Guanylate-binding protein 1 inhibits Hantaan virus infection by restricting virus entry.

Hantaan virus (HTNV) infection can cause hemorrhagic fever with renal syndrome (HFRS) in humans, and currently, there are no long-standing protective vaccines or specific antivirals available. Guanylate-binding protein 1 (GBP1) is an interferon-stimulated gene that defends against various pathogen infections. However, the function of GBP1 in HTNV infection remains unknown. Here, we describe how GBP1 prevents HTNV infection by obstructing virus entry. We found that HTNV infection induced GBP1 expression and that overexpression of GBP1 inhibited HTNV infection, while knockout of GBP1 had the opposite effect. Interestingly, GBP1 did not affect interferon (IFN) signaling during HTNV infection. Instead, GBP1 prevented HTNV from entering cells through clathrin-mediated endocytosis (CME). We also discovered that GBP1 specifically interacted with actin but not dynamin 2 (DNM2) and made it difficult for DNM2 to be recruited by actin, which may account for the suppression of CME during HTNV infection. These findings establish an antiviral role for GBP1 in inhibiting HTNV infection and help us better understand how GBP1 regulates HTNV entry and could potentially aid in developing treatments for this virus.

T-cell immunoglobulin and mucin 1 (TIM-1) mediates infection of Hantaan virus in Jurkat T cells.

Hantaan virus (HTNV) is a major public health concern due to its ability to cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia. Symptoms of HFRS include fever, hemorrhage, immune dysfunction and renal impairment, and severe cases can be fatal. T cell-mediated adaptive immune responses play a pivotal role in countering HTNV infection. However, our understanding of HTNV and T cell interactions in the disease progression is limited. In this study, we found that human CD4+ T cells can be directly infected with HTNV, thereby facilitating viral replication and production. Additionally, T-cell immunoglobulin and mucin 1 (TIM-1) participated in the process of HTNV infection of Jurkat T cells, and further observed that HTNV enters Jurkat T cells via the clathrin-dependent endocytosis pathway. These findings not only affirm the susceptibility of human CD4+ T lymphocytes to HTNV but also shed light on the viral tropism. Our research elucidates a mode of the interaction between the virus infection process and the immune system. Critically, this study provides new insights into the pathogenesis of HTNV and the implications for antiviral research.

Investigation of a subunit protein vaccine for HFRS based on a consensus sequence between envelope glycoproteins of HTNV and SEOV.

Due to the global resurgence of hemorrhagic fever with renal syndrome (HFRS), more attention is being focused on this dangerous illness. In China and Korea, the only vaccines available are the virus-inactivated vaccine against Hantaan virus (HTNV) or Seoul virus (SEOV), but their efficacy and safety are inadequate. Therefore, it is important to develop new vaccines that are safer and more efficient to neutralize and regulate areas with a high prevalence of HFRS. We employed bioinformatics methods to design a recombinant protein vaccine based on conserved regions of protein consensus sequences in HTNV and SEOV membranes. The S2 Drosophila expression system was utilized to enhance protein expression, solubility and immunogenicity. After the Gn and Gc proteins of HTNV and SEOV were successfully expressed, mice were immunized, and the humoral immunity, cellular immunity, and in vivo protection of the HFRS universal subunit vaccine were systematically evaluated in mouse models. These results indicated that the HFRS subunit vaccine generated elevated levels of binding and neutralizing antibodies, particularly IgG1, compared to that of the traditional inactivated HFRS vaccine. Additionally, the spleen cells of immunized mice secreted IFN-r and IL-4 cytokines effectively. Moreover, the HTNV-Gc protein vaccine successfully protected suckling mice from HTNV infection and stimulated GC responses. In this research, a new scientific approach is investigated to develop a universal HFRS subunit protein vaccine that is capable of producing effective humoral and cellular immunity in mice. The results suggest that this vaccine could be a promising candidate for preventing HFRS in humans.

In vitro Anti-Hantavirus Activity of Protein Kinase Inhibitor 8G1 Targeting AKT/mTOR/eIF4E Signaling Pathway.

Hantaan virus (HTNV) is the main cause of hemorrhagic fever with renal syndrome (HFRS) around the world, which results in profound morbidity and mortality. However, there are currently no FDA-approved therapeutics or vaccines against HFRS. To find new anti-HTNV drugs, the inhibitory activity of 901 small molecule kinase inhibitors against HTNV is analyzed. Among these compounds, compound 8G1 inhibits HTNV with a relatively high inhibition rate and lower toxicity. The viral titer and nucleocapsid protein of HTNV are reduced after compound 8G1 treatment in a dose-dependent manner at concentrations ranging from 1 to 20 µM. In addition, the administration of compound 8G1 at the early stage of HTNV infection can inhibit the replication of HTNV. The molecular docking result reveals that compound 8G1 forms interactions with the key amino acid residues of serine/threonine-protein kinase B (Akt), which is responsible for the observed affinity. Then, the mammalian target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E (eIF4E) signaling pathways are inhibited. Our results may help to design novel targets for therapeutic intervention against HTNV infection and to understand the anti-HTNV mechanism of protein kinase inhibitors.

