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1.
Arch Virol ; 166(9): 2495-2504, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34232400

RESUMO

Short beak and dwarfism syndrome (SBDS) emerged in Cherry Valley duck flocks in China in 2015, and novel goose parvovirus (NGPV) was shown to be the etiological agent of SBDS. To date, it is not known whether SBDS-related NGPV isolates possess common molecular characteristics. In this study, three new NGPV strains (namely, SDHT16, SDJN19, and SDLC19) were isolated from diseased ducks showing typical signs of SBDS and successfully passaged in embryonated goose or Cherry Valley duck eggs. The complete genome sequences of these NGPV strains were 98.9%-99.7% identical to each other but showed slightly less similarity (95.2%-96.1% identity) to classical GPV strains. A total of 16 common amino acid substitutions were present in the VP1 proteins of six NGPV strains (SDHT16, SDJN19, SDLC19, QH, JS1, and SDLC01) compared with the classical Chinese GPV strains, nine of which were identical to those found in European GPV strain B. The non-structural protein Rep1 of the six NGPV strains had 12 common amino acid substitutions compared with the classical GPV strains. Phylogenetic analysis indicated that the Chinese NGPV strains clustered with the European SBDS-related NGPV strains, forming a separate branch that was distinct from the group formed by the classical GPV strains. The present study shows the common molecular characteristics of NGPV isolates and suggests that the Chinese NGPV isolates probably share a common ancestor with European SBDS-related NGPV strains.


Assuntos
Nanismo/veterinária , Nanismo/virologia , Parvovirinae/classificação , Parvovirinae/genética , Filogenia , Doenças das Aves Domésticas/virologia , Animais , China , Patos/virologia , Gansos/virologia , Genoma Viral , Infecções por Parvoviridae/virologia , Parvovirus/genética , Alinhamento de Sequência , Sequenciamento Completo do Genoma
2.
Nat Commun ; 12(1): 4219, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244505

RESUMO

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA Guia/genética , Staphylococcus/enzimologia , Animais , Proteína 9 Associada à CRISPR/isolamento & purificação , Linhagem Celular Tumoral , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Parvovirinae/genética , Engenharia de Proteínas , Ribonucleases , Staphylococcus/genética , Especificidade por Substrato , Síndromes de Usher/genética , Síndromes de Usher/terapia
3.
Arch Virol ; 166(7): 2011-2016, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34080052

RESUMO

Recently, a novel duck-origin goose parvovirus (N-GPV) was reported to cause short beak and dwarfism syndrome in ducks. In this study, we performed complete genome sequencing and analyzed three different duck-derived parvoviruses that infected different breeds of ducks. Phylogenetic trees based on gene sequences indicated that they were classical goose parvovirus (C-GPV), Muscovy duck parvovirus (MDPV), and N-GPV. Furthermore, potential recombination events were found. These results improve our understanding of the diversity of duck-derived parvoviruses in Anhui province, eastern China, and provide a reference for the prevention of associated diseases.


Assuntos
Patos/virologia , Infecções por Parvoviridae/virologia , Parvovirinae/genética , Parvovirus/genética , Animais , Bico/virologia , China , Filogenia , Doenças das Aves Domésticas/virologia , Análise de Sequência de DNA
4.
Res Vet Sci ; 137: 68-76, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33933710

RESUMO

In this study, the effects of Goose parvovirus (GPV) infection as well as the possible role of NS1 protein on apoptosis induction in goose embryo fibroblast (GEF) cells were examined. Flow cytometry analysis and TUNEL assays revealed that GPV infection and NS1 transfection induced significant apoptosis in GEF cells compared to what was observed in mock-infected cells. Interestingly, the increase in the rate of apoptosis detected in GPV-infected GEFs was accompanied by an increased viral load in the cells. In addition, the apoptotic pathway was mediated by apoptosis-inducing factors (AIFs) and internal factors that influence the release of AIFs. The results indicated that the mitochondrial membrane potential was decreased, and AIF expression was increased in the nucleus (P < 0.01). Reactive oxygen species (ROS) increased gradually within 48 h (P < 0.001). Cathepsin D activities were also increased (P < 0.05). The results demonstrated that the AIF-mediated pathway is a new mitochondrial apoptotic pathway and that mitochondrial depolarization, ROS content, and cathepsin D activities are the key factors influencing apoptosis in GEF cells.


