In Vivo Kinetics and Biodistribution of a Hantaan Virus DNA Vaccine after Intramuscular Injection in Mice

To study the kinetics in vivo of a Hantaan virus DNA vaccine, we constructed a fusion DNA vaccine, pEGFP/S, by cloning the S segment of Hantavirus into the vector, pEGFP-C1, which encodes Green fluorescent protein EGFP. In this report, we provide evidence that pEGFP/S was distributed and persistently expressed for more than 60 days in several organs after inoculation. Our findings suggest that the persistent immune responses induced by a Hantaan virus DNA vaccine are likely due to the plasmid pEGFP/S deposited in vivo, which acts as a booster immunization.

Separation and Anti-Hantaan Virus Activity of Extracts from Alternanthera philoxcroides in vitro and in vivo

A water-soluble substance was extracted from the Chinese herb, Alternantheraphiloxcroides with hot water and alcohol. Aliquots of this initial extract were further fractionated by treatment with ether, ethyl acetate and alcohol respectively. The four extracts were assayed for anti-viral activity against three serum, Hantaan virus 114 （HV114）, HV435 and A9 strains. Results show that the four extracts are capable of inhibiting Hantaan virus propagation, of which extract No. 1 has the best efficiency. The three dosage of extract No. 1, which are used upon three Hantaan virus serum IC50, are 153, 157, 154 μg/mL. New-born mice were made to be infected with HV114 and then fed in vivo with extract No.l on the 3rd, 10th and 14th days after being infected by the virus. The treatment continued for 8 days with a dosage of 2.5 g/kg. Result shows that survival rates of mice were 75%, 50% and 0, respectively. The median time to death （MTDs） of the three groups were 37, 30, 23 days.

Expression of Hantaan virus 26 kD fragment of nucleocapsid protein in insect cells and prelimimary study on its immunogenicity

Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.

加拿大伴肾脏综合征出血热汉坦病毒(Hantaan virus)的血清流行病学研究

伴肾脏综合征出血热(HFRS)在世界大部分地区呈地方性流行。病源因子是汉坦病毒(Hantaan virus)。用间接免疫荧光抗体技术对采自全国各省所有年龄组人群的2000余份血清进行了本病病毒抗体的测定。血清阳性率的范围在0.3%至4.0%之间,全国平均阳性率是1.4%。大西洋地区的纽芬兰省(Newfoundland)和新伯伦瑞克省(New Brunswick)阳性率最高,高于朝鲜高度地方性流行区的阳性率。

用单克隆抗体鉴定Hantaan virus和HFRS苏联毒株之间的抗原差异性

1983年在苏联西部Bashkiria地区捕获了欧洲棕背(鼠平)(Cletrionomys glareolus),将ELISA法检出的HFRS抗原阳性(鼠平)肺悬液在实验室Wistar大白鼠体内传代。用欧洲棕背(鼠平)以及接种后的大白鼠鼠肺悬液接种E6细胞,每隔10天将完整的活受染E6细胞连同新鲜E6细胞混合进行传代。

Study on the Cytotoxic T Lymphocytes Clone Specific for the Nucleocapsid Protein of Hantaan Virus from Peripheral Blood in Patients with Hemorrhagic Fever with Renal Syndrome

In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from peripheral blood mononuclear cells (PBMC) of patients with HFRS. The activities of CTL were detected as usual with EBV-transformed lymphoblastoid cell line (BLCL) as target cells. The results showed that the CTL clone could recognized and killed the target cells with specificity of nucleocapsid protein of Hantaan virus (HTNVNP) with the cytotoxicity percentages of 50.2%, 25.4% and 39.0% respectively. These results demonstrated that the antigenic epitopes of HTNVNP mainly located on the C-terminal of the viral nucleocapsid protein.

