Diagnostics for Viral Pathogens in Veterinary Diagnostic Laboratories.

Laboratory testing is one part of clinical diagnosis, and quick and reliable testing results provide important data to support treatment decision and develop control strategies. Clinical viral testing has been shifting from traditional virus isolation and electron microscopy to molecular polymerase chain reaction and point-of-care antigen tests. This shift in diagnostic methodology also means change from looking for infectious virions or viral particles to hunting viral antigens and genomes. With technological development, it is predicted that metagenomic sequencing will be commonly used in veterinary clinical diagnosis for unveiling the whole picture of microbes involved in diseases in the future.

Recent Developments in Nanotechnology-Based Biosensors for the Diagnosis of Coronavirus.

The major challenge in today's world is that medical research is facing the existence of a vast number of viruses and their mutations, which from time to time cause outbreaks. Also, the continuous and spontaneous mutations occurring in the viruses and the emergence of resistant virus strains have become serious medical hazards. So, in view of the growing number of diseases, like the recent COVID-19 pandemic that has caused the deaths of millions of people, there is a need to improve rapid and sensitive diagnostic strategies to initiate timely treatment for such conditions. In the cases like COVID-19, where a real cure due to erratic and ambiguous signs is not available, early intervention can be life-saving. In the biomedical and pharmaceutical industries, nanotechnology has evolved exponentially and can overcome multiple obstacles in the treatment and diagnosis of diseases. Nanotechnology has developed exponentially in the biomedical and pharmaceutical fields and can overcome numerous challenges in the treatment and diagnosis of diseases. At the nano stage, the molecular properties of materials such as gold, silver, carbon, silica, and polymers get altered and can be used for the creation of reliable and accurate diagnostic techniques. This review provides insight into numerous diagnostic approaches focused on nanoparticles that could have been established for quick and early detection of such diseases.

Virus Detection and Identification in Minutes Using Single-Particle Imaging and Deep Learning.

The increasing frequency and magnitude of viral outbreaks in recent decades, epitomized by the COVID-19 pandemic, has resulted in an urgent need for rapid and sensitive diagnostic methods. Here, we present a methodology for virus detection and identification that uses a convolutional neural network to distinguish between microscopy images of fluorescently labeled intact particles of different viruses. Our assay achieves labeling, imaging, and virus identification in less than 5 min and does not require any lysis, purification, or amplification steps. The trained neural network was able to differentiate SARS-CoV-2 from negative clinical samples, as well as from other common respiratory pathogens such as influenza and seasonal human coronaviruses. We were also able to differentiate closely related strains of influenza, as well as SARS-CoV-2 variants. Additional and novel pathogens can easily be incorporated into the test through software updates, offering the potential to rapidly utilize the technology in future infectious disease outbreaks or pandemics. Single-particle imaging combined with deep learning therefore offers a promising alternative to traditional viral diagnostic and genomic sequencing methods and has the potential for significant impact.

Chapter 7 - Testing viral infections

Viral diagnostics was previously confined to research purposes, but with the advent of modern modalities it has transcended the limits and forayed into main stream diagnostics. It is now a significant contributor towards prognosis, management and prevention strategies. Since the onslaught of the COVID-19 pandemic, it has become more important to accurately diagnose and differentiate various viral infections as the clinical features can be overlapping and confusing many a times. A misdiagnosed viral infection can play havoc on a patient’s health, as the severity of the infection and its clinical features may not always be in sync. So, it is of utmost importance to diagnose a viral infection using either point of care tests or advanced expert systems based on the available resources rather than going with the instincts, in this era of diagnostic revolution. This chapter aims to provide an overview of the various diagnostic modalities and developments in the field of virology that may augment our decision-making capacity for the best possible patient outcome.

Synthetically recoded virus sCPD9 - A tool to accelerate SARS-CoV-2 research under biosafety level 2 conditions.

Research with infectious SARS-CoV-2 is complicated because it must be conducted under biosafety level 3 (BSL-3) conditions. Recently, we constructed a live attenuated SARS-CoV-2 virus by rational design through partial recoding of the SARS-CoV-2 genome and showed that the attenuated virus, designated sCPD9, was highly attenuated in preclinical animal models. The recoded sequence was designed by codon pair deoptimization and is located at the distal end of gene ORF1ab. Codon pair deoptimization involves recoding of the viral sequence with underrepresented codon pairs but without altering the amino acid sequence of the encoded proteins. Thus, parental and attenuated viruses produce exactly the same proteins. In Germany, the live attenuated SARS-CoV-2 mutant sCPD9 was recently classified as a BSL-2 pathogen based on its genetic stability and strong attenuation in preclinical animal models. Despite its high attenuation in vivo, sCPD9 grows to high titers in common cell lines, making it suitable as substitute for virulent SARS-CoV-2 in many experimental setups. Consequently, sCPD9 can ease and accelerate SARS-CoV-2 research under BSL-2 conditions, particularly in experiments requiring replicating virus, such as diagnostics and development of antiviral drugs.

