Expression of Gn protein of severe fever with thrombocytopenia syndrome virus and its immunogenicity / 中国生物制品学杂志

@#Objective To express the Gn protein of severe fever with thrombocytopenia syndrome virus(SFTSV) through adeno-associated virus 9(AAV9) expression system and evaluate its immunogenicity.Methods SFTSV Gn gene was inserted into viral vector pAAV-CMV-FH and the recombinant plasmid was transfected into HEK293T cells to obtain recombinant virus AAV9-Gn.The expression of Gn protein was determined by immunofluorescence and Western blot.Eighteen fernale BALB/c mice were randomly divided into three groups:Mock group(serum-free DMEM),AAV9-GFP group(1 × 10~(11) vg) and AAV9-Gn group(1 × 10~(11) vg),all of which were injected intramuscularly into the right hind limb at a dose of 100 μL per mouse.The body mass,diet,behavior and mental state of mice in each group were monitored continuously for 21 d,and the change rate of body mass was calculated;At 2,4,8 and 16 weeks after immunization,the levels of SFTSV neutralizing antibody in serum of mice in each group were detected by fluorescent reduction neutralization test(FRNT),and the levels of specific IgGl and IgG2a in serum of mice in AAV9-Gn group were detected by ELISA.Results After incubation with specific antibody,Vero cells transfected with AAV9-Gn showed specific green fluorescence under fluorescence microscope,and had specific binding to mouse anti-SFTSV Gn monoclonal antibody,and the specific binding bands were found at a relative molecular mass of about 61 000.The body mass of the three groups showed an increasing trend,there was no significant difference between the three groups(F=0.158—2.621,P> 0.05),and the diet,behavior and mental state were normal.At 2,4,8 and 16 weeks after immunization,the titer of SFTSV neutralizing antibody in serum of mice in AAV9-Gn group was significantly higher than that of Mock group and AAV9-GFP group(H=13.332—14.538,each P <0.001),and the titer peak appeared at 8 weeks;The level of specific IgG1 in serum of mice was significantly higher than that of IgG2a(F=4.373—12.975,each P <0.05) at different time points.Conclusion SFTSV Gn protein can be expressed correctly through AAV9 expression system,and has low toxicity to mice with good immunogenicity,which is expected to be a candidate component of SFTSV vaccine.

Comparison Between the SFTS-QS Kit and the PowerChek SFTSV Real-time PCR Kit for the Detection of Severe Fever With Thrombocytopenia Syndrome Virus

The recent increase in severe fever with thrombocytopenia syndrome (SFTS) cases has led to the development of the SFTS-QS kit (MiCoBioMed, Seongnam, Korea) for detecting the SFTS virus (SFTSV, now renamed Huaiyangshan banyangvirus). SFTS-QS is a qualitative real-time reverse transcription PCR assay based on lab-on-a-chip technology. We evaluated the performance of the SFTS-QS kit and compared it with that of the PowerChek SFTSV Real-time PCR kit (PowerChek; Kogene Biotech, Seoul, Korea). A total of 117 serum samples were simultaneously assayed using the SFTS-QS and PowerChek kits. Sanger sequencing targeting the S and M segments of SFTSV was performed as the reference method. The total turnaround time of the two kits was compared. The SFTS-QS results agreed with those of PowerChek with a kappa value of 0.92. The diagnostic sensitivity and specificity of the SFTS-QS kit were both 100% (14/14 and 103/103, respectively), whereas those of the PowerChek kit were 100% (14/14) and 98.1% (101/103), respectively. The results of SFTS-QS and PowerChek were comparable; however, the SFTS-QS kit required a shorter total turnaround time. The SFTS-QS kit produced accurate and fast results and thus could serve as a useful tool for detecting SFTSV.

Prevalence of severe fever with thrombocytopenia syndrome virus infection in Zhoushan island and duration of serum positive IgG antibody in infected patients / 中华临床感染病杂志

