Search Results (63)

In recent years, the persistent emergence of novel infectious pathogens (epitomized by the global coronavirus disease-2019 (COVID-2019) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) has propelled nucleic acid testing (NAT) into an unprecedented phase of rapid development. As a key technology in modern molecular diagnostics, NAT achieves precise pathogen identification through specific nucleic acid sequence recognition, establishing itself as an indispensable diagnostic tool across diverse scenarios, including public health surveillance, clinical decision-making, and food safety control. The reliability of NAT systems fundamentally depends on reference materials (RMs) that authentically mimic the biological characteristics of natural viruses. This critical requirement reveals significant limitations of current RMs in the NAT area: naked nucleic acids lack the structural authenticity of viral particles and exhibit restricted applicability due to stability deficiencies, while inactivated viruses have biosafety risks and inter-batch heterogeneity. Notably, pseudovirus has emerged as a novel RM that integrates non-replicative viral vectors with target nucleic acid sequences. Demonstrating superior performance in mimicking authentic viral structure, biosafety, and stability compared to conventional RMs, the pseudovirus has garnered substantial attention. In this comprehensive review, we critically summarize the engineering strategies of pseudovirus platforms and their emerging role in ensuring the reliability of NAT systems. We also discuss future prospects for standardized pseudovirus RMs, addressing key challenges in scalability, stability, and clinical validation, aiming to provide guidance for optimizing pseudovirus design and practical implementation, thereby facilitating the continuous improvement and innovation of NAT technologies. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. The development of antiviral drugs for COVID-19 has been hampered by the requirement of a biosafety level 3 (BSL3) laboratory for experiments related to SARS-CoV-2, and by the lack of easy and precise methods for quantification of infection. Here, we developed a SARS-CoV-2 viral vector composed of all four SARS-CoV-2 structural proteins constitutively expressed in lentivirally transduced cells, combined with an RNA replicon deleted for SARS-CoV-2 structural protein genes S, M, and E, and expressing a luciferase–GFP fusion protein. We show that, after concentrating viral stocks by ultracentrifugation, the SARS-CoV-2 viral vector is able to infect two human cell lines expressing receptors ACE2 and TMPRSS2. Both luciferase activity and GFP fluorescence were detected, and transduction was remdesivir-sensitive. We also show that this vector is inhibited by three type I interferon (IFN-I) subtypes. Although improvements are needed to increase infectious titers, this vector system may prove useful for antiviral drug screening and SARS-CoV-2-related investigations. Full article

Background: The COVID-19 pandemic significantly disrupted forensic science, exposing vulnerabilities and introducing unprecedented challenges. Five years later, its impact persists, necessitating ongoing adaptations in forensic practice. This study examines key transformations, persistent issues, and emerging challenges in forensic science post-pandemic. Methods: A critical analysis of forensic science’s response to the pandemic was conducted, focusing on operational disruptions, methodological advancements, educational shifts, and technological integration. Results: Forensic operations faced delays due to case backlogs, restricted in-person work, and postponed court proceedings. Forensic pathology evolved with increased reliance on molecular autopsy techniques to clarify COVID-19-related deaths. Educational methods shifted toward virtual learning, prompting discussions on standardized digital training. Additionally, artificial intelligence and automation gained prominence in forensic investigations, enhancing crime scene analysis and predictive modeling. Discussion: While forensic science demonstrated adaptability, challenges remain in international collaboration, resource distribution, and professional training. The pandemic accelerated technological integration but also raised ethical and procedural concerns, particularly regarding AI applications in legal contexts. Virtual learning innovations necessitate further development to ensure competency in forensic training. Conclusions: Forensic science continues to evolve in response to post-pandemic realities. Addressing gaps in cooperation, technology implementation, and training will be crucial to strengthening the field. By assessing these changes, this study underscores forensic science’s resilience and adaptability, offering insights into its future trajectory amid ongoing challenges. Full article

