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1.
Biotechnol J ; 19(3): e2300667, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38479987

RESUMO

The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.


Assuntos
Capsídeo , Parvovirinae , Humanos , Células HEK293 , Vetores Genéticos/genética , Dependovirus/genética , Plasmídeos/genética , Proteínas do Capsídeo/genética , Parvovirinae/genética
2.
Arch Virol ; 169(3): 52, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38378929

RESUMO

Parvoviruses are responsible for multiple diseases, and there is a critical need for effective antiviral therapies. Specific antiviral treatments for parvovirus infections are currently lacking, and the available options are mostly supportive and symptomatic. In recent years, significant research efforts have been directed toward understanding the molecular mechanisms of parvovirus replication and identifying potential targets for antiviral interventions. This review highlights the structure, pathogenesis, and treatment options for major viruses of the subfamily Parvovirinae, such as parvovirus B19 (B19V), canine parvovirus type 2 (CPV-2), and porcine parvovirus (PPV) and also describes different approaches in the development of antiviral alternatives against parvovirus, including drug repurposing, serendipity, and computational tools (molecular docking and artificial intelligence) in drug discovery. These advances greatly increase the likelihood of discoveries that will lead to potent antiviral strategies against different parvovirus infections.


Assuntos
Infecções por Parvoviridae , Parvovirinae , Parvovirus B19 Humano , Parvovirus , Animais , Suínos , Antivirais/farmacologia , Antivirais/uso terapêutico , Inteligência Artificial , Simulação de Acoplamento Molecular , Infecções por Parvoviridae/tratamento farmacológico
3.
Gene Ther ; 31(3-4): 175-186, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38200264

RESUMO

Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken ß-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5' and 3' ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.


Assuntos
Infecções por Citomegalovirus , Parvovirinae , Humanos , Células Ganglionares da Retina/metabolismo , Terapia Genética , Transgenes , Nervo Óptico , Dependovirus/genética , Parvovirinae/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Vetores Genéticos/genética
4.
Int J Mol Sci ; 24(21)2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37958544

RESUMO

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is an inborn error of metabolism caused by inactivating mutations in SGPL1, the gene encoding sphingosine-1-phosphate lyase (SPL), an essential enzyme needed to degrade sphingolipids. SPLIS features include glomerulosclerosis, adrenal insufficiency, neurological defects, ichthyosis, and immune deficiency. Currently, there is no cure for SPLIS, and severely affected patients often die in the first years of life. We reported that adeno-associated virus (AAV) 9-mediated SGPL1 gene therapy (AAV-SPL) given to newborn Sgpl1 knockout mice that model SPLIS and die in the first few weeks of life prolonged their survival to 4.5 months and prevented or delayed the onset of SPLIS phenotypes. In this study, we tested the efficacy of a modified AAV-SPL, which we call AAV-SPL 2.0, in which the original cytomegalovirus (CMV) promoter driving the transgene is replaced with the synthetic "CAG" promoter used in several clinically approved gene therapy agents. AAV-SPL 2.0 infection of human embryonic kidney (HEK) cells led to 30% higher SPL expression and enzyme activity compared to AAV-SPL. Newborn Sgpl1 knockout mice receiving AAV-SPL 2.0 survived ≥ 5 months and showed normal neurodevelopment, 85% of normal weight gain over the first four months, and delayed onset of proteinuria. Over time, treated mice developed nephrosis and glomerulosclerosis, which likely resulted in their demise. Our overall findings show that AAV-SPL 2.0 performs equal to or better than AAV-SPL. However, improved kidney targeting may be necessary to achieve maximally optimized gene therapy as a potentially lifesaving SPLIS treatment.


