Exploring the potential mechanisms of impairment on genitourinary system associated with coronavirus disease 2019 infection:Bioinformatics and molecular simulation analyses

Objective:The novel coronavirus(severe acute respiratory syndrome coronavirus 2)has been spreading worldwide since December 2019,posing a serious danger to human health and socioeconomic development.A large number of clinical trials have revealed that coronavirus disease 2019(COVID-19)results in multi-organ damage including the urogenital system.This study aimed to explore the potential mechanisms of genitourinary damage associated with COVID-19 infection through bioinformatics and molecular simulation analysis.Methods:We used multiple publicly available databases to explore the expression patterns of angiotensin-converting enzyme 2(ACE2),transmembrane serine protease 2(TMPRSS2),and CD147 in major organs in the healthy and disease-specific populations,particularly the genitourinary organs.Single-cell RNA sequencing was used to analyze the cell-specific expression patterns of ACE2,TMPRSS2,CD147,cytokine receptors,and cytokine interacting proteins in genitourinary organs,such as the bladder,kidney,prostate,and testis.Additionally,gene set enrichmentanalysis was used to investigate the relationship between testosterone levels and COVID-19 vulnerability in patients with prostate cancer.Results:The results revealed that ACE2,TMPRSS2,and CD147 were highly expressed in normal urogenital organs.Then,they were also highly expressed in multiple tumors and chronic kidney diseases.Additionally,ACE2,TMPRSS2,and CD147 were significantly expressed in a range of cells in urogenital organs according to single-cell RNA sequencing.Cytokine receptors and cytokine interacting proteins,especially CCL2,JUN,and TIMP1,were commonly highly expressed in urogenital organs.Finally,gene set enrichment analysis results showed that high testosterone levels in prostate cancer patients were significantly related to the JAK-STAT signaling pathway and the Toll-like receptor signaling pathway which were associated with COVID-19.Conclusion:Our study provides new insights into the potential mechanisms of severe acute respiratory syndrome coronavirus 2 damage to urogenital organs from multiple perspectives,which may draw the attention of urologists to COVID-19 and contribute to the development of targeted drugs.

A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus(2019-nCoV) infected pneumonia(standard version)

In December 2019, a new type viral pneumonia cases occurred in Wuhan, Hubei Province;and then named "2019 novel coronavirus(2019-nCoV)" by the World Health Organization(WHO) on 12 January 2020. For it is a never been experienced respiratory disease before and with infection ability widely and quickly, it attracted the world’s attention but without treatment and control manual. For the request from frontline clinicians and public health professionals of 2019-nCoV infected pneumonia management, an evidence-based guideline urgently needs to be developed. Therefore, we drafted this guideline according to the rapid advice guidelines methodology and general rules of WHO guideline development;we also added the first-hand management data of Zhongnan Hospital of Wuhan University. This guideline includes the guideline methodology, epidemiological characteristics, disease screening and population prevention, diagnosis, treatment and control(including traditional Chinese Medicine), nosocomial infection prevention and control, and disease nursing of the 2019-nCoV. Moreover, we also provide a whole process of a successful treatment case of the severe 2019-nCoV infected pneumonia and experience and lessons of hospital rescue for 2019-nCoV infections. This rapid advice guideline is suitable for the first frontline doctors and nurses, managers of hospitals and healthcare sections, community residents, public health persons, relevant researchers, and all person who are interested in the 2019-nCoV.

The origin, transmission and clinical therapies on coronavirus disease 2019(COVID-19) outbreak——an update on the status

