Etiological Analysis of Viral Encephalitis in Children in Zhejiang Province from 2018 to 2019.

OBJECTIVE: To investigate the common pathogens of viral encephalitis (VE) in children, and to provide guidance for the empirical diagnosis and treatment of patients with VE. METHODS: A total of 227 cerebrospinal fluid (CSF) samples were collected from pediatric patients with VE in Zhejiang province from January 2018 to December 2019. The samples were tested using multiplex and singleplex Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with primers specific to enterovirus (EV), varicella-zoster virus (VZV), mumps virus (MuV), cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1)/type 2 (HSV-2), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6). The data of the two analyses were compared and then verified using Sanger sequencing. RESULTS: Of the 227 CSF samples, 90 were shown to be positive for multiplex RT-PCR with a positivity rate of 39.65% and a 95% confidence interval (33.2%, 46.1%). EV was the most common cause of VE, followed by EBV, HHV-6, MuV, CMV, VZV, and HSV-1. Most included cases occurred in summer, accounting for 49.78% of all cases. For EV, EBV, and HSV-2, multiplex RT-PCR showed a positivity rate of 34.36%, which was not statistically different from that of 30.4% shown by singleplex RT-PCR. The sequences of EV, EBV, VZV, MuV, CMV, HSV-1, HHV-6, and HSV-2 were confirmed by sequencing the PCR products obtained from multiplex and singleplex PCR. CONCLUSIONS: In children, VE is more prevalent in the summer than in other seasons in Zhejiang province, and EV may be the most common causative pathogen.

Hetero-bivalent Nanobodies Provide Broad-spectrum Protection against SARS-CoV-2 Variants of Concern including Omicron (preprint)

Following Delta, Omicron variant triggered a new wave of SARS-CoV-2 infection globally, adaptive evolution of the virus may not stop, the development of broad-spectrum antivirals is still urgent. We previously developed two hetero-bivalent nanobodies with potent neutralization against original WT SARS-CoV-2, termed aRBD-2-5 and aRBD-2-7, by fusing aRBD-2 with aRBD-5 or aRBD-7, respectively. Here, we resolved crystal structures of these nanobodies in complex with RBD, and found the epitope of aRBD-2 differs from that of aRBD-5, aRBD-7. aRBD-2 binds to a conserved epitope which renders its binding activity to all variants of concern (VOCs) including Omicron. Interestingly, although monovalent aRBD-5 and aRBD-7 lost binding to some variants, they effectively improved the overall affinity when transformed into the hetero-bivalent form after being fused with aRBD-2. Consistent with the high binding affinities, aRBD-2-5-Fc and aRBD-2-7-Fc exhibited ultra-potent neutralization to all five VOCs; particularly, aRBD-2-5-Fc neutralized authentic virus of Beta, Delta and Omicron with the IC50 of 5.98~9.65 ng/mL or 54.3~87.6 pM. Importantly, aRBD-2-5-Fc provided in vivo prophylactic protection for mice against WT and mouse-adapted SARS-CoV-2, and provided full protection against Omicron in hamster model when administrated either prophylactically or therapeutically. Taken together, we found a conserved epitope on RBD, and hetero-bivalent nanobodies had increased affinity for VOCs over its monovalent form, and provided potent and broad-spectrum protection both in vitro and in vivo against all tested major variants, and potentially future emerging variants. Our strategy provides a new solution in the development of therapeutic antibodies for COVID-19 caused by newly emergent VOCs.

Can dimensional reduction technology make better use of the information of uncertainty indices when predicting volatility of Chinese crude oil futures?

In this paper, we try to forecast the volatility of Chinese crude oil futures (COF) using multiple economic policy uncertainty indicators. MIDAS-RV model is combined with the principal component analysis (PCA), scaled PCA (SPCA) and partial least squares (PLS) techniques in this work, construct the newly MIDAS-RV-PCA, MIDAS-RV-PLS and MIDAS-RV-SPCA models, their prediction performance is compared with the common combination forecasting methods. The in-sample estimation analysis on MIDAS-RV-X models show the that it is necessary to consider multiple economic policy uncertainty indices when predicting the Chinese COF volatility and the in-sample analysis on dimensionality reduction model further demonstrate the rationality of using dimensionality reduction techniques. The out-of-sample evaluation results show that the MIDAS-RV-SPCA is a superior model when forecasting the short-term volatility of Chinese COF using multiple economic policy uncertainty indicators, especially during the periods of high volatility and COVID-19 pandemic. The results also indicates that the DMSPE(0.9) method in the combination forecasting method shows its superior forecasting ability in long-term volatility of Chinese COF, especially during the low volatility and pre-pandemic period.

