Influenza epidemics occur worldwide and impose a substantial socioeconomic burden. In our previous studies, epidemic influenza virus strains isolated from seasonal influenza patient specimens exhibited a wide range of growth capabilities and demonstrated growth-dependent cell death in vitro. Based on these findings, we hypothesized that diversity in growth capabilities contributes to the severity of influenza infection. In this study, we investigated the relationship between the growth capabilities of epidemic influenza virus strains and the severity of inflammation in the bronchoalveolar cavity of inoculated mice. Our results demonstrated that high growth capability influenza A virus (IAV) strains replicated significantly faster in mice compared to low growth capability strains. High growth capability strains also induced earlier and stronger production of pro-inflammatory cytokines and promoted more rapid infiltration of inflammatory cells into the lungs. Moreover, mice infected with high growth capability strains exhibited greater weight loss and a higher mortality than those infected with low growth capability strains. These findings suggest that high growth capability IAV strains circulating in the community may induce rapid and pronounced inflammatory responses in infected individuals, leading to severe illness. Our findings may facilitate the identification of high-risk strains and support the development of improved therapeutic strategies for severe influenza cases, thereby providing new insights into the mechanisms underlying influenza severity.

Persistent SARS-CoV-2 infections involve prolonged viral replication and immune system interactions, potentially driving viral evolution and immune escape. This study examines viral characteristics and host gene expression changes in persistent infections. The nasopharyngeal samples from four patients with persistent SARS-CoV-2 infections at Tottori University Hospital, Japan, were analyzed. Viral isolates were cultured, and infectivity was assessed using TCID50 assays. To investigate host responses, RNA sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs), and Gene Ontology (GO) enrichment analysis mapped affected biological pathways. Viral genome sequencing detected mutations associated with prolonged infection. The results showed significant infectivity differences between early- and late-phase infection. Gene expression analysis revealed a strong early phase of pro-inflammatory response (IL6, TNF, IL1B, CXCL10) followed by immune suppression. GO enrichment analysis highlighted inflammation and cytokine-mediated immune pathways. Genomic sequencing identified mutations in ORF1ab and the spike (S) protein, potentially aiding immune escape. The findings underscore that SARS-CoV-2 adapts during persistent infections, altering infectivity and immune responses. These highlight the need for continued monitoring of prolonged infections to mitigate immune escape and viral evolution.

It has been postulated from a combination of evidence that a sudden increase in COVID-19 cases among pediatric patients after onset of the Omicron wave was attributed to a reduced requirement for TMPRSS2-mediated entry in pediatric airways with lower expression levels of TMPRSS2. Epidemic strains were isolated from the indigenous population in an area, and the levels of TMPRSS2 required for Delta and Omicron variants were assessed. As a result, Delta variants proliferated fully in cultures of TMPRSS2-positive Vero cells but not in TMPRSS2-negative Vero cell culture (350-fold, Delta vs 9.6-fold, Omicron). There was no obvious age-dependent selection of Omicron strains affected by the TMPRSS2 (9.6-fold, Adults vs. 12-fold, Children). A phylogenetic tree was generated and Blast searches (up to 100 references) for the spread of strains in the study area showed that each strain had almost identical homology (>99.5%) with foreign isolates, although indigenous strains had obvious differences from each other. This suggested that the differences had been present abroad for a long period. Therefore, the lower requirement for TMPRSS2 by Omicron strains might be applicable to epidemic strains globally. In conclusion, the property of TMPRSS2-independent cleavage makes Omicron proliferate with ease and allows epidemics among children with fewer TMPRSS2 on epithelial surfaces of the respiratory organs.

