Biosafety Level Research Articles

Identification of circular RNAs expression pattern in caprine fetal fibroblast cells exposed to a chronic non-cytotoxic dose of graphene oxide-silver nanoparticle nanocomposites.

The widespread use of graphene oxide-silver nanoparticle nanocomposites (GO-AgNPs) in biomedical sciences is increasing the chances of human and animal exposure to its chronic non-toxic doses. Exposure to AgNPs-related nanomaterials may result in the negative effect on the dam, fetus and offspring. However, there are only little available information for profound understanding of the epigenetic alteration in the cells and animals caused by low-dose chronic exposure of GO-AgNPs. The present study investigated the effect of 0.5μg/mL GO-AgNPs for 10weeks on the differential expression of circular RNAs (circRNAs) in caprine fetal fibroblast cells (CFFCs), and this dose of GO-AgNPs did not affect cell viability and ROS level. We predicted the functions of those differentially expressed (DE) circRNAs in CFFCs by bioinformatics analysis. Furthermore, we validated the expression of ten DE circRNAs using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) to ensure the reliability of the sequencing data. Our results showed that the DE circRNAs may potentially regulate the GO-AgNPs-inducing epigenetic toxicity through a regulatory network consisted of circRNAs, miRNAs and messenger RNAs (mRNAs). Therefore, the epigenetics toxicity is essential to assess the biosafety level of GO-AgNPs.

Vaccine Candidates against Arenavirus Infections.

The viral family Arenaviridae contains several members that cause severe, and often lethal, diseases in humans. Several highly pathogenic arenaviruses are classified as Risk Group 4 agents and must be handled in the highest biological containment facility, biosafety level-4 (BSL-4). Vaccines and treatments are very limited for these pathogens. The development of vaccines is crucial for the establishment of countermeasures against highly pathogenic arenavirus infections. While several vaccine candidates have been investigated, there are currently no approved vaccines for arenavirus infection except for Candid#1, a live-attenuated Junin virus vaccine only licensed in Argentina. Current platforms under investigation for use include live-attenuated vaccines, recombinant virus-based vaccines, and recombinant proteins. We summarize here the recent updates of vaccine candidates against arenavirus infections.

Characterisation of the Antibody Response in Sinopharm (BBIBP-CorV) Recipients and COVID-19 Convalescent Sera from the Republic of Moldova.

The early availability of effective vaccines against SARS-CoV-2, the aetiologic cause of COVID-19, has been at the cornerstone of the global recovery from the pandemic. This study aimed to assess the antispike RBD IgG antibody titres and neutralisation potential of COVID-19 convalescent plasma and the sera of Moldovan adults vaccinated with the Sinopharm BBIBP-CorV vaccine. An IgG ELISA with recombinant SARS-CoV-2 spike RBD and two pseudovirus-based neutralisation assays have been developed to evaluate neutralising antibodies against SARS-CoV-2 in biosafety level 2 containment facilities. A significant moderate correlation was observed between IgG titres and the overall neutralising levels for each neutralisation assay (ρ = 0.64, p < 0.001; ρ = 0.52, p < 0.001). A separate analysis of convalescent and vaccinated individuals showed a higher correlation of neutralising and IgG titres in convalescent individuals (ρ = 0.68, p < 0.001, ρ = 0.45, p < 0.001) compared with vaccinated individuals (ρ = 0.58, p < 0.001; ρ = 0.53, p < 0.001). It can be concluded that individuals who recovered from infection developed higher levels of antispike RBD IgG antibodies. In comparison, the Sinopharm-vaccinated individuals produced higher levels of neutralising antibodies than convalescent plasma.

Development of a sensitive, high-throughput extraction protocol for qPCR detection of African swine fever virus in formalin-fixed, paraffin-embedded tissues.

