Search Results (70)

Yeasts possess a range of environmental adaptations that allow them to colonize soil and sand. They can circulate seasonally between different components of lake ecosystems, including beach sand, water, and the coastal phyllosphere. The accumulation of people on beaches promotes the development and transmission of yeasts, posing an increasing sanitary and epidemiological risk. The aim of this study was to determine the species and quantitative composition of potentially pathogenic and pathogenic yeasts for humans present in the sand of supervised and unsupervised beaches along the shores of lakes in the city of Olsztyn (northeastern Poland). The study material consisted of sand samples collected during two summer seasons (2019; 2020) from 12 research sites on sandy beaches of four lakes located within the administrative boundaries of Olsztyn. Standard isolation and identification methods used in diagnostic mycological laboratories were applied and are described in detail in the following sections of this study. A total of 259 yeast isolates (264, counting species in two-species isolates separately) belonging to 62 species representing 47 genera were obtained during the study. Among all the isolates, five were identified as mixed (two species from a single colony). Eight isolated species were classified into biosafety level 2 (BSL-2) and risk group 2 (RG-2). The highest average number of viable yeast cells was found in sand samples collected in July 2019 (5.56 × 10² CFU/g), August, and September 2020 (1.03 × 10³ CFU/g and 1.94 × 10³ CFU/g, respectively). The lowest concentrations were in samples collected in April, September, and October 2019, and October 2020 (1.48 × 10² CFU/g, 1.47 × 10² CFU/g, 1.40 × 10² CFU/g, and 1.40 × 10² CFU/g, respectively). The results indicate sand contamination with yeasts that may pose etiological factors for human mycoses. In light of these findings, continuous sanitary-epidemiological monitoring of beach sand and further studies on its mycological cleanliness are warranted, along with actions leading to appropriate legal regulations. Full article

Hendra virus (HeV) and Nipah virus (NiV) are two highly pathogenic RNA viruses with zoonotic potential, which can cause severe diseases with high mortality rates (50–100%) in humans and animals. Given this context, these viruses are classified as Biosafety Level 4 (BSL-4) pathogens, thus limiting research studies. Despite the high case fatalities, there are currently no human vaccines available for either virus, owing in part to the limitations in research and hesitancy in funding. In the absence of widespread vaccination, diagnostic tests are crucial for the rapid identification of cases and disease surveillance. This review synthesizes current knowledge on the epidemiology, transmission dynamics, and pathogenesis of NiV and HeV to contextualize a detailed assessment of the available diagnostic tools. We examined molecular and serological assays, including RT-PCR, ELISA, and LAMP, highlighting sample sources, detection windows, and performance. Diagnostic considerations across human and animal hosts are discussed, with emphasis on outbreak applicability and field-readiness, given the need for diagnostic assays that are suitable for use in low-income areas. Further development of diagnostic assays, including isothermal amplification tests and other next-generation approaches, is recommended to fill the gap in rapid, point-of-care diagnostics. Full article

Research on highly pathogenic biosafety level 4 (BSL-4) viruses that are classified as Select Agents involves transferring inactivated materials to lower containment levels for further analysis. Compliance with Select Agent and BSL-4 safety regulations necessitates the validation and verification of inactivation procedures. To streamline this process, it would be beneficial to use surrogate BSL-2 viruses for inactivation studies. This not only simplifies BSL-4 work but also enables the testing and validation of inactivation procedures in research facilities that lack access to high-containment laboratories yet may receive samples containing highly pathogenic viruses that require efficient and complete inactivation. In this study, we used Hazara virus (HAZV) as a surrogate virus for Crimean–Congo hemorrhagic fever virus to show the efficacy of various inactivation methods. We demonstrate the successful inactivation of HAZV using TRIzol/TRIzol LS and aldehyde fixation. Importantly, the parameters of the aldehyde inactivation of cell pellets differed from those of the monolayers, highlighting the importance of inactivation validation. As part of this study, we also defined specific criteria that must be met by a BSL-2 virus to be used as a surrogate for a closely related BSL-4 virus. Defining these criteria helps identify suitable nonpathogenic surrogates for developing inactivation procedures for highly pathogenic viruses. 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

Nipah virus (NiV), a highly lethal zoonotic pathogen causing encephalitis and respiratory diseases with mortality rates up to 40–70%, faces research limitations due to its strict biosafety level 4 (BSL-4) containment requirements, hindering antiviral development. To address this, we generated two NiV minigenome replicons (Fluc- and EGFP-based) expressed via helper plasmids encoding viral N, P, and L proteins, enabling replication studies under BSL-2 conditions. The minigenome replicon recapitulated the cytoplasmic inclusion body (IB) formation observed in live NiV infections. We further demonstrated that IB assembly is driven by liquid–liquid phase separation (LLPS), with biochemical analyses identifying the C-terminal N core domain of the N protein, as well as N₀ and XD domains and the intrinsically disordered region (IDR) of the P protein, as essential structural determinants for LLPS-mediated IB biogenesis. The targeted siRNA silencing of the 5′ and 3′ untranslated regions (UTRs) significantly reduced replicon-derived mRNA levels, validating the regulatory roles of these regions. Importantly, the minigenome replicon demonstrated sensitivity to type I/II/III interferons and antivirals (remdesivir, azvudine, molnupiravir), establishing its utility for drug screening. This study provides a safe and efficient platform for investigating NiV replication mechanisms and accelerating therapeutic development, circumventing the constraints of BSL-4 facilities while preserving key virological features. Full article