CLINICAL SIGNIFICANCE OF DETECTING SERUM ANTIBODIES TO NUCLEOPROTEIN AND GLYCOPROTEIN G_2 OF HANTAAN VIRUS IN PATIENTS WITH HEMORRHAGIC FEVER WITH RENAL SYNDROME

Antibody blocking enzyme linked immunosorbent assays, respectively detecting antibodies to Hantaan virus nucleoprotein (NPAb) and glycoprotein GZ (G,Ab), were developed using monoclonal antibody L133, L13r3, LV48A and LVZB28B NPAb and GZAb in 291 serum samples from 65 patientswith kemorrkagic fever with renal syudrome (HFRS) were detfrmlned by these methods. The positive rates or NPAb were 90N on day 2-3 and 100 % on day 8-9 arter onset of disease, respectively.NPAb titers Increased during fever period and reached Peak levels during kypotensive and oliguric periods of HFRS. It was suggested that NPAb might be an important component Involved in the immunopathogenlc lin'alrmeut of HFRS and the detection of NPAb might be useful for the early diagnosis or HFRS. The I,osltlve rates and titers of GZAh were very low during the rirst three periods,namely rever, hypoteuslve and ollgurlc periods, and reached high levels during the convalescent period. GRAb titers were negatively related to the I,rotelnurla levels during the course of HFRS. It wasIndicated that GZAb might be the main component or neutralizing autlhodles to Hantaan virus Infection and the efrlclent production or GZAb was a good marker ror predicting the recovery and betterprognosis of HFRS.

HLA-E-restricted Hantaan virus-specific CD8^(+)T cell responses enhance the control of infection in hemorrhagic fever with renal syndrome

Infection with the Hantaan virus(HTNV)may result in severe hemorrhagic fever with renal syndrome(HFRS).The functions of HLA-E-restricted CD8^(+)T lymphocytes in virus control and vaccine development have recently received increased attention.The purpose of this research is to discover HLA-E-restricted CD8^(+)T cell epitopes on HTNV as well as the features of these epitope-specific CD8^(+)T cells in HFRS patients.To anticipate HLA-Erestricted HTNV epitopes,the NetMHCpan servers were utilized.The K562/HLA-E cell binding test and the enzyme-linked immunospot assay were used to confirm epitope binding to HLA-E.The number and features of HLA-E-restricted epitope-specific CD8^(+)T lymphocytes in HFRS patients were investigated using tetramer staining,intracellular cytokine labeling,proliferation,and cytotoxicity assays.Six HTNV-derived HLA-Erestricted CD8^(+)T cell epitopes were found in this study.In mild/moderate HFRS patients,the frequency of HLA-E-restricted epitope-specific CD8^(+)T cells was greater than in severe/critical patients.CD38+HLA-DR+HLA-E-restricted CD8^(+)T cells were identified.Meanwhile,CD45RA^(+)CCR7^(-)effector memory-re-expressing CD45RA T cells with early and intermediate maturation and differentiation characteristics predominated.Notably,CD8^(+)T cells from milder HFRS patients produced more interferon-γ,interleukin-2,and granzyme B,had a stronger proliferative potential,and were inversely linked with the amount of plasma HTNV virus load.Furthermore,HLA-E-restricted epitope-specific CD8^(+)T cells demonstrated improved cytotoxic activity in vitro during the acute stage of HFRS.Taken together,the findings demonstrate the protective effects of HLA-E-restricted CD8^(+)T cells during HTNV infection,suggesting that HLA-E-targeted vaccines against HTNV might be developed for HLA-diverse populations.

Increased expression of Hsp70 and co-localization with nuclear protein in cells infected with the Hantaan virus

Objective To investigate the effect of Hantaan virus infection on the expression of stress genes. Methods Techniques of virus infection, Western blot, immunohistochemistry, dual-immunofluorecsence staining, laser scanning confocal microscopy, RNA dot blot and in situ hybridization were used.Results Expression of heat shock protein 70 (Hsp70) was observed in cells infected with HTV as well as the translocation of Hsp70 from cytoplasm to nucleoli following virus infection. The variable distribution of Hsp70 was related to the various time after infection. Double-label indirect immunofluorescence of nuclear protein (NP) and Hsp70 in infected cells demonstrated co-localization of these proteins in the cytoplasm. Conclusion Overexpression of Hsp70 can be induced directly by Hantaan virus, which may be associated with virus protein assembly. The Hantaan virus proteins co-localize with, and possibly form a physical complex with cellular Hsp70 in infected cells.

