Staffs

Professor: Seiji Kageyama, MD, PhD. E-mail; skageyama (add an additional part)
Associate Professor: Kyosuke Kanai, PhD. E-mail; kkanai
Assistant Professor: Akeno Tsuneki-Tokunaga. E-mail: atsuneki

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VISION AND MISSION

Vision:
•To contribute to prevention and control of viral infectious diseases

GOALS AND OUTCOMES FOR EDUCATION/TRAINING, RESEARCH, AND SOCIAL DEVELOPMENT:

1. Studies on human immunodeficiency viruses and AIDS

1.1  Hinay AA, Kanai K, Tsuneki-Tokunaga A, Komatsu M, Telan EO, Kageyama S. In vitro susceptibility of HIV isolates with high growth capability to antiretroviral drugs. Int. J. Mol. Sci., 23: 15380, 2022.

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.

1.2. Kageyama S, Hinay AA, Telan EFO, Samonte GMJ, Leano PSA, Tsuneki-Tokunaga A, and Kanai Intrinsic replication competences of HIV strains after Zidovudine/ Lamivudine/Nevirapine treatment in the Philippines. J Int Assoc Provid AIDS Care 18: 1-8, 2019.

While 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 anti-retroviral therapy. Among 11,306 participants in a drug resistance surveillance in the Philippines, 2,629 plasma samples were obtained from individuals after a 12-month treatment with Zidovudine/Lamivudine/Nevirapine. The replication competence of HIV isolates was then assessed by re-inoculation 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 - 3.4×10⁹ copies/mL) in primary culture of peripheral blood mononuclear cells. The viral yields were higher than the corresponding plasma viral load (1,300 - 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.

2. Studies on Epstein-Barr viruses and associated diseases

2.1. Kanai K, Park AM, Watanabe A, Arikawa T, Yasui T, Yoshida H, Tsunoda I, Yoshie O. Murine γ-Herpesvirus 68 Induces Severe Lung Inflammation in IL-27-Deficient Mice with Liver Dysfunction Preventable by Oral Neomycin. J Immunol. 200: 2703-2713, 2018.

IL-27 is an immunoregulatory cytokine consisting of p28 and EBI3. Its receptor also has two subunits, WSX1 and gp130. Although IL-27 promotes Th1 differentiation in naive T cells, it also induces IL-10 expression in effector Th1 cells to curtail excessive immune responses. By using p28-deficient mice and WSX1-deficient mice (collectively called IL-27-deficient mice), we examined the role of IL-27 in primary infection by murine γ-herpesvirus 68 (MHV68), a murine model of EBV. Upon airway infection with MHV68, IL-27-deficient mice had more aggravated lung inflammation than wild-type mice, although MHV68 infection per se was better controlled in IL-27-deficient mice. Although epithelial cells and alveolar macrophages were primarily infected by MHV68, interstitial macrophages and dendritic cells were the major producers of IL-27. The lung inflammation of IL-27-deficient mice was characterized by more IFN-γ-producing CD8+ T cells and fewer IL-10-producing CD8+ T cells than that of wild-type mice. An infectious mononucleosis-like disease was also aggravated in IL-27-deficient mice, with prominent splenomegaly and severe hepatitis. Infiltration of IFN-γ-producing effector cells and upregulation of the CXCR3 ligand chemokines CXCL9, CXCL10, and CXCL11 were noted in the liver of MHV68-infected mice. Oral neomycin effectively ameliorated hepatitis, with decreased production of these chemokines in the liver, suggesting that the intestinal microbiota plays a role in liver inflammation through upregulation of these chemokines. Collectively, IL-27 is essential for the generation of IL-10-producing effector cells in primary infection by MHV68. Our findings may also provide new insight into the mechanism of hepatitis associated with infectious mononucleosis.

2.2. Kanai K, Park AM, Yoshida H, Tsunoda I, Yoshie O. IL-35 Suppresses Lipopolysaccharide-Induced Airway Eosinophilia in EBI3-Deficient Mice. J Immunol. 198: 119-127, 2017.