Assuntos
Fibroblastos/virologia , Gansos/embriologia , Parvovirinae/patogenicidade , Proteínas não Estruturais Virais/farmacologia , Animais , Apoptose , Fator de Indução de Apoptose/metabolismo , Fator de Indução de Apoptose/farmacologia , Catepsina D/metabolismo , Morte Celular , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Arch Virol ; 166(7): 1931-1942, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33934195

RESUMO

Since its first recognition in the early 1960s, Derzsy's disease has caused significant economic losses in the goose meat industry through the world. Today, Derzsy's disease still maintains its importance for small-scale waterfowl farming, despite not having a significant impact on public health. In the present study, we investigated the distribution of goose parvovirus (GPV) and its potential variants from a 2019 outbreak in Turkey. Tissue samples were obtained from infected eggs and goslings that were raised in distinct farming areas of the various provinces. For this purpose, a novel primer set for amplification of a 630-bp region of VP3 was designed to confirm GPV infection by conventional PCR method. A 4709-base nucleotide sequence including the structural, non-structural, and 5' inverted terminal repeat regions was obtained from three samples from the Central Anatolian region. Multiple sequence comparisons and phylogenetic analysis demonstrated that the field strains clustered with European group 2 and contained a series of unique amino acid substitutions that might affect the virulence of the virus. These results confirmed that European-related field strains caused the outbreak in Asia Minor, and this might assist in understanding the circulation of GPV in Asia and Europe.


Assuntos
Gansos/virologia , Parvovirinae/genética , Parvovirus/genética , Virulência/genética , Substituição de Aminoácidos/genética , Animais , Ásia , Surtos de Doenças , Europa (Continente) , Infecções por Parvoviridae/virologia , Filogenia , Doenças das Aves Domésticas/virologia
6.
J Neurosci Methods ; 359: 109221, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34004203

RESUMO

BACKGROUND: Retrograde and anterograde transsynaptic viral vectors are useful tools for studying the input and output organization of neuronal circuitry, respectively. While retrograde transsynaptic viral vectors are widely used, viral vectors that show anterograde transsynaptic transduction are not common. NEW METHOD: We chose recombinant avian adeno-associated virus (A3V) carrying the mCherry gene and injected it into the eyeball, cochlear duct, and midbrain auditory center of chickens. We observed different survival times to examine the virus transcellular transport and the resulting mCherry expression. To confirm the transcellular transduction mode, we co-injected A3V and cholera toxin B subunit. RESULTS: Injecting A3V into the eyeball and cochlea labeled neurons in the visual and auditory pathways, respectively. Second-, and third-order labeling occurred approximately two and seven days, respectively, after injection into the midbrain. The distribution of labeled neurons strongly suggests that A3V transport is preferentially anterograde and transduces postsynaptic neurons. COMPARISON WITH EXISTING METHOD(S): A3V displays no extrasynaptic leakage and moderate speed of synapse passage, which is better than other viruses previously reported. Compared with AAV1&9, which have been shown to pass one synapse anterogradely, A3V passes several synapses in the anterograde direction. CONCLUSIONS: A3V would be a good tool to study the topographic organization of projection axons and their target neurons.


Assuntos
Galinhas , Parvovirinae , Animais , Neurônios , Sinapses
7.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802760

RESUMO

Neurotrophic factors (NTFs) are essential for cell growth, survival, synaptic plasticity, and maintenance of specific neuronal population in the central nervous system. Multiple studies have demonstrated that alterations in the levels and activities of NTFs are related to the pathology and symptoms of neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease. Hence, the key molecule that can regulate the expression of NTFs is an important target for gene therapy coupling adeno-associated virus vector (AAV) gene. We have previously reported that the Ras homolog protein enriched in brain (Rheb)-mammalian target of rapamycin complex 1 (mTORC1) axis plays a vital role in preventing neuronal death in the brain of AD and PD patients. AAV transduction using a constitutively active form of Rheb exerts a neuroprotective effect through the upregulation of NTFs, thereby promoting the neurotrophic interaction between astrocytes and neurons in AD conditions. These findings suggest the role of Rheb as an important regulator of the regulatory system of NTFs to treat neurodegenerative diseases. In this review, we present an overview of the role of Rheb in neurodegenerative diseases and summarize the therapeutic potential of AAV serotype 1 (AAV1)-Rheb(S16H) transduction in the treatment of neurodegenerative disorders, focusing on diseases, such as AD and PD.


