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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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00141-0) contains supplementary material, which is available to authorized users.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV. The isolation and characterization of glycoprotein-specific monoclonal antibodies from a patient with Rift Valley fever (RVF) provide insights into the humoral immune responses elicited by the RVF virus as well as the therapeutic potential of antibody-based strategies.

The genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened.

BACKGROUNDCoronavirus disease 2019 (COVID-19) triggers distinct patterns of pneumonia progression with multiorgan disease, calling for cell- and/or tissue-type specific host injury markers.METHODSAn integrated hypothesis-free single biomarker analysis framework was performed on nasal swabs (n = 484) from patients with COVID-19 in GSE152075. The origin of candidate biomarker was assessed in single-cell RNA data (GSE145926). The candidate biomarker was validated in a cross-sectional cohort (n = 564) at both nucleotide and protein levels.RESULTSPhospholipase A2 group VII (PLA2G7) was identified as a candidate biomarker in COVID-19. PLA2G7 was predominantly expressed by proinflammatory macrophages in lungs emerging with progression of COVID-19. In the validation stage, PLA2G7 was found in patients with COVID-19 and pneumonia, especially in severe pneumonia, rather than patients suffered mild H1N1 influenza infection. Up to 100% positive rates of PLA2G7 were positively correlated with not only viral loads in patients with COVID-19 but also severity of pneumonia in non-COVID-19 patients. Although Ct values of PLA2G7 in severe pneumonia was significantly lower than that in moderate pneumonia (P = 7.2e-11), no differences were observed in moderate pneumonia with COVID-19 between severe pneumonia without COVID-19 (P = 0.81). Serum protein levels of PLA2G7, also known as lipoprotein-associated phospholipase A2 (Lp-PLA2), were further found to be elevated and beyond the upper limit of normal in patients with COVID-19, especially among the re-positive patients.CONCLUSIONSWe firstly identified and validated PLA2G7, a biomarker for cardiovascular diseases (CVDs), was abnormally enhanced in COVID-19 patients at both nucleotide and protein aspects. These findings provided indications into the prevalence of cardiovascular involvements seen in COVID-19 patients. PLA2G7 could be a hallmark of COVID-19 for monitoring disease progress and therapeutic response.FUNDINGThis study was supported by grants from China Mega-Projects for Infectious Disease (2018ZX10711001), National Natural Science Foundation of China (82041023).

Dengue fever, a mosquito-borne viral disease in humans, has been endemic in many Southeast Asian countries. Since its first outbreak in 1978 in Foshan, Guangdong province, China, dengue has been continually epidemic in recent years in Guangdong, which raised the concern whether dengue infection is endemic in Guangdong. In this study, we performed phylogenetic, recombinant, and nucleotide variation analyses of 114 complete genome sequences of dengue virus serotypes 1–4 (DENV1-4) collected from 2013 to 2017 in 18 of 21 cities of Guangdong. Phylogenetic analyses revealed that DENV sequences did not form a single cluster, indicating that dengue fever was not endemic in Guangdong, although DENV1-4 co-circulated in Guangdong. Twenty intra-serotype recombinant isolates involving DENV1-4 were detected, but no inter-serotype recombinant events were identified in this study. Additionally, the most recombinant events were detected simultaneously in the gene NS3 of DENV1-4. Nucleotide variation analyses showed that no significant intra-serotype differences were observed, whereas more significant inter-subtype differences were discovered in non-structural genes than in structural genes. Our investigation will facilitate the understanding of the current prevalent status of dengue fever in Guangdong and contribute to designing more effective preventive and control strategies for dengue infection., Author summary In 1978, dengue fever was first reported in Guangdong province, China, and this has been continuously prevalent in Guangdong in recent years. This is responsible for the heavy burden on the control of dengue, and raises the concern about whether dengue outbreaks have become endemic in Guangdong. Previous studies based on single E gene or few full-length genome sequences were inconclusive. In this study, we sequenced 114 DENV complete genomes of DENV1-4 obtained from 2013 to 2017 in Guangdong and further analyzed the epidemiological and molecular characteristics. Phylogenetic analyses revealed that dengue fever was not endemic in Guangdong, which was indirectly supported by results of our recombination analyses. Nucleotide variation analyses indicated that purification selection shaped dengue virus population. Our investigation will facilitate the development of more effective epidemiological surveillance strategies for dengue infection.