African swine fever (ASF) causes fatal disease in pigs and is an escalating threat to the global swine industry. ASF has re-emerged from Africa as a transcontinental epidemic spreading through the Caucasus into Europe, Russia, China, numerous Asian countries, and the Caribbean. ASF virus (ASFV) is a U.S. select agent requiring handling in high-containment biosafety level 3 (BSL-3) laboratories for pathogen work. Formalin-fixation eliminates infectivity and preserves the genome, providing noninfectious specimens for BSL-2 work. Recovery of DNA from formalin-fixed, paraffin-embedded tissue (FFPET) is challenging and cumbersome. A reliable and easy-to-perform method for DNA recovery from FFPET would facilitate surveillance. To meet this objective, we developed a high-throughput protocol for the recovery of ASFV DNA from FFPET. Deparaffinization, tissue lysis, and reversal of cross-linking were performed in a single tube, followed by DNA purification via automated magnetic bead extraction. Quantitative PCR (qPCR) detection was used to determine the copy number of the B646L gene that encodes for the ASFV p72 protein in tissues (5 pigs, 4 tissues) from pigs with lesions consistent with acute ASF. Copy numbers obtained from FFPET were within one log of copy numbers obtained from fresh tissue, thus enabling ASF qPCR surveillance from formalin-inactivated and preserved tissues at BSL-2 at diagnostic sensitivity similar to fresh tissues tested at BSL-3.

Quantitative 16S rRNA Gene Amplicon Sequencing for Comprehensive Pathogenic Bacterial Tracking in a Municipal Wastewater Treatment Plant

Quantification of bacterial pathogens in the environment is crucial for determining their potential risk of pathogenicity and infection. Here, we applied quantitative sequencing (qSeq) based on the 16S rRNA gene with spike-in internal standards for comprehensive pathogen quantification in a municipal wastewater treatment plant (WWTP). A novel bacterial pathogen database based on biosafety levels (BSLs) was constructed for rapid pathogen identification. Pathogen taxonomies were obtained from the constructed database using a basic local alignment search tool, and amounts of pathogens were quantified by regression analysis of spike-in internal standards. The total pathogen concentrations were gradually decreased from 6.8 ± 4.9 × 107 copies/mL in the influent to 1.6 ± 1.4 × 105 copies/mL in the effluent. BSL2 pathogens (e.g., Aeromonas spp., Mycobacterium spp., Klebsiella spp., and pathogenic Escherichia spp.) were detected more than 4.0 × 103 copies/mL at the effluent. The genus Arcobacter (BSL1) showed the highest abundance throughout the WWTP (8.8 ± 8.1 × 104 to 2.5 ± 3.5 × 107 copies/mL). BSL2 Mycobacterium spp. noticeably increased at the anaerobic treatment (2.3 × 106 copies/mL) and showed least removal at the effluent. The qSeq analysis with the pathogen database provided a holistic view of the microbial pathogen dynamics and ecology in the WWTP.

Study on bioaerosol diffusion and deposition in a mobile BSL-4 laboratory based on air age analysis

Biosafety issues have aroused global concern, especially after the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron strain of corona virus disease 2019 (COVID-19) caused incalculable human and property losses. Laboratory-acquired infections (LAIs) caused by improper operations or accidents are frequently reported. Research is urgently needed for a mobile biosafety level-4 (BSL-4) laboratory with a high risk for exposure. Deposition characteristics and the spatial distribution of bioaerosols under two typical cases were studied in this paper. Based on the age of air and simulation of airflow pattern, a detailed analysis of infection risk and the distribution of bioaerosols was conducted. The deposition characteristics of particles on different surfaces were analyzed based on particle tracking technology. The results showed that the removal rate of bioaerosols was lower in the space area of the laboratory from 1.6 m above the ground. The distribution of high-risk areas is affected by the coupling of equipment layout and pollution sources, mainly located downstream of the main airflow in the laboratory, and the particle concentration was eight times that of the low-risk areas. More than half of bioaerosol particles are deposited on laboratory equipment and walls. The number of particles deposited on the wall was the largest, accounting for 25.02% of the total. The unit area deposition ratio of the experimental table was the highest, which was 6.14 %/m2. The main deposition area of each surface was determined, which could be of guiding significance to the determination of the key disinfection location of the mobile BSL-4 laboratory.

Are we ready to fight the Nipah virus pandemic? An overview of drug targets, current medications, and potential leads.

