Pathogenicity of genotype 2.1 classical swine fever virus isolated from Japan in 2019 in pigs.

Classical swine fever (CSF) re-emerged in Japan in 2018 for the first time in 26 years. The disease has been known to be caused by a moderately pathogenic virus, rather than the highly pathogenic virus that had occurred in the past. However, the underlying pathophysiology remains unknown. This study conducted an experimental challenge on specific pathogen-free (SPF) pigs in a naïve state for 2, 4, and 6 weeks and confirmed the disease state during each period by clinical observation, virus detection, and pathological necropsy. We revealed the pathological changes and distribution of pathogens and virus-specific antibodies at each period after virus challenge. These results were comprehensively analyzed and approximately 70% of the pigs recovered, especially at 4- and 6-week post-virus challenge. This study provides useful information for future countermeasures against CSF by clarifying the pathogenicity outcomes in unvaccinated pigs with moderately pathogenic genotype 2.1 virus.

Pathogen detection via inductively coupled plasma mass spectrometry analysis with nanoparticles.

Infections caused by viruses and bacteria pose a significant threat to global public health, emphasizing the critical importance of timely and precise detection methods. Inductively coupled plasma mass spectrometry (ICP-MS), a contemporary approach for pathogen detection, offers distinct advantages such as high sensitivity, a wide linear range, and multi-index capabilities. This review elucidates the underexplored application of ICP-MS in conjunction with functional nanoparticles (NPs) for the identification of viruses and bacteria. The review commences with an elucidation of the underlying principles, procedures, target pathogens, and NP requirements for this innovative approach. Subsequently, a thorough analysis of the advantages and limitations associated with these techniques is provided. Furthermore, the review delves into a comprehensive examination of the challenges encountered when utilizing NPs and ICP-MS for pathogen detection, culminating in a forward-looking assessment of the potential pathways for advancement in this domain. Thus, this review contributes novel perspectives to the field of pathogen detection in biomedicine by showcasing the promising synergy of ICP-MS and NPs.

Advancements in SARS-CoV-2 detection: Navigating the molecular landscape and diagnostic technologies.

According to information from the World Health Organization, the world has experienced about 430 million cases of COVID-19, a world-wide health crisis caused by the SARS-CoV-2 virus. This outbreak, originating from China in 2019, has led to nearly 6 million deaths worldwide. As the number of confirmed infections continues to rise, the need for cutting-edge techniques that can detect SARS-CoV-2 infections early and accurately has become more critical. To address this, the Federal Drug Administration (FDA) has issued emergency use authorizations (EUAs) for a wide range of diagnostic tools. These include tests based on detecting nucleic acids and antigen-antibody reactions. The quantitative real-time reverse transcription PCR (qRT-PCR) assay stands out as the gold standard for early virus detection. However, despite its accuracy, qRT-PCR has limitations, such as complex testing protocols and a risk of false negatives, which drive the continuous improvement in nucleic acid and serological testing approaches. The emergence of highly contagious variants of the coronavirus, such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529), has increased the need for tests that can specifically identify these mutations. This article explores both nucleic acid-based and antigen-antibody serological assays, assessing the performance of recently approved FDA tests and those documented in scientific research, especially in identifying new coronavirus strains.

Development of a dual channel detection system for pan-genotypic simultaneous quantification of hepatitis B and delta viruses.

Hepatitis B virus (HBV) infection remains a major public health problem and, in associated co-infection with hepatitis delta virus (HDV), causes the most severe viral hepatitis and accelerated liver disease progression. As a defective satellite RNA virus, HDV can only propagate in the presence of HBV infection, which makes HBV DNA and HDV RNA the standard biomarkers for monitoring the virological response upon antiviral therapy, in co-infected patients. Although assays have been described to quantify these viral nucleic acids in circulation independently, a method for monitoring both viruses simultaneously is not available, thus hampering characterization of their complex dynamic interactions. Here, we describe the development of a dual fluorescence channel detection system for pan-genotypic, simultaneous quantification of HBV DNA and HDV RNA through a one-step quantitative PCR. The sensitivity for both HBV and HDV is about 10 copies per microliter without significant interference between these two detection targets. This assay provides reliable detection for HBV and HDV basic research in vitro and in human liver chimeric mice. Preclinical validation of this system on serum samples from patients on or off antiviral therapy also illustrates a promising application that is rapid and cost-effective in monitoring HBV and HDV viral loads simultaneously.

Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope in situ hybridization signal amplification.

RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.

Nano-PCR for the early detection of tomato leaf curl virus.

Nano-PCR is a potential tool for the early detection of plant viruses. In the current study, different concentrations of silver nanoparticles (20 nm) and magnesium oxide nanoparticles (50 nm) were included in the PCR mixture to improve the sensitivity of PCR for the detection of tomato leaf curl virus. The inclusion of nanoparticles in single or combination in PCR mixture has resulted in improvement of PCR sensitivity. Four-fold improvement was exhibited by the inclusion of 3 ng/µL silver nanoparticles, whereas the combination of silver and magnesium oxide nanoparticles (3 ng/µL and 200 ng/µL, respectively), resulted in a 4.5-fold improvement. The inclusion of 200 ng/µL of magnesium oxide nanoparticles in the PCR mixture exhibited a 7.6-fold increase in PCR sensitivity. Replacement of magnesium chloride with a combination of silver and magnesium oxide nanoparticles (3 ng/µL and 275 ng/µL, respectively) resulted in a 12-fold increase. A 13-fold improvement in PCR sensitivity was observed by the replacement of magnesium chloride in PCR buffer with 275 ng/µL of magnesium oxide nanoparticles. This could also produce detectable amplicon in PCR with a minimum of 25 cycles, resulting in a 26.5% reduction in the duration of PCR. This is the first report on the use of magnesium oxide nanoparticles in PCR for the early detection and better management of tomato leaf curl virus. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03842-2.

Prokaryotic Argonaute Proteins: A New Frontier in Point-of-Care Viral Diagnostics.

The recent pandemic of SARS-CoV-2 has underscored the critical need for rapid and precise viral detection technologies. Point-of-care (POC) technologies, which offer immediate and accurate testing at or near the site of patient care, have become a cornerstone of modern medicine. Prokaryotic Argonaute proteins (pAgo), proficient in recognizing target RNA or DNA with complementary sequences, have emerged as potential game-changers. pAgo present several advantages over the currently popular CRISPR/Cas systems-based POC diagnostics, including the absence of a PAM sequence requirement, the use of shorter nucleic acid molecules as guides, and a smaller protein size. This review provides a comprehensive overview of pAgo protein detection platforms and critically assesses their potential in the field of viral POC diagnostics. The objective is to catalyze further research and innovation in pAgo nucleic acid detection and diagnostics, ultimately facilitating the creation of enhanced diagnostic tools for clinic viral infections in POC settings.

Hepatitis A Virus and Hepatitis E Virus as Food- and Waterborne Pathogens-Transmission Routes and Methods for Detection in Food.

Foodborne viruses are an important threat to food safety and public health. Globally, there are approximately 5 million cases of acute viral hepatitis due to hepatitis A virus (HAV) and hepatitis E virus (HEV) every year. HAV is responsible for numerous food-related viral outbreaks worldwide, while HEV is an emerging pathogen with a global health burden. The reported HEV cases in Europe have increased tenfold in the last 20 years due to its zoonotic transmission through the consumption of infected meat or meat products. HEV is considered the most common cause of acute viral hepatitis worldwide currently. This review focuses on the latest findings on the foodborne transmission routes of HAV and HEV and the methods for their detection in different food matrices.

Designing of rapid assay for the detection of RdRp/Orf1ab specific to SARS-CoV-2.

