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.     

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.     

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.     

The nucleoprotein of severe fever with thrombocytopenia syndrome virus processes a stable hexameric ring to facilitate RNA encapsidation

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 ? resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identifi ed through mutational analysis and identifi ed to have a signifi cant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The fi ndings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.     

Isolation,characterization, and phylogenic analysis of three new severe fever with thrombocytopenia syndrome bunyavirus strains derived from Hubei Province,China

Hubei Province is a major epidemic area of severe fever with thrombocytopenia syndrome bunyavirus(SFTSV) in China. However, to date, a few SFTSV strains have been isolated from Hubei Province, preventing effective studies of epidemic outbreaks. Here, we report three confirmed patients(2015–2016) with typical symptoms of severe fever with thrombocytopenia syndrome disease(SFTS) who were farmers resident in different regions in Hubei Province. Three new SFTSV strains were isolated from the serum samples of each patient. Characterization of viral growth properties showed that there were no significant differences in virus production. All strains were completely sequenced, and phylogenetic analysis showed that unlike the other strains from Hubei province, which belonged to the SFTSV C3 genotype, one of the three strains belonged to the SFTSV C2 genotype. These results suggested that multiple SFTSV genotypes have been circulating in Hubei Province, providing insights into SFTSV evolution and improving our understanding of SFTSV prevalence in Hubei Province.     

Molecular Evolution and Spatial Transmission of Severe Fever with Thrombocytopenia Syndrome Virus Based on Complete Genome Sequences

Severe fever with thrombocytopenia syndrome virus (SFTSV) was a novel tick-borne bunyavirus that caused hemorrhagic fever with a high fatality rate in East Asia. In this study we analyzed the complete genome sequences of 122 SFTSV strains to determine the phylogeny, evolution and reassortment of the virus. We revealed that the evolutionary rate of three genome segments were different, with highest in the S segment and lowest in the L segment. The SFTSV strains were phylogenetically classified into 5 lineages (A, B, C, D and E) with each genome segment. SFTSV strains from China were classified in all 5 lineages, strains from South Korea were classified into 3 lineages (A, D, and E), and all strains from Japan were classified in only linage E. Using the average evolutionary rate of the three genome segments, we found that the extant SFTSV originated 20–87 years ago in the Dabie Mountain area in central China. The viruses were then transmitted to other areas of China, Japan and South Korea. We also found that six SFTSV strains were reassortants. Selection pressure analysis suggested that SFTSV was under purifying selection according to the four genes (RNA-dependent RNA polymerase, glycoprotein, nucleocapsid protein, non-structural protein), and two sites (37, 1033) of glycoproteins were identified as being under strong positive selection. We concluded that SFTSV originated in central China and spread to other places recently and the virus was under purifying selection with high frequency of reassortment.     

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.     

An emerging hemorrhagic fever in China caused by a novel bunyavirus SFTSV

Severe fever with thrombocytopenia syndrome(SFTS) is an emerging hemorrhagic fever in rural areas of China and is caused by a new bunyavirus,SFTSV,named after the disease.The transmission vectors and animal hosts of SFTSV are unclear.Ticks are the most likely transmission vectors and domestic animals,including goats,dogs,and cattle,are potential amplifying hosts of SFTSV.The clinical symptoms of SFTS are nonspecific,but major symptoms include fever,gastrointestinal symptoms,myalgia,dizziness,joint pain,chills,and regional lymphadenopathy.The most common abnormalities in laboratory test results are thrombocytopenia(95%),leukocytopenia(86%),and elevated levels of serum alanine aminotransferase,aspartate aminotransferase,creatine kinase,and lactate dehydrogenase.The fatality rate for SFTS is 12% on average,and the annual incidence of the disease is approximately five per 100000 of the rural population.     

SFTSV infection in rodents and their ectoparasitic chiggers

SFTSV, a tick-borne bunyavirus causing a severe hemorrhagic fever termed as severe fever with thrombocytopenia syndrome (SFTS). To evaluate the potential role of rodents and its ectoparasitic chiggers in the transmission of SFTSV, we collected wild rodents and chiggers on their bodies from a rural area in Qingdao City, Shandong Province, China in September 2020. PCR amplification of the M and L segments of SFTSV showed that 32.3% (10/31) of rodents and 0.2% (1/564) of chiggers (Leptotrombidium deliense) from the rodents were positive to SFTSV. Our results suggested that rodents and chiggers may play an important role in the transmission of SFTSV, although the efficiency of chiggers to transmit SFTSV needs to be further investigated experimentally.     

Depletion but Activation of CD56dimCD16+ NK Cells in Acute Infection with Severe Fever with Thrombocytopenia Syndrome Virus

Virologica Sinica - Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high mortality (12%–30%). The mechanism by which the SFTS bunyavirus (SFTSV)...     

Establishment of a Reverse Genetic System of Severe Fever with Thrombocytopenia Syndrome Virus Based on a C4 Strain

Virologica Sinica - Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes hemorrhagic fever-like disease (SFTS) in humans with a case fatality...     

