Bunyavirus SFTSV exploits autophagic flux for viral assembly and egress.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging negatively stranded enveloped RNA bunyavirus that causes SFTS with a high case fatality rate of up to 30%. Macroautophagy/autophagy is an evolutionarily conserved process involved in the maintenance of host homeostasis, which exhibits anti-viral or pro-viral responses in reaction to different viral challenges. However, the interaction between the bunyavirus SFTSV and the autophagic process is still largely unclear. By establishing various autophagy-deficient cell lines, we found that SFTSV triggered RB1CC1/FIP200-BECN1-ATG5-dependent classical autophagy flux. SFTSV nucleoprotein induced BECN1-dependent autophagy by disrupting the BECN1-BCL2 association. Importantly, SFTSV utilized autophagy for the viral life cycle, which not only assembled in autophagosomes derived from the ERGIC and Golgi complex, but also utilized autophagic vesicles for exocytosis. Taken together, our results suggest a novel virus-autophagy interaction model in which bunyavirus SFTSV induces classical autophagy flux for viral assembly and egress processes, suggesting that autophagy inhibition may be a novel therapy for treating or releasing SFTS.

Development of a Nucleocapsid Protein-Based ELISA for Detection of Human IgM and IgG Antibodies to SARS-CoV-2.

SARS-CoV-2 is the etiologic agent of COVID-19, which has led to a dramatic loss of human life and presents an unprecedented challenge to public health worldwide. The gold standard assay for SARS-CoV-2 identification is real-time polymerase chain reaction; however, this assay depends on highly trained personnel and sophisticated equipment and may suffer from false results. Thus, a serological antibody test is a supplement to the diagnosis or screening of SARS-CoV-2. Here, we develop and evaluate the diagnostic performance of an IgM/IgG indirect ELISA method for antibodies against SARS-CoV-2 in COVID-19. The ELISA was constructed by coating with a recombinant nucleocapsid protein of SARS-CoV-2 on an enzyme immunoassay plate, and its sensitivity and specificity for clinical diagnosis of SARS-CoV-2 infection was assessed by detecting the SARS-CoV-2-specific IgM and IgG antibodies in COVID-19 patient's sera or healthy person's sera. The SARS-CoV-2 positive serum samples (n = 168) were collected from confirmed COVID-19 patients. A commercial nucleocapsid protein-based chemiluminescent immunoassay (CLIA) kit and a colloidal gold immunochromatography kit were compared with those of the ELISA assay. The specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of IgM were 100, 95.24, 100, and 91.84%, whereas those of IgG were 100, 97.02, 100, and 94.74%, respectively. We developed a highly sensitive and specific SARS-CoV-2 nucleocapsid protein-based ELISA method for the diagnosis and epidemiologic investigation of COVID-19 by SARS-CoV-2 IgM and IgG antibody detection.

Misdiagnosis of scrub typhus as hemorrhagic fever with renal syndrome and potential co-infection of both diseases in patients in Shandong Province, China, 2013-2014.

BACKGROUND: Scrub typhus, caused by Orientia tsutsugamushi, an obligate intracellular gram-negative bacterium, along with hemorrhagic fever with renal syndrome (HFRS), caused by hantaviruses, are natural-focus infectious diseases prevalent in Shandong Province, China. Both diseases have similar clinical manifestations in certain disease stages and similar epidemic seasons, which has caused difficulties for physicians in distinguishing them. The aim of this study was to investigate whether misdiagnosis of scrub typhus as HFRS occurred in patients in Shandong Province. METHODS: Serum samples (N = 112) of clinically suspected HFRS patients from 2013 to 2014 in Shandong Province were analyzed with enzyme-linked immunosorbent assay (ELISA) for antibodies to both hantavirus and Orientia tsutsugamushi. RESULTS: ELISA showed that 56.3% (63/112) and 8.0% (9/112) of clinically suspected HFRS patients were IgM antibody positive to hantavirus and O. tsutsugamushi, respectively. Among the hantavirus IgM antibody positive patients, 7.9% (5/63) were also IgM antibody positive to O. tsutsugamushi. Among the hantavirus IgM antibody negative sera, 8.2% (4/49) of sera were positive to O. tsutsugamushi. CONCLUSIONS: We concluded that some scrub typhus patients were misdiagnosed as HFRS and co-infection of scrub typhus and HFRS might exist in China. Due to the different treatments for scrub typhus and HFRS, physicians should carefully differentiate between scrub typhus and HFRS and consider administering anti-rickettsia antibiotics if treatment for HFRS alone does not work.