SARS-CoV-2 is still threat and mostly used detection method is real time reverse transcriptase polymerase chain reaction (rRT-PCR) for the open reading frame (Orf1ab), RNA-dependent RNA polymerase (RdRp), nucleocapsid (N) and envelope (E) genes of virus. However, rRT-PCR may have false negative rate for the nucleic acid detection. Since the RdRp/Orf1ab has high sensitivity for the molecular detection, two sandwich models, Model 1A-Model 1B, based on hybridization on lateral flow assay (LFA) were designed here and applied with the synthetic and clinical samples of RdRp/Orf1ab. In this purpose colloidal gold nanoparticles (AuNPs) were used as label. Membranes having different flow rate, three oligonucleotide probe concentrations and running buffers were used. Although synthetic target sequence was recognized by all the LFAs, PCR products obtained from either the synthetic plasmid DNA or oro/nasopharyngeal swabs were detected by Model 1 A using W12 membrane. Designed strip assays detected the RdRp/Orf1ab of the clinical samples as 100% sensitivity and specifity. It means that they might be used for the detection of virus and can be modified for the recognition of mutant genes of virus. These findings also demonstrated the importance of membranes, sandwich models, probe concentrations and sample contents for developing LFAs for viral detection.

Highly Selective Aptamer-Molecularly Imprinted Polymer Hybrids for Recognition of SARS-CoV-2 Spike Protein Variants.

Virus recognition has been driven to the forefront of molecular recognition research due to the COVID-19 pandemic. Development of highly sensitive recognition elements, both natural and synthetic is critical to facing such a global issue. However, as viruses mutate, it is possible for their recognition to wane through changes in the target substrate, which can lead to detection avoidance and increased false negatives. Likewise, the ability to detect specific variants is of great interest for clinical analysis of all viruses. Here, a hybrid aptamer-molecularly imprinted polymer (aptaMIP), that maintains selective recognition for the spike protein template across various mutations, while improving performance over individual aptamer or MIP components (which themselves demonstrate excellent performance). The aptaMIP exhibits an equilibrium dissociation constant of 1.61 nM toward its template which matches or exceeds published examples of imprinting of the spike protein. The work here demonstrates that "fixing" the aptamer within a polymeric scaffold increases its capability to selectivity recognize its original target and points toward a methodology that will allow variant selective molecular recognition with exceptional affinity.

Rational Design of Profile HMMs for Sensitive and Specific Sequence Detection with Case Studies Applied to Viruses, Bacteriophages, and Casposons.

Profile hidden Markov models (HMMs) are a powerful way of modeling biological sequence diversity and constitute a very sensitive approach to detecting divergent sequences. Here, we report the development of protocols for the rational design of profile HMMs. These methods were implemented on TABAJARA, a program that can be used to either detect all biological sequences of a group or discriminate specific groups of sequences. By calculating position-specific information scores along a multiple sequence alignment, TABAJARA automatically identifies the most informative sequence motifs and uses them to construct profile HMMs. As a proof-of-principle, we applied TABAJARA to generate profile HMMs for the detection and classification of two viral groups presenting different evolutionary rates: bacteriophages of the Microviridae family and viruses of the Flavivirus genus. We obtained conserved models for the generic detection of any Microviridae or Flavivirus sequence, and profile HMMs that can specifically discriminate Microviridae subfamilies or Flavivirus species. In another application, we constructed Cas1 endonuclease-derived profile HMMs that can discriminate CRISPRs and casposons, two evolutionarily related transposable elements. We believe that the protocols described here, and implemented on TABAJARA, constitute a generic toolbox for generating profile HMMs for the highly sensitive and specific detection of sequence classes.

Viral Metagenomics as a Tool to Track Sources of Fecal Contamination: A One Health Approach.