Assuntos
Terapia Genética , Parvovirinae , Esfingosina , Animais , Humanos , Camundongos , Aldeído Liases/genética , Aldeído Liases/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos Knockout , Parvovirinae/metabolismo , Fosfatos , Esfingosina/metabolismo
5.
J Virol ; 97(10): e0078023, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37702486

RESUMO

IMPORTANCE: AAVs are extensively studied as promising therapeutic gene delivery vectors. In order to circumvent pre-existing antibodies targeting primate-based AAV capsids, the AAAV capsid was evaluated as an alternative to primate-based therapeutic vectors. Despite the high sequence diversity, the AAAV capsid was found to bind to a common glycan receptor, terminal galactose, which is also utilized by other AAVs already being utilized in gene therapy trials. However, contrary to the initial hypothesis, AAAV was recognized by approximately 30% of human sera tested. Structural and sequence comparisons point to conserved epitopes in the fivefold region of the capsid as the reason determinant for the observed cross-reactivity.


Assuntos
Antígenos Virais , Capsídeo , Parvovirinae , Animais , Humanos , Capsídeo/química , Proteínas do Capsídeo/química , Dependovirus/química , Vetores Genéticos , Primatas/genética , Antígenos Virais/química , Parvovirinae/química
6.
J Virol Methods ; 321: 114792, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37591371

RESUMO

The Adeno-Associated Virus (AAV) has emerged as a promising candidate for delivery of genetic material, exhibiting significant potential in various clinical applications. Although multiple AAV serotypes have been shown to transduce ocular tissues, there have been few studies of AAV transduction of lens epithelial cells (LECs) in the ocular. In this study, we compared the efficiency of intravitreal injection of six AAV serotypes (AAV2, AAV5, AAV6, AAV8, AAV9, and AAVDJ) to transduce lens and retina in rats, The expression and localization of the reporter gene ZsGreen in the lens and retina were examined using immunofluorescence staining, and the relative expression of ZsGreen mRNA was detected using RT-qPCR. Our results demonstrated that AAV2 had the highest efficiency in transducing LECs. All six AAV serotypes could transduce the retina. To validate this observation, we further constructed an AAV2 vector with exogenous gene senescence marker protein 30 (SMP30) and performed intravitreal injection to successfully overexpress SMP30 in LECs of rats. our results provide a basis for the use of AAV vector-mediated gene therapy for lens diseases.


Assuntos
Parvovirinae , Animais , Ratos , Dependovirus/genética , Células Epiteliais , Genes Reporter
7.
Nat Commun ; 14(1): 4762, 2023 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-37553329

RESUMO

Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel vector of which capsid was composed of capsid proteins derived from both of the AAV serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for AAV1 vector) and neuron specificity (for AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.


Assuntos
Dependovirus , Parvovirinae , Animais , Dependovirus/metabolismo , Transdução Genética , Vetores Genéticos/genética , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Transgenes , Primatas/genética , Parvovirinae/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Neurônios/metabolismo
8.
Gene Ther ; 30(9): 723-735, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37386155

RESUMO

Adeno-associated virus serotype 2 (AAV2) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the retina. One strategy for altering AAV2 vectors involves the mutation of phosphodegron residues, which are thought to be phosphorylated/ubiquitinated in the cytosol, facilitating degradation of the vector and the inhibition of transduction. As such, mutation of phosphodegron residues have been correlated with increased transduction of target cells, however, an assessment of the immunobiology of wild-type and phosphodegron mutant AAV2 vectors following intravitreal (IVT) delivery to immunocompetent animals is lacking in the current literature. In this study, we show that IVT of a triple phosphodegron mutant AAV2 capsid is associated with higher levels of humoral immune activation, infiltration of CD4 and CD8 T-cells into the retina, generation of splenic germinal centre reactions, activation of conventional dendritic cell subsets, and elevated retinal gliosis compared to wild-type AAV2 capsids. However, we did not detect significant changes in electroretinography arising after vector administration. We also demonstrate that the triple AAV2 mutant capsid is less susceptible to neutralisation by soluble heparan sulphate and anti-AAV2 neutralising antibodies, highlighting a possible utility for the vector in terms of circumventing pre-existing humoral immunity. In summary, the present study highlights novel aspects of rationally-designed vector immunobiology, which may be relevant to their application in preclinical and clinical settings.


