Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein

Background

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas.

Methodology/Principal Findings

We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID₅₀/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>10⁵ copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<10⁵ copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV.

Conclusions

The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia.     

Age Is a Critical Risk Factor for Severe Fever with Thrombocytopenia Syndrome

Background

Severe Fever with Thrombocytopenia Syndrome (SFTS) is an emerging infectious disease in East Asia. SFTS is a tick borne hemorrhagic fever caused by SFTSV, a new bunyavirus named after the syndrome. We investigated the epidemiology of SFTS in Laizhou County, Shandong Province, China.

Methods

We collected serum specimens of all patients who were clinically diagnosed as suspected SFTS cases in 2010 and 2011 in Laizhou County. The patients'' serum specimens were tested for SFTSV by real time fluorescence quantitative PCR (RT-qPCR). We collected 1,060 serum specimens from healthy human volunteers by random sampling in Laizhou County in 2011. Healthy persons'' serum specimens were tested for specific SFTSV IgG antibody by ELISA.

Results

71 SFTS cases were diagnosed in Laizhou County in 2010 and 2011, which resulted in the incidence rate of 4.1/100,000 annually. The patients ranged from 15 years old to 87 years old and the median age of the patients were 59 years old. The incidence rate of SFTS was significantly higher in patients over 40 years old and fatal cases only occurred in patients over 50 years old. 3.3% (35/1,060) of healthy people were positive to SFTSV IgG antibody. The SFTSV antibody positive rate was not significantly different among people at different age groups.

Conclusion

Our results revealed that seroprevalence of SFTSV in healthy people in Laizhou County was not significantly different among age groups, but SFTS patients were mainly elderly people, suggesting that age is the critical risk factor or determinant for SFTS morbidity and mortality.     

A new luciferase immunoprecipitation system assay provided serological evidence for missed diagnosis of severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV) infection, was first reported in 2010 in China with an initial fatality of up to 30%. The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy. In this study, a new luciferase immunoprecipitation system (LIPS) assay based on pREN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase (Rluc), was established and used to investigate the levels of antibody responses to SFTSV. Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019. The results showed that 82 of the 464 patients (17.7%) had antibody response to SFTSV, which were further supported by immunofluorescence assays (IFAs). Further, qRT-PCR and microneutralization tests showed that among the 82 positive cases, 15 patients had viremia, 10 patients had neutralizing antibody, and one had both (totally 26 patient). However, none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations. All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases. The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019, and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.     

测定呼吸道合胞病毒抗体方法敏感性的比较

用ELISA、间接免疫荧光和中和试验三种方法测定兔免疫血清的呼吸道合胞病毒抗体。结果中和试验测定的免疫血清几何平均抗体滴度为1:32;间接免疫荧光试验为1:508,比中和试验高16倍;ELISA测得的抗体滴度1:4,000～1:5,000,为中和试验的125～156倍。以ELISA敏感性最高,间接免疫荧光试验次之,中和试验景差。     

Deep sequencing and phylogenetic analysis of severe fever with thrombocytopenia syndrome virus from the tick,Haemaphysalis longicornis,in Korea

The Asian longhorned tick, Haemaphysalis longicornis, is widely distributed in China, Japan, and Korea and may transmit infectious diseases. Severe fever with thrombocytopenia syndrome (SFTS) is an important tick‐borne disease caused by the SFTS virus (SFTSV). Deep sequencing to confirm the presence of SFTSV in ticks has not been reported in Korea. To detect SFTSV, RNA was extracted from tick samples and analyzed using high‐throughput deep sequencing. Based on BLASTN results, numerous SFTSV reads were identified. Moreover, a nearly complete genome of SFTSV (JNU‐1 isolate) was obtained using Sanger sequencing. The genome of the JNU‐1 isolate includes three segments of 6,286, 3,299 and 1,642 nucleotides (nt) termed large (L), medium (M), and small (S), respectively. Also, phylogenetic and recombination analyses for each segment of SFTSV were performed using the JNU‐1 isolate. The three segments of JNU‐1 isolate were closely related to the genotype B known human‐derived Korean SFTSV isolate; the JNU‐1 isolate showed no recombination sites with other isolates. This study is the first report of detection of SFTSV from ticks using deep sequencing in Korea and provides information on the genetic diversity of SFTSV in East Asia.     

