Antibody signatures in hospitalized hand, foot and mouth disease patients with acute enterovirus A71 infection.

Enterovirus A71 (EV-A71) infection is a major cause of severe hand, foot and mouth disease (HFMD) in young children. The characteristics of EV-A71 neutralizing antibodies in HFMD patients are not well understood. In this study, we identified and cloned EV-A71-neutralizing antibodies by single cell RNA and B cell receptor sequencing of peripheral blood mononuclear cells. From 145 plasmablasts, we identified two IgG1 monoclonal antibodies (mAbs) and six IgM mAbs that neutralized EV-A71. Four of the IgM mAbs harbor germline variable sequences and neutralize EV-A71 potently. Two genetically similar IgM antibodies from two patients have recurrent heavy chain variable domain gene usage and similar complementarity-determining region 3 sequences. We mapped the residues of EV-A71 critical for neutralization through selection of virus variants resistant to antibody neutralization in the presence of neutralizing mAbs. The residues critical for neutralization are conserved among EV-A71 genotypes. Epitopes for the two genetically similar antibodies overlap with the SCARB2 binding site of EV-A71. We used escape variants to measure the epitope-specific antibody response in acute phase serum samples from EV-A71 infected HFMD patients. We found that these epitopes are immunogenic and contributed to the neutralizing antibody response against the virus. Our findings advance understanding of antibody response to EV-A71 infection in young children and have translational potential: the IgM mAbs could potentially be used for prevention or treatment of EV-A71 infections.

Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays.

The scientific community has responded to the coronavirus disease 2019 (COVID-19) pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undertaken in high-containment laboratories; however, it is often desirable to work with samples at lower-containment levels. To facilitate the transfer of infectious samples from high-containment laboratories, we have tested methods commonly used to inactivate virus and prepare the sample for additional experiments. Incubation at 80°C, a range of detergents, Trizol reagents, and UV energies were successful at inactivating a high titer of SARS-CoV-2. Methanol and paraformaldehyde incubation of infected cells also inactivated the virus. These protocols can provide a framework for in-house inactivation of SARS-CoV-2 in other laboratories, ensuring the safe use of samples in lower-containment levels.

Structural Study of the C-Terminal Domain of Nonstructural Protein 1 from Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to other emerging viral pathogens, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout Southeast Asia, the Pacific Islands, and the Far East, causing around 68,000 cases of infection worldwide each year. In this report, we present a 2.1-Å-resolution crystal structure of the C-terminal ß-ladder domain of JEV nonstructural protein 1 (NS1-C). The surface charge distribution of JEV NS1-C is similar to those of WNV and ZIKV but differs from that of DENV. Analysis of the JEV NS1-C structure, with in silico molecular dynamics simulation and experimental solution small-angle X-ray scattering, indicates extensive loop flexibility on the exterior of the protein. This, together with the surface charge distribution, indicates that flexibility influences the protein-protein interactions that govern pathogenicity. These factors also affect the interaction of NS1 with the 22NS1 monoclonal antibody, which is protective against West Nile virus infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 mediates interaction with membranes and that sulfate binding sites common to NS1 structures are not glycosaminoglycan binding interfaces. This report highlights several differences between flavivirus NS1 proteins and contributes to our understanding of their structure-pathogenic function relationships.IMPORTANCE JEV is a major cause of viral encephalitis in Asia. Despite extensive vaccination, epidemics still occur. Nonstructural protein 1 (NS1) plays a role in viral replication, and, because it is secreted, it can exhibit a wide range of interactions with host proteins. NS1 sequence and protein folds are conserved within the Flavivirus genus, but variations in NS1 protein-protein interactions among viruses likely contribute to differences in pathogenesis. Here, we compared characteristics of the C-terminal ß-ladder domain of NS1 between flaviviruses, including surface charge, loop flexibility, epitope cross-reactivity, membrane adherence, and glycosaminoglycan binding. These structural features are central to NS1 functionality and may provide insight into the development of diagnostic tests and therapeutics.

Diagnostic performance of different specimens in detecting enterovirus A71 in children with hand, foot and mouth disease.

Hand, foot and mouth disease (HFMD) is a major public health problem among children in the Asia-Pacific region. The optimal specimen for HFMD virological diagnosis remains unclear. Enterovirus A71 (EV-A71) neutralizing antibody titres detected in paired sera were considered the reference standard for calculating the sensitivity, specificity, positive and negative predictive value of throat swabs, rectal swabs, stool, blood samples and cerebrospinal fluid (CSF) by RT-PCR or ELISA assay. In this study, clinical samples from 276 HFMD patients were collected for analysing the sensitivity of different kind of specimens. Our results showed that stool had the highest sensitivity (88%, 95% CI: 74%-96%) and agreement with the reference standard (91%). The order of diagnostic yield for EV-A71 infection was stool sample â€‹≥ â€‹rectal swab â€‹> â€‹throat swab â€‹> â€‹blood sample â€‹> â€‹CSF sample, and using a combination of clinical samples improved sensitivity for enterovirus detection. The sensitivity of ELISA for IgM antibody detection in sterile-site specimens was significantly higher than that of RT-PCR (serum/plasma: 62% vs. 2%, CSF: 47% vs. 0%) (P â€‹< â€‹0.002). In conclusion, our results suggest that stool has the highest diagnostic yield for EV-A71-infected HFMD. If stool is unavailable, rectal swabs can be collected to achieve a similar diagnostic yield. Otherwise, throat swabs may be useful in detecting positive samples. Although IgM in blood or CSF is diagnostically accurate, it lacks sensitivity, missing 40%-50% of cases. The higher proportion of severe cases and shorter interval between onset and sampling contributed to the increase in congruency between clinical testing and the serological reference standard.

