Unveiling the seroprevalence of human papillomavirus in Guangdong, China: Implications for vaccination strategies.

Seroepidemiological characteristics of human papillomavirus (HPV) in community residents reflect natural infection and can guide the reform of vaccination programs. A population-based serological survey was conducted in Guangdong Province. Serum anti-HPV IgG antibody levels were determined by an ELISA. Neutralizing antibodies against HPV6, 11, 16, and 18 were detected via a pseudovirus-based neutralization assay (PBNA). A total of 5122 serum samples were collected from community residents, including 1989 males and 3133 females, in three cities of Guangdong Province. The rate of HPV IgG antibody positivity in females was 5.39% (95% CI: 4.6-6.2), which was greater than that in males (2.36%; 95% CI: 1.7-3.1). HPV IgG antibodies were more frequently detected in females aged 51-60 years (11.30%; 95% CI: 7.6-16.0), whereas in males, the detection increased with age and reached 4.94% (95% CI: 2.8-6.9) in the group aged ≥71 years. The seropositivity of neutralizing antibodies against HPV6 and 11 was greater than that against HPV16 and 18. The serum neutralizing antibody titers in individuals who received three doses of a vaccine were 7- to 12-fold greater than those in individuals who did not receive the vaccine. The neutralizing antibody titers slightly decreased within 40 months and ranged from 0.038 to 0.057 log ED50 per month. A moderate consistency between the HPV ELISA and PBNA results was observed (Kappa score = 0.49, r = 0.249, 0.635, 0.382, and 0.466 for HPV6, 11, 16, and 18, respectively). The HPV seropositivity rate among healthy residents of Guangdong Province was found to be low among children and adolescents and to increase with age. The serum neutralizing antibody titers were significantly greater in the vaccine group than that in the control group, and this difference persisted over time, which indicated promising protection against HPV infection.

A Pseudovirus Nanoparticle Displaying the Vaccinia Virus L1 Protein Elicited High Neutralizing Antibody Titers and Provided Complete Protection to Mice against Mortality Caused by a Vaccinia Virus Challenge.

The recent worldwide incidence of mpox infection and concerns about future emerging variants of mpox viruses highlight the need for the development of a new generation of mpox vaccines. To achieve this goal, we utilized our norovirus S nanoparticle vaccine platform to produce and evaluate two pseudovirus nanoparticles (PVNPs), S-L1 and S-J1. These PVNPs displayed the L1 neutralizing antigen target of the vaccinia virus and a yet-untested J1 antigen of the mpox virus, respectively, with the aim of creating an effective nanoparticle-based mpox vaccine. Each self-assembled PVNP consists of an inner shell resembling the interior layer of the norovirus capsid and multiple L1 or J1 antigens on the surface. The PVNPs improved the antibody responses toward the displayed L1 or J1 antigens in mice, resulting in significantly greater L1/J1-specific IgG and IgA titers than those elicited by the corresponding free L1 or J1 antigens. After immunization with the S-L1 PVNPs, the mouse sera exhibited high neutralizing antibody titers against the vaccinia virus, and the S-L1 PVNPs provided mice with 100% protection against mortality caused by vaccinia virus challenge. In contrast, the S-J1 PVNPs induced low neutralizing antibody titers and conferred mice weak protective immunity. These data confirm that the L1 protein is an excellent vaccine target and that the readily available S-L1 PVNPs are a promising mpox vaccine candidate worthy of further development.

Neutralization potency of the 2023-24 seasonal influenza vaccine against circulating influenza H3N2 strains.

Seasonal influenza is a severe disease that significantly impacts public health, causing millions of infections and hundreds of thousands of deaths each year. Seasonal influenza viruses, particularly the H3N2 subtype, exhibit high antigenic variability, often leading to mismatch between vaccine strains and circulating strains. Therefore, rapidly assessing the alignment between existing seasonal influenza vaccine and circulating strains is crucial for enhancing vaccine efficacy. This study, based on a pseudovirus platform, evaluated the match between current influenza H3N2 vaccine strains and circulating strains through cross-neutralization assays using clinical human immune sera against globally circulating influenza virus strains. The research results show that although mutations are present in the circulating strains, the current H3N2 vaccine strain still imparting effective protection, providing a scientific basis for encouraging influenza vaccination. This research methodology can be sustainably applied for the neutralization potency assessment of subsequent circulating strains, establishing a persistent methodological framework.