Ecology of the Tick-Borne Phlebovirus Causing Severe Fever with Thrombocytopenia Syndrome in an Endemic Area of China

Background

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a tick-borne phlebovirus in family Bunyaviridae. Studies have found that humans, domestic and wildlife animals can be infected by SFTSV. However, the viral ecology, circulation, and transmission remain largely unknown.

Methodology/Principal Findings

Sixty seven human SFTS cases were reported and confirmed by virus isolation or immunofluorescence assay between 2011 and 2014. In 2013–2014 we collected 9,984 ticks from either vegetation or small wild mammals in the endemic area in Jiangsu, China, and detected SFTSV-RNA by real-time RT-PCR in both questing and feeding Haemaphysalis longicornis and H. flava. Viral RNA was identified in larvae of H. longicornis prior to a first blood meal, which has never been confirmed previously in nature. SFTSV-RNA and antibodies were also detected by RT-PCR and ELISA, respectively, in wild mammals including Erinaceus europaeus and Sorex araneus. A live SFTSV was isolated from Erinaceus europaeus captured during the off tick-feeding season and with a high SFTSV antibody titer. Furthermore, SFTSV antibodies were detected in the migratory birds Anser cygnoides and Streptopelia chinensis using ELISA.

Conclusions/Significance

The detection of SFTSV-RNA in non-engorged larvae indicated that vertical transmission of SFTSV in H. longicornis might occur in nature, which suggests that H. longicornis is a putative reservoir host of SFTSV. Small wild mammals such as Erinaceus europaeus and Sorex araneus could be infected by SFTSV and may serve as natural amplifying hosts. Our data unveiled that wild birds could be infected with SFTSV or carry SFTSV-infected ticks and thus might contribute to the long-distance spread of SFTSV via migratory flyways. These findings provide novel insights for understanding SFTSV ecology, reservoir hosts, and transmission in nature and will help develop new measures in preventing its rapid spread both regionally and globally.     

Current status of severe fever with thrombocytopenia syndrome in China

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus (SFTSV). SFTSV is associated with a high mortality rate and has been reported in China, South Korea and Japan. SFTSV undergoes rapid changes owing to evolution, gene mutations, and reassortment between different strains of SFTSV. In this review, we summarize the recent cases and general properties of SFTS, focusing on the epidemiology, genetic diversity, clinical features, and diagnostics of SFTSV in China. From 2010 to October 2016, SFTS cases were reported in 23 provinces of China, with increased numbers yearly. Infection and death cases are mainly found in central China, where the Haemaphysalis longicornis ticks are spread. The national average mortality rate of SFTS infection was 5.3%, with higher risk to elder people. The main epidemic period was from May to July, with a peak in May. Thus, SFTS reminds a significant public health problem, and development of prophylactic vaccines and effective antiviral drugs will be highly needed.

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Comparison of commercially available and novel West Nile virus immunoassays for detection of seroconversion in pig-tailed macaques (Macaca nemestrina)

We report the assessment and validation of an NS1 epitope-blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to West Nile virus (WNV) in macaques. Sera from naturally infected Macaca nemestrina were tested by ELISA and plaque reduction neutralization test (PRNT). Results were correlated with hemagglutination inhibition (HAI) data. Our results demonstrate that the blocking ELISA rapidly and specifically detects WNV infection in M. nemestrina. In addition, the diagnostic value of 7 commercially available immunoassays (PanBio immunoglobulin [Ig] M ELISA, PanBio IgG ELISA, PanBio immunofluorescence assay (IFA), InBios IgG ELISA, InBios IgM ELISA, Focus Diagnostics IgG ELISA, and Focus Diagnostics IgM ELISA) in M. nemestrina was evaluated and compared with that of the epitope-blocking ELISA. The PanBio IgG ELISA was found to effectively diagnose WNV exposure in M. nemestrina. Further, PanBio IFA slides are fast and reliable screening tools for diagnosing flaviviral exposure in M. nemestrina.     

