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Biomolecular NMR Assignments Jun 2024Parvovirus B19 (B19V) is a human pathogen that is the causative agent of several diseases in infants and adults. Due to a lack of antivirals against this virus,...
Parvovirus B19 (B19V) is a human pathogen that is the causative agent of several diseases in infants and adults. Due to a lack of antivirals against this virus, treatment options are limited. The minor capsid protein of B19V has a unique N terminus, named VP1u, which is essential for infection. The VP1u encodes a receptor binding domain (RBD), necessary for host cell entry, and a phospholipase A2 (PLA) domain, crucial for endosomal escape during cellular trafficking. Both domains are indispensable for infection, making the RBD a plausible drug target for inhibitors against B19V, as it is located on the exterior surface of the virus. To date, no experimental structural information has been available for the VP1u component for any Parvovirus. Here we report the backbone NMR resonance assignments for the RBD of B19V and demonstrate it forms a stable structure. The backbone chemical shifts are in good agreement with a structure predicted by AlphaFold, validating that the RBD contains three helices connected by tight turns. This RBD construct can now be used for further NMR studies, including assignment of full-length VP1u, determination of protein-protein interaction interfaces, and development of B19 antivirals specific to the RBD domain.
PubMed: 38904726
DOI: 10.1007/s12104-024-10181-7 -
Euro Surveillance : Bulletin Europeen... Jun 2024From April 2023 to May 2024, an unusual epidemic of parvovirus B19 (B19V) infections occurred in France. The number of B19V IgM-positive serologies was four times higher...
From April 2023 to May 2024, an unusual epidemic of parvovirus B19 (B19V) infections occurred in France. The number of B19V IgM-positive serologies was four times higher than in the previous epidemic in 2019. Clinical data from emergency networks corroborated this observation. Morbidity and mortality consequences were observed in children through all data sources. In adults, the increase was only observed in laboratory-confirmed data. Physicians and decisionmakers should be informed in order to better prevent, diagnose and manage at-risk patients.
Topics: Humans; France; Parvovirus B19, Human; Disease Outbreaks; Adult; Female; Male; Child; Parvoviridae Infections; Immunoglobulin M; Adolescent; Child, Preschool; Middle Aged; Antibodies, Viral; Erythema Infectiosum; Young Adult; Infant; Aged
PubMed: 38904112
DOI: 10.2807/1560-7917.ES.2024.29.25.2400339 -
The Lancet. Infectious Diseases Jun 2024
PubMed: 38901439
DOI: 10.1016/S1473-3099(24)00373-6 -
Biotechnology Journal Jun 2024Recombinant adeno-associated virus (rAAV) is the leading vector for the delivery of gene therapies. However, low viral genome (VG) titers are common and the proportion...
Recombinant adeno-associated virus (rAAV) is the leading vector for the delivery of gene therapies. However, low viral genome (VG) titers are common and the proportion of "full" capsids containing the therapeutic gene payload can be highly variable. The coordinated molecular design of plasmids encoding viral components and Helper functions remains a major challenge for rAAV manufacturing. Here we present the design of improved Rep/Cap and Helper plasmids for rAAV2/8 production, (i) a Rep/Cap expression vector harboring independently controllable rep and cap genes and (ii) an improved Helper plasmid harboring E4 gene deletion variants. First, an optimized Rep/Cap vector utilized a truncated p5 promoter, a p5 cis-regulatory element at the 3' end in combination with a heterologous promoter to drive Cap expression and an additional copy of the rep52/40 gene to overexpress short Rep proteins. We demonstrate that Rep78 is essential for efficient rAAV2/8 production in HEK293 cells, and a higher ratio of short Rep to long Rep proteins enhances genome packaging. Second, we identified regulators and open reading frames within the Helper plasmid that contribute to increased rAAV2/8 production. While L4-33k/22k is integral to optimal production, the use of E4orf6-6/7 subset significantly enhanced VG titer. Together, an optimal combination of engineered Rep/Cap and Helper plasmid variants increased VG titer by 3.1-fold. This study demonstrates that configuring and controlling the expression of the different AAV genetic elements contributes toward high rAAV production and product quality (full/empty capsid ratio).