Prevalence and trend of low vision among primary and secondary school students in Wuhan from 2019 to 2020

Objective: To provide a large-scale assessment the prevalence of poor vision in 2020 among children and adolescents in Wuhan City, Hubei province and to provide basis for healthy vision promotion.

Clinical features of COVID-19 in cancer patients within Wuhan, China.

BACKGROUND: There have been few reports on cancer patients with COVID-19 since its outbreak. Our study aimed to understand the clinical features of cancer patients with COVID-19 and determine the impact of surgery and chemotherapy on the patients' conditions. METHODS: Seventy COVID-19 patients from Renmin Hospital of Wuhan University, including 18 cancer patients, were enrolled in this study. Patients were classified into moderate or severe cases of COVID-19 and as well as non-cancer or cancer patients. Cancer patients were further grouped into Group A (prevalent cases with cancer history) and Group B (incident cases who underwent cancer treatment recently). Laboratory results were analyzed to determine whether cancer-related surgery and chemotherapy worsened the condition of cancer patients. The patients presented with clinical symptoms of COVID-19, including fever, dry cough, and polypnea; blood tests also revealed decreased lymphocyte counts and cellular immune function, and examination of CT scans revealed patchy ground-glass opacity of lungs. RESULTS: The results showed a significant difference (P<0.05) in levels of CD3 CD4 T lymphocytes and D-dimer between non-cancer and cancer patients with moderate COVID-19; there was also a significant difference (P<0.05) in levels of D-dimer between non-cancer and cancer patients with severe COVID-19. Except for liver function, there was no significant difference (P>0.05) between cancer patients in Group A and B with moderate COVID-19. A significant difference (P<0.05) in neutrophil-to-lymphocyte ratio (NLR) and CD4 T lymphocytes was observed between cancer patients with moderate COVID-19 and those with severe COVID-19. CONCLUSIONS: The results indicated that chemotherapy and surgery might not worsen the conditions of COVID-19 patients. NLR and CD4 T lymphocyte might be used as effective indicators for the conditions of cancer patients with COVID-19.

Management of Endovascular Treatment for Acute Ischemic Stroke During the COVID-19 Pandemic at a Single Institution in Beijing, China: A Brief Report.

BACKGROUND: The coronavirus disease (COVID-19) pandemic is currently a major challenge for health care systems around the world. For a time-sensitive emergency such as acute ischemic stroke (AIS), streamlined workflow times are essential to ensure good clinical outcomes. METHODS: The aim of this single-center, retrospective, observational study was to describe changes in stroke workflow patterns and clinical care during the COVID-19 pandemic. Data from AIS patients undergoing emergent endovascular treatment (EVT) between 23 January and 8 April 2020 were retrospectively collected and compared with data from patients admitted during a similar period in 2019. The primary outcome was difference in time from symptom onset to recanalization. Secondary outcomes included workflow times, clinical management, discharge outcomes, and health-economic data. RESULTS: In all, 21 AIS patients were admitted for emergent EVT during the 77-day study period, compared with 42 cases in 2019. Median time from symptom onset to recanalization was 132 minutes longer during the pandemic compared with the previous year (672 vs. 540 min, P=0.049). Patients admitted during the pandemic had a higher likelihood of endotracheal intubation (84.6% vs. 42.4%, P<0.05) and a higher incidence of delayed extubation after EVT (69.2% vs. 45.5%, P<0.05). National Institutes of Health Stroke Scale at hospital discharge was similar in the 2 cohorts, whereas neurointensive care unit stay was longer in patients admitted during the pandemic (10 vs. 7 days, P=0.013) and hospitalization costs were higher (123.9 vs. 95.2 thousand Chinese Yuan, P=0.052). CONCLUSION: Disruptions to medical services during the COVID-19 pandemic has particularly impacted AIS patients undergoing emergent EVT, resulting in increased workflow times. A structured and multidisciplinary protocol should be implemented to minimize treatment delays and maximize patient outcomes.