The Zika virus (ZIKV) is an arbovirus that has spread rapidly worldwide within recent times. There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome (GBS) and microcephaly in humans. The ZIKV is genetically diverse and can be separated into Asian and African lineages. A rapid, sensitive, and specific assay is needed for the detection of ZIKV across various pandemic regions. So far, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains. To this end, we have developed a one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based on conserved sequences in the ZIKV envelope (E) gene. The detection limit of the assay was determined to be five RNA transcript copies and 2.94 × 10(–3) 50% tissue culture infectious doses (TCID(50)) of live ZIKV per reaction. The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification, when tested against other flaviviruses. The assay was also successful in testing for ZIKV in clinical samples. Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by a phlebovirus of the Bunyaviridae family, which is designated as SFTS virus (SFTSV). To our knowledge, no efficient SFTSV vaccine exists. Here, we report the identification of a standard virus strain for the eight major SFTSV strains circulating in China for use in evaluating the SFTSV vaccine. Rabbits were immunized with the SFTSV strains and the cross-neutralization capacities of SFTSV anti-sera were determined in microculture cytopathic effect (CPE)-inhibition assays. The mean cross-neutralization capacity of the eight SFTSV anti-sera ranged from 62.4 to 142.6%, compared to autologous strains. The HB29 strain demonstrated strong cross-reactivity with heterologous antibodies, and 33 serum samples from SFTS patients efficiently neutralized HB29, suggesting its broad cross-reactivity. In addition, HB29 demonstrated good replication in Vero and MRC-5 cells (8.0 and 6.0 lg 50% cell culture-infectious dose/mL, respectively) and significant CPE, which satisfied the requirements for a standard virus strain. The HB29 isolate was proven identical to the reported HB29 strain by DNA sequencing, and showed high homology in the S segments with other SFTSV strains (94.8–99.7%). Our results suggest that HB29 may be the best candidate standard strain for use in SFTS vaccine development in China.

We report the first imported case of Rift Valley fever (RVF) in China. The patient returned from Angola, a non-epidemic country, with an infection of a new reassortant from different lineages of Rift Valley fever viruses (RVFVs). The patient developed multiorgan dysfunction and gradually recovered with continuous renal replacement therapy and a short regimen of methylprednisolone treatment. The disordered cytokines and chemokines in the plasma of the patient revealed hypercytokinemia, but the levels of protective cytokines were low upon admission and fluctuated as the disease improved. Whole-genome sequencing and phylogenetic analysis revealed that the imported strain was a reassortant comprising the L and M genes from lineage E and the S gene from lineage A. This case highlights that RVFV had undergone genetic reassortment, which could potentially alter its biological properties, cause large outbreaks and pose a serious threat to global public health as well as the livestock breeding industry.

Yellow fever virus (YFV) and Rift Valley fever virus (RVFV) are important vector-borne pathogens, which all originated in Africa. Now both viruses spread globally and pose a wider threat to public health. Recent studies have shown that YFV was detected in serum and urine samples and viable YFV was isolated from urine samples. In contrast, RVFV has been detected in urine samples, but the isolation of variable virus has not been reported. Here, we report results from a comparative analysis of the detection window for YFV in patient sera and urine samples. Moreover, viable YFV and RVFV were isolated from clinical samples, verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and sequencing. YFV persisted in urine until 32 days after disease onset, which was longer than other reports on natural YFV infections. Overall, our data demonstrated that urine may be considered as an alternative non-invasive source of samples for clinical detection of YFV, RVFV, and other vector-borne pathogens, which should improve diagnosis, help with surveillance efforts against the spread of emerging or re-emerging arboviruses, and reduce the risk of spreading viruses through the urine.

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection.

Yellow fever (YF) is a viral disease endemic to the tropical regions of Africa and South America. An outbreak of YF has been occurring in Angola, since the beginning of 2016. In March 2016, a 32-year-old Chinese man who returned from Angola was hospitalized and diagnosed with the first case of imported YF in China. Clinical observations, blood viral RNA detection, serological testing and treatments for the patient were performed daily. The virus was isolated in Vero cells, and the complete viral genome was sequenced and analyzed using the next-generation genomic sequencing platform. The patient presented with hemorrhagic fever, jaundice and oliguria at day 3 after onset, which rapidly progressed to multisystem organ failure with extremely elevated liver, pancreatic and myocardial enzymes. The patient died despite the intensive supportive treatments that were performed. A liver biopsy showed severe and multilobular necrosis. Viral RNA was detectable throughout the clinical course of the disease. Whole-genomic sequence analysis revealed that the virus belongs to the Angola71 genotype. Although the virus has been circulating in Angola for 45 years, only 14 amino-acid substitutions and no amino-acid changes were observed in the membrane and envelope proteins compared with the virus collected in 1971. The presence of this imported YF case in China indicated that with the increase in business travel among countries, YF outbreaks in Africa can lead to the international spread of the disease. The production and use of YF vaccines is, therefore, an urgent issue.