The One Health framework recognizes that human, animal, and environmental health are linked and highly interdependent. Fecal contamination of water, soil, foodstuff, and air may impact many aspects of One Health, and culture, PCR-based, and sequencing methods are utilized in the detection of fecal contamination to determine source, load, and risk to inform targeted mitigation strategies. Viruses, particularly, have been considered as fecal contamination indicators given the narrow host range many exhibit and their association with other biological contaminants. Culture- and molecular-based methods are considered the gold-standards for virus detection and for determining specific sources of fecal contamination via viral indicators. However, viral metagenomics is also being considered as a tool for tracking sources of fecal contamination. In the present review, studies tracking potential sources of fecal contamination in freshwaters, marine waters, foodstuff, soil, and air using viral metagenomics are discussed to highlight the potential of viral metagenomics for optimizing fecal source tracking. Limitations of the use of viral metagenomics to track fecal contamination sources, including sample processing, nucleic acid recovery, sequencing depth, and bioinformatics are also discussed. Finally, the present review discusses the potential of viral metagenomics as part of the toolbox of methods in a One Health approach.

Monkeypox viral detection in semen specimens of confirmed cases: A systematic review and meta-analysis.

The current literature shows increasing concerns about potential seminal transmission of monkeypox virus (MPXV). Accordingly, we aimed to understand better the potential presence of MPXV in the seminal fluids and others specimens obtained from MPX cases. On June 26, 2022, a systematic search of the literature was conducted to find articles that examine the presence of MPXV in the seminal fluid of confirmed cases. The search was updated once on August 12 and another on October 12, 2022, to include newly published articles. The prevalence of MPXV DNA presence in the seminal fluid and other specimens was pooled in a meta-analysis (from studies with sample size > 5 to reduce overestimation) and results were presented as effect sizes (ES) and their corresponding 95% confidence intervals (CI). Nine articles were included. Only five studies were eligible for a meta-analysis, and the pooled prevalence of MPXV DNA in semen specimens was 72.4% (95% CI: 55.7%-84.5%) among 115 patients. The positive rate of MPXV viral polymerase chain reaction (PCR) was higher among skin samples (89%; 95% CI: 78.2%-94.8%; N = 62; studies = 2), followed by anogenital/rectal samples (74.3%; 95% CI: 60.4%-84.5%; N = 54; studies = 2). On the other hand, the positivity rate was lower in nasopharyngeal (62.4%; 95% CI: 20.4%-91.5%; N = 587; studies = 3), urine (21.1%; 95% CI: 4.3%-61.1%; N = 617; studies = 4), and blood/plasma (14.3%; 95% CI: 11.3%-18.1%; N = 609; studies = 3) samples. Besides, MPXV can be detected in semen early from Day 1 and up to 19 days after symptoms onset. Finally, two articles investigated the infectivity of MPXV particles detected in seminal specimens by testing their replication competence. Culturing MPXV was successful in two out of four patients included in these studies. MPXV is highly prevalent in seminal specimens of MPX cases, further corroborating the role of sexual transmission of the disease. However, further evidence is still needed to shed more light on the replication competence of these particles.

Rapid, Sensitive and Simultaneous Detection of Two Wheat RNA Viruses Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA).

In China, wheat yellow mosaic disease is mostly caused by wheat yellow mosaic virus (WYMV) and Chinese wheat mosaic virus (CWMV). If wheat is co-infected with these two viruses, it can cause severe yellow mosaic symptoms and yield losses. Early detection of viruses is crucial for preventing disease in the field. In this study, we optimized a sensitive, specific reverse transcription recombinase polymerase amplification (RT-RPA) detection method for two viruses, WYMV and CWMV. Two sets of primers were designed based on the capsid protein (CP)-encoding genes of the two viruses, and the reaction conditions were determined. The RT-RPA method, which amplified the target amplicon by a handheld reaction mixture for 20 min, was more sensitive than PCR-CP in the detection of WYMV. Finally, the RT-RPA method was performed on 110 randomly selected field samples, demonstrating its applicability to samples from different regions and specificity for co-infected samples. This study not only describes an improved method for detecting WYMV and CWMV using RT-RPA but also demonstrates the potential of this method, which could be applied under field conditions.