Kinetics of the neutralising antibody response in patients with hand, foot, and mouth disease caused by EV-A71: A longitudinal cohort study in Zhengzhou during 2017-2019.

BACKGROUND: Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) poses a serious threat to children's health. Kinetics of the neutralising antibody (NAb) response in EV-A71 infected HFMD patients remains unclear. The ideal sampling time of paired serum samples for serological diagnosis of EV-A71 infection is not well defined. METHODS: HFMD inpatients admitted to Henan Children's Hospital between February 15, 2017 and February 15, 2018 were enrolled. Serial serum samples collected during hospitalisation and up to 1.5 years after discharge were tested for NAb against EV-A71. Random intercept modelling with B-spline was conducted to characterize the kinetics of the EV-A71 NAb response over time after illness onset. FINDINGS: A total of 524 serum samples from 264 EV-A71 RNA positive HFMD inpatients were collected. NAb titres of EV-A71 infected patients were estimated to increase from 40 (95% CI: 9-180) at the day of onset to the peak of 2417 (95% CI: 1859-3143) at day 13, then remained above 1240 until 26 months. For serological diagnosis of EV-A71 infection, if at least a 4-fold rise in titre was used as the criteria, the acute phase serum should be collected at 0-4 days, the corresponding convalescent serum should be collected 14.9 days (95% CI: 9.1-23.8) after illness onset. INTERPRETATION: EV-A71 infection induced a strong and persistent humoral immune response in HFMD patients. The findings provide a scientific support for determining the collection time of paired serum samples for serological diagnosis of EV-A71 infected HFMD patients. FUNDING: National Science Fund for Distinguished Young Scholars.

Long-term neurodevelopment outcomes of hand, foot and mouth disease inpatients infected with EV-A71 or CV-A16, a retrospective cohort study.

Hand, foot and mouth disease (HFMD) is a common infectious disease in western Asia area and the full range of the long-term sequelae of HFMD remains poorly described. We conducted a retrospective hospital-based cohort study of HFMD patients with central nervous system (CNS) complications caused by EV-A71 or CV-A16 between 2010 and 2016. Patients were classified into three groups, including CNS only, autonomic nervous system (ANS) dysregulation, and cardiorespiratory failure. Neurologic examination, neurodevelopmental assessments, Magnetic Resonance Imaging (MRI) and lung function, were performed at follow up. Of the 176 patients followed up, 24 suffered CNS only, 133 ANS dysregulation, and 19 cardiorespiratory failure. Median follow-up period was 4.3 years (range [1.4-8.3]). The rate of neurological abnormalities was 25% (43 of 171) at discharge and 10% (17 of 171) at follow-up. The rates of poor outcome were significantly different between the three groups of complications in motor (28%, 38%, 71%) domain (p=0.020), but not for cognitive (20%, 24%, 35%), language (25%, 36%, 41%) and adaptive (24%, 16%, 26%) domains (p = 0.537, p = 0.551, p = 0.403). For children with ventilated during hospitalization, 41% patients (14 of 34) had an obstructive ventilatory defect, and one patient with scoliosis had mixed ventilatory dysfunction. Persistent abnormalities on brain MRI were 0% (0 of 7), 9% (2 of 23) and 57% (4 of 7) in CNS, ANS and cardiorespiratory failure group separately. Patients with HFMD may have abnormalities in neurological, motor, language, cognition, adaptive behaviour and respiratory function. Long-term follow-up programmes for children's neurodevelopmental and respiratory function may be warranted.

Enterovirus genomic load and disease severity among children hospitalised with hand, foot and mouth disease.

BACKGROUND: Examining associations between viral genomic loads of enteroviruses and clinical severity is important for promoting and improving development of antiviral drugs related to hand, foot and mouth disease (HFMD). METHODS: Throat swabs were collected from HFMD cases at acute phase of illness using a standardized technique in a prospective study. The viral genomic load was categorized into low, medium, and high groups using parameters of real-time reverse transcription-polymerase chain reaction. The clinical severities were assessed with four indicators, respectively. FINDINGS: We analysed 1109 HFMD cases, including 538 children with CV-A6, 231 with CV-A16, 156 with EV-A71, 78 with CV-A10, 59 with CV-A4, and 47 with CV-A2. EV-A71 genomic load categories were associated with risks of diagnoses of CNS complications (p = 0.016), requiring systemic corticosteroids or IVIG (p = 0.011), intensive care unit admission (p = 0.002) and length of hospital stay over 5 days (p = 0.048). In the multivariate analyses, point estimates of adjusted odds ratio (OR) tended to increase with viral genomic loads for all four severe outcomes and ORs of highest viral genomic load were all significantly larger than one for EV-A71. INTERPRETATION: HFMD clinical severities positively associate with viral genomic loads of EV-A71 in throat swabs. Specific antiviral drugs should be developed to reduce enterovirus load and to alleviate the clinical severities for HFMD cases. FUNDING: National Science Fund for Distinguished Young Scholars.