SFTSV Infection Induced Interleukin-1ß Secretion Through NLRP3 Inflammasome Activation.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus that causes hemorrhagic fever. Previous studies showed that SFTSV-infected patients exhibited elevated levels of pro-inflammatory cytokines like interleukin-1ß (IL-1ß), indicating that SFTSV infection may activate inflammasomes. However, the detailed mechanism remains poorly understood. Herein, we found that SFTSV could stimulate the IL-1ß secretion in the infected human peripheral blood mononuclear cells (PBMCs), human macrophages, and C57/BL6 mice. We demonstrate that the maturation and secretion of IL-1ß during SFTSV infection is mediated by the nucleotide and oligomerization domain, leucine-rich repeat-containing protein family, pyrin-containing domain 3 (NLRP3) inflammasome. This process is dependent on protease caspase-1, a component of the NLRP3 inflammasome complex. For the first time, our study discovered the role of NLRP3 in response to SFTSV infection. This finding may lead to the development of novel drugs to impede the pathogenesis of SFTSV infection.

Development of a lateral flow immunoassay strip for rapid detection of IgG antibody against SARS-CoV-2 virus.

The ongoing worldwide SARS-CoV-2 epidemic clearly has a tremendous influence on public health. Molecular detection based on oral swabs was used for confirmation of SARS-CoV-2 infection. However, high false negative rates were reported. We describe here the development of a point-of-care (POC) serological assay for the detection of IgG antibody against SARS-CoV-2. The principle of a lateral flow immunoassay strip (LFIAs) consists of fixing SARS-CoV-2 nucleocapsid protein to the surface of the strip and coupling anti-human IgG with colloidal gold nanoparticles (Au NPs). A series of parameters of this method were optimized, including the concentration of coating antigen, BSA blocking concentration and pH value for conjugation. The entire detection process took 15-20 min with a volume of 80 µL of the analyte solution containing 10 µL of serum and 70 µL sample diluent. The performance of the established assay was evaluated using serum samples of the clinically diagnosed cases of Coronavirus Disease 2019 (COVID-19). Our results indicated that the LFIAs for SARS-CoV-2 had satisfactory stability and reproducibility. As a result, our fast and easy LFIAs could provide a preliminary test result for physicians to make the correct diagnosis of SARS-CoV-2 infections along with alternative testing methods and clinical findings, as well as seroprevalence determination, especially in low-resource countries.

Rapid Detection of IgM Antibodies against the SARS-CoV-2 Virus via Colloidal Gold Nanoparticle-Based Lateral-Flow Assay.

Last year, the novel coronavirus disease (COVID-19) emerged in Wuhan, and it has rapidly spread to many other countries and regions. COVID-19 exhibits a strong human-to-human transmission infectivity and could cause acute respiratory diseases. Asymptomatic carriers are able to infect other healthy persons, and this poses a challenge for public health; the World Health Organization (WHO) has already announced COVID-19 as a global pandemic. Nucleic acid testing, considered as the current primary method for diagnosing COVID-19, might lead to false negatives and is difficult to be applied for every suspected patient because of the existence of asymptomatic carriers. Meanwhile, detecting specific antibodies in blood, such as the IgM antibody, against the SARS-CoV-2 virus is another choice for COVID-19 diagnosis, as it is widely accepted that IgM is an important indicator in the acute infection period. In this study, a colloidal gold nanoparticle-based lateral-flow (AuNP-LF) assay was developed to achieve rapid diagnosis and on-site detection of the IgM antibody against the SARS-CoV-2 virus through the indirect immunochromatography method. For preparing AuNP-LF strips, the SARS-CoV-2 nucleoprotein (SARS-CoV-2 NP) was coated on an analytical membrane for sample capture, and antihuman IgM was conjugated with AuNPs to form the detecting reporter. Optimization of AuNP-LF assay was carried out by altering the pH value and the amount of antihuman IgM. The performance of AuNP-LF assay was evaluated by testing serum samples of COVID-19 patients and normal humans. The results were compared with the real-time polymerase chain reaction. The sensitivity and specificity of AuNP-LF assay were determined to be 100 and 93.3%, respectively, and an almost perfect agreement was exhibited by Kappa statistics (κ coefficient = 0.872). AuNP-LF assay showed outstanding selectivity in the detection of IgM against the SARS-CoV-2 virus with no interference from other viruses such as severe fever with thrombocytopenia syndrome virus (SFTSV) and dengue virus (DFV). AuNP-LF assay was able to achieve results within 15 min and needed only 10-20 µL serum for each test. As a whole, in the light of its advantages such as excellent specificity and stability, easy operation, low cost, and being less time-consuming, AuNP-LF assay is a feasible method for the diagnosis of COVID-19 in primary hospitals and laboratories, especially in emergency situations in which numerous samples need to be tested on time.

