Viral Diagnostics Research Articles

Digital Quantification and Ultrasensitive Detection of Single Influenza Virus Using Microgel-in-Droplet Enzyme-Linked Immunosorbent Assay.

Detection and quantification of viral particles (VPs) facilitate both diagnostics of pathogenic viruses and quality control testing of virus-based products. However, existing technologies fail to afford concurrent ultrasensitive detection and large-scale absolute quantification of VPs. Here, we propose a digital Microgel-in-Droplet enzyme-linked immunosorbent assay (ELISA) system that enables the processing and monitoring of millions of ELISA reactions at the single-VP level by incorporating droplet microfluidics with sandwich ELISA. Upon validating the microfluidic workflow and optimizing ELISA parameters, we demonstrate ultrasensitive VP detection at a limit of detection of 56 PFU/test. Leveraging a fluorescence-based screening platform, we further realize high-throughput digital counting of VPs with a linear detection range of 500-64 000 PFU/test. The precision is comparable to that of the gold standard, the plaque assay, across a wide range of virus concentrations. We anticipate that our system will provide a novel paradigm for the absolute enumeration of various types of viral particles.

CRISPR-Cas Innovative Strategies for Combating Viral Infections and Enhancing Diagnostic Technologies

Background: CRISPR-Cas technology has transformed molecular diagnostics and therapeutic strategies for viral infections, particularly COVID-19. Its ability to precisely edit viral genomes and detect viral RNA/DNA offers a novel approach to combating persistent viral infections.Objective: This study aimed to evaluate the diagnostic accuracy and therapeutic potential of CRISPR-Cas systems in viral infections, with a focus on COVID-19.Methods: A systematic review and meta-analysis of 25 peer-reviewed studies were conducted, including clinical trials and experimental research. Data collection involved searching PubMed, Scopus, and Google Scholar using keywords such as "CRISPR-Cas," "viral diagnostics," and "COVID-19." Statistical analysis was performed using SPSS version 25, with pooled sensitivity and specificity estimates calculated for CRISPR-based diagnostics.Results: CRISPR-based diagnostics, including SHERLOCK and DETECTR, achieved pooled sensitivity of 94% (95% CI: 92%-96%) and specificity of 97% (95% CI: 95%-99%) for SARS-CoV-2 detection. Therapeutic interventions using CRISPR-Cas9 showed an 84% reduction in viral replication across HIV and Hepatitis B studies (95% CI: 80%-88%).Conclusion: CRISPR-Cas technologies demonstrate high diagnostic accuracy and therapeutic potential, particularly in resource-limited settings. Further clinical validation is needed to enhance global healthcare applications.

Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements

This study presents a graphene field-effect transistor (gFET) biosensor with dual detection capabilities for SARS-CoV-2: one RNA detection assay to confirm viral positivity and the other for nucleocapsid (N-)protein detection as a proxy for infectiousness of the patient. This technology can be rapidly adapted to emerging infectious diseases, making an essential tool to contain future pandemics. To detect viral RNA, the highly conserved E-gene of the virus was targeted, allowing for the determination of SARS-CoV-2 presence or absence using nasopharyngeal swab samples. For N-protein detection, specific antibodies were used. Tested on 213 clinical nasopharyngeal samples, the gFET biosensor showed good correlation with RT-PCR cycle threshold values, proving its high sensitivity in detecting SARS-CoV-2 RNA. Specificity was confirmed using 21 pre-pandemic samples positive for other respiratory viruses. The gFET biosensor had a limit of detection (LOD) for N-protein of 0.9 pM, establishing a foundation for the development of a sensitive tool for monitoring active viral infection. Results of gFET based N-protein detection corresponded to the results of virus culture in all 16 available clinical samples and thus it also proved its capability to serve as a proxy for infectivity. Overall, these findings support the potential of the gFET biosensor as a point-of-care device for rapid diagnosis of SARS-CoV-2 infection and indirect assessment of infectiousness in patients, providing additional information for clinical and public health decision-making.