Assuntos
Capsídeo , Parvovirinae , Camundongos , Animais , Capsídeo/metabolismo , Sorogrupo , Transdução Genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Parvovirinae/genética , Dependovirus/metabolismo , Vetores Genéticos/genética
9.
J Virol ; 97(7): e0177222, 2023 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-37310260

RESUMO

Adeno-associated virus (AAV) is a nonenveloped single-stranded DNA (ssDNA) icosahedral T=1 virus being developed as a vector for clinical gene delivery systems. Currently, there are approximately 160 AAV clinical trials, with AAV2 being the most widely studied serotype. To further understand the AAV gene delivery system, this study investigates the role of viral protein (VP) symmetry interactions on capsid assembly, genome packaging, stability, and infectivity. A total of 25 (seven 2-fold, nine 3-fold, and nine 5-fold symmetry interface) AAV2 VP variants were studied. Six 2-fold and two 5-fold variants did not assemble capsids based on native immunoblots and anti-AAV2 enzyme-linked immunosorbent assays (ELISAs). Seven of the 3-fold and seven of the 5-fold variants that assembled capsids were less stable, while the only 2-fold variant that assembled had ~2°C higher thermal stability (Tm) than recombinant wild-type AAV2 (wtAAV2). Three of the 3-fold variants (AAV2-R432A, AAV2-L510A, and N511R) had an approximately 3-log defect in genome packaging. Consistent with previous reports of the 5-fold axes, the region of the capsid is important for VP1u externalization and genome ejection, and one 5-fold variant (R404A) had a significant defect in viral infectivity. The structures of wtAAV2 packaged with a transgene (AAV2-full) and without a transgene (AAV2-empty) and one 5-fold variant (AAV2-R404A) were determined by cryo-electron microscopy and three dimensional (3D)-image reconstruction to 2.8, 2.9, and 3.6 Å resolution, respectively. These structures revealed the role of stabilizing interactions on the assembly, stability, packaging, and infectivity of the virus capsid. This study provides insight into the structural characterization and functional implications of the rational design of AAV vectors. IMPORTANCE Adeno-associated viruses (AAVs) have been shown to be useful vectors for gene therapy applications. Consequently, AAV has been approved as a biologic for the treatment of several monogenic disorders, and many additional clinical trials are ongoing. These successes have generated significant interest in all aspects of the basic biology of AAV. However, to date, there are limited data available on the importance of the capsid viral protein (VP) symmetry-related interactions required to assemble and maintain the stability of the AAV capsids and the infectivity of the AAV capsids. Characterizing the residue type and interactions at these symmetry-driven assembly interfaces of AAV2 has provided the foundation for understanding their role in AAV vectors (serotypes and engineered chimeras) and has determined the residues or regions of the capsid that can or cannot tolerate alterations.


Assuntos
Capsídeo , Parvovirinae , Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Sorogrupo , Microscopia Crioeletrônica , Proteínas do Capsídeo/metabolismo , Parvovirinae/genética , Parvovirinae/metabolismo , Proteínas Virais/metabolismo , Vetores Genéticos , Montagem de Vírus
11.
Arch Virol ; 168(6): 163, 2023 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-37198283

RESUMO

Birds carry a large number of viruses that may cause diseases in animals or humans. At present, information about the virome of zoo birds is limited. In this study, using viral metagenomics, we investigated the fecal virome of zoo birds collected from a zoo in Nanjing, Jiangsu Province, China. Three novel parvoviruses were obtained and characterized. The genomes of the three viruses are 5,909, 4,411, and 4,233 nt in length, respectively, and contain four or five ORFs. Phylogenetic analysis showed that these three novel parvoviruses clustered with other strains and formed three different clades. Pairwise comparison of NS1 amino acid sequences showed that Bir-01-1 shared 44.30-74.92% aa sequence identity with other parvoviruses belonging to the genus Aveparvovirus, while Bir-03-1 and Bir-04-1 shared less than 66.87% and 53.09% aa sequence identity, respectively, with other parvoviruses belonging to the genus Chaphamaparvovirus. Each of these three viruses was identified as a member of a novel species based on the species demarcation criteria for parvoviruses. These findings broaden our knowledge of the genetic diversity of parvoviruses and provide epidemiological data regarding potential outbreaks of parvovirus disease in birds.


