Virus load and virus shedding of SARS-CoV-2 and their impact onpatient outcomes

BACKGROUND Understanding a virus shedding patterns in body fluids/secretions is importantto determine the samples to be used for diagnosis and to formulate infectioncontrol measures.AIM To investigate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)shedding patterns and its risk factors.METHODS All laboratory-confirmed coronavirus disease 2019 patients with completemedical records admitted to the Shenzhen Third People’s Hospital from January28, 2020 to March 8, 2020 were included. Among 145 patients (54.5% males;median age, 46.1 years), three (2.1%) died. The bronco-alveolar lavage fluid(BALF) had the highest virus load compared with the other samples. The viralload peaked at admission (3.3 × 108 copies) and sharply decreased 10 d afteradmission.RESULTS The viral load was associated with prolonged intensive care unit (ICU) duration.Patients in the ICU had significantly longer shedding time compared to those inthe wards (P < 0.0001). Age > 60 years [hazard ratio (HR) = 0.6;95% confidenceinterval (CI): 0.4-0.9] was an independent risk factor for SARS-CoV-2 shedding,while chloroquine (HR = 22.8;95%CI: 2.3-224.6) was a protective factor.CONCLUSION BALF had the highest SARS-CoV-2 load. Elderly patients had higher virus loads,which was associated with a prolonged ICU stay. Chloroquine was associatedwith shorter shedding duration and increased the chance of viral negativity.

Clinical diagnosis of severe COVID-19:A derivation and validation of a prediction rule

BACKGROUND The widespread coronavirus disease 2019(COVID-19)has led to high morbidity and mortality.Therefore,early risk identification of critically ill patients remains crucial.AIM To develop predictive rules at the time of admission to identify COVID-19 patients who might require intensive care unit(ICU)care.METHODS This retrospective study included a total of 361 patients with confirmed COVID-19 by reverse transcription-polymerase chain reaction between January 19,2020,and March 14,2020 in Shenzhen Third People’s Hospital.Multivariate logistic regression was applied to develop the predictive model.The performance of the predictive model was externally validated and evaluated based on a dataset involving 126 patients from the Wuhan Asia General Hospital between December 2019 and March 2020,by area under the receiver operating curve(AUROC),goodness-of-fit and the performance matrix including the sensitivity,specificity,and precision.A nomogram was also used to visualize the model.RESULTS Among the patients in the derivation and validation datasets,38 and 9 participants(10.5%and 2.54%,respectively)developed severe COVID-19,respectively.In univariate analysis,21 parameters such as age,sex(male),smoker,body mass index(BMI),time from onset to admission(>5 d),asthenia,dry cough,expectoration,shortness of breath,asthenia,and Rox index<18(pulse oxygen saturation,SpO2)/(FiO2×respiratory rate,RR)showed positive correlations with severe COVID-19.In multivariate logistic regression analysis,only six parameters including BMI[odds ratio(OR)3.939;95%confidence interval(CI):1.409-11.015;P=0.009],time from onset to admission(≥5 d)(OR 7.107;95%CI:1.449-34.849;P=0.016),fever(OR 6.794;95%CI:1.401-32.951;P=0.017),Charlson index(OR 2.917;95%CI:1.279-6.654;P=0.011),PaO2/FiO2 ratio(OR 17.570;95%CI:1.117-276.383;P=0.041),and neutrophil/lymphocyte ratio(OR 3.574;95%CI:1.048-12.191;P=0.042)were found to be independent predictors of COVID-19.These factors were found to be significant risk factors for severe patients confirmed with COVID-19.The AUROC was 0.941(95%CI:0.901-0.981)and 0.936(95%CI:0.886-0.987)in both datasets.The calibration properties were good.CONCLUSION The proposed predictive model had great potential in severity prediction of COVID-19 in the ICU.It assisted the ICU clinicians in making timely decisions for the target population.

Type Ⅰ interferon receptor knockout mice as models for infection of highly pathogenic viruses with outbreak potential

Due to their inability to generate a complete immune response, mice knockout for type I interferon （IFN） receptors （Ifnar-/-） are more susceptible to viral infections, and are thus commonly used for pathogenesis studies. This mouse model has been used to study many diseases caused by highly pathogenic viruses from many families, including the Flaviviridae, Filoviridae, Arenaviridae, Bunyaviridae, Henipaviridae, and Togaviridae. In this review, we summarize the findings from these animal studies, and discuss the pros and cons of using this model versus other known methods for studying pathogenesis in animals.