Nipah virus (NiV) is a high-lethality RNA virus from the family of Paramyxoviridae and genus Henipavirus, classified under Biosafety Level-4 (BSL-4) pathogen due to the severity of pathogenicity and lack of medications and vaccines. Direct contacts or the body fluids of infected animals are the major factor of transmission of NiV. As it is not an airborne infection, the transmission rate is relatively low. Still, mutations of the NiV in the animal reservoir over the years, followed by zoonotic transfer, can make the deadliness of the virus manifold in upcoming years. Therefore, there is no denial of the possibility of a pandemic after COVID-19 considering the severe pathogenicity of NiV, and that is why we need to be prepared with possible drugs in upcoming days. Considering the time constraints, computational aided drug design (CADD) is an efficient way to study the virus and perform the drug design and test the HITs to lead experimentally. Therefore, this review focuses primarily on NiV target proteins (covering NiV and human), experimentally tested repurposed drug details, and latest computational studies on potential lead molecules, which can be explored as potential drug candidates. Computationally identified drug candidates, including their chemical structures, docking scores, amino acid level interaction with corresponding protein, and the platform used for the studies, are thoroughly discussed. The review will offer a one-stop study to access what had been performed and what can be performed in the CADD of NiV.

Nipah Virus Bangladesh Infection Elicits Organ-Specific Innate and Inflammatory Responses in the Marmoset Model.

The common marmoset (Callithrix jacchus) is increasingly recognized as an ideal nonhuman primate (NHP) at high biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets at biosafety level 4. Infection via the intranasal and intratracheal route resulted in fatal disease in all 4 infected marmosets. Three developed pulmonary edema and hemorrhage as well as multifocal hemorrhagic lymphadenopathy, while 1 recapitulated neurologic clinical manifestations and cardiomyopathy on gross pathology. Organ-specific innate and inflammatory responses were characterized by RNA sequencing in 6 different tissues from infected and control marmosets. Notably, a unique transcriptome was revealed in the brainstem of the marmoset exhibiting neurological signs. Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.

The Stability Study of Electrochemical Aptasensor to Detect SARS-CoV-2 Spike Protein and Its Application for Clinical Samples of Nasopharyngeal Swab

The stability characteristics associated with the shelf life of a biosensor are rarely investigated, however, they are important factors for real applications. Stability is the variation in the detection signal over a long period of storage. This study aims to determine the effect of storage time on the stability of SARS-CoV-2 receptor binding domain (RBD) spike protein aptamers related to shelf life and the performance of an electrochemical aptasensor on clinical samples. The research method includes a stability study conducted using the accelerated stability method based on the Arrhenius equation at three variations of temperature and storage time. The electrochemical aptasensor's performance was evaluated on clinical samples of 32 nasopharyngeal swabs at biosafety level 3 and its potential on clinical saliva samples. The results indicated that the developed electrochemical aptasensor was stable for ± 15 days with a shelf life of 18, 17 and 16 days, respectively, at 25, 40 and 50 °C. This electrochemical aptasensor has the potential to be a Point of Care (POC) device for the clinical detection of SARS-CoV-2 because it can be tested on clinical samples of nasopharyngeal swabs and the results show its potential application to detect in clinical saliva samples.

Scrub Typhus In A Tertiary Care Hospital In Chengalpattu District

Introduction: Orientia tsutsugamushi is the bacterium that causes scrub typhus. By being bitten by a larval trombiculid mite(chigger), it is transmitted. The incubation period is 10-14 days. Fever, headache, body aches, and sometimes rashes are the most common symptoms. The hallmark is the characteristic eschar lesion-scab at the site of chigger bite and regional lymphadenopathy. The severity of the illness varies from person to person ranging from fever to multi-organ involvement. Scrub typhus is diagnosed in the laboratory using serological tests such as ELISA IgM and Immunofluorescence IgM and molecular test -real-time PCR. Though the organism can be isolated using egg and cell culture is confined to the referral or research laboratories having biosafety level 3 facilities. The disease is managed with doxycycline or chloramphenicol and the organism does not respond to penicillins. Aim: To eloborate a case report of scrub typhus in a 41-year-old female patient in our tertiary care hospital. Materials and Methods: laboratory work up of a single patient with clinical suspicion of scrub typhus was carried out using TruNat,Molbio for detection of Orientia tsutsugamushi. All other differential diagnosis of fever including Dengue, were also evaluated to confirm the diagnosis Conclusion: Considering scrub typhus as differential diagnosis of fever is suggested.

Development and characterization of a chronic hepatitis B murine model with a mutation in the START codon of an HBV polymerase.

