NSs of the mildly virulent sandfly fever Sicilian virus is unable to inhibit interferon signaling and upregulation of interferon-stimulated genes.

Phleboviruses (order Bunyavirales, family Phenuiviridae) are globally emerging arboviruses with a wide spectrum of virulence. Sandfly fever Sicilian virus (SFSV) is one of the most ubiquitous members of the genus Phlebovirus and associated with a self-limited, incapacitating febrile disease in travellers and military troops. The phleboviral NSs protein is an established virulence factor, acting as antagonist of the antiviral interferon (IFN) system. Consistently, we previously reported that SFSV NSs targets the induction of IFN mRNA synthesis by specifically binding to the DNA-binding domain of the IFN transcription factor IRF3. Here, we further characterized the effect of SFSV and its NSs towards IFN induction, and evaluated its potential to affect the downstream IFN-stimulated signalling and the subsequent transactivation of antiviral interferon-stimulated genes (ISGs). We found that SFSV dampened, but did not entirely abolish type I and type III IFN induction. Furthermore, SFSV NSs did not affect IFN signalling, resulting in substantial ISG expression in infected cells. Hence, although SFSV targets IRF3 to reduce IFN induction, it is not capable of entirely disarming the IFN system in the presence of high basal IRF3 and/or IRF7 levels, and we speculate that this significantly contributes to its low level of virulence.

Clinically Important Phleboviruses and Their Detection in Human Samples.

The detection of phleboviruses (family: Phenuiviridae) in human samples is challenged by the overall diversity and genetic complexity of clinically relevant strains, their predominantly nondescript clinical associations, and a related lack of awareness among some clinicians and laboratorians. Here, we seek to inform the detection of human phlebovirus infections by providing a brief introduction to clinically relevant phleboviruses, as well as key targets and approaches for their detection. Given the diversity of pathogens within the genus, this report focuses on diagnostic attributes that are generally shared among these agents and should be used as a complement to, rather than a replacement of, more detailed discussions on the detection of phleboviruses at the individual virus level.

Early-Warning Immune Predictors for Invasive Pulmonary Aspergillosis in Severe Patients With Severe Fever With Thrombocytopenia Syndrome.

Aspergillus-related disease was confirmed to be associated with immune disorders in patients, severe patients with severe fever with thrombocytopenia syndrome (SFTS) infected by novel phlebovirus were confirmed to have severe immune damage including cellular immunosuppression and cytokine storms. Secondary invasive pulmonary aspergillosis (IPA) in severe SFTS patients can increase fatality rate. This study investigated early-warning predictive factors of secondary IPA in severe SFTS patients. Receiver operating characteristic analysis was used to assess the value of immune parameters to predict IPA in SFTS patients. The cut-off values of CD4+ and CD8+ T-cell counts to predict IPA were 68 and 111 cells/mm3, with sensitivities of 82.6% and 72%, and specificities of 56.7% and 83.3%, respectively. Cut-off values of IL-6, TNF-α, IL-8, and IL-10 to predict IPA incidence in critically ill SFTS patients were 99 pg/mL, 63 pg/mL, 120 pg/mL, and 111 pg/mL, with sensitivities of 90.0%, 86.7%, 83.3% and 90.0% and specificities of 80.4%, 71.7%, 82.6% and 65.2%, respectively. Lower CD4+ and CD8+ T-cells counts, higher levels of IL-6, TNF-α, IL-8 and IL-10, higher incidence of pancreatic and renal damage, early antibacterial therapy of carbapenems, and intensive care unit admission were risk factors of IPA in SFTS patients. Multivariate logistic regression analysis indicated counts of CD4+ T-cells <68 cells/mm3 combined with CD8+ T-cells <111 cells/mm3 (odds ratio [OR] 0.218, 95% confidence interval [CI] 0.059-0.803, p=0.022), IL-6 >99 pg/ml combined with IL-10 >111 pg/ml (OR 17.614, 95% CI 2.319-133.769, p=0.006), and brain natriuretic peptide level >500 pg/ml (OR 13.681, 95% CI 1.994-93.871, p=0.008) were independent risk factors for IPA in SFTS patients. The mortality in the IPA group was significantly higher than in the non-IPA group (p=0.001). Early antifungal treatment of IPA patients was significantly associated with improved survival (log-rank, p=0.022). Early diagnosis of IPA and antifungal treatment can improve the prognosis of SFTS patients. Besides, we speculate SFTS may be as a host factor for IPA.