An acute respiratory disease,caused by a novel coronavirus(SARS-CoV-2,previously known as 2019-nCoV),the coronavirus disease 2019(COVID-19)has spread throughout China and received worldwide attention.On 30 January 2020,World Health Organization(WHO)officially declared the COVID-19 epidemic as a public health emergency of international concern.The emergence of SARS-CoV-2,since the severe acute respiratory syndrome coronavirus(SARSCoV)in 2002 and Middle East respiratory syndrome coronavirus(MERS-CoV)in 2012,marked the third introduction of a highly pathogenic and large-scale epidemic coronavirus into the human population in the twenty-first century.As of 1 March 2020,a total of 87,137 confirmed cases globally,79,968 confirmed in China and 7169 outside of China,with 2977 deaths(3.4%)had been reported by WHO.Meanwhile,several independent research groups have identified that SARS-CoV-2 belongs toβ-coronavirus,with highly identical genome to bat coronavirus,pointing to bat as the natural host.The novel coronavirus uses the same receptor,angiotensin-converting enzyme 2(ACE2)as that for SARS-CoV,and mainly spreads through the respiratory tract.Importantly,increasingly evidence showed sustained human-tohuman transmission,along with many exported cases across the globe.The clinical symptoms of COVID-19 patients include fever,cough,fatigue and a small population of patients appeared gastrointestinal infection symptoms.The elderly and people with underlying diseases are susceptible to infection and prone to serious outcomes,which may be associated with acute respiratory distress syndrome(ARDS)and cytokine storm.Currently,there are few specific antiviral strategies,but several potent candidates of antivirals and repurposed drugs are under urgent investigation.In this review,we summarized the latest research progress of the epidemiology,pathogenesis,and clinical characteristics of COVID-19,and discussed the current treatment and scientific advancements to combat the epidemic novel coronavirus.

Zoonotic origins of human coronavirus 2019(HCoV-19/SARS-CoV-2):why is this work important?

The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by infection with human coronavirus 2019 (HCoV-19/SARS-CoV-2/2019-nCoV), is a global threat to the human population. Here, we briefly summarize the available data for the zoonotic origins of HCoV-19, with reference to the other two epidemics of highly virulent coronaviruses, SARSCoV and MERS-CoV, which cause severe pneumonia in humans. We propose to intensify future efforts for tracing the origins of HCoV-19, which is a very important scientific question for the control and prevention of the pandemic.

Fear can be more harmful than the severe acute respiratory syndrome coronavirus 2 in controlling the corona virus disease 2019 epidemic

The current corona virus disease 2019 outbreak caused by severe acute respiratory syndrome coronavirus 2 started in Wuhan,China in December 2019 and has put the world on alert.To safeguard Chinese citizens and to strengthen global health security,China has made great efforts to control the epidemic.Many in the global community have joined China to limit the epidemic.However,discrimination and prejudice driven by fear or misinformation have been flowing globally,superseding evidence and jeopardizing the anti-severe acute respiratory syndrome coronavirus 2 efforts.We analyze this phenomenon and its underlying causes and suggest practical solutions.

Traditional Chinese medicine for treatment of coronavirus disease 2019:a review

prevalence.A number of clinical workers and researchers have made great efforts to understand the pathogenesis and clinical characteristics and develop effective drugs for treatment.However,no effective drugs with antiviral effects on severe acute respiratory syndrome coronavirus 2 have been discovered currently.Traditional Chinese medicine(TCM)has gained abundant experience in the treatment of infectious diseases for thousands of years.In this review,the authors summarized the clinical outcome,pathogensis and current application of TCM on coronavirus disease 2019.Further,we discussed the potential mechanisms and the future research directions of TCM against severe acute respiratory syndrome coronavirus 2.

Network pharmacology studies on the effect of Chai-Ling decoction incoronavirus disease 2019

Background:Chai-Ling decoction(CLD),derived from a modification of Xiao-Chai-Hu(XCH)decoction and Wu-Ling-San(WLS)decoction,has been used to treat the early-stage of coronavirus disease 2019(COVID-19).However,the mechanisms of CLD in COVID-19 remain unknown.In this study,the potential mechanisms of CLD in COVID-19 were preliminarily investigated based on network pharmacology and molecular docking method.Methods:Initially,the active components and targets of CLD were screened based on Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and PharmMapper database.The targets of COVID-19 were obtained from GeneCards database.The protein-protein interaction network was established using STRING database to analyze the key targets.Gene Oncology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes analysis were also conducted to evaluate the pathways related to the targets of CLD on COVID-19.Moreover,the compound-target-pathway network was established using Cytoscape 3.2.7.Subsequently,the molecular docking method was performed to select the active compounds with high binding affinity on severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and angiotensin-converting enzyme 2(ACE2),which is the key target of SARS-CoV-2 in entering target cells.The possible binding sites were also visualized by a three-dimensional graph.Results:Network pharmacology analysis showed that there were 106 active components and 160 targets of CLD.Additionally,251 targets related to COVID-19 were identified,and 24 candidates of CLD on COVID-19 were selected.A total of 283 GO terms of CLD on COVID-19 were identified,and 181 pathways were screened based on GO and Kyoto Encyclopedia of Genes and Genomes analyses.CLD might alleviate the inflammatory response and improve lung injury to treat COVID-19 through interleukin 17 signaling,T helper cell 17 differentiation,tumor necrosis factor signaling,and hypoxia inducible factor-1 signaling.Besides,molecular docking indicated that beta-sitosterol,kaempferol,and stigmasterol were the top three candidates in CLD with the highest affinity to SARS-CoV-2 and ACE2.Conclusion:Our study identifies the potential mechanisms of CLD on COVID-19 and beta-sitosterol,kaempferol,and stigmasterol may be the key compounds that exert antiviral effects against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic: A review and an update on cases in Africa

