The rapid spread of African swine fever virus (ASFV), causing severe and often lethal disease in domestic pigs and Eurasian wild boar, continues to be a threat to pig populations and dependent industries. Despite scientific achievements that have deepened our understanding of ASFV pathogenesis, alternative transmission routes for ASFV remain to be elucidated. We previously demonstrated the efficient transmission of ASFV from infected boars to naïve recipient gilts via artificial insemination, thereby highlighting the importance of surveillance of boar semen prior to its shipment. Since the accurate and reliable detection of even low amounts of ASFV in boar semen is key to disease prevention and control, we established a suitable diagnostic workflow to efficiently detect the ASFV genome in boar semen. Here, we assessed the sensitivity of various routine nucleic acid extraction kits as well as qPCR protocols in detecting the ASFV genome in the blood and semen of infected boars. The feasibility of the respective kits and methods for future use in boar studs was also considered. Variability in sensitivity mostly concerned samples with low to very low amounts of the ASFV genome. Ultimately, we defined a well-suited workflow for precisely detecting the ASFV genome in boar semen as early as 2 days post ASFV infection.

Epidemics caused by multiple viruses continue to emerge, which have brought a terrible impact on human society. Identification of viral infections with high sensitivity and portability is of significant importance for the screening and management of diseases caused by viruses. Herein, a microfluidic chip (MFC)-assisted upconversion luminescence biosensing platform is designed and fabricated for point-of-care virus detection. Upconversion nanoparticles with excellent stability are successfully synthesized as luminescent agents for optical signal generation in the portable virus diagnostic platform. The relevant investigation results illustrate that the MFC-assisted virus diagnostic platform possesses outstanding performance such as good integration, high sensitivity (1.12 pg mL-1), ease of use, and portability. In addition, clinical sample test result verifies its more prominent virus diagnostic properties than commercially available rapid test strips. All of these thrilling capabilities imply that the designed portable virus diagnostic platform has great potential for future virus detection applications.

Plants growing in open airfields can be infected by several viruses even as a multiple infection. Virus infection in crops can lead to a serious damage to the harvest. In addition, virus presence in grapevine, fruit trees, and tuberous vegetables, propagated vegetatively affects the phytosanitary status of the propagation material (both the rootstock and the variety) having profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Small interfering (si) RNAs produced by the RNA silencing-based host immune system during viral infection can be sequenced by high-throughput techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample and therefore opening new avenues in virus diagnostics. This method is based on Illumina sequencing and bioinformatics analysis of virus-derived siRNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, to ensure success for every user.

Our growing ability to tailor healthcare to the needs of individuals has the potential to transform clinical treatment. However, the measurement of multiple biomarkers to inform clinical decisions requires rapid, effective, and affordable diagnostics. Chronic diseases and rapidly evolving pathogens in a larger population have also escalated the need for improved diagnostic capabilities. Current chemical diagnostics are often performed in centralized facilities and are still dependent on multiple steps, molecular labeling, and detailed analysis, causing the result turnaround time to be over hours and days. Rapid diagnostic kits based on lateral flow devices can return results quickly but are only capable of detecting a handful of pathogens or markers. Herein, we present the use of disposable plasmonics with chiroptical nanostructures as a platform for low-cost, label-free optical biosensing with multiplexing and without the need for flow systems often required in current optical biosensors. We showcase the detection of SARS-CoV-2 in complex media as well as an assay for the Norovirus and Zika virus as an early developmental milestone toward high-throughput, single-step diagnostic kits for differential diagnosis of multiple respiratory viruses and any other emerging diagnostic needs. Diagnostics based on this platform, which we term \"disposable plasmonics assays,\" would be suitable for low-cost screening of multiple pathogens or biomarkers in a near-point-of-care setting.

Yam (Dioscorea spp.) productivity is constrained significantly by the lack of a formal seed system. Vegetative propagation, through tuber setts as \'seed\' yams, encourages the recycling of virus-infected planting materials, contributing to high virus incidence and yield losses. Efforts are ongoing to increase the production of high-quality seed yams in a formal seed system to reduce virus-induced yield losses and enhance the crop\'s productivity and food security. Specific and sensitive diagnostic tests are imperative to prevent the multiplication of virus-infected materials contributing to a sustainable seed yam certification system. During routine indexing of yam accessions, discrepancies were observed between the results obtained from the reverse transcription loop-mediated isothermal amplification (RT-LAMP) test and those from reverse transcription polymerase chain reaction (RT-PCR); RT-LAMP failed to detect Yam mosaic virus (YMV) in some samples that tested positive by RT-PCR. This prompted the design of a new set of LAMP primers, YMV1-OPT primers. These primers detected as little as 0.1 fg/µL of purified RNA obtained from a YMV-infected plant, a sensitivity equivalent to that obtained with RT-PCR. RT-LAMP using YMV1-OPT primers is recommended for all future virus-indexing of seed yams for YMV, offering a rapid, sensitive, and cost-effective approach.

