Search Results (51)

African swine fever virus (ASFV) is a high-consequence pathogen that causes African swine fever (ASF), for which mortality rates can reach 90–100%, with death typically occurring within 14 days. ASF is currently a highly contagious pandemic disease responsible for extensive losses in pig production in multiple affected countries suffering from extended outbreaks. While a limited number of vaccines to prevent ASF are in use in south-east Asia, vaccines are not widely available, are only effective against highly homologous strains of ASFV, and must be used prior to an outbreak on a farm. Currently, there is no treatment for ASF and culling affected farms is the only response to outbreaks on farms to try and prevent spreading. CRISPR/Cas systems evolved as an adaptive immune response in bacteria and archaea that function by cleaving and disrupting the genomes of invading bacteriophage pathogens. CRISPR technology has since been leveraged into an array of endonuclease-based systems used for nucleic acid detection, targeting, genomic cleavage, and gene editing, making them particularly well-suited for development as sequence-specific therapeutic modalities. The programmability of CRISPR-based therapeutics offers a compelling new way to rapidly and specifically target pathogenic viral genomes simply by using different targeting guide RNAs (gRNA) as an adaptable antiviral modality. Here, we demonstrate for the first time a specific CRISPR/Cas9 multiplexed gRNA system that targets the African swine fever viral genome, resulting in sequence-specific cleavage, leading to the reduction in the viral load in infected animals, and subsequent recovery from an otherwise lethal dose of ASFV. Moreover, animals that recovered had protective immunity to subsequent homologous ASFV infection. Full article

Sweetpotato (Ipomoea batatas L. Lam) production is threatened by complex viral diseases, notably sweet potato virus disease (SPVD) worldwide, which results from co-infection by sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV). This study provides virus-specific transcriptomic insights into the immune responses of three sweetpotato cultivars, ‘Beauregard’, ‘Tanzania’, and ‘New Kawogo’, to SPFMV, SPCSV, and SPVD. Using RNA-seq profiling across three timepoints post-infection at 3, 6, and 12 weeks, we identified distinct virus- and genotype-specific gene expression responses. ‘New Kawogo’ activated early and sustained immune pathways involving redox regulation, transcriptional control, and hormonal signaling in response to both SPCSV and SPFMV, while showing minimal transcriptional disruption under SPVD, reflecting robust tolerance. ‘Beauregard’ exhibited early suppression of immune and metabolic genes, with delayed and disorganized recovery efforts, particularly under SPVD. Defense-related pathways including NBS-LRR signaling, RNA silencing, and hormonal regulation were consistently upregulated in ‘New Kawogo’ and to a lesser extent in ‘Tanzania’, but remained inactive in ‘Beauregard’. This study highlights candidate resistance and susceptibility genes for each virus, providing a molecular basis for developing virus-resilient sweetpotato cultivars through functional genomics and marker-assisted breeding. These findings elucidate the molecular basis of virus resistance in sweetpotato and identify candidate genes for marker-assisted breeding, despite limitations arising from the use of a diploid reference genome and discrete sampling intervals. Full article

Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding. Full article

Background: Since liquid-based cytology (LBC) has replaced the conventional Papanicolaou test in cervical cancer screening programs, pre-analytical procedures—particularly the choice of LBC collection media—have become crucial to ensure the accuracy of high-risk (HR) HPV DNA testing. This study aims to evaluate whether the newly developed CytoPath^® LBC medium can serve as a reliable alternative to standard solutions. Methods: This study exploited cell lines to evaluate the stability, integrity, and recovery rate of genomic DNA at different fixation time points (1, 7, 14 and 40 days) and serial dilutions (1:5, 1:10 and 1:20) extracted from cell lines. These samples have also undergone quantitative Real-Time PCR (qPCR) based HR-HPV test to assess the relative performance of the new preservative solution in detecting viral DNA with respect to the standard reference. Results: Cervical cell lines preserved in both media demonstrated consistent DNA stability over time. DNA yields were comparable between the two media. Notably, the DNA Integrity Number (DIN) was higher in samples fixed with the CytoPath^® solution. HR-HPV detection by qPCR showed equivalent performance, regardless of the fixative used. Conclusions: The CytoPath^® fixative solution represents a valid alternative to standard preservation media, offering improved DNA integrity while maintaining equivalent performance in HR-HPV qPCR testing. Full article

