Cross-reactive antibody responses to coronaviruses elicited by SARS-CoV-2 infection or vaccination (preprint)

Background: The newly emerged SARS-CoV-2 possesses shared antigenic epitopes with other human coronaviruses. We investigated if COVID-19 vaccination or SARS-CoV-2 infection may boost cross-reactive antibodies to other human coronaviruses. Methods Pre- and post-vaccination sera from SARS-CoV-2 naïve healthy subjects who received three doses of the mRNA vaccine (BioNTech, BNT) or the inactivated vaccine (CoronaVac, CV) were used to monitor the level of cross-reactive antibodies raised against other human coronaviruses by enzyme-linked immunosorbent assay. In comparison, convalescent sera from COVID-19 patients with or without prior vaccination history were also tested. Pseudoparticle neutralization assay was performed to detect neutralization antibody against MERS-CoV. Results Among SARS-CoV-2 infection naïve subjects, BNT or CV significantly increased the anti-S2 antibodies against Betacoronaviruses (OC43 and MERS-CoV) but not Alphacoronaviruses (229E). The pre-vaccination antibody response to the common cold human coronaviruses did not negatively impact the post-vaccination antibody response to SARS-CoV-2. Cross-reactive antibodies that binds to the S2 protein of MERS-CoV were similarly detected from the convalescent sera of COVID-19 patients with or without vaccination history. However, these anti-S2 antibodies do not possess neutralizing activity in MERS-CoV pseudoparticle neutralisation tests. Conclusions Our results suggest that SARS-CoV-2 infection or vaccination may potentially modulate population immune landscape against previously exposed or novel human coronaviruses. The findings have implications for future sero-epidemiological studies on MERS-CoV.

Identification of Age-Related Characteristic Genes Involved in Severe COVID-19 Infection Among Elderly Patients using Machine Learning and Immune Cell Infiltration Analysis (preprint)

Background Elderly patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are at higher risk of severe clinical manifestation, extended hospitalization, and increased mortality. Those patients are more likely to experience persistent symptoms and exacerbate the condition of basic diseases with long COVID-19 syndrome. However, the molecular mechanisms underlying severe COVID-19 in the elderly patients remain unclear. Our study aims to investigate the function of the interaction between disease-characteristic genes and immune cell infiltration in patients with severe COVID-19 infection.Methods COVID-19 datasets (GSE164805 and GSE180594) and aging dataset (GSE69832) were obtained from the Gene Expression Omnibus (GEO) database. The combined different expression genes (DEGs) were subjected to Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Diseases Ontology (DO) functional enrichment analysis, Gene Set Enrichment Analysis (GSEA), machine learning, and immune cell infiltration analysis.Results GO and KEGG enrichment analysis revealed that the eight DEGs (IL23A, PTGER4, PLCB1, IL1B, CXCR1, C1QB, MX2, ALOX12) were mainly involved in inflammatory mediator regulation of TRP channels, coronavirus disease-COVID-19, and cytokine activity signaling pathways. Two-degree algorithm (LASSO and SVM-RFE) and correlation analysis showed that the seven DEGs upregulated the immune cells of macrophages M0/M1, memory B cells, gramma delta T cell, dendritic cell resting and master cell resisting.Conclusion Our study identified seven hallmark genes that can serve as disease-characteristic genes and target immune cells infiltrated in severe COVID-19 patients among the elderly population, which may contribute to the study of pathogenesis and the evaluation of diagnosis and prognosis in aging patients infected with severe COVID-19.

Glycyrrhetinic acid: A potential drug for the treatment of COVID-19 cytokine storm.