Seroprevalence of Toscana Virus and Sandfly Fever Sicilian Virus in European Bat Colonies Measured Using a Neutralization Test.

Toscana phlebovirus (TOSV) and Sicilian phlebovirus (SFSV) are endemic in the Mediterranean area where they are transmitted to humans by infected sandflies. Vertebrates of several species have been postulated to act as reservoirs of these viruses, but convincing evidence is still awaited. Among them, bats have been suggested, however documented evidence is lacking. Here we tested a total of 329 bats belonging to eight species collected from twelve localities in southern Spain for the presence of neutralizing antibodies specific to TOSV and SFSV. Positive sera were detected in Schreiber's long-fingered bat (Miniopterus schreibersii), mouse-eared Myotis (Myotis myotis), European free-tailed bat (Tadarida teniotis), and common serotine (Eptesicus serotinus) with the latter showing the highest prevalence rates for SFSV (22.6%) and TOSV (10%). There was no difference between females and males. Results suggest that bats are not likely to play a major role in the natural cycle of these two sandfly-borne phleboviruses. However, large breeding colonies of bats can be used as sentinels for surveillance of the presence of such viruses in a given locality. In addition, capture-recapture studies should be initiated in order to understand better the dynamics of TOSV and SFSV in bat populations.

Severe fever with thrombocytopenia syndrome virus targets B cells in lethal human infections.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever caused by a tick-borne banyangvirus and is associated with high fatality. Despite increasing incidence of SFTS and serious public health concerns in East Asia, the pathogenesis of lethal SFTS virus (SFTSV) infection in humans is not fully understood. Numbers of postmortem examinations to determine target cells of the viral infection have so far been limited. Here we showed that B cells differentiating into plasmablasts and macrophages in secondary lymphoid organs were targets for SFTSV at the end stage of lethal infection, and the majority of SFTSV-infected cells were B cell-lineage lymphocytes. In affected individuals, B cell-lineage lymphocytes with SFTSV infection were widely distributed in both lymphoid and nonlymphoid organs, and infiltration of these cells into the capillaries of the organs could be observed occasionally. Moreover, a human plasmablastic lymphoma cell line, PBL-1, was susceptible to SFTSV propagation and had a similar immunophenotype to that of target cells of SFTSV in fatal SFTS. PBL-1 can therefore provide a potential in vitro model for human SFTSV infection. These results extend our understanding of the pathogenesis of human lethal SFTSV infection and can facilitate the development of SFTSV countermeasures.

Quality by Design-Driven Process Development of Severe Fever With Thrombocytopenia Syndrome Vaccine.

Owing to the biological activity of the vaccine, the complicated production process, sterility, and uniformity of the product, the producing process of the vaccine is complicated and the product quality hard to control. In recent years, with the development of basic science such as cell biology, molecular biology, and metabolic engineering, bioprocess engineering research has developed rapidly. Therefore, U.S. Food and Drug Administration and European Medicines Agency conduct stringent control over the development of biomedical process engineering and product quality. This case study describes an example of Quality by Design-driven process development for manufacturing a human vaccine produced with Vero cells. Cell density in harvest fermentation broth and antigenic titer were chosen as 2 critical quality attributes. The study through 3 rounds design of experiment revealed that H2O2 and cell boost 4 had a significant effect on antigenic titer. Ethanolamine had significant improvement in the final concentration of cells. Through the Monte Carlo simulation, the design spaces and control space of process parameters were determined. A successful validation in a bioreactor was executed to verify the results of a spinner flask. Our investigation presents a successful case of Quality by Design principle, which encourages other researchers to combine the methodology into other biopharmaceutical manufacturing process.

Development of a SFTSV DNA vaccine that confers complete protection against lethal infection in ferrets.