Fast Viral Diagnostics: FTIR-Based Identification, Strain-Typing, and Structural Characterization of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered an ongoing global pandemic, necessitating rapid and accurate diagnostic tools to monitor emerging variants and preparedness for the next outbreak. This study introduces a multidisciplinary approach combining Fourier Transform Infrared (FTIR) microspectroscopy and Machine learning to comprehensively characterize and strain-type SARS-CoV-2 variants. FTIR analysis of pharyngeal swabs from different pandemic waves revealed distinct vibrational profiles, particularly in nucleic acid and protein vibrations. The spectral wavenumber range between 1150 and 1240 cm-1 was identified as the classification marker, distinguishing Healthy (noninfected) and infected samples. Machine learning algorithms, with neural networks exhibiting superior performance, successfully classified SARS-CoV-2 variants with a remarkable accuracy of 98.6%. Neural networks were also able to identify and differentiate a small cohort infected with influenza A variants, H1N1 and H3N2, from SARS-CoV-2-infected and Healthy samples. FTIR measurements further show distinct red shifts in vibrational energy and secondary structural alterations in the spike proteins of more transmissible forms of SARS-CoV-2 variants, providing experimental validation of the computational data. This integrated approach presents a promising avenue for rapid and reliable SARS-CoV-2 variant identification, enhancing our understanding of viral evolution and aiding in diagnostic advancements, particularly for an infectious disease with unknown etiology.

Novel Pestiviruses Detected in Cattle Interfere with Bovine Viral Diarrhea Virus Diagnostics.

Since the start of the mandatory nationwide bovine viral diarrhea (BVD) eradication program in Germany in 2011, the number of persistently infected (PI) animals has decreased considerably, resulting in a continuous decrease in seroprevalence. The increasingly BVD-naive cattle population could facilitate spillover infections with non-BVDV ruminant pestiviruses. Here, we report two cases in which novel pestiviruses were isolated from cattle; in both cases, the whole genome sequence showed the highest level of identity to strain "Pestivirus reindeer-1". Both novel viruses gave positive results in BVDV diagnostic test systems, confirming that cross-reactivity is an important issue in pestivirus diagnostics. In the first case, the pestivirus was probably transmitted from sheep kept with the affected cattle, suggesting that the co-housing of small ruminants and cattle is a risk factor. The source of infection could not be determined in the second case. The occurrence of these two cases in independent cattle holdings within a relatively short time frame suggests that it would be useful to determine the presence of pestiviruses in small ruminants or even wild ruminants to better assess risk factors, especially for BVDV-free populations.

Acoustofluidic Virus Isolation via Bessel Beam Excitation Separation Technology.

The isolation of viruses from complex biological samples is essential for creating sensitive bioassays that assess the efficacy and safety of viral therapeutics and vaccines, which have played a critical role during the COVID-19 pandemic. However, existing methods of viral isolation are time-consuming and labor-intensive due to the multiple processing steps required, resulting in low yields. Here, we introduce the rapid, efficient, and high-resolution acoustofluidic isolation of viruses from complex biological samples via Bessel beam excitation separation technology (BEST). BEST isolates viruses by utilizing the nondiffractive and self-healing properties of 2D, in-plane acoustic Bessel beams to continuously separate cell-free viruses from biofluids, with high throughput and high viral RNA yield. By tuning the acoustic parameters, the cutoff size of isolated viruses can be easily adjusted to perform dynamic, size-selective virus isolation while simultaneously trapping larger particles and separating smaller particles and contaminants from the sample, achieving high-precision isolation of the target virus. BEST was used to isolate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from human saliva samples and Moloney Murine Leukemia Virus from cell culture media, demonstrating its potential use in both practical diagnostic applications and fundamental virology research. With high separation resolution, high yield, and high purity, BEST is a powerful tool for rapidly and efficiently isolating viruses. It has the potential to play an important role in the development of next-generation viral diagnostics, therapeutics, and vaccines.