Objective@#To analyze the prevalence of severe fever with thrombocytopenia syndrome virus(SFTSV)infection in Zhoushan island of Zhejiang province and the duration of serum positive IgG antibody in patients infected with SFTSV.@*Methods@#One thousand one hundred and twenty-two healthy people from Zhoushan island of Zhejiang province were recruited for cross-sectional study in August 2019, including 641 from non-epidemic areas and 481 from epidemic areas. The serum SFTSV-IgG antibody was detected by enzyme-linked immunosorbent assay (ELISA), and the positive rates of SFTSV-IgG antibody were compared between people from the epidemic areas and non epidemic areas. Meanwhile, the antibody titer of SFTSV-IgG in 19 patients confirmed between July 2011 and June 2018 was detected by indirect ELISA. SPSS 17.0 software was used to analyze data.@*Results@#The positive rate of SFTSV-IgG antibody was 1.5% (7/481) in the epidemic area, which was higher than that in the non-epidemic area (0/641) (χ2=7.187, P<0.01). The positive rates of SFTSV-IgG antibody in 2019 were lower than those in the epidemic area (11.7%) and non-epidemic area (2.5%) in 2013 (χ2=22.556 and 10.352, both P<0.01). The serum SFTSV-IgG antibody of 18 patients with previous infection was still positive, and the longest one lasted for 8 years.@*Conclusions@#There is a SFTSV latent infection in population from epidemic area of Zhoushan island. The SFTSV-IgG antibody can last for a long time in patients with SFTS and it may have certain protective effect.

Research advances on animal infection and animal models of severe fever with thrombocytopenia syndrome virus / 中国实验动物学报

Severe fever with thrombocytopenia syndrome virus( SFTSV) is a novel Bunyavirus which was first repor-ted in China in 2009. Tick is its important reservoir host and vector. Both wild and domestic animals can be naturally infec-ted. IFNAR-/ - immunodeficient mice are ideal experimental animal models. In this article, the research result of animal infection and animal models of SFTSV are summarized, which provide the basis for the control of SFTSV natural epidemic and for animal experiments.

Study on the relationship between viral load of severe fever with thrombocytopenia syndrome bunyavirus and patient's condition / 中华传染病杂志

Objective To explore the severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) existence time in the body,and the correlation between viral load and the severity and prognosis of disease.Methods The clinical data of 125 SFTS patients from May 2015 to October 2016 in Weihai Central Hospital in Shandong province were analyzed retrospectively.Patients were divided into low viral load group and high viral load group according to the SFTSV RNA levels.Neurological symptoms,bleeding tendency,the incidence of myocardial damage and severe pneumonia,laboratory biochemical index and prognosis of two groups were compared.SFTSV RNA of 46 cases were detected dynamically.Data with homogeneity of variance were tested by t test,and data with heterogeneity of variance was tested by rank sum test.Results Among the 125 cases,64 were male and 61 were female.The mean age was (59.0±3.6) years old.One hundred and one cases were cured,and 24 died.SFTSV RNA loads in low viral load group(81 cases) were (3.08± 1.01) copies/mL,and those in high viral load group (44 cases) were (5.69 ± 0.99) copies/mL,with statically significant difference (t =11.78,P＜0.05).By the dynamic detection of SFTSV RNA load in 46 patients,viral loads in most patients were gradually declined after 1 week of onset,and cleared after 23 days.The incidence of neurological symptoms,bleeding tendency,severe myocardial damage and pneumonia of two groups showed significant difference (x2 =92.987,38.711,75.889 and 54.680,respectively,all P＜0.05).The viral loads of patients who died varied from 1.06× 104 copies/mL to 5.78 × 107 copies/mL.White blood counts of two groups showed no significant difference (t =0.181,P＞ 0.05).The platelet counts of two groups had significant difference (t =2.869,P＜0.05).AST and γ-GT of two groups also had significant difference (P＜0.01 and 0.05,respectively).creatine kinase,creatine kinase isoenzyme,lactic dehydrogenase and hydroxybutyrate dehydrogenase of two groups all had significant difference (P＜0.01 or 0.05).Serum sodium,blood calcium and glucose of the two groups had significant difference (P＜0.01 or 0.05).activated partial thromboplastin time of the two groups showed significant difference as well (t=5.623,P＜0.01).Conclusions After the onset of SFTSV infection,the virus existence in the body may less than 4 weeks.Viral loads are closely associated with disease severity and prognosis.The higher the viral loads are,the heavier organ dysfunction could be and the higher mortality is.

Research advances on animal infection and animal models of severe fever with thrombocytopenia syndrome virus / 中国实验动物学报

Severe fever with thrombocytopenia syndrome virus( SFTSV) is a novel Bunyavirus which was first repor-ted in China in 2009. Tick is its important reservoir host and vector. Both wild and domestic animals can be naturally infec-ted. IFNAR-/ - immunodeficient mice are ideal experimental animal models. In this article, the research result of animal infection and animal models of SFTSV are summarized, which provide the basis for the control of SFTSV natural epidemic and for animal experiments.