Although the incidence of severe fever with thrombocytopenia syndrome virus (SFTSV) infection has increased from its discovery with a mortality rate of 10-20%, no effective vaccines are currently available. Here we describe the development of a SFTSV DNA vaccine, its immunogenicity, and its protective efficacy. Vaccine candidates induce both a neutralizing antibody response and multifunctional SFTSV-specific T cell response in mice and ferrets. When the vaccine efficacy is investigated in aged-ferrets that recapitulate fatal clinical symptoms, vaccinated ferrets are completely protected from lethal SFTSV challenge without developing any clinical signs. A serum transfer study reveals that anti-envelope antibodies play an important role in protective immunity. Our results suggest that Gn/Gc may be the most effective antigens for inducing protective immunity and non-envelope-specific T cell responses also can contribute to protection against SFTSV infection. This study provides important insights into the development of an effective vaccine, as well as corresponding immune parameters, to control SFTSV infection.

Severe Fever With Thrombocytopenia Syndrome Virus-Induced Macrophage Differentiation Is Regulated by miR-146.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever with a high mortality rate in humans, which is caused by SFTS virus (SFTSV), a novel phlebovirus in the Bunyaviridae family, is tick borne and endemic in Eastern Asia. Previous study found that SFTSV can infect and replicate in macrophages in vivo and in vitro. However, the role of macrophages in virus replication and the potential pathogenic mechanisms of SFTSV in macrophage remain unclear. In this study, we provided evidence that the SFTSV infection drove macrophage differentiation skewed to M2 phenotype, facilitated virus shedding, and resulted in viral spread. We showed evidence that miR-146a and b were significantly upregulated in macrophages during the SFTSV infection, driving the differentiation of macrophages into M2 cells by targeting STAT1. Further analysis revealed that the elevated miR-146b but not miR-146a was responsible for IL-10 stimulation. We also found that SFTSV increased endogenous miR-146b-induced differentiation of macrophages into M2 cells mediated by viral non-structural protein (NSs). The M2 skewed differentiation of macrophages may have important implication to the pathogenesis of SFTS.

The proportion, origin and pro-inflammation roles of low density neutrophils in SFTS disease.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a novel emerging viral infectious disease. We explored the percentage, origins and functional roles of low density neutrophils (LDNs), one of the neutrophils subsets, in SFTS. METHODS: The LDNs and normal density neutrophils (NDNs) from blood of SFTS and normal volunteers which were collected separately. The percentage, origins and the phagocytic capability of SFTS viral (SFTSV) of LDNs were investigated by flow cytometry and real time PCR. The capacity of LDNs to secrete cytokines and to damage endothelial cells was assessed by ELISA and flow cytometry. RESULTS: We observed that the proportion of LDNs increased dramatically compared with the healthy donors and became the dominant circulating neutrophil population in SFTS patients. Interestingly, the NDNs from the normal donors could switch to LDNs under the SFTS environment. Moreover, SFTSV load in LDNs was significantly higher than that of NDNs in the severe SFTS patients. In addition, the LDNs secreted much higher levels of pro-inflammatory cytokines than NDNs in SFTS and could induce endothelial cell injury. CONCLUSION: The NDNs can be converted to LDNs. This conversion mechanism could become the source of LDNs. The LDNs in severe SFTS patient could engulf more SFTSV and exhibit pro-inflammation functions. TRIAL REGISTRATION: The Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IORG No: IORG0003571) gave a final APPROVAL for the study.

Neutralizing antibodies to Severe Fever with Thrombocytopenia Syndrome Virus in general population, Shandong Province, China.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus (SFTSV) in East Asia. The research on seroprevalence of SFTSV in healthy people and risk factors had been detailed. However, the levels of neutralizing antibodies against SFTSV in general population were currently unclear. In the present study, we tested 1375 healthy persons from Penglai County, eastern China, for SFTSV neutralizing antibodies; 0.58% (8/1,375) was positive and the positive rates were not significantly different among people at different age groups, occupations and genders. Besides, a follow-up study was conducted and the titer of neutralizing antibodies decreased over time in all eight people but one, and the neutralizing antibodies of five lasted for the entire study period of seven years. Our results suggesting that subclinical infection or a relatively mild form of SFTS illness is occurring in this population, but a small percentage of sera have neutralizing capacity to SFTSV. Hence, most people are just susceptible to SFTSV infection.