家养动物体表蜱中发热伴血小板减少综合征布尼亚病毒分离及鉴定

为了解发热伴血小板减少综合征布尼亚病毒(SFTSV)的传播机制,采集了山东疫区家养牛、羊和狗等动物体表蜱,分类鉴定后,通过Real-time PCR筛查、病毒分离培养和基因组序列分析等方法分离鉴定蜱中的病毒。所采集的蜱,以长角血蜱为主,占91.4%。其中3头SFTSV核酸检测阳性,阳性率为2.14%,并在其中一份羊体表蜱标本中分离到SFTSV病毒,命名为SDLZTick12。序列分析显示与我国在不同省份患者标本中分离的病毒全基因序列具有高度同源性,且病毒的抗原性和生长特性与人源病毒相同。本研究首次在山东疫区蜱中分离到新型布尼亚病毒,并与人源病毒进行了系统比较研究,提示蜱可能为该新病原体的传播媒介,对疾病的防控具有重要的指导意义。     

Novel SFTSV Phylogeny Reveals New Reassortment Events and Migration Routes

Severe fever with thrombocytopenia syndrome virus(SFTSV), the causative agent of a febrile human disease, was first identified from central and eastern provinces in China, and later in Japan and South Korea. Hubei Province is one of the major SFTS epidemic areas in the central part of China. This study reported the isolation of 11 new SFTSV strains from patients in Hubei Province collected in 2017. Extensive phylogenetic analyses were conducted based on the complete coding sequences of SFTSV segments including the new strains. It was suggested that five different SFTSV genotypes were circulating in Hubei, and 15 reassortment patterns and migration pathways correlated with each genotype were identified, which was more than previously recognized. Hubei Province was more involved in the evolutionary events of SFTSV than that previously thought in which the evolutionary events of SFTSV were reported to be independent from those in other epidemic regions. Further divergence of SFTSV strains was suggested by pairwise comparison of SFTSV sequences from each genotype and sequence identity normalized to representative strain in genotype C1. Subsequently,amino acid variations specific for genotype(s), strain(s), or cluster(s) were inspected, which may be related to differential biological activity of SFTSV strains/genotypes. In conclusion, we analyzed the current status of SFTSV phylogeny in Hubei Province and discussed the possible events correlated to SFTSV evolution. It provided an in-depth insight into SFTSV evolution, raising concerns for the use of proper SFTSV strains in future studies.     

Epidemiologic Features and Environmental Risk Factors of Severe Fever with Thrombocytopenia Syndrome,Xinyang, China

Background

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease discovered in rural areas of Central China in 2009, caused by a novel bunyavirus, SFTS virus (SFTSV). The disease usually presents as fever, thrombocytopenia, and leukocytopenia, with case-fatality rates ranging from 2.5% to 30%. Haemaphysalis longicornis was suspected to be the most likely vector of SFTSV. By the end of 2012, the disease had expanded to 13 provinces of China. SFTS patients have been reported in Japan and South Korea, and a disease similar to SFTS has been reported in the United States.

Methodology/Principal Findings

We characterized the epidemiologic features of 504 confirmed SFTS cases in Xinyang Region, the most severely SFTS-afflicted region in China from 2011 to 2012, and assessed the environmental risk factors. All cases occurred during March to November, with the epidemic peaking from May to July. The patients'' ages ranged from 7 to 87 years (median 61 years), and the annual incidence increased with age (χ² test for trend, P<0.001). The female-to-male ratio of cases was 1.58, and 97.0% of the cases were farmers who resided in the southern and western parts of the region. The Poisson regression analysis revealed that the spatial variations of SFTS incidence were significantly associated with the shrub, forest, and rain-fed cropland areas.