The world has experienced several epidemics posing serious threat to global public health,including the 2002 severe acute respiratory syndrome(SARS)epidemic that caused 800 deaths out of about 8000 cases,the 2009 H1N1 pandemic with 18500 deaths,the 2012 Middle East respiratory syndrome(MERS)epidemic that caused 800 deaths out of 2500 cases,the 2014 Ebola outbreak with 28616 cases and 11310 deaths,and the current coronavirus disease(COVID-19)pandemic with more than 35000 deaths out of over 730000 confirmed cases till now.Emerging infectious diseases continue to infect and reduce human populations.The COVID-19 pandemic has spread to more than 114 countries before it was officially declared as a pandemic by the WHO on the 11th March 2020.Here,the first set of index cases inAfrica,and the differences between SARS-CoV-2 and other coronaviruses in addition to the preventive strategies on the emergence of COVID-19 were reviewed.

Comparison of Clinical Characteristics and Outcomes of Pediatric and Adult Patients with Coronavirus Disease 2019 in Shenzhen,China

Objective Here we aimed to investigate the difference in clinical characteristics and outcomes between pediatric and adult patients with COVID-19.Methods A total of 333 consecutive patients with laboratory-confirmed SARS-CoV-2 infection treated in the departments of Internal medicine of Shenzhen Third People’s Hospital from January 11 th to February 10 th,2020 were included.The data were obtained from electronic medical records.The epidemiological data,clinical characteristics,length of hospital stays,and outcomes of pediatric and adult patients were compared.Results Compared with adult patients,pediatric patients had a shorter time of symptom onset to hospitalization than adults[median time,1(IQR,1.0-1.0)d vs.3(IQR,2.0-6.0)d,P<0.001],milder or fewer symptoms,less severe chest CT findings.The clinical severity classification of children was less severe than adults.Up to 15 th March,the end of the follow-up,33(100%)children and 292(97.3%)adult patients had been discharged from hospital.Only 2(0.7%)adult patients died,with an overall case mortality of 0.6%.The median length of hospital stay of pediatric patients was shorter than that of adult patients[19(95%CI:16.6-21.4)d vs.21(95%CI:19.9-22.1)d,P=0.024].Conclusion Pediatric patients with COVID-19 had milder or less clinical symptoms,less evident pulmonary imaging changes,better prognosis,and shorter length of hospital stay.

Home‑based treatment and nursing plan of a patient diagnosed with mild‑to‑common coronavirus disease 2019

To date,there are no known specific antiviral medicines recommended for coronavirus disease 2019(COVID‑19).The treatment mainly focuses on the improvement of the immunity of the individual and the application of palliative treatment.A case of a patient diagnosed with mild COVID‑19 and then later developed common COVID‑19 which was treated and closely monitored at home will be presented.The patient was advised for self‑quarantine with several prescribed medications.Nursing home and self‑care advice were given to the patient,including a change of lifestyle for the duration of the isolation,medication advice,symptomatic care,change of diet,how to monitor the progress of the disease,psychological care,and follow‑up instructions.During the course of the nursing plan,the patient showed significant improvements,and normal life functions were restored.On February 05,2020,novel coronavirus nucleic acid test showed negative after re‑examination;the patient was cured.Therefore,providing adequate nursing plan for home‑based self‑care treatment can effectively cure mild and common cases of COVID‑19.

