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Viruses May 2024Viral co-infections are frequently observed among children, but whether specific viral interactions enhance or diminish the severity of respiratory disease is still...
Viral co-infections are frequently observed among children, but whether specific viral interactions enhance or diminish the severity of respiratory disease is still controversial. This study aimed to investigate the type of viral mono- and co-infections by also evaluating viral correlations in 3525 respiratory samples from 3525 pediatric in/outpatients screened by the Allplex Respiratory Panel Assays and with a Severe Acute Respiratory Syndrome-COronaVirus 2 (SARS-CoV-2) test available. Overall, viral co-infections were detected in 37.8% of patients and were more frequently observed in specimens from children with lower respiratory tract infections compared to those with upper respiratory tract infections (47.1% vs. 36.0%, = 0.003). SARS-CoV-2 and influenza A were more commonly detected in mono-infections, whereas human bocavirus showed the highest co-infection rate (87.8% in co-infection). After analyzing viral pairings using Spearman's correlation test, it was noted that SARS-CoV-2 was negatively associated with all other respiratory viruses, whereas a markedly significant positive correlation ( < 0.001) was observed for five viral pairings (involving adenovirus/human bocavirus/human enterovirus/metapneumoviruses/rhinovirus). The correlation between co-infection and clinical outcome may be linked to the type of virus(es) involved in the co-infection rather than simple co-presence. Further studies dedicated to this important point are needed, since it has obvious implications from a diagnostic and clinical point of view.
Topics: Humans; Coinfection; Respiratory Tract Infections; Italy; Child, Preschool; Child; Infant; Female; Male; Tertiary Care Centers; COVID-19; SARS-CoV-2; Hospitals, Pediatric; Adolescent; Human bocavirus; Virus Diseases; Hospitalization; Viruses; Infant, Newborn; Metapneumovirus
PubMed: 38793631
DOI: 10.3390/v16050750 -
Viruses Apr 2024A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel papillomavirus...
A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.
Topics: Animals; Fish Diseases; China; Phylogeny; Flatfishes; Parvoviridae Infections; Parvovirus; Genome, Viral; Papillomaviridae; Papillomavirus Infections; In Situ Hybridization, Fluorescence
PubMed: 38793587
DOI: 10.3390/v16050705 -
International Journal of Molecular... May 2024Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the...
Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze-thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.
Topics: Dependovirus; Genome, Viral; Genetic Vectors; Humans; Polymerase Chain Reaction; HEK293 Cells; Genetic Therapy; Viral Load
PubMed: 38791184
DOI: 10.3390/ijms25105149 -
Veterinary Sciences Apr 2024Duck hepatitis B virus (DHBV) is widely prevalent in global ducks and has been identified in Chinese geese with a high prevalence; the available detection techniques are...
Duck hepatitis B virus (DHBV) is widely prevalent in global ducks and has been identified in Chinese geese with a high prevalence; the available detection techniques are time-consuming and require sophisticated equipment. In this study, an assay combining multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) was developed for the efficient and rapid detection of DHBV. The primary reaction condition of the MIRA assay for DHBV detection was 10 min at 38 °C without a temperature cycler. Combined with the LFD assay, the complete procedure of the newly developed MIRA assay for DHBV detection required only 15 min, which is about one-fourth of the reaction time for routine polymerase chain reaction assay. And electrophoresis and gel imaging equipment were not required for detection and to read the results. Furthermore, the detection limit of MIRA was 45.6 copies per reaction, which is approximately 10 times lower than that of a routine polymerase chain reaction assay. The primer set and probe had much simpler designs than loop-mediated isothermal amplification, and they were only specific to DHBV, with no cross-reactivity with duck hepatitis A virus subtype 1 and duck hepatitis A virus subtype 3, goose parvovirus, duck enteritis virus, duck circovirus, or . In this study, we offer a simple, fast, and accurate assay method to identify DHBV in clinical serum samples of ducks and geese, which would be suitable for widespread application in field clinics.
PubMed: 38787163
DOI: 10.3390/vetsci11050191 -
Euro Surveillance : Bulletin Europeen... May 2024In France, blood donations are tested in pools of 96 samples for parvovirus B19 (B19V) DNA to discard plasma for fractionation when it contains high viral loads. Between...
In France, blood donations are tested in pools of 96 samples for parvovirus B19 (B19V) DNA to discard plasma for fractionation when it contains high viral loads. Between January 2015 and March 2024, B19V-positive donations decreased during the COVID-19 pandemic, followed by a strong rebound in 2023 and unusually high circulation during winter 2023/24 (ca 10 times higher December 2023-March 2024 vs the pre-pandemic period). Variations over time are probably related to measures implemented to limit SARS-CoV-2 spread.
Topics: Humans; Blood Donation; Blood Donors; COVID-19; DNA, Viral; France; Mass Screening; Pandemics; Parvoviridae Infections; Parvovirus B19, Human; Seasons; Viral Load
PubMed: 38785091
DOI: No ID Found -
Genome Biology May 2024Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with...
BACKGROUND
Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with severe DCM. However, the exact association between multiple mutations of Nexn and DCM remains unclear. Moreover, it is critical for the development of precise and effective therapeutics in treatments of DCM.