[Sandfly fever-a "neglected" disease]. / Sandmückenfieber ­ eine "vernachlässigte" Krankheit.

A 45-year-old woman presented at the outpatient department of a center for tropical diseases with fever, diarrhea, headache, myalgia, malaise, and an itchy papular rash. She had been on holiday with her family for 11 days in a mountain village in northern Cyprus. The place was infested with a lot of small, stinging flies or mosquitoes. She and her family became sick after they returned home. The physical examination was normal apart from the rash on the inside of the extremities. Significantly elevated transaminases and a slightly increased C­reactive protein level were found in the blood examination. Considering the country of travel, the report of the "stinging flies" and the clinical presentation, sandfly fever was also taken into account as a differential diagnosis for the hepatitis. Antibodies to the sandfly fever Sicilian virus (SFSV) were detected. They showed the typical dynamics during the course of the illness and thus "pappataci fever" was diagnosed. The case report and a short review of up-to-date literature is meant encourage consideration of phlebovirus infection as a possible differential diagnosis in travelers or refugees suffering from severe febrile hepatitis and rash or aseptic viral meningitis after their stay in the Mediterranean area.

Two Conserved Amino Acids within the NSs of Severe Fever with Thrombocytopenia Syndrome Phlebovirus Are Essential for Anti-interferon Activity.

The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) sequesters TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures to inhibit the phosphorylation and nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-ß) production. Although the C-terminal region of SFTSV NSs (NSs66-249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-ß responses, the role of the N-terminal region in antagonizing host antiviral responses remains to be defined. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the SFTSV and heartland virus (HRTV) NSs are essential for suppression of IRF3 phosphorylation and IFN-ß mRNA expression following infection with SFTSV or recombinant influenza virus lacking the NS1 gene. Surprisingly, formation of SFTSV/HRTV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, an association between SFTSV/HRTV NSs and TBK1 is required for suppression of mitochondrial antiviral signaling protein (MAVS)-mediated activation of IFN-ß promoter activity. Although SFTSV NSs did not prevent the ubiquitination of TBK1, it associates with TBK1 through its N-terminal kinase domain (residues 1 to 307) to block the autophosphorylation of TBK1. Furthermore, we found that both wild-type NSs and the 21/23A mutant (NSs in which residues at positions 21 and 23 were replaced with alanine) of SFTSV suppressed NLRP3 inflammasome-dependent interleukin-1ß (IL-1ß) secretion, suggesting that the importance of these residues is restricted to TBK1-dependent IFN signaling. Together, our findings strongly implicate the two conserved amino acids at positions 21 and 23 of SFTSV/HRTV NSs in the inhibition of host interferon responses.IMPORTANCE Recognition of viruses by host innate immune systems plays a critical role not only in providing resistance to viral infection but also in the initiation of antigen-specific adaptive immune responses against viruses. Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus. The 294-amino-acid nonstructural protein (NSs) of SFTSV associates with TANK-binding kinase 1 (TBK1), a key regulator of host innate antiviral immunity, to inhibit interferon beta (IFN-ß) production and enhance viral replication. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the NSs of SFTSV and heartland virus, another tick-borne phlebovirus, are essential for association with TBK1 and suppression of IFN-ß production. Our results provide important insight into the molecular mechanisms by which SFTSV NSs helps to counteract host antiviral strategies.

Low Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus Antibodies in Individuals Living in an Endemic Area in Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infection with a high mortality rate. It is caused by the SFTS virus (SFTSV) and is endemic in some areas in western Japan, including the Kagoshima prefecture. In the present study, healthy individuals living in this prefecture were examined to assess for anti-SFTSV seroprevalence. An initial study was performed using the serum samples collected from a total of 646 individuals living in Kagoshima. At the same time, a questionnaire was used to collect information (such as occupation and a history of tick bite). Enzyme-linked immunosorbent assay and indirect immunofluorescence assay were used for the screening. Finally, the seroprevalence of anti-SFTSV antibodies was confirmed using a neutralization assay. Only 2 (0.3%) out of 646 study participants were positive for anti-SFTSV antibodies. No significant difference was observed between individuals who are at a high or low risk of tick bite in terms of seropositivity. Next, a total of 1,000 serum samples collected from general blood donors by the Japanese Red Cross Kyushu Block Blood Center were tested. None of these samples tested positive for anti-SFTSV antibodies. These results suggest a low seroprevalence of anti-SFTSV antibodies in healthy individuals living in an endemic area in Japan.