Reliability of Self-Sampling for Accurate Assessment of Respiratory Virus Viral and Immunologic Kinetics.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic demonstrates the need for accurate and convenient approaches to diagnose and therapeutically monitor respiratory viral infections. We demonstrated that self-sampling with mid-nasal foam swabs is well-tolerated and provides quantitative viral output concordant with flocked swabs. Using longitudinal home-based self-sampling, we demonstrate that nasal cytokine levels correlate and cluster according to immune cell of origin. Periods of stable viral loads are followed by rapid elimination, which could be coupled with cytokine expansion and contraction. Nasal foam swab self-sampling at home provides a precise, mechanistic readout of respiratory virus shedding and local immune responses.

A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread.

Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP (reverse transcription loop-mediated isothermal amplification) workflow for viral detection in saliva, and to provide more information regarding its potential in curbing COVID-19 transmission. Clinical and contrived specimens were used to optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate the clinical performance of the test and to characterize saliva based on age, gender and time from onset of symptoms. Our workflow achieved an overall sensitivity of 77.2% (n = 90), with 93.2% sensitivity, 97% specificity, and 0.895 Kappa for specimens containing >102 copies/µL (n = 77). Further analyses in saliva showed that viral load peaks in the first days of symptoms and decreases afterwards, and that viral load is ~10 times lower in females compared to males, and declines following symptom onset. NOP RT-PCR data did not yield relevant associations. This work suggests that saliva reflects the transmission dynamics better than NOP specimens, and reveals gender differences that may reflect higher transmission by males. This saliva RT-LAMP workflow can be applied to track viral spread and, to maximize detection, testing should be performed immediately after symptoms are presented, especially in females.

SARS-CoV-2 RNA Detection by a Cellphone-Based Amplification-Free System with CRISPR/CAS-Dependent Enzymatic (CASCADE) Assay.

CRISPR (Clustered regularly interspaced short palindromic repeats)-based diagnostic technologies have emerged as a promising alternative to accelerate delivery of SARS-CoV-2 molecular detection at the point of need. However, efficient translation of CRISPR-diagnostic technologies to field application is still hampered by dependence on target amplification and by reliance on fluorescence-based results readout. Herein, an amplification-free CRISPR/Cas12a-based diagnostic technology for SARS-CoV-2 RNA detection is presented using a smartphone camera for results readout. This method, termed Cellphone-based amplification-free system with CRISPR/CAS-dependent enzymatic (CASCADE) assay, relies on mobile phone imaging of a catalase-generated gas bubble signal within a microfluidic channel and does not require any external hardware optical attachments. Upon specific detection of a SARS-CoV-2 reverse-transcribed DNA/RNA heteroduplex target (orf1ab) by the ribonucleoprotein complex, the transcleavage collateral activity of the Cas12a protein on a Catalase:ssDNA probe triggers the bubble signal on the system. High analytical sensitivity in signal detection without previous target amplification (down to 50 copies µL-1) is observed in spiked samples, in ≈71 min from sample input to results readout. With the aid of a smartphone vision tool, high accuracy (AUC = 1.0; CI: 0.715 - 1.00) is achieved when the CASCADE system is tested with nasopharyngeal swab samples of PCR-positive COVID-19 patients.

Extraction of Viral Nucleic Acids with Carbon Nanotubes Increases SARS-CoV-2 Quantitative Reverse Transcription Polymerase Chain Reaction Detection Sensitivity.

The global SARS-CoV-2 coronavirus pandemic has led to a surging demand for rapid and efficient viral infection diagnostic tests, generating a supply shortage in diagnostic test consumables including nucleic acid extraction kits. Here, we develop a modular method for high-yield extraction of viral single-stranded nucleic acids by using "capture" ssDNA sequences attached to carbon nanotubes. Target SARS-CoV-2 viral RNA can be captured by ssDNA-nanotube constructs via hybridization and separated from the liquid phase in a single-tube system with minimal chemical reagents, for downstream quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. This nanotube-based extraction method enables 100% extraction yield of target SARS-CoV-2 RNA from phosphate-buffered saline in comparison to ∼20% extraction yield when using a commercial silica-column kit. Notably, carbon nanotubes enable extraction of nucleic acids directly from 50% human saliva with a similar efficiency as achieved with commercial DNA/RNA extraction kits, thereby bypassing the need for further biofluid purification and avoiding the use of commercial extraction kits. Carbon nanotube-based extraction of viral nucleic acids facilitates high-yield and high-sensitivity identification of viral nucleic acids such as the SARS-CoV-2 viral genome with a reduced reliance on reagents affected by supply chain obstacles.

Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic.

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/µL for a single guide RNA to 106 copies/µL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/µL and is completed using patient samples in less than 30 min.

Detect and destroy: CRISPR-based technologies for the response against viruses.

Despite numerous viral outbreaks in the last decade, including a devastating global pandemic, diagnostic and therapeutic technologies remain severely lacking. CRISPR-Cas systems have the potential to address these critical needs in the response against infectious disease. Initially discovered as the bacterial adaptive immune system, these systems provide a unique opportunity to create programmable, sequence-specific technologies for detection of viral nucleic acids and inhibition of viral replication. This review summarizes how CRISPR-Cas systems-in particular the recently discovered DNA-targeting Cas12 and RNA-targeting Cas13, both possessing a unique trans-cleavage activity-are being harnessed for viral diagnostics and therapies. We further highlight the numerous technologies whose development has accelerated in response to the COVID-19 pandemic.