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Generation of nanobodies from transgenic 'LamaMice' lacking an endogenous immunoglobulin repertoire.Nature Communications Jun 2024Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in...
Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.
Topics: Animals; Single-Domain Antibodies; Camelids, New World; Immunoglobulin Heavy Chains; Mice, Transgenic; Mice; Spike Glycoprotein, Coronavirus; Lectins, C-Type; SARS-CoV-2; Immunoglobulin E; Humans; Dependovirus; Immunoglobulin G; COVID-19; B-Lymphocytes
PubMed: 38830864
DOI: 10.1038/s41467-024-48735-x -
The Science of the Total Environment Aug 2024No single microbial source tracking (MST) marker can be applied to determine the sources of fecal pollution in all water types. This study aimed to validate a...
No single microbial source tracking (MST) marker can be applied to determine the sources of fecal pollution in all water types. This study aimed to validate a high-throughput quantitative polymerase chain reaction (HT-qPCR) method for the simultaneous detection of multiple MST markers. A total of 26 fecal-source samples that had been previously collected from human sewage (n = 6) and ruminant (n = 3), dog (n = 6), pig (n = 6), chicken (n = 3), and duck (n = 2) feces in the Kathmandu Valley, Nepal, were used to validate 10 host-specific MST markers, i.e., Bacteroidales (BacHum, gyrB, BacR, and Pig2Bac), mitochondrial DNA (mtDNA) (swine, bovine, and Dog-mtDNA), and viral (human adenovirus, porcine adenovirus, and chicken/turkey parvovirus) markers, via HT-qPCR. Only Dog-mtDNA showed 100 % accuracy. All the tested bacterial markers showed a sensitivity of 100 %. Nine of the 10 markers were further used to identify fecal contamination in groundwater sources (n = 54), tanker filling stations (n = 14), drinking water treatment plants (n = 5), and river water samples (n = 6). The human-specific Bacteroidales marker BacHum and ruminant-specific Bacteroidales marker BacR was detected at a high ratio in river water samples (83 % and 100 %, respectively). The results of HT-qPCR were in agreement with the standard qPCR. The comparable performances of HT-qPCR and standard qPCR as well as the successful detection of MST markers in the fecal-source and water samples demonstrated the potential applicability of these markers for detecting fecal contamination sources via HT-qPCR.
Topics: Environmental Monitoring; Feces; Water Microbiology; Animals; Nepal; Real-Time Polymerase Chain Reaction; Humans; Sewage; Water Pollution
PubMed: 38821279
DOI: 10.1016/j.scitotenv.2024.173604 -
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 -
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