BACKGROUND: The cytokine storm (CS) triggered by coronavirus disease 2019 (COVID-19) has caused serious harm to health of humanity and huge economic burden to the world, and there is a lack of effective methods to treat this complication. PURPOSE: In this research, we used network pharmacology and molecular docking to reveal the interaction mechanism in the glycyrrhetinic acid (GA) for the treatment of CS, and validated the effect of GA intervention CS by experiments. STUDY DESIGN: First, we screened corresponding target of GA and CS from online databases, and obtained the action target genes through the Venn diagram. Then, protein-protein interaction (PPI) network, Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of the action target genes were acquired by R language to predict its mechanism. Next, molecular docking was performed on core targets. Finally, experiments in which GA intervened in lipopolysaccharide (LPS)-induced CS were implemented. RESULTS: 84 action target genes were obtained from online database. The PPI network of target genes showed that TNF, IL6, MAPK3, PTGS2, ESR1 and PPARG were considered as the core genes. The results of GO and KEGG showed that action target genes were closely related to inflammatory and immune related signaling pathways, such as TNF signaling pathway, IL-17 signaling pathway, Human cytomegalovirus infection, PPAR signaling pathway and so on. Molecule docking results prompted that GA had fine affinity with IL6 and TNF proteins. Finally, in vivo and in vitro experimental results showed that GA could significantly inhibit LPS-induced CS. CONCLUSION: GA has a potential inhibitory effect on CS, which is worthy of further exploration.

Roles and Challenges for Village Doctors in COVID-19 Pandemic Prevention and Control in Rural Beijing, China: A Qualitative Study

Objectives Rural areas in China are more vulnerable to COVID-19 pandemic than urban areas, due to their far fewer health care resources. Village doctors, as rural grassroots health workers in China, have been actively engaged in the pandemic prevention and control. This study aims to describe the roles of village doctors in rural China, and the challenges they have faced during the prevention and control of the COVID-19 pandemic. Setting This study was conducted in three towns in Huairou District, Beijing, China. Design We carried out semi-structured interviews with 75 key informants. All the interviews were audio-recorded and transcribed verbatim. We employed thematic analysis to define themes and sub-themes from the qualitative data. Results We reported four themes. First, the village doctor guided the village committee to carry out decontamination, monitored home-isolated residents, and disseminated knowledge on prevention of the COVID-19 pandemic during the rural pandemic prevention and control. Second, they took pandemic prevention measures in village clinics, distributed pandemic prevention materials, and undertook pre-screening triage. Third, village doctors provided basic medical care, including treatment of common diseases as well as the purchase and delivery of medicines to villagers. Fourth, village doctors faced difficulties and challenges, such as inadequate medical skills, aging staff structure, and lack of pandemic prevention materials. Conclusions Despite many difficulties and challenges, village doctors have actively participated in rural pandemic prevention and control, and made outstanding contributions to curbing spread of COVID-19 pandemic in rural areas. Village doctors provide basic health care while participating in various non-medical tasks.

Total virome characterizations of game animals in China reveals a spectrum of emerging viral pathogens (preprint)

Game animals are wildlife species often traded and consumed as exotic food, and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1725 game animals, representing 16 species and five mammalian orders, sampled across China. From this we identified 71 mammalian viruses, with 45 described for the first time. Eighteen viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high risk viruses. We identified the transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We similarly identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence.

Willingness to Receive COVID-19 Vaccination Among People Living With HIV and AIDS in China: A Nationwide Online Survey (preprint)