Assuntos
Doenças Neurodegenerativas/terapia , Parvovirinae/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Transdução Genética , Animais , Humanos , Modelos Biológicos , Fatores de Crescimento Neural/metabolismo
8.
Arch Virol ; 166(5): 1517-1520, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33694004

RESUMO

The subfamily Parvovirinae within the family Parvoviridae consists of viruses that can infect a wide range of vertebrate hosts and cause effects ranging from severe disease to asymptomatic infection. In the present study, high-throughput sequencing (HTS) was utilized to analyze samples obtained from an abortion outbreak in a sheep flock to identify a putative viral etiology. A highly divergent nearly complete parvovirid genome sequence, approximately 4.9 kb in length, was determined. The nonstructural protein (NS1) amino acid (aa) sequence of this virus shared less than 30% identity with those of other copiparvoviruses and less than 22% identity with those of members of other genera in the subfamily Parvovirinae. Phylogenetically, this virus, which we have provisionally named "sheep copiparvovirus 1", formed a cluster with copiparvovirus sequences and should be classified as a member of a new species in the genus Copiparvovirus.


Assuntos
Infecções por Parvoviridae/veterinária , Parvovirinae/genética , Doenças dos Ovinos/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Brasil/epidemiologia , DNA Viral/genética , Feminino , Genoma Viral/genética , Masculino , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Parvovirinae/classificação , Filogenia , Ovinos , Doenças dos Ovinos/epidemiologia , Proteínas Virais/genética
9.
Poult Sci ; 100(4): 101021, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33677399

RESUMO

Duck-origin parvovirus disease is an epidemic disease mainly caused by duck-origin goose parvovirus (D-GPV), which is characterized by beak atrophy and dwarfism syndrome. Its main symptoms are persistent diarrhea, skeletal dysplasia, and growth retardation. However, the pathogenesis of Cherry Valley ducks infected by D-GPV has not been studied thoroughly. To perceive the distribution of D-GPV in the intestinal tract, intestinal morphological development, intestinal permeability, inflammatory cytokines in Cherry Valley ducks, and expression of tight junction protein, the D-GPV infection was given intramuscularly. Illumina MiSeq sequencing technology was used to analyze the diversity and structure of ileum flora and content of short-chain fatty acids of its metabolites. To investigate the relationship between intestinal flora changes and intestinal barrier function after D-GPV infection on Cherry Valley ducks is of great theoretical and practical significance for further understanding the pathogenesis of D-GPV and the structure of intestinal flora in ducks. The results showed that D-GPV infection was accompanied by intestinal inflammation and barrier dysfunction. At this time, the decrease of a large number of beneficial bacteria and the content of short-chain fatty acids in intestinal flora led to the weakening of colonization resistance of the intestinal flora and the accumulation of potentially pathogenic bacteria, which would aggravate the negative effect of D-GPV damage to the intestinal tract. Furthermore, a significant increase in Unclassified_S24-7 and decrease in Streptococcus was observed in D-GPV persistent, indicating the disruption in the structure of gut microbiota. Notably, the shift of microbiota was associated with the transcription of tight-junction protein and immune-associated cytokines. These results indicate that altered ileum microbiota, intestinal barrier, and immune dysfunction are associated with D-GPV infection. Therefore, there is a relationship between the intestinal barrier dysfunction and dysbiosis caused by D-GPV, but the specific mechanism needs to be further explored.