Extrahepatic viral infections are often accompanied by acute hepatitis, as evidenced by elevated serum liver enzymes and intrasinusoidal infiltration of CD8+ T cells, without direct infection of the liver. An example is infectious mononucleosis caused by primary infection with EBV. Previously, we demonstrated that airway infection of mice with murine γ-herpesvirus 68 (MHV68), a murine model of EBV, caused liver inflammation with elevated serum liver enzymes and intrahepatic infiltration of IFN-γ-producing CD8+ T cells and NK cells. Mechanistically, the expression of the CXCR3-ligand chemokines, which are commonly induced by IFN-γ and attract IFN-γ-producing Th1-type cells via CXCR3, was upregulated in the liver. Importantly, the liver inflammation was suppressed by oral neomycin, an intestine-impermeable aminoglycoside, suggesting an involvement of some products from the intestinal microbiota. In this study, we showed that the liver inflammation and the expression of the CXCR3-ligand chemokines in the liver were effectively ameliorated by i.p. administration of anti-TLR4 mAb or C34, a TLR4 blocker, as well as in TLR4-deficient mice. Conversely, intrarectal inoculation of Escherichia coli as an extraintestinal source of LPS aggravated liver inflammation in MHV68-infected mice with increased expression of the CXCR3-ligand chemokines in the liver. In contrast, the lung inflammation in MHV68-infected mice was not affected by oral neomycin, i.p. administration of C34, or TLR4 deficiency. Collectively, the LPS-TLR4 pathway plays a pivotal role in the liver inflammation of MHV68-infected mice at least in part by upregulating the CXCR3-ligand chemokines in the liver.

It has been considered that reduced susceptibility to antiretroviral drugs is influenced by drug adherence, drug tolerance and drug-resistance-related mutations in the HIV genome. In the present study, we assessed the intrinsic high viral growth capability as a potential viral factor that may influence their susceptibility to antiretroviral drugs using an in vitro model. Phytohemagglutinin-activated peripheral blood mononuclear cells (1.5 × 106 cells) were infected with HIV isolates (106 copies/mL). The culture was carried out at different concentrations (0.001-20 μM) of 13 synthetic antiretroviral compounds (six nucleoside/nucleotide reverse transcriptase inhibitors, one non-nucleoside reverse transcriptase inhibitor, four integrase inhibitors, and two protease inhibitors), and HIV production was assessed using HIV-RNA copies in culture. The 90% inhibitory concentration (IC90) and pharmacokinetics of an antiretroviral agent were used as parameters to determine the reduced antiretroviral drug susceptibility of HIV isolates with high growth capability to synthetic antiretroviral compounds. The high growth capability of HIV isolates without any known drug resistance-related mutation affected their susceptibility to tenofovir (IC90 = 2.05 ± 0.40 μM), lamivudine (IC90 = 6.83 ± 3.96 μM), emtricitabine (IC90 = 0.68 ± 0.37 μM), and efavirenz (IC90 = 3.65 ± 0.77 μM). These antiretroviral drugs showed IC90 values close to or above the maximum plasma concentration against HIV isolates with high growth capability without any known drug resistance-related mutation. Our results may contribute to the development of effective strategies to tailor and individualize antiretroviral therapy in patients harboring HIV isolates with high growth capability.

In a previous study, we described the diverse growth capabilities of circulating seasonal influenza A viruses (IAVs) with low to high viral copy numbers in vitro. In this study, we analyzed the cause of differences in growth capability by evaluating pro-inflammatory cytokines (TNF-α, IL-6, IFN-β) and antiviral interferon-stimulated genes (ISG-15, IFIM1, and TRIM22). A549 cells (3.0 × 105 cells) were inoculated with circulating seasonal IAV strains and incubated for 6 and 24 h. In cells inoculated for 6 h, IAV production was assessed using IAV-RNA copies in the culture supernatant and cell pellets to evaluate gene expression. At 24 h post-infection, cells were collected for IFN-β and ISG-15 protein expression. A549 cells inoculated with seasonal IAV strains with a high growth capability expressed lower levels of IFN-β and ISGs than strains with low growth capabilities. Moreover, suppression of the JAK/STAT pathway enhanced the viral copies of seasonal IAV strains with a low growth capability. Our results suggest that the expression of ISG-15, IFIM1, and TRIM22 in seasonal IAV-inoculated A549 cells could influence the regulation of viral replication, indicating the existence of strains with high and low growth capability. Our results may contribute to the development of new and effective therapeutic strategies to reduce the risk of severe influenza infections.

