Search Results (66)

The CRISPR/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4–103.5%. This dual-amplification strategy—operating without target pre-amplification—featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases. Full article

Human parvovirus B19 (B19V) follows a well-documented cyclical epidemiology, with peaks occurring every 3–4 years. However, recent reports indicate an unusual resurgence in B19V infections across multiple countries, prompting increased surveillance. This study analyzed molecular diagnostic assay results from 826 unique-patient samples tested for B19V DNA between 2019 and 2024 at a large Italian tertiary-care hospital, covering pre-, during, and post-COVID-19 years. Overall, 80 of 826 patients (9.7%) tested positive for B19V DNA. A significant increase in positivity was observed in 2024 (23.4%), with a peak in May, representing an eightfold rise compared to 2019–2020. Despite this surge, the distribution of positive cases across population categories remained consistent with previous years, with 32 of 80 (40.0%) positive samples from pregnant women and 27 of 80 (33.8%) from hematology/oncology patients. Among 66 B19V DNA-positive patients with available serology, 4 of 66 (6.1%)—all immunocompromised—lacked detectable IgM/IgG despite high B19V DNA levels (7.8 log₁₀ IU/mL). These findings highlight the importance of integrating molecular and serological diagnostics, particularly in high-risk populations. Given the potential impact of the COVID-19 pandemic on B19V circulation, continued surveillance is essential to determine whether this resurgence represents a temporary fluctuation or a sustained epidemiological shift. Full article

Human parvovirus B19 (B19V) is a major human pathogen in which the ssDNA genome is replicated within the nucleus of infected human erythroid progenitor cells (EPCs) through a process involving both cellular and viral proteins, including the non-structural protein (NS)1. We previously characterized the interaction between NS1 classical nuclear localization signal (cNLS: GACHAKKPRIT-182) and host cell importin (IMP)α and proposed it as a potential target for antiviral drug development. Here, we further extend on such findings. First, we demonstrate that NS1 nuclear localization is required for viral production since introducing the K177T substitution in a cloned, infectious viral genome resulted in a non-viable virus. Secondly, we demonstrate that the antiparasitic drug ivermectin (IVM), known to inhibit the IMPα/β dependent nuclear import pathway, could impair the NS1-NLS:IMPα interaction and suppress viral replication in UT7/EpoS1 cells in a dose-dependent manner. We also show that a panel of structurally related avermectins (AVMs) can dissociate the NS1-NLS:IMPα complex with half-maximal inhibitory concentrations in the nanomolar range. Among them, Eprinomectin emerged as the most selective inhibitor of B19V replication, with a selectivity index of c. 5.0. However, when tested in EPCs generated from peripheral blood mononuclear cells, which constitute a cellular population close to the natural target cells in bone marrow, the inhibitory effect of IVM and Eprinomectin was demonstrated to a lesser extent, and both compounds exhibited high toxicity, thus highlighting the need for more specific inhibitors of the NS1-NLS:IMPα interaction. Full article

Ancient human viruses have been detected in ancient DNA (aDNA) samples of both Anatomically Modern Humans and Neanderthals. Reconstructing genomes from aDNA using reference mapping presents numerous problems due to the unique nature of ancient samples, their degraded state, smaller read sizes and the limitations of current methodologies. The spurious alignments of reads to reference sequences (mapping) are a main source of false positives in aDNA assemblies and the assessment of signal-to-noise ratios is essential to differentiate bona fide reconstructions from random, noisy assemblies. Here, we analyzed the statistical distributions of viral genome assemblies, ancient and modern, and their respective random “mock” controls used to evaluate the signal-to-noise ratio. We tested if differences between real and random assemblies could be detected from their statistical distributions. Our analysis shows that the coverage distributions of (1) real viral aDNA assemblies of adenovirus (ADV), herpesvirus (HSV) and papillomavirus (HPV) do not follow power laws nor log-normal laws, (2) (ADV) and control aDNA assemblies are well approximated by log-normal laws, (3) negative control parvovirus B19 (real and random) follow a power law with infinite variance and (4) the mapDamage negative control with non-ancient DNA (modern ADV) and the mapDamage positive control (human mtDNA) are well approximated by the negative binomial distribution, consistent with the Lander–Waterman model. Our results show that the tails of the distributions of aDNA and their controls reveal the weight of random effects and can differentiate spurious assemblies, or false positives, from bona fide assemblies. Full article