Chronic hepatitis B (CHB) is caused by the Hepatitis B virus (HBV) and affects millions of people worldwide. Developing an effective CHB therapy requires using in vivo screening methods, such as mouse models reflecting CHB based on hydrodynamic delivery of plasmid vectors containing a replication-competent HBV genome. However, long-term expression of HBV proteins is accompanied by production of progeny virions, thereby requiring a Biosafety Level (BSL) 3 animal facility. In the present study, we introduced a point mutation in the START codon of the HBV polymerase to develop a mouse model reflecting chronic hepatitis B infection without formation of viral progeny. We induced the mouse model by hydrodynamic injection of adeno-associated virus plasmid vector (pAAV) and minicircle plasmid (pMC) constructs into C57Bl/6 and C3H/HeN mouse strains, monitoring HBV antigens and antibodies in blood by enzyme-linked immunosorbent assay and analyzing liver expression of HBV core antigen by immunohistology. Persisting expression of viral antigens over 140 days (study endpoint) was observed only in the C3H/HeN mouse strain when using pAAV/1.2HBV-A and pMC/1.0HBV-D with pre-C and pre-S recombination sites. In addition, pAAV/1.2HBV-A in C3H/HeN sustained HBV core antigen positivity up to the study endpoint in C3H/HeN mice. Moreover, introducing the point mutation in the START codon of polymerase effectively prevented the formation of viral progeny. Our study establishes an accessible and affordable experimental paradigm for developing a robust mouse model reflecting CHB suitable for preclinical testing of anti-HBV therapeutics in a BSL2 animal facility.

Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells.

Coronavirus disease (COVID-19) is an infectious disease caused by the SARS coronavirus 2 (SARS-CoV-2) virus. Direct assessment, detection, and quantitative analysis using high throughput methods like single-cell RNA sequencing (scRNAseq) is imperative to understanding the host response to SARS-CoV-2. One barrier to studying SARS-CoV-2 in the laboratory setting is the requirement to process virus-infected cell cultures, and potentially infectious materials derived therefrom, under Biosafety Level 3 (BSL-3) containment. However, there are only 190 BSL3 laboratory facilities registered with the U.S. Federal Select Agent Program, as of 2020, and only a subset of these are outfitted with the equipment needed to perform high-throughput molecular assays. Here, we describe a method for preparing non-hazardous RNA samples from SARS-CoV-2 infected cells, that enables scRNAseq analyses to be conducted safely in a BSL2 facility-thereby making molecular assays of SARS-CoV-2 cells accessible to a much larger community of researchers. Briefly, we infected African green monkey kidney epithelial cells (Vero-E6) with SARS-CoV-2 for 96 hours, trypsin-dissociated the cells, and inactivated them with methanol-acetone in a single-cell suspension. Fixed cells were tested for the presence of infectious SARS-CoV-2 virions using the Tissue Culture Infectious Dose Assay (TCID50), and also tested for viability using flow cytometry. We then tested the dissociation and methanol-acetone inactivation method on primary human lung epithelial cells that had been differentiated on an air-liquid interface. Finally, we performed scRNAseq quality control analysis on the resulting cell populations to evaluate the effects of our virus inactivation and sample preparation protocol on the quality of the cDNA produced. We found that methanol-acetone inactivated SARS-CoV-2, fixed the lung epithelial cells, and could be used to obtain noninfectious, high-quality cDNA libraries. This methodology makes investigating SARS-CoV-2, and related high-containment RNA viruses at a single-cell level more accessible to an expanded community of researchers.

Method for the Rapid Detection of SARS-CoV-2-Neutralizing Antibodies Using a Nanogel-Based Surface Plasmon Resonance Biosensor.

The efficacy of coronavirus disease 2019 (COVID-19) vaccination is closely related to the serum levels of SARS-CoV-2-neutralizing antibodies (NAb) that bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Therefore, the rapid and quantitative measurement of SARS-CoV-2 NAb in the sera of vaccinated individuals is essential to develop an effective vaccine and further achieve population immunity, that is, herd immunity. The plaque reduction neutralization test, the gold standard for NAb effectiveness in serological tests, is accurate but requires biosafety level 3 facilities because of the use of the virus, which hampers its application in common laboratories and clinical practice. Here, we developed a bioresponsive nanogel-based surface plasmon resonance (nSPR) platform that detects SARS-CoV-2 NAb in clinical samples without complicated pretreatment. We found that multivalent protein binding (MPB) between the nanogel-conjugated RBD protein and SARS-CoV-2 NAb yields significantly enhanced SPR signals compared to the nonspecific interference from serum proteins in the nSPR assay. The excellence of our nanogel-based SARS-CoV-2 NAb test is due to its selectivity for NAb, with resistance to all other proteins, allowing the rapid detection and quantification of NAbs in each individual. Importantly, this nSPR assay provides a NAb detection platform for easier and safer COVID-19 vaccination strategies.

