Assessing the evidence for effective biosafety risk management in Coxiella burnetii research.

Genomic insights into pyrazinamide and fluoroquinolone resistance in multidrug-resistant tuberculosis in Khyber Pakhtunkhwa, Pakistan.

The present study aimed to analyze the biosafety measures implemented in autopsy laboratories, with emphasis on identifying the main occupational risks and evaluating the preventive strategies adopted to reduce professionals’ exposure to infectious agents and potentially hazardous biological materials. An integrative literature review was conducted with a search in the Medline/PubMed database. Eight articles that directly addressed biosafety measures in autopsies were selected. The analysis revealed a diversity of risks, highlighting the generation of aerosols, sharps accidents, and exposure to chemical agents and pathogens, including SARS-CoV-2. The analysis of the studies evidenced that biological risks, particularly the inhalation of aerosols generated by oscillating saws and liquid aspiration, are predominant. Inadequate infrastructure, such as the lack of negative pressure rooms (Biosafety Level 3 - BSL-3), and failures in the use of Personal Protective Equipment (PPE) and waste management amplify these dangers. Effective strategies include proper facility design, the adoption of additional physical barriers (such as the “craniotomy box”), controlled ventilation, and permanent staff education. It is concluded that the rigorous adoption of biosafety measures, aligned with international guidelines and supported by adequate infrastructure and continuous training, is imperative to mitigate occupational risks in autopsy laboratories. Such actions are essential to ensure professional safety, the quality of forensic procedures, and the protection of public health.

Langat virus (LGTV) is a tick-borne member of theFlaviviridaefamily and a biosafety level 2 surrogate for studying tick-borne encephalitis virus (TBEV) replication and pathogenesis. Here, we report the construction of a plasmid encoding a cytomegalovirus (CMV) promoter-driven LGTV cDNA that initiates infection following direct transfection of mammalian cells. Incorporation of three introns eliminated viral cDNA-associated toxicity in bacteria, enabling stable propagation of the full-length plasmid. Transfection of this construct resulted in high-level production of infectious LGTV, which exhibited robust replication kinetics, though slightly slower growth compared to a patient-derived isolate. We further engineered mCherry and Gaussia luciferase reporter versions of the clone, which yielded viruses expressing high levels of their respective reporters while retaining efficient replication. These LGTV infectious clones provide versatile tools for investigating viral replication, gene function, and pathogenesis, and may facilitate screening for antiviral inhibitors.HighlightsSingle-plasmid CMV-driven system launches infectious Langat virus (LGTV)Three introns stabilize full-length LGTV cDNA in E. coliDNA transfection yields high-titer infectious virus in mammalian cellsReporter LGTVs enable fluorescent and luminescent infection readoutsProvides a versatile platform for LGTV genetics and pathogenesis studies

Magnetic bead-based electrochemical surrogate virus neutralization test for quantification of antibody neutralizing efficiency.

BackgroundStudies on the tuberculosis pathogen Mycobacterium tuberculosis bacillus require a biosafety level 3 laboratory. This study aims to investigate the gender distribution of researchers working in the most hazardous tuberculosis laboratory.MethodsIn this study, the original articles indexed in Web of Science between 1970 and 2021, which include phenotypic testing methods in which the susceptibility results of drugs used in the treatment of tuberculosis are determined, were reviewed.ResultsAs a result of our analysis, it was found that female researchers were included as first name in the publications at a rate of 49%, unlike other fields of science, and 51% came from lower-middle-income and upper-middle-income countries. According to the incidence rates announced by the World Health Organization in the years when the articles were published, it was seen that female researchers participated in more studies in regions with a high incidence of tuberculosis to ('x̄' = 89.94).ConclusionAccording to our data, it was found that the proportion of female researchers was high in the laboratory with a high risk of contamination of biosafety level 3. The high proportion of female researchers in hazardous areas is striking.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13010-025-00177-z.

