Rapid identification of SARS-CoV-2 variants using stable high-frequency mutation sites.

Respiratory infectious viruses, including SARS-CoV-2, undergo rapid genetic evolution, resulting in diverse subtypes with complex mutations. Detecting and differentiating these subtypes pose significant challenges in respiratory virus surveillance. To address these challenges, we integrated ARMS-PCR with molecular beacon probes, allowing selective amplification and discrimination of subtypes based on adjacent mutation sites. The method exhibited high specificity and sensitivity, detecting as low as 104 copies/mL via direct fluorescence analysis and ~106 copies/mL using real-time PCR. Our robust detection approach offers a reliable and efficient solution for monitoring evolving respiratory infections, aiding early diagnosis and control measures. Further research could extend its application to other respiratory viruses and optimize its implementation in clinical settings.

Salivary Glands and Viral Pathogenesis.

The oral cavity is an epidemiologically relevant route of viral transmission due to the shedding of viruses in saliva. With advancements in salivary diagnostics, an increasing number of viruses have been detected. However, the anatomic source of virus in saliva is still largely unknown. Some viruses have a well-established tropism for the salivary glands (SGs), and recent studies have emphasized the importance of the glands as potential reservoirs for infectious viruses. Viral infections of the SGs have been linked to acute and chronic SG pathology and may be associated with SG dysfunction, with phenotypes similar to those seen in SjÖgren's disease (SjD), an autoimmune condition that affects the salivary and lacrimal glands. Understanding the breadth of viruses that infect the SG and the conserved or distinct host responses to these infections may provide insights into the pathogenesis of virus-mediated SG diseases. There is a need for further research to fully understand the molecular mechanisms by which viruses enter and replicate in the glands, their physiologic impact on SG function, and whether the SGs can serve as a long-term reservoir for infectious viral particles. The purpose of this review is to highlight a group of viruses that infect the salivary gland: hepatitis C virus, hepatitis D virus, severe acute respiratory syndrome coronavirus 2, enteric viruses, human T-cell leukemia virus type I, human immunodeficiency virus, human cytomegalovirus, and BK polyomavirus. We focus on the effects of viral infection on salivary gland (SG) inflammation, function, and its association with SjD.

Magnetic biosensors for identification of SARS-CoV-2, Influenza, HIV, and Ebola viruses: a review.

Infectious diseases such as novel coronavirus (SARS-CoV-2), Influenza, HIV, Ebola, etc kill many people around the world every year (SARS-CoV-2 in 2019, Ebola in 2013, HIV in 1980, Influenza in 1918). For example, SARS-CoV-2 has plagued higher than 317 000 000 people around the world from December 2019 to January 13, 2022. Some infectious diseases do not yet have not a proper vaccine, drug, therapeutic, and/or detection method, which makes rapid identification and definitive treatments the main challenges. Different device techniques have been used to detect infectious diseases. However, in recent years, magnetic materials have emerged as active sensors/biosensors for detecting viral, bacterial, and plasmids agents. In this review, the recent applications of magnetic materials in biosensors for infectious viruses detection have been discussed. Also, this work addresses the future trends and perspectives of magnetic biosensors.

Toward a next-generation diagnostic tool: A review on emerging isothermal nucleic acid amplification techniques for the detection of SARS-CoV-2 and other infectious viruses.

As the COVID-19 pandemic continues to affect human health across the globe rapid, simple, point-of-care (POC) diagnosis of infectious viruses such as SARS-CoV-2 remains challenging. Polymerase chain reaction (PCR)-based diagnosis has risen to meet these demands and despite its high-throughput and accuracy, it has failed to gain traction in the rapid, low-cost, point-of-test settings. In contrast, different emerging isothermal amplification-based detection methods show promise in the rapid point-of-test market. In this comprehensive study of the literature, several promising isothermal amplification methods for the detection of SARS-CoV-2 are critically reviewed that can also be applied to other infectious viruses detection. Starting with a brief discussion on the SARS-CoV-2 structure, its genomic features, and the epidemiology of the current pandemic, this review focuses on different emerging isothermal methods and their advancement. The potential of isothermal amplification combined with the revolutionary CRISPR/Cas system for a more powerful detection tool is also critically reviewed. Additionally, the commercial success of several isothermal methods in the pandemic are highlighted. Different variants of SARS-CoV-2 and their implication on isothermal amplifications are also discussed. Furthermore, three most crucial aspects in achieving a simple, fast, and multiplexable platform are addressed.

