Zhikai Xu

Hantaan Virus RNA Load in Patients Having Hemorrhagic Fever With Renal Syndrome: Correlation With Disease Severity

To investigate the role of viral load in the pathogenesis of hemorrhagic fever with renal syndrome, the Hantaan virus RNA load in plasma from 101 patients was quantified, and the relationships between viral load and disease course, severity, and level of specific humoral immunity were analyzed. The viral load, detectable in 79 patients, ranged from 3.43 to 7.33 log10 copies/mL of plasma. In the early stage of disease, patients in severe/critical group were found to have higher viral loads than those in the mild/moderate group (5.90 vs 5.03 log10 copies/mL; P = .001), suggesting an association between Hantaan virus load and disease severity.

Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model

The completion of Mycobacterium tuberculosis genome sequence has opened a new way for the identification and characterization of bacterial antigens, such as ESAT-6, CFP10, MPT64, and Ag85 complex, which are helpful for tuberculosis control. In this work, genes of ESAT-6 and MPT64 were fused and expressed in Escherichia coli in form of inclusion bodies with a histidine tag. The expressed fusion protein was purified by nitrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, and the yield was 18mg/L of culture. In mice, the purified ESAT-6-MPT64 fusion protein elicited stronger humoral response, greater splenic lymphocyte stimulated index, and higher levels of IFN-gamma and IL-12 production than that of the single MPT64 inoculation group, and rendered modest protection on the experimental tuberculosis mouse models. In short, the ESAT-6-MPT64 fusion protein might be a potential candidate vaccine for tuberculosis.

The Long Noncoding RNA NEAT1 Exerts Anti-Hantaviral Effects by Acting as a Positive Feedback for RIG-I Signaling.

ABSTRACT Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon (IFN-β) production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.

Downregulation of NDRG1 promotes invasion of human gastric cancer AGS cells through MMP-2

The N-myc downstream-regulated gene-1 (NDRG1) has recently been proposed as a metastasis suppressor, but its precise role remains unclear. To investigate whether NDRG1 can indeed influence the metastasis progress, expression of endogenous NDRG1 was knocked down in human AGS gastric adenocarcinoma cells using RNA interference. Stable NDRG1 “silenced” transfectants showed similar growth rates as their control counterparts. By contrast, invasive ability in Matrigel invasion activity and Gelatinolytic activity by matrix metalloproteinase-2 (MMP-2) were markedly increased in NDRG1 “silenced” cells. Moreover, re-expression of NDRG1 by recombinant adenovirus Ad-NDRG1 in NDRG1 “silenced” cells inhibited the increased invasive ability. Further study, we found the induction of MMP-2 by downregulation of NDRG1 was mediated by MT1-MMP. Altogether, our results imply that NDRG-1 could play a key role in the regulation of cellular invasion and metastasis, which may involve the upregulation of matrix metalloproteinases.

Induction of Specific Humoral and Cellular Immune Responses in a Mouse Model following Gene Fusion of HSP70C and Hantaan Virus Gn and S0.7 in an Adenoviral Vector

Heat shock proteins (HSPs) display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV) glycoprotein (GP) and nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359–610 aa, HSP70C) to the Gn and 0.7 kb fragment of the NP (aa1–274-S0.7). C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7) and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV.

A recombinant pseudotyped lentivirus expressing the envelope glycoprotein of Hantaan virus induced protective immunity in mice

BackgroundHantaviruses cause acute hemorrhagic fever with renal syndrome (HFRS). Currently, several types of inactivated HFRS vaccines are widely used, however the limited ability of these immunogen to elicit neutralizing antibodies restricts vaccine efficacy. Development of an effective vaccine to overcome this weakness is must.MethodsIn the present study, a recombinant pseudotyped lentivirus bearing the hantaan virus (HTNV) envelope glycoproteins (GP), rLV-M, was constructed. C57BL/6 mice were immunized with the rLV-M and a series of immunological assays were conducted to determine the immunogenicity of the recombinant pseudotyped lentivirus. The humoral and cell-mediated immune responses induced by rLV-M were compared with those of the inactivated HFRS vaccine.ResultsIndirect immunofluorescence assay (IFA) showed the rLV-M expressed target proteins in HEK-293cells. In mice, the rLV-M efficiently induced GP-specific humoral responses and protection against HTNV infection. Furthermore, the rLV-M induced higher neutralizing antibody titers than the inactivated HFRS vaccine control.ConclusionsThe results indicated the potential of using a pseudotyped lentivirus as a delivery vector for a hantavirus vaccine immunogen.