Human Parvovirus B19 (hB19V) is a widespread virus, causing erythema infectiosum in children and several clinical manifestations from acute to persistent infections in adults. In early 2024, ECDC reported an increased human Parvovirus B19 circulation in 14 European countries. A hB19V outbreak was also reported in Tuscany, Italy, prompting a detailed investigation of its genetic characteristics. In this context, through strict monitoring of circulating strains via next-generation sequencing (NGS), we carried out a phylogenetic analysis based on the whole of hB19V genomes. Phylogenetic clustering assigned all isolates to the G1a genotype, although with some mutations in NS1, VP1, and VP2, compared to the reference strains. Further characterization of these variants is necessary to fully assess their potential implications for public health. This study provides valuable insights into the spread of Parvovirus B19 and underlines the importance of continuous genomic surveillance to monitor and respond to possible hB19V epidemics that could impact public health. Full article

Gene II Protein (Gp2/P2) is a nicking enzyme of the M13 bacteriophage that plays a role in the DNA replication of the viral genome. P2 recognizes a specific sequence at the f1 replication origin and nicks one of the strands and starts replication. This study was conducted to address the limitations of previous experiments, improve methodologies, and precisely determine the biochemical activity conditions of the P2 enzyme in vitro. For these purposes, the gene encoding P2 was cloned in Escherichia coli and expressed as a hybrid protein together with a green fluorescent protein (P2-GFP). P2-GFP was purified via metal affinity chromatography, and its nicking activity was determined by conversion of supercoiled DNA to open circular or linear forms. We discovered that, among the two loops of the f1 origin defined previously, P2 can recognize just the A1 loop. When a supercoiled plasmid containing the f1 origin was treated with P2-GFP, the plasmid was present in an open circular form, indicating that a nick was created on only one of the strands. However, when the A1 loop sequence was inserted into the 3′ ends of both strands by cloning a PCR product obtained by primers with the A1 loop sequence, the plasmid was linearized by treatment with P2-GFP, indicating that nicks were created on both strands. Certain infectious diseases are caused by single-stranded DNA viruses, and some of them have specific nicking enzymes that enable strand displacement and free 3′ end of a single strand that works as a primer for their replication mechanisms like M13 bacteriophages, such as parvovirus B19. Despite there being different host viruses such as bacteria and humans, their DNA replication mechanisms are very similar in this concept. Investigating the features of the P2-nicking enzyme may deepen the understanding of human pathogenic single-stranded viruses and facilitate the development of drugs that inhibit viral replication. Full article

Neurological manifestations associated with human parvovirus B19 (B19V) infections are rare and varied. Acute encephalitis and encephalopathy are the most common, accounting for 38.8% of all neurological manifestations associated with human B19V. Herein, we report on the clinical features of 13 laboratory-confirmed human cases of B19V-associated encephalitis in Senegal in the framework of a hospital-based surveillance of acute viral encephalitis conducted from 2021 to 2023. Overall, B19V was detected from 13 cerebrospinal fluid samples using specific real time PCR. The mean age was 16.7 years among B19V-positive patients, with a higher prevalence in 0–5-year-old children and the sex ratio (male/female) was 2.25. The B19V-positive patients mainly exhibited hypoleukocytosis, normal glycorrhachia, and normal proteinorrachia in the cerebrospinal fluid. While the main neurological symptoms included meningeal and infectious syndromes. Furthermore, three complete B19V genome sequences were successfully characterized using next-generation sequencing. The newly characterized sequences belonged to the genotype 1a and represent, to date, the first complete B19V genome sequences from Senegal. These sequences could be useful not only in future phylodynamic studies of B19V but also in the development of prevention or treatment countermeasures. Our study is noteworthy for the identification of acute B19V-associated encephalitis in Senegal More investigations on the risk factors associated with B19V transmission in Africa are warranted. Full article