Freestanding Laser-Induced Graphene Ultrasensitive Resonative Viral Sensors.

Early and reliable detection of an infectious viral disease is critical to accurately monitor outbreaks and to provide individuals and health care professionals the opportunity to treat patients at the early stages of a disease. The accuracy of such information is essential to define appropriate actions to protect the population and to reduce the likelihood of a possible pandemic. Here, we show the fabrication of freestanding laser-induced graphene (FLIG) flakes that are highly sensitive sensors for high-fidelity viral detection. As a case study, we show the detection of SARS-CoV-2 spike proteins. FLIG flakes are nonembedded porous graphene foams ca. 30 µm thick that are generated using laser irradiation of polyimide and can be fabricated in seconds at a low cost. Larger pieces of FLIG were cut forming a cantilever, used as suspended resonators, and characterized for their electromechanics behavior. Thermomechanical analysis showed FLIG stiffness comparable to other porous materials such as boron nitride foam, and electrostatic excitation showed amplification of the vibrations at frequencies in the range of several kilo-hertz. We developed a protocol for aqueous biological sensing by characterizing the wetting dynamic response of the sensor in buffer solution and in water, and devices functionalized with COVID-19 antibodies specifically detected SARS-CoV-2 spike protein binding, while not detecting other viruses such as MS2. The FLIG sensors showed a clear mass-dependent frequency response shift of ∼1 Hz/pg, and low nanomolar concentrations could be detected. Ultimately, the sensors demonstrated an outstanding limit of detection of 2.63 pg, which is equivalent to as few as ∼5000 SARS-CoV-2 viruses. Thus, the FLIG platform technology can be utilized to develop portable and highly accurate sensors, including biological applications where the fast and reliable protein or infectious particle detection is critical.

Is SARS-CoV-2 a Concern for Food Safety? A Very Low Prevalence from a Food Survey during the COVID-19 Pandemic in Northern Italy.

In 2019, SARS-CoV-2 was identified as the cause of an easily transmissible disease that was declared as a world pandemic. Foodborne transmission was never reported. However, early studies suggested that food could be involved in SARS-CoV-2 entry in the human gastrointestinal tract leading to possible infection, and highlighting the importance of further studies to inspect possible issues linked to food consumption. In this perspective, this work aimed at monitoring SARS-CoV-2 presence in some food and mains water samples in Northern Italy during the COVID-19 pandemic (2020-2022). A total of 1806 foods, 112 mains water samples, and 580 swabs on meat and dairy product surfaces were analyzed for SARS-CoV-2 RNA detection by Real-time PCR. All the analyzed samples were negative to viral RNA detection with the exception of one vegetable sample. Even if data on foodborne coronavirus transmission suggested a limited importance of this pathway, the impact of the current pandemic in Northern Italy deserved a rigorous investigation to rule out such possibility. Indeed, gaining insight on all SARS-CoV-2 possible transmission pathways, including the foodborne route, seemed of interest to maintain consumers' confidence and trust in food safety, and for the effective management of the current, and future, possible pandemics.

Recent advances of the biological and biomedical applications of CRISPR/Cas systems.

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas) system, referred to as CRISPR/Cas system, has attracted significant interest in scientific community due to its great potential in translating into versatile therapeutic tools in biomedical field. For instance, a myriad of studies has demonstrated that the CRISPR/Cas system is capable of detecting various types of viruses, killing antibiotic-resistant bacteria, treating inherited genetic diseases, and providing new strategies for cancer therapy. Furthermore, CRISPR/Cas systems are also exploited as research tools such as genome engineering tool that allows researchers to interrogate the biological roles of unexplored genes or uncover novel functions of known genes. Additionally, the CRISPR/Cas system has been employed to edit the genome of a wide range of eukaryotic, prokaryotic organisms and experimental models, including but not limited to mammalian cells, mice, zebrafish, plants, yeast, and Escherichia coli. The present review mainly focuses on summarizing recent discoveries regarding the type II CRISPR/Cas9 and type VI CRISPR/Cas13a systems to give researchers a glimpse of their potential applications in the biological and biomedical field.

