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Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS), which is a zoonosis endemic in eastern Asia, especially in China. The reservoir host of HTNV is field mouse (Apodemus agraricus). The main manifestation of HFRS, including acute kidney injury, increases vascular permeability, and coagulation abnormalities. In this paper, we review the current knowledge of the pathogenesis of HFRS including virus factor, immunity factor and host genetic factors. Furthermore, the treatment and prevention will be discussed.

Hantaan virus (HTNV), which belongs to the genus Hantavirus, causes hemorrhagic fever with renal syndrome (HFRS) mainly in China. The diagnosis of HFRS depends on clinical manifestations and serological tests. A SYBR Green I based one-step real-time PCR assay was established in this study to detect HTNV. The HTNV standard curves were generated by plotting mean cycle threshold (Ct) values versus 10-fold serial dilutions of a previous titrated HTNV stock over a wide range of concentrations (1×10(7) to 1PFU/ml). The minimum detection limit of the assay was 1PFU/ml, and it was 100-fold more sensitive than conventional RT-PCR. Melting curve analysis indicated that there were no primer-dimers and non-specific products in the assay. No cross-reaction was observed with Seoul virus (SEOV). The specificity of the asssay was also verified by nuleotide sequencing of the PCR products. Intra- and inter-assay variability data were analyzed to examine the reproducibility of the assay. HTNV viral loads in HFRS patients were also investigated with the assay. These results indicated that the one-step real-time PCR assay is useful for detecting HTNV and for monitoring the viral loads., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hantaan virus (HTNV) is a major zoonotic pathogen that causes hemorrhagic fever with renal syndrome (HFRS) in Asia, especially in China. Shaanxi province, which is located in northwest of China, is one of the areas in China most severely afflicted with HFRS epidemics annually. This study aims to establish a quantitative RT-PCR (qRT-PCR) assay to detect HTNV both in cell culture and clinical serum samples. We established a SYBR Green I-based one-step qRT-PCR assay that targets the S segment of the HTNV genome for rapid detection and quantification. The HTNV cRNA standards were constructed by in vitro transcription, and the copy numbers of the HTNV cRNA were quantified. Standard curve was generated by determining the mean cycle threshold (Ct) values versus 10-fold serial dilutions of the HTNV cRNA over a range of 1 × 10(8) to 1 × 10(3) copies/μl. The standard curve had a reaction efficiency of 102.1%, a correlation coefficient (R(2)) of 0.998, and a slope of -3.273. The coefficient of variation (CV) of the intra- and inter-assays ranged from 0.68% to 3.00% and from 0.86% to 3.21%, respectively. The cycle intervals of the qRT-PCR assay between each dilution ranged from 2.9 to 3.8 cycles, and the lowest detection limit of the qRT-PCR assay was 10 copies/μl. The assay exhibited high specificity that was confirmed by melting curve analysis, and no cross reaction with the Seoul virus (SEOV) and other viruses (HBV, HCV and HIV) was observed. HTNV RNA was also detected in the 27 serum samples of clinical HFRS patients using the assay, and the HTNV RNA viral load ranged from 2.06 × 10(1) to 1.95 × 10(5) copies/μl. The SYBR Green I-based one-step qRT-PCR assay is a sensitive, specific, reproducible, and simple method for detecting and quantifying HTNV in cell culture and clinical samples.

The innate immune response induced by Hantavirus is responsible for endothelial cell dysfunction and viral pathogenicity. Recent studies demonstrate that TLR4 expression is upregulated and mediates the secretion of several cytokines in Hantaan virus (HTNV)-infected endothelial cells. To examine viral interactions with host endothelial cells and characterize the innate antiviral responses associated with Toll-like receptors, we selected TLR4 as the target molecule to investigate anti-hantavirus immunity. TLR4 mRNA-silenced EVC-304 (EVC-304 TLR4-) cells and EVC-304 cells were used to investigate signaling molecules downstream of TLR4. The expression of the adaptor protein TRIF was higher in HTNV-infected EVC-304 cells than in EVC-304 TLR4- cells. However, there was no apparent difference in the expression of MyD88 in either cell line. The transcription factors for NF-κB and IRF-3 were translocated from the cytoplasm into the nucleus in HTNV-infected EVC-304 cells, but not in HTNV-infected EVC-304 TLR4- cells. Our results demonstrate that TLR4 may play an important role in the antiviral immunity of the host against HTNV infection through an MyD88-independent signaling pathway.

