Improved detection of Little cherry virus-2 using a hydrolysis probe to manage the Pacific Northwest little cherry disease epidemic.

Little cherry virus-2 (LChV-2) is a viral pathogen that is reaching epidemic levels in Washington state. This virus is insect-vectored and has significant impacts on sweet cherry production. To aid growers in making informed management decisions we sought to develop a diagnostic assay to better detect isolates of LChV-2 currently found in Washington, allowing for more accurate estimations of disease occurrence. This study showed that there were two distinct genotypes of LChV-2 present in Washington state. This information was used to develop an up-to-date reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) assay, which was then optimized, validated, and compared to four previously published assays using a panel of field samples. This comparison demonstrated that the newly developed assay provided greater sensitivity, accurately detecting less than 10 copies per reaction and could detect both LChV-2 genotypes. Finally, we examined the effect of potential inhibitors in various tissue types from cherry, finding that young leaf tissue affected sensitivity of detection less than root tissues.

Evaluation of Three Automated Extraction Systems for the Detection of SARS-CoV-2 from Clinical Respiratory Specimens

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly contagious and causes coronavirus disease 2019 (COVID-19). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the most accurate and reliable molecular assay to detect active SARS-CoV-2 infection. However, a rapid increase in test subjects has created a global bottleneck in testing capacity. Given that efficient nucleic acid extraction greatly affects reliable and accurate testing results, we compared three extraction platforms: MagNA Pure 96 DNA and Viral NA Small Volume kit on MagNA Pure 96 (Roche, Basel, Switzerland), careGENETM Viral/Pathogen HiFi Nucleic Acid Isolation kit (WELLS BIO Inc., Seoul, Korea) on KingFisher Flex (Thermo Fisher Scientific, Rocklin, CA, USA), and SGRespiTM Pure kit (Seegene Inc., Seoul, Korea) on Maelstrom 9600 (Taiwan Advanced Nanotech Inc., Taoyuan, Taiwan). RNA was extracted from 245 residual respiratory specimens from the different types of samples (i.e., NPS, sputum, and saliva) using three different kits. The 95% limits of detection of median tissue culture infectious dose per milliliter (TCID50/mL) for the MagNA Pure 96, KingFisher Flex, and Maelstrom 9600 were 0.37–3.15 × 101, 0.41–3.62 × 101, and 0.33–1.98 × 101, respectively. The KingFisher Flex platform exhibited 99.2% sensitivity and 100% specificity, whereas Maelstrom 9600 exhibited 98.3–100% sensitivity and 100% specificity. Bland–Altman analysis revealed a 95.2% concordance between MagNA Pure 96 and KingFisher Flex and 95.4% concordance between MagNA Pure 96 and Maelstrom 9600, indicating that all three platforms provided statistically reliable results. This suggests that two modifying platforms, KingFisher Flex and Maelstrom 9600, are accurate and scalable extraction platforms for large-scale SARS-CoV-2 clinical detection and could help the management of COVID-19 patients.

COVID-19 and RAAS inhibitors: is there a final conclusion?

Coronavirus disease 2019 (COVID-19), the first pandemic caused by a human infecting coronavirus, has drawn global attention from the first time it appeared in Wuhan city of China in late December 2019. Detection of the responsible viral pathogen, named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by WHO, and its possible pathogenesis lead to the forming of many hypotheses about the factors that may affect the patients’ outcome. One of the SARS-CoV-2 infection concerns was the potential role of angiotensin-converting enzyme (ACE) inhibitors and angiotensin-receptor blockers (ARBs) in COVID-19 patients’ morbidity and mortality. Studies demonstrated that because SARS-CoV-2 uses human ACE2 cell receptors as an entry receptor to invade the cells, there might be an association between antihypertensive drugs such as RAAS inhibitors (specifically ACEIs and ARBs) and the COVID-19 disease. Data are scarce and conflicting regarding ACEI or ARB consumption and how it influences disease outcomes, and a single conclusion has not been reached yet. According to the literature review in our article, the most evidentially supported theory about the use of RAAS inhibitors in COVID-19 is that these medications, including ACEI/ARB, are not associated with the increased risk of infection, disease severity, and patient prognosis. However, further studies are needed to support the hypothesis.