Recent Advances in Plasma-Engineered Polymers for Biomarker-Based Viral Detection and Highly Multiplexed Analysis.

Infectious diseases remain a pervasive threat to global and public health, especially in many countries and rural urban areas. The main causes of such severe diseases are the lack of appropriate analytical methods and subsequent treatment strategies due to limited access to centralized and equipped medical centers for detection. Rapid and accurate diagnosis in biomedicine and healthcare is essential for the effective treatment of pathogenic viruses as well as early detection. Plasma-engineered polymers are used worldwide for viral infections in conjunction with molecular detection of biomarkers. Plasma-engineered polymers for biomarker-based viral detection are generally inexpensive and offer great potential. For biomarker-based virus detection, plasma-based polymers appear to be potential biological probes and have been used directly with physiological components to perform highly multiplexed analyses simultaneously. The simultaneous measurement of multiple clinical parameters from the same sample volume is possible using highly multiplexed analysis to detect human viral infections, thereby reducing the time and cost required to collect each data point. This article reviews recent studies on the efficacy of plasma-engineered polymers as a detection method against human pandemic viruses. In this review study, we examine polymer biomarkers, plasma-engineered polymers, highly multiplexed analyses for viral infections, and recent applications of polymer-based biomarkers for virus detection. Finally, we provide an outlook on recent advances in the field of plasma-engineered polymers for biomarker-based virus detection and highly multiplexed analysis.

SARS-CoV-2 RNA detection in Formalin-Fixed Paraffin-Embedded (FFPE) tissue by droplet digital PCR (ddPCR).

BACKGROUND: SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. MATERIALS AND METHODS: A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. RESULTS: SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. CONCLUSION: This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.

A Novel Method for Training the Interdiction of Restricted and Hazardous Biological Materials by Detection Dogs.

The interdiction of restricted and hazardous biological agents presents challenges for any detection method due to the inherent complexity of sample type and accessibility. Detection capabilities for this category of agents are limited and restricted in their mobility, adaptability and efficiency. The potential for identifying biological agents through a volatile organic compound (VOC) signature presents an opportunity to use detection dogs in a real-time mobile capacity for surveillance and screening strategies. However, the safe handling and access to the materials needed for training detection dogs on restricted or hazardous biological agents prevents its broader application in this field. This study evaluated the use of a polymer-based training aid in a viral detection model using bovine viral diarrhea virus mimicking biosafety level 3+ agent conditions. After the biological agent-based odor was absorbed into the polymer, the aid was rendered safe for handling through a rigorous sterilization process. The viral culture-based training aid was then used to train a cohort of detection dogs (n = 6) to discriminate agent-based target odor in culture from relevant distractor odors including non-target biological agent-based odors. Following culture-based training, dogs were tested for generalization to aids with infected animal sample-based odors across five sample types (fecal, blood, nasal, saliva, and urine). Within the context of the polymer-based training aid system, dogs were successfully trained to detect and discriminate a representative biological viral agent-based odor from distractor odors with a 97.22% (±2.78) sensitivity and 97.11% (±1.94) specificity. Generalization from the agent-based odor to sample-based odors ranged from 65.40% (±8.98) to 91.90 % (±6.15) sensitivity and 88.61% (±1.46) to 96.00% (±0.89) specificity across the sample types. The restrictive nature for mimicking the access and handling of a BSL 3+ agent presented challenges that required a strict study design uncommon to standard detection dog training and odor presentation. This study demonstrates the need to further evaluate the utility and challenges of training detection dogs to alert to biological samples using safe and manageable training aids.