Point-of-Care Multiplex Detection of Respiratory Viruses.

The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.

A Review of Probe-Based Enrichment Methods to Inform Plant Virus Diagnostics.

Modern diagnostic techniques based on DNA sequence similarity are currently the gold standard for the detection of existing and emerging pathogens. Whilst individual assays are inexpensive to use, assay development is costly and carries risks of not being sensitive or specific enough to capture an increasingly diverse range of targets. Sequencing can provide the entire nucleic acid content of a sample and may be used to identify all pathogens present in the sample when the depth of coverage is sufficient. Targeted enrichment techniques have been used to increase sequence coverage and improve the sensitivity of detection within virus samples, specifically, to capture sequences for a range of different viruses or increase the number of reads from low-titre virus infections. Vertebrate viruses have been well characterised using in-solution hybridisation capture to target diverse virus families. The use of probes for genotyping and strain identification has been limited in plants, and uncertainty around sensitivity is an impediment to the development of a large-scale virus panel to use within regulatory settings and diagnostic pipelines. This review aims to compare significant studies that have used targeted enrichment of viruses to identify approaches to probe design and potential for use in plant virus detection and characterisation.

Improving grapevine virus diagnostics: Comparative analysis of three dsRNA enrichment methods for high-throughput sequencing

The extraction of double stranded (ds) RNA is a common enrichment method for the study, characterization, and detection of RNA viruses. In addition to RNA viruses, viroids, and some DNA viruses, can also be detected from dsRNA enriched extracts which makes it an attractive method for detecting a wide range of viruses when coupled with HTS. Several dsRNA enrichment strategies have been developed. The oldest utilizes the selective binding properties of dsRNA to cellulose. More recent methods are based on the application of anti-dsRNA antibodies and viral proteins with a specific affinity for dsRNA. All three methods have been used together with HTS for plant virus detection and study. To our knowledge, this is the first comparative study of three alternative dsRNA enrichment methods for virus and viroid detection through HTS using virus-infected, and healthy grapevine test plants. Extracts were performed in triplicate using methods based on, the anti-dsRNA antibody mAb rJ2 (Millipore Sigma Canada Ltd, Oakville, ON, Canada), the B2 dsRNA binding protein, and ReliaPrep™ Resin (Promega Corporation, Madison, WI, USA). The results show that the workflows for all three methods are effectively comparable, apart from purification steps related to antibody and binding protein construct. Both the cellulose resin and dsRNA binding protein construct methods provide highly enriched dsRNA extracts suitable for HTS with the B2 method providing a 36× and the ReliaPrep™ Resin a 163× increase in dsRNA enrichment compared to the mAb rJ2 antibody. The overall consistency and cost effectiveness of the ReliaPrep™ cellulose resin-based method and the potentially simpler adaptation to robotics made it the method of choice for future transfer to a semi-automated workflow.

Preamplification-free viral RNA diagnostics with single-nucleotide resolution using MARVE, an origami paper-based colorimetric nucleic acid test.

The evolution and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgent concerns as they pose the risk of vaccine failure and increased viral transmission. However, affordable and scalable tools allowing rapid identification of SARS-CoV-2 variants are not readily available, which impedes diagnosis and epidemiological surveillance. Here we present a colorimetric nucleic acid assay named MARVE (multiplexed, preamplification-free, single-nucleotide-resolved viral evolution) that is convenient to perform and yields single-nucleotide resolution. The assay integrates nucleic acid strand displacement reactions with enzymatic amplification to colorimetrically sense viral RNA using a metal ion-incorporated DNA probe (TEprobe). We provide detailed guidelines to design TEprobes for discriminating single-nucleotide variations in viral RNAs, and to fabricate a test paper for the detection of SARS-CoV-2 variants of concern. Compared with other nucleic acid assays, our assay is preamplification-free, single-nucleotide-resolvable and results are visible via a color change. Besides, it is smartphone readable, multiplexed, quick and cheap ($0.30 per test). The protocol takes ~2 h to complete, from the design and preparation of the DNA probes and test papers (~1 h) to the detection of SARS-CoV-2 or its variants (30-45 min). The design of the TEprobes requires basic knowledge of molecular biology and familiarity with NUPACK or the Python programming language. The fabrication of the origami papers requires access to a wax printer using the CAD and PDF files provided or requires users to be familiar with AutoCAD to design new origami papers. The protocol is also applicable for designing assays to detect other pathogens and their variants.