RESULTS
In our study, Nexn global knockout mice and mice carrying human equivalent G645del mutation are studied using functional gene rescue assays. AAV-mediated gene delivery is conducted through systemic intravenous injections at the neonatal stage. Heart tissues are analyzed by immunoblots, and functions are assessed by echocardiography. Here, we identify functional components of Nexilin and demonstrate that exogenous introduction could rescue the cardiac function and extend the lifespan of Nexn knockout mouse models. Similar therapeutic effects are also obtained in G645del mice, providing a promising intervention for future clinical therapeutics.
CONCLUSIONS
In summary, we demonstrated that a single injection of AAV-Nexn was capable to restore the functions of cardiomyocytes and extended the lifespan of Nexn knockout and G645del mice. Our study represented a long-term gene replacement therapy for DCM that potentially covers all forms of loss-of-function mutations in NEXN.
Topics: Animals; Cardiomyopathy, Dilated; Mice, Knockout; Mice; Genetic Therapy; Humans; Dependovirus; Myocytes, Cardiac; Disease Models, Animal; Mutation; Genetic Vectors; Gene Transfer Techniques
PubMed: 38783323
DOI: 10.1186/s13059-024-03283-x -
rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination.Nucleic Acids Research Jun 2024Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor...
Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.
Topics: Dependovirus; Humans; Promoter Regions, Genetic; Gene Editing; Homologous Recombination; HEK293 Cells; Capsid Proteins; Capsid; Mutation; Genetic Vectors; Transcription, Genetic; CRISPR-Cas Systems; Recombinational DNA Repair; Terminal Repeat Sequences; DNA Replication
PubMed: 38783157
DOI: 10.1093/nar/gkae401 -
Cell Jun 2024Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic...
Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.
Topics: Animals; Mice; Gene Regulatory Networks; Single-Cell Analysis; Forkhead Transcription Factors; Cerebral Cortex; Neurons; Female; Dependovirus; Humans; Nerve Tissue Proteins; CRISPR-Cas Systems; Genetic Vectors; Mice, Inbred C57BL; Transcriptome
PubMed: 38772369
DOI: 10.1016/j.cell.2024.04.050 -
Frontiers in Microbiology 2024Pseudorabies (PR) is a multi-animal comorbid disease caused by pseudorabies virus (PRV), which are naturally found in pigs. At the end of 2011, the emergence of PRV...
INTRODUCTION
Pseudorabies (PR) is a multi-animal comorbid disease caused by pseudorabies virus (PRV), which are naturally found in pigs. At the end of 2011, the emergence of PRV variant strains in many provinces in China had caused huge economic losses to pig farms. Rapid detection diagnosis of pigs infected with the PRV variant helps prevent outbreaks of PR. The immunochromatography test strip with colloidal gold nanoparticles is often used in clinical testing due to its low cost and high throughput.
METHODS
This study was designed to produce monoclonal antibodies targeting PRV through immunization of mice using the eukaryotic system to express the gE glycoprotein. Subsequently, paired monoclonal antibodies were screened based on their sensitivity and specificity for use in the preparation of test strips.
RESULTS AND DISCUSSION
The strip prepared in this study was highly specific, only PRV was detected, and there was no cross-reactivity with glycoprotein gB, glycoprotein gC, glycoprotein gD, and glycoprotein gE of herpes simplex virus and varicellazoster virus, porcine epidemic diarrhea virus, Senecavirus A, classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus. Moreover, it demonstrated high sensitivity with a detection limit of 1.336 × 10 copies/μL (the number of viral genome copies per microliter); the coincidence rate with the RT-PCR detection method was 96.4%. The strip developed by our laboratory provides an effective method for monitoring PRV infection and controlling of PR vaccine quality.
PubMed: 38765685
DOI: 10.3389/fmicb.2024.1399123 -
Frontiers in Microbiology 2024There are three major categories of waterfowl parvoviruses, namely goose parvovirus (GPV), Muscovy duck parvovirus, and novel goose parvovirus (NGPV). NGPV can infect...
INTRODUCTION
There are three major categories of waterfowl parvoviruses, namely goose parvovirus (GPV), Muscovy duck parvovirus, and novel goose parvovirus (NGPV). NGPV can infect both Cherry Valley ducks and mule ducks, resulting in short beaks and dwarfism syndrome, and the incidence of short beaks and dwarfism syndrome rises annually, posing a significant threat to the waterfowl breeding and the animal husbandry. Therefore, clarifying the biological characteristics and genetic evolution of NGPV is very important for the prevention and control of NGPV.
METHODS
Ducks with short beaks and dwarfism syndrome from Shandong and Henan Province were investigated by dissection and the tissue samples were collected for study. The NGPV genome was amplified by PCR, and the genome was analyzed for genetic evolution.
RESULTS
Eight strains of NGPV were isolated, which were designated as HZ0512, HZ0527, HZ0714, HZ0723, HZ0726, HZ0811, HZ0815, and HN0403. The nucleotide homology among these strains ranged from 99.9% to 100%. The eight strains, along with other NGPVs, belong to GPV. The eight strains showed a 92.5%-98.9% nucleotide homology with the classical GPV, while a 96.0%-99.9% homology with NGPV.Therefore, it can be deduced that there have been no major mutations of NGPV in Shandong and Henan provinces in recent years.
DISCUSSION
This study lays a theoretical foundation for further studying the genetic evolution and pathogenicity of NGPV, thereby facilitating the prevention and control of NGPV.
PubMed: 38765684
DOI: 10.3389/fmicb.2024.1373601