Bomidin: An Optimized Antimicrobial Peptide With Broad Antiviral Activity Against Enveloped Viruses.

The rapid evolution of highly infectious pathogens is a major threat to global public health. In the front line of defense against bacteria, fungi, and viruses, antimicrobial peptides (AMPs) are naturally produced by all living organisms and offer new possibilities for next-generation antibiotic development. However, the low yields and difficulties in the extraction and purification of AMPs have hindered their industry and scientific research applications. To overcome these barriers, we enabled high expression of bomidin, a commercial recombinant AMP based upon bovine myeloid antimicrobial peptide-27. This novel AMP, which can be expressed in Escherichia coli by adding methionine to the bomidin sequence, can be produced in bulk and is more biologically active than chemically synthesized AMPs. We verified the function of bomidin against a variety of bacteria and enveloped viruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), herpes simplex virus (HSV), dengue virus (DENV), and chikungunya virus (CHIKV). Furthermore, based on the molecular modeling of bomidin and membrane lipids, we elucidated the possible mechanism by which bomidin disrupts bacterial and viral membranes. Thus, we obtained a novel AMP with an optimized, efficient heterologous expression system for potential therapeutic application against a wide range of life-threatening pathogens.

Coumarin Derivative N6 as a Novel anti-hantavirus Infection Agent Targeting AKT.

Hantaviruses are globally emerging zoonotic viruses that can cause hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe, which is primarily caused by Hantaan virus (HTNV) infection, results in profound morbidity and mortality. However, no specific treatment is available for this disease. Coumarin derivatives have been reported as antiviral molecules, while studies about the bioactivity of coumarin derivatives against HTNV infection are limited. To study the potential antiviral activity of coumarin derivatives, 126 coumarin derivatives are synthesized, and their inhibitory activity against HTNV is analyzed in vitro. Among these compounds, N6 inhibits HTNV with relatively high selectivity index at 10.9, and the viral titer of HTNV is reduced significantly after 5, 10, and 20 µM N6 treatments. Furthermore, the administration of N6 at the early stage of HTNV infection can inhibit the replication and production of infectious HTNV in host cell, this therapeutic efficacy is confirmed in HTNV-infected newborn mice at the early stage of infection. The molecular docking results show that N6 forms interactions with the key amino acid residues at its active site, and reveals several molecular interactions responsible for the observed affinity, and the treatment of N6 can inhibit the expression of p (Ser473)Akt and HTNV nucleocapsid protein significantly. As such, these observations demonstrate that coumarin derivative N6 might be used as a potential agent against HTNV infection.

Nlrc3 Knockout Mice Showed Renal Pathological Changes After HTNV Infection.

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.

HTNV infection of CD8+ T cells is associated with disease progression in HFRS patients.

Hantaan viruses (HTNVs) are zoonotic pathogens transmitted mainly by rodents and capable of infecting humans. Increasing knowledge of the human response to HTNV infection can guide the development of new preventative vaccines and therapeutic strategies. Here, we show that HTNV can infect CD8+ T cells in vivo in patients diagnosed with hemorrhagic fever with renal syndrome (HFRS). Electron microscopy-mediated tracking of the life cycle and ultrastructure of HTNV-infected CD8+ T cells in vitro showed an association between notable increases in cytoplasmic multivesicular bodies and virus production. Notably, based on a clinical cohort of 280 patients, we found that circulating HTNV-infected CD8+ T cell numbers in blood were proportional to disease severity. These results demonstrate that viral infected CD8+ T cells may be used as an adjunct marker for monitoring HFRS disease progression and that modulating T cell functions may be explored for new treatment strategies.

[Construction of lentivirus plasmid pCDH-NLRX1 and stable expression of NLRX1 in A549 cells].