Assuntos
Infecções por Parvoviridae , Parvovirinae , Parvovirus , Vírus , Animais , Humanos , Filogenia , Parvovirus/genética , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/veterinária , Aves , Parvovirinae/genética
12.
Mol Ther ; 31(7): 2028-2041, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-37056049

RESUMO

In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development.


Assuntos
Parvovirinae , Retinite Pigmentosa , Humanos , Animais , Cães , Camundongos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Retinite Pigmentosa/metabolismo , Retina/metabolismo , Eletrorretinografia , Rodopsina/metabolismo
13.
Gene Ther ; 30(6): 503-519, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36635457

RESUMO

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken ß-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken ß-actin/short ß-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.


Assuntos
Infecções por Citomegalovirus , Parvovirinae , Camundongos , Animais , Humanos , Células Ganglionares da Retina/metabolismo , Actinas/genética , Actinas/metabolismo , Transdução Genética , Camundongos Endogâmicos C57BL , Transgenes , Dependovirus/genética , Dependovirus/metabolismo , Parvovirinae/genética , Proteínas de Fluorescência Verde/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Vetores Genéticos/genética
14.
Poult Sci ; 102(2): 102396, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36565640

RESUMO

Goose astrovirus (GoAstV), goose parvovirus (GPV), and goose circovirus (GoCV) infections have similar symptoms, such as severe diarrhea, and cause serious economic losses to the goose industry globally. Therefore, it is necessary to develop a rapid and accurate method for the differential diagnosis of the 3 viruses. In this study, a TaqMan probe-based multiplex reverse transcription-qualitative polymerase chain reaction (RT-qPCR) method was established and optimized for simultaneous detection of the three viruses. Three pairs of specific primers and probes were designed considering the conserved sequences of ORF2, VP3, and Rep of GoAstV, GPV, and GoCV, respectively. Singleplex real-time RT-qPCR detected a minimum of 10 copies of these genes, while multiplex real-time RT-qPCR detected a minimum of 100 copies. The correlation coefficients exceeded 0.99, and the amplification efficiency was 80 to 100%. The assay had high sensitivity, specificity, and repeatability. In 85 tissue samples, GoAstV and GPV were the main pathogens and demonstrated co-infection. This assay provides a rapid, efficient, specific, and sensitive tool for the detection of GoAstV, GPV, and GoCV. This can facilitate disease management and epidemiological surveillance.


Assuntos
Galinhas , Parvovirinae , Animais , Gansos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Taq Polimerase/metabolismo
15.
Am J Ophthalmol ; 249: 108-125, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36496192