Eucalyptus Essential Oil Inhibits Cell Infection by SARS-CoV-2 Spike Pseudotyped Lentivirus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a public health concern due to infections with new SARS-CoV-2 variants. Therefore, finding effective preventive and therapeutic treatments against all SARS-CoV-2 variants is of great interest. In this study, we examined the capacity of eucalyptus essential oil (EEO) and eucalyptol (EOL) to prevent SARS-CoV-2 infection, using as a model SARS-CoV-2 Spike pseudotyped lentivirus (SARS-CoV-2 pseudovirus) and 293T cells transfected with human angiotensin-converting enzyme 2 (hACE2-293T cells). First, we determined the cytotoxicity of EEO and EOL using the MTT colorimetric assay, selecting non-cytotoxic concentrations ≤ 0.1% (v/v) for further analysis. Subsequently, we evaluated the capacity of EEO and EOL in cell cultures to preclude infection of hACE2-293T cells by SARS-CoV-2 pseudovirus, using a luciferase-based assay. We found that EEO and EOL significantly reduced SARS-CoV-2 pseudovirus infection, obtaining IC50 values of 0.00895% and 0.0042% (v/v), respectively. Likewise, EEO and EOL also reduced infection by vesicular stomatitis virus (VSV) pseudovirus, although higher concentrations were required. Hence, EEO and EOL may be able to inhibit SARS-CoV-2 infection, at least partially, through a Spike-independent pathway, supporting the implementation of aromatherapy with these agents as a cost-effective antiviral measure.

Engineering a NanoBiT biosensor for detecting angiotensin-converting enzyme-2 (hACE2) interaction with SARS-CoV-2 spike protein and screening the inhibitors to block hACE2 and spike interaction.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is facilitated by its trimeric surface spike protein, which binds to the human angiotensin-converting enzyme 2 (hACE2) receptor. This critical interaction facilitates viral entry and is a primary target for therapeutic intervention against COVID-19. However, it is difficult to fully optimize viral infection using existing protein-protein interaction methods. Herein, we introduce a nano-luciferase binary technology (NanoBiT)-based pseudoviral sensor designed to stimulate the dynamics of viral infection in both living cells and animals. Infection progression can be dynamically visualized via a rapid increase in luminescence within 3 h using an in vivo imaging system (IVIS). Inhibition of viral infection by baicalein and baicalin was evaluated using a NanoBiT-based pseudoviral sensor. These results indicate that the inhibitory efficacy of baicalein was strengthened by targeting the spike protein, whereas baicalin targeted the hACE2 protein. Additionally, under optimized conditions, baicalein and baicalin provided a synergistic combination to inhibit pseudoviral infection. Live bioluminescence imaging was used to evaluate the in vivo effects of baicalein and baicalin treatment on LgBiT-hACE2 mice infected with the BA.2-SmBiT spike pseudovirus. This innovative bioluminescent system functions as a sensitive and early-stage quantitative viral transduction in vitro and in vivo. This platform provides novel opportunities for studying the molecular biology of animal models.

A Dynamic and Effective Peptide-Based Strategy for Promptly Addressing Emerging SARS-CoV-2 Variants of Concern.

