Better therapeutic effect of oral administration of GS441524 compared with GC376.

FIP is a fatal feline disease caused by FIPV. Two drugs (GS441524 and GC376) target FIPV and have good therapeutic effect when administered by subcutaneous injection. However, subcutaneous injection has limitations compared with oral administration. Additionally, the oral efficacy of the two drugs has not been determined. Here, GS441524 and GC376 were shown to efficiently inhibit FIPV-rQS79 (recombination virus with a full-length field type I FIPV and the spike gene replaced with type II FIPV) and FIPV II (commercially available type II FIPV 79-1146) at a noncytotoxic concentration in CRFK cells. Moreover, the effective oral dose was determined via the in vivo pharmacokinetics of GS441524 and GC376. We conducted animal trials in three dosing groups and found that while GS441524 can effectively reducing the mortality of FIP subjects at a range of doses, GC376 only reducing the mortality rate at high doses. Additionally, compared with GC376, oral GS441524 has better absorption, slower clearance and a slower rate of metabolism. Furthermore, there was no significant difference between the oral and subcutaneous pharmacokinetic parameters. Collectively, our study is the first to evaluate the efficacy of oral GS441524 and GC376 using a relevant animal model. We also verified the reliability of oral GS441524 and the potential of oral GC376 as a reference for rational clinical drug use. Furthermore, the pharmacokinetic data provide insights into and potential directions for the optimization of these drugs.

Food Retail Network Spatial Matching and Urban Planning Policy Implications: The Case of Beijing, China

Food is the core of urban daily life and socio-economic activities but is rarely the focus of urban planning. The spatial layout of food retail outlets is important for optimizing the urban food system, improving land resource allocation, and encouraging healthy food consumption. Based on food retail POI data, this study employed kernel density estimation, road network centrality, spatial autocorrelation analysis, and locational entropy to analyze the spatial characteristics of supermarkets, produce markets, and small stores in an urban center in Beijing, and explored street coupling and supply-demand matching. The results indicated that within the study area: (1) supermarkets had an obvious “core-periphery” distribution, produce markets had a polycentric distribution, and small stores had a relatively uniform distribution;(2) road network centrality indices revealed a differentiated multi-core-edge distribution;(3) streets with high locational entropy values for supermarkets and produce markets were mostly concentrated in the central area, whereas the matching distribution of small stores was relatively balanced. From the perspective of urban planning, policy implications are proposed based on spatial and social equity, urban-rural differences, population structure and distribution status, and a resilient supply chain. The study findings have practical significance for guiding the development of urban food systems in a healthy, just, and sustainable direction, as well as rational urban land planning.

Identification of a Novel Neutralizing Epitope on the N-Terminal Domain of the Human Coronavirus 229E Spike Protein.

The receptor binding domain (RBD) of the coronavirus spike protein (S) has been verified to be the main target for potent neutralizing antibodies (nAbs) in most coronaviruses, and the N-terminal domain (NTD) of some betacoronaviruses has also been indicated to induce nAbs. For alphacoronavirus HCoV-229E, its RBD has been shown to have neutralizing epitopes, and these epitopes could change over time. However, whether neutralizing epitopes exist on the NTD and whether these epitopes change like those of the RBD are still unknown. Here, we verified that neutralizing epitopes exist on the NTD of HCoV-229E. Furthermore, we characterized an NTD targeting nAb 5H10, which could neutralize both pseudotyped and authentic HCoV-229E VR740 in vitro. Epitope mapping indicated that 5H10 targeted motif E1 (147-167 aa) and identified F159 as critical for 5H10 binding. More importantly, our results revealed that motif E1 was highly conserved among clinical isolates except for F159. Further data proved that mutations at position 159 gradually appeared over time and could completely abolish the neutralizing ability of 5H10, supporting the notion that position 159 may be under selective pressure during the human epidemic. In addition, we also found that contemporary clinical serum has a stronger binding capacity for the NTD of contemporary strains than historic strains, proving that the epitope on the NTD could change over time. In summary, these findings define a novel neutralizing epitope on the NTD of HCoV-229E S and provide a theoretical basis for the design of vaccines against HCoV-229E or related coronaviruses. IMPORTANCE Characterization of the neutralizing epitope of the spike (S) protein, the major invasion protein of coronaviruses, can help us better understand the evolutionary characteristics of these viruses and promote vaccine development. To date, the neutralizing epitope distribution of alphacoronaviruses is not well known. Here, we identified a neutralizing antibody that targeted the N-terminal domain (NTD) of the alphacoronavirus HCoV-229E S protein. Epitope mapping revealed a novel epitope that was not previously discovered in HCoV-229E. Further studies identified an important residue, F159. Mutations that gradually appeared over time at this site abolished the neutralizing ability of 5H10, indicating that selective pressure occurred at this position in the spread of HCoV-229E. Furthermore, we found that the epitopes within the NTD also changed over time. Taken together, our findings defined a novel neutralizing epitope and highlighted the role of the NTD in the future prevention and control of HCoV-229E or related coronaviruses.