Topics: Dependovirus; HEK293 Cells; Humans; Genetic Vectors; Plasmids; Promoter Regions, Genetic; Genome, Viral; Viral Proteins
PubMed: 38900035
DOI: 10.1002/biot.202300685 -
Journal of Veterinary Internal Medicine Jun 2024Equine parvovirus hepatitis (EqPV-H) can cause Theiler's disease and subclinical hepatitis in horses.
BACKGROUND
Equine parvovirus hepatitis (EqPV-H) can cause Theiler's disease and subclinical hepatitis in horses.
OBJECTIVES
Assess the frequency of subclinical EqPV-H infection in hospitalized horses and to study viral transmission by investigating potential shedding routes.
ANIMALS
One hundred sixteen equids, that presented to the University Equine Hospital of the University of Veterinary Medicine Vienna between February 2021 and March 2022, for causes other than hepatopathy.
METHODS
In this cross-sectional study, samples (serum, feces, nasal, and buccal swabs) of hospitalized horses were collected. Sera were screened for the presence of anti-EqPV-H antibodies by a luciferase immunoprecipitation system assay. Quantitative PCR was used for the detection of EqPV-H DNA in the samples and a nested PCR was used for further validation.
RESULTS
Seroprevalence was 10.3% (12/116) and viremia occurred in 12.9% (15/116) of the serologically positive horses. The detected viral load in serum varied from non-quantifiable amount to 1.3 × 10 genome equivalents per milliliter of serum. A low viral load of EqPV-H DNA was detected in 2 nasal swabs and 1 fecal sample.
CONCLUSION AND CLINICAL IMPORTANCE
EqPV-H DNA was detected in nasal secretions and feces of viremic horses, which could pose a risk to naive hospitalized horses. It is advisable to screen hospitalized horses that are potential donors of blood or plasma to reduce the risk of iatrogenic EqPV-H transmission.
PubMed: 38899610
DOI: 10.1111/jvim.17129 -
Applied Microbiology and Biotechnology Jun 2024Recombinant adeno-associated virus (rAAV) is a major gene delivery vehicle. We have constructed a stable rAAV producer cell line by integrating essential rAAV genome,... (Comparative Study)
Comparative Study
Recombinant adeno-associated virus (rAAV) is a major gene delivery vehicle. We have constructed a stable rAAV producer cell line by integrating essential rAAV genome, viral and helper genes into the genome of HEK293 cell under the control of inducible promoters. Upon induction, the cell line produces transducing rAAV. To gain insight into enhancing rAAV productivity and vector quality, we performed a comparative transcriptomic and proteomic analysis of our synthetic cell line GX2 and two wild-type AAV (wtAAV) production systems, one by virus co-infection and the other by multi-plasmid transfection. The three systems had different kinetics in viral component synthesis but generated comparable copies of AAV genomes; however, the capsid titer of GX2 was an order of magnitude lower compared to those two wtAAV systems, indicating that its capsid production may be insufficient. The genome packaging efficiency was also lower in GX2 despite it produced higher levels of Rep52 proteins than either wtAAV systems, suggesting that Rep52 protein expression may not limit genome packaging. In the two wtAAV systems, VP were the most abundant AAV proteins and their levels continued to increase, while GX2 had high level of wasteful cargo gene expression. Furthermore, upregulated inflammation, innate immune responses, and MAPK signaling, as well as downregulated mitochondrial functions, were commonly observed in either rAAV or wtAAV systems. Overall, this comparative multi-omics study provided rich insights into host cell and viral factors that are potential targets for genetic and process intervention to enhance the productivity of synthetic rAAV producer cell lines. KEY POINTS: • wtAAV infection was more efficient in producing full viral particles than the synthetic cell GX2. • Capsid protein synthesis, genome replication, and packaging may limit rAAV production in GX2. • wtAAV infection and rAAV production in GX2 elicited similar host cell responses.