Hepatitis B virus (HBV) produces large (L), middle (M), and small (S) envelope proteins, alternatively referred to as hepatitis B surface antigen (HBsAg). Currently, yeast-derived S protein serves as the preventive vaccine, while hepatitis B immune globulin (HBIG) concentrated from pooled plasma of vaccine recipients is employed for post-exposure prophylaxis. However, only a small proportion of the antibodies in HBIG are HBV specific. In the present study, a human monoclonal anti-S antibody (G12) was developed, produced under GLP conditions, and subjected to a panel of functional assays. In vitro results demonstrated high affinity of G12 for the S protein (KD = 7.56 nM). It reacted with envelope proteins of all 7 HBV genotypes tested (A-F, H) by immunofluorescent staining, and more than 97% of HBsAg-positive patient serum samples by enzyme-linked immunosorbent assay. G12 recognized a conformational epitope, although the exact sequence remains unknown. Strikingly, G12 was at least 1,000-fold more potent than HBIG in neutralizing HBV infectivity in both HepaRG cell line and HepG2 cells reconstituted with the HBV receptor. In a transgenic mouse model of HBV persistence, a single peritoneal injection of G12 markedly diminished serum HBsAg titers in all 7 mice, which was sustained for the observation period of 144 d in mice with low pre-treatment levels. While the therapeutic potential of G12 warrants further investigation using a large number of animals, G12 is a potent neutralizing human monoclonal antibody and a promising candidate to replace or supplement HBIG in the prevention of HBV infection.

Background Severe fever with thrombocytopenia syndrome (SFTS) is a newly identified severe infectious disease caused by SFTS phlebovirus (SFTSV). SFTS monitoring has been carried out since 2010 in mainland China. We analysed the detection results of SFTSV RNA and antibody in SFTS surveillance cases to provide basic data for SFTS diagnosis. Methods This study was conducted in Shandong Province. Sera of SFTS surveillance cases were collected to detect SFTSV RNA and antibody by real-time RT-PCR and enzyme-linked immunosorbent assay, respectively. Detection rates were calculated. SPSS 18.0 (Chicago, IL, USA) was used for statistical analysis to compare the detection rates of SFTSV RNA and antibodies among different sera groups. Results A total of 374 SFTS surveillance cases were enrolled. Overall, 93.3% (349/374) of the sera samples were collected within 2 weeks after onset, and 6.7% (25/374) were collected between 15 days and 45 days. Of these, 183 (48.9%) were positive for SFTSV RNA. The SFTSV RNA-positive rate peaked (52.2%) in samples collected ≤7 days after onset and then showed a decreasing trend. The detection rate of SFTSV-specific IgM antibody was 30.5% (46/151) and was highest in samples collected between 8 and 14 days (43.3%, 26/60). The positive rate of SFTSV-specific IgG antibody (17.9%, 27/151) showed an increasing trend with the specimen collection time. In total, 74.8% (113/151) of sera samples had the same SFTSV RNA and IgM antibody detection results. However, 23.2% (29/125) of SFTSV RNA-negative cases were IgM antibody-positive, and 8.6% (9/105) of IgM antibody-negative cases were SFTSV RNA-positive. Conclusions SFTSV RNA detection was preferred for SFTSV infection during disease surveillance. For highly suspected SFTS cases, IgM antibody is suggested to make a comprehensive judgement.

Background Severe fever with thrombocytopenia syndrome (SFTS) is a severe viral disease caused by SFTSV. It is important to estimate the rate of missed SFTS diagnosis and to further understand the actual incidence in high endemic areas in China. Methods This study was conducted in two high SFTS endemic provinces in 2015. Patients hospitalized in 2014 or within 1 year before investigation were selected after considering their clinical manifestations, specifically, fever, platelet, and white blood cell. During retrospective investigation, sera were collected to detect SFTSV antibodies to assess SFTSV infection. To further understand SFTSV infection, acute phase sera were detected; SFTSV infection rate among a healthy population was also investigated to determine the basic infection level. Results In total, 246 hospitalized cases were included, including 83 cases (33.7%) with fever, thrombocytopenia and leukopenia, 38 cases (15.4%) with fever and thrombocytopenia but without leukopenia, and 125 cases (50.8%) without fever but with thrombocytopenia and leukopenia. In total, 13 patients (5.3%) were SFTSV IgM antibody-positive, 48 (19.5%) were IgG-positive. Of the 13 IgM-positive cases, 11 (84.6%) were IgG-positive (9 with titres ≥1:400). Seropositive rates of antibodies were high (8.4% for IgM and 30.1% for IgG) in patients with fever, thrombocytopenia and leukopenia. Furthermore, among IgG-positive cases in this group, 76% (19/25) of patients’ IgG antibody titres were ≥1:400. Additionally, 28 of 246 cases were initially diagnosed with suspected SFTS and were then excluded, and 218 patients were never diagnosed with SFTS; the seropositive rates of IgM and IgG in these two groups were 25% and 67.9% and 2.8% and 13.3%, respectively. These rates were 64.3% and 21.4% in 14 sera collected during acute phase of the 28 cases mentioned above. Seropositive rate of SFTSV IgG was only 1.3% in the patient-matched healthy group, and no IgM antibody was detected. A preliminary estimate of 8.3% of SFTS cases were missed in SFTS high endemic provinces. Conclusions The actual SFTS incidence was underestimated. Effective measures such as adding a new SFTS case category - “SFTS clinical diagnosis cases” or using serological detection methods during acute phase should be considered to avoid missed diagnoses. Electronic supplementary material The online version of this article (10.1186/s12879-018-2970-7) contains supplementary material, which is available to authorized users.