Emergence of Zika virus infection in China.

Currently, Zika virus (ZIKV) is spreading across the world and no ZIKV infection cases have ever been reported in China. Here, we aimed to determine whether ZIKV infection exists in China. Blood samples of 273 healthy individuals were collected from Nanning City, Guangxi Province, China in March 2019. We found that 9.5% (26/273) and 1.8% (5/273) of healthy persons were positive to ZIKV total antibody (IgG and/or IgM) IgM antibody, respectively. All ZIKV positive plasma samples were negative to Dengue virus and West Nile virus. Among the ZIKV antibody positive plasma samples, 65.4% (17/26) exhibited neutralizing activity to ZIKV. Followed up studies showed that none had clinical symptoms of ZIKV infection and oversea experience. Together, our study indicates that endemic ZIKV infections emerge in China, which not only suggested that ZIKV posed a potential threat to public health in China, but also expand the ZIKV epidemic areas in East and Southeast Asia.

Rapid detection of Shiga toxin type II using lateral flow immunochromatography test strips of colorimetry and fluorimetry.

Two types of lateral flow immunochromatographic test strips (LFITS) using gold nanoparticles and fluorescent CdTe quantum dots (QDs) as signal labels, respectively, were developed for Shiga toxin type II (STX2) assays. Under optimal conditions, the corresponding visual detection limits were 25 ng mL-1 and 5 ng mL-1, respectively. The test results of gold based LFITS can be recognized directly by the naked eye, whereas the visualized results of CdTe QDs based LFITS can be observed by the aid of a UV lamp. Both assays showed good specificity and stability. The inexpensive LFITS were promising for the rapid clinical detection of STX2.

Rapid Detection of Severe Fever with Thrombocytopenia Syndrome Virus via Colloidal Gold Immunochromatography Assay.

To develop the point-of-care testing method to facilitate the clinical detection of severe fever with thrombocytopenia syndrome virus (SFTSV), colloidal gold paper-based lateral flow immunochromatography test strips (LFITSs) have been fabricated for the rapid detection for the first time. The pH value and the amount of monoclonal antibody to prepare colloidal gold nanoparticle-labeled monoclonal antibody bioconjugates were optimized. In addition, 0.4% bovine serum albumin was considered to be the best concentration for blocking nitrocellulose membranes. Under optimal conditions, the limit of detection for SFTSV was as low as 1 ng/mL depending on a visual line. Meanwhile, the entire detection process required no more than 10 min with a volume of only 50 µL of the analyte solution. Moreover, paper-based LFITSs were evaluated in real samples of human serum of patients with satisfactory results. In addition, all strips were of high stability and specificity. In the light of advantages such as simple, portable, rapid, and low cost, the developed LFITSs will extensively come into service, especially in remote areas.

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway.

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A1, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.

A family cluster of infections by a newly recognized bunyavirus in eastern China, 2007: further evidence of person-to-person transmission.