A Landscape of CRISPR/Cas Technique for Emerging Viral Disease Diagnostics and Therapeutics: Progress and Prospects.

Viral diseases have emerged as a serious threat to humanity and as a leading cause of morbidity worldwide. Many viral diagnostic methods and antiviral therapies have been developed over time, but we are still a long way from treating certain infections caused by viruses. Acquired immunodeficiency syndrome (AIDS) is one of the challenges where current medical science advancements fall short. As a result, new diagnostic and treatment options are desperately needed. The CRISPR/Cas9 system has recently been proposed as a potential therapeutic approach for viral disease treatment. CRISPR/Cas9 is a specialised, effective, and adaptive gene-editing technique that can be used to modify, delete, or correct specific DNA sequences. It has evolved into an advanced, configurable nuclease-based single or multiple gene-editing tool with a wide range of applications. It is widely preferred simply because its operational procedures are simple, inexpensive, and extremely efficient. Exploration of infectious virus genomes is required for a comprehensive study of infectious viruses. Herein, we have discussed the historical timeline-based advancement of CRISPR, CRISPR/Cas9 as a gene-editing technology, the structure of CRISPR, and CRISPR as a diagnostic tool for studying emerging viral infections. Additionally, utilizing CRISPR/Cas9 technology to fight viral infections in plants, CRISPR-based diagnostics of viruses, pros, and cons, and bioethical issues of CRISPR/Cas9-based genomic modification are discussed.

The Use of CRISPR/Cas9 as a Tool to Study Human Infectious Viruses.

Clustered regularly interspaced short palindromic repeats (CRISPR) systems are a set of versatile gene-editing toolkit that perform diverse revolutionary functions in various fields of application such as agricultural practices, food industry, biotechnology, biomedicine, and clinical research. Specially, as a novel antiviral method of choice, CRISPR/Cas9 system has been extensively and effectively exploited to fight against human infectious viruses. Infectious diseases including human immunodeficiency virus (HIV), hepatitis B virus (HBV), human papillomavirus (HPV), and other viruses are still global threats with persistent potential to probably cause pandemics. To facilitate virus removals, the CRISPR/Cas9 system has already been customized to confer new antiviral capabilities into host animals either by modifying host genome or by directly targeting viral inherent factors in the form of DNA. Although several limitations and difficulties still need to be conquered, this technology holds great promises in the treatment of human viral infectious diseases. In this review, we will first present a brief biological feature of CRISPR/Cas9 systems, which includes a description of CRISPR/Cas9 structure and composition; thereafter, we will focus on the investigations and applications that employ CRISPR/Cas9 system to combat several human infectious viruses and discuss challenges and future perspectives of using this new platform in the preclinical and clinical settings as an antiviral strategy.

Assessment of airborne enteric viruses emitted from wastewater treatment plant: Atmospheric dispersion model, quantitative microbial risk assessment, disease burden.

From a health prospective, it is critical to provide a comprehensive model which integrates all the parameters involved in virus transmission and its consequences on human body. In order to estimate the health risks, for workers and residents, associated with an exposure airborne viruses emitted from a wastewater treatment (WWTP), the concentration levels of viruses in emitted bioaerosols over a twelve-month period were measured by real-time polymerase chain reaction (RT-PCR). A combined Gaussian plum dispersion model and quantitative microbial risk assessment (QMRA) with Monte-Carlo simulation served as suitable explanatory tools to estimate the risk of acquiring gastrointestinal illness (GI) due to exposure to air containing Rotavirus (RoV) and Norovirus (NoV) bioaerosols. Additionally, DALY metric was applied to quantify the disability and mortality for workers and residents. RoV and NoV were detected above aeration tank with annual mean concentration 27 and 3099 (Viruses/m3.h), respectively. The medium calculated DALY indicator based on viral loads in contaminant source (RoV:5.76 × 10-2 and NoV:1.23 × 10-1) and estimated in different distances away (300-1000 m) (RoV:2.87 × 10-2- 2.75 × 10-2 and NoV:1.14 × 10-1-1.13 × 10-1) were markedly higher than the threshold values recommended by US EPA (10-4 DALY pppy) and WHO (10-6 DALY pppy). The sensitivity analysis highlighted dose exposure and disease burden per case (DBPC) as two most influential factors for both workers and residents following exposure to two pathogens of concern. Due to high resistance and high concentration in the environment, the presence of RoV and NoV can intensify the consequences of diarrhea especially for children under five years of age; A comprehensible and transparent presentation of DALYs and QMRA can help decision makers and responsibilities to justify the priorities of exposure to wastewater in comparison with other risks of daily life.