RESUMO

PURPOSE: To evaluate the safety profile of lenadogene nolparvovec (Lumevoq) in patients with Leber hereditary optic neuropathy. DESIGN: Pooled analysis of safety data from 5 clinical studies. METHODS: A total of 189 patients received single unilateral or bilateral intravitreal injections of a recombinant adeno-associated virus 2 (rAAV2/2) vector encoding the human wild-type ND4 gene. Adverse events (AEs) were collected throughout the studies, up to 5 years. Intraocular inflammation and increased intraocular pressure (IOP) were ocular AEs of special interest. Other assessments included ocular examinations, vector bio-dissemination, and systemic immune responses against rAAV2/2. RESULTS: Almost all patients (95.2%) received 9 × 1010 viral genomes and 87.8% had at least 2 years of follow-up. Most patients (75.1%) experienced at least one systemic AE, but systemic treatment-related AEs occurred in 3 patients; none were serious. Intraocular inflammation was reported in 75.6% of lenadogene nolparvovec-treated eyes. Almost all intraocular inflammations occurred in the anterior chamber (58.8%) or in the vitreous (40.3%), and were of mild (90.3%) or moderate (8.8%) intensity; most resolved with topical corticosteroids alone. All IOP increases were mild to moderate in intensity. No AE led to study discontinuation. Bio-dissemination of lenadogene nolparvovec and systemic immune response were limited. The safety profile was comparable for patients treated bilaterally and unilaterally. CONCLUSIONS: Lenadogene nolparvovec had a good overall safety profile with excellent systemic tolerability, consistent with limited bio-dissemination. The product was well tolerated, with mostly mild ocular side effects responsive to conventional ophthalmologic treatments.


Assuntos
Atrofia Óptica Hereditária de Leber , Parvovirinae , Humanos , Atrofia Óptica Hereditária de Leber/tratamento farmacológico , Atrofia Óptica Hereditária de Leber/genética , Vetores Genéticos , Parvovirinae/genética , Terapia Genética , Inflamação/etiologia
16.
Virol J ; 19(1): 212, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36494863

RESUMO

The COVID-19 pandemic, caused by the SARS-CoV-2 virus and its variants, has posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against SARS-CoV-2 variants. Therefore, novel vaccines to match mutated viral lineages by providing long-term protective immunity are urgently needed. We designed a recombinant adeno-associated virus 5 (rAAV5)-based vaccine (rAAV-COVID-19) by using the SARS-CoV-2 spike protein receptor binding domain (RBD-plus) sequence with both single-stranded (ssAAV5) and self-complementary (scAAV5) delivery vectors and found that it provides excellent protection from SARS-CoV-2 infection. A single-dose vaccination in mice induced a robust immune response; induced neutralizing antibody (NA) titers were maintained at a peak level of over 1:1024 more than a year post-injection and were accompanied by functional T-cell responses. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines produced high levels of serum NAs against the circulating SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta. A SARS-CoV-2 virus challenge showed that the ssAAV5-RBD-plus vaccine protected both young and old mice from SARS-CoV-2 infection in the upper and lower respiratory tracts. Whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genomes of vaccinated mice one year after vaccination, demonstrating vaccine safety. These results suggest that the rAAV5-based vaccine is safe and effective against SARS-CoV-2 and several variants as it provides long-term protective immunity. This novel vaccine has a significant potential for development into a human prophylactic vaccination to help end the global pandemic.


Assuntos
COVID-19 , Parvovirinae , Animais , Humanos , Camundongos , SARS-CoV-2/genética , COVID-19/prevenção & controle , Pandemias , Vacinas Sintéticas/genética , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Neutralizantes , Anticorpos Antivirais
17.
Virol J ; 19(1): 175, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36320007

RESUMO

BACKGROUND: Theiler's disease, a.k.a. equine serum hepatitis, is a devastating, highly fatal disease of horses. Equine parvovirus-hepatitis (EqPV-H) has been identified as the likely cause of this disease. While the incidence of Theiler's disease is low, the prevalence of EqPV-H DNA in horses is high, with up to 37% in some regions, suggesting that subclinical or persistent infection is common. METHODS: To determine the prevalence and pathogenicity of EqPV-H infection at New York racetracks, DNA was extracted from archived formalin-fixed, paraffin-embedded liver tissues from racehorses submitted for necropsy to the Animal Health Diagnostic Center as part of the New York State Gaming Commission-Cornell University postmortem examination program. A total of 191 liver samples from horses between 2 and 13 years old were evaluated. Extracted DNA was tested for EqPV-H using PCR and gel electrophoresis. PCR-positive samples were further assessed for tissue morphology using histology and detection of viral nucleic acid using in situ hybridization. RESULTS: Forty-two samples were PCR positive (22%). Of those, 31 samples had positive viral nucleic acid hybridization in hepatocytes with 11 samples showing positive hybridization in necrotic hepatocytes associated with inflammatory cells, indicating active hepatitis. Both individual hepatocyte necrosis and hepatitis were positively associated with EqPV-H detection (p < 0.0001 and p = 0.0005, respectively). CONCLUSION: These findings indicate that presence of EqPV-H in the liver and parvoviral-associated hepatitis are prevalent in racehorses from New York racetracks, thus warranting additional studies examining potential associations between EqPV-H infection and racehorse performance.


