the liver, bone marrow, and intestinal tract exists through EVs secreted from HBV-infected cells.

PD-L1 is induced on the hepatocyte surface via CKLF-like MARVEL transmembrane domain-containing protein 6 up-regulation by the anti-HBV drug Entecavir.

Chronic hepatitis B is now controllable when treated with nucleoside reverse transcriptase inhibitors (NRTIs), which inhibit hepatitis B virus (HBV) replication. However, once the NRTIs are discontinued, most patients relapse, necessitating lifelong NRTIs treatment. HBV infection relapse is assumed to be caused by the persistent existence of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. The mechanism by which cccDNA-positive hepatocytes escape immune surveillance during NRTIs treatment remains elusive. Entecavir (ETV), a commonly used NRTI, post-transcriptionally up-regulates programmed cell death-ligand 1 (PD-L1), an immune checkpoint molecule, on the cell surface of hepatocytes regardless of HBV infection. Up-regulation by ETV depends on up-regulation of CKLF-like MARVEL transmembrane domain-containing 6, a newly identified potent regulator of PD-L1 expression on the cell surface. ETV-treated hepatic cells suppressed the activity of primary CD3 T cells and programmed cell death protein-1 (PD-1)-over-expressed Jurkat cells. Finally, ETV induces PD-L1 in primary hepatocytes infected by HBV. These results provide evidence that ETV considerably up-regulates PD-L1 on the cell surface of infected hepatocytes, which may be one of the mechanisms by which infected hepatocytes subvert immune surveillance.

PD-L1/L2 protein levels rapidly increase on monocytes via trogocytosis from tumor cells in classical Hodgkin lymphoma.

In classical Hodgkin lymphoma (cHL)-characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells-tumor-associated macrophages (TAMs) play a pivotal role in tumor formation. However, the significance of direct contact between HRS cells and TAMs has not been elucidated. HRS cells and TAMs are known to express PD-L1, which leads to PD-1+ CD4+ T cell exhaustion in cHL. Here, we found that PD-L1/L2 expression was elevated in monocytes co-cultured with HRS cells within 1 h, but not in monocytes cultured with supernatants of HRS cells. Immunofluorescence analysis of PD-L1/L2 revealed that their upregulation resulted in membrane transfer called "trogocytosis" from HRS cells to monocytes. PD-L1/L2 upregulation was not observed in monocytes co-cultured with PD-L1/L2-deficient HRS cells, validating the hypothesis that there is a direct transfer of PD-L1/L2 from HRS cells to monocytes. In the patients, both ligands (PD-L1/L2) were upregulated in TAMs in contact with HRS cells, but not in TAMs distant from HRS cells, suggesting that trogocytosis occurs in cHL patients. Taken together, trogocytosis may be one of the mechanisms that induces rapid upregulation of PD-L1/L2 in monocytes to evade antitumor immunity through the suppression of T cells as mediated by MHC antigen presentation.

[Exosomes in the hematopoietic system].

The secretion of extracellular vesicles (EVs) from cells has been observed. Recently, because EVs were found to contain functional molecules such as micro RNAs (miRNAs) and possess the ability to transfer them to other cells, its functions were expanded as an "intracellular communicator." The exosome is one such EV that has been extensively investigated, particularly in cancer research because cancer cells abundantly secrete exosomes, suggesting their potential as promising diagnostic markers. Research on exosomes in the hematopoietic system has just begun. We recently reported that the exosome secreted from the EBV-infected lymphoma cells has critical functions in lymphomagenesis and maintenance. Moreover, EVs in HBV infection are now being investigated to generalize their functions.

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma.

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

The immunological function of extracellular vesicles in hepatitis B virus-infected hepatocytes.

Hepatitis B virus (HBV) generates large amounts of complete and incomplete viral particles. Except for the virion, which acts as infectious particles, the function of those particles remains elusive. Extracellular vesicles (EVs) have been revealed to have biological functions. The EVs which size are less than 100 nm in diameter, were collected from HBV infected-patients. These vesicles contain, complete and incomplete virions, and exosomes, which have been recently shown to be critical as intercellular communicators. Here, the effects of the exosome, the complete, and the incomplete particles on the target cells were investigated. These particles are endocytosed by monocyte/macrophages and function primarily to upregulate PD-L1. The functions and composition of the EVs were affected by nucleotide reverse transcriptase inhibitors (NRTIs), suggesting that the EVs are involved in the pathogenesis of HBV hepatitis and clinical course of those patients treated by NRTIs.

IL-10 expression in pyramidal neurons after neuropathogenic coronaviral infection.

The apoptosis of pyramidal neurons in CA2 and CA3 subregions of the hippocampus is induced after infection with Mu-3 virus (Mu-3), a neuropathogenic strain of the JHM virus (JHMV), at 4-5 days post-inoculation (dpi). The viral antigens in the hippocampus are mainly found in the CD11b-positive cells distributed in the stratum oriens located outside the pyramidal layer, and only a few pyramidal neurons are infected. Furthermore, the apoptotic cells, indicated as showing caspase 3 (Cas3) activation, consist of a high number of uninfected cells. Therefore, it is considered that the apoptotic lesions occur through the indirect effects of infection, and not as a result of direct infection with Mu-3, similar to the reported neuronal apoptosis in the hippocampus after other types of infection. The apoptosis in the pyramidal neurons is accompanied by various types of proinflammatory cytokines depending on the causative agents. Thus, the local expression of proinflammatory cytokines was studied, revealing no correlation in the distribution of cytokine expression with the subregions showing apoptosis. However, the anti-inflammatory cytokine IL-10 was produced by pyramidal neurons of CA2 and CA3 at 3 dpi when there is no destructive change or viral invasion in the hippocampus.

Extracellular matrix in the CNS induced by neuropathogenic viral infection.

During the early phase of infection with an extremely neurovirulent murine coronavirus, cl-2, the ER-TR7 antigen (ERag)-positive fibers (ERfibs) associated with laminin and collagen III show a rapid increase in expression levels in the meninges, followed by an appearance of the antigens in the ventricle and brain parenchyma. Then, cl-2 invades the ventricle and ventricular wall along the newly assembled ERfibs after infection, using them as a pathway from the meninges, the initial site of infection. In the lymph nodes and spleen, ERag is mainly produced by fibroblastic reticular cells (FRCs), which play a key role in nursing the ERfibs to form a fibroblastic reticular network (FRN). The FRN functions as a conduit system to transfer antigens, cytokines or leukocytes in the lymphoid organs. In the brain parenchyma, astrocytes were found to produce the main components of mature ERfibs, such as collagen, laminin and ERag, which have been identified in the lymphoid organs. The producibility of these extracellular matrices (ECMs) by astrocytes was further confirmed by primary brain cultures, which disclosed the dissociation of laminin and ERag production, and the close association of ERag production with that of collagen, forming a fibrous structure. The pattern of ECM production in vitro indicated the process of forming mature ERfibs in the brain, that is, fibers made of collagen fibers and ERag are wrapped by laminin prepared as a sheet structure. In addition, the brain parenchymal cells that produce interferon ß after infection in spite of their residence away from the sites of viral invasion were surrounded by ERfibs, which were closely associated with astrocytic fibers. These findings indicate that astrocytes play a central role in forming the astrocytic reticular network (ARN) in the brain parenchyma, as FRCs do to form FRN in the lymphoid organs.