Human Coronavirus OC43 as a Low-Risk Model to Study COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has had irreversible and devastating impacts on every aspect of human life. To better prepare for the next similar pandemic, a clear understanding of coronavirus biology is a prerequisite. Nevertheless, the high-risk nature of the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), requires the use of a cumbersome biosafety level-3 (BSL-3) confinement facility. To facilitate the development of preventive and therapeutic measures against SARS-CoV-2, one of the endemic strains of low-risk coronaviruses has gained attention as a useful research alternative: human coronavirus OC43 (HCoV-OC43). In this review, its history, classification, and clinical manifestations are first summarized. The characteristics of its viral genomes, genes, and evolution process are then further explained. In addition, the host factors necessary to support the life cycle of HCoV-OC43 and the innate, as well as adaptive, immunological responses to HCoV-OC43 infection are discussed. Finally, the development of in vitro and in vivo systems to study HCoV-OC43 and its application to the discovery of potential antivirals for COVID-19 by using HCoV-OC43 models are also presented. This review should serve as a concise guide for those who wish to use HCoV-OC43 to study coronaviruses in a low-risk research setting.

Research and development of prevention and control measures on the transmission of pathogens in compartments of passenger transport

Abstract Contagious pathogens like COVID-19 transmitted via respiratory droplets spread effortlessly in the passenger compartments of transport, significantly jeopardizing passengers’ safety when taking public transportation. To date, studies on the fundamental theories of airborne droplet transmission and the engineering application of decontamination techniques are insufficient for the prevention and control of pathogens transmitting in the compartments of passenger transport. It is essential to systematically investigate the control approaches to restrain pathogens from transmitting in passenger compartments. Herein, a theoretical framework for calculating the transmission of pathogens in a complex compartment environment was proposed, and experimental platforms that satisfy the Biosafety Level-2 Laboratory safety level for compartment environment simulations were built based on a set of real train cabins. On these bases, numerical investigations on the motion of pathogen-laden droplets were conducted, and decontamination techniques were examined experimentally. Thereby, control measures on the pathogen transmission and pathogen decontamination schemes were proposed. Moreover, highly efficient decontamination devices were developed, and coping strategies for epidemic emergencies were devised. The outcomes provide theoretical and technical support for developing the next generation of transportation and the prevention and control measures cooperatively considering regular and pandemic times.

Evaluation of the interaction between potent small molecules against the Nipah virus Glycoprotein in Malaysia and Bangladesh strains, accompanied by the human Ephrin-B2 and Ephrin-B3 receptors; a simulation approach.

Malaysia reported the first human case of Nipah virus (NiV) in late September 1998 with encephalitis and respiratory symptoms. As a result of viral genomic mutations, two main strains (NiV-Malaysia and NiV-Bangladesh) have spread around the world. There are no licensed molecular therapeutics available for this biosafety level 4 pathogen. NiV attachment glycoprotein plays a critical role in viral transmission through its human receptors (Ephrin-B2 and Ephrin-B3), so identifying small molecules that can be repurposed to inhibit them is crucial to developing anti-NiV drugs. Consequently, in this study annealing simulations, pharmacophore modeling, molecular docking, and molecular dynamics were used to evaluate seven potential drugs (Pemirolast, Nitrofurantoin, Isoniazid Pyruvate, Eriodictyol, Cepharanthine, Ergoloid, and Hypericin) against NiV-G, Ephrin-B2, and Ephrin-B3 receptors. Based on the annealing analysis, Pemirolast for efnb2 protein and Isoniazid Pyruvate for efnb3 receptor were repurposed as the most promising small molecule candidates. Furthermore, Hypericin and Cepharanthine, with notable interaction values, are the top Glycoprotein inhibitors in Malaysia and Bangladesh strains, respectively. In addition, docking calculations revealed that their binding affinity scores are related to efnb2-pem (- 7.1kcal/mol), efnb3-iso (- 5.8kcal/mol), gm-hyp (- 9.6kcal/mol), gb-ceph (- 9.2kcal/mol). Finally, our computational research minimizes the time-consuming aspects and provides options for dealing with any new variants of Nipah virus that might emerge in the future.