Genomic surveillance based on sequencing the entire genetic code of SARS-CoV-2 involves monitoring and studying genetic changes and variations in disease-causing organisms such as viruses and bacteria. By tracing the virus, it is possible to prevent epidemic spread in the community, ensuring a 'precision public health' strategy. A peptide-based design was applied to provide an efficacious strategy that is able to counteract any emerging viral variant of concern dynamically and promptly to affect the outcomes of a pandemic at an early stage while waiting for the production of the anti-variant-specific vaccine, which require longer times. The inhibition of the interaction between the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and one of the cellular receptors (DPP4) that its receptors routinely bind to infect human cells is an intriguing therapeutic approach to prevent the virus from entering human cells. Among the other modalities developed for this purpose, peptides surely offer unique advantages, including ease of synthesis, serum stability, low immunogenicity and toxicity, and small production and distribution chain costs. Here, we obtained a potent new inhibitor based on the rearrangement of a previously identified peptide that has been rationally designed on a cell dipeptidyl peptidase 4 (DPP4) sequence, a ubiquitous membrane protein known to bind the RBD-SPIKE domain of the virus. This novel peptide (named DPP4-derived), conceived as an endogenous "drug", is capable of targeting the latest tested variants with a high affinity, reducing the VSV* DG-Fluc pseudovirus Omicron's infection capacity by up to 14%, as revealed by in vitro testing in human Calu-3 cells. Surface plasmon resonance (SPR) confirmed the binding affinity of the new DPP4-derived peptide with Omicron variant RBD.

Functional Characterisation of Surfactant Protein A as a Novel Prophylactic Means against Oncogenic HPV Infections.

Human papillomavirus (HPV) infection poses a significant health challenge, particularly in low- and middle-income countries (LMIC), where limited healthcare access and awareness hinder vaccine accessibility. To identify alternative HPV targeting interventions, we previously reported on surfactant protein A (SP-A) as a novel molecule capable of recognising HPV16 pseudovirions (HPV16-PsVs) and reducing infection in a murine cervicovaginal HPV challenge model. Building on these findings, our current study aimed to assess SP-A's suitability as a broad-spectrum HPV-targeting molecule and its impact on innate immune responses. We demonstrate SP-A's ability to agglutinate and opsonise multiple oncogenic HPV-PsVs types, enhancing their uptake and clearance by RAW264.7 murine macrophages and THP-1 human-derived immune cells. The SP-A opsonisation of HPV not only led to increased lysosomal accumulation in macrophages and HaCaT keratinocytes but also resulted in a decreased infection of HaCaT cells, which was further decreased when co-cultured with innate immune cells. An analysis of human innate immune cell cytokine profiles revealed a significant inflammatory response upon SP-A exposure, potentially contributing to the overall inhibition of HPV infection. These results highlight the multi-layered impact of SP-A on HPV, innate immune cells and keratinocytes and lay the basis for the development of alternative prophylactic interventions against diverse HPV types.

Development and utility of a SARS-CoV-2 pseudovirus assay for compound screening and antibody neutralization assays.

Background: The highly infectious nature of SARS-CoV-2 necessitates using bio-containment facilities to study viral pathogenesis and identify potent antivirals. However, the lack of high-level bio-containment laboratories across the world has limited research efforts into SARS-CoV-2 pathogenesis and the discovery of drug candidates. Previous research has reported that non-replicating SARS-CoV-2 Spike-pseudotyped viral particles are effective tools to screen for and identify entry inhibitors and neutralizing antibodies. Methods: To generate SARS-CoV-2 pseudovirus, a lentiviral packaging plasmid p8.91, a luciferase expression plasmid pCSFLW, and SARS-CoV-2 Spike expression plasmids (Wild-type (D614G) or Delta strain) were co-transfected into HEK293 cells to produce a luciferase-expressing non-replicating pseudovirus which expresses SARS-CoV-2 spike protein on the surface. For relative quantitation, HEK293 cells expressing ACE2 (ACE2-HEK293) were infected with the pseudovirus, after which luciferase activity in the cells was measured as a relative luminescence unit. The ACE2-HEK293/Pseudovirus infection system was used to assess the antiviral effects of some compounds and plasma from COVID-19 patients to demonstrate the utility of this assay for drug discovery and neutralizing antibody screening. Results: We successfully produced lentiviral-based SARS-CoV2 pseudoviruses and ACE2-expressing HEK293 cells. The system was used to screen compounds for SARS-CoV-2 entry inhibitors and identified two compounds with potent activity against SARS-CoV-2 pseudovirus entry into cells. The assay was also employed to screen patient plasma for neutralizing antibodies against SARS-CoV-2, as a precursor to live virus screening, using successful hits. Significance: This assay is scalable and can perform medium-to high-throughput screening of antiviral compounds with neither severe biohazard risks nor the need for higher-level containment facilities. Now fully deployed in our resource-limited laboratory, this system can be applied to other highly infectious viruses by swapping out the envelope proteins in the plasmids used in pseudovirus production.

