Mifang Liang

Person-to-Person Transmission of Severe Fever With Thrombocytopenia Syndrome Bunyavirus Through Blood Contact

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.

Clinical Progress and Risk Factors for Death in Severe Fever with Thrombocytopenia Syndrome Patients

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.

Host Cytokine Storm Is Associated With Disease Severity of Severe Fever With Thrombocytopenia Syndrome

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.

Pathogenesis of emerging severe fever with thrombocytopenia syndrome virus in C57/BL6 mouse model

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.

Severe Fever with Thrombocytopenia Syndrome Virus among Domesticated Animals, China

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.

Early diagnosis of novel SFTS bunyavirus infection by quantitative real-time RT-PCR assay

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.

Generation, Characterization and Epitope Mapping of Two Neutralizing and Protective Human Recombinant Antibodies against Influenza A H5N1 Viruses

Background The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development. Methodology/Principal Findings We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model. Conclusions/Significance Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.

Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus

Zika virus–specific antibodies isolated from a single infected patient show postexposure protection in mice and reveal targets for therapy. Stopping Zika virus in its tracks Zika virus is a global concern because of its association with fetal microcephaly and neurological complications, and there are no approved countermeasures. In new work, Wang et al. isolated 13 antibodies from a patient infected with Zika virus, two of which (Z3L1 and Z23) showed potent neutralizing activity without cross-reactivity to dengue virus strains 1 to 4. Moreover, the Z3L1 and Z23 antibodies conferred postexposure protection against Zika virus in a murine model. Structural studies indicated that the antibodies bound to different viral epitopes, suggesting that these antibodies could be used as a therapeutic cocktail. The 2015–2016 outbreak of Zika virus (ZIKV) disease has affected many countries and is a major public health concern. ZIKV is associated with fetal microcephaly and neurological complications, and countermeasures are needed to treat and prevent ZIKV infection. We report the isolation of 13 specific human monoclonal antibodies from a single patient infected with ZIKV. Two of the isolated antibodies (Z23 and Z3L1) demonstrated potent ZIKV-specific neutralization in vitro without binding or neutralizing activity against strains 1 to 4 of dengue virus, the closest relative to ZIKV. These two antibodies provided postexposure protection to mice in vivo. Structural studies revealed that Z23 and Z3L1 bound to tertiary epitopes in envelope protein domain I, II, or III, indicating potential targets for ZIKV-specific therapy. Our results suggest the potential of antibody-based therapeutics and provide a structure-based rationale for the design of future ZIKV-specific vaccines.

Suppression of the Interferon and NF-κB Responses by Severe Fever with Thrombocytopenia Syndrome Virus

ABSTRACT 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.

Human recombinant neutralizing antibodies against hantaan virus G2 protein

Old world hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS), still present a public health problem in Asia and Eastern Europe. The majority of cases has been recorded in China. The aim of our study was to generate human recombinant neutralizing antibodies to a hantavirus by phage display technology. To preserve the structural identity of viral protein, the panning procedure was performed on native Hantaan (HTN) (76-118) virus propagated in Vero-E6 cells. In total, five complete human recombinant IgG antibodies were produced in a baculovirus expression system. All of them were able to completely neutralize HTN, and Seoul (SEO) virus in a plaque reduction neutralization test (PRNT). Three of these antibodies could also completely neutralize Dobrava (DOB) virus but not Puumala (PUU) virus. All antibodies bind to Hantaan virus G2 protein localized in the virus envelope. The sequence areas within the HTN (76-118)-G2 protein detected by five selected antibodies were mapped using peptide scans. Two partial epitopes, 916-KVMATIDSF-924 and 954-LVTKDIDFD-963, were recognized, which presumably are of paramount importance for docking of the virus to host cell receptors. A consensus motif 916-KVXATIXSF-924 could be identified by mutational analysis. The neutralizing antibodies to the most widely distributed hantaviruses causing HFRS might be promising candidates for the development of an agent for prevention and treatment of HFRS in patients.