An emerging infectious disease, severe fever with thrombocytopenia syndrome (SFTS), was identified to be associated with a novel SFTS virus (SFTSV). Transmission of the disease among humans has been described, but clinical impact factors and transmission mechanisms still need further study. An outbreak of person-to-person transmission of SFTS in a cluster of nine patients that occurred in an SFTS endemic area, Penglai County, Shandong province, China, was investigated. We found that the onset date of all eight secondary SFTS patients ranged from 7 to 13 days after exposure to the corpse of the index patient, and clinical incubation time was mostly focused on 9–10 days (n = 6). The two dead patients, including the index patient and one secondary infected patient, presented unusually high levels of viral load (6 × 108-9 copies/mL), low levels of platelets count (

The pathogenesis of clinical manifestations caused by newly emerging tick-borne phleboviruses [i.e., Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV)], such as severe thrombocytopenia and lymphocytopenia, are not yet fully understood. In the present study, to establish an animal model mimicking the profile of fatal human cases, we examined the susceptibilities of adult mice from 12 strains, aged mice from two strains, and cynomolgus macaques to SFTSV and/or HRTV infections. However, none of these immunocompetent animals developed lethal diseases after infection with SFTSV or HRTV. Thus, we tested a lethal animal model of SFTSV infection using interferon-α/β receptor knock-out (IFNAR-/-) mice to identify the target cell(s) of virus infection, as well as lesions that are potentially associated with hematological changes. IbaI-positive macrophages and Pax5-positive immature B cells overlapped with SFTSV-positive cells in the spleen and lymph nodes of IFNAR-/- mice, and IbaI-SFTSV-double positive cells were also observed in the liver and kidney, thereby suggesting crucial roles for macrophages in the pathogenesis of SFTSV infection in mice. In the mandibular lymph nodes and spleens of infected mice, we observed extensive necrosis comprising B220-positive B cells, which may be associated with severe lymphocytopenia. The results of this study suggest a resemblance between the IFNAR-/- mouse model and lethal infections in humans, as well as roles for multiple cells during pathogenesis in mice.

Viral hemorrhagic fevers (VHFs) are worldwide diseases caused by several kinds of viruses. With the emergence of new viruses, advanced diagnostic methods are urgently needed for identification of VHFs. Based on Luminex xMAP technology, a rapid, sensitive, multi-pathogen and high-throughput method which could simultaneously detect hemorrhagic fever viruses (HFVs) specific IgG antibodies was developed. Recombinant antigens of nine HFVs including Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), Andes virus (ANDV), Sin Nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) and dengue virus (DENV) were produced and purified from a prokaryotic expression system and the influence of the coupling amount was investigated. Cross-reactions among antigens and their rabbit immune sera were evaluated. Serum samples collected from 51 laboratory confirmed hemorrhagic fever with renal syndrome (HFRS) patients, 43 confirmed SFTS patients and 88 healthy donors were analyzed. Results showed that recombinant nucleocapsid protein of the five viruses belonging to the genus Hantavirus, had serological cross-reactivity with their corresponding rabbit immune sera, but not apparent with immune sera of other four viruses. Evaluation of this new method with clinical serum samples showed 98.04% diagnostic sensitivity for HFRS, 90.70% for SFTS detection and the specificity was ranging from 66.67% to 100.00%. The multiplexed Luminex-based immunoassay has firstly been established in our study, which provides a potentially reliable diagnostic tool for IgG antibody detection of VHFs.