Development and characterization of a guinea pig model for Marburg virus

The Angolan strain of Marburg virus （MARV/Ang） can cause lethal disease in humans with a case fatality rate of up to 90%, but infection of immunocompetent rodents do not result in any observable symptoms. Our previous work includes the development and characterization of a MARV/Ang variant that can cause lethal disease in mice （MARV/Ang-MA）, with the aim of using this tool to screen for promising prophylactic and therapeutic candidates. An intermediate animal model is needed to confirm any findings from mice studies before testing in the gold-standard non-human primate （NHP） model. In this study, we serially passaged the clinical isolate of MARV/Ang in the livers and spleens of guinea pigs until a variant emerged that causes 100% lethality in guinea pigs （MARV/Ang- GA）. Animals infected with MARV/Ang-GA showed signs of filovirus infection including lymphocytopenia, thrombocytopenia, and high viremia leading to spread to major organs, including the liver, spleen, lungs, and kidneys. The MARV/Ang-GA guinea pigs died between 7-9 days after infection, and the LD50 was calculated to be 1.1x10-1 TCID50 （median tissue culture infective dose）. Mutations in MARV/Ang-GA were identified and compared to sequences of known rodent-adapted MARV/Ang variants, which may benefit future studies characterizing important host adaptation sites in the MARV/Ang viral genome.

Mpox(formerly monkeypox):pathogenesis,prevention,and treatment

In 2022,a global outbreak of Mpox(formerly monkeypox)occurred in various countries across Europe and America and rapidly spread to more than 100 countries and regions.The World Health Organization declared the outbreak to be a public health emergency of international concern due to the rapid spread of the Mpox virus.Consequently,nations intensified their efforts to explore treatment strategies aimed at combating the infection and its dissemination.Nevertheless,the available therapeutic options for Mpox virus infection remain limited.So far,only a few numbers of antiviral compounds have been approved by regulatory authorities.Given the high mutability of the Mpox virus,certain mutant strains have shown resistance to existing pharmaceutical interventions.This highlights the urgent need to develop novel antiviral drugs that can combat both drug resistance and the potential threat of bioterrorism.Currently,there is a lack of comprehensive literature on the pathophysiology and treatment of Mpox.To address this issue,we conducted a review covering the physiological and pathological processes of Mpox infection,summarizing the latest progress of anti-Mpox drugs.Our analysis encompasses approved drugs currently employed in clinical settings,as well as newly identified small-molecule compounds and antibody drugs displaying potential antiviral efficacy against Mpox.Furthermore,we have gained valuable insights from the process of Mpox drug development,including strategies for repurposing drugs,the discovery of drug targets driven by artificial intelligence,and preclinical drug development.The purpose of this review is to provide readers with a comprehensive overview of the current knowledge on Mpox.

Diagnostic approaches for monkeypox virus

Mpox(formerly Monkeypox)is a zoonotic infection caused by Monkeypox virus(MPXV).Since 2022,Mpox epidemics have occurred in many nonendemic countries and regions,leading the World Health Organization to declare a public health emergency of international concern.With the persistent transmission and evolution of MPXV,symptoms of Mpox have become milder,with some infections being asymptomatic.In addition,MPXV has become more contagious.Therefore,rapid and accurate diagnosis and screening of MPXV is vital to prevent and control MPXV epidemics.Here,we review and summarize the technical details,application scenarios,and the advantages and disadvantages of MPXV‐specific diagnostic methods.