Assuntos
Microbioma Gastrointestinal , Infecções por Parvoviridae , Doenças das Aves Domésticas , Animais , Galinhas , Patos , Infecções por Parvoviridae/veterinária , Parvovirinae
10.
J Vet Sci ; 22(1): e1, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33522153

RESUMO

BACKGROUND: Goslings in several Taiwanese farms experienced gosling feather loss disease (GFL) at 21-35 days and goose broke feather disease (GBF) at 42-60 days. The prevalence ranges from a few birds to 500 cases per field. It is estimated that about 12,000 geese have been infected, the morbidity is 70-80% and the mortality is 20-30%. OBJECTIVES: This study aims to investigate the pathogens that cause GFL and GBF. Focus on the study of the correlation between goose circovirus (GoCV) and goose parvovirus (GPV) with the goose feather loss in southern Taiwan. Furthermore, a phylogenetic tree was established to align the differences between southern and northern Taiwan and compare with virus strains from China and Europe. METHODS: Samples were collected from animal hospitals. Molecular and microscopy diagnostics were used to examine 92 geese. Specific quantitative polymerase chain reaction (Q-PCR) assays are performed to evaluate GPV and GoCV viral loads and simultaneously evaluated the feather loss conditions in geese with the scoring method. RESULTS: High prevalence of GoCV and GPV infection in geese showing signs of GFL and GBF. Inclusion body was detected in the feather follicles and Lieberkühn crypt epithelial cells. The Q-PCR showed the high correlation between feather loss and viruses during 3rd-5th week. However, the infection was not detected using the same test in 60 healthy geese. CONCLUSIONS: Thus, GFL and GBF appear to be significantly closely related to GoCV and GPV. The geese feathers showed increasing recovery after being quarantined and disinfected.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/isolamento & purificação , Plumas/patologia , Gansos , Infecções por Parvoviridae/veterinária , Parvovirinae/isolamento & purificação , Doenças das Aves Domésticas/virologia , Animais , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/patologia , Infecções por Circoviridae/virologia , Plumas/virologia , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/patologia , Infecções por Parvoviridae/virologia , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/patologia , Prevalência , Taiwan/epidemiologia
11.
Arch Virol ; 166(2): 427-438, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33389172

RESUMO

The leopard cat (Prionailurus bengalensis) was listed as an endangered species under the Wildlife Conservation Act in Taiwan in 2009. However, no study has evaluated the possible direct or indirect effects of pathogens on the Taiwanese leopard cat population. Here, we targeted viral pathogens, including carnivore protoparvovirus 1 (genus Protoparvovirus), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), coronaviruses (CoVs), and canine distemper virus (CDV), through molecular screening. The spatial and temporal dynamics of the target pathogens were evaluated. Through sequencing and phylogenetic analysis, we clarified the phylogenetic relationship of viral pathogens isolated from leopard cats and domestic carnivores. Samples from 23 live-trapped leopard cats and 29 that were found dead were collected from 2015 to 2019 in Miaoli County in northwestern Taiwan. Protoparvoviruses and CoVs were detected in leopard cats, and their prevalence (95% confidence interval) was 63.5% (50.4%-76.6%) and 8.8% (0%-18.4%), respectively. Most of the protoparvovirus sequences amplified from Taiwanese leopard cats and domestic carnivores were identical. All of the CoV sequences amplified from leopard cats were identified as feline CoV. No spatial or temporal aggregation of protoparvovirus infection in leopard cats was found in the sampling area, indicating a wide distribution of protoparvoviruses in the leopard cat habitat. We consider sympatric domestic carnivores to be the probable primary reservoir for the identified pathogens. We strongly recommend management of protoparvoviruses and feline CoV in the leopard cat habitat, particularly vaccination programs and population control measures for free-roaming dogs and cats.


Assuntos
Doenças do Gato/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/veterinária , Panthera/virologia , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/veterinária , Animais , Doenças do Gato/virologia , Gatos , Coronavirus Felino/genética , Coronavirus Felino/isolamento & purificação , Vírus da Cinomose Canina/genética , Vírus da Cinomose Canina/isolamento & purificação , Doenças do Cão/epidemiologia , Doenças do Cão/virologia , Cães , Feminino , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/isolamento & purificação , Vírus da Leucemia Felina/genética , Vírus da Leucemia Felina/isolamento & purificação , Masculino , Programas de Rastreamento , Parvovirinae/genética , Parvovirinae/isolamento & purificação , Taiwan/epidemiologia
12.
Nat Commun ; 12(1): 686, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514718

RESUMO

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.