The co-assembly of spike silica nanoparticles with high affinity to nucleic acid for airborne virus detection

The global pandemic of coronavirus disease 2019 (COVID-19) highlights the shortcomings of the current testing paradigm for viral disease diagnostics. Though the Polymerase Chain Reaction (PCR) technique is currently used for nucleic acid detection in liquid samples, its application in airborne virus has not yet been developed, due to low viral load in the air, lack of efficient capture probe and supporting equipment. Herein, we developed an ultrasensitive biosensing nanoplatform for airborne virus detection. The extraction and concentration of nucleic acid from test samples has been accomplished by novel spike silica nanoparticles (SSNPs)-based nucleic acid capture probe with high spike length to nanoparticle diameter ratio and appropriate surface potential. When integrated with air sampler and Quantitative Real-Time PCR (qPCR) equipment, nucleic acid enrichment can be achieved significantly. Our results demonstrated a successful detection of the virus in the air with the limit of detection (LoD) of 500 copies/L, which greatly surpasses the LoD of 1 × 106 copies/L. This detection method effectively addresses the obstacle posed by the low abundance of pathogens in the environment and could be applied for the detection of pathogens in air or water, aiming to achieve the goal of disease prevention and control prior to its onset.

LinearAlifold: Linear-time consensus structure prediction for RNA alignments

Predicting the consensus structure of a set of aligned RNA homologs is a convenient method to find conserved structures in an RNA genome, which has many applications including viral diagnostics and therapeutics. However, the most commonly used tool for this task, RNAalifold, is prohibitively slow for long sequences, due to a cubic scaling with the sequence length, taking over a day on 400 SARS-CoV-2 and SARS-related genomes (∼30,000nt). We present LinearAlifold, a much faster alternative that scales linearly with both the sequence length and the number of sequences, based on our work LinearFold that folds a single RNA in linear time. Our work is orders of magnitude faster than RNAalifold (0.7 h on the above 400 genomes, or ∼36× speedup) and achieves higher accuracies when compared to a database of known structures. More interestingly, LinearAlifold’s prediction on SARS-CoV-2 correlates well with experimentally determined structures, substantially outperforming RNAalifold. Finally, LinearAlifold supports two energy models (Vienna and BL*) and four modes: minimum free energy (MFE), maximum expected accuracy (MEA), ThreshKnot, and stochastic sampling, each of which takes under an hour for hundreds of SARS-CoV variants. Our resource is at:https://github.com/LinearFold/LinearAlifold (code) and http://linearfold.org/linear-alifold (server).

Comparison of SERS spectra of intact and inactivated viruses via machine learning algorithms for the viral disease’s diagnosis application

In this work, Surface-enhanced Raman spectroscopy (SERS) along with machine learning algorithms (MLA) were used to detect and classify the viral particles to assess the possibility of using the spectra of inactivated influenza A viruses for MLA training and spectra database compilation for further study and diagnosis of intact forms of the virus. Viral particles inactivation was performed by formalin, ultraviolet and beta-propiolactone. Support vector method and principal component analysis allowed to classify intact and inactivated viral particles spectra with an accuracy of 80.0–96.7 %. The results obtained suggest that it is not advisable to create a spectral database and train machine learning algorithms for their further application in SERS diagnostics of intact viruses based on the spectra of the inactivated virus particles.