Assuntos
Hepatite Viral Animal , Hepatite , Doenças dos Cavalos , Parvovirinae , Parvovirus , Cavalos , Animais , Prevalência , New York , Doenças dos Cavalos/epidemiologia
18.
J Gen Virol ; 103(11)2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36367762

RESUMO

Over the past few months there have been reports of severe acute hepatitis in several hundred, otherwise healthy, immunocompetent young children. Several deaths have been recorded and a relatively large proportion of the patients have needed liver transplants. Most of the cases, so far, have been seen in the UK and in North America, but it has also been reported in many other European countries, the Middle East and Asia. Most common viruses have been ruled out as a causative agent; hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) were not detected, nor were Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV) in many cases. A small proportion of the children had been infected with SARS-CoV-2 but these seem to be in a minority; similarly, almost none of the children had been vaccinated against COVID-19. Significantly, many of the patients were infected with adenovirus 41 (HAdV-F41). Previously, HAdV-41 had not been linked to hepatitis and is usually considered to cause gastroenteritis in both immunocompetent and immunocompromised patients. In two most recent studies, adeno-associated virus 2 (AAV2) was detected in almost all patients, together with species C and F HAdVs and human herpesvirus 6B (HHV6B). Here, I discuss the possibility that a change in tropism of HAdV-41 and changes in AAV2 may be responsible for their links to acute hepatitis.


Assuntos
Infecções por Adenoviridae , COVID-19 , Infecções por Vírus Epstein-Barr , Hepatite , Parvovirinae , Criança , Humanos , Pré-Escolar , Adenoviridae , Herpesvirus Humano 4 , SARS-CoV-2 , Hepatite/complicações
19.
Transbound Emerg Dis ; 69(6): e3455-e3461, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36271506

RESUMO

Equine parvovirus-hepatitis (EqPV-H) is one of the etiological agents of Theiler's disease, causing fulminant hepatitis; however, its transmission route and pathogenesis remain unclear. In the present study, we aimed to determine EqPV-H shedding in oral/nasal/vaginal swabs or semen samples from horses living in Korea using nested polymerase chain reaction. We then used the data obtained to investigate various risk factors associated with EqPV-H including viral shedding, hepatopathological changes, and genetic diversity. Our data revealed occurrence of EqPV-H shedding in these animals (oral: 3/102 [2.9%]; nasal: 3/102 [2.9%]; semen: 1/9 [11.1%]) and identified that both age and country of foaling were significantly associated with EqPV-H shedding (p < .05). In addition, we noted that one of the newly isolated strains clustered separately from the other strains in the phylogenetic tree, revealing unique nucleotide and amino acid substitutions. This is a field surveillance study providing evidence of natural and venereal shedding of EqPV-H and describing its presence in both oral/nasal fluids and semen. This epidemiological and clinical analysis may help specify the clinicopathological features of EqPV-H and facilitate the development of novel disease prevention strategies.