Early and Sensitive Detection of Pathogens for Public Health and Biosafety:An Example of Surveillance and Genotyping of SARS-CoV-2 in Sewage Water by Cas12a-Facilitated Portable Plasmonic Biosensor

Infectious diseases severely threaten public health and global biosafety.In addition to transmission through the air,pathogenic microorganisms have also been detected in environmental liquid samples,such as sewage water.Conventional biochemical detection methodologies are time-consuming and cost-ineffective,and their detection limits hinder early diagnosis.In the present study,ultrafine plasmonic fiber probes with a diameter of 125μm are fabricated for clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)-12a-mediated sensing of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Single-stranded DNA exposed on the fiber surface is trans-cleaved by the Cas12a enzyme to release gold nanoparticles that are immobilized onto the fiber surface,causing a sharp reduction in the surface plasmon resonance(SPR)wavelength.The proposed fiber probe is virus-specific with the limit of detection of~2,300 copies/ml,and genomic copy numbers can be reflected as shifts in wavelengths.A total of 21 sewage water samples have been examined,and the data obtained are consistent with those of quantitative polymerase chain reaction(qPCR).In addition,the Omicron variant and its mutation sites have been fast detected using S gene-specific Cas12a.This study provides an accurate and convenient approach for the real-time surveillance of microbial contamination in sewage water.

Lycorine derivative effectively inhibits the replication of coronaviruses both in vitro and in vivo

The established and ongoing prevalence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and seasonal human coronaviruses(HCoV)like HCoV-OC43,HCoV-NL63,and HCoV-229E,pose a continuous threat to public health.Therefore,it is urgently needed to explore antiviral drugs with broad-spectrum anti-coronavirus activity.Our previous studies have revealed that lycorine is a potent broad-spectrum anti-coronavirus drug,a natural alkaloid extracted from Amaryllidaceae with various pharmacological and microbiological effects.However,it is unsafe to directly use lycorine as a clinical antiviral drug due to the cytotoxicity and induction of cell apoptosis.In this study,a series of lycorine derivatives were designed and synthesized.One of them,named Ly-8,was found to effectively inhibit the replication of different coronavirus strains in vitro,including SARS-CoV-2.Moreover,Ly-8 was also shown to effectively inhibit HCoVOC43 replication in the central nervous system,and provide effective protection against HCoV-OC43 infection in mice with low drug toxicity.Furthermore,Ly-8-resistant mutants were not observed during the 30 times sequential passages in cell culture.Collectively,these findings suggest that Ly-8 may be a potential candidate drug for the future development of broad-spectrum anti-coronavirus drugs.

Animal models for filovirus infections

The family Filoviridae, which includes the genera Marburgvirus and Ebolavirus, contains some of the most pathogenic viruses in humans and non-human primates （NHPs）, causing severe hemorrhagic fevers with high fatality rates. Small animal models against filoviruses using mice, guinea pigs, hamsters, and ferrets have been developed with the goal of screening candidate vaccines and antivirals, before testing in the gold standard NHP models. In this review, we summarize the different animal models used to understand filovirus pathogenesis, and discuss the advantages and disadvantages of each model with respect to filovirus disease research.

Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect

The global pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus has necessitated rapid,easy-to-use,and accurate diagnostic methods to monitor the virus infection.Herein,a ratiometric fluorescence enzyme-linked immunosorbent assay(ELISA)was developed using Si-fluorescein isothiocyanate nanoparticles(FITC NPs)for detecting SARSCoV-2 nucleocapsid(N)protein.Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane(APTES)-FITC as the Si source.This method did not need post-modification and avoided the reduction in quantum yield and stability.The p-nitrophenyl(pNP)produced by the alkaline phosphatase(ALP)-mediated hydrolysis of pnitrophenyl phosphate(pNPP)could quench Si fluorescence in Si-FITC NPs via the inner filter effect.In ELISA,an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody.ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs.The change in fluorescence intensity ratio could be used for detecting N protein,with a wide linearity range(0.01-10.0 and 50-300 ng/mL)and low detection limit(0.002 ng/mL).The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum.Moreover,this proposed method can accurately distinguish coronavirus disease 2019(COVID-19)and non-COVID-19 patient samples.Therefore,this simple,sensitive,and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection.