(1) Background: A safe and effective nucleic acid sample transportation method was developed that is suitable for underdeveloped areas which lack advanced sequencing capabilities, specifically for virus genomic sequencing and infectious disease monitoring. (2) Methods: This study evaluated the use of Flinders Technology Associates (FTA) cards for transporting amplified whole-genome DNA from 120 SARS-CoV-2-positive nasopharyngeal swab samples in Sierra Leone. Nucleic acid extraction and whole-genome amplification were conducted at a local laboratory. Amplified products were applied to FTA Elute cards for room temperature shipment to China CDC for elution and sequencing. (3) Results: The FTA card method achieved a 9.6% recovery rate for amplicons, sufficient for viral genome sequencing. In total, 86 (71.7%) high-quality SRAS-CoV-2 genomic sequences were obtained, with the majority reaching depths exceeding 100X. Sequence analysis revealed co-circulation of Delta, Omicron, and B.1 lineages. Higher Ct values in the original sample significantly reduced coverage and depth, with Ct ≤ 27; 73.6% of samples yielded effective sequences. (4) Conclusions: Transportation of amplified nucleic acid samples using FTA cards enables virus genomic sequencing in resource-limited areas. This approach can potentially improve local virus surveillance and outbreak response capabilities. Further optimizations could improve sequence recovery rate. Implementing this method could significantly enhance sequencing accessibility in underdeveloped regions. Full article

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has profoundly impacted global health, with pneumonia emerging as a major complication in severe cases. The pathogenesis of COVID-19 is marked by the overproduction of reactive oxygen species (ROS) and an excessive inflammatory response, resulting in oxidative stress and significant tissue damage, particularly in the respiratory system. Antioxidants have garnered considerable attention for their potential role in managing COVID-19 pneumonia by mitigating oxidative stress and modulating immune responses. This review provides a comprehensive overview of the literature on the use of antioxidants in hospitalized patients with mild-to-moderate COVID-19. Studies exploring antioxidants, including vitamins, trace elements, nitric oxide (NO), ozone (O₃), glutathione (GSH), L-carnitine, melatonin, bromelain, N-acetylcysteine (NAC), and numerous polyphenols, have yielded promising outcomes. Through their ROS-scavenging properties, these molecules support endothelial function, reduce the thrombosis risk, and may help mitigate the effects of the cytokine storm, a key contributor to COVID-19 morbidity and mortality. Clinical evidence suggests that antioxidant supplementation may improve patient outcomes by decreasing inflammation, supporting immune cell function, and potentially shortening recovery times. Furthermore, these molecules may mitigate the symptoms of COVID-19 by exerting direct antiviral effects that inhibit the infection process and genomic replication of SARS-CoV-2 in host cells. Moreover, antioxidants may work synergistically with standard antiviral treatments to reduce viral-induced oxidative damage. By integrating findings from the literature with real-world data from our clinical experience, we gain a more profound understanding of the role of antioxidants in managing COVID-19 pneumonia. Further research combining comprehensive literature reviews with real-world data analysis is crucial to validate the efficacy of antioxidants and establish evidence-based guidelines for their use in clinical practice. Full article

The increasingly widespread application of next-generation sequencing (NGS) in clinical diagnostics and epidemiological research has generated a demand for robust, fast, automated, and user-friendly bioinformatics workflows. To guide the choice of tools for the assembly of full-length viral genomes from NGS datasets, we assessed the performance and applicability of four open-source bioinformatics pipelines (shiver—for which we created a user-friendly Dockerized version, referred to as dshiver; SmaltAlign; viral-ngs; and V-pipe) using both simulated and real-world HIV-1 paired-end short-read datasets and default settings. All four pipelines produced consensus genome assemblies with high quality metrics (genome fraction recovery, mismatch and indel rates, variant calling F1 scores) when the reference sequence used for assembly had high similarity to the analyzed sample. The shiver and SmaltAlign pipelines (but not viral-ngs and V-Pipe) also showed robust performance with more divergent samples (non-matching subtypes). With empirical datasets, SmaltAlign and viral-ngs exhibited an order of magnitude shorter runtime compared to V-Pipe and shiver. In terms of applicability, V-Pipe provides the broadest functionalities, SmaltAlign and dshiver combine user-friendliness with robustness, while the use of viral-ngs requires less computational resources compared to other pipelines. In conclusion, if a closely matched reference sequence is available, all pipelines can reliably reconstruct viral consensus genomes; therefore, differences in user-friendliness and runtime may guide the choice of the pipeline in a particular setting. If a matched reference sequence cannot be selected, we recommend shiver or SmaltAlign for robust performance. The new Dockerized version of shiver offers ease of use in addition to the accuracy and robustness of the original pipeline. Full article

