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PLoS Biology May 2024The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the...
The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.
Topics: Dependovirus; Humans; Gene Editing; Genetic Vectors; CRISPR-Cas Systems; Animals; HEK293 Cells; Genetic Therapy; CRISPR-Associated Proteins; Mice; Endodeoxyribonucleases; Bacterial Proteins
PubMed: 38814985
DOI: 10.1371/journal.pbio.3002619 -
Turkish Journal of Medical Sciences 2023Dorsal root ganglia (DRG) are structures containing primary sensory neurons. Intraganglionic (IG) and intrathecal (IT) applications are the most common methods used for... (Comparative Study)
Comparative Study
BACKGROUND/AIM
Dorsal root ganglia (DRG) are structures containing primary sensory neurons. Intraganglionic (IG) and intrathecal (IT) applications are the most common methods used for viral vector transfer to DRG. We aim to compare the efficiencies and pathological effects of IT and IG viral vector delivery methods to DRG, through in vivo imaging.
MATERIALS AND METHODS
Mice were divided into four groups of six each: IT, IG, IT-vehicle, and IG-vehicle. Adeno-associated virus (AAV) injection was performed for EGFP expression in IT/IG groups. DRGs were made visible through vertebral window surgery and visualized with multiphoton microscopy. After imaging, spinal cords and DRGs were removed and cleared, then imaged with light sheet microscopy.
RESULTS
No neuronal death was observed after IT injection, while the death rate was 17% 24 h after IG injection. EGFP expression efficiencies were 90%-95% of neurons in both groups. EGFP expression was only observed in targeted L2 DRG after IG injection, while it was observed in DRGs located between L1-L5 levels after IT injection.
CONCLUSION
IT injection is a more suitable method for labeling DRG neurons in neurodegenerative injury models. However, when the innervation of DRG needs to be specifically studied, IT injection reduces this specificity due to its spread. In these studies, IG injection is the most suitable method for labeling single DRG neurons.
Topics: Animals; Ganglia, Spinal; Injections, Spinal; Mice; Dependovirus; Green Fluorescent Proteins; Genetic Vectors; Male
PubMed: 38813001
DOI: 10.55730/1300-0144.5702 -
BMC Cardiovascular Disorders May 2024Sudden cardiac death (SCD) is a major public health issue worldwide. In the young (< 40 years of age), genetic cardiomyopathies and viral myocarditis, sometimes in...
Sudden cardiac death (SCD) is a major public health issue worldwide. In the young (< 40 years of age), genetic cardiomyopathies and viral myocarditis, sometimes in combination, are the most frequent, but underestimated, causes of SCD. Molecular autopsy is essential for prevention. Several studies have shown an association between genetic cardiomyopathies and viral myocarditis, which is probably underestimated due to insufficient post-mortem investigations. We report on four autopsy cases illustrating the pathogenesis of these combined pathologies. In two cases, a genetic hypertrophic cardiomyopathy was diagnosed in combination with Herpes Virus Type 6 (HHV6) and/or Parvovirus-B19 (PVB19) in the heart. In the third case, autopsy revealed a dilated cardiomyopathy and virological analyses revealed acute myocarditis caused by three viruses: PVB19, HHV6 and Epstein-Barr virus. Genetic analyses revealed a mutation in the gene coding for desmin. The fourth case illustrated a channelopathy and a PVB19/HHV6 coinfection. Our four cases illustrate the highly probable deleterious role of cardiotropic viruses in the occurrence of SCD in subjects with genetic cardiomyopathies. We discuss the pathogenetic link between viral myocarditis and genetic cardiomyopathy. Molecular autopsy is essential in prevention of these SCD, and a close collaboration between cardiologists, pathologists, microbiologists and geneticians is mandatory.
Topics: Humans; Myocarditis; Death, Sudden, Cardiac; Autopsy; Male; Adult; Female; Herpesvirus 6, Human; Parvovirus B19, Human; Cardiomyopathy, Dilated; Roseolovirus Infections; Cardiomyopathy, Hypertrophic; Parvoviridae Infections; Young Adult; Genetic Predisposition to Disease; Fatal Outcome; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Coinfection; Cause of Death; Mutation; Middle Aged
PubMed: 38811883
DOI: 10.1186/s12872-024-03913-z -
Microbial Pathogenesis Jul 2024This study prepared a novel monoclonal antibody (MAb) against mink enteritis parvovirus (MEV) and identified its antigen epitope. The antibody subclass is identified as...