Epidemiological survey of severe fever with thrombocytopenia syndrome virus in ticks in Nagasaki, Japan

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that is endemic in East Asia. The SFTS virus (SFTSV) is transmitted to other organisms by infected ticks and is endemic to Nagasaki in western Japan. However, epidemiological information regarding SFTSV in Nagasaki ticks has not been elucidated. In this study, we first examined the sensitivities of SFTSV gene detection by real-time RT-PCR and virus isolation in cultured cells and mice. These methods could detect SFTSV in the samples containing more than 4 × 10⁰ ffu. Next, we attempted to isolate SFTSV and to detect viral gene in 2,222 nymph and adult ticks collected from May to August 2013 among seven regions of Nagasaki. However, neither virus isolation nor viral gene detection were confirmed in those tick pools. SFTSV positivity rates are considered very low in ticks and viral loads in ticks are also very limited. Further investigation by increasing the number of ticks and including larval samples in the investigation, as well as improved detection methods, may be required to find SFTSV-positive ticks in this region.

Epidemiological Survey of Severe Fever with Thrombocytopenia Syndrome Virus in Ticks in Nagasaki, Japan

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease endemic in East Asia. Transmitted to other organisms by infected ticks, the SFTS virus (SFTSV) and is endemic to Nagasaki in western Japan. However, epidemiological information regarding SFTSV in Nagasaki ticks has not been available to date. In this study, we began by examining the sensitivities of SFTSV gene detection by real-time RT-PCR and virus isolation in cultured cells and mice. These methods could detect SFTSV in the samples containing more than 4 × 10⁰ ffu. Next, we attempted to isolate SFTSV and to detect viral gene in 2,222 nymph and adult ticks collected from May to August 2013 among seven regions of Nagasaki. However, neither virus isolation nor viral gene detection were confirmed in the tick pools. SFTSV positivity rates are considered to be very low in ticks, and viral loads are also very limited. Further investigations increasing the number of ticks and including larval samples as well as improved detection methods, may be required to find SFTSV-positive ticks in this region.

Analyses of Entry Mechanisms of Novel Emerging Viruses Using Pseudotype VSV System

Emerging infectious diseases include newly identified diseases caused by previously unknown organisms or diseases found in new and expanding geographic areas. Viruses capable of causing clinical disease associated with fever and bleeding are referred to as viral hemorrhagic fevers (VHFs). Arenaviruses and Bunyaviruses, both belonging to families classified as VHFs are considered major etiologies of hemorrhagic fevers caused by emerging viruses; having significant clinical and public health impact. Because these viruses are categorized as Biosafety Level (BSL) 3 and 4 pathogens, restricting their use, biological studies including therapeutic drug and vaccine development have been impeded. Due to these restrictions and the difficulties in handling such live viruses, pseudotype viruses bearing envelope proteins of VHF viruses have been developed using vesicular stomatitis virus (VSV) as a surrogate system. Here, we report the successful developments of two pseudotype VSV systems; bearing the envelope proteins of Lujo virus and severe fever with thrombocytopenia syndrome (SFTS) virus, both recently identified viruses of the family Arenaviridae and Bunyaviridae, respectively. My presentation will summarize the characterization of the envelope proteins of Lujo virus including its cellular receptor use and cell entry mechanisms. In addition, I will also present a brief introduction of SFTS reported in Japan and the diagnostic studies in progress using these newly pseudotype VSV system.

Analyses of entry mechanisms of novel emerging viruses using pseudotype VSV system

Emerging infectious diseases include newly identified diseases caused by previously unknown organisms or diseases found in new and expanding geographic areas. Viruses capable of causing clinical disease associated with fever and bleeding are referred to as viral hemorrhagic fevers (VHFs). Arenaviruses and Bunyaviruses, both belonging to families classified as VHFs are considered major etiologies of hemorrhagic fevers caused by emerging viruses; having significant clinical and public health impact. Because these viruses are categorized as Biosafety Level (BSL) 3 and 4 pathogens, restricting their use, biological studies including therapeutic drug and vaccine development have been impeded. Due to these restrictions and the difficulties in handling such live viruses, pseudotype viruses bearing envelope proteins of VHF viruses have been developed using vesicular stomatitis virus (VSV) as a surrogate system. Here, we report the successful developments of two pseudotype VSV systems; bearing the envelope proteins of Lujo virus and severe fever with thrombocytopenia syndrome (SFTS) virus, both recently identified viruses of the family Arenaviridae and Bunyaviridae, respectively. My presentation will summarize the characterization of the envelope proteins of Lujo virus including its cellular receptor use and cell entry mechanisms. In addition, I will also present a brief introduction of SFTS reported in Japan and the diagnostic studies in progress using these newly pseudotype VSV system.