Background: People living with HIV and AIDS (PLWHA) could benefit from COVID-19 vaccination. There is a dearth of evidence on willingness to receive COVID-19 vaccination (WTR) among PLWHA.Methods: The study is a multicenter cross-sectional online survey conducted in eight conveniently selected Chinese metropolitan cities between January and February 2021. Participants were PLWHA aged 18-65 years and living in the study cities. Eight community-based organizations (CBO) providing services to PLWHA, one in each city, facilitated the recruitment through their networks. Eligible PLWHA completed an online survey developed using Golden Data, a widely used encrypted web-based survey platform in China. Logistic regression models and path analysis were used in data analysis.Findings: Out of 10,845 PLWHA approached by the CBO, 2740 completed the survey. This analysis was performed among 2570 participants who had never received COVID-19 vaccination. Over half of the participants reported WTR (57·2%, 1470/2570). After adjusting for potential confounders, all four constructs of the Theory of Planned Behavior (TPB) were significantly associated with WTR, including positive attitudes (adjusted odds ratio, aOR: 1·11, 95%CI: 1·09, 1·12), negative attitudes (aOR: 0·96, 95%CI: 0·94, 0.97), perceived support from significant others (perceived subjective norm) (aOR: 1·53, 95%CI: 1·46, 1·61), and perceived higher behavioral control (aOR: 1·13, 95%CI: 1·11, 1·14). At the interpersonal level, receiving advice supportive of COVID-19 vaccination from doctors (aOR: 1·99, 95%CI: 1·65, 2·40), CBO staff (aOR: 1·89, 95%CI: 1·51, 2·36), friends and/or family members (aOR: 3·22, 95%CI: 1·93, 5·35), and PLWHA peers (aOR: 2.38, 95%CI: 1·85, 3·08) were associated with higher WTR. Overall opinion supporting COVID-19 vaccination for PLWHA on Internet or social media was also positively associated with WTR (aOR: 1·59, 95%CI: 1·31, 1·94). Path analysis indicated that interpersonal-level variables was indirectly associated with WTR through TPB-related perceptions (β=0·43, 95% CI=0·37, 0·51).Interpretation: PLWHA in China reported a relatively low WTR. Interpersonal communication and Internet/social media may be the major sources to convey information and promote COVID-19 vaccination in the population.Funding Statement: This work was supported by the Academic technology leader project of Changzhi Medical College (Grant No. XSQ201902), he Beijing Excellent Talent Plan (2018000021223ZK04) and the National Institute of Mental Health of the National Institutes of Health under Award Numbers R34MH119963.Declaration of Interests: The authors have no conflicts of interest to disclose.Ethics Approval Statement: The Institutional Review Boards of Changzhi Medical College (RT2021003) approved this study.

Impact of Home Confinement Due to COVID-19 Pandemic on Mental Health in Young Children: A Natural Experiment (preprint)

Background: The outbreak of coronavirus disease in 2019 (COVID-19) has had an enormous impact on people’s health, and profoundly changed our lives and society. However, the impact of pandemic-related home confinement on children’s life and their mental health remains unknown. We took advantage of a natural experiment due to the onset of COVID-19 and examined this issue in two consecutive cohorts. Methods: This study compared mental health, family environment and lifestyles of two representative cohorts of young children in Shanghai, China. Both prospective cohorts recruited children at entry to kindergarten. Surveys were conducted at both entry and graduation of kindergarten using the same assessment instruments. Children in the non-exposed cohort experienced a normal period of kindergarten study and graduated in June, 2019; those in the exposed cohort were under confinement for five months and graduated in June, 2020. Findings: Compared with the non-exposed cohort, the exposed cohort had a significant increased percentage of children classified as “at-risk for mental health problems”, with an adjusted absolute risk difference of 3·1% (95% CI, 1·9% to 4·4%) for total difficulties as measured by the Strength and Difficulty Questionnaire. The effect of pandemic related confinement on mental health was larger in children with lower parental education. The exposed cohort had longer media time, sleep duration and a less favorable home environment. Interpretation: The pandemic related confinement negatively affected children's mental health and home environment. Strategies and actions are urgently needed to improve parenting skills and mitigate the negative impact by strengthening the family environment.Funding Statement: National Natural Science Foundation of China, Shanghai Committee of Science and Technology, Shanghai Municipal Health Commission, Shanghai Education CommitteeDeclaration of Interests: We declare no competing interests.Ethics Approval Statement: The study was approved by the Institutional Review Board of the Shanghai Children’s Medical Center, the Shanghai Jiao Tong University School of Medicine (SCMCIRB-K2016022-01)

Homology analysis of ACE2 among different species and its implications for the host range of 2019-nCoV

In order to explore the possibility of cross-species transmission of 2019-nCoV between human and animals, the homology of angiotensin-converting enzyme 2(ACE2)receptor among different species were analyzed. The amino acid sequences of ACE2 in different species were downloaded from NCBI and analyzed by BLAST, iTOL and MEGA 7 software. The results showed that the full-length amino acid sequence of ACE2 (1-805) was highly homologous in mammals, especially in non-human primates. Comparing with the full-length amino acid sequence of ACE2, the recognition rate with ACE2-PD of human (peptidase domain 19-615, which binds to the S protein of 2019-nCoV) was further improved in cats and ruminants, while bats was declined. Alignment of 20 key amino acids (interface amino acids) directly interacting with S protein in ACE2 protein showed that the interface amino acid sequence of rhesus monkey was identical to that of human, while that of ruminants (cattle, sheep) and cats was similar to that of human. In conclusion, the primates can be used as good animal infection models, ruminants (cattle, sheep) and cats may also be potentially infected with 2019-nCoV. Therefore. It suggest that the etiological and serological detection of 2019-nCoV among animals should be actively promoted.

Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2).

PURPOSE: Lianhuaqingwen (LH) as traditional Chinese medicine (TCM) formula has been used to treat influenza and exerted broad-spectrum antiviral effects on a series of influenza viruses and immune regulatory effects Ding et al. (2017). The goal of this study is to demonstrate the antiviral activity of LH against the novel SARS-CoV-2 virus and its potential effect in regulating host immune response. METHODS: The antiviral activity of LH against SARS-CoV-2 was assessed in Vero E6 cells using CPE and plaque reduction assay. The effect of LH on virion morphology was visualized under transmission electron microscope. Pro-inflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. RESULTS: LH significantly inhibited SARS-CoV-2 replication in Vero E6 cells and markedly reduced pro-inflammatory cytokines (TNF-α, IL-6, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Furthermore, LH treatment resulted in abnormal particle morphology of virion in cells. CONCLUSIONS: LH significantly inhibits the SARS-COV-2 replication, affects virus morphology and exerts anti-inflammatory activity in vitro. These findings indicate that LH protects against the virus attack, making its use a novel strategy for controlling the COVID-19 disease.

Snapshot of the evolution and mutation patterns of SARS-CoV-2 (preprint)

The COVID-19 pandemic is the most important public health threat in recent history. Here we study how its causal agent, SARS-CoV-2, has diversified genetically since its first emergence in December 2019. We have created a pipeline combining both phylogenetic and structural analysis to identify possible human-adaptation related mutations in a data set consisting of 4,894 SARS-CoV-2 complete genome sequences. Although the phylogenetic diversity of SARS-CoV-2 is low, the whole genome phylogenetic tree can be divided into five clusters/clades based on the tree topology and clustering of specific mutations, but its branches exhibit low genetic distance and bootstrap support values. We also identified 11 residues that are high-frequency substitutions, with four of them currently showing some signal for potential positive selection. These fast-evolving sites are in the non-structural proteins nsp2, nsp5 (3CL-protease), nsp6, nsp12 (polymerase) and nsp13 (helicase), in accessory proteins (ORF3a, ORF8) and in the structural proteins N and S. Temporal and spatial analysis of these potentially adaptive mutations revealed that the incidence of some of these sites was declining after having reached an (often local) peak, whereas the frequency of other sites is continually increasing and now exhibit a worldwide distribution. Structural analysis revealed that the mutations are located on the surface of the proteins that modulate biochemical properties. We speculate that this improves binding to cellular proteins and hence represents fine-tuning of adaptation to human cells. Our study has implications for the design of biochemical and clinical experiments to assess whether important properties of SARS-CoV-2 have changed during the epidemic.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Household transmission of COVID-19, Shenzhen, January-February 2020 (preprint)

Coronavirus disease 2019 has led to more than three million cases globally. Since the first family cluster of COVID-19 cases identified in Shenzhen in early January, most of the local transmission occurred within household contacts. Identifying the factors associated with household transmission is of great importance to guide preventive measures.

Comparison of SARS-CoV-2 spike protein binding to human, pet, farm animals, and putative intermediate hosts ACE2 and ACE2 receptors (preprint)

The emergence of a novel coronavirus, SARS-CoV-2, resulted in a pandemic. Here, we used recently released X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptor allows to define residues important for binding. From the 20 amino acids in ACE2 that contact S up to seven can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S-binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or the acquisition of N-glycosylation sites located near the S-interface. Of note, pigs and dogs which are not or not effectively infected, respectively, have only a few changes in the binding site have relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with viruses from bat and pangolin revealed that the latter contains only one substitution, whereas the bat virus exhibits five. However, ACE2 of pangolin exhibit seven changes relative to human ACE2, a similar number of substitutions is present in ACE2 of bats, raccoon, and civet suggesting that SARS-CoV-2 may not especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-COV-2. IMPORTANCESARS-CoV-2 is threatening people worldwide and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes indicating that the species barrier might be low. However, the lower expression of ACE2 in the upper respiratory tract of some pets and livestock means more research and monitoring should be done to explore the animal source of infection and the risk of potential cross-species transmission. Finally, the analysis also showed that SARS-CoV-2 may not specifically adapted to any of its putative intermediate hosts.