Ratiometric fluorescent Si-FITC nanoprobe for immunoassay of SARS-CoV-2 nucleocapsid protein

Coronavirus disease 2019(COVID-19)highlights the importance of rapid and reliable diagnostic assays for the management of virus transmission.Here,we developed a one-pot hydrothermal method to prepare Si-FITC nanoparticles(NPs)for the fluorescent immunoassay of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)nucleocapsid protein(N protein).The synthesis of Si-FITC NPs did not need post-modification,which addressed the issue of quantum yield reduction during the coupling reaction.Si-FITC NPs showed two distinct peaks,Si fluorescence atλem=385 nm and FITC fluorescence atλem=490 nm.In the presence of KMnO_(4),Si fluorescence was decreased and FITC fluorescence was enhanced.Briefly,in the presence of N protein,catalase(CAT)-linked secondary antibody/reporter antibody/N protein/capture antibody immunocomplexes were formed on microplates.Subsequently,hydrogen peroxide(H_(2)O_(2))and Si-FITC NPs/KMnO_(4)were injected into the microplate together.The decomposition of H_(2)O_(2)by CAT resulted in remaining of KMnO_(4),which changed the fluorescence intensity ratio of Si-FITC NPs.The fluorescence intensity ratio correlated significantly with the N protein concentration ranging from 0.02 to 50.00 ng/mL,and the detection limit was 0.003 ng/mL,which was more sensitive than the commercial ELISA kit with a detection limit of 0.057 ng/mL.The N protein concentration can be accurately determined in human serum.Furthermore,the COVID-19 and non-COVID-19 patients were distinguishable by this method.Therefore,the ratiometric fluorescent immunoassay can be used for SARS-CoV-2 infection diagnosis with a high sensitivity and selectivity.

Distinct and relatively mild clinical characteristics of SARS-CoV-2 BA.5 infections against BA.2

Dear Editor,The global pandemic of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Omicron variant has resulted in its continuous evolution and the emergence of numerous subvariants of Omicron(https://gisaid.org/).Currently,possibly owing to the increased evasion of neutralizing antibodies elicited by previous infection and vaccination and the higher transmissibility,the BA.5 variant has replaced BA.2 variant and dominated the pandemic.

Preclinical characterization and anti-SARS-CoV-2 efficacy of ATV014:an oral cyclohexanecarboxylate prodrug of 1′-CN-4-aza-7,9-dideazaadenosine C-nucleoside

Dear Editor,Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of the global pandemic coronavirus disease 2019(COVID-19),has proven itself to be a highly virulent respiratory pathogen with an unpredictable evolutionary capacity,posing a persistent threat to mankind.At the time of this manuscript’s publication.

Automated robot and artificial intelligence-powered wastewater surveillance for proactive mpox outbreak prediction

In the wake of the largest‐ever recorded outbreak of mpox in terms of magnitude and geographical spread in human history since May 2022,we innovatively developed an automated online sewage virus enrichment and concentration robot for disease tracking.Coupled with an artificial intelligence(AI)model,our research aims to estimate mpox cases based on the concentration of the monkeypox virus(MPXV)in wastewater.Our research has revealed a compelling link between the levels of MPXV in wastewater and the number of clinically confirmed mpox infections,a finding that is reinforced by the ability of our AI prediction model to forecast cases with remarkable precision,capturing 87%of the data’s variability.However,it is worth noting that this high precision in predictions may be related to the relatively high frequency of data acquisition and the relatively non‐mobile isolated environment of the hospital itself.In conclusion,this study represents a significant step forward in our ability to track and respond to mpox outbreaks.It has the potential to revolutionize public health surveillance by utilizing innovative technologies for disease surveillance and prediction。

S-Allylmercaptocysteine improves alcoholic liver disease partly through a direct modulation of insulin receptor signaling

Alcoholic liver disease(ALD) causes insulin resistance, lipid metabolism dysfunction, and inflammation. We investigated the protective effects and direct regulating target of S-allylmercaptocysteine(SAMC) from aged garlic on liver cell injury. A chronic ethanol-fed ALD in vivo model(the NIAAA model) was used to test the protective functions of SAMC. It was observed that SAMC(300 mg/kg, by gavage method) effectively ameliorated ALD-induced body weight reduction, steatosis,insulin resistance, and inflammation without affecting the health status of the control mice, as demonstrated by histological, biochemical, and molecular biology assays. By using biophysical assays and molecular docking, we demonstrated that SAMC directly targeted insulin receptor(INSR) protein on the cell membrane and then restored downstream IRS-1/AKT/GSK3 b signaling. Liver-specific knock-down in mice and siRNA-mediated knock-down in AML-12 cells of Insr significantly impaired SAMC(250 mmol/L in cells)-mediated protection. Restoration of the IRS-1/AKT signaling partly recovered hepatic injury and further contributed to SAMC’s beneficial effects. Continuous administration of AKT agonist and recombinant IGF-1 in combination with SAMC showed hepato-protection in the mice model.Long-term(90-day) administration of SAMC had no obvious adverse effect on healthy mice. We conclude that SAMC is an effective and safe hepato-protective complimentary agent against ALD partly through the direct binding of INSR and partial regulation of the IRS-1/AKT/GSK3 b pathway.

