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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.

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.

Vaccines has long been the focus of antiviral immunotherapy research. Viral epitopes are thought to be useful biomarkers for immunotherapy (both antibody-based and cellular). In this study, we designed a novel vaccine molecule, the Hantaan virus (HTNV) glycoprotein (GP) tandem Th epitope molecule (named the Gnc molecule), in silico. Subsequently, computer analysis was used to conduct a comprehensive and in-depth study of the various properties of the molecule and its effects as a vaccine molecule in the body. The Gnc molecule was designed for DNA vaccines and optimized with a lysosomal-targeting membrane protein (LAMP) strategy. The effects of GP-derived Th epitopes and multiepitope vaccines were initially verified in animals. Our research has resulted in the design of two vaccines based on effective antiviral immune targets. The effectiveness of molecular therapies has also been preliminarily demonstrated in silico and in laboratory animals, which lays a foundation for the application of a vaccines strategy in the field of antivirals.

MHC-I antigen processes and presentation trigger host-specific anti-viral cellular responses during infection, in which epitope-recognizing cytotoxic T lymphocytes eliminate infected cells and contribute to viral clearance through a cytolytic killing effect. In this study, Hantaan virus (HTNV) GP-derived 9-mer dominant epitopes were obtained with high affinity to major HLA-I and H-2 superfamilies. Further immunogenicity and conservation analyses selected 11 promising candidates, and molecule docking (MD) was then simulated with the corresponding MHC-I alleles. Two-way hierarchical clustering revealed the interactions between GP peptides and MHC-I haplotypes. Briefly, epitope hotspots sharing good affinity to a wide spectrum of MHC-I molecules highlighted the biomedical practice for vaccination, and haplotype clusters represented the similarities among individuals during T-cell response establishment. Cross-validation proved the patterns observed through both MD simulation and public data integration. Lastly, 148 HTNV variants yielded six types of major amino acid residue replacements involving four in nine hotspots, which minimally influenced the general potential of MHC-I superfamily presentation. Altogether, our work comprehensively evaluates the pan-MHC-I immunoreactivity of HTNV GP through a state-of-the-art workflow in light of comparative immunology, acknowledges present discoveries, and offers guidance for ongoing HTNV vaccine pursuit.

Striped field mice (Apodemus agrarius) are the main host for the Hantaan virus (HTNV), the cause of hemorrhagic fever with renal syndrome (HFRS) in central China. It has been shown that host population density is associated with pathogen dynamics and disease risk. Thus, a higher population density of A. agrarius in an area might indicate a higher risk for an HFRS outbreak. Here, we surveyed the A. agrarius population density between 2005 and 2012 on the Weihe Plain, Shaanxi Province, China, and used this monitoring data to examine the relationships between the dynamics of A. agrarius populations and environmental conditions of crop-land, represented by remote sensing based indicators. These included the normalized difference vegetation index, leaf area index, fraction of photosynthetically active radiation absorbed by vegetation, net photosynthesis (PsnNet), gross primary productivity, and land surface temperature. Structural equation modeling (SEM) was applied to detect the possible causal relationship between PsnNet, A. agrarius population density and HFRS risk. The results showed that A. agrarius was the most frequently captured species with a capture rate of 0.9 individuals per hundred trap-nights, during 96 months of trapping in the study area. The risk of HFRS was highly associated with the abundance of A. agrarius, with a 1–5-month lag. The breeding season of A. agrarius was also found to coincide with agricultural activity and seasons with high PsnNet. The SEM indicated that PsnNet had an indirect positive effect on HFRS incidence via rodents. In conclusion, the remote sensing-based environmental indicator, PsnNet, was highly correlated with HTNV reservoir population dynamics with a 3-month lag (r = 0.46, p < 0.01), and may serve as a predictor of potential HFRS outbreaks.

Hantaan viruses cause two severe diseases lacking efficient treatment, yet no effective prophylactic vaccines are available. Continued exploration of alternative antiviral agents to treat hantavirus-related syndromes remains compulsory. The fluorescence-based quantitative real-time PCR (qPCR) has become the touchstone for target gene quantification. In the present study, standard curves for Hantaan virus (HTNV), mouse, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were generated by serial 10-fold dilutions of the constructed recombinant plasmid pGEM-T/HTNV, pGEM-T/mouse-GAPDH, and pGEM-T/human-GAPDH, respectively. Comparisons between the indirect immunofluorescence assay and qPCR assay in the detection of HTNV-infected Vero E6 cells showed improved detection limit and sensitivity of latter method. To characterize the inhibitory effect of several conventional antivirals (arbidol and ribavirin) and unconventional antivirals (indomethacin and curcumin) on HTNV, the levels of viral RNAs were measured for 4 days post-treatment of HTNV-infected Vero E6 cells and 18 days post-inoculation of HTNV-infected suckling mice. Our results validated that HTNV was sensitive to ribavirin and arbidol treatment, while indomethacin and curcumin may also be therapeutically effective in treating HTNV infection. As a result, the establishment and application of qPCR may be a useful tool for the evaluation of potential antivirals for Hantaan virus infection in vitro and in vivo.

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

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.

•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.