Comprehensive high-throughput sequence-based detection and RT-LAMP confirmation of RNA viruses associated with dragon fruit (Selenicereus spp.) in India.

Molecular Diagnosis of Common Viral Infectious Diseases Based on Real Time PCR

BackgroundFew data exist regarding respiratory virus quantitation in lower respiratory samples and detection in serum from hematopoietic cell transplantation (HCT) recipients with respiratory virus-associated pneumonia.MethodsWe retrospectively identified HCT recipients with respiratory syncytial virus (RSV), parainfluenza (PIV), influenza, metapneumovirus (MPV), and coronavirus (CoV) detected in BAL samples, and tested stored BAL and/or serum samples using quantitative PCR.ResultsIn 85 BAL samples from 82 patients, median viral load in copies/ml for RSV (n=35) was 2.6×106, PIV (n=35) 4.9×107, influenza (n=9) 6.8×105, MPV (n=7) 3.9×107, and CoV (n=4) 1.8×105. Quantitative viral load was not associated with mechanical ventilation or death. Viral RNA was detected in serum of 6 of 66 patients: 4 of 41 with RSV pneumonia, 1 with influenza B, and 1 with MPV/influenza A/CoV co-infection (influenza A and MPV RNA detected). RSV detection in serum was associated with high viral load in BAL (p=0.05). Detection of viral RNA in serum was significantly associated with death (adjusted RR 1.8, p=0.02).ConclusionQuantitative PCR detects high viral load in BAL samples from HCT recipients with respiratory virus pneumonia. Viral RNA is also detectable in serum of patients with RSV, influenza, and MPV pneumonia, and may correlate with disease severity.

The main objective of this paper is to develop a driving circuit for a QCM sensor based on the detection of RNA virus. The circuit uses negative capacitance compensation technology, automatic gain control circuit and differential operation circuit, and this structure will improve the starting conditions of the resonant circuit and achieve continuous stability of the circuit oscillation. Thus the QCM front-end oscillation driving circuit can oscillate stably in a liquid phase environment. By connecting the Bessel band-pass filter, The circuit is stable under three harmonics at 15MHz, Removing some clutter in the oscillation process, and the obtained frequency is limited to a certain range, ensuring that the single-chip machine can read out the most accurate crystal frequency. Through the rail-to-rail operational amplifier chip, the frequency signal is fixed within a certain interval to ensure that the microcontroller can read the valid output frequency signal. Further, a frequency counter is used to count and display the read frequencies. Finally, the stability of the circuit is verified experimentally, and the experimental results and the theoretical calculation results of the formula are analyzed for error. When the experiment requires high parameters, the temperature fuzzy self-correcting PID control algorithm is used to control the operating environment temperature of the instrument within a basically constant range to reduce the error.

A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function. A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function.

Developing efficient and highly sensitive diagnostic techniques for early detections of pathogenic viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) is vitally important for preventing its widespread. However, the conventional polymerase chain reaction (PCR)‐based detection features high complexity, excessive time‐consumption, and labor‐intensiveness, while viral protein‐based detections suffer from moderate sensitivity and specificity. Here, a non‐PCR but ultrasensitive viral RNA detection strategy is reported based on a facile nanoprobe‐coupling strategy without enzymatic amplification, wherein PCR‐induced bias and other shortcomings are successfully circumvented. This approach endows the viral RNA detection with ultra‐low background to maximum signal ratio in the linear signal amplification by using Au nanoparticles as reporters. The present strategy exhibits 100% specificity toward SARS‐CoV‐2 N gene, and ultrasensitive detection of as low as 52 cp mL−1 of SARS‐CoV‐2 N gene without pre‐PCR amplification. This approach presents a novel ultrasensitive tool for viral RNA detections for fighting against COVID‐19 and other types of pathogenic virus‐caused diseases.

The NucliSens easyMAG and BioRot 9604 automated nucleic acid extraction systems were evaluated and compared with the manual QIAamp (Qiagen) extraction method for their abilities to extract nucleic acid from nasopharyngeal aspirate samples for the detection of RNA and DNA respiratory viruses. The nucleic acids recovered by all three methods gave comparable sensitivities in PCR tests, and the three methods gave comparable viral loads. There was no evidence of residual PCR inhibitors and no evidence of PCR cross-contamination.

A Cell Line That Expresses a Reporter Gene in Response to Infection by Sindbis Virus: A Prototype for Detection of Positive Strand RNA Viruses

Extra-pulmonary viral shedding in H7N9 Avian Influenza patients

The COVID‐19 crisis has defined the need of novel approaches for a straightforward management of pandemic and epidemic conditions. Here, an approach based on electronic sensing of viral RNA is proposed for both virus detection and variant tracking. The sensing strategy of the Organic Charge‐Modulated Field‐Effect Transistor (OCMFET) is employed for the development of a multi‐sensing platform, capable of parallel evaluation of control references and investigated samples. PCR‐free detection of viral genomic RNA is demonstrated at different target concentrations, with optimal discrimination with respect to negative control. Moreover, the same approach is feasible for virus variant tracking, as a screening strategy for mutation detection in PCR‐amplified samples: the approach can be used for a rationalization of RNA sequencing procedures, which are difficult to perform in the acute phases of pandemics, but are fundamental for an effective pandemic management. The results pave the way for novel strategies to enhance cost‐effectiveness, easiness‐of‐use in combination with selectivity and sensitivity in virus detection during severe pandemic conditions, and a more effective variant tracking by optimizing the access to sequencing resources even during the acute phases of pandemic.

Plant viruses are destructive pathogens of various crop species. Rapid, sensitive, and specific detection is crucial for the effective containment of emerging and resistance-breaking viruses. CRISPR/Cas has been established as a new tool for plant virus identification. However, its application for direct detection of viruses in the field is still limited. In this study, we present a CRISPR/Cas13a-based method for rapid detection of different viruses directly from RNA of several crop species, including tomato, cucumber, and rapeseed. This method was used to identify the emerging tomato brown rugose fruit virus (ToBRFV), a prominent pathogen in tomato cultivation, and distinguish it from closely related viruses in infected tomato plants. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Finally, we developed a user-friendly, extraction-free, 15 min protocol for on-site virus detection using a portable fluorescence viewer and a mobile phone camera. This protocol was successfully applied to identifyToBRFV in several commercial greenhouses. These results demonstrate that CRISPR/Cas13a is a robust technology for on-site detection of multiple viruses in different crop plants. This method could be swiftly adapted to identify newly emerging pests, which threaten global food security.

Comparison of three different techniques for the isolation of viral RNA in sputum

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control, 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL), and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of a co-inoculants of PRRSV (1 × 105 TCID50 per mL) and PEDV (1 × 105 TCID50 per mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one minute, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values were analyzed using SAS GLIMMIX v 9.4 (SAS, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P > 0.10). An interaction between disinfectant × surface impacted (P < 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon was greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P > 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces.

Viral RNA detection by RIG-I-like receptors

Detection of swine vesicular disease virus RNA by reverse transcription-polymerase chain reaction

High prevalence of SARS-CoV-2 detection and prolonged viral shedding in stools: A systematic review and cohort study