Serum AZD7442 (tixagevimab-cilgavimab) concentrations and in vitroIC50 values predict SARS-CoV-2 neutralising antibody titres.

Objectives: The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates rapid methods for assessing monoclonal antibody (mAb) potency against emerging variants. Authentic virus neutralisation assays are considered the gold standard for measuring virus-neutralising antibody (nAb) titres in serum. However, authentic virus-based assays pose inherent practical challenges for measuring nAb titres against emerging SARS-CoV-2 variants (e.g. storing infectious viruses and testing at biosafety level-3 facilities). Here, we demonstrate the utility of pseudovirus neutralisation assay data in conjunction with serum mAb concentrations to robustly predict nAb titres in serum. Methods: SARS-CoV-2 nAb titres were determined via authentic- and lentiviral pseudovirus-based neutralisation assays using serological data from three AZD7442 (tixagevimab-cilgavimab) studies: PROVENT (NCT04625725), TACKLE (NCT04723394) and a phase 1 dose-ranging study (NCT04507256). AZD7442 serum concentrations were assessed using immunocapture. Serum-based half-maximal inhibitory concentration (IC50) values were derived from pseudovirus nAb titres and serum mAb concentrations, and compared with in vitro IC50 measurements. Results: nAb titres measured via authentic- and lentiviral pseudovirus-based neutralisation assays were strongly correlated for the ancestral SARS-CoV-2 virus and SARS-CoV-2 Alpha. Serum AZD7442 concentrations and pseudovirus nAb titres were strongly correlated for multiple SARS-CoV-2 variants with all Spearman correlation coefficients ≥ 0.78. Serum-based IC50 values were similar to in vitro IC50 values for AZD7442, for ancestral SARS-CoV-2 and Alpha, Delta, Omicron BA.2 and Omicron BA.4/5 variants. Conclusions: These data highlight that serum mAb concentrations and pseudovirus in vitro IC50 values can be used to rapidly predict nAb titres in serum for emerging and historical SARS-CoV-2 variants.

Andrographolide suppresses SARS-CoV-2 infection by downregulating ACE2 expression: A mechanistic study.

Angiotensin-converting enzyme 2 (ACE2) is the receptor that enables SARS-CoV-2 to invade host cells. Previous studies have reported that reducing ACE2 expression may have an anti-SARS-CoV-2 effect. In this study, we constructed a pGL4.10-F2-ACE2 vector with double luciferase genes (firefly and Renilla luciferase) under the control of the ACE2 promoter and used it to screen compounds from Chinese traditional medicinal herbs (CTMHs) that can inhibit ACE2 transcription in human cells. We transfected HEK293T cells with pGL4.10-F2-ACE2 and treated them with CTMH compounds and then measured fluorescence to evaluate the indirect inhibition of ACE2 transcription. Out of 37 compounds tested, andrographolide demonstrated a dose-dependent inhibition of ACE2 transcription. We further confirmed by RT-qPCR and Western blot assays that andrographolide also reduced ACE2 expression in BEAS-2B cells in a dose-dependent manner. Moreover, pseudovirus infection assays in BEAS-2B cells demonstrated that andrographolide can inhibit SARS-CoV-2 infection in a dose-dependent manner. These results suggest that andrographolide has potential anti-SARS-CoV-2 activity and could be a candidate drug for COVID-19 prevention and treatment.

Validation of a Pseudovirus Neutralization Assay for Severe Acute Respiratory Syndrome Coronavirus 2: A High-Throughput Method for the Evaluation of Vaccine Immunogenicity.