Microglia do not restrict SARS-CoV-2 replication following infection of the central nervous system of K18-hACE2 transgenic mice

Unlike SARS-CoV-1 and MERS-CoV, infection with SARS-CoV-2, the viral pathogen responsible for COVID-19, is often associated with neurologic symptoms that range from mild to severe, yet increasing evidence argues the virus does not exhibit extensive neuroinvasive properties. We demonstrate SARS-CoV-2 can infect and replicate in human iPSC-derived neurons and that infection shows limited anti-viral and inflammatory responses but increased activation of EIF2 signaling following infection as determined by RNA sequencing. Intranasal infection of K18 human ACE2 transgenic mice (K18-hACE2) with SARS-CoV-2 resulted in lung pathology associated with viral replication and immune cell infiltration. In addition, ∼50% of infected mice exhibited CNS infection characterized by wide-spread viral replication in neurons accompanied by increased expression of chemokine ( Cxcl9, Cxcl10, Ccl2, Ccl5 and Ccl19 ) and cytokine ( Ifn-λ and Tnf-α ) transcripts associated with microgliosis and a neuroinflammatory response consisting primarily of monocytes/macrophages. Microglia depletion via administration of colony-stimulating factor 1 receptor inhibitor, PLX5622, in SARS-CoV-2 infected mice did not affect survival or viral replication but did result in dampened expression of proinflammatory cytokine/chemokine transcripts and a reduction in monocyte/macrophage infiltration. These results argue that microglia are dispensable in terms of controlling SARS-CoV-2 replication in in the K18-hACE2 model but do contribute to an inflammatory response through expression of pro-inflammatory genes. Collectively, these findings contribute to previous work demonstrating the ability of SARS-CoV-2 to infect neurons as well as emphasizing the potential use of the K18-hACE2 model to study immunological and neuropathological aspects related to SARS-CoV-2-induced neurologic disease. Importance Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the role of microglia in aiding in host defense following experimental infection of the central nervous system (CNS) of K18-hACE2 with SARS-CoV-2, the causative agent of COVID-19. Neurologic symptoms that range in severity are common in COVID-19 patients and understanding immune responses that contribute to restricting neurologic disease can provide important insight into better understanding consequences associated with SARS-CoV-2 infection of the CNS.

Effects of Cigarette Smoking on Influenza Virus/Host Interplay

Cigarette smoking has been shown to increase the risk of respiratory infection, resulting in the exacerbation of infectious disease outcomes. Influenza viruses are a major respiratory viral pathogen, which are responsible for yearly epidemics that result in between 20,000 and 50,000 deaths in the US alone. However, there are limited general summaries on the impact of cigarette smoking on influenza pathogenic outcomes. Here, we will provide a systematic summarization of the current understanding of the interplay of smoking and influenza viral infection with a focus on examining how cigarette smoking affects innate and adaptive immune responses, inflammation levels, tissues that contribute to systemic chronic inflammation, and how this affects influenza A virus (IAV) disease outcomes. This summarization will: (1) help to clarify the conflict in the reports on viral pathogenicity; (2) fill knowledge gaps regarding critical anti-viral defenses such as antibody responses to IAV; and (3) provide an updated understanding of the underlying mechanism behind how cigarette smoking influences IAV pathogenicity.

Is It Possible to Produce Certified Hazelnut Plant Material in Sicily? Identification and Recovery of Nebrodi Genetic Resources, in vitro Establishment, and Innovative Sanitation Technique From Apple Mosaic Virus

Eight Sicilian cultivars of hazelnut (Corylus avellana L.), namely-Curcia, Nociara Collica, Panottara Collica, Panottara Galati Grande, Parrinara, Panottara Baratta Piccola, Enzo, and Rossa Galvagno, registered into the Italian Cultivar Register of fruit tree species in 2017 were selected from Nebrodi area and established in vitro. The aim of the work was to carry out the sanitation of the cultivars and get virus-free plants from the most important viral pathogen threat, the apple mosaic virus. Virus-free plant material is essential for the production of certified plants from Sicilian hazelnut cultivars, complying the CE (cat. CAC) quality and the technical standards established in 2017 for voluntary certification by the Italian Ministry of Agricultural, Food and Forestry Policies (MIPAAF). In this study, we investigated the possibility of establishing in vitro true-to-type and virus-free hazelnut plantlets via the encapsulation technology of apexes. The in vitro shoot proliferation rates were assessed for the different cultivars, sampling periods, temperature treatments, and type of explant used for culture initiation. Viability, regrowth, and conversion rates of both conventional meristem tip culture (MTC) and not conventional (MTC combined with the encapsulation technology) sanitation techniques were evaluated.

Clinical presentation and disease course in patients with flu-like illness: does microbiological aetiology matter?