Close observation of the local transmission of influenza A(H1N1) viruses enabled an estimation of the transmission period of the virus without a mutation. Of 4,448 isolates from 11 consecutive years, 237 isolates could be categorized into 57 strain groups with identical hemagglutinin genes. Transmission of these 57 strains was chased for the maximum duration of an epidemic season. In addition, 35 identical strains were recognized at the study site and other countries within 147 days. Consequently, it can be postulated that once an influenza virus enters a temperate country, the strain rarely mutates until the end of the season.

The correlation of viral growth capability (n = 156) with the viral load in nasopharyngeal swabs (n = 76) was assessed. Epidemic influenza A/H1N1, A/H3N2, and B viruses showed a wide range of growth capability (104-1011 copies/mL) in Madin-Darby canine kidney cells. The growth was correlated with the nasopharyngeal viral load (r = 0.53). Six selected strains showed growth-dependent cell death (r = 0.96) in a growth kinetics assay. Epidemic influenza viruses exhibit a wide range of growth capability. Growth capability should be considered one of the key factors in disease prognosis.

Although drug-resistant HIV variants are considered to be less fit than drug-susceptible viruses, replication competence of these variants harbored by patients has not yet been elucidated in detail. We herein assessed the replication competence of strains obtained from individuals receiving antiretroviral therapy. Among 11 306 participants in a drug resistance surveillance in the Philippines, 2629 plasma samples were obtained from individuals after a 12-month treatment with zidovudine (ZDV)/lamivudine (3TC)/nevirapine (NVP). The replication competence of HIV isolates was then assessed by reinoculation into seronegative peripheral blood mononuclear cells in the absence of drugs in vitro. The drug resistance rate was estimated to be 9.2%. Drug-resistant strains were still a minority of closely related strains in a phylogenetic cluster. Among the available 295 samples, 37 HIV strains were successfully isolated. Progeny viruses were produced at a wide range (5.1 × 10⁶ to 3.4 × 10⁹ copies/mL) in primary culture of peripheral blood mononuclear cells. The viral yields were higher than the corresponding plasma viral load (1300 to 3.4 × 10⁶ copies/mL) but correlated with those ( r = 0.4). These results suggest that strains with higher intrinsic replication competence are one of the primary targets of newly selected drugs at the increasing phase of the plasma viral load during antiretroviral therapy.

Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor β1 (TGF-β1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-β1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases.

Post-transplant lymphoproliferative disorder (PTLD) is one of the most important complications of solid organ transplantation or hematopoietic stem cell transplantation. Most PTLDs are associated with Epstein-Barr virus (EBV) infection. Although post-transplant Hodgkin lymphoma (HL) is included in PTLD, there have been no studies in the literature on adult cases of post-transplant HL after cord blood stem cell transplantation (CBSCT). This is due to the fact that EBV infection of cord blood cells usually does not occur, and EBV-infected lymphocytes of the recipient should be eradicated by preconditioning therapy. We report a 26-year-old woman case of post-transplant HL, which occurred after CBSCT for relapsed acute lymphoblastic leukemia. Three years and eight months after CBSCT, the enlarged cervical lymph node was histologically diagnosed as EBV associated post-transplant HL, which showed immunophenotypes of classical HL and latency type II EBV infection. She underwent chemotherapy, and has survived 4 years and 6 months after CBSCT. Differential diagnosis of post-transplant HL with good prognosis and HL-like PTLD with aggressive behavior is important, and immunohistochemical methods were useful and essential for it. The source of EBV associated HL in this case will be discussed.

EB virus causes infectious mononucleosis (IM) with hepatitis in primary infected adults, but the mechanism of pathogenesis is not well understood. We have previously shown that intestinal bacterial products may be associated with the hepatitis using MHV68-infected mice. In the present study, we evaluated the impact of lypopolysaccaride (LPS) and peptideglycan (PDG) as candidates of intestinal bacterial products. As the result, Neutralizing antibodies to TLR4, LPS receptor, suppressed hepatitis. However, neutralizing antibodies to TLR2, PDG receptor, did not inhibit. Furthermore, the TLR4 inhibitor C34 suppressed hepatitis. These results suggest that the LPS-TLR4 pathway may be associated with the hepatitis in IM.