The evaluation of coronavirus disease 2019 (COVID-19) vaccine immunogenicity remains essential as the severe acute respiratory syncytial virus 2 (SARS-CoV-2) pandemic continues to evolve and as additional variants emerge. Neutralizing antibodies are a known correlate of protection for SARS-CoV-2 vaccines. A pseudovirus neutralization (PNT) assay was developed and validated at Novavax Clinical Immunology Laboratories to allow for the detection of neutralizing antibodies in vaccine clinical trial sera. The PNT assay was precise, accurate, linear, and specific in measuring SARS-CoV-2 neutralization titers in human serum for ancestral strain and the Omicron subvariants BA.5 and XBB.1.5, with an overall geometric coefficient of variation of ≤43.4%, a percent relative bias within the expected range of -60% to 150%, and a linearity value of R2 > 0.98 for all three strains. This pseudovirus assay will be useful for the analysis of vaccine clinical trial samples to assess vaccine immunogenicity. Future work will focus on modifying the assay for emerging variants, including XBB.1.16, EG.5.1, BA.2.86, and any other variants that emerge in the ongoing pandemic.

Electrochemical profiling of poliovirus particles inactivated by chemical method and ionizing radiation.

Electrochemical profiling of formaldehyde-inactivated poliovirus particles demonstrated a relationship between the D-antigen concentration and the intensity of the maximum amplitude currents of the poliovirus samples. The resultant signal was therefore identified as electrochemical oxidation of the surface proteins of the poliovirus. Using registration of electrooxidation of amino acid residues of the capsid proteins, a comparative electrochemical analysis of poliovirus particles inactivated by electrons accelerated with doses of 5 kGy, 10 kGy, 15 kGy, 25 kGy, 30 kGy at room temperature was carried out. An increase in the radiation dose was accompanied by an increase in electrooxidation signals. A significant increase in the signals of electrooxidation of poliovirus capsid proteins was detected upon irradiation at doses of 15-30 kGy. The data obtained suggest that the change in the profile and increase in the electrooxidation signals of poliovirus capsid proteins are associated with an increase in the degree of structural reorganization of surface proteins and insufficient preservation of the D-antigen under these conditions of poliovirus inactivation.

Electrochemical biosensor for sensitive detection of SARS-CoV-2 gene fragments using Bi2Se3 topological insulator.

In this study, we have designed an electrochemical biosensor based on topological material Bi2Se3 for the sensitive detection of SARS-CoV-2 in the COVID-19 pandemic. Flake-shaped Bi2Se3 was obtained directly from high-quality single crystals using mechanical exfoliation, and the single-stranded DNA was immobilized onto it. Under optimal conditions, the peak current of the differential pulse voltammetry method exhibited a linear relationship with the logarithm of the concentration of target-complementary-stranded DNA, ranging from 1.0 × 10-15 to 1.0 × 10-11 M, with a detection limit of 3.46 × 10-16 M. The topological material Bi2Se3, with Dirac surface states, enhanced the signal-to-interference plus noise ratio of the electrochemical measurements, thereby improving the sensitivity of the sensor. Furthermore, the electrochemical sensor demonstrated excellent specificity in recognizing RNA. It can detect complementary RNA by amplifying and transcribing the initial DNA template, with an initial DNA template concentration ranging from 1.0 × 10-18 to 1.0 × 10-15 M. Furthermore, the sensor also effectively distinguished negative and positive results by detecting splitting-synthetic SARS-CoV-2 pseudovirus with a concentration of 1 copy/µL input. Our work underscores the immense potential of the electrochemical sensing platform based on the topological material Bi2Se3 in the detection of pathogens during the rapid spread of acute infectious diseases.

Design and Application of Biosafe Coronavirus Engineering Systems without Virulence.

In the last twenty years, three deadly zoonotic coronaviruses (CoVs)-namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2-have emerged. They are considered highly pathogenic for humans, particularly SARS-CoV-2, which caused the 2019 CoV disease pandemic (COVID-19), endangering the lives and health of people globally and causing unpredictable economic losses. Experiments on wild-type viruses require biosafety level 3 or 4 laboratories (BSL-3 or BSL-4), which significantly hinders basic virological research. Therefore, the development of various biosafe CoV systems without virulence is urgently needed to meet the requirements of different research fields, such as antiviral and vaccine evaluation. This review aimed to comprehensively summarize the biosafety of CoV engineering systems. These systems combine virological foundations with synthetic genomics techniques, enabling the development of efficient tools for attenuated or non-virulent vaccines, the screening of antiviral drugs, and the investigation of the pathogenic mechanisms of novel microorganisms.