RIG-I-Like Receptor and Toll-Like Receptor Signaling Pathways Cause Aberrant Production of Inflammatory Cytokines/Chemokines in a Severe Fever with Thrombocytopenia Syndrome Virus Infection Mouse Model.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a tick-borne phlebovirus of the family Bunyaviridae, SFTS virus (SFTSV). Wild-type and type I interferon (IFN-I) receptor 1-deficient (IFNAR1-/-) mice have been established as nonlethal and lethal models of SFTSV infection, respectively. However, the mechanisms of IFN-I production in vivo and the factors causing the lethal disease are not well understood. Using bone marrow-chimeric mice, we found that IFN-I signaling in hematopoietic cells was essential for survival of lethal SFTSV infection. The disruption of IFN-I signaling in hematopoietic cells allowed an increase in viral loads in serum and produced an excess of multiple inflammatory cytokines and chemokines. The production of IFN-I and inflammatory cytokines was abolished by deletion of the signaling molecules IPS-1 and MyD88, essential for retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) and Toll-like receptor (TLR) signaling, respectively. However, IPS-1-/- MyD88-/- mice exhibited resistance to lethal SFTS with a moderate viral load in serum. Taken together, these results indicate that adequate activation of RLR and TLR signaling pathways under low to moderate levels of viremia contributed to survival through the IFN-I-dependent antiviral response during SFTSV infection, whereas overactivation of these signaling pathways under high levels of viremia resulted in abnormal induction of multiple inflammatory cytokines and chemokines, causing the lethal disease.IMPORTANCE SFTSV causes a severe infectious disease in humans, with a high fatality rate of 12 to 30%. To know the pathogenesis of the virus, we need to clarify the innate immune response as a front line of defense against viral infection. Here, we report that a lethal animal model showed abnormal induction of multiple inflammatory cytokines and chemokines by an uncontrolled innate immune response, which triggered the lethal SFTS. Our findings suggest a new strategy to target inflammatory humoral factors to treat patients with severe SFTS. Furthermore, this study may help the investigation of other tick-borne viruses.

Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis.

The presence of the endogenous Leishmania RNA virus 1 (LRV1) replicating stably within some parasite species has been associated with the development of more severe forms of leishmaniasis and relapses after drug treatment in humans. Here, we show that the disease-exacerbatory role of LRV1 relies on type I IFN (type I IFNs) production by macrophages and signaling in vivo. Moreover, infecting mice with the LRV1-cured Leishmania guyanensis (LgyLRV1- ) strain of parasites followed by type I IFN treatment increased lesion size and parasite burden, quantitatively reproducing the LRV1-bearing (LgyLRV1+ ) infection phenotype. This finding suggested the possibility that exogenous viral infections could likewise increase pathogenicity, which was tested by coinfecting mice with L. guyanensis and lymphocytic choriomeningitis virus (LCMV), or the sand fly-transmitted arbovirus Toscana virus (TOSV). The type I IFN antiviral response increased the pathology of L. guyanensis infection, accompanied by down-regulation of the IFN-γ receptor normally required for antileishmanial control. Further, LCMV coinfection of IFN-γ-deficient mice promoted parasite dissemination to secondary sites, reproducing the LgyLRV1+ metastatic phenotype. Remarkably, LCMV coinfection of mice that had healed from L. guyanensis infection induced reactivation of disease pathology, overriding the protective adaptive immune response. Our findings establish that type I IFN-dependent responses, arising from endogenous viral elements (dsRNA/LRV1), or exogenous coinfection with IFN-inducing viruses, are able to synergize with New World Leishmania parasites in both primary and relapse infections. Thus, viral infections likely represent a significant risk factor along with parasite and host factors, thereby contributing to the pathological spectrum of human leishmaniasis.

Neutralizing Antibodies to Severe Fever with Thrombocytopenia Syndrome Virus 4 Years after Hospitalization, China.