Machupo virus, a member of the Mammarenavirus genus within the Arenaviridae family, is the etiological agent of Bolivian Hemorrhagic Fever (BHF). This enveloped virus possesses a bi-segmented, single-stranded ambisense RNA genome. The small (S) segment encodes the glycoprotein precursor (GPC) and nucleoprotein (NP), while the large (L) segment encodes the RNA-dependent RNA polymerase (L) and the matrix protein (Z). The GPC is cleaved into GP1, which binds the human transferrin receptor 1 (hTfR1) for cell entry, and GP2, which mediates membrane fusion. Transmission to humans occurs primarily via contact with or inhalation of aerosols from the excreta of the natural rodent reservoir, Calomys callosus. The disease is endemic to the Bolivian plains, with outbreaks often linked to increased rodent populations. Following an incubation period of 7-14 days, BHF presents with non-specific symptoms including fever, headache, myalgia, and anorexia. The illness can progress to severe hemorrhagic manifestations such as epistaxis, hematemesis, and neurological signs like tremors and convulsions, with a mortality rate of 5-30%. Diagnosis is achieved through RT-PCR, antigen-capture ELISA, or serology, though testing requires high-containment biosafety level-4 facilities. Treatment relies on the antiviral ribavirin, which is most effective when administered early. Prevention focuses on public health measures, including rodent control, safe food storage, and the use of personal protective equipment to prevent nosocomial transmission. Currently, no vaccine is available for human use.

Arenaviruses are a genus of enveloped, negative-sense or ambisense RNA viruses within the family Arenaviridae, known as significant zoonotic pathogens causing severe hemorrhagic fevers in humans. The virions are spherical and pleomorphic, ranging from 60–300 nm in diameter, and contain host-derived ribosomes, giving them a sandy (“arena”) appearance. Their genome has two single-stranded RNA segments—the small (S) and large (L) segments—using an ambisense coding strategy to encode four main proteins: nucleoprotein (NP), glycoprotein precursor (GPC), RNA-dependent RNA polymerase (L), and matrix protein (Z). Arenaviruses persist in specific rodent reservoirs, forming two major complexes: the Old World (e.g., Lassa, Lujo viruses) and the New World (e.g., Junin, Machupo, Guanarito viruses). Human infection occurs mainly through inhalation of aerosols or contact with fomites contaminated with rodent excreta. Clinical manifestations range from mild febrile illness to severe viral hemorrhagic fever (VHF) with high fever, coagulopathy, vascular leakage, and multi-organ failure. Diagnosis requires biosafety level 4 (BSL-4) containment and relies on serology (ELISA, IFA), reverse transcription-polymerase chain reaction (RT-PCR), or virus isolation. Treatment is supportive, though ribavirin shows limited efficacy for Lassa and Junin viruses when given early. Prevention focuses on rodent control, public health education, and infection control in healthcare settings. Despite promising vaccine candidates, none are commercially available, underscoring arenaviruses as persistent and emerging global health threats.

CCHFV causes severe hemorrhagic fever outbreaks, with a mortality rate of up to 40%. Countries generally list CCHFV as one of the pathogens that requires the highest biosafety level 4 (BSL-4) of containment, which hinders the study of its cell biology and pathogenesis. LDLR was recently identified as a receptor for CCHFV, but other receptors or co-factors remain to be explored. We perform genome-wide CRISPR screens using a safe replication-competent CCHFV pseudovirus and identify a secretory MFGE8 protein that functions as an entry mediator by binding to both the Gc protein and PtdSer on the viral envelope and to the integrins on the cells. Cell entry mediated by a soluble protein may greatly expand the tissue tropism, and the strategies developed to disturb the interaction of MFGE8 with virions or with integrins may help to mitigate the fatal disease induced by CCHFV.

Nipah virus (NiV) is a pathogen to be handled in BSL-4 facilities. Multiple surrogate systems such as virus-like particles (VLPs) and pseudoviruses enable carrying out NiV neutralization assays and study of virus entry pathways in biosafety level-2 (BSL-2) facilities. These are dual protein expression and surface display systems comprising NiV structural glycoproteins F and G as the key components. During generation of NiV VLPs or pseudovirions, ensuring batch to batch uniformity is a major concern due to the lack of proportionate incorporation of these proteins in the producer cells as well as their consistent incorporation in the particles. We established HEK293 pseudovirion producer cells that stably co-incorporated NiV F and G proteins to address the issue. Fluorescence-activated cell sorting (FACS) analysis of clonally selected cells for high and uniform level F and G protein co-incorporation; and their further expansion were carried out to further refine the system. High titer vesicular stomatitis virus (VSV)-based pseudoviruses which showed consistent incorporation of both the glycoprotein were reproducibly generated from these producer cells. In functional assays, these Nipah pseudovirions (NiV) exhibited a dose-dependent neutralization by commercial anti-NiV F and G antibodies as well as by convalescent serum from Nipah recovered patients. A pseudovirus neutralization test (PVNT) with a secreted alkaline phosphatase (SEAP) as the reporter was established in the study. The assay supports high-throughput adaptability with a quick turn-around time. It will aid large-scale human and animal serosurveillance studies in Nipah endemic regions as well as screening of virus entry inhibitors and monoclonal antibodies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-025-04375-9.