Cellular immunogenicity of a multi-epitope peptide vaccine candidate based on hepatitis C virus NS5A, NS4B and core proteins in HHD-2 mice

To develop a vaccine against hepatitis C virus (HCV), a multi-epitope peptide was synthesized from nonstructural proteins containing HLA-A2 epitopes inducing mainly responses in natural infection. The engineered vaccine candidate, VAL-44, consists of multiple epitopes from the HCV NS5A, NS4B and core proteins. Immunization with the VAL-44 peptide induced higher CTL responses than those by the smaller VL-20 peptide. VAL-44 induced antigen-specific IFN-γ-producing CD4+ T cells and CD8+ T cells. VAL-44 elicited a Th1-biased immune response with secretion of high amounts of IFN-γ and IL-2, compared with VL-20. These results suggest that VAL-44 can elicit strong cellular immune responses. The VAL-44 peptide stimulated IFN-γ production from viral-specific peripheral blood mononuclear cells (PBMCs) of patients infected with HCV. These results suggest that VAL-44 could be developed as a potential HCV multi-epitope peptide vaccine.

Production and characterization of a recombinant single-chain antibody against Hantaan virus envelop glycoprotein.

Hantaan virus (HTNV) is the type of Hantavirus causing hemorrhagic fever with renal syndrome, for which no specific therapeutics are available so far. Cell type-specific internalizing antibodies can be used to deliver therapeutics intracellularly to target cell and thus, have potential application in anti-HTNV infection. To achieve intracellular delivery of therapeutics, it is necessary to obtain antibodies that demonstrate sufficient cell type-specific binding, internalizing, and desired cellular trafficking. Here, we describe the prokaryotic expression, affinity purification, and functional testing of a single-chain Fv antibody fragment (scFv) against HTNV envelop glycoprotein (GP), an HTNV-specific antigen normally located on the membranes of HTNV-infected cells. This HTNV GP-targeting antibody, scFv3G1, was produced in the cytoplasm of Escherichia coli cells as a soluble protein and was purified by immobilized metal affinity chromatography. The purified scFv possessed a high specific antigen-binding activity to HTNV GP and HTNV-infected Vero E6 cells and could be internalized into HTNV-infected cells probably through the clathrin-dependent endocytosis pathways similar to that observed with transferrin. Our results showed that the E. coli-produced scFv had potential applications in targeted and intracellular delivery of therapeutics against HTNV infections.

Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.

Hantaan virus can infect human keratinocytes and activate an interferon response through the nuclear translocation of IRF-3.

Hantaan virus (HTNV) is a rodent-borne virus that causes hemorrhagic fever with renal syndrome (HFRS) in Asia and can be transmitted to humans through bites or the inhalation of aerosolized urine, droppings, or saliva of infected rodents. Keratinocytes predominate in the epidermis and reportedly serve as a replication site for multiple vector-borne viruses, little is known about the susceptibility of human skin cells to HTNV infection. Therefore, we aimed to evaluate whether human keratinocytes support HTNV replication and elicit an immune response against HTNV infection. We found that a human keratinocyte cell line, HaCaT, supports HTNV replication. In addition, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation associated gene-5 (MDA5) play key roles in the detection of HTNV infection in HaCaT cells and in the up-regulation of interferon (IFN)-β expression, which subsequently leads to the production of a large amount of antiviral interferon-stimulated genes (ISGs) and other chemokines used for immune cell recruitment. Furthermore, we suggest that interferon regulatory factor (IRF)-3, as opposed to NF-κB/p65 or IRF-7, is translocated to the nucleus to induce IFN-β. However, the early induction of chemokine CXCL10 was a direct result of HaCaT cells counteracting HTNV infection and was not due to the induction of IFN. Overall, our data demonstrate, for the first time, the permissiveness of human keratinocytes to HTNV infection.