Flocked swab might be one main reason causing the high false-negative rate in COVID-19 screening----the advantages of a novel silicone swab (preprint)

RNA testing using RT-PCR can provide direct evidence for diagnoses of COVID-19 which has brought unexpected disasters and changes to our human society. However, the absorption of cotton swab for RNA lysates may lead to a low concentration of detectable RNA, which might be one of the main reasons for the unstable positive detecting rate. We designed and manufactured a kind of silicone swab with concave-convex structure, and further compared the effects of silicone and cotton swab on RNA extraction. Principal component analysis and Paired Wilcoxcon test suggested that a higher RNA concentration and A260/A280 would be obtained using silicone swab. The results indicated that our silicone swab had a more excellent ability to sample than the cotton swab, characterized by the higher quantity and quality of extracted RNA. Thus, we advised that the current cotton swabs need to be improved urgently in COVID-19 diagnoses and the process of "sample collection" and "sample pre-processing" must be standardized and emphasized. HighlightsThe current cotton swabs need to be improved urgently in COVID-19 screening.

Serological immunochromatographic approach in diagnosis with SARS-CoV-2 infected COVID-19 patients (preprint)

An outbreak of new coronavirus SARS-CoV-2 was occurred in Wuhan, China and rapidly spread to other cities and nations. The standard diagnostic approach that widely adopted in the clinic is nuclear acid detection by real-time RT-PCR. However, the false-negative rate of the technique is unneglectable and serological methods are urgently warranted. Here, we presented the colloidal gold-based immunochromatographic (ICG) strip targeting viral IgM or IgG antibody and compared it with real-time RT-PCR. The sensitivity of ICG assay with IgM and IgG combinatorial detection in nuclear acid confirmed cases were 11.1%, 92.9% and 96.8% at the early stage (1-7 days after onset), intermediate stage (8-14 days after onset), and late-stage (more than 15 days), respectively. The ICG detection capacity in nuclear acid-negative suspected cases was 43.6%. In addition, the consistencies of whole blood samples with plasma were 100% and 97.1% in IgM and IgG strips, respectively. In conclusion, serological ICG strip assay in detecting SARS-CoV-2 infection is both sensitive and consistent, which is considered as an excellent supplementary approach in clinical application.

Can routine laboratory tests discriminate 2019 novel coronavirus infected pneumonia from other community-acquired pneumonia? (preprint)

Background. The clinical presentation of 2019 Novel Coronavirus (2019-nCov) infected pneumonia (NCIP) resembles that of other etiologies of community-acquired pneumonia (CAP). We aimed to identify clinical laboratory features to distinguish NCIP from CAP. Methods. We compared the ability of the hematological and biochemical features of 84 patients with NCIP at hospital admission and 316 patients with CAP. Parameters independently predictive of NCIP were calculated by multivariate logistic regression. The receiver operating characteristic (ROC) curves were generated and the area under the ROC curve (AUC) was measured to evaluate the discriminative ability. Results. Most hematological and biochemical indexes of patients with NCIP were significantly different from patients with CAP. Nine laboratory parameters were identified to be highly predictive of a diagnosis of NCIP by multivariate analysis. The AUCs demonstrated good discriminatory ability for red cell distribution width (RDW) with an AUC of 0.88 and Hemoglobin (HGB) with an AUC of 0.82. Red blood cell (RBC), albumin (ALB), eosinophil (EO), hematocrit (HCT), alkaline phosphatase (ALP), and white blood cell (WBC) had fair discriminatory ability. Combinations of any two parameters performed better than did the RDW alone. Conclusions. Routine laboratory examinations may be helpful for the diagnosis of NCIP. Application of laboratory tests may help to optimize the use of isolation rooms for patients when they present with unexplained febrile respiratory illnesses.