Conclusions

The distribution of SFTS showed highly significant temporal and spatial heterogeneity in Xinyang Region, with the majority of SFTS cases being elderly farmers who resided in the southern and western parts of the region, mostly acquiring infection between May and July when H. longicornis is highly active. The shrub, rain-fed, and rain-fed cropland areas were associated with high risk for this disease.     

Severe fever with thrombocytopenia syndrome and its pathogen SFTSV

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever in East Asia with case fatality up to 50%. SFTS is caused by SFTSV, a tick borne bunyavirus. In endemic area in China 1%–3% population was infected with SFTSV, but age is critical risk factor for hospitalization and death of SFTS patients.     

Tilorone confers robust in vitro and in vivo antiviral effects against severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging pathogen, is a tick-borne bunyavirus belonging to the genus Bandavirus in the family Phenuiviridae (Kuhn et al., 2020). This pathogen was first identified in China during the heightened surveillance of acute febrile illness in 2009, and has been reported to cause several outbreaks in eastern Asia areas, including China, Japan, and Korea (Yu et al., 2011). Besides, Vietnam has also reported several confirmed SFTS cases (Tran et al., 2019). The mortality rate in hospitalised patients with SFTSV infection is up to 10%–30%. Moreover, SFTSV has been reported to possibly transmitted by the contact of body fluids from person-to-person, and extensive SFTSV contamination was detected in the patient rooms (Kim et al., 2015). These reports suggest that more stringent isolation measures are needed for the prevention of massive SFTSV outbreak.     

Detection of postvaccination mumps virus antibody by neutralization test, enzyme-linked immunosorbent assay and sensitive hemagglutination inhibition test

A group of 251 children aged 2-3 years given live attenuated mumps virus vaccine PAVIVAC of Czechoslovak production were tested for antiparotitis antibody levels in pre- and postvaccination sera by neutralization test (NT), enzyme-linked immunosorbent assay (ELISA) and sensitive hemagglutination inhibition test, enhanced by heterologous antibody to human immunoglobulin G (E-HIT). The prevaccination findings were as follows: positive ELISA IgG titres, neutralization antibodies and hemagglutination inhibition antibodies were present in, respectively, 35%, 25.9% and 27.9% of the sera. Postvaccination seroconversions were evaluated in 159 susceptible vaccinees whose prevaccination sera had been negative by all three tests. The lowest seroconversion was detected by NT (74.2%), seroconversions by ELISA and E-HIT were appreciably higher (82.4% and 86.8%, respectively). The seven children showing a seroconversion by E-HIT but not by ELISA had a 4 fold increase of anti-mumps ELISA IgG antibodies as well, but the rise of antibody titres was at a level falling in the range below the positivity criterion for ELISA. The statistically evaluated detection rate for antibodies was significantly higher (significance test "t") by ELISA as compared with neutralization test. However, antibody levels (geometric mean titres) were 8-10 times lower in postvaccination sera than in convalescent sera of 30 children with mumps in all three tests.     

Borna disease virus: evidence of naturally-occurring infection in cats in Australia

In Europe, Borna disease virus (BDV) infection has been linked with staggering disease. The aim of this study was serological investigation for BDV infection in Australian cats. De-identified sera were obtained from domestic cats presented at various veterinary clinics. BDV antigen levels were measured by a monoclonal antibody-based ELISA. Antibody to BDV measured semiquantitatively by ELISA was detected in 0.8% of cats from South Australia and 3.2% of animals from NSW Confirmatory assays for ELISA positive samples included Western blot and immunofluorescence assay (IFA) with BDV-specific staining. Seven BDV-antigen positive sera (2.4%) were identified in sera from cats from New South Wales (NSW). In blinded testing, amongst a large number of negative results, repeat submissions over a seven-month period from a cat co-infected with Feline Immunodeficiency Virus (FIV) were BDV-antigen positive. Anti-BDV antibody detected in this cat by ELISA was confirmed by Western blot (p24/ p40/p56) and IFA. For 4 other anti-BDV ELISA-positive samples, specific reactions with BDV proteins were observed by Western blot. Ten other anti-BDV ELISA-positive samples were IFA positive. These data provide consistent serological evidence that, while horses in Australia are free of BDV infection, there may be a low rate of BDV infection in cats.     