High-throughput sequencing (HTS) technologies may be a useful tool for testing imported plant germplasm for multiple pathogens present in a sample, offering strain-generic detection not offered by most PCR-based assays. Metatranscriptomics (RNAseq) and tiled amplicon PCR (TA-PCR) were tested as HTS-based techniques to detect viruses present in low titres. Strawberry mottle virus (SMoV), an RNA virus, and strawberry vein banding virus (SVBV), a DNA virus, were selected for comparison of RNAseq and TA-PCR with quantitative PCR assays. RNAseq of plant ribosomal RNA-depleted samples of low viral titre was used to obtain datasets from 3 M to 120 M paired-end (PE) reads. RNAseq demonstrated PCR-like sensitivity, able to detect as few as 10 viral copies/µL when 60 million (M) PE reads were generated. The custom TA-PCR primer panels designed for each virus were successfully used to recover most of the reference genomes for each virus. Single- and multiple-target TA-PCR allowed the detection of viruses in samples with around 10 viral copies/µL with a minimum continuous sequence length recovery of 500 bp. The limit of detection of the HTS-based protocols described here is comparable to that of quantitative PCR assays. This work lays the groundwork for an increased flexibility in HTS detection of plant viruses. Full article

Human noroviruses (HuNoVs), the most prevalent viral contaminant in food, account for a substantial proportion of nonbacterial gastroenteritis cases. Extensive work has been focused on the diagnosis of HuNoVs in clinical samples, whereas the availability of sensitive detection methods for their detection in food is lacking. Here, we developed a virus enrichment approach utilizing graphene-based nanocomposites (CTAB-rGO-Fe₃O₄) that does not rely on large instruments and is suitable for on-site food pretreatment. The recovery efficiency of the developed virus enrichment procedure for serially diluted GII.4 norovirus ranged from 10.06 to 72.67% in strawberries and from 2.66 to 79.65% in oysters. Furthermore, we developed a real-time recombinase polymerase amplification (real-time RPA) assay, which can detect as low as 1.22 genome copies µL⁻¹ of recombinant plasmid standard and has no cross-reactivity with genomes of astrovirus, rotavirus, adenovirus, and MS2 bacteriophage. Notably, the combined virus enrichment and real-time RPA detection assay enhanced the detection limits to 2.84 and 37.5 genome copies g⁻¹ in strawberries and oysters, respectively, compared to those of qPCR. Our strategy, the graphene-based virus enrichment method combined with real-time RPA, presents a promising tool for sensitively detecting HuNoVs in food samples. Full article

Wastewater surveillance is crucial for the epidemiological monitoring of SARS-CoV-2. Various concentration techniques, such as skimmed milk flocculation (SMF) and polyethylene glycol (PEG) precipitation, are employed to isolate the virus effectively. This study aims to compare these two methods and determine the one with the superior recovery rates. From February to December 2021, 24-h wastewater samples were collected from the Ioannina Wastewater Treatment Plant’s inlet and processed using both techniques. Subsequent viral genome isolation and a real-time RT-qPCR detection of SARS-CoV-2 were performed. The quantitative analysis demonstrated a higher detection sensitivity with a PEG-based concentration than SMF. Moreover, when the samples were positive by both methods, PEG consistently yielded higher viral loads. These findings underscore the need for further research into concentration methodologies and the development of precise protocols to enhance epidemiological surveillance through wastewater analysis. Full article

Background: Newly generated cardiomyocytes (NGCs) concur with the recovery of human myocarditis occurring spontaneously in around 50% of cases. However, NGCs decline with age, and their modality of myocardial homing and integration are still unclear. Methods: We retrospectively assessed NGCs in 213 consecutive patients with endomyocardial biopsy denoting acute myocarditis, with normal coronaries and valves. Tissue samples were processed for histology (H&E), immunohistochemistry for the evaluation of inflammatory infiltrates, immunostaining for alpha-sarcomeric-actin, junctional connexin-43, Ki-67, and phosphorylated STAT3 (p-STAT3), and Western blot (WB) for HMGB1. Frozen samples were analyzed using polymerase chain reaction (PCR) for cardiotropic viruses. Controls included 20 normal surgical biopsies. Results: NGCs were defined as small myocytes (diameter < 10 µm) with nuclear positivity to Ki-67 and p-STAT3 and positive immunostaining for cytoplasmic α-sarcomeric actin and connexin-43. Their number/mm² in relation to age and pathway of integration was evaluated. NGCs crossed the membrane and grew integrated within the empty necrotic myocytes. NGC mean diameter was 6.6 ± 3.34 vs. 22.5 ± 3.11 µm adult cells; their number, in comparison to LVEF, was 86.3 ± 10.3/mm² in patients between 18 and 40 years, 50.4 ± 13.8/mm² in those between 41 and 60, and 15.1 ± 5.7/mm² in those between 61 and 80. Control NGCs’ mean diameter was 0.2 ± 0.2 mm². PCR was positive for viral genomes in 16% of cases; NGCs were not statistically different in viral and non-viral myocarditis. WB analysis revealed a higher expression of HMGB1 in myocarditis compared to myocardial controls. Conclusions: NGCs are constantly recognizable in acute human myocarditis. Their number declines with age. Their integration within necrotic myocytes allows for the preservation of the cardiac structure and function. Full article