Neutralization assays have become an important tool since the beginning of the COVID-19 pandemic for testing vaccine responses and therapeutic antibodies as well as for monitoring humoral immunity to SARS-CoV-2 in epidemiological studies. The spike glycoprotein (S) present on the viral surface contains a receptor binding domain (RBD) that recognizes the angiotensin-converting enzyme 2 receptor (ACE2) in host cells, allowing virus entry. The gold standard for determining SARS-CoV-2 neutralizing antibodies is the plaque reduction neutralization test (PRNT), which relies on live-virus replication performed exclusively in biosafety level 3 (BSL-3) laboratories. Here, we report the development of a surrogate live-cell imaging-based fluorescent SARS-CoV-2 neutralization assay, applicable to BSL-1 or BSL-2 laboratories, by antibody-mediated blockage of the interaction between recombinant RBD with overexpressed ACE2 receptor in a genetically modified HEK 293T stable cell line. Our approach was able to detect neutralizing antibodies both in COVID-19-positive human serum samples and polyclonal equine formulations against SARS-CoV-2. This new cell-based surrogate neutralization assay represents a virus-free fluorescence imaging alternative to the reported approaches, which can be used to detect antibody-neutralizing capabilities toward SARS-CoV-2. This assay could also be extrapolated in the future to other established and emergent viral agents. Full article

In 2021, at the height of the COVID-19 pandemic, coronavirus research spiked, with over 83,000 original research articles related to the word “coronavirus” added to the online resource PubMed. Just 2 years later, in 2023, only 30,900 original research articles related to the word “coronavirus” were added. While, irrefutably, the funding of coronavirus research drastically decreased, a possible explanation for the decrease in interest in coronavirus research is that projects on SARS-CoV-2, the causative agent of COVID-19, halted due to the challenge of establishing a good cellular or animal model system. Most laboratories do not have the capabilities to culture SARS-CoV-2 ‘in house’ as this requires a Biosafety Level (BSL) 3 laboratory. Until recently, BSL 2 laboratory research on endemic coronaviruses was arduous due to the low cytopathic effect in isolated cell culture infection models and the lack of means to quantify viral loads. The purpose of this review article is to compare the human coronaviruses and provide an assessment of the latest techniques that use the endemic coronaviruses—HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1—as lower-biosafety-risk models for the more pathogenic coronaviruses—SARS-CoV-2, SARS-CoV, and MERS-CoV. Full article

Dental students are frequently exposed to percutaneous injuries (PCIs) due to the nature of their clinical work, which involves sharp instruments and close patient contact. The COVID-19 pandemic further emphasized the need for stringent biosafety measures and the use of personal protective equipment (PPE). Despite these precautions, injuries remain prevalent, highlighting the need for comprehensive education and training in biosafety and infection control. This study investigates the incidence and causes of injuries among undergraduate dental students during clinical sessions. This study was conducted at the Department of Dentistry, National and Kapodistrian University of Athens, focusing on injuries reported from 2021 to 2024. Data were collected through self-reported clinical records. The primary variables assessed included the type of injury, the instrument involved, the clinical procedure performed, and the immediate actions taken post-injury. Serological testing was conducted on students and patients to assess the risk of the transmission of bloodborne pathogens. The findings reveal a high prevalence of injuries, with needles being the most common cause (51.4%), followed by other tools such as dental probes (25.7%) and burs (8.6%). The most frequent injury type is piercing (74.2%), primarily affecting the fingers (88.6%). Periodontal treatments, restorative procedures, and endodontic treatments are the main activities leading to injuries, with 17.1% of injuries being caused by each. No statistically significant results are recorded. Despite regular medical records for most patients treated by injured students, serological testing shows significant positivity rates for HCV and HBV. Notably, most injured students demonstrate their commitment to safety by adhering to recommended post-exposure protocols, including wound cleaning, disinfecting, and serological testing. Furthermore, the impact of COVID-19 heightened the importance of personal protective equipment (PPE) and reinforced occupational health standards. Our study highlights the critical need for enhanced biosafety awareness and training among undergraduate dental students to reduce injury risks. Full article

The COVID-19 pandemic posed significant challenges to public health, exposing first responders to high biosafety risks during medical assistance and containment efforts. The PANDEM-2 study aimed to address these critical biosafety issues by emphasising the importance of frequently updated, harmonised guidelines. This study reviewed scientific publications, lessons learned, and real-world experiences from the COVID-19 pandemic to identify biorisk gaps in three critical areas: (i) patient transportation and management, (ii) sample handling and testing, and (iii) data management and communication by laboratory staff. At the onset of the pandemic, first responders faced several challenges, including the rapid expansion of emergency medical services, conversion of non-medical structures, increased internal and cross-border transport of infected patients, frequent changes in biosafety protocols, and a shortage of personal protective equipment. In response, this study developed a versatile and easily adaptable toolkit, including biosafety guidance and recommendations linked to updated national and international online repositories. It establishes the groundwork for a minimum standard that can be tailored to various pandemic response scenarios, using monkeypox as a fictive test case. The toolkit enables rapid access to updated information via QR codes and mobile devices, improving biorisk response by providing an adaptable and standardised approach for caregivers involved in national and cross-border responses. Full article