Objective To construct nucleotide-binding oligomerization domain-like receptors family member X1 (NLRX1) lentivirus plasmid and stable NLRX1-overexpressing A549 cell line. Methods Full-length cDNA encoding human NLRX1 was amplified from pCMV3-NLRX1 plasmid and then was inserted into pCDH-CMV-MCS-EF1-Puro vector to obtain NLRX1-overexpressing plasmid pCDH-NLRX1. pCDH-NLRX1 lentivirus particles were obtained by co-transfecting pCDH-NLRX1 with packaging plasmids (pMD2 and pAX2) into HEK293T cells. A549 cells were infected with concentrated pCDH-NLRX1 lentivirus particles and then were screened by puromycin to obtain stable NLRX1-overexpressed A549 cell line. The mRNA transcription and protein expression of NLRX1 were detected by real-time quantitative PCR, Western blot analysis and immunofluorescence. Results The lentivirus plasmid pCDH-NLRX1 was successfully constructed. After pCDH-NLRX1 and packaging plasmids were co-transfected into HEK293T cells, we obtained NLRX1 lentivirus particles with the titer of 1×105 TU/mL. After A549 cells were infected with lentivirus particles and screened by puromycin, a stable over-expression cell line of NLRX1 was obtained. NLRX1 was obviously expressed in the lentivirus-infected A549 cells and its mRNA and protein levels significantly increased. Conclusion NLRX1 lentivirus plasmid and stable NLRX1-overexpressing A549 cell line have been successfully constructed.

Incorporation of CD40 ligand or granulocyte-macrophage colony stimulating factor into Hantaan virus (HTNV) virus-like particles significantly enhances the long-term immunity potency against HTNV infection.

PURPOSE: Hantavirus infections cause severe haemorrhagic fever with renal syndrome (HFRS) in humans and are associated with high fatality rates. In 2017, numerous outbreaks were reported in China and Germany. This represents a significant public-healthcare issue with no effective HFRS vaccines that offer a long-term immune response. In this study, we investigated the long-term humoral and cellular immune responses and protective immunity of Hantaan virus (HTNV) granulocyte-macrophage colony stimulating factor (GM-CSF) and CD40 ligand (CD40L) virus-like particles (VLPs) in mice. METHODOLOGY: GM-CSF and CD40L VLPs were constructed via co-transfection of pCI-S and pCI-M-CD40L, and pCI-S and pCI-M-GM-CSF, into dihydrofolatereductase (dhfr)-deficient Chinese hamster ovary cells, respectively. Mice were immunized with HTNV VLPs 2 weeks apart. The animals were challenged 6 months after immunization. Specific and neutralizing antibodies were assessed by ELISA; IFN-γ was measured by enzyme-linked immunospot (ELISpot) assay and effectiveness by cytotoxic T lymphocyte (CTL) cytotoxicity assays. Nucleic acid loads of HTNV were tested by quantitative real-time PCR and viral antigen was detected via indirect ELISA. Pathological alterations were detected via haematoxylin-eosin staining. RESULTS: GM-CSF and CD40L VLPs provided stable, long-term protection with a high titre of neutralizing antibody in mice 6 months after immunization. Furthermore, VLPs increased HTNV-specific cellular immune responses via higher expression of IFN-γ and CTL responses. HTNV challenge assay results showed long-term protection against HFRS. No significant pathological alteration was observed in the organs of mice after immunization. CONCLUSION: This is, to the best of our knowledge, the first report demonstrating the long-term potency of HTNV VLP vaccines against HTNV infection and offers new insights into HTNV vaccine development.

Development of a Simple and Cost-Effective Method Based on T7 Endonuclease Cleavage for Detection of Single Nucleotide Polymorphisms.

AIMS: Single nucleotide polymorphisms (SNP) can be used as genetic markers and for risk assessment of allele-linked diseases, which can provide information for clinical diagnosis. Large-scale microarray and next-generation sequencing methods have made genome-wide SNP genotyping possible. However, in addition to their high cost, these techniques are dependent on having specialized equipment. Thus, there is a need for a simple genotyping method that can be implemented in a resource-limited environment. METHODS: We developed a strategy for SNP genotyping based on T7 Endonuclease I cleavage and an enzyme-linked microparticle immune assay. Using this method, we genotyped two common SNP sites (rs11526468 and rs12979860). The quality of the genotyping process was validated. RESULTS: Although a 70% false-negative rate was observed, no false-positive reactions were found. Therefore, multiple parallel repeat reactions can offset the possibility of mutation detection failure. DISCUSSION: This method employs a duplicate reagent-dependent procedure, and therefore has the potential for integration into a portable kit for field utilization.

Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus.