Retinitis pigmentosa (RP) poses a significant threat to eye health worldwide, with prevalence rates of 1 in 5000 worldwide. This genetically diverse retinopathy is characterized by the loss of photoreceptor cells and atrophy of the retinal pigment epithelium. Despite the involvement of more than 3000 mutations across approximately 90 genes in its onset, finding an effective treatment has been challenging for a considerable time. However, advancements in scientific research, especially in gene therapy, are significantly expanding treatment options for this most prevalent inherited eye disease, with the discovery of new compounds, gene-editing techniques, and gene loci offering hope for more effective treatments. Gene therapy, a promising technology, utilizes viral or non-viral vectors to correct genetic defects by either replacing or silencing disease-causing genes, potentially leading to complete recovery. In this review, we primarily focus on the latest applications of gene editing research in RP. We delve into the most prevalent genes associated with RP and discuss advancements in genome-editing strategies currently employed to correct various disease-causing mutations. Full article

Before December 2020, Antarctica had remained free of COVID-19 cases. The main concern during the pandemic was the limited health facilities available at Antarctic stations to deal with the disease as well as the potential impact of SARS-CoV-2 on Antarctic wildlife through reverse zoonosis. In December 2020, 60 cases emerged in Chilean Antarctic stations, disrupting the summer campaign with ongoing isolation needs. The SARS-CoV-2 RNA was detected in the wastewater of several scientific stations. In Antarctica, treated wastewater is discharged directly into the seawater. No studies currently address the recovery of infectious virus particles from treated wastewater, but their presence raises the risk of infecting wildlife and initiating new replication cycles. This study highlights the initial virus detection in wastewater from Antarctic stations, identifying viral RNA via RT-qPCR targeting various genomic regions. The virus’s RNA was found in effluent from two wastewater plants at Maxwell Bay and O’Higgins Station on King George Island and the Antarctic Peninsula, respectively. This study explores the potential for the reverse zoonotic transmission of SARS-CoV-2 from humans to Antarctic wildlife due to the direct release of viral particles into seawater. The implications of such transmission underscore the need for continued vigilance and research. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the worldwide COVID-19 pandemic. Animal models are extremely helpful for testing vaccines and therapeutics and for dissecting the viral and host factors that contribute to disease severity and transmissibility. Here, we report the assessment and comparison of intranasal and small particle (~3 µm) aerosol SARS-CoV-2 exposure in ferrets. The primary endpoints for analysis were clinical signs of disease, recovery of the virus in the upper respiratory tract, and the severity of damage within the respiratory tract. This work demonstrated that ferrets were productively infected with SARS-CoV-2 following either intranasal or small particle aerosol exposure. SARS-CoV-2 infection of ferrets resulted in an asymptomatic disease course following either intranasal or small particle aerosol exposure, with no clinical signs, significant weight loss, or fever. In both aerosol and intranasal ferret models, SARS-CoV-2 replication, viral genomes, and viral antigens were detected within the upper respiratory tract, with little to no viral material detected in the lungs. The ferrets exhibited a specific IgG immune response to the SARS-CoV-2 full spike protein. Mild pathological findings included inflammation, necrosis, and edema within nasal turbinates, which correlated to positive immunohistochemical staining for the SARS-CoV-2 virus. Environmental sampling was performed following intranasal exposure of ferrets, and SARS-CoV-2 genomic material was detected on the feeders and nesting areas from days 2–10 post-exposure. We conclude that both intranasal and small particle aerosol ferret models displayed measurable parameters that could be utilized for future studies, including transmission studies and testing SARS-CoV-2 vaccines and therapeutics. Full article

Wolbachia are obligate intracellular bacteria that occur in insects and filarial worms. Strains that infect insects have genomes that encode mobile genetic elements, including diverse lambda-like prophages called Phage WO. Phage WO packages an approximately 65 kb viral genome that includes a unique eukaryotic association module, or EAM, that encodes unusually large proteins thought to mediate interactions between the bacterium, its virus, and the eukaryotic host cell. The Wolbachia supergroup B strain, wStri from the planthopper Laodelphax striatellus, produces phage-like particles that can be recovered from persistently infected mosquito cells by ultracentrifugation. Illumina sequencing, assembly, and manual curation of DNA from two independent preparations converged on an identical 15,638 bp sequence that encoded packaging, assembly, and structural proteins. The absence of an EAM and regulatory genes defined for Phage WO from the wasp, Nasonia vitripennis, was consistent with the possibility that the 15,638 bp sequence represents an element related to a gene transfer agent (GTA), characterized by a signature head–tail region encoding structural proteins that package host chromosomal DNA. Future investigation of GTA function will be supported by the improved recovery of physical particles, electron microscopic examination of potential diversity among particles, and rigorous examination of DNA content by methods independent of sequence assembly. Full article