应用PEPSCAN技术研究单克隆抗体L13F3识别的抗原表位

目的 分析汉滩病毒核衣壳蛋白 (NP)主要线性表位免疫学反应的最小抗原多肽的组成 .方法 在应用多株国产单克隆抗体和噬菌体文库技术基本确定了病毒 NP氨基端的一个主要的 B细胞线性抗原表位的基础上 ,进一步用单克隆抗体 L 13F3对 6肽或 15肽随机多肽文库进行筛选 ,并根据病毒 NP氨基端 aa15 - 6 7的氨基酸序列 ,在纤维素膜上每间隔 1或 2个氨基酸合成了 15肽和 8肽的阵列 ,用 L13F3对这两组多肽阵列进行免疫学检测 .结果 对随机多肽文库进行筛检未获阳性结果 ,而对两组多肽阵列的检测确定了 2个分别由 5肽和 3肽组成的最小的抗原反应多肽 .结论 多肽阵列检测技术即肽扫描 (peptide scan)

Monitoring Neutralization Property Change of Evolving Hantaan and Seoul Viruses with a Novel Pseudovirus-Based Assay

The Hantaan virus(HTNV)and Seoul virus(SEOV)mutants have accumulated over time.It is important to determine whether their neutralizing epitopes have evolved,thereby making the current vaccine powerless.However,it is impossible to determine by using traditional plaque reduction neutralization test(PRNT),because it requires large numbers of live mutant strains.Pseudovirus-based neutralization assays(PBNA)were developed by employing vesicular stomatitis virus(VSV)backbone incorporated with HTNV or SEOV glycoproteins(VSVDG*-HTNVG or VSVDG*-SEOVG).56 and 51 single amino acid substitutions of glycoprotein(GP)in HTNV and SEOV were selected and introduced into the reference plasmid.Then the mutant pseudoviruses were generated and tested by PBNA.The PBNA results were highly correlated with PRNT ones with R2 being 0.91 for VSVDG*-HTNVG and 0.82 for VSVDG*-SEOVG.53 HTNV mutant pseudoviruses and 46 SEOV mutants were successfully generated.Importantly,by using PBNA,we found that HTNV or SEOV immunized antisera could neutralize all the corresponding 53 HTNV mutants or the 46 SEOV mutants respectively.The novel PBNA enables us to closely monitor the effectiveness of vaccines against large numbers of evolving HTNV and SEOV.And the current vaccine remains to be effective for the naturally occurring mutants.

Hantavirus Infection during Pregnancy

Hantavirus infection is a global health challenge,causing widespread public concern.In recent years,cases of hantavirus infection in pregnant women have been reported in many countries.The infected pregnant women and their fetuses appear to have more severe clinical symptoms and worse clinical outcomes.Hence,to study the prevalence of hantavirus infection in pregnant women,this study will focus on the epidemiological distribution of the virus,different virus species penetrating the placental barrier,and factors affecting the incidence and clinical outcome of the infection in pregnant women and their fetuses.In addition,this review will also discuss the diagnostic tools and treatments for pregnant patients and provide an overview of the relevant future research.

Screening and identification of HTNV_(pv)entry inhibitors with high-throughput pseudovirus-based chemiluminescence

Hantaviruses,such as Hantaan virus(HTNV)and Seoul virus,are the causative agents of Hantavirus cardiopulmonary syndrome(HCPS)and hemorrhagic fever with renal syndrome(HFRS),and are important zoonotic pathogens.China has the highest incidence of HFRS,which is mainly caused by HTNV and Seoul virus.No approved antiviral drugs are available for these hantaviral diseases.Here,a chemiluminescence-based highthroughput-screening(HTS)assay was developed and used to screen HTNV pseudovirus(HTNVpv)inhibitors in a library of 1813 approved drugs and 556 small-molecule compounds from traditional Chinese medicine sources.We identified six compounds with in vitro anti-HTNVpvactivities in the low-micromolar range(EC50values of0.1–2.2μmol/L;selectivity index of 40–900).Among the six selected compounds,cepharanthine not only showed good anti-HTNVpvactivity in vitro but also inhibited HTNVpv-fluc infection in Balb/c mice 5 h after infection by94%(180 mg/kg/d,P<0.01),93%(90 mg/kg/d,P<0.01),or 92%(45 mg/kg/d,P<0.01),respectively,in a bioluminescent imaging mouse model.A time-of-addition analysis suggested that the antiviral mechanism of cepharanthine involves the membrane fusion and entry phases.Overall,we have established a HTS method for antiviral drugs screening,and shown that cepharanthine is a candidate for HCPS and HFRS therapy.These findings may offer a starting point for the treatment of patients infected with hantaviruses.