Background: There is little evidence about the relation between aetiology, illness severity and clinical course of respiratory tract infections (RTI) in primary care. Understanding these associations would aid to develop effective management strategies for these infections. Aim: To investigate whether the clinical presentation and illness course differ between RTI in whom a viral pathogen was detected and those in whom a potential bacterial pathogen was found. Design and setting: Post hoc analysis of data from a pragmatic randomised trial on the effects of oseltamivir in patients with influenza-like illness (ILI) in primary care (n=3266) in 15 European countries. Methods: Patient characteristics, signs and symptoms were registered at baseline. Naso-pharyngeal (adults) or nasal and pharyngeal (children) swabs were taken for PCR analysis. Patients were followed up until 28 days after inclusion. Regression models and Kaplan-Meier curves were used to analyse the relation between aetiology, clinical presentation at baseline and course of disease including complications. Results: Except for a less prominent congested nose (OR 0.55, CI 0.35 – 0.86) and acute cough (OR 0.52, CI 0.27 – 0.65) in ILI patients in whom a possible bacterial pathogen was isolated, there were no clear clinical differences in presentations between those with a possible bacterial aetiology than in those with a viral one. Also the course of disease and complications were not related to aetiology. Conclusion: Given the currently available microbiological tests and antimicrobial treatments, and outside pandemics like COVID-19, microbiological testing in primary care patients with ILI seems to have limited value.

Rapid screening and identification of viral pathogens in metagenomic data

Background Virus screening and viral genome reconstruction are urgent and crucial for the rapid identification of viral pathogens, i.e., tracing the source and understanding the pathogenesis when a viral outbreak occurs. Next-generation sequencing (NGS) provides an efficient and unbiased way to identify viral pathogens in host-associated and environmental samples without prior knowledge. Despite the availability of software, data analysis still requires human operations. A mature pipeline is urgently needed when thousands of viral pathogen and viral genome reconstruction samples need to be rapidly identified. Results In this paper, we present a rapid and accurate workflow to screen metagenomics sequencing data for viral pathogens and other compositions, as well as enable a reference-based assembler to reconstruct viral genomes. Moreover, we tested our workflow on several metagenomics datasets, including a SARS-CoV-2 patient sample with NGS data, pangolins tissues with NGS data, Middle East Respiratory Syndrome (MERS)-infected cells with NGS data, etc. Our workflow demonstrated high accuracy and efficiency when identifying target viruses from large scale NGS metagenomics data. Our workflow was flexible when working with a broad range of NGS datasets from small (kb) to large (100 Gb). This took from a few minutes to a few hours to complete each task. At the same time, our workflow automatically generates reports that incorporate visualized feedback (e.g., metagenomics data quality statistics, host and viral sequence compositions, details about each of the identified viral pathogens and their coverages, and reassembled viral pathogen sequences based on their closest references). Conclusions Overall, our system enabled the rapid screening and identification of viral pathogens from metagenomics data, providing an important piece to support viral pathogen research during a pandemic. The visualized report contains information from raw sequence quality to a reconstructed viral sequence, which allows non-professional people to screen their samples for viruses by themselves (Additional file 1).

Designing a multi-epitope vaccine to provoke the robust immune response against influenza A H7N9

A new strain of Influenza A Virus (IAV), so-called "H7N9 Avian Influenza", is the first strain of this virus in which a human is infected by transmitting the N9 of influenza virus. Although continuous human-to-human transmission has not been reported, the occurrence of various H7N9-associated epidemics and the lack of production of strong antibodies against H7N9 in humans warn of the potential for H7N9 to become a new pandemic. Therefore, the need for effective vaccination against H7N9 as a life-threatening viral pathogen has become a major concern. The current study reports the design of a multi-epitope vaccine against Hemagglutinin (HA) and Neuraminidase (NA) proteins of H7N9 Influenza A virus by prediction of Cytotoxic T lymphocyte (CTL), Helper T lymphocyte (HTL), IFN-γ and B-cell epitopes. Human β-defensin-3 (HβD-3) and pan HLA DR-binding epitope (PADRE) sequence were considered as adjuvant. EAAAK, AAY, GPGPG, HEYGAEALERAG, KK and RVRR linkers were used as a connector for epitopes. The final construct contained 777 amino acids that are expected to be a recombinant protein of about ~ 86.38 kDa with antigenic and non-allergenic properties after expression. Modeled protein analysis based on the tertiary structure validation, docking studies, and molecular dynamics simulations results like Root-mean-square deviation (RMSD), Gyration, Root-mean-square fluctuation (RMSF) and Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) showed that this protein has a stable construct and capable of being in interaction with Toll-like receptor 7 (TLR7), TLR8 and m826 antibody. Analysis of the obtained data the demonstrates that suggested vaccine has the potential to induce the immune response by stimulating T and Bcells, and may be utilizable for prevention purposes against Avian Influenza A (H7N9).