Viruses are the most common congenital infections in humans and an important cause of foetal malformations, neonatal morbidity, and mortality. The effects of these infections, which are transmitted in utero (transplacentally), during childbirth or in the puerperium depend on the timing of the infections. These vary from miscarriages (usually with infections in very early pregnancy), congenital malformations (when the infections occur during organogenesis) and morbidity (with infections occurring late in pregnancy, during childbirth or after delivery). The most common of these viruses are cytomegalovirus, hepatitis, herpes simplex type-2, parvovirus B19, rubella, varicella zoster and zika viruses. There are currently very few efficacious antiviral agents licensed for use in pregnancy. For most of these infections, therefore, prevention is mainly by vaccination (where there is a vaccine). The administration of immunoglobulins to those exposed to the virus to offer passive immunity or appropriate measures to avoid being infected would be options to minimise the infections and their consequences. In this review, we discuss some of the congenital and perinatal infections and their consequences on both the mother and fetus and their management focusing mainly on prevention. Full article

Neutrophil extracellular traps (NETs) formation, namely NETosis, is implicated in antiphospholipid syndrome (APS)-related thrombosis in various autoimmune disorders such as systemic lupus erythematosus (SLE) and APS. Human parvovirus B19 (B19V) infection is closely associated with SLE and APS and causes various clinical manifestations such as blood disorders, joint pain, fever, pregnancy complications, and thrombosis. Additionally, B19V may trigger the production of autoantibodies, including those against nuclear and phospholipid components. Thus, exploring the connection between B19V, NETosis, and thrombosis is highly relevant. An in vitro NETosis model using differentiated HL-60 neutrophil-like cells (dHL-60) was employed to investigate the effect of B19V-VP1u IgG on NETs formation. A venous stenosis mouse model was used to test how B19V-VP1u IgG-mediated NETs affect thrombosis in vivo. The NETosis was observed in the dHL-60 cells treated with rabbit anti-B19V-VP1u IgG and was inhibited in the presence of either 8-Br-cAMP or CGS216800 but not GSK484. Significantly elevated reactive oxygen species (ROS), myeloperoxidase (MPO), and citrullinated histone (Cit-H3) levels were detected in the dHL60 treated with phorbol myristate acetate (PMA), human aPLs IgG and rabbit anti-B19V-VP1u IgG, respectively. Accordingly, a significantly larger thrombus was observed in a venous stenosis-induced thrombosis mouse model treated with PMA, human aPLs IgG, rabbit anti-B19V-VP1u IgG, and human anti-B19V-VP1u IgG, respectively, along with significantly increased amounts of Cit-H3-, MPO- and CRAMP-positive infiltrated neutrophils in the thrombin sections. This research highlights that anti-B19V-VP1u antibodies may enhance the formation of NETosis and thrombosis and implies that managing and treating B19V infection could lower the risk of thrombosis. Full article

Respiratory viral infections pose a significant public health threat, particularly in children and older adults, with high mortality rates. Some of these pathogens are the human respiratory syncytial virus (hRSV), severe acute respiratory coronavirus-2 (SARS-CoV-2), influenza viruses (IV), human parvovirus B19 (B19V), and human bocavirus 1 (HBoV1). These viruses cause various respiratory symptoms, including cough, fever, bronchiolitis, and pneumonia. Notably, these viruses can also impact the central nervous system (CNS), leading to acute manifestations such as seizures, encephalopathies, encephalitis, neurological sequelae, and long-term complications. The precise mechanisms by which these viruses affect the CNS are not fully understood. Glial cells, specifically microglia and astrocytes within the CNS, play pivotal roles in maintaining brain homeostasis and regulating immune responses. Exploring how these cells interact with viral pathogens, such as hRSV, SARS-CoV-2, IVs, B19V, and HBoV1, offers crucial insights into the significant impact of respiratory viruses on the CNS. This review article examines hRSV, SARS-CoV-2, IV, B19V, and HBoV1 interactions with microglia and astrocytes, shedding light on potential neurological consequences. Full article