Viral infections can pose a major threat to public health by causing serious illness, leading to pandemics, and burdening healthcare systems. The global spread of such infections causes disruptions to every aspect of life including business, education, and social life. Fast and accurate diagnosis of viral infections has significant implications for saving lives, preventing the spread of the diseases, and minimizing social and economic damages. Polymerase chain reaction (PCR)-based techniques are commonly used to detect viruses in the clinic. However, PCR has several drawbacks, as highlighted during the recent COVID-19 pandemic, such as long processing times and the requirement for sophisticated laboratory instruments. Therefore, there is an urgent need for fast and accurate techniques for virus detection. For this purpose, a variety of biosensor systems are being developed to provide rapid, sensitive, and high-throughput viral diagnostic platforms, enabling quick diagnosis and efficient control of the virus\'s spread. Optical devices, in particular, are of great interest due to their advantages such as high sensitivity and direct readout. The current review discusses solid-phase optical sensing techniques for virus detection, including fluorescence-based sensors, surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS), optical resonators, and interferometry-based platforms. Then, we focus on an interferometric biosensor developed by our group, the single-particle interferometric reflectance imaging sensor (SP-IRIS), which has the capability to visualize single nanoparticles, to demonstrate its application for digital virus detection.

Certain viral pathogens can be shed into the human breast milk and cause infections in the infant upon breastfeeding. Thus, it is important to clarify whether viral RNA as well as infectious virus can be found in breast milk. The complexity of this body fluid poses several challenges for viral RNA isolation and detection of infectious virus. We here provide a protocol that allowed the identification of SARS-CoV-2 RNA in breast milk and the isolation of infectious virus after the virus has been artificially spiked into milk samples.

As part of a special edition for MDPI on plant virology in Australia, this review provides a brief high-level overview on the evolution of diagnostic techniques used in Australian government Post-Entry Quarantine (PEQ) facilities for testing imported plants for viruses. A comprehensive range of traditional and modern diagnostic approaches have historically been employed in PEQ facilities using bioassays, serological, and molecular techniques. Whilst these techniques have been effective, they are time consuming, resource intensive and expensive. The review highlights the importance of ensuring the best available science and diagnostic developments are constantly tested, evaluated, and implemented by regulators to ensure primary producers have rapid and safe access to new genetics to remain productive, sustainable and competitive.

Millions of people are tested for COVID-19 daily during the pandemic, and a lack of evidence to guide optimal nasal swab testing can increase the risk of false-negative test results. This study aimed to determine the optimal insertion depth for nasal mid-turbinate and nasopharyngeal swabs. The measurements were made with a flexible endoscope during the collection of clinical specimens with a nasopharyngeal swab at a public COVID-19 test center in Copenhagen, Denmark. Participants were volunteer adults undergoing a nasopharyngeal SARS-CoV-2 rapid antigen test. All 109 participants (100%) completed the endoscopic measurements; 52 (48%) women; 103 (94%) white; mean age 34.39 (SD, 13.2) years; and mean height 176.7 (SD, 9.29) cm. The mean swab length to the posterior nasopharyngeal wall was 9.40 (SD, 0.64) cm. The mean endoscopic distance to the anterior and posterior end of the inferior turbinate was 1.95 (SD, 0.61) cm and 6.39 (SD, 0.62) cm, respectively. The mean depth to nasal mid-turbinate was calculated as 4.17 (SD, 0.48) cm. The optimal depths of insertion for nasal mid-turbinate swabs are underestimated in current guidelines compared with our findings. This study provides clinical evidence to guide the performance of anatomically correct nasal and nasopharyngeal swab specimen collection for virus testing.

One of the most widely used methods to detect an acute viral infection in clinical specimens is diagnostic real-time polymerase chain reaction. However, because of the COVID-19 pandemic, mass-spectrometry-based proteomics is currently being discussed as a potential diagnostic method for viral infections. Because proteomics is not yet applied in routine virus diagnostics, here we discuss its potential to detect viral infections. Apart from theoretical considerations, the current status and technical limitations are considered. Finally, the challenges that have to be overcome to establish proteomics in routine virus diagnostics are highlighted.