Development and evaluation of one-step RT-qPCR TaqMan multiplex panels applied to six viruses occurring in lily and tulip bulbs

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics.

COVID-19 pandemic – Cocktail of variants, a study from Northern India

ABSTRACT Context: The aim of the study was to identify and monitor the circulating strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the samples received at our center and update the existing national and international genomic surveillance data. Introduction: SARS-CoV-2 is no exception to the basic nature of the viruses ability to change and evolve. Since its first report in December 2019 from Wuhan, China, multiple variants of the virus have emerged and been reported. Five variants of concern have been recognized and reported by the Centers for Disease Control and Prevention, which are associated with variable degrees of transmissibility and mortality. Materials and Methods: Nasopharyngeal and oropharyngeal swabs received in viral transport medium at the Viral Research Diagnostic Laboratory were processed for reverse transcription-polymerase chain reaction for SARS-CoV-2. Whole genome sequencing (WGS) was performed for selective positive samples using Oxford Nanopore sequencing technology, using MinKNOW software for data acquisition. Statistical Analysis: The clades were assigned using Nextclade v2.4.1 software. The statistical analysis was calculated using OpenEpi version 3, an open-source calculator, and two by two. Results: Variants reported over the study period included Alpha, Kappa, Delta, and Omicron. Delta dominated in the year 2021, while Omicron was the dominant variant in 2022. In both the dominant variants, asymptomatics contributed to around 30–40% of positives. Intensive care unit admissions and mortality were higher in the Delta variant, while vaccination history and travel history were higher in the patients with Omicron variant. Conclusion: The trend tracking of these variants has been important in view of public health, enabling early interventions to control the spread of the disease and foresight in preparation for the situation.

Single-tube, single-strip lateral flow assays utilizing loop-mediated isothermal amplification for simultaneous hepatitis B and C viral detection.

Globally, hepatitis B virus (HBV) affects over 250 million people, whereas hepatitis C virus (HCV) affects approximately 70 million people, posing major public health challenges. Despite the availability of vaccines and treatments, a lack of comprehensive diagnostic coverage has left many cases undiagnosed and untreated. To address the need for sensitive, specific, and accessible diagnostics, this study introduced a multiplex loop-mediated isothermal amplification assay with lateral flow detection for simultaneous HBV and HCV testing. This assay achieved exceptional sensitivity and was capable of detecting HBV and HCV concurrently in a single tube and on a single strip within 25 min, achieving the required clinical sensitivity (10 and 103 genomic copies/reaction for HBV and HCV, respectively). The method was validated in clinical samples of various viral genotypes, achieving an equivalent limit of detection. Additionally, a custom portable heating device was developed for field use. The assay developed here, capable of direct viral detection on the strip, shows promise in supplanting current methods that solely identify antibodies and necessitate additional qPCR for viral activity assessment. This economical and rapid assay aligns with point-of-care testing needs, offering significant advancements in enhancing viral hepatitis diagnostics in settings with limited resources.

Viral infections and inborn errors of immunity.

The purpose of this focused review is to discuss unusual presentations of viral infections in the context of specific inborn errors of immunity. We will discuss hyper immunoglobulin E (IgE) syndromes, epidermodysplasia verruciformis, and X-linked agammaglobulinemia as examples of inborn errors of immunity associated with specific presentations of viral infection and disease. Advances in both genetic and viral diagnostics have broadened our understanding of viral pathogenesis in the setting of immune dysfunction and the variable phenotype of inborn errors of immunity. Dedicator of cytokinesis 8 (DOCK8) deficiency is now recognized as an inborn error of immunity within the hyper IgE syndrome phenotype and is associated with unusually aggressive cutaneous disease caused by herpes simplex and other viruses. Studies of patients with epidermodysplasia verruciformis have proven that rarely detected human papillomavirus subtypes may cause malignancy in the absence of adequate host defenses. Finally, patients with X-linked agammaglobulinemia may remain at risk for severe and chronic viral infections, even as immune globulin supplementation reduces the risk of bacterial infection. Susceptibility to viral infections in patients with inborn errors of immunity is conferred by specific, molecular defects. Recurrent, severe, or otherwise unusual presentations of viral disease should prompt investigation for an underlying genetic defect.