Structure of the multiple functional domains from coronavirus nonstructural protein 3.

Coronaviruses (CoVs) are potential pandemic pathogens that can infect a variety of hosts and cause respiratory, enteric, hepatic and neurological diseases. Nonstructural protein 3 (nsp3), an essential component of the replication/transcription complex, is one of the most important antiviral targets. Here, we report the first crystal structure of multiple functional domains from porcine delta-coronavirus (PDCoV) nsp3, including the macro domain (Macro), ubiquitin-like domain 2 (Ubl2) and papain-like protease (PLpro) catalytic domain. In the asymmetric unit, two of the subunits form the head-to-tail homodimer with an interaction interface between Macro and PLpro. However, PDCoV Macro-Ubl2-PLpro mainly exists as a monomer in solution. Then, we conducted fluorescent resonance energy transfer-based protease assays and found that PDCoV PLpro can cleave a peptide by mimicking the cognate nsp2/nsp3 cleavage site in peptide substrates and exhibits deubiquitinating and de-interferon stimulated gene(deISGylating) activities by hydrolysing ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) and ISG15-AMC substrates. Moreover, the deletion of Macro or Macro-Ubl2 decreased the enzyme activity of PLpro, indicating that Macro and Ubl2 play important roles in maintaining the stability of the PLpro domain. Two active sites of PLpro, Cys260 and His398, were determined; unexpectedly, the conserved site Asp412 was not the third active site. Furthermore, the motif "NGYDT" (amino acids 409-413) was important for stabilizing the enzyme activity of PLpro, and the N409A mutant significantly decreased the enzyme activity of PLpro. These results provide novel insights into the replication mechanism of CoV and new clues for future drug design.

Lysine 164 is critical for SARS-CoV-2 Nsp1 inhibition of host gene expression.

The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469 000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.

Interpretation of Novel Coronavirus Infection ：Expert Consensus on Guidance and Prevention Strategies for Hospi tal Pharmacists and the Pharmacy Workforce / 中国药房

OBJECTIVE：To provid e reference for pharmaceutical workers to better understand Novel Coronavirus Infection ： Expert Consensus on Guidance and Prevention Strategies for Hospital Pharmacists and the Pharmacy Workforce （hereinafter referred to as “expert consensus ”），and to apply and practice in specific work ，so as to give full play to the role of pharmacists to help fight the epidemic.METHODS ：The background of the formulation and revision of the expert consensus were introduced ，and its main contents and viewpoints were interpreted. RESULTS & CONCLUSIONS ：The text of expert consensus is divided into 8 parts，mainly including disease diagnosis and treatment [SARS-CoV- 2 infection related background ，clinical manifestations and diagnosis， treatment]，hospital pharmacy （prevention and control strategy ，work guidance ），drug and facility support management（key drug/facility/equipment support ，management and use of the drug in special circumstances ），information sources and related resources ，etc.，which comprehensively and detailedly provide information ，guidance and strategies for coronavirus SARS-CoV-2 infection prevention and control to play the role of pharmacists in hospital pharmacy well ，do well in the protection of staff in different pharmaceutical posts ，drug security work in response to epidemic situation ，and develop pharmaceutical care. So far，the understanding of SARS-CoV- 2 in the pharmaceutical industry is relatively limited. Based on the accumulated experience and progress in epidemic prevention and control ，the expert consensus will be updated and improved continuously ，so as to provide guidance and help for hospital pharmaceutical personnel.