The cytotoxic T lymphocyte (CTL) response plays a key role in controlling viral infection, but only a few epitopes within the HTNV glycoprotein (GP) that are recognized by CTLs have been reported. In this study, we identified one murine HTNV GP-derived H2-Kb-restricted CTL epitope in C57BL/6 mice, which could be used to design preclinical studies of vaccines for HTNV infection. First, 15 8-mer peptides were selected from the HTNV GP amino acid sequence based on a percentile rank of <=1% by IEDB which is the most comprehensive collection of epitope prediction and analysis tool. A lower percentile rank indicates higher affinity and higher immune response. In the case of the consensus method, we also evaluated the binding score of peptide-binding affinity by the BIMAS software to confirm that all peptides were able to bind H2-Kb. Second, one novel GP-derived CTL epitope, GP6 aa456-aa463 (ITSLFSLL), was identified in the splenocytes of HTNV-infected mice using the IFN-γ ELISPOT assay. Third, a single peptide vaccine was administered to C57BL/6 mice to evaluate the immunogenic potential of the identified peptides. ELISPOT and cell-mediated cytotoxicity assays showed that this peptide vaccine induced a strong IFN-γ response and potent cytotoxicity in immunized mice. Last, we demonstrated that the peptide-vaccinated mice had partial protection from challenge with HTNV. In conclusion, we identified an H2-Kb-restricted CTL epitope with involvement in the host immune response to HTNV infection.

Construction and immunological characterization of CD40L or GM-CSF incorporated Hantaan virus like particle.

Infection of Hantaan virus (HTNV) usually causes hemorrhagic fever with renal syndrome (HFRS). China has the worst epidemic incidence of HFRS as well as high fatality. Inactivated whole virus has been used for HFRS vaccination, however there are still problems such as safety concerns. CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GM-CSF) are well-known immune stimulating molecules that can enhance antigen presenting, lymphocytes activation and maturation, incorporation of CD40L and GM-CSF to the surface of virus like particles (VLPs) can greatly improve the vaccination effect. We constructed eukaryotic vectors expressing HTNV M segment and S segment, as well as vectors expressing HTNV M segment with CD40L or GM-CSF, our results showed successful production of CD40L or GM-CSF incorporated HTNV VLPs. In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. CD40L/GM-CSF incorporated VLP can induce higher level of HTNV specific antibody and neutralizing antibody in mice. Immunized mice splenocytes showed higher ability of secreting IFN-γ and IL-2, as well as enhancing CTL activity. These results suggest CD40L/GM-CSF incorporated VLP can serve as prospective vaccine candidate.

Development of a SYBR-Green â quantitative PCR assay for the detection and genotyping of different hantaviruses.

Hemorrhagic fever with renal syndrome (HFRS) is a severe, viral zoonotic disease which occurs worldwide, particularly in Asia and Europe. In China, the Hantaan virus (HTNV) and the Seoul virus (SEOV) are known to be the most prevalent causative agents of HFRS. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for disease surveillance. In the present study, the viral loads in cell culture supernatant, infected mice blood and clinical serum samples were quantified using the SYBR­Green I-based reverse transcription-quantitiative polymerase chain reaction (RT-qPCR) assay, which targeted the S gene sequence of the HTNV and SEOV genomes. The cRNA of these two viruses were synthesized as a positive control and 10-fold serially diluted from 1x105 to 1x100 copies/µl. Standard curves were generated by plotting the mean cycle threshold (Ct) values versus copy numbers. The standard curve of HTNV had a correlation coefficient (R2) of 0.994, efficiency of amplification (E) of 101.9%, and the slope of -3.278, whereas that of SEOV had an R2 of 0.993, E of 104.8%, and the slope of -3.212. The minimum detection limit of the RT-qPCR assay for HTNV and SEOV was 101 copies/µl. Two qPCR assays were successfully established for the detection of HTNV and SEOV, respectively. Taken together, these findings demonstrate that using the SYBR­Green I-based RT-qPCR assay, the HTNV and SEOV may be genotyped precisely without cross-reactivity. Furthermore, viral RNA may be detected and quantified in cells, mice and infected individuals, which may be useful in epidemiological studies as well as for early monitoring and further preventative treatment against SEOV and HTNV-induced diseases.

Incorporation of GM-CSF or CD40L Enhances the Immunogenicity of Hantaan Virus-Like Particles.

A safe and effective Hantaan virus (HTNV) vaccine is highly desirable because HTNV causes an acute and often fatal disease (hemorrhagic fever with renal syndrome, HFRS). Since the immunity of the inactivated vaccine is weak and the safety is poor, HTNV virus-like particles (VLPs) offer an attractive and safe alternative. These particles lack the viral genome but are perceived by the immune system as virus particles. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this enhancement, we generated chimeric HTNV VLPs containing glycosylphosphatidylinositol (GPI)-anchored granulocyte macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity in vitro. The immunization of mice with chimeric HTNV VLPs containing GM-CSF or CD40L induced stronger humoral immune responses and cellular immune responses compared to the HTNV VLPs and Chinese commercial inactivated hantavirus vaccine. Chimeric HTNV VLPs containing GM-CSF or CD40L also protected mice from an HTNV challenge. Altogether, our results suggest that anchoring immunostimulatory molecules into HTNV VLPs can be a potential approach for the control and prevention of HFRS.