Background. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease in China, caused by SFTS virus (SFTSV). Severe SFTS patients can quickly proceed to multiorgan dysfunction and death; however, underlying pathogenic mechanisms remain unclear. Methods. Serum samples from 15 fatal and 44 nonfatal SFTS cases were subjected to multiplex-microbead immunoassays to detect a broad spectrum of cytokines. The viral load and virus-specific IgG titers were also tested by real-time PCR and ELISA, respectively. Results. Cytokines IL-1RA, IL-6, IL-10, G-CSF, IP-10, and MCP-1 were elevated in SFTS patients and produced at robust levels in fatal cases. In contrast, cytokines PDGF-BB and RANTES decreased in SFTS patients. These cytokines reverted to normal ranges during the convalescent phase of SFTSV infection. Cytokines IL-1β, IL-8, MIP-1α, and MIP-1β showed a unique pattern of elevation in fatal cases but not in nonfatal cases. However, these cytokines increased in the convalescent phase of nonfatal SFTS cases. Our regression analysis revealed that the serum viral load correlated with these cytokines. Moreover, levels of these cytokines correlated with various clinical parameters and virus-specific IgG titers. Conclusion. The study demonstrates that SFTSV infection induces a cytokine storm with abnormally expressed cytokine profiles, which are associated with the disease severity.

Background Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV) with an average fatality rate of 12%. The clinical factors for death in SFTS patients remain unclear. Methods Clinical features and laboratory parameters were dynamically collected for 11 fatal and 48 non-fatal SFTS cases. Univariate logistic regression was used to evaluate the risk factors associated with death. Results Dynamic tracking of laboratory parameters revealed that during the initial fever stage, the viral load was comparable for the patients who survived as well as the ones that died. Then in the second stage when multi-organ dysfunction occurred, from 7-13 days after disease onset, the viral load decreased in survivors but it remained high in the patients that died. The key risk factors that contributed to patient death were elevated serum aspartate aminotransferase, lactate dehydrogenase, creatine kinase, and creatine kinase fraction, as well as the appearance of CNS (central nervous system) symptoms, hemorrhagic manifestation, disseminated intravascular coagulation, and multi-organ failure. All clinical markers reverted to normal in the convalescent stage for SFTS patients who survived. Conclusions We identified a period of 7-13 days after the onset of illness as the critical stage in SFTS progression. A sustained serum viral load may indicate that disease conditions will worsen and lead to death.

The discovery of an emerging viral disease, severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV), has prompted the need to understand pathogenesis of SFTSV. We are unique in establishing an infectious model of SFTS in C57/BL6 mice, resulting in hallmark symptoms of thrombocytopenia and leukocytopenia. Viral RNA and histopathological changes were identified in the spleen, liver, and kidney. However, viral replication was only found in the spleen, which suggested the spleen to be the principle target organ of SFTSV. Moreover, the number of macrophages and platelets were largely increased in the spleen, and SFTSV colocalized with platelets in cytoplasm of macrophages in the red pulp of the spleen. In vitro cellular assays further revealed that SFTSV adhered to mouse platelets and facilitated the phagocytosis of platelets by mouse primary macrophages, which in combination with in vivo findings, suggests that SFTSV-induced thrombocytopenia is caused by clearance of circulating virus-bound platelets by splenic macrophages. Thus, this study has elucidated the pathogenic mechanisms of thrombocytopenia in a mouse model resembling human SFTS disease.

Hepatitis A virus (HAV) is a wide spread pathogenic agent and is the common cause of acute Hepatitis A worldwide. Passive immunization of HAV plays an extremely important role in post-exposure prophylaxis with clinical applications often requiring large amounts of antibody. As an alternative to the in vitro production of recombinant proteins, expression of monoclonal antibodies (mAbs) in the milk of transgenic animals is currently used being associated with low production costs and high activity. In this paper, eight founder lines of transgenic mice were generated by co-microinjection of the two cassettes encoding the heavy- and light-chains of a neutralizing anti-HAV antibody, respectively. The expressed heavy- and light-chains of the mAb were correctly assembled and modified in the mammary gland as detected by western blotting. High expression levels of the antibody were achieved during the lactation period and found to be independent of the copy numbers of integrated transgenes. The highest level was up to 32.2 mg/ml. The binding specificity and neutralizing activity of the expressed mAb were assayed by ELISA and neutralizing test, showing that it is capable to neutralize the JN strain of Hepatitis A virus efficiently. Therefore, our results suggest that a large-scale and efficient production of the anti-HAV mAb in the milk of transgenic farm animals would be feasible in the future.