Topics: Dependovirus; Humans; HEK293 Cells; Proteomics; Transcriptome; Genetic Vectors; Kinetics; Genome, Viral; Gene Expression Profiling; Proteome
PubMed: 38896252
DOI: 10.1007/s00253-024-13203-5 -
Translational Vision Science &... Jun 2024To report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a...
PURPOSE
To report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector.
DESIGN
Single-center, retrospective chart review.
METHODS
In this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated.
RESULTS
Three out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector.
CONCLUSIONS
We observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes.
TRANSLATIONAL RELEVANCE
Caution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies.
Topics: Humans; Retrospective Studies; Genetic Vectors; Genetic Therapy; Male; Female; Child; Visual Acuity; Tomography, Optical Coherence; cis-trans-Isomerases; Dependovirus; Atrophy; Visual Fields
PubMed: 38888288
DOI: 10.1167/tvst.13.6.11 -
Protein Science : a Publication of the... Jul 2024Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of...
Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.
Topics: Dependovirus; Capsid Proteins; Genetic Vectors; Genetic Therapy; Capsid; Hydrogen Deuterium Exchange-Mass Spectrometry; Protein Stability; Humans; Protein Conformation; Models, Molecular
PubMed: 38888268
DOI: 10.1002/pro.5074 -
Journal of Medical Virology Jun 2024The diversity and evolution of the genomes of human bocavirus (HBoV), which causes respiratory diseases, have been scarcely studied. Here, we aimed to obtain and...
The diversity and evolution of the genomes of human bocavirus (HBoV), which causes respiratory diseases, have been scarcely studied. Here, we aimed to obtain and characterize HBoV genomes from patients's nasopharyngeal samples collected between 2017 and 2022 period (5 years and 7 months). Next-generation sequencing (NGS) used Illumina technology after having implemented using GEMI an in-house multiplex PCR amplification strategy. Genomes were assembled and analyzed with CLC Genomics, Mafft, BioEdit, MeV, Nextclade, MEGA, and iTol. A total of 213 genomes were obtained. Phylogeny classified them all as of Bocavirus 1 (HBoV1) species. Five HBoV1 genotypic clusters determined by hierarchical clustering analysis of 27 variable genome positions were scattered over the study period although with differences in yearly prevalence. A total of 167 amino acid substitutions were detected. Besides, coinfection was observed for 52% of the samples, rhinoviruses then adenoviruses (HAdVs) being the most common viruses. Principal component analysis showed that HBoV1 genotypic cluster α tended to be correlated with HAdV co-infection. Subsequent HAdV typing for HBoV1-positive samples and negative controls demonstrated that HAdVC species predominated but HAdVB was that significantly HBoV1-associated. Overall, we described here the first HBoV1 genomes sequenced for France. HBoV1 and HAdVB association deserves further investigation.
Topics: Humans; Human bocavirus; Genome, Viral; France; Phylogeny; Parvoviridae Infections; Female; Genotype; High-Throughput Nucleotide Sequencing; Child, Preschool; Male; Child; Adult; Infant; Middle Aged; Coinfection; Adolescent; Nasopharynx; Young Adult; Aged; Sequence Analysis, DNA; Genetic Variation; DNA, Viral
PubMed: 38888111
DOI: 10.1002/jmv.29706 -
PLoS Pathogens Jun 2024Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of...
Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy.
Topics: Dependovirus; Humans; Animals; Evolution, Molecular; Capsid Proteins; Selection, Genetic; Genetic Variation; Genetic Therapy
PubMed: 38885242
DOI: 10.1371/journal.ppat.1012260