This study prepared a novel monoclonal antibody (MAb) against mink enteritis parvovirus (MEV) and identified its antigen epitope. The antibody subclass is identified as IgG1, the titers of the MAb is up to 1:1 × 10 and keeps stably after low-temperature storage for 9 months or 11 passages of the MAb cells. The MAb can specifically recognize MEV in the cells in IFA, but not Aleutian disease virus (ADV) or canine distemper virus (CDV). Its antigen epitope was identified as a polypeptide containing 5 key amino acids (YAFGR) and the homology in 20 MEV strains, 4 canine parvovirus strains, and 4 feline panleukopenia virus strains was 100%. This study supplies a biological material for developing new methods to detect MEV.
Topics: Animals; Antibodies, Monoclonal; Epitopes; Mink enteritis virus; Distemper Virus, Canine; Antibodies, Viral; Antigens, Viral; Mink; Immunoglobulin G; Aleutian Mink Disease Virus; Parvovirus, Canine; Feline Panleukopenia Virus; Epitope Mapping; Mice; Mice, Inbred BALB C; Mink Viral Enteritis
PubMed: 38810766
DOI: 10.1016/j.micpath.2024.106709 -
Journal of Virological Methods Jul 2024In this report, a multiplex PCR method was developed for the detection of three diarrhea-associated viruses in mink, including circovirus (MCV), bocavirus (MBoV), and...
In this report, a multiplex PCR method was developed for the detection of three diarrhea-associated viruses in mink, including circovirus (MCV), bocavirus (MBoV), and enteritis virus (MEV). Three compatible sets of primers specific for each virus were designed respectively based on their conserved sequences. After optimization of the crucial factors such as primer concentration and annealing temperature in single and multiple amplification, three specific fragments were simultaneously amplified with the highest sensitivity and specificity in one PCR reaction. The fragments amplified were 259 bp (MCV),455 bp (MBoV) and 671 bp (MEV). The sensibility of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There were no cross-reactions with some relevant pathogens like mink coronavirus, canine distemper virus, and aleutian mink disease virus. In our study we analyzed viral DNA in mink fecal samples by multiplex PCR assay from China, which revealed the occurrence of MCV, MBoV, and MEV as 3.1 %, 5.7 %, and 9.8 %, respectively. The testing results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100 %. These results indicated that the method could provide technical support for rapid detection of the three diarrhea-associated viruses, and epidemiological investigation of mink viral diarrhea.
Topics: Animals; Mink; Multiplex Polymerase Chain Reaction; Sensitivity and Specificity; China; Diarrhea; DNA Primers; Feces; Circovirus; Bocavirus; Mink enteritis virus; Molecular Diagnostic Techniques
PubMed: 38801834
DOI: 10.1016/j.jviromet.2024.114958 -
Environmental Pollution (Barking, Essex... Aug 2024Environmental viruses in wastewater and sludge are widely recognized for their roles in waterborne diseases. However, previous studies mainly focused on RNA viruses, and...
Environmental viruses in wastewater and sludge are widely recognized for their roles in waterborne diseases. However, previous studies mainly focused on RNA viruses, and little is known about the diversity of DNA viral communities and their driving factors in municipal wastewater treatment environments. Herein, we conducted a pilot study to explore DNA virus profiles in municipal wastewater and recycled sludge by metagenomics method, and track their temporal changes in northern China. Results showed that 467 viral species were co-shared among all the samples. We identified six families of human viruses with a prevalence of 0.1%, which were rare but relatively stable in wastewater and sludge for six months. Adenoviridae, Parvoviridae, and Herpersviridae were the most dominant human viral families in municipal wastewater and recycled sludge. A time series of samples revealed that the dynamic changes of human DNA viruses were stable based on qPCR results, particularly for high-risk fecal-oral transmission viruses of adenovirus, bocavirus, polyomavirus, human gamma herpesvirus, human papillomavirus, and hepatitis B virus. Concentrations of Adenovirus (5.39-7.48 log copies/L) and bocavirus (4.36-7.48 log copies/L) were observed to be the highest in these samples compared to other viruses. Our findings demonstrated the DNA viruses' high prevalence and persistence in municipal wastewater treatment environments, highlighting the value of enhancing public health responses based on wastewater-based epidemiology.