Background Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease recently identified to be caused by a novel bunyavirus (SFTSV). The clinical diagnosis is urgently needed to differentiate the disease from other infections. Objective To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of SFTSV viral RNA and evaluate potential use for clinical diagnosis of SFTS. Study design Primers and probes were designed to target the L, M, and S segments of SFTSV, and standard curves were established based on serial dilutions of in vitro transcribed viral RNA or viral RNA extracts. The serum samples collected from 70 laboratory confirmed SFTS patients, 114 non-SFTS patients, and 400 healthy donors were analyzed. Results Based on three optimized primer–probe sets to detect L, M, S genes of SFTSV, the quantitative real-time RT-PCR assay could discriminate SFTSV infection from other vector-borne viral diseases in human with potential detection limit of 10 viral RNA copies/μl or 10 TCID 50 /ml virus load. Strong linear correlations ( r 2 >0.99) between the C t values and viral RNA standards over a liner range were obtained. The assay specificity was determined by sequence alignment and experimentally tested on various related viruses. Evaluation of the study method with clinical serum samples showed 98.6% clinical diagnostic sensitivity and over 99% specificity. Conclusion The quantitative real-time RT-PCR assay established in this study can be used as a reliable method for early diagnosis of SFTSV infection.

Severe fever with thrombocytopenia syndrome bunyavirus is a newly discovered bunyavirus with high pathogenicity to human. The transmission model has been largely uncharacterized. Investigation on a cluster of severe fever with thrombocytopenia syndrome cases provided evidence of person-to-person transmission through blood contact to the index patient with high serum virus load.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that was first reported in China in 2009. Phylogenetic analysis of the viral genome showed that SFTS virus represents a new lineage within the Phlebovirus genus, distinct from the existing sandfly fever and Uukuniemi virus groups, in the family Bunyaviridae . SFTS disease is characterized by gastrointestinal symptoms, chills, joint pain, myalgia, thrombocytopenia, leukocytopenia, and some hemorrhagic manifestations with a case fatality rate of about 2 to 15%. Here we report the development of reverse genetics systems to study STFSV replication and pathogenesis. We developed and optimized functional T7 polymerase-based M- and S-segment minigenome assays, which revealed errors in the published terminal sequences of the S segment of the Hubei 29 strain of SFTSV. We then generated recombinant viruses from cloned cDNAs prepared to the antigenomic RNAs both of the minimally passaged virus (HB29) and of a cell culture-adapted strain designated HB29pp. The growth properties, pattern of viral protein synthesis, and subcellular localization of viral N and NSs proteins of wild-type HB29pp (wtHB29pp) and recombinant HB29pp viruses were indistinguishable. We also show that the viruses fail to shut off host cell polypeptide production. The robust reverse genetics system described will be a valuable tool for the design of therapeutics and the development of killed and attenuated vaccines against this important emerging pathogen. IMPORTANCE SFTSV and related tick-borne phleboviruses such as Heartland virus are emerging viruses shown to cause severe disease in humans in the Far East and the United States, respectively. Study of these novel pathogens would be facilitated by technology to manipulate these viruses in a laboratory setting using reverse genetics. Here, we report the generation of infectious SFTSV from cDNA clones and demonstrate that the behavior of recombinant viruses is similar to that of the wild type. This advance will allow for further dissection of the roles of each of the viral proteins in the context of virus infection, as well as help in the development of antiviral drugs and protective vaccines.

SFTS virus (SFTSV) is a highly pathogenic bunyavirus that causes severe fever with thrombocytopenia syndrome (SFTS), an emerging infectious disease in China. Laboratory mice have been reported to be susceptible to SFTSV infection, but the infection in nonhuman primates has not been investigated. This study is the first to report that, in rhesus macaques, SFTSV does not cause severe symptoms or death but causes fever, thrombocytopenia, leukocytopenia, and increased levels of transaminases and myocardial enzymes in blood. Viremia, virus-specific immunoglobulin M and immunoglobulin G antibodies, and neutralizing antibodies were identified in all infected macaques. Levels of the cytokines interferon γ, eotaxin, tumor necrosis factor α, and macrophage inflammatory protein 1β were significantly elevated in the blood. Minor pathological lesions were observed in the liver and kidney during the late stages of infection. Overall, SFTSV infection in rhesus macaques resembled mild SFTS in humans.

Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging bunyavirus that causes a hemorrhagic fever with a high mortality rate. The virus is likely tick-borne and replicates primarily in hemopoietic cells, which may lead to disregulation of proinflammatory cytokine induction and loss of leukocytes and platelets. The viral genome contains L, M, and S segments encoding a viral RNA polymerase, glycoproteins G(n) and G(c), nucleoprotein (NP), and a nonstructural S segment (NSs) protein. NSs protein is involved in the regulation of host innate immune responses and suppression of IFNβ-promoter activities. In this article, we demonstrate that NSs protein can form viroplasm-like structures (VLSs) in infected and transfected cells. NSs protein molecules interact with one another, interact with NP, and were associated with viral RNA in infected cells, suggesting that NSs protein may be involved in viral replication. Furthermore, we observed that NSs-formed VLS colocalized with lipid droplets and that inhibitors of fatty acid biosynthesis decreased VLS formation or viral replication in transfected and infected cells. Finally, we have demonstrated that viral dsRNAs were also localized in VLS in infected cells, suggesting that NSs-formed VLS may be implicated in the replication of SFTS bunyavirus. These findings identify a novel function of nonstructural NSs in SFTSV-infected cells where it is a scaffolding component in a VLS functioning as a virus replication factory. This function is in addition to the role of NSs protein in modulating host responses that will broaden our understanding of viral pathogenesis of phleboviruses.

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.

To investigate the infections of severe fever with thrombocytopenia syndrome virus (SFTSV) in domesticated animals, we sampled a total of 3,039 animals in 2 counties in Shandong Province, People’s Republic of China, from April to November 2011. SFTSV-specific antibodies were detected in 328 (69.5%) of 472 sheep, 509 (60.5%) of 842 cattle, 136 (37.9%) of 359 dogs, 26 (3.1%) of 839 pigs, and 250 (47.4%) of 527 chickens. SFTSV RNA was detected in all sampled animal species, but the prevalence was low, ranging from 1.7% to 5.3%. A cohort study in 38 sheep was conducted to determine when seroconversion to SFTSV occured. SFTSVs were isolated from sheep, cattle, and dogs and shared >95% sequence homology with human isolates from the same disease-endemic regions. These findings demonstrate that natural infections of SFTSV occur in several domesticated animal hosts in disease-endemic areas and that the virus has a wide host range.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, multiorgan dysfunction, and a high fatality rate between 12 and 30%. It is caused by SFTS virus (SFTSV), a novel Phlebovirus in family Bunyaviridae . Although the viral pathogenesis remains largely unknown, hemopoietic cells appear to be targeted by the virus. In this study we report that human monocytes were susceptible to SFTSV, which replicated efficiently, as shown by an immunofluorescence assay and real-time reverse transcription-PCR. We examined host responses in the infected cells and found that antiviral interferon (IFN) and IFN-inducible proteins were induced upon infection. However, our data also indicated that downregulation of key molecules such as mitochondrial antiviral signaling protein (MAVS) or weakened activation of interferon regulatory factor (IRF) and NF-κB responses may contribute to a restricted innate immunity against the infection. NSs, the nonstructural protein encoded by the S segment, suppressed the beta interferon (IFN-β) and NF-κB promoter activities, although NF-κB activation appears to facilitate SFTSV replication in human monocytes. NSs was found to be associated with TBK1 and may inhibit the activation of downstream IRF and NF-κB signaling through this interaction. Interestingly, we demonstrated that the nucleoprotein (N), also encoded by the S segment, exhibited a suppressive effect on the activation of IFN-β and NF-κB signaling as well. Infected monocytes, mainly intact and free of apoptosis, may likely be implicated in persistent viral infection, spreading the virus to the circulation and causing primary viremia. Our findings provide the first evidence in dissecting the host responses in monocytes and understanding viral pathogenesis in humans infected with a novel deadly Bunyavirus .

The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

Increasing evidences suggest that the type I interferon α (IFN α) plays a critical role in the etiopathogenesis of systemic lupus erythematosus (SLE), which makes it a promising therapeutic target for the treatment of the disease. By screening a large size non-immune human antibody library, we have developed a human single-chain antibody (ScFv) AIFN α 1bScFv01 and corresponding whole antibody AIFN α 1bIgG01 to human interferon α 1b (IFN α 1b) with high specificity and high affinity. The IgG antibody could down-regulate the expression of ISG15 and IFIT-1 induced by either recombinant IFN α 1b or naïve IFN α from SLE patients' sera, and reduced total serum IgG and IgM antibodies level in a pristane-primed lupus-like mouse model. The crystal structure of AIFN α 1bScFv01-IFN α 1b complex solved to 2.8 Å resolution revealed that both Pro26-Gln40 region in loop AB and Glu147-Arg150 region in helix E of IFN α 1b contribute to binding with AIFN α 1bScFv01. Four residues of above two regions (Leu30, Asp32, Asp35 and Arg150) are critical for the formation of antigen-antibody complexes. AIFN α 1bScFv01 shares partial epitopes of IFN α 1b with its receptor IFNAR2 but with much higher binding affinity to IFN α 1b than IFNAR2. Thus, AIFN α 1bIgG01 exhibits its neutralizing activity through competition with IFNAR2 to bind with IFN α and prevents the activation of IFN α-mediated signaling pathway. Our results highlight the potential use of the human antibody for modulating the activity of IFN α in SLE.