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has filled a gap in our knowledge regarding the prevention of CoVs. Swine coronavirus (CoV) is a significant pathogen that causes huge economic losses to the global swine industry. Until now, anti-CoV prevention and control have been challenging due to the rapidly generated variants. Silver nanoparticles (AgNPs) with excellent antimicrobial activity have attracted great interest for biosafety prevention and control applications. In this study, we synthesized chitosan-modified AgNPs (Chi-AgNPs) with good biocompatibility to investigate their antiviral effects on swine CoVs. In vitro assays showed that Chi-AgNPs could significantly impaired viral entry. The direct interaction between Chi-AgNPs and CoVs can destroy the viral surface spike (S) protein secondary structure associated with viral membrane fusion, which is caused by the cleavage of disulfide bonds in the S protein. Moreover, the mechanism showed that Chi-AgNPs reduced the virus-induced apoptosis of Vero cells via the ROS/p53 signaling activation pathway. Our data suggest that Chi-AgNPs can serve as a preventive strategy for CoVs infection and provide a molecular basis for the viricidal effect of Chi-AgNPs on CoVs. Full article

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. Full article

Numerous hospital laboratories in Indonesia need to implement improved bio-risk management (BRM) systems. There are many potential biohazards in laboratory activities that can impact health and the environment, leading to laboratory incidents. To minimize the impact and occurrence of such incidents, it is necessary to evaluate the implementation of BRM in every hospital laboratory that uses biological agents. This study was conducted in eight COVID-19 reference hospitals in Indonesia in the regions of Sumatra, Kalimantan, and Java, which have committed to implementing BRM systems in their laboratory activities. This research employed a descriptive study design and quantitative methods, with the aim of analyzing and evaluating the implementation of BRM systems in laboratories by assessing the achievements and gap analysis obtained from each laboratory. This research utilized primary data in the form of checklist forms referencing ISO 35001:2019 for the laboratory BRM system. Then, the assessments were based on virtual interviews conducted by the researcher with laboratory personnel as the primary data. The evaluation conducted on gap analysis from the seven clauses in ISO 35001:2019 across all hospitals revealed large gaps, particularly in three clauses: leadership, support, and performance. However, the aspects concerning organization, improvement, and performance evaluation were relatively satisfactory. Hence, there is a need for further improvement in leadership, support, and performance evaluation clauses. Additionally, it is essential to highlight the importance of comprehensive performance assessment, including proactive audits and continuous enhancements to achieve optimal bio-risk management. Full article

Neutralizing antibody responses from COVID-19 vaccines are pivotal in conferring protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Effective COVID-19 vaccines and assays measuring neutralizing antibodies against emerging variants (i.e., XBB.1.5, XBB.1.16, and XBB.2.3) are needed. The use of biosafety level (BSL)-3 laboratories for live virus assays results in higher costs and a longer turnaround time; therefore, a BSL-2–based pseudovirus neutralization assay (PNT) was developed. The pseudoviruses were produced by cotransfecting cells with plasmids encoding a lentiviral backbone-expressing luciferase reporter; non-surface proteins for lentiviral production; and ancestral or Omicron (BA.1 and BA.5) SARS-CoV-2 spike (S) proteins. The PNT was developed and optimized in dose and kinetics experiments. The representative serum samples (COVID-19–convalescent or NVX-CoV2373–vaccinated participants enrolled in the 2019nCoV-101 trial) demonstrated a wide dynamic range. The neutralization data showed robust correlation with validated anti-recombinant spike IgG levels and angiotensin-converting enzyme 2 inhibition titers (ancestral). This assay is suitable for measurement of the neutralization ability in clinical samples from individuals infected with SARS-CoV-2 or immunized with a COVID-19 vaccine. The results suggest that this PNT provides a lower cost, high-throughput, rapid turnaround alternative to BSL-3–based microneutralization assays and enables the discovery and development of effective vaccines against emerging variants. Full article