The Study of Molecular Mechanisms of Xuanbai Chengqi Decoction(宣白承气汤)in the Treatment of Coronavirus Disease 2019 Based on Network Pharmacology and Molecular Docking Method

Objective:To explore the molecular mechanism of Xuanbai Chengqi Decoction(宣白承气汤)in the treatment of Coronavirus Disease 2019(COVID-19)based on network pharmacology,and to verify by molecular docking technology.Methods:The components and targets of Xuanbai Chengqi Decoction(宣白承气汤)were obtained by TCMSP,targets’information was corrected based on the databases such as UniP rot and DrugBank,and the software Cytoscape3.7.1 was adopted to construct TCM-Component-Target and Component-Target network.The main targets were mapped to the KEGG pathway and the GO biological process with the help of DAVID to further elucidate the potential relationship between the main targets and Xuanbai Chengqi Decoction(宣白承气汤)therapy for COVID-19.In the end,the Swiss Dock platform was adopted for the molecular docking verification of key components and targets.Results:The Component-Target network consists of 35 components and 106 corresponding targets,the main targets include COX-2,NCOA2,PTGS1,HSP90 AB1,PRKACA and PGR,etc.There are 561 GO entries of target mapping(P 0.05),including 155 entries for Biological Processes(BP),147 entries for Cell Composition(CC),and 259 entries for Molecular Function(MF).There are 38 KEGG mapping pathways(P 0.05),including many aspects of infectious disease,immune system and endocrine system,as well as Calcium signaling pathway,VEGF signaling pathway,PI3K-Akt signaling pathway and other processes.Conclusion:The result of molecular docking shows that the affinity of the key components such as beta-sitosterol and stigmasterol are similar to recommended medications for COVID-19.Its effect in the treatment of middle stage of COVID-19 may be related to the blocking of the binding of COVID-19 virus and ACE2,antivirus,and relieving inflammatory storm.

Corticosteroids Treatment of Patients with Coronavirus Disease 2019:A Propensity Score Matching Study

The role of corticosteroids in the treatment of coronavirus disease 2019(COVID-19)is controversial.In the present study,we evaluated the effects of adjuvant corticosteroids treatment on the outcome of patients with COVID-19(n=966),using Propensity Score Matching to adjust for potential differences between the corticosteroids group(n=289)and the non-corticosteroids group(n=677).Analysis of data without adjusting differences in baseline characteristics indicated that the proportion of mechanical ventilation and the mortality was higher in the corticosteroids treatment group in total or severe/critical patients.The duration of viral shedding was longer in the non-corticosteroids treatment group in total or general/mild patients.After adjusting the difference between the corticosteroids and non-corticosteroids treatment group,the analysis revealed that the use of corticosteroids had no effect on the duration of viral shedding,in-hospital mortality or 28-day mortality.

Clinical features and potential mechanism of coronavirus disease 2019-associated liver injury

Coronavirus disease 2019(COVID-19),caused by severe acute respiratory syndrome coronavirus 2,has posed a serious threat to global public health security.With the increase in the number of confirmed cases globally,the World Health Organization has declared the outbreak of COVID-19 an international public health emergency.Despite atypical pneumonia as the primary symptom,liver dysfunction has also been observed in many clinical cases and is associated with the mortality risk in patients with COVID-19,like severe acute respiratory syndrome and Middle East respiratory syndrome.Here we will provide a schematic overview of the clinical characteristics and the possible mechanisms of liver injury caused by severe acute respiratory syndrome coronavirus 2 infection,which may provide help for optimizing the management of liver injury and reducing mortality in COVID-19 patients.