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax and its use in a seroepidemiological survey of the Eastern Caribbean Basin

An enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against a South American (New World) strain of Trypanosoma vivax was developed and used for mass screening of cattle from 20 islands in the Eastern Caribbean Basin. The sensitivity and specificity of antigens prepared from a bovine-derived field strain and a murine-adapted laboratory strain of T. vivax, both of New World origin, were compared using an indirect fluorescent antibody (IFA) test, and an antigen prepared from the murine-adapted strain was subsequently used to develop an ELISA test. The results of the ELISA test were then compared with the results of a concurrently run IFA test. There was no cross-reactivity with either test using serum from a Trypanosoma theileri-infected cow. Both tests were weakly cross-reactive with sera from a T. brucei-infected steer, and the IFA test was moderately cross-reactive with several serum samples from a T. evansi-infected steer. For bovine sera collected from herds on islands in the Eastern Caribbean region, only five of 640 tested positive with the ELISA test. Thirty five of 653 sera tested were positive by IFA although the fluorescence elicited was weak as compared to that elicited by sera from known infected animals. Sera collected from 27 cattle in a region known to be free of T. vivax (OH, U.S.A) were negative with the ELISA test, whereas seven of 30 sera from a herd in French Guiana known to be infected with T. vivax were positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

SARS-CoV-2重组S1和S蛋白疫苗诱导保护性免疫的研究*

目的:评价新型冠状病毒(SARS-CoV-2)重组S1蛋白和S蛋白疫苗对SARS-CoV-2的免疫保护效果。方法:将SARS-CoV-2重组S1蛋白和S蛋白分别联合氢氧化铝佐剂以0.1 μg/只、1 μg/只、5 μg/只、10 μg/只不同剂量接种6~8周BALB/c纯系健康雌性小鼠。第二次免疫后采血通过酶联免疫吸附试验(ELISA)检测血清中IgG抗体效价,通过假病毒中和试验比较免疫小鼠血清对SARS-CoV-2野生型株(WT)、英国株(B.1.1.7)、巴西株(P.1)、印度株(B.1.617.2)、Mu毒株(B.1.621)和南非株(501Y.V2-1)六种假病毒毒株中和活性效价,取脾细胞通过酶联免疫斑点技术(ELISpot)检测免疫小鼠的细胞免疫水平。结果:SARS-CoV-2重组S和S1蛋白都能诱导小鼠产生较强的IgG抗体水平。免疫S1蛋白的小鼠血清对SARS-CoV-2野生型株、英国株、巴西株有明显的中和活性,免疫S蛋白的小鼠血清除了对SARS-CoV-2野生型株、英国株、巴西株有明显中和活性之外,对印度株也有明显的中和活性,两种蛋白质免疫的小鼠血清均对野生型株中和效果最强。S蛋白免疫的小鼠脾细胞能够显著诱导出γ干扰素(IFN-γ)和白介素-4(IL-4)的产生。S蛋白诱导产生的IgG抗体、中和抗体、细胞免疫水平均高于S1。结论:SARS-CoV-2重组S蛋白疫苗能够诱导产生较强的保护性免疫应答。     

不同载体表达的人类细小病毒B19壳蛋白VP2的抗原性比较研究

构建表达质粒pcDNA3 VP2,将其转染CHO细胞建立了稳定表达的细胞系;用间接免疫荧光法和Western印迹证明了表达的VP2蛋白的特异性。对昆虫杆状病毒系统表达的VP2蛋白作初步纯化。分别用由大肠杆菌、CHO细胞和昆虫杆状病毒表达系统表达的VP2蛋白,以间接免疫荧光法和ELISA法检测人群血清中的VP2抗体,结果表明,间接免疫荧光法的敏感性高于ELISA法。