Sputum cultures are the gold standard for tuberculosis (TB) diagnosis and treatment monitoring. However, cultures in MGIT liquid media are susceptible to microbial contamination, often rendering them uninterpretable. Research has shown that maintaining strict cold chains and supervised sample collection can reduce contamination rates, but few longitudinal studies with weekly sampling have explored this. Here, we evaluated whether (i) the time between specimen collection and laboratory processing and (ii) unsupervised specimen collection are associated with contamination rates. Additionally, we estimated contamination rates over the first 12 weeks of treatment and assessed the clinical and behavioral predictors of contamination. We collected 3,155 sputum specimens from 301 participants undergoing TB treatment. Contamination was lowest (12.3%) at treatment initiation, increased over the first few weeks, and stabilized around 30% from week 8 onwards. Samples collected without supervision were more likely to be contaminated at treatment initiation (P = 0.048) and over 12 weeks (P = 0.028). We observed an inverse relationship between smear grade and contamination risk throughout the sampling period. These findings underscore the importance of supervised sputum collection to reduce contamination and provide ways to enhance the clinical and research values of weekly cultures, particularly those collected later in treatment. This is especially relevant for community-collected specimens used in monitoring treatment response.IMPORTANCEIt is essential to understand how we can minimize sputum specimen contamination rates, as culture contamination may lead to false negative or indeterminate results that require repeat sampling and testing, increasing the burden on healthcare systems and potentially delaying treatment initiation. This research underscores the importance of maintaining a stringent cold chain and highlights the need for participant education and supervision during sample collection. The findings from this study have important implications for TB diagnosis and treatment outcome programs in low- and middle-income countries where biosafety level 3 facilities are located within centralized national reference laboratories or tertiary care hospitals.

Qualification of a reporter virus microneutralization assay for evaluation of influenza specific antibodies in human clinical trials.

Energy efficiency analysis and life cycle assessment of a biosafety level 4 laboratory

Efficacy of a candidate vaccine against Leishmania infantum on naturally exposed dogs to sand flies.

Highly pathogenic for humans orthopoxviruses (OPV) - Variola virus (VARV) and Monkeypox virus (MPXV) can cause systemic diseases characterized by high contagiousness and often leading to death. In previous publications (https://doi.org/10.3390/v14112580), we reported on the development of a sensitive, rapid and easy-to-use immunochemical test potentially suitable for use in the infection foci and in laboratory conditions with a high level of biosecurity. The prototype of the diagnostic kit was tested on non-pathogenic and low-pathogenic for humans OPV and specifically detected them with a sensitivity within the range of 103 to 104 PFU/mL. The aim of this work was to evaluate the sensitivity of this assay in detecting highly pathogenic for humans MPXV and VARV, as well as the applicability of the assay in a laboratory with a high level of biosafety (BSL-4). The efficiency of virus detection in cryolysates of CV-1 cell culture samples infected with VARV and MPXV was assessed using a one-step dot immunoassay method in a laboratory with biosafety level BSL-4. It was shown that the one-step dot assay allows detection of MPXV at a concentration of 2.5 × 103 PFU/mL, and VARV at a concentration of 1.0 × 104 PFU/mL. It was noted that vapors from disinfectants (hydrogen peroxide/formaldehyde) applied in biosafety cabinet decontamination may interfere with assay performance and affect result interpretation. The one-step dot immunoassay can be performed in a BSL-4 laboratory. When limiting the contact of the detecting system with formaldehyde vapors, the sensitivity of the test for detection of VARV and MPXV falls within the previously declared range of 103-104 PFU/mL.

ABSTRACT The New World alphaviruses, including Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV), and Venezuelan Equine Encephalitis Virus (VEEV), are known to cause neurological diseases that pose a significant threat to public health concerns and bioterrorism preparedness challenges due to their potential for aerosol transmission. Currently, no FDA-approved vaccines or antiviral drugs are available for humans, although ongoing studies are exploring potential solutions. Most vaccine evaluation methods rely on live virus models, which require handling in biosafety level 3 (BSL-3) facilities. In this study, we constructed pseudoviruses for NW alphaviruses using the vesicular stomatitis virus (VSV) system by expressing the glycoproteins E3-E2-E1 on the surface of the VSV vector. In vitro cell infection experiments revealed that the pseudovirus titres of EEEV and VEEV were comparatively higher. Bioluminescence imaging in a mouse model was used to assess infection in vivo. When injected into the brain, this was the main site of infection for NW alphavirus-based pseudoviruses. When administered via the tail vein, the pseudovirus primarily infected the spleen, while intraperitoneally injected pseudoviruses mainly infected the intestines and thymus. Furthermore, we systematically evaluated the correlation between neutralizing antibody titres induced by DNA immunization and the protection against homologous virus strains. This study establishes a safe, convenient, and efficient system for evaluating the protective effects of vaccines against NW alphaviruses, which can be operated in a BSL-2 facility.