Clinical Features and Organ System Dysfunction in Coronavirus Disease 2019 in China

Objectives: To enhance understanding of COVID-19 in clinical characteristics, related organ system dysfunctions and therapies. Methods: We enrolled patients with COVID-19 admitted to two Chinese hospitals from 19 January 2020 to 15 March 2020 and collected demographic and clinical data. Results: We retrospectively enrolled 130 patients: 7 mild, 103 moderate, and 20 severe cases. Severe patients were older than mild/moderate patients (60.1 vs 52.9 years;P = 0.028) and had more comorbidities (85.0% vs 52.7%;p = 0.006). 116 patients (89.2%) were 2019-noval coronavirus RNA-positive with a median detectable time of 10 days. Obvious absorbed of pneumonia in chest imaging occurred in 20 days. The most common symptoms were fever (n = 87, 66.9%), cough (n = 74, 56.9%), fatigue (n = 73, 56.2%), and chest tightness (n = 54, 41.5%). The incidence of liver, kidney, heart, and coagulation dysfunction was 38.4%, 24.5%, 30.1%, and 24.6%, respectively. The most frequently used antiviral therapies were arbidol (n = 64, 49.2%), oseltamivir (n = 73, 56.2%), and Lianhua Qingwen (n = 73, 56.2%). All patients recovered being hospitalized with a median duration of 15 days. Conclusions: Middle-aged and elderly with comorbidities are more likely to be infected with COVID-19 and develop severe symptoms. The lung, liver, heart, kidney, and coagulation system all may sustain injury, especially in severe cases.

Screening key target genes for coronavirus disease 2019 based on bioinformatics and gene network

Objective:To use bioinformatics and gene networks to screen key target genes of coronavirus disease 2019,which provides references for clinical research and development of drugs for coronavirus disease 2019.Methods:Target genes related to coronavirus disease 2019 were screened in the GeneCards and National Center for Biotechnology Information databases,and the obtained gene data were imported into the Database for Annotation,Visualization and Integrated Discovery(Version 6.8)database to collect the related information about pathways and genes.The genes enriched in the first 20 pathways and the genes whose occurrence frequency≥5 were imported into the String database respectively to construct protein-protein interaction network diagram and compare the two network diagrams.Results:TNF,IL-6,IL-2,IL-8,CXCL8,IL1B,CCL2,IFNG,STAT1,MAPK1,MAPK3,MAPK8,TP53 and RELA are ranked top in the two network diagrams,and the frequency of occurrence in the first 20 pathways was≥5.Conclusion:The incidence of coronavirus disease 2019 is associated with multiple signaling pathways,including influenza A,pathways in cancer,toll-like receptor signaling pathway,hypoxia-inducible factor-1 signaling pathway,et al.TNF,IL-6,IL-2,IL-8,CXCL8,IL1B,CCL2,IFNG,STAT1,MAPK1,MAPK3,MAPK8,TP53 and RELA are closely related to coronavirus disease 2019,which needs to be further studied.By analyzing the pathways of the genes related to coronavirus disease 2019 and the interactive network diagrams between the genes,it is helpful to understand the pathogenesis of the disease and provide a reference for clinical research and development of effective drugs for coronavirus disease 2019.

A network pharmacology approach to explore the mechanism of Kangguan decoction in the treatment of coronavirus disease 2019 with preliminary verification

Objective:To explore the mechanism of Kangguan decoction in the treatment of coronavirus disease 2019(COVID-19)and then perform preliminary verification.Methods:The effective compounds and target genes of Kangguan decoction were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.COVID-19 related target genes were searched in the GeneCards database.The active target genes of Kangguan decoction acting on COVID-19 were identified to perform GO function enrichment and KEGG pathway enrichment analysis.The compound-target network and protein-protein interaction were constructed;Molecular docking simulations of macromolecular protein target receptors and their corresponding compounds were performed.The clinical data of COVID-19 patients were retrieved from their electronic medical records of Nantong Third People's Hospital.Results:We screened out 137 effective compounds and 274 effective target genes of Kangguan decoction from TCMSP.The active target genes of Kangguan decoction were compared with the COVID-19 related target genes,and 63 active target genes for Kangguan decoction acting on COVID-19 were identified.GO function enrichment and KEGG pathway enrichment analysis were performed.The compound-target network and PPI network were constructed and the key compounds and key targets were selected to construct a key compound-target network.Finally,the binding of the target and its corresponding components was verified by molecular docking and two clinical cases with obvious clinical efficacy after Kangguan decoction application were demonstrated.Conclusion:The pharmacological mechanism of Kangguan decoction acting on COVID-19 has been explored,and the active compounds and targets of Kangguan decoction acting on COVID-19 and clinical efficacy for Kangguan decoction treating COVID-19 patients have been preliminarily verified.