Parvovirus B19 (B19V) is a human pathogen belonging to the Parvoviridae family. It is widely diffused in the population and responsible for a wide range of diseases, diverse in pathogenetic mechanisms, clinical course, and severity. B19V infects and replicates in erythroid progenitor cells (EPCs) in the bone marrow leading to their apoptosis. Moreover, it can also infect, in an abortive manner, a wide set of different cell types, normally non-permissive, and modify their normal physiology. Differences in the characteristics of virus–cell interaction may translate into different pathogenetic mechanisms and clinical outcomes. Joint involvement is a typical manifestation of B19V infection in adults. Moreover, several reports suggest, that B19V could be involved in the pathogenesis of some autoimmune rheumatologic diseases such as rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), systemic sclerosis (SSc), systemic lupus erythematosus (SLE), or vasculitis. This review provides basic information on the B19 virus, highlights characteristics of viral infection in permissive and non-permissive systems, and focuses on recent findings concerning the pathogenic role of B19V in rheumatologic diseases. Full article

Brucellosis is a global problem, with the causative agent being the genus Brucella. B. canis can cause undulant fever in dogs, which is a zoonotic disease that can spread not only among dogs but also to humans. This poses a public health threat to society. In this study, a rapid and straightforward immune colloidal gold test strip was developed for the diagnosis of canine brucellosis through the detection of anti-LPS antibodies in serum samples. Rabbit anti-canine IgG conjugated with colloidal gold was employed as the colloidal gold-labeled antibody. The extracted high-purity R-LPS was employed as the capture antigen in the test line (T-line), while goat anti-rabbit IgG was utilized as the capture antibody in the control line (C-line). The colloidal gold strip exhibited high specificity in the detection of brucellosis, with no cross-reaction observed with the common clinical canine diseases caused by Canine coronavirus (CCV), Canine distemper virus (CDV), and Canine parvovirus (CPV). In comparison to the commercial iELISA kit, the sensitivity and specificity of the colloidal gold test strip were found to be 95.23% and 98.76%, respectively. The diagnostic coincidence rate was 98.47%. The findings of this study indicate that colloidal gold test strips may be employed as a straightforward, expeditious, sensitive, and specific diagnostic instrument for the identification of canine brucellosis, particularly in resource-limited regions. Full article

Autoimmune thyroiditis (Hashimoto’s thyroiditis) is the most common autoimmune disease. It most often manifests itself as hypothyroidism but may also present with euthyroidism or even hyperthyroidism. The etiopathogenesis of autoimmune thyroiditis is still unclear. However, in addition to genetic and epigenetic factors, many environmental factors are known to increase the risk of developing AIT. In this review, we aimed to collect and analyze data connected with environmental factors and autoimmune thyroiditis development. Our review indicates iodine intake, vitamin D deficiency, selenium deficiency, viral infections caused by Epstein–Barr Virus (EBV), Human parvovirus B19 (PVB19), Human herpesvirus 6A (HHV-6A) and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), bacterial infection caused by Helicobacter pylori, microbiome disruption, medications such as interferon-alpha and tyrosine kinase inhibitors, as well as stress, climate, and smoking can influence the risk of the occurrence of autoimmune thyroiditis. Having knowledge of risk factors allows for making changes to one’s diet and lifestyle that will reduce the risk of developing the disease and alleviate the course of autoimmune thyroiditis. Full article

Human parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2 (PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape. Full article

Animals, including wildlife, are part of One-Health concept since many infectious diseases can affect both humans and animals. In this study, 126 red foxes (Vulpes vulpes) from Northern Italy in 2022–2023 were tested by molecular assays for Protoparvovirus carnivoran 1 (PPVC-1), Canine adenovirus type 1 and 2 (CAdV-1 and CAdV-2), Circovirus canine (CanineCV), Canine distemper virus (CDV), and Leptospira spp. A total of 39 of 126 (30.9%) red foxes were infected with at least one pathogen and five of these were coinfected: 20/126 (15.9%) red foxes tested positive for PPVC-1, 3/126 (2.4%) for CAdV, 20/126 (15.9%) for CanineCV, and 2/126 (1.6%) for Leptospira spp. DNA. No foxes tested positive for CDV RNA. The pathogens identified were genetically analysed. New findings were reported such as a fox with multiple feline panleukopenia virus (FPV) and canine parvovirus type 2b (CPV-2b) infection associated with quasispecies dynamics, typical genetic characteristics of the identified CanineCV, and the first detection in red foxes of Leptospira ST198 related to L. interrogans serogroup Australis. Further studies are necessary to investigate the transmission between domestic animals and wildlife and to understand the role of red foxes in the maintenance of these pathogens not only in the wild but also in urban and peri-urban environments. Full article