Antiviral activity of quercetin-3-β-O-D-glucoside against Zika virus infection

Dear Editor, The 2015-2016 outbreak of Zika virus （ZIKV） fever, first reported in Brazil during early 2015 （Zanluca et al., 2015）, has infected millions of people and is a global public health concern. ZIKV infections are associated with fetal microcephaly, as well as neurological complications in humans. The virus can be shed in the semen and vaginal secretions of humans, leading to sexual transmission, and unexpectedly ZIKV infections cause severe damage to the male reproductive organs in male mice （Govero et al., 2016; Ma et al., 2016）.

Genetic and Phylogenetic Characterization of a Chikungunya Virus Imported into Shenzhen, China

Dear Editor,Chikungunya virus(CHIKV)is a mosquito-borne virus belonging to the family Togaviridae,genus Alphavirus,and was first isolated in Tanzania in the 1950s(Silva and Dermody 2017;Weaver and Lecuit 2015).Human infections with CHIKV typically result in a rapid-onset febrile disease,with symptoms that include fever,headache,rash,severe joint and muscle pain,as well as prolonged periods of disability in some patients(Weaver and Lecuit 2015;Silva and Dermody 2017).

Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages

The Zika virus(ZIKV) is an arbovirus that has spread rapidly worldwide within recent times. There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome(GBS) and microcephaly in humans. The ZIKV is genetically diverse and can be separated into Asian and African lineages. A rapid, sensitive, and specific assay is needed for the detection of ZIKV across various pandemic regions. So far, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains. To this end, we have developed a one-step quantitative reverse transcription polymerase chain reaction(qRT-PCR) assay based on conserved sequences in the ZIKV envelope(E) gene. The detection limit of the assay was determined to be five RNA transcript copies and 2.94 × 10^(–3) 50% tissue culture infectious doses(TCID50) of live ZIKV per reaction. The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification, when tested against other flaviviruses. The assay was also successful in testing for ZIKV in clinical samples. Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

Rift Valley Fever Virus and Yellow Fever Virus in Urine: A Potential Source of Infection

Dear Editor,In recent years,the incidence of human infections caused by emerging or re-emerging pathogens has rapidly increased.Diseases that were once regional now have the ability to spread globally in a short amount of time and pose a wider threat to public health(Weaver et al.2018).Yellow fever virus(YFV,family Flaviviridae,genus Flavivirus)is a mosquito-borne flavivirus that causes yellow fever in humans and has been endemic in Africa and Latin America for many years(Domingo et al.2018).

Laboratory Diagnosis and Monitoring the Viral Shedding of SARS-CoV-2 Infection

The worldwide epidemic of coronavirus disease 2019(COVID-19)is ongoing.Rapid and accurate detection of the causative virus SARSCoV-2 is vital for the treatment and control of COVID-19.In this study,the comparative sensitivity of different respiratory specimen types were retrospectively analyzed using 3,552 clinical samples from 410 COVID-19 patients confirmed by Guangdong CDC(Center for Disease Control and Prevention).Except for bronchoalveolar lavage fluid(BALF),the sputum possessed the highest positive rate(73.4%–87.5%),followed by nasal swabs(53.1%–85.3%)for both severe and mild cases during the first 14 days after illness onset(d.a.o.).Viral RNA could be detected in all BALF samples collected from the severe group within 14 d.a.o.and lasted up to 46 d.a.o.Moreover,although viral RNA was negative in the upper respiratory samples,it was also positive in BALF samples in most cases from the severe group during treatment.Notably,no viral RNA was detected in BALF samples from the mild group.Despite typical ground-glass opacity observed via computed tomographic scans,no viral RNA was detected in the first three or all upper respiratory tract specimens from some COVID-19 patients.In conclusion,sputum is most sensitive for routine laboratory diagnosis of COVID-19,followed by nasal swabs.Detection of viral RNA in BALF improves diagnostic accuracy in severe COVID-19 patients.