ABO血型与SARS-CoV-2感染的相关性及其机制假说

ABO血型与新型冠状病毒SARS-CoV-2感染及其病情程度具有相关性,其中O型人群的SARS-CoV-2易感性较低,A型人群的SARS-CoV-2易感性较高,且病情较重。这种易感性和致病性差异可能有多种机制,抗A和(或)抗B抗体与病毒包膜相应抗原结合中和病毒;SARS-CoV-2刺突蛋白与抗A抗体结合,阻断病毒与血管紧张素转换酶2受体相互作用,阻止病毒进入肺上皮细胞;A型人群血管性血友病因子、凝血因子Ⅷ及炎症因子、黏附分子水平较高,血栓栓塞风险更高、炎症反应更强。上述假说尚需前瞻性研究进一步验证,以指导相应防治策略的制订。

Digestive system involvement of infections with SARS-CoV-2 and other coronaviruses:Clinical manifestations and potential mechanisms

Although coronavirus(CoV)infection is often characterized by respiratory symptoms,the virus can also result in extrapulmonary symptoms,especially the symptoms related to the digestive system.The outbreak of coronavirus disease 2019(COVID-19)is currently the world’s most pressing public health threat and has a significant impact on civil societies and the global economy.The occurrence of digestive symptoms in patients with COVID-19 is closely related to the development and prognosis of the disease.Moreover,thus far,there are no specific antiviral drug or vaccine approved for the treatment or prevention of COVID-19.Therefore,we elaborate on the effects of CoVs on the digestive system and the potential underlying mechanisms.

Novel coronavirus"Omicron subtype variant strain BA.2":The latest research progress

Since the outbreak of COVID-19,with the emergence of a series of novel coronavirus variants of different types,COVID-19 has continued to spread across the globe,posing a huge threat to the lives and health of people around the world and posing a severe challenge to global public health security.Recently,the World Health Organization(WHO)announced that novel coronavirus variant strain"Omicron"subtype variant strain BA.2 has been found in 57 countries and regions.It has higher vaccine and antibody tolerance and is more infectious than omicron(B.1.1.529).In this paper,we briefly review the recent studies on the omicron subtype variant strain BA.2.

Biology of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and the humoral immunoresponse:a systematic review of evidence to support global policy-level actions and research

Background Both population-level epidemiological data and individual-level biological data are needed to control the coronavirus disease 2019(COVID-19)pandemic.Population-level data are widely available and efforts to combat COVID-19 have generated proliferate data on the biology and immunoresponse to the causative pathogen,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).However,there remains a paucity of systemized data on this subject.Objective In this review,we attempt to extract systemized data on the biology and immuno-response to SARS-CoV-2 from the most up-to-date peer-reviewed studies.We will focus on the biology of the virus and immunological variations that are key for determining long-term immunity,transmission potential,and prognosis.Data Sources and Methods Peer-reviewed articles were sourced from the PubMed database and by snowballing search of selected publications.Search terms included:“Novel Coronavirus”OR“COVID-19”OR“SARS-CoV-2”OR“2019-nCoV”AND“Immunity”OR“Immune Response”OR“Antibody Response”OR“Immunologic Response”.Studies published from December 31,2019 to December 31,2020 were included.To ensure validity,papers in pre-print were excluded.Results Of 2889 identified papers,36 were included.Evidence from these studies suggests early seroconversion in patients infected with SARS-CoV-2.Antibody titers appear to markedly increase two weeks after infection,followed by a plateau.A more robust immune response is seen in patients with severe COVID-19 as opposed to mild or asymptomatic presentations.This trend persists with regard to the length of antibody maintenance.However,overall immunity appears to wane within two to three months post-infection.Conclusion Findings of this study indicate that immune responses to SARS-CoV-2 follow the general pattern of viral infection.Immunity generated through natural infection appears to be short,suggesting a need for long-term efforts to control the pandemic.Antibody testing will be essential to gauge the epidemic and inform decision-making on effective strategies for treatment and prevention.Further research is needed to illustrate immunoglobulin-specific roles and neutralizing antibody activity.