The highly pathogenic Nipah virus (NiV), a World Health Organization priority pathogen with pandemic potential, remains a critical public health threat due to its capacity to cause fatal encephalitis and respiratory disease. Despite its 1998 emergence, no approved therapeutics exist against NiV infection, underscoring the urgent need to identify genomic regulatory elements as antiviral targets. Our study focuses on the extended 5′ UTRs characteristic of NiV transcripts, a distinctive genomic feature whose functional significance remained enigmatic. Comparative reporter assays showed these UTRs strongly inhibit downstream ORF translation through mechanisms distinct from internal ribosome entry site activity. Mutagenesis studies identified upstream ATG elements in multiple transcripts as critical regulators of translational efficiency, with the C 5′ UTR exhibiting maximal suppression. A functional hotspot spanning within the C 5′ UTR was mapped as the primary ribosomal initiation blockade, and ribosome leaky scanning was confirmed as the mechanism enabling dual-protein expression. Notably, therapeutic targeting of this regulatory element with antisense oligonucleotides significantly impaired viral replication. These findings provide fundamental insights into henipaviral translation regulation while identifying concrete antiviral targets, particularly the druggable C 5′ UTR element, advancing efforts to combat this biosafety level 4 pathogen.

ObjectiveThe aim of this study was to identify and analyze biosafety risk points in biosafety level 3 (BSL-3) laboratory facilities to bring awareness to the attention of administrative staff, reduce the biosafety risks, and improve the risk management.MethodsThe biosafety risk points in BSL-3 facilities were identified by literature searches and field research methods, and the identified biosafety risk points subsequently analyzed using the fault analysis event tree method.Results & conclusionThroughout the comprehensive screening and identification of biosafety risk points in BSL-3 laboratory facilities, risk assessments were performed to rank their seriousness. This will help effectively reduce the biosafety risk level of BSL-3 laboratory facilities.

Nipah virus (NiV), a highly lethal RNA virus from the Paramyxoviridae family, causes severe neurological and respiratory diseases in humans. First identified during the 1990s outbreak in Malaysia, NiV remains a significant global health threat due to the absence of approved vaccines or antiviral treatments. Since its discovery, more than 754 cases have been reported, with a mortality rate exceeding 50%. Despite its classification as a biosafety level 4 pathogen, the molecular mechanisms underlying NiV pathogenesis remain poorly understood. Two surface glycoproteins—the attachment (G) and fusion (F) proteins—play crucial roles in facilitating early stages of cell entry and determining host specificity. While naturally occurring mutations in the G glycoprotein are limited, experimental studies involving engineered mutations have provided critical insights into receptor binding, fusion activation, and immune evasion. This review summarizes current knowledge of these antigenic and mutational findings, highlighting their implications for viral entry and host specificity, and providing valuable insights for the development of vaccines and therapeutics.

BackgroundThe management of biosafety laboratories for pathogenic microorganisms is directly related to public health and the effectiveness of biological experiments. However, persistent non-compliance issues in biosafety level 2 (BSL-2) laboratory management remain a challenge. This study aimed to assess the quality control of BSL-2 laboratories for pathogenic microorganisms in Lishui.MethodsBy combining the Biosafety Online Supervision Systems and on-site inspections, this study assessed the quality control of 73 medical institutions and the 128 biosafety laboratories under their management in Lishui.ResultsThe results discovered that the 73 medical institutions had low compliance rates in several fields: Responsibilities of the biosafety management (80.82%), development of system documentation (65.75%), risk assessment (84.56%), training of laboratory personnel (82.19%), and biosafety labeling (52.05%). Additionally, 128 laboratories had low pass rates for the access control management system (85.94%), hand/eye wash and shower stations (85.94%), and biosafety cabinet operations (89.06%).ConclusionThis study demonstrates that future efforts should focus on strengthening laboratory personnel training and implementing biosafety management responsibilities to ensure safe and regulation-compliant operations in laboratories handling pathogenic microorganisms.