EBI3 functions as the subunit of immune-regulatory cytokines, such as IL-27 and IL-35, by pairing with p28 and p35, respectively. We treated wild-type and EBI3-deficient mice with intratracheal administration of LPS and obtained bronchoalveolar lavage fluid (BALF) 24 h later. Although neutrophils were the predominant cells in BALF from both groups of mice, eosinophils were highly enriched and there was increased production of eosinophil-attracting chemokines CCL11 and CCL24 in BALF of EBI3-deficient mice. The bronchial epithelial cells and alveolar macrophages were the major producers of CCL11 and CCL24. Because no such increases in eosinophils were seen in BALF of p28/IL-27-deficient mice or WSX-1/IL-27Rα subunit-deficient mice upon intratracheal stimulation with LPS, we considered that the lack of IL-35 was responsible for the enhanced airway eosinophilia in EBI3-deficient mice. In vitro, IL-35 potently suppressed production of CCL11 and CCL24 by human lung epithelial cell lines treated with TNF-α and IL-1β. IL-35 also suppressed phosphorylation of STAT1 and STAT3 and induced suppressor of cytokine signaling 3. In vivo, rIL-35 dramatically reduced LPS-induced airway eosinophilia in EBI3-deficient mice, with concomitant reduction of CCL11 and CCL24, whereas neutralization of IL-35 significantly increased airway eosinophils in LPS-treated wild-type mice. Collectively, our results suggest that IL-35 negatively regulates airway eosinophilia, at least in part by reducing the production of CCL11 and CCL24.

3. Studies on influenza viruses and influenza

3.1. Hinay A A, Kakee S, Kageyama S, Tsuneki-Tokunaga A, Perdana WY, Akena Y, Kanai K. Pro-inflammatory cytokines and interferon-stimulated gene responses induced by seasonal influenza A virus with varying growth capabilities in human lung epithelial cell lines. Vaccines, 10:1507, 2022.

Background: In a previous study, we described the diverse growth capabilities of circulating seasonal influenza A virus (IAV) with low to high viral copies in vitro. In this study, we analyzed the cause of differences in growth capability by evaluating Interferon-β and antiviral interferon-stimulated genes (ISGs) expression.

Methods: A549 cells (3.0x10⁵ cells) were inoculated with circulating seasonal IAV strains for 6 and 24 h. In cells inoculated for 6 h, IAV production was assessed by IAV-RNA copies in culture supernatant and cell pellets for gene expression evaluation. At 24 hours post-infection, cells were collected for IFN-β and ISG-15 protein expression. JAK/STAT pathway inhibitor, pyridone 6 (P6) was used to evaluate the influence of ISGs on the growth capability of seasonal IAV.

Results: A549 cells inoculated with seasonal IAV strains with high growth capability expressed less IFN-β and ISGs, whereas low growth capability strains expressed more IFN-β and ISGs. The use of a P6, a JAK/STAT inhibitor, suppressed the expression of ISGs and enhanced the viral copies of seasonal IAV strains with low growth capability. This result suggests that growth capability is regulated by ISG-mediated IFN-β production.

Conclusion: In this study, we provide a new insight into the mechanism of circulating seasonal IAV replication. Our study showed that the difference in the growth capability of circulating seasonal IAV strains is due to the regulation of antiviral ISGs. Inhibition of the JAK/STAT pathway permits seasonal IAV strains with low growth capability to enhance their viral copies by suppressing antiviral ISGs. Our results may contribute in developing new effective therapeutic strategies and reduce the risk of developing severe influenza disease.

 3.2. Tsuneki-Tokunaga A, Takanori K, Kanai K, Itagaki^, A, Tsuchie H, Okada T, Kasagi M, Tanaka K, Aoki M, Hinay AA and Kageyama S. Local spread of influenza A (H1N1) viruses without a mutation for the maximum duration of an epidemic season in Japan. Arch Virol. 2022; 167:195–199.

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.

3.3. Tsuneki-Tokunaga A, Kanai K, Itagaki^, A, Tsuchie H, Okada T, Kasagi M, Tanaka K, Aoki M, Hinay AA and Kageyama S. Growth capability of epidemic influenza viruses in Japan since the 2009 H1N1 pandemic. Arch Virol. 2021; 166:1193-1196.

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.

4. Ethical consideration

     Studies are still ongoing based on the reports described above. These studies are conducted as collaborative research projects with the approval and under the control of the institutional review boards of Faculty of Medicine, Tottori University, Japan and other institutes associated with the research(s). Those are carried out in an unlinked and anonymous manner. Therefore, if research participants have interests in and/or concerns about the results, please contact us by e-mail.

(Updated on April 22, 2023)