Microbead-based extracorporeal immuno-affinity virus capture: a feasibility study to address the SARS-CoV-2 pandemic

In this paper, we report on the utilization of micro-technology based tools to fight viral infections. Inspired by various hemoperfusion and immune-affinity capture systems, a blood virus depletion device has been developed that offers highly efficient capture and removal of the targeted virus from the circulation, thus decreasing virus load. Single-domain antibodies against the Wuhan (VHH-72) virus strain produced by recombinant DNA technology were immobilized on the surface of glass micro-beads, which were then utilized as stationary phase. For feasibility testing, the virus suspension was flown through the prototype immune-affinity device that captured the viruses and the filtered media left the column. The feasibility test of the proposed technology was performed in a Biosafety Level 4 classified laboratory using the Wuhan SARS-CoV-2 strain. The laboratory scale device actually captured 120,000 virus particles from the culture media circulation proving the feasibility of the suggested technology. This performance has an estimated capture ability of 15 million virus particles by using the therapeutic size column design, representing three times over-engineering with the assumption of 5 million genomic virus copies in an average viremic patient. Our results suggested that this new therapeutic virus capture device could significantly lower virus load thus preventing the development of more severe COVID-19 cases and consequently reducing mortality rate.Graphical

Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers.

Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results.

On-Site Remote Monitoring System with NIR Signal-Based Detection of Infectious Disease Virus in Opaque Salivary Samples.

Infectious disease viruses, such as foot-and-mouth disease virus (FMDV), are highly contagious viruses that cause significant socioeconomic damage upon spreading. Developing an on-site diagnostic tool for early clinical detection and real-time surveillance of FMDV outbreaks is essential to prevent the further spread of the disease. However, early diagnosis of FMDV is still challenging due to the limited sensitivity and time-consuming manual result entry of commercial on-site tests for salivary samples. Here, we report a near-infrared (NIR) signal nanoprobe-based highly accurate detection and remote monitoring system toward FMDVs, which automates the analysis and reporting of diagnosis data. The NIR signal lateral flow immunoassay (LFA) was assembled with a nanoprobe with a stable emission intensity at 800 nm, minimizing the interference signal of opaque salivary samples. We investigated the clinical applicability of the NIR signal LFA at biosafety level 3 (BSL-3) laboratories using 147 opaque salivary samples. The NIR signal LFA achieved a 32-fold lower limit of detection (LOD) than a commercial LFA in detecting live FMDVs, including all isolates occurring in the Republic of Korea during 2010-2017. Our results showed that the NIR signal LFA successfully discriminated the FMDV-positive clinical salivary samples from healthy controls with a sensitivity of 96.9%, specificity of 100.0%, and AUC (area under the receiver operator characteristic curve) value of 0.999. Finally, we substantiated the real-time collection of diagnostic results using a customized portable NIR reader at nine different laboratories of government-certified quarantine institutions for foot-and-mouth disease (FMD).

Rapid, sensitive, and specific, low-resource molecular detection of Hendra virus

Efficient and accurate diagnosis of Hendra virus (HeV), a biosafety level 4 (BSL-4) pathogen and zoonotic disease, is of primary importance for surveillance and outbreak control in the Australian equine industry. Sporadic HeV spillover events pose a serious public health concern and are predicted to expand geographically, aligning with the moving distribution of the main reservoir hosts, the flying-foxes. Here we describe the development of a low-resource rapid Hendra test. The test used a fast and simple sample processing protocol followed by reverse transcription isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results were obtained in 30 min and required only a heating block, ice, and pipettes for liquid handling. The one-step sample processing protocol inactivated HeV in 2 min, providing a simple protocol that could enable safe testing outside of a laboratory. Analytical sensitivity testing demonstrated a detection limit of 1000 copies/μL of synthetic HeV RNA, and analytical specificity testing indicated assays did not detect other pathogens. Gamma-irradiated HeV-spiked in viral transport medium was detected with high sensitivity, down to 10,000 TCID50/mL, the equivalent of 18 RNA copies per reaction. Collectively, our data suggests that our rapid Hendra test offers a potential first-line screening on-site alternative to gold-standard RT-PCR detection, which requires samples to be shipped to central containment laboratories, thermocyclers and labour-intensive viral RNA purification, with testing time of approximately four hours. Our rapid Hendra test provided performance and speed without compromising sensitivity and specificity, and could become a promising more accessible tool for testing under resource-limited conditions for the veterinary community and thoroughbred industry.