Assessing the effect of sample storage time on viral detection using a rapid and cost-effective CTAB-based extraction method

BackgroundCassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce.ResultsThe effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.–vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p < 0.005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods.ConclusionOur method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions.

Common cold viruses circulating in children threaten wild chimpanzees through asymptomatic adult carriers

Reverse zoonotic respiratory diseases threaten great apes across Sub-Saharan Africa. Studies of wild chimpanzees have identified the causative agents of most respiratory disease outbreaks as “common cold” paediatric human pathogens, but reverse zoonotic transmission pathways have remained unclear. Between May 2019 and August 2021, we conducted a prospective cohort study of 234 children aged 3–11 years in communities bordering Kibale National Park, Uganda, and 30 adults who were forest workers and regularly entered the park. We collected 2047 respiratory symptoms surveys to quantify clinical severity and simultaneously collected 1989 nasopharyngeal swabs approximately monthly for multiplex viral diagnostics. Throughout the course of the study, we also collected 445 faecal samples from 55 wild chimpanzees living nearby in Kibale in social groups that have experienced repeated, and sometimes lethal, epidemics of human-origin respiratory viral disease. We characterized respiratory pathogens in each cohort and examined statistical associations between PCR positivity for detected pathogens and potential risk factors. Children exhibited high incidence rates of respiratory infections, whereas incidence rates in adults were far lower. COVID-19 lockdown in 2020–2021 significantly decreased respiratory disease incidence in both people and chimpanzees. Human respiratory infections peaked in June and September, corresponding to when children returned to school. Rhinovirus, which caused a 2013 outbreak that killed 10% of chimpanzees in a Kibale community, was the most prevalent human pathogen throughout the study and the only pathogen present at each monthly sampling, even during COVID-19 lockdown. Rhinovirus was also most likely to be carried asymptomatically by adults. Although we did not detect human respiratory pathogens in the chimpanzees during the cohort study, we detected human metapneumovirus in two chimpanzees from a February 2023 outbreak that were genetically similar to viruses detected in study participants in 2019. Our data suggest that respiratory pathogens circulate in children and that adults become asymptomatically infected during high-transmission times of year. These asymptomatic adults may then unknowingly carry the pathogens into forest and infect chimpanzees. This conclusion, in turn, implies that intervention strategies based on respiratory symptoms in adults are unlikely to be effective for reducing reverse zoonotic transmission of respiratory viruses to chimpanzees.

Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses.

Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers prohibit the widespread adoption of this technology in clinical laboratories. We validate an end-to-end mNGS assay for detection of respiratory viruses. Our assay is optimized to reduce turnaround time, lower cost-per-sample, increase throughput, and deploy secure and actionable bioinformatic results. We validated our assay using residual nasopharyngeal swab specimens from Vancouver General Hospital (n = 359), RT-PCR-positive, or negative for Influenza, SARS-CoV-2, and RSV. We quantified sample stability, assay precision, the effect of background nucleic acid levels, and analytical limits of detection. Diagnostic performance metrics were estimated. We report that our mNGS assay is highly precise, semi-quantitative, with analytical limits of detection ranging from 103-104 copies/mL. Our assay is highly specific (100%) and sensitive (61.9% Overall: 86.8%; RT-PCR Ct < 30). Multiplexing capabilities enable processing of up to 55-specimens simultaneously on an Oxford Nanopore GridION device, with results reported within 12-hours. This study outlines the diagnostic performance and feasibility of mNGS for respiratory viral diagnostics, infection control, and public health surveillance. We addressed translational barriers to widespread mNGS adoption.