The drug resistance rate was estimated to be 9.2% among the 2,629 referred samples from patients receiving antiretroviral treatment in 2015-2016 in the Philippines. Thirty-seven HIV strains were isolated from 142 HIV-positive serum/plasma samples of patients harboring drug-resistant strains with detectable viral load after a 12-month treatment of drug-naive patients. The replication competence was then assessed by re-inoculation into seronegative peripheral blood mononuclear cells in vitro. The viral yield correlated with the plasma viral load. However, more progeny viruses were produced after the in vitro re-inoculation than the corresponding plasma viral load. Intrinsic replication competence could be assessed by the in vitro re-inoculation system, but not be estimated from in vivo plasma viral load. These results suggest that the replication competence of HIV become an important marker especially when a drug regimen should be altered.

CCL28 is expressed in the mucosal tissues and to attract IgA-antibody secreting cells (IgA-ASCs) via CCR10. CCL28 has an antimicrobial activity against microbes in vitro. However, in vivo evidence for the role of CCL28 in the mucosal immunity remains scanty. We generated CCL28-deficient mice and demonstrated that CCL28-deficient mice showed reduced numbers and altered distribution of IgA-ASCs in the colon. The IgA contents in the fecal extracts were low. The average amounts of IgA secreted by a single IgA-ASC isolated from the lamina propria of the colon was reduced. Furthermore, the 16S rRNA sequencing analysis of feces revealed an increase of the Class Bacilli. Consistent with the low IgA production and altered microbiota in the colon, CCL28-deficient mice had aggravated colitis upon treatment with dextran sulfate, which was ameliorated by oral antibiotics. Therefore, CCL28 has an role in the mucosal immunity of the colon as a chemoattractant with a direct antimicrobial activity.

Cell-mediated immunity (CMI) is thought to be crucial for protection against Varicella-Zoster virus (VZV) infection. We screened for major CMI-inducible VZV protein by Enzyme-Linked Immuno-Spot (ELISPOT) assay. Because already-known CMI antigens of other herpesviruses are localized in cell nuclei, nuclear distributed VZV proteins were examined as candidates for major CMI antigens. VZV protein(s) inducing CMI were searched among peripheral blood mononuclear cell specimens derived from healthy volunteers. As the result, ORF8, 61 and 66 induced CMI in more than 80% of specimens. About ORF61, epitope sequences were identified using oligo peptides. Our findings contribute to establish a new standardized CMI-quantification system.

EBウイルス関連疾患の病態・病因解明および治療法開発のため、EBウイルス感染モデル動物による実験系の構築を目的とした研究が、様々な動物種を用いてなされている。ウサギ(Japanese White種)は有望な動物種候補のひとつであり、EBウイルスが静脈注射により感染することがこれまでに知られている。EBウイルス感染ウサギは一過性の脾腫、末梢血中でのEBV-DNAの検出がなされ、これらの症状は次第に緩和する。本研究は、EBウイルス感染ウサギにおけるEBウイルスの感染状態のより詳細な解析を目的とした以下の検討を行った. EBウイルス感染ウサギの末梢血におけるEBV遺伝子発現の経時的解析を行ったところ、EBウイルス遺伝子の発現が長期間継続して確認された。また、死後の剖検で脾臓からウイルス遺伝子の発現が確認されただけでなく、血球貪食像や異型リンパ球が認められた。このことから、ウサギにおけるEBウイルス感染症状は一時的なものだけではなく、終生継続していることが示され、またEBウイルスが長期的な影響を宿主に及ぼしている可能性が示唆された。 試験管内における末梢血リンパ球、脾臓リンパ球のEBV感染の解析を行ったところ、EBウイルス遺伝子発現が確認され、試験管内でも感染が成立することが示された。また、これまで検討に用いられたB95-8株EBVとは異なるEBV株であるP3HR-1株EBVを用いてウサギへの感染の検討を行い、P3HR-1株EBVでもウサギへの感染が成立することが明らかにした。これらの事実は、ウサギを用いてのEBウイルスの病態・病因解析が、試験管内で行える可能性を示す。 本研究により得られた事実は、EBウイルス感染モデル動物としてのウサギの有用性が改めて支持した。今後、これらEBウイルス感染ウサギが、EBウイルス関連疾患の病態・病因の解析に役立つことが期待される。