Pseudovirus-Based Systems for Screening Natural Antiviral Agents: A Comprehensive Review.

Since the outbreak of COVID-19, researchers have been working tirelessly to discover effective ways to combat coronavirus infection. The use of computational drug repurposing methods and molecular docking has been instrumental in identifying compounds that have the potential to disrupt the binding between the spike glycoprotein of SARS-CoV-2 and human ACE2 (hACE2). Moreover, the pseudovirus approach has emerged as a robust technique for investigating the mechanism of virus attachment to cellular receptors and for screening targeted small molecule drugs. Pseudoviruses are viral particles containing envelope proteins, which mediate the virus's entry with the same efficiency as that of live viruses but lacking pathogenic genes. Therefore, they represent a safe alternative to screen potential drugs inhibiting viral entry, especially for highly pathogenic enveloped viruses. In this review, we have compiled a list of antiviral plant extracts and natural products that have been extensively studied against enveloped emerging and re-emerging viruses by pseudovirus technology. The review is organized into three parts: (1) construction of pseudoviruses based on different packaging systems and applications; (2) knowledge of emerging and re-emerging viruses; (3) natural products active against pseudovirus-mediated entry. One of the most crucial stages in the life cycle of a virus is its penetration into host cells. Therefore, the discovery of viral entry inhibitors represents a promising therapeutic option in fighting against emerging viruses.

Exploring immune evasion of SARS-CoV-2 variants using a pseudotyped system.

In the United States, coronavirus disease 2019 (COVID-19) cases have consistently been linked to the prevailing variant XBB.1.5 of SARS-CoV-2 since late 2022. A system has been developed for producing and infecting cells with a pseudovirus (PsV) of SARS-CoV-2 to investigate the infection in a Biosafety Level 2 (BSL-2) laboratory. This system utilizes a lentiviral vector carrying ZsGreen1 and Firefly luciferase (Fluc) dual reporter genes, facilitating the analysis of experimental results. In addition, we have created a panel of PsV variants that depict both previous and presently circulating mutations found in circulating SARS-CoV-2 strains. A series of PsVs includes the prototype SARS-CoV-2, Delta B.1.617.2, BA.5, XBB.1, and XBB.1.5. To facilitate the study of infections caused by different variants of SARS-CoV-2 PsV, we have developed a HEK-293T cell line expressing mCherry and human angiotensin converting enzyme 2 (ACE2). To validate whether different SARS-CoV-2 PsV variants can be used for neutralization assays, we employed serum from rats immunized with the PF-D-Trimer protein vaccine to investigate its inhibitory effect on the infectivity of various SARS-CoV-2 PsV variants. According to our observations, the XBB variant, particularly XBB.1.5, exhibits stronger immune evasion capabilities than the prototype SARS-CoV-2, Delta B.1.617.2, and BA.5 PsV variants. Hence, utilizing the neutralization test, this study has the capability to forecast the effectiveness in preventing future SARS-CoV-2 variants infections.

Antibiotics daptomycin interacts with S protein of SARS-CoV-2 to promote cell invasion of Omicron (B1.1.529) pseudovirus.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed enormous challenges to global public health. The use of antibiotics has greatly increased during the SARS-CoV-2 epidemic owing to the presence of bacterial co-infection and secondary bacterial infections. The antibiotics daptomycin (DAP) is widely used in the treatment of infectious diseases caused by gram-positive bacteria owing to its highly efficient antibacterial activity. It is pivotal to study the antibiotics usage options for patients of coronavirus infectious disease (COVID-19) with pneumonia those need admission to receive antibiotics treatment for bacterial co-infection in managing COVID-19 disease. Herein, we have revealed the interactions of DAP with the S protein of SARS-CoV-2 and the variant Omicron (B1.1.529) using the molecular docking approach and Omicron (B1.1.529) pseudovirus (PsV) mimic invasion. Molecular docking analysis shows that DAP has a certain degree of binding ability to the S protein of SARS-CoV-2 and several derived virus variants, and co-incubation of 1-100 µM DAP with cells promotes the entry of the PsV into human angiotensin-converting enzyme 2 (hACE2)-expressing HEK-293T cells (HEK-293T-hACE2), and this effect is related to the concentration of extracellular calcium ions (Ca2+). The PsV invasion rate in the HEK-293T-hACE2 cells concurrently with DAP incubation was 1.7 times of PsV infection alone. In general, our findings demonstrate that DAP promotes the infection of PsV into cells, which provides certain reference of antibiotics selection and usage optimization for clinicians to treat bacterial coinfection or secondary infection during SARS-CoV-2 infection.