During the COVID-19 pandemic, first responders faced significant biosafety challenges, especially while handling patient transport, potentially exposing them to infection. The PANDEM-2 (European project on pandemic preparedness and response) project, funded by the Horizon 2020 program, sought to investigate the challenges confronting Emergency Medical Systems throughout the EU. First responders from Portugal’s National Institute of Medical Emergency (INEM) were considered as a representative operational model of the national first responder agencies of European member states because they played a critical role during the COVID-19 pandemic. As a result, they were asked to complete an online survey about their COVID-19 pandemic-related professional activities. The survey focused on their perspectives on current biosafety guidelines and their operational practices. It covered opinions on existing protocols, technical concerns during patient transport, and issues after the patients arrived at the hospital. The key findings revealed concerns about risk assessment, the inadequacy of guidelines, and disparities in equipment access. This survey emphasizes the importance of developing streamlined, adaptable biosafety protocols, better coordination between prehospital and in-hospital services, and the development of scalable, cost-effective biosafety solutions. Based on our findings, we propose improvements to national and European biosafety directives and advocate for streamlined adaptation during pandemics. Full article

A reliable and efficient serological test is crucial for monitoring neutralizing antibodies against SARS-CoV-2 and its variants of concern (VOCs). Here, we present an integrated research–clinical platform for a live SARS-CoV-2 neutralization assay, utilizing highly attenuated SARS-CoV-2 (Δ3678_WA1-spike). This strain contains mutations in viral transcription regulation sequences and deletion in the open-reading-frames 3, 6, 7, and 8, allowing for safe handling in biosafety level 2 (BSL-2) laboratories. Building on this backbone, we constructed a genetically stable reporter virus (mGFP Δ3678_WA1-spike) by incorporating a modified green fluorescent protein sequence (mGFP). We also constructed mGFP Δ3678_BA.5-spike and mGFP Δ3678_XBB.1.5-spike by substituting the WA1 spike with variants BA.5 and XBB.1.5 spike, respectively. All three viruses exhibit robust fluorescent signals in infected cells and neutralization titers in an optimized fluorescence reduction neutralization assay that highly correlates with a conventional plaque reduction assay. Furthermore, we established that a streamlined robot-aided Bench-to-Clinics COVID-19 Neutralization Test workflow demonstrated remarkably sensitive, specific, reproducible, and accurate characteristics, allowing the assessment of neutralization titers against SARS-CoV-2 variants within 24 h after sample receiving. Overall, our innovative approach provides a valuable avenue for large-scale testing of clinical samples against SARS-CoV-2 and VOCs at BSL-2, supporting pandemic preparedness and response strategies. Full article

Due to the COVID-19 pandemic, researchers have focused on new preventive measures to limit the spread of SARS-CoV-2. One promising application is the usage of antimicrobial materials on often-touched surfaces to reduce the load of infectious virus particles. Since tests with in vitro-propagated SARS-CoV-2 require biosafety level 3 (BSL-3) laboratories with limited capacities and high costs, experiments with an appropriate surrogate like the bacteriophage ɸ6 are preferred in most studies. The aim of this study was to compare ɸ6 and SARS-CoV-2 within antiviral surface tests. Different concentrations of copper coatings on polyethylene terephthalate (PET) were used to determine their neutralizing activity against ɸ6 and SARS-CoV-2. The incubation on the different specimens led to similar inactivation of both SARS-CoV-2 and ɸ6. After 24 h, no infectious virus particles were evident on any of the tested samples. Shorter incubation periods on specimens with high copper concentrations also showed a complete inactivation. In contrast, the uncoated PET foils resulted only in a negligible reduced inactivation during the one-hour incubation. The similar reduction rate for ɸ6 and SARS-CoV-2 in our experiments provide further evidence that the bacteriophage ɸ6 is an adequate model organism for SARS-CoV-2 for this type of testing. Full article

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains widely pandemic around the world. Animal models that are sensitive to the virus are therefore urgently needed to evaluate potential vaccines and antiviral agents; however, SARS-CoV-2 requires biosafety level 3 containment. To overcome this, we developed an animal model using the intranasal administration of SARS-CoV-2 pseudovirus. As the pseudovirus contains the firefly luciferase reporter gene, infected tissues and the viral load could be monitored by in vivo bioluminescent imaging. We used the model to evaluate the protective efficacy of monoclonal antibodies and the tissue tropism of different variants. The model may also be a useful tool for the safe and convenient preliminary evaluation of the protective efficacy of vaccine candidates against SARS-CoV-2, as well as the treatment efficacy of anti-viral drugs. Full article