Hantaviruses infect human endothelial cells (ECs) and are known to cause vascular-permeability-based diseases, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The αvβ3 integrins, which are highly expressed on the surface of ECs, serve as hantavirus receptors. Specifically, the β3 integrin and vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) form a functional complex and interact with each other. Signaling through this complex causes cytoskeletal reorganization, which is one of the most important mechanisms underlying hyperpermeability. In this study, we show that VEGF dramatically enhances Hantaan virus (HTNV)-directed permeability and increases the reorganization of the cytoskeleton and the disruption of junctional organizations in an EC monolayer at 3 days postinfection. HTNV infection reduced the effect of VEGF on adhesion, migration, and the upregulation of β3 expression, but the infection alone upregulated the expression of β3 and VEGFR2. These results indicate that in addition to its role in blocking β3 integrin activation as reported previously, HTNV blocks the function of the complex of VEGFR2 and β3 integrin, and the dysfunction of the complex may contribute to cytoskeletal reorganization in an HTNV-directed hyperpermeability response to VEGF.

Objective: To investigate the role of vascular endothelial growth factor (VEGF) in haemorrhagic fever with renal syndrome (HFRS)., Methods: VEGF, soluble VEGF receptor (sVEGFR)-2, angiopoietin (Ang)-1, tumour necrosis factor (TNF)-α and interferon (IFN)-γ levels were measured in serum samples from 68 patients with HFRS. Cultured human umbilical vein endothelial cells (HUEVCs) were infected by Hantaan virus (HTNV) and/or stimulated with recombinant VEGF; dextran permeability of the cells was determined. Claudin-1 and vascular endothelial (VE)-cadherin levels were determined by real-time reverse transcription-polymerase chain reaction and Western blot analyses., Results: Serum VEGF, TNF-α and IFN-γ levels were significantly elevated, whereas sVEGFR2 and Ang-1 levels were reduced, during the acute phase of HFRS. In vitro cell permeability was unaffected by HTNV infection or VEGF stimulation alone, but the combination of HTNV infection and VEGF treatment significantly increased the permeability of endothelial cell monolayers in a time-dependent manner. Claudin-1 and VE-cadherin were downregulated at both the mRNA and protein level by combined HTNV infection and VEGF stimulation., Conclusions: Elevated VEGF induced by HTNV infection may play an important role in the vascular hyperpermeability that is characteristic of HFRS.

Hantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS), which is characterized by capillary leakage, hemorrhage, and renal injury, and is an important public health problem in China. Some kinds of immune cells, particularly CD8(+) T cells, are involved in the pathogenesis of Hantavirus infection. The nucleocapsid protein (NP) of the Hantavirus is the most conserved structural protein and the most abundant viral protein produced during infection. It is one of the important target antigens that induce the CD8(+) T-cell response. In this study, we examined the CD8(+) T-cell response to HTNV NP C-terminal polypeptides. We synthesized 23 overlapping C-terminal polypeptides and detected the antigen-specific CD8(+) T cell response in 15 patients with HFRS. The results demonstrated that there were NP-specific T-cell responses in bulk cultures of peripheral blood mononuclear cells (PBMCs) from 9 of 15 patients. The peptide 51 (aa 301-315: SPSSIWVFAGAPDRC), peptide 60 (aa 355-369: LRKKSSFYQSYLRRT), and peptide 70 (aa 415-429: DVKVKEISNQEPLKL) induced strong CD8(+) T-cell responses. Among them, peptide 70 induced CTL responses in donors 7, 9, and 11, and the strongest responses were seen in donor 11. Depletion of CD8(+) T cells from PBMCs completely abrogated the peptide-specific T-cell response, while depletion of CD4(+) T cells did not diminish the number of IFN-gamma spot-forming cells. These data suggest that infection with HTNV results in CTL responses to immunodominant regions on the NP.

Hantaan virus (HTNV) is an Old World hantavirus associated with hemorrhagic fever with renal syndrome (HFRS). To visualize the localization of the L protein of HTNV strain 84FLi within cells, a fusion protein composed of enhanced green fluorescent protein and L protein, EGFP-L, was expressed in Vero cells. The 273 KDa expressed fusion protein of EGFP-L localized in the perinuclear region. We also described the development of a reverse genetics system for HTNV strain 84FLi. The RNA polymerase I (pol I)-mediated transcription system was used to generate artificial viral RNA genome segments (minigenomes), which contained the chloramphenicol acetyltransferase (CAT) reporter gene in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of HTNV 84FLi L segment. CAT could be detected in cells after transfection, indicating the successful encapsidation, transcription and replication of the pol I-derived minigenomes. The passaged transfer of CAT demonstrates that recombinant virus containing packaged pol I-derived minigenomes has been produced. This system may be helpful in studying the gene function and pathogenesis of HTNV.