RIPK3 promotes hantaviral replication by restricting JAK-STAT signaling without triggering necroptosis

Hantaan virus(HTNV)is a rodent-borne virus that causes hemorrhagic fever with renal syndrome(HFRS),resulting in a high mortality rate of 15%.Interferons(IFNs)play a critical role in the anti-hantaviral immune response,and IFN pretreatment efficiently restricts HTNV infection by triggering the expression of a series of IFNstimulated genes(ISGs)through the Janus kinase-signal transducer and activator of transcription 1(JAK-STAT)pathway.However,the tremendous amount of IFNs produced during late infection could not restrain HTNV replication,and the mechanism remains unclear.Here,we demonstrated that receptor-interacting protein kinase 3(RIPK3),a crucial molecule that mediates necroptosis,was activated by HTNV and contributed to hantavirus evasion of IFN responses by inhibiting STAT1 phosphorylation.RNA-seq analysis revealed the upregulation of multiple cell death-related genes after HTNV infection,with RIPK3 identified as a key modulator of viral replication.RIPK3 ablation significantly enhanced ISGs expression and restrained HTNV replication,without affecting the expression of pattern recognition receptors(PRRs)or the production of type I IFNs.Conversely,exogenously expressed RIPK3 compromised the host's antiviral response and facilitated HTNV replication.RIPK3^(-/-)mice also maintained a robust ability to clear HTNV with enhanced innate immune responses.Mechanistically,we found that RIPK3 could bind STAT1 and inhibit STAT1 phosphorylation dependent on the protein kinase domain(PKD)of RIPK3 but not its kinase activity.Overall,these observations demonstrated a noncanonical function of RIPK3 during viral infection and have elucidated a novel host innate immunity evasion strategy utilized by HTNV.

STING strengthens host anti-hantaviral immunity through an interferon-independent pathway

Hantaan virus(HTNV),the prototype virus of hantavirus,could escape innate immunity by restraining type I interferon(IFN)responses.It is largely unknown whether there existed other efficient anti-hantaviral tactics in host cells.Here,we demonstrate that the stimulator of interferon genes(STING)strengthens the host IFNindependent anti-hantaviral immunity.HTNV infection activates RIG-I through IRE1-XBP 1-mediated ER stress,which further facilitates the subcellular translocation and activation of STING.During this process,STING triggers cellular autophagy by interacting with Rab7A,thus restricting viral replication.To note,the anti-hantaviral effects of STING are independent of canonical IFN signaling.Additionally,neither application of the pharmacological antagonist nor the agonist targeting STING could improve the outcomes of nude mice post HTNV challenge in vivo.However,the administration of plasmids exogenously expressing the mutant C-terminal tail(ΔCTT)STING,which would not trigger the type I IFN responses,protected the nude mice from lethal HTNV infection.In summary,our research revealed a novel antiviral pathway through the RIG-I-STING-autophagy pathway,which offered novel therapeutic strategies against hantavirus infection.

Research Progress on the Pathogenesis of Hemorrhagic Fever with Renal Syndrome

Hemorrhagic fever with renal syndrome(HFRS)is an acute natural focus epidemic disease characterized by fever,shock,hemorrhage and kidney injury caused by hantavirus infection.Hantavirus mainly infects human vascular endothelial cells,and induces extensive damage to small blood vessels and capillaries.Increased vascular permeability is the pathological basis for clinical manifestations of HFRS.Although domestic and foreign scholars have carried out many studies on the hantavirus pathogenesis,such as the immune pathological response induced by hantavirus,host genetics and apoptosis,thrombocytopenia,coagulation and fibrinolysis dysfunction,and the vascular endothelial damage,the pathogenesis of HFRS has not been fully elucidated and there is no effective drug yet.In-depth discussion of the molecular mechanism of HFRS and finding effective therapeutic drugs are still the research hotspots on the field of hantavirus/HFRS.This review will elaborate the research progress on the pathogenesis of HFRS in recent years.