Severe fever with thrombocytopenia syndrome is an emerging hemorrhagic fever disease in eastern Asia, caused by a tickborne bunyavirus. Of 25 patients hospitalized with this disease in China, 100% produced and maintained neutralizing antibodies to severe fever with thrombocytopenia syndrome virus for the study period of 4 years.

Seroprevalence and risk factors of severe fever with thrombocytopenia syndrome virus infection in endemic areas.

BACKGROUND: To determine the seroprevalence, latent infection rate and risk factors for severe fever with thrombocytopenia syndrome virus (SFTSV) infection, a cross-sectional study was conducted in the general population of the Western region of Anhui Province of China from 1 September to 31 December 2014. METHODS: Twelve villages with the highest rates of endemic SFTS infection were selected from six towns in two counties in the western region of Anhui Province. Blood samples were collected and tested for the presence of SFTSV-IgG antibodies by ELISA. Each participant was interviewed using a structured questionnaire before blood collection. Participants with seropositive specimens were further investigated using another structured questionnaire. RESULTS: Of 2126 blood specimens collected, 99 (4.66%) were seropositive for SFTSV. None of the participants had been diagnosed with SFTS before the blood collection or were accompanied by fever, thrombocytopenia and leukocytopenia after blood collection. Multivariate logistic regression model analysis revealed living in areas of uncontrolled vegetation growth, long-term residents of the locality and tick bites as high risk factors for SFTSV infection. CONCLUSIONS: The overall seroprevalence of SFTSV is higher in the western region of Anhui, possibly due to latent infection, with the main risk factors being living in areas of uncontrolled vegetation growth, long-term residents of the locality and tick bites. Further investigations are warranted to clarify the modes of SFTS virus transmission, while vector management, education on tick bite prevention and personal hygiene management should be implemented for high risk groups in endemic areas.

Isolation, full genomic characterization and neutralization-based human seroprevalence of Medjerda Valley virus, a novel sandfly-borne phlebovirus belonging to the Salehabad virus complex in northern Tunisia.

A new phlebovirus, Medjerda Valley virus (MVV), was isolated from one pool of Phlebotomus sp. (Diptera; Psychodidae) sandflies trapped in the vicinity of the Utique site, northern Tunisia. Genetic analysis based on complete coding of genomic sequences of the three RNA segments indicated that MVV is most closely related to members of the Salehabad virus species, where it is the fourth virus for which the complete sequence is available. A seroprevalence study was performed to search for neutralizing antibodies in human sera in the same region. The results demonstrate that in this area, MVV can readily infect humans despite low seroprevalence rates. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our human seroprevalence results, together with high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece. Further studies are needed to investigate the capacity of each specific member of the Salehabad virus species to cause human or animal diseases.

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients. METHODS: The full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system. RESULTS: The native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively. CONCLUSIONS: The rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations.

Characterization of immunological responses in patients with severe fever with thrombocytopenia syndrome: a cohort study in China.

BACKGROUND: The immunological responses of patients with severe fever with thrombocytopenia syndrome (SFTS) remain largely unknown. We aim to study the magnitude and sustainability of host immune responses and their correlation with clinical, virological and hematological parameters. METHODS: A longitudinal cohort study was performed in a SFTS reference hospital. The sequential immunological evaluation was determined for SFTSV infected patients, including anti-SFTSV IgM, IgG antibodies and the lymphocyte subsets. RESULTS: Altogether 298 laboratory-confirmed SFTS cases were analyzed, from whom 55 patients were followed after convalescence. SFTSV specific IgM antibody could be detected at medium of 9 days, surged to peak levels by 4 weeks, and remained persistent until 6 months after disease onset. SFTSV specific IgG antibody could be detected at medium of 6 weeks; surged to peak levels by 6 months, and remained positive in most of the patients even at 3 years after infection. SFTS patients experienced obvious T cell, B cell and NK cells loss during the first week of infection, which was rapidly restored to normal levels. A significantly lower level of humoral immunity was identified concurrently from severe disease, especially in acute phase of the infection. These abnormalities can be used as a potential indicator in the prediction of an adverse clinical outcome. CONCLUSIONS: Information gained from this study have clinical significance in enhancing our understanding of SFTS immunological characteristics and the disease pathogenesis.