Efficient collection of viable virus aerosol through laminar-flow, water-based condensational particle growth.

AIMS: State-of-the-art bioaerosol samplers have poor collection efficiencies for ultrafine virus aerosols. This work evaluated the performance of a novel growth tube collector (GTC), which utilizes laminar-flow water-based condensation to facilitate particle growth, for the collection of airborne MS2 viruses. METHODS AND RESULTS: Fine aerosols (<500 nm) containing MS2 coliphage were generated from a Collison nebulizer, conditioned by a dilution dryer and collected by a GTC and a BioSampler. The GTC effectively condensed water vapour onto the virus particles, creating droplets 2-5 µm in diameter, which facilitated collection. Comparison of particle counts upstream and downstream revealed that the GTC collected >93% of the inlet virus particles, whereas the BioSampler's efficiency was about 10%. Viable counts of the GTC-collected viruses were also one order of magnitude higher than those of the BioSampler (P = 0·003). CONCLUSION: The efficiency of the GTC for the viable collection of MS2 viruses exceeds that of industry standard instrument, the BioSampler, by a factor of 10-100. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that the GTC is an effective collector of viable MS2 aerosols, and concludes the instrument will be an effective tool for studying viable virus aerosols and the inhalation risks posed by airborne viruses.

Molecular Methods in Food Safety Microbiology: Interpretation and Implications of Nucleic Acid Detection.

Because of increasing demand for rapid results, molecular techniques are now applied for the detection of microorganisms in foodstuffs. However, interpretation problems can arise for the results generated by molecular methods in relation to the associated public health risk. Discrepancies between results obtained by molecular and conventional culture methods stem from the difference in target, namely nucleic acids instead of actively growing microorganisms. Nucleic acids constitute 5% to 15% of the dry weight of all living cells and are relatively stable, even after cell death, so they may be present in a food matrix after the foodborne microorganisms have been inactivated. Therefore, interpretation of the public health significance of positive results generated by nucleic acid detection methods warrants some additional consideration. This review discusses the stability of nucleic acids in general and highlights the persistence of microbial nucleic acids after diverse food-processing techniques based on data from the scientific literature. Considerable amounts of DNA and RNA (intact or fragmented) persist after inactivation of bacteria and viruses by most of the commonly applied treatments in the food industry. An overview of the existing adaptations for molecular assays to cope with these problems is provided, including large fragment amplification, flotation, (enzymatic) pretreatment, and various binding assays. Finally, the negligible risks of ingesting free microbial nucleic acids are discussed and this review ends with the future perspectives of molecular methods such as next-generation sequencing in diagnostic and source attribution food microbiology.

Study on Detection, Isolation and Identifications of Infectious Viruses in Various Water Samples

Various factors using cell lines can effect kinds and frequencies of infectious viruses obtained in the detection tests on various water samples. We tried to find out technical problems for the maximum virus isolations from water samples and characterize the virus isolates from waters in nature and in various purification stages. Fourteen viruses were isolated from 169 water samples by virus monitoring protocol for the information collection requirements rule, US EPA. The morphological changes caused by viruses and mycoplasma infections were compared with for increasing the specificity of tests employed. Cytopathic effects of slow growing viruses were found very similar with those by toxic effects in water samples and mycoplasma infections. Five of 6 stream water samples tested (83.33%) showed virus contaminations with the range of 1.03 to 5.75 MPNs/100 liter. Eight of 24 source water samples (33.35%) showed viral contaminations. One water sample of 24 water samples during precipitation stages was shown to include infectious viruses. It was confirmed that infectious viruses were significantly decreased by purification stages from streams. The titers (TCID50) of virus isolates were ranged as 10(-6.8) ~ 10(-6.925)/ml. The virus isolates were identified by immune fluorescent antibody (IFA) method using virus specific immune sera and serotyped using serotype specific reference sera. Of 14 virus isolates, 7 samples were identified as poliovirus and the other 7 were identified as coxsakie virus. Of 7 polioviruses, one was serotyped as type I, 3 viruses as type II and another 3 as type III. Conclusively, BGM cell lines must be free of mycoplasma for the strict examination of infectious viruses in water and highly sensitive for mainly enteroviruses. In addition, most of infectious viruses showing typical cytopathic effect from water samples were confirmed as coxsackie B and live attenuated vaccine strains of 3 polio types when BGM cells were used for virus isolations.