Assuntos
Anemia Falciforme/terapia , Eritropoese/genética , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Globinas beta/genética , Anemia Falciforme/sangue , Anemia Falciforme/diagnóstico , Anemia Falciforme/genética , Animais , Sistemas CRISPR-Cas/genética , Terapia Combinada/métodos , Modelos Animais de Doenças , Feminino , Edição de Genes/métodos , Técnicas de Introdução de Genes , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Transgênicos , Parvovirinae/genética , Transplante Autólogo/métodos
13.
Nat Commun ; 12(1): 697, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514733

RESUMO

Mutations in voltage-gated potassium channel KCNE1 cause Jervell and Lange-Nielsen syndrome type 2 (JLNS2), resulting in congenital deafness and vestibular dysfunction. We conducted gene therapy by injecting viral vectors using the canalostomy approach in Kcne1-/- mice to treat both the hearing and vestibular symptoms. Results showed early treatment prevented collapse of the Reissner's membrane and vestibular wall, retained the normal size of the semicircular canals, and prevented the degeneration of inner ear cells. In a dose-dependent manner, the treatment preserved auditory (16 out of 20 mice) and vestibular (20/20) functions in mice treated with the high-dosage for at least five months. In the low-dosage group, a subgroup of mice (13/20) showed improvements only in the vestibular functions. Results supported that highly efficient transduction is one of the key factors for achieving the efficacy and maintaining the long-term therapeutic effect. Secondary outcomes of treatment included improved birth and litter survival rates. Our results demonstrated that gene therapy via the canalostomy approach, which has been considered to be one of the more feasible delivery methods for human inner ear gene therapy, preserved auditory and vestibular functions in a dose-dependent manner in a mouse model of JLNS2.


Assuntos
Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Síndrome de Jervell-Lange Nielsen/terapia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais Semicirculares/cirurgia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Vetores Genéticos/genética , Audição/genética , Humanos , Injeções/métodos , Síndrome de Jervell-Lange Nielsen/genética , Masculino , Camundongos , Camundongos Knockout , Parvovirinae/genética , Propriocepção/genética
14.
Arch Virol ; 166(3): 779-788, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33433693

RESUMO

Ungulate protoparvovirus 1, also known as porcine parvovirus 1 (PPV1), is considered to be one of the major causes of reproductive failure in pig breeding herds. Other parvoviruses have also been identified in pigs, including ungulate tetraparvovirus 3, or PPV2, ungulate tetraparvovirus 2, or PPV3, and ungulate copiparvovirus 2, or PPV4, but their significance for pigs is unknown. In the present study, the prevalence of PPV1-4 was investigated using a total of 231 lung and serum samples collected from slaughterhouses in 13 provinces throughout Vietnam. The overall prevalence was 54.5% (126/231) for PPV1, 28.0% (65/231) for PPV2, 17.7% (41/231) for PPV3, and 7.8% (18/231) for PPV4. While PPV1 and PPV2 were found in 11 provinces, PPV4 was detected in only three provinces. Co-circulation of PPV1, PPV2 and PPV3 was frequently observed, with PPV1/PPV2 coinfection predominating, with 20.8% (48/231). All four PPVs were detected together in only one sample from Thua Thien Hue. Three nearly complete PPV4 genome sequences of 5,453 nt were determined and deposited in the GenBank database. Alignment and comparison of the three genome sequences showed 99.5-99.6% nucleotide sequence identity, and the deduced amino acid sequences of open reading frames 1-3 were 99.6-99.9% identical to each other, 98.9-99.3% identical to those of other Vietnamese strains and 99.4-99.7% identical to those of Chinese strains). Phylogenetic analysis further confirmed a close relationship between Vietnamese and Chinese PPV4 strains. These results are the first to report the prevalence of PPV1, PPV2, PPV3, and PPV4 and nearly complete genomic sequences of PPV4 in pigs from slaughterhouses in Vietnam.


Assuntos
Infecções por Parvoviridae/epidemiologia , Parvovirinae/classificação , Parvovirinae/genética , Doenças dos Suínos/epidemiologia , Matadouros , Sequência de Aminoácidos/genética , Animais , DNA Viral/genética , Genoma/genética , Genoma Viral/genética , Infecções por Parvoviridae/patologia , Parvovirinae/isolamento & purificação , Análise de Sequência de DNA , Sus scrofa/virologia , