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Hantaan virus (HTNV), a Hantavirus serotype that is prevalent in Asia, causes hemorrhagic fever with renal syndrome (HFRS) with high mortality in human race. However, the pathogenesis of HTNV infection remains elusive. Circular RNAs (circRNAs), a new type of non-coding RNAs, play a crucial role in various pathogenic processes. Nevertheless, circRNA expression profiles and their effects on pathogenesis of HTNV infection are still completely unknown. In the present study, RNA sequencing was performed to analyze the circRNA, microRNA (miRNA), and mRNA expression profiles in HTNV-infected and mock-infected human umbilical vein endothelial cells (HUVECs). A total of 70 circRNAs, 66 miRNAs, and 788 mRNAs were differently expressed. Several differentially expressed RNAs were validated by RT-qPCR. Moreover, we verified that some differentially expressed RNAs, such as circ_0000479, miR-149-5p, miR-330-5p, miR-411-3p, RIG-I, CMPK2, PARP10, and GBP1, promoted or inhibited HTNV replication. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis demonstrated that the host genes of differentially expressed circRNAs were principally involved in the innate immune response, the type I interferon (IFN) signaling pathway, and the cytokine-mediated signaling pathway. Additionally, the circRNA-miRNA-mRNA regulatory network was integrally analyzed. The data showed that there were many circRNA-miRNA-mRNA interactions in HTNV infection. By dual-luciferase reporter assay, we confirmed that circ_0000479 indirectly regulated RIG-I expression by sponging miR-149-5p, hampering viral replication. This study for the first time presents a comprehensive overview of circRNAs induced by HTNV and reveals that a network of enriched circRNAs and circRNA-associated competitive endogenous RNAs (ceRNAs) is involved in the regulation of HTNV infection, thus offering new insight into the mechanisms underlying HTNV-host interaction.

Hemorrhagic fever with renal syndrome (HFRS) is a zoonotic disease with high mortality. Almost 90% of global cases of HFRS are induced by Hantaan virus (HTNV) infection. Although lymphocyte dysfunction is a critical factor in HFRS progression, the specific immune dynamics of HTNV remain unexplored, and current analyses predominantly depend on single-time point sampling. Therefore, comprehensive longitudinal studies are needed to characterize circulating lymphocyte dynamics during HTNV-induced HFRS progression. In this study, we conducted a flow cytometric analysis of circulating lymphocytes in 39 patients with HTNV-induced HFRS across different clinical phases. The analysis encompassed conventional T cells, unconventional T cells, B cells, NK cells and their respective repertoires. Here, we revealed phase-specific immune patterns: CD8 T, CD8 Tems, and activated CD8 T, MAIT and NKT cells peaked during febrile/oliguric phases before declining in polyuria/recovery, while CD4 T and MAIT cells showed inverse fluctuation patterns. Higher frequencies of CD8 Tem, B, and CD56 NK cells during the febrile phase correlated with severe disease, enabling early risk stratification. Lower CD4 Tcm levels in the oliguric phase marked progression to severe HFRS, indicating potential therapeutic strategies aimed at enhancing CD4 Tcm generation or inhibiting effector differentiation. Additionally, CD38 and CD161 expression predicted specific lymphocyte subset dynamics, offering novel biomarkers for immunomodulatory strategies. Our study thus provides the first comprehensive atlas of lymphocyte evolution in HTNV-induced HFRS, connecting immune dysregulation with clinical outcomes.

Endoplasmic reticulum stress (ER stress) can be induced by virus infection. In this part, we explored whether Hantaan virus (HTNV) infection could induce ER stress in differentiated THP-1 (dTHP-1) cells. It showed that the mRNA and protein levels of ER stress-related 78 kDa glucose-regulated protein (GRP78, HSPA5) and mRNA levels of X box-binding protein 1 (XBP-1), activating transcription factor 6(ATF6) and PKR-like ER kinase (PERK) after HTNV infection, were significantly higher than that in uninfected control group. However, the mRNA levels of C/EBP homologous protein (CHOP), glucose-regulated protein 94 (GRP94, HSPC4), and inositol-requiring enzyme1 (IRE1) were not significantly different between the infected group and the untreated group in 2 h after virus infection. It is unusual in activating GRP78 but not GRP94. Meanwhile, dTHP-1 cells infected with HTNV at 12 h did not show obvious apoptosis. These results indicated that the HTNV infection could induce the unfolded protein response (UPR) in dTHP-1 cells, without directly leading to cell apoptosis during 12 h after virus infection.

•HV004 strain showed higher proliferation efficiency and pathogenicity.•The inactivated HTNV vaccine was administered safely in immunized BALB/C mice.•The vaccine induced sufficient neutralizing antibodies and cellular immune response. Hantaan virus (HTNV, Orthohantavirus hantanensae species, Hantaviridae family) is the main etiological agent responsible for hemorrhagic fever with renal syndrome (HFRS). The novel HTNV may pose a potential danger to the control and prevention of HFRS in China, which highlights the importance of vaccine development in public health management. In previous studies, our laboratory discovered and successfully isolated a new HTNV strain, HV004 strain, from Apodemus agrarius captured in an epidemic area in Hubei, China. An initial biological and pathogenicity characterization of HTNV 76–118 (standard train), HV114 strain (a clinical isolate from Hubei province in 1986), and the novel isolate HV004 strain from the epidemic areas of Hubei province were performed in susceptible cells and in vivo. An experimental HV004 strain inactivated vaccine was prepared, and its corresponding immunogenicity was analyzed in BALB/c mice. HV004 strain had a similar but higher pathogenicity than HTNV 76–118 and HV114 in suckling mice. A subcutaneous vaccination (s.c.) with the inactivated HTNV vaccine adjuvanted with aluminum, followed by a challenge intraperitoneally with 106 FFU/ml HTNV, afforded full protection against an HTNV challenge. All immunized mice in every group elicited serum neutralizing antibodies with increasing dosages, which may protect mice from HTNV infection. A dose-dependent stimulation index of splenocytes was also observed in immunized mice. The percentage of IFN-γ-producing CD3+CD8+ T cells was significantly higher in the spleens of immunized mice than in those of control mice. These findings suggest that the inactivated HTNV vaccine may stimulate mice to produce high levels of antibodies with neutralization activity and elicit specific anti-HTNV humoral and cellular immune responses in BALB/c mice against the prevalent strain of HTNV in south central China.

Hantaan virus (HTNV) infection causes an epidemic of hemorrhagic fever with renal syndrome (HFRS) mainly in Asia. It is well known that T cells mediated anti-viral immune response. Although previous studies showed that double positive T (DP T) cells, a little portion of T lymphocytes, were involved in adaptive immune response during virus infection, their kinetic changes and roles in HTNV infection have not yet been explored. In this study, we characterized DP T cells from HFRS patients based on flow cytometry data combined with scRNA-seq data. We showed that HTNV infection caused the upregulation of DP T cells in the peripheral blood, which were correlated with disease stage. The scRNA-seq data clustered DP T cells, unraveled their gene expression profile, and estimated the ordering of these cells. The production of granzyme B and CD107a from DP T cells and the abundant TCR distribution indicated the anti-viral property of DP T cells. In conclusion, this study identified, for the first time, an accumulation of DP T cells in the peripheral blood of HFRS patients and suggested these DP T cells belonging to CD8+T cells lineage. The DP T cells shared the similar characteristics with cytotoxic T cells (CTL) and exerted an anti-viral role in HFRS.