Targeting Viral and Cellular Cysteine Proteases for Treatment of New Variants of SARS-CoV-2.

The continuous emergence of SARS-CoV-2 variants caused the persistence of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. The viral proteases are the most attractive targets for developing antiviral drugs. In this scenario, our study explores the use of HIV-1 protease inhibitors against SARS-CoV-2. An in silico screening of a library of HIV-1 proteases identified four anti-HIV compounds able to interact with the 3CLpro of SARS-CoV-2. Thus, in vitro studies were designed to evaluate their potential antiviral effectiveness against SARS-CoV-2. We employed pseudovirus technology to simulate, in a highly safe manner, the adsorption of the alpha (α-SARS-CoV-2) and omicron (ο-SARS-CoV-2) variants of SARS-CoV-2 and study the inhibitory mechanism of the selected compounds for cell-virus interaction. The results reported a mild activity against the viral proteases 3CLpro and PLpro, but efficient inhibitory effects on the internalization of both variants mediated by cathepsin B/L. Our findings provide insights into the feasibility of using drugs exhibiting antiviral effects for other viruses against the viral and host SARS-CoV-2 proteases required for entry.

Development of a SARS-CoV-2 neutralization assay based on a pseudotyped virus using a HIV system.

Regarding the extensive global attention to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that constitutes an international public health emergency, pseudovirus neutralization assays have been widely applied due to their advantages of being able to be conducted in biosafety level 2 laboratories and having a high safety factor. In this study, by adding a blue fluorescent protein (AmCyan) gene to the HIV system pSG3-△env backbone plasmid HpaI and truncating the C-terminal 21 amino acids of the SARS-CoV-2 spike protein (S), high-titer SARS-CoV-2-Sdel21-AmCyan fluorescent pseudovirus was successfully packaged. The fluorescent pseudovirus was used to establish a neutralization assay in a 96-well plate using 293T cells stably transfected with the AF cells. Then, parameters such as the ratio of backbone and membrane plasmid, sensitive cells, inoculation of cells and virus, as well as incubation and detection time were optimized. The pseudovirus neutralization assay demonstrated high accuracy, sensitivity, repeatability, and a strong correlation with the luminescent pseudovirus neutralization assay. Additionally, we scaled up the neutralizing antibody determination method by increasing the plate size from 96 wells to 384 wells. We have established a robust fluorescent pseudotyped virus neutralization assay for SARS-CoV-2 using the HIV system, providing a foundation for serum neutralization antibody detection, monoclonal antibody screening, and vaccine development.

Quantitative and Standardized Pseudovirus Neutralization Assay for COVID-19.

COVID-19 is a global pandemic caused by the highly infectious SARS-CoV-2 virus. Efforts to combat SARS-CoV-2 infection include mass vaccination and development of monoclonal and convalescent plasma therapeutics that require precise measurements of correlative, functional neutralizing antibodies that prevent virus infection. Developing rapid, safe, easy-to-use, and high-quality neutralization assays are essential for the success of the massive effort. Here, we developed a vesicular stomatitis virus-based neutralization assay that was capable of quantifying varying degrees of neutralization in patient serum samples. This assay has two detection readouts, flow cytometry and live cell imaging. The two readout methods produced consistent values of all 50% neutralization titers, further enhancing measurement confidence on the assay. Moreover, the use of available reference standards such as the World Health Organization International Standard (NIBSC code 20/136) enables quantification and standardization of the pseudovirus neutralization assay with neutralizing antibody titers measured in International Units/mL. Quantitative and standardized neutralization assays are critical for reliable efficacy evaluation and comparison of numerous vaccines and therapeutics.