Crimean–Congo hemorrhagic fever virus (CCHFV) causes severe or fatal infections in humans and is geographically widespread. The virus has coevolved with its tick vectors, establishing persistent infections critical to its transmission. This study explored the mechanisms underpinning these persistent infections, using tick cell lines and the Hazara virus (HAZV) as a biosafety level 2 (BSL-2) model for CCHFV. Initially, an RT-qPCR protocol was developed to detect HAZV in tick cells. The study then focused on the production of virus-derived DNA (vDNAs) by tick cells as a defensive response to infection. These vDNAs regulate viral particle production, enabling tick cells to maintain viability and establish persistent infections. The experiments characterized vDNAs production, viral titers, and subcellular localization, and they examined the effect of the reverse transcriptase inhibitor azidothymidine triphosphate (AZT). The results showed that all tested tick cell lines supported HAZV replication, achieving persistent infections without cytopathic effects. vDNAs was detected in both the cytoplasm and nucleus, and its formation was dependent on HAZV infection. Importantly, vDNAs presence was linked to infection persistence; cells treated with AZT exhibited a marked reduction in vDNAs production and an associated increase in viral particle production, which correlated with higher cell death. These findings underscore the critical role of vDNAs in balancing viral replication and promoting long-term cell survival in tick cells, highlighting their importance in the coevolution of tick-borne viruses and their vectors. Full article

Laboratory-acquired infections (LAIs) pose significant risks to laboratory personnel, public health, and the environment, despite the implementation of biosafety measures. This study provides a comprehensive analysis of global LAIs reported from 1974 to 2024, identifying trends, causes, and pathogen distributions to address gaps in biosafety knowledge. A systematic literature review was conducted using databases such as PubMed, Cochrane, Google Scholar, and the American Biological Safety Association (ABSA). A total of 234 studies meeting strict inclusion criteria were analyzed. Bacterial pathogens accounted for 58.6% of reported incidents, followed by viruses at 36.1%. Procedural errors and accidents were the predominant causes of LAIs, with Brucella spp. being the most frequently reported pathogen, primarily in China. Temporal trends indicated a decline in incidents coinciding with the implementation of international biosafety regulations. However, disparities in incident reporting and compliance remain evident across countries. This study underscores the urgent need for a global regulatory framework, mandatory biosafety audits, a centralized incident database, and standardized training for high-containment laboratory personnel. Enhancing global collaboration, transparency in research, and adherence to ethical standards will further reduce LAI risks and strengthen public health security worldwide. 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

Cedar henipavirus (CedV), which was isolated from the urine of pteropodid bats in Australia, belongs to the genus Henipavirus in the family of Paramyxoviridae. It is closely related to the Hendra virus (HeV) and Nipah virus (NiV), which have been classified at the highest biosafety level (BSL4) due to their high pathogenicity for humans. Meanwhile, CedV is apathogenic for humans and animals. As such, it is often used as a model virus for the highly pathogenic henipaviruses HeV and NiV. In this study, we challenged eight Rousettus aegyptiacus fruit bats of different age groups with CedV in order to assess their age-dependent susceptibility to a CedV infection. Upon intranasal inoculation, none of the animals developed clinical signs, and only trace amounts of viral RNA were detectable at 2 days post-inoculation in the upper respiratory tract and the kidney as well as in oral and anal swab samples. Continuous monitoring of the body temperature and locomotion activity of four animals, however, indicated minor alterations in the challenged animals, which would have remained unnoticed otherwise. Full article

High-consequence pathogens such as the Ebola, Marburg, and Lassa viruses are handled in maximum-containment biosafety level 4 (BSL-4) laboratories. Genetic material is often isolated from such viruses and subsequently removed from BSL-4 laboratories for a multitude of downstream analyses using readily accessible technologies and equipment available at lower-biosafety level laboratories. However, it is essential to ensure that these materials are free of viable viruses before removal from BSL-4 laboratories to guarantee sample safety. This study details the in-house procedure used for validating the inactivation of Ebola, Marburg, and Lassa virus cultures after incubation with AVL lysis buffer (Qiagen) and ethanol. This study’s findings show that no viable virus was detectable when high-titer cultures of Ebola, Marburg, and Lassa viruses were incubated with AVL lysis buffer for 10 min, followed by an equal volume of 95% ethanol for 3 min, using a method with a sensitivity of ≤0.8 log₁₀ TCID₅₀ as the limit of detection. Full article

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that affects cloven-hoofed animals and causes severe economic losses in the livestock industry. Given that this high-risk pathogen has to be handled in a biosafety level (BSL)-3 facility for safety reasons and the limited availability of BSL-3 laboratories, experiments on FMDV call for more attention. Therefore, we aimed to develop an FMDV experimental model that can be handled in BSL-2 laboratories. The NanoBiT luciferase (Nano-luc) assay is a well-known assay for studying protein–protein interactions. To apply the NanoBiT split luciferase assay to the diagnosis and evaluation of FMD, we developed an inactivated HiBiT-tagged Asia1 Shamir FMDV (AS-HiBiT), a recombinant Asia1 shamir FMDV with HiBiT attached to the VP1 region of Asia1 shamir FMDV. In addition, we established LgBiT-expressing LF-BK cell lines, termed LgBit-LF-BK cells. It was confirmed that inactivated AS-HiBiT infected LgBiT-LF-BK cells and produced a luminescence signal by binding to the intracellular LgBiT of LgBiT-LF-BK cells. In addition, the luminescence signal became stronger as the number of LgBiT-LF-BK cells increased or the concentration of inactivated AS-HiBiT increased. Moreover, we confirmed that inactivated AS-HiBiT can detect seroconversion in sera positive for FMDV-neutralizing antibodies. This NanoBiT split luciferase assay system can be used for the diagnosis and evaluation of FMD and expanded to FMD-like virus models to facilitate the evaluation of FMDV vaccines and antibodies. Full article

While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases. 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

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