Zoonotic viral diseases have continued to threaten global public health in recent decades, with rodent-borne viruses being significant contributors. Infection by rodent-carried hantaviruses (HV) can result in hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) in humans, with varying degrees of morbidity and mortality. However, no Food and Drug Administration (FDA) vaccines or therapeutics have been approved for the treatment of these diseases. In an effort to identify antiviral bioactive molecules, we isolated four oligomeric phloroglucinols from Callistemon rigidus leaves, including two new phloroglucinol trimers, callistemontrimer A and B, along with two previously characterized phloroglucinol dimers, rhodomyrtosone B and rhodomyrtone. We evaluated the anti-Hantaan virus (HTNV) activity of these compounds. Notably, callistemontrimer A demonstrated higher anti-HTNV activity compared to ribavirin. Mechanistic studies revealed that callistemontrimer A exerted its antiviral effects by inhibiting viral replication, likely through interaction with RNA-dependent RNA polymerase (RdRp) of HTNV, as supported by molecular docking analysis. These results highlight oligomeric phloroglucinols as promising lead candidates for the development of anti-HV therapeutics.

Hantaan orthohantavirus (HTNV) is responsible for severe hemorrhagic fever with renal syndrome (HFRS), which has a case fatality rate of 1% to 10%. Currently, the inactive vaccine licensed in endemic areas elicit low levels of neutralizing antibodies (NAbs). Early NAbs administration is helpful for patients recovery from HFRS. Therefore, measuring NAbs is crucial for evaluating the immune response following infection or vaccination. The golden standard for HTNV NAbs measurement is the focus reduction neutralization test (FRNT), which typically requires skilled technicians and is performed under high biosafety containment facility. Here, we established a surrogate NAbs titration method with replication-competent vesicular stomatitis virus (VSV) bearing HTNV glycoprotein (rVSV-HTNV-GP) based plaque reduction neutralization test (PRNT). Then compared and correlated this method with the authentic HTNV based FRNT, and applied it to measure the NAbs level in 47 serum samples from HFRS patients, healthy donors and inactive vaccine recipients. We observed positive correlations between two neutralization assays among HFRS patients and inactive vaccine recipients (R 2  = 0.5994 and 0.3440, respectively) and confirmed the clear specificity with healthy donors without vaccinated and reproducibility with three more assays. Our results suggest that rVSV-HTNV-GP based PRNT is a reliable lower-biosafety level surrogate for HTNV NAbs evaluation, which is easy to perform with higher sensitivity. Graphical abstract

Hantaan virus (HTNV) infects humans and causes hemorrhagic fever with renal syndrome (HFRS). The development of well-characterized animal models of HFRS could accelerate the testing of vaccine candidates and therapeutic agents and provide a useful tool for studying the pathogenesis of HFRS. Because NLRC3 has multiple immunoregulatory roles, we investigated the susceptibility of Nlrc3 −/− mice to HTNV infection in order to establish a new model of HFRS. Nlrc3 −/− mice developed weight loss, renal hemorrhage, and tubule dilation after HTNV infection, recapitulating many clinical symptoms of human HFRS. Moreover, infected Nlrc3 −/− mice showed higher viral loads in serum, spleen, and kidney than wild type C57BL/6 (WT) mice, and some of them manifested more hematological disorders and significant pathological changes within multiple organs than WT mice. Our results identify that HTNV infected Nlrc3 −/− mice can develop clinical symptoms and pathological changes resembling patients with HFRS, suggesting a new model for studying the pathogenesis and testing of candidate vaccines and therapeutics.

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

China is one of the main epidemic areas for hemorrhagic fever with renal syndrome (HFRS). Currently, there is no human antibody specific to Hantaan virus (HTNV) for the emergency prevention and treatment of HFRS. To prepare human antibodies with neutralizing activity, we established an anti-HTNV phage antibody library using phage display technology by transforming peripheral blood mononuclear cells (PBMCs) of patients with HFRS into B lymphoblastoid cell lines (BLCLs) and extracting cDNA from BLCLs that secreted neutralizing antibodies. Based on the phage antibody library, we screened HTNV-specific Fab antibodies with neutralizing activities. Our study provides a potential way forward for the emergency prevention of HTNV and specific treatment of HFRS.