Assuntos
Hepatite Viral Animal , Hepatite , Doenças dos Cavalos , Infecções por Parvoviridae , Parvovirinae , Parvovirus , Feminino , Animais , Cavalos , Infecções por Parvoviridae/veterinária , Filogenia , Sêmen , Hepatite Viral Animal/epidemiologia , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/patologia , República da Coreia
20.
Avian Dis ; 66(3): 1-12, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36106907

RESUMO

The aim of this study is to identify and characterize virus isolates (which are named for Bacgiang Agriculture and Forestry University [BAFU]) from diseased Cherry Valley duck and mule duck flocks and investigate the damage caused by a novel parvovirus-related virus (DuPV) to tissues and organs, including the brain, cerebellum, kidney, liver, lung, spleen, and spinal cord. The results of phylogenetic analysis show that DuPV-BAFU evolved from a goose lineage and duck parvoviruses rather than from Muscovy duck parvoviruses. In the genetic lineages, DuPVs were identified from the DuPV samples analyzed, and DuPV-BAFU was found to be closely clustered with two known goose origin parvoviruses (GPVa2006 and GPV1995) and a duck GPVs. Finally, structural modeling revealed that DuPV-BAFU and the closely related viruses GPVa2006 and GPV1995 possessed identical clusters of receptor-interacting amino acid residues in the VP3 protein, a major determinant of viral receptor binding and host specificity. Significantly, these three viruses differed from DuPVs, Muscovy duck parvoviruses, and other goose parvoviruses at these positions. These results also demonstrated that DuPV-BAFU represents a new variant of goose-origin parvovirus that currently circulates in ducklings and causes beak atrophy and dwarfism syndrome, as noted in the previous reports in Europe, Taiwan, and China. This new finding highlights the need for future surveillance of DuPV-BAFU in waterfowl in order to gain a better understanding of both the evolution and the biology of this emerging parvovirus in waterfowl.


Identificación molecular y patogenicidad de un nuevo parvovirus de ganso de origen en pato aislado del síndrome de atrofia del pico y enanismo de las aves acuáticas en el norte de Vietnam. El objetivo de este estudio es identificar y caracterizar aislados de virus detectados en la Universidad de Agricultura y Silvicultura de Bacgiang (BAFU) de parvadas de patos enfermos Cherry Valley e híbridos y también investigar el daño causado por un nuevo virus relacionado con parvovirus del pato (DuPV) en tejidos y órganos, incluidos el cerebro, el cerebelo, los riñones, el hígado, los pulmones, el bazo y la médula espinal. Los resultados del análisis filogenético mostraron que el virus DuPV-BAFU evolucionó a partir de un linaje de parvovirus de patos y gansos en lugar del parvovirus de patos reales. En los linajes genéticos, se identificaron virus DuPV a partir de las muestras de DuPV analizadas, y se encontró que el DuPV-BAFU estaba estrechamente agrupado con dos parvovirus conocidos de origen de ganso (GPVa2006 y GPV1995) y con parvovirus de pato. Finalmente, el modelado estructural reveló que el virus DuPV-BAFU y los virus estrechamente relacionados GPVa2006 y GPV1995 poseían grupos idénticos de residuos de aminoácidos que interactúan con el receptor en la proteína VP3, que es un determinante importante de la unión al receptor viral y la especificidad del huésped. Significativamente, estos tres virus diferían de los DuPV, los parvovirus del pato real y de otros parvovirus del ganso en estas posiciones. Estos resultados también demostraron que el virus DuPV-BAFU representa una nueva variante del parvovirus de origen ganso que actualmente circula en patitos y causa atrofia del pico y síndrome de enanismo, como se señaló en reportes anteriores en Europa, Taiwán y China. Este nuevo hallazgo destaca la necesidad de una vigilancia futura para el virus DuPV-BAFU en las aves acuáticas para comprender mejor tanto la evolución como la biología de este parvovirus emergente en las aves acuáticas.


Assuntos
Nanismo , Infecções por Parvoviridae , Parvovirus , Doenças das Aves Domésticas , Aminoácidos , Animais , Atrofia/veterinária , Bico/patologia , Patos , Nanismo/patologia , Nanismo/veterinária , Infecções por Parvoviridae/veterinária , Parvovirinae , Parvovirus/genética , Filogenia , Vietnã , Virulência
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