Topics: China; Wastewater; DNA Viruses; Sewage; Humans; Metagenomics; Waste Disposal, Fluid; Environmental Monitoring
PubMed: 38797349
DOI: 10.1016/j.envpol.2024.124215 -
Poultry Science Jul 2024Short-beak and dwarf syndrome (SBDS) is caused by infection with novel goose parvovirus (NGPV), which leads to intestinal dysbiosis, developmental delay, short beak,...
Short-beak and dwarf syndrome (SBDS) is caused by infection with novel goose parvovirus (NGPV), which leads to intestinal dysbiosis, developmental delay, short beak, lameness, and paralysis in ducks and is the cause of skeletal health problems. NGPV infection can cause intestinal microbial disturbances, but it is still unclear whether the intestinal microbiota affects the pathogenicity of NGPV. Here, the effects of intestinal microbiota on NGPV-induced SBDS in Cherry Valley ducks were assessed by establishing a duck model for gut microflora depletion/reestablishment through antibiotics (ABX) treatment/fecal microbiota transplanted (FMT). By measuring body weight, beak length, beak width and tarsal length, we found that SBDS clinical symptoms were alleviated in ducks treated with ABX, but not in FMT ducks. Next, we conducted a comprehensive analysis of bone metabolism, gut barrier integrity, and inflammation levels using quantitative real-time PCR (qPCR), enzyme linked immunosorbent assay (ELISA), biochemical analysis and histological analysis. The results showed that ABX treatment improved bone quality reduced bone resorption, mitigated tissue lesions, protected intestinal barrier integrity, and inhibited systemic inflammation in NGPV-infected ducks. Moreover, cecal microflora composition and short-chain fatty acids (SCFAs) production were examined by bacterial 16S rRNA sequencing and gas chromatography. The results revealed that ABX treatment mitigated the decreased abundance of Firmicutes and Bacteroidota in NGPV-infected ducks, as well as increased SCFAs production. Furthermore, ABX treatment reduced the mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) and nuclear factor κB (NF-κB) expression, which are correlated with systemic inflammation in SBDS ducks. These findings suggested that intestinal microflora depletion alleviated NGPV-induced SBDS by maintaining intestinal homeostasis, inhibiting inflammatory response and alleviating bone resorption. These results provide evidence for the pivotal role of intestinal microbiota in the process of SBDS and contribute a theoretical basis for the feasibility of microecological preparation as a method to control SBDS.
Topics: Animals; Gastrointestinal Microbiome; Ducks; Poultry Diseases; Parvoviridae Infections; Parvovirinae; Anti-Bacterial Agents; Fecal Microbiota Transplantation
PubMed: 38795515
DOI: 10.1016/j.psj.2024.103853 -
Viruses May 2024Based on several clinical observations it was hypothesized that herpesviruses may influence the replication of human bocaviruses, the second known parvoviruses that have...
Based on several clinical observations it was hypothesized that herpesviruses may influence the replication of human bocaviruses, the second known parvoviruses that have been confirmed as human pathogens. While several cell lines support the growth of HSV-1, HBoV-1 was exclusively cultivated on air-liquid interface cultures, the latter being a rather complicated, slow, and low throughput system. One of the cell lines are T84 cells, which are derived from the lung metastasis of a colorectal tumor. In this study, we provide evidence that T84 also supports HBoV replication when cultivated as monolayers, while simultaneously being permissive for HSV-1. The cell culture model thus would enable co-infection studies of both viruses and is worth being optimized for high throughput studies with HBoV-1. Additionally, the study provides evidence for a supporting effect of HSV-1 on the replication and packaging of HBoV-1 progeny DNA into DNase-resistant viral particles.
Topics: Herpesvirus 1, Human; Humans; Virus Replication; Coinfection; Human bocavirus; Cell Line; Cell Line, Tumor; Cell Culture Techniques; Herpes Simplex; Parvoviridae Infections; Chlorocebus aethiops; Virus Cultivation
PubMed: 38793654
DOI: 10.3390/v16050773 -
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