发热伴血小板减少综合征布尼亚病毒结构和非结构蛋白表达研究

发热伴血小板减少综合征布尼亚病毒(SFTSV)是我国2010年新发现的新布尼亚病毒,可导致人类严重发热伴血小板减少综合征。SFTS新布尼亚病毒全基因组已解析,但病毒分子生物学结构蛋白特征及功能尚需更多研究。本文通过蔗糖密度梯度离心确定发热伴血小板减少综合征布尼亚病毒(HB29株)病毒颗粒的沉降密度及超离纯化条件,得出该病毒颗粒在蔗糖中的沉降密度为1.135g/mL。利用PCR方法扩增SFTSV病毒株HB29株病毒RNA聚合酶(RdRp)、糖蛋白前体蛋白(M)、包膜糖蛋白(Gn)、包膜糖蛋白(Gc)、核蛋白(NP)及非结构蛋白(NSs)的编码区基因片段,分别克隆入真核表达载体pcDNA5/FRT或VR1012,在293T细胞上获得上述基因表达。通过SDS-PAGE分析纯化病毒颗粒和重组蛋白,并通过免疫印迹(Western blotting)和间接免疫荧光(IFA)确定蛋白活性和分子量。本研究结果将有利于对新布尼亚病毒分子生物学特征的认识,为后期研究提供基础。     

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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A multicenter non-randomized,uncontrolled single arm trial for evaluation of the efficacy and the safety of the treatment with favipiravir for patients with severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS) is a bunyavirus infection with high mortality. Favipiravir has shown effectiveness in preventing and treating SFTS virus (SFTSV) infection in animal models. A multicenter non-randomized, uncontrolled single arm trial was conducted to collect data on the safety and the effectiveness of favipiravir in treatment of SFTS patients. All participants received favipiravir orally (first-day loading dose of 1800 mg twice a day followed by 800 mg twice a day for 7–14 days in total). SFTSV RT-PCR and biochemistry tests were performed at designated time points. Outcomes were 28-day mortality, clinical improvement, viral load evolution, and adverse events (AEs). Twenty-six patients were enrolled, of whom 23 were analyzed. Four of these 23 patients died of multi-organ failure within one week (28-day mortality rate: 17.3%). Oral favipiravir was well tolerated in the surviving patients. AEs (abnormal hepatic function and insomnia) occurred in about 20% of the patients. Clinical symptoms improved in all patients who survived from a median of day 2 to day10. SFTSV RNA levels in the patients who died were significantly higher than those in the survivors (p = 0.0029). No viral genomes were detectable in the surviving patients a median of 8 days after favipiravir administration. The 28-day mortality rate in this study was lower than those of the previous studies in Japan. The high frequency of hepatic dysfunction as an AE was observed. However, it was unclear whether this was merely a side effect of favipiravir, because liver disorders are commonly seen in SFTS patients. The results of this trial support the effectiveness of favipiravir for patients with SFTS.     

Infection of humanized mice with a novel phlebovirus presented pathogenic features of severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne emerging phlebovirus with high mortality rates of 6.0 to 30%. SFTSV infection is characterized by high fever, thrombocytopenia, leukopenia, hemorrhage and multiple organ failures. Currently, specific therapies and vaccines remain elusive. Suitable small animal models are urgently needed to elucidate the pathogenesis and evaluate the potential drug and vaccine for SFTSV infection. Previous models presented only mild or no pathogenesis of SFTS, limiting their applications in SFTSV infection. Therefore, it is an urgent need to develop a small animal model for the investigation of SFTSV pathogenesis and evaluation of therapeutics. In the current report, we developed a SFTSV infection model based on the HuPBL-NCG mice that recapitulates many pathological characteristics of SFTSV infection in humans. Virus-induced histopathological changes were identified in spleen, lung, kidney, and liver. SFTSV was colocalized with macrophages in the spleen and liver, suggesting that the macrophages in the spleen and liver could be the principle target cells of SFTSV. In addition, histological analysis showed that the vascular endothelium integrity was severely disrupted upon viral infection along with depletion of platelets. In vitro cellular assays further revealed that SFTSV infection increased the vascular permeability of endothelial cells by promoting tyrosine phosphorylation and internalization of the adhesion molecule vascular endothelial (VE)–cadherin, a critical component of endothelial integrity. In addition, we found that both virus infection and pathogen-induced exuberant cytokine release dramatically contributed to the vascular endothelial injury. We elucidated the pathogenic mechanisms of hemorrhage syndrome and developed a humanized mouse model for SFTSV infection, which should be helpful for anti-SFTSV therapy and pathogenesis study.     

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.     

A highly attenuated vaccinia virus strain LC16m8-based vaccine for severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 10³ TCID₅₀ and 10⁵ TCID₅₀. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS.     

SNX11 Identified as an Essential Host Factor for SFTS Virus Infection by CRISPR Knockout Screening

Severe fever with thrombocytopenia syndrome virus(SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome(SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. Using a genome-wide CRISPR-based screening strategy, we identified a host cellular protein, sorting nexin 11(SNX11) which is involved in the intracellular endosomal trafficking pathway, as an essential cell factor for SFTSV infection. An SNX11-KO HeLa cell line was established, and SFTSV replication was significantly reduced. The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum(ER) or Golgi apparatus. pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells, and lysosomal-associated membrane protein 1(LAMP1) expression was significantly elevated in the SNX11-KO cells. Overall,these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11. Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting, membrane fusion, and other endocytic machinery.