Background The incidence of dengue, an infectious disease caused by dengue virus (DENV), has dramatically increased around the world in recent decades and is becoming a severe public health threat. However, there is currently no specific treatment for dengue fever, and licensed vaccine against dengue is not available. Vaccination with virus-like particles (VLPs) has shown considerable promise for many viral diseases, but the effect of DENV VLPs to induce specific immune responses has not been adequately investigated. Results By optimizing the expression plasmids, recombinant VLPs of four antigenically different DENV serotypes DENV1-4 were successfully produced in 293T cells. The vaccination effect of dengue VLPs in mice showed that monovalent VLPs of each serotype stimulated specific IgG responses and potent neutralizing antibodies against homotypic virus. Tetravalent VLPs efficiently enhanced specific IgG and neutralizing antibodies against all four serotypes of DENV. Moreover, vaccination with monovalent or tetravalent VLPs resulted in the induction of specific cytotoxic T cell responses. Conclusions Mammalian cell expressed dengue VLPs are capable to induce VLP-specific humoral and cellular immune responses in mice, and being a promising subunit vaccine candidate for prevention of dengue virus infection.

Hepatitis A virus (HAV) is the major pathogen responsible for acute infectious hepatitis A, a disease that is prevalent worldwide. Although HAV immunization effectively prevents infection, primary immunizations must be administered at least 2 weeks prior to HAV exposure. In contrast, passive immunization with pooled human immunoglobulin (Ig) can provide immediate and rapid protection from HAV infection. Because the use of human sera-derived Igs carries the risk of contamination, we sought to develop recombinant HAV-neutralizing human antibodies. We prepared a combinatorial phage display library of recombinant human anti-HAV antibodies from RNA extracted from the blood lymphocytes of a convalescent hepatitis A patient. Two recombinant human IgG antibodies, HAIgG16 and HAIgG78, were screened from the antibody library by their ability to bind with high affinity to purified, inactivated HAV virions. These antibodies recognized different epitopes of the HAV virion capsid, and competed with both patient sera and well-characterized neutralizing mouse monoclonal antibodies. A cocktailed mixture of HAIgG16 and HAIgG78 at a 3:1 ratio was prepared to compare its combined biological activity with that conferred by each antibody individually. The cocktailed antibodies displayed a stronger neutralizing activity in vitro than that observed with either HAIgG16 and HAIgG78 alone. To determine the in vivo neutralizing abilities of these antibodies, rhesus monkeys were inoculated with cocktailed antibodies and challenged with HAV. Whereas control animals developed hepatitis A and seroconverted to the HAV antibody, animals receiving cocktailed antibodies were protected either from viral infection or from developing clinical hepatitis. These results demonstrate that recombinant human antibody preparations could be used to prevent or treat early-stage HAV infection.

BACKGROUND: SFTS virus (SFTSV) is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS) in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. METHODOLOGY/PRINCIPAL FINDINGS: We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs) recognized the nucleocapsid (N) protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. CONCLUSIONS/SIGNIFICANCE: The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection.

Background Chikungunya virus (CHIKV) has caused large outbreaks worldwide in recent years, especially on the islands of the Indian Ocean and India. The virus is transmitted by mosquitoes (Aedes aegypti), which are widespread in China, with an especially high population density in southern China. Analyses of full-length viral sequences revealed the acquisition of a single adaptive mutation providing a selective advantage for the transmission of CHIKV by this species. No outbreaks due to the local transmission of CHIKV have been reported in China, and no cases of importation were detected on mainland China before 2008. We followed the spread of imported CHIKV in southern China and analyzed the genetic character of the detected viruses to evaluate their potential for evolution. Results The importation of CHIKV to mainland China was first detected in 2008. The genomic sequences of four of the imported viruses were identified, and phylogenetic analysis demonstrated that the sequences were clustered in the Indian Ocean group; however, seven amino acid changes were detected in the nonstructural protein-coding region, and five amino acid changes were noted in the structural protein-coding regions. In particular, a novel substitution in E2 was detected (K252Q), which may impact the neurovirulence of CHIKV. The adaptive mutation A226V in E1 was observed in two imported cases of chikungunya disease. Conclusions Laboratory-confirmed CHIKV infections among travelers visiting China in 2008 were presented, new mutations in the viral nucleic acids and proteins may represent adaptive mutations for human or mosquito hosts.