Discovery and evolution of 12N-substituted aloperine derivatives as anti-SARS-CoV-2 agents through targeting late entry stage.

So far, there is still no specific drug against COVID-19. Taking compound 1 with anti-EBOV activity as the lead, fifty-four 12N-substituted aloperine derivatives were synthesized and evaluated for the anti-SARS-CoV-2 activities using pseudotyped virus model. Among them, 8a exhibited the most potential effects against both pseudotyped and authentic SARS-CoV-2, as well as SARS-CoV and MERS-CoV, indicating a broad-spectrum anti-coronavirus profile. The mechanism study disclosed that 8a might block a late stage of viral entry, mainly via inhibiting host cathepsin B activity rather than directly targeting cathepsin B protein. Also, 8a could significantly reduce the release of multiple inflammatory cytokines in a time- and dose-dependent manner, such as IL-6, IL-1ß, IL-8 and MCP-1, the major contributors to cytokine storm. Therefore, 8a is a promising agent with the advantages of broad-spectrum anti-coronavirus and anti-cytokine effects, thus worthy of further investigation.

Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay (preprint)

The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLpro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PLpro inhibitors with IC50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50 values ranging from 0.56 to 0.90 {micro}M. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PLpro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLpro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50 values of 8.89 and 8.32 {micro}M, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 {micro}M). The X-ray crystal structures of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PLpro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLpro assay might be a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.

Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain (preprint)

Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL ([~]0.043 nM) and 3.18 ng/mL ([~]0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19. ImportanceTo date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics including SARS-CoV-2 targeting antibodies remains critical. Due to their small size (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.

An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity (preprint)

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent Mpro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 microM and EC50 of 1.27 microM, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the -methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.

Association of time to diagnosis with socioeconomic position and geographical accessibility to healthcare among symptomatic COVID-19 patients: A retrospective study in Hong Kong.

Early diagnosis is important to control COVID-19 outbreaks. This study aimed to assess how individual and area socioeconomic position and geographical accessibility to healthcare services were associated with the time to diagnosis among symptomatic COVID-19 patients in Hong Kong. Multivariable generalized linear regression was used to estimate the associations while adjusting for sociodemographic characteristics and case classification. This study found living in public rental housing and living in an area with low education were associated with longer time to diagnosis in the first wave of infections. Specifically, the risk of delayed diagnosis for public rental housing residents was mitigated by the higher density of public clinics/hospitals but was slightly increased by the higher density of private medical practitioners nearby. No such relations were found in the second wave of infections when the surveillance measures were enhanced. Given the grave impact of pandemics around the world, our findings call on taking inequalities into account when public health policies are being devised.

The Perioperative Management of Subarachnoid Hemorrhage During the Coronavirus Disease 2019 Pandemic in China.

BACKGROUND: For most of the international community outside the epicenter, coronavirus disease 2019 (COVID-19) containment is normalizing, and daily medical practice runs parallel to preventing and treating COVID-19. This experience of simultaneously conducting emergent surgery and infection control for COVID-19 disease is useful outside the epicenter during the pandemic. CASE DESCRIPTION: In this single-center retrospective observational study, we enrolled patients with subarachnoid hemorrhage (SAH) who were emergently admitted from January 23 to April 8, 2020. Based on the COVID-19 triage, patients with SAH were divided into 3 categories: positive, negative, and under investigation. During 77 days, 90 patients with SAH were admitted at the center. The median age was 55 years (range, 18-80 years) and 40 patients (44.4%) were male. None was positive, 42 patients were negative, and 48 patients were under investigation for COVID-19 before surgery. During the same period, 9 patients were diagnosed with COVID-19 without nosocomial infection. CONCLUSIONS: Rescuing patients with SAH and containment of COVID-19 benefit from joint prevention and control, a centralized system of equipment distribution and personnel assignment, and quick workflow establishment.

Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L (preprint)

The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a {gamma}-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1 pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1 pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the -ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.