Hantaan virus (HTNV) infects endothelial cells and is associated with increased vascular permeability during hemorrhagic fever with renal syndrome (HFRS). The pattern of increased vascular permeability is mediated by immune response. Therefore, it is necessary to characterize the mechanism of HTNV involvement in the host's innate immune. In this study, the expression of five toll-like receptors (TLRs) was analyzed in Endothelial vein cells (EVC-304) following HTNV infection in vitro. TLR4 showed an altered expression after HTNV infection. HTNV infection significantly increased IFN-beta, IL-6 and TNF-alpha secretion from EVC-304 cells, particularly after lipopolysaccharide stimulation. The increased IFN-beta, IL-6 and TNF-alpha production was mediated by TLR4 induction, since the introduction of the small interfering RNA against TLR4 specifically inhibited the HTNV-induced cytokine production. In conclusion, HTNV infection directly induces TLR4 expression and thereby enhanced production of IFN-beta, IL-6 and TNF-alpha, which may contribute to the host's innate immune response.

The G1 cytoplasmic tail of Hantaan virus (HTNV) harbors a highly conserved region, which is homologous to immunoreceptor tyrosine-based activation motifs (ITAM) and is termed the ITAM-like sequence. To demonstrate the potential signal-transducing activity of G1 ITAM-like sequence resembling the canonical ITAM within immune and endothelial cells, a series of experiments were performed to define its interaction with cellular kinases. The synthesized G1 ITAM-like peptide was shown to coprecipitate with cellular phosphoprotein complexes by an immune-complex kinase assay. Mutational analyses showed that this ITAM-like sequence was a substrate for the Src family kinase Fyn, and two conserved tyrosine residues were required for coprecipitating Lyn, Syk, and ZAP-70 kinases. These findings demonstrated that HTNV envelope glycoprotein G1 contains a functional ITAM-like sequence in its cytoplasmic tail, which can bind critical cellular kinases that regulate immune and endothelial cell functions.

Cellular entry of pathogenic hantaviruses had been shown to be mediated by beta3 integrins. However, no direct evidence exists that hantavirus binds to beta3 integrins, and integrin beta3 subunit is not expressed on some cells permissive to hantavirus infection. In this report, utilizing beta3-integrin-transfected CHO cells, we demonstrated that integrin beta3 subunit renders CHO cells susceptible to Chinese Hantaan virus (HTN) strain A9 (isolated in China), and the viral infection was correspondingly inhibited by antibodies to alphavbeta3, alphaIIbbeta3, beta3, and alphav integrins. Furthermore, virus overlay protein-binding assay and 'quarternary Western' analysis indicate that HTN A9 directly interacts with beta3 integrins and an unidentified 70kDa protein. These findings indicate that beta3 integrins play a crucial role in cellular entry of HTN A9 via specific interactions with the virus. In addition, a novel 70kDa protein may serves as a candidate receptor or alternative cellular component for interaction with HTN.

Objective: To investigate the relationship between cellular entry of Hantaan virus (HTNV) and expression of beta3 integrin in beta3-integrin-deficient and HTNV-insusceptible China hamster ovary (CHO) cells., Methods: Eukaryotic expression vector encoding human integrin beta3 and eukaryotic expression vector harboring human integrin alphav or alphaIIb subunit cDNA were transfected into HTNV non-permissive CHO cells individually or collectively. Screening for stable transfectant clones was performed using G418 selective (culture medium. The exogenous gene expression was analyzed qualitatively and quantitatively by immunofluorescence assay (IFA) and flow cytometry (FCM). Various modified CHO cells and untransfected CHO cells were infected using HTNV A9. At various time points after infection, HTNV antigens in infected cells were detected qualitatively and quantitatively by IFA, FCM., Results: Highly-effective surface expression of beta3 integrin was measured in CHO/alphavbeta3 and CHO/alphaIIbbeta3, while weaker surface expression was detected in CHO/beta3 (P < 0.05). Expression of alphav or alphaIIb integrin in the individually transfected group was significantly lower than in the cotransfected group (P < 0.01) and the sites of localization changed. In contrast, effective surface expression was not seen when pcDNA3 was transfected alone. The infection rate of CHO/alphavbeta3 (60.1%) and CHO/alphaIIbbeta3 (55. 9%) cells were significantly higher than that of CHO/beta3 (38.7%) cells, while the infection rate of CHO/beta3 was significantly higher than that of CHO/alphav, CHO/pcDNA3 and CHO cells respectively. There was a close relationship between the positive percentage of HTNV A9-infected cells and expression of beta3 integrin., Conclusion: These results indicated that cellular entry of HTNV was related to the expression of beta3 integrin.