BACKGROUND: Seven persons in one family living in eastern China developed fever and thrombocytopenia during May 2007, but the initial investigation failed to identify an infectious etiology. In December 2009, a novel bunyavirus (designated severe fever with thrombocytopenia syndrome bunyavirus [SFTSV]) was identified as the cause of illness in patients with similar clinical manifestations in China. We reexamined this family cluster for SFTSV infection. METHODS: We analyzed epidemiological and clinical data for the index patient and 6 secondary patients. We tested stored blood specimens from the 6 secondary patients using real time reverse transcription polymerase chain reaction (RT-PCR), viral culture, genetic sequencing, micro-neutralization assay (MNA), and indirect immunofluorescence assay (IFA). RESULTS: An 80-year-old woman with fever, leucopenia, and thrombocytopenia died on 27 April 2007. Between 3 and 7 May 2007, another 6 patients from her family were admitted to a local county hospital with fever and other similar symptoms. Serum specimens collected in 2007 from these 6 patients were positive for SFTS viral RNA through RT-PCR and for antibody to SFTSV through MNA and IFA. SFTSV was isolated from 1 preserved serum specimen. The only shared characteristic between secondary patients was personal contact with the index patient; none reported exposure to suspected animals or vectors. CONCLUSIONS: Clinical and laboratory evidence confirmed that the patients of fever and thrombocytopenia occurring in a family cluster in eastern China in 2007 were caused by a newly recognized bunyavirus, SFTSV. Epidemiological investigation strongly suggests that infection of secondary patients was transmitted to family members by personal contact.

[Preparation and identification of monoclonal antibody against human growth differentiation factor 15].

AIM: To prepare the monoclonal antibody (mAb) against human growth differentiation factor 15 (GDF15). METHODS: The expression vector pGEX-4T-2-gdf15 was constructed and transformed into E.coli Top10F' for expression. Then the purified fusion protein was used to immunize the BALB/c mice. The mouse myeloma cells (Sp2/0) were fused with spleen cells from the BALB/c immunized by the purified protein. Subsequently, limited dilution method was used three times to screen hybridoma cell lines. The titer of the mAb was detected by ELISA and its specificity was analyzed by Western blot. The serum level of GDF15 in hepatocellular carcinoma (HCC) and health people was measured by co-immunoprecipitation (IP) method. RESULTS: The GST-GDF15 fusion protein was successfully expressed and purified. One hybridoma cell line designated 9G3 against GDF15 was obtained. IP and mass spectrometric analysis revealed that the mAb recognized GDF15 in human sera with high specificity. The level of GDF15 in HCC patients was much higher than that in health people. CONCLUSION: The success in mouse anti-GDF15 mAb preparation provides the basis for further developing HCC diagnose kit.

[Construction and expression of eukaryotic expression vector of NS1 protein of influenza A (H1N1)].

AIM: To insert the full-length NS1 gene of influenza A (H1N1) into an eukaryotic expression vector PXJ40-HA, and to evaluate the expression of NS1 gene in transfected 293T cells. METHODS: The NS1 gene of influenza A (H1N1) was amplified by RT-PCR and cloned into pMD18-T vector to construct a plasmid, named pMD18-T-NS1.The pMD18-T-NS1 and the PXJ40-HA were both digested using the same restrict enzymes and ligated, yielding the recombinant eukaryotic expression vector PXJ40-HA-NS1. The expression of the NS1 gene in transfected 293T cells was tested by Western blot. RESULTS: The recombinant eukaryotic expression vector PXJ40-HA-NS1 was successfully constructed. The NS1 protein was observed to be expressed in 293T cells. CONCLUSION: The full-length NS1 gene is obtained and its recombinant eukaryotic expression plasmid is successfully constructed. This study is of help to further understanding the biological function of NS1 protein and the mechanism of diseases induced by influenza A virus.

[Screening and identification of human anti-c-Met Fab from a phage antibody library].

OBJECTIVE: To screen anti-c-Met Fab from a phage antibody library and identify its binding activity. METHODS: The expression of c-Met of HCC lines was identified by Western blot and immunofluorescence. Antibodies against c-Met were screened with immobilized antigen. After five rounds of panning, 30 randomly selected clones were identified by phage ELISA to select specific clones with high affinity. The positive clones were selected for Fab soluble expression in TOP10F and the binding activities were analysed in HCC lines. RESULTS: c-Met expressed in HCC membrane was confirmed by Western blot and immunofluorescence. A Fab fragment named AM2-26 with fine activity to c-Met was selected. AM2-26 binding specificity was confirmed by IP, FACS and immunofluorescence. CONCLUSION: The anti-c-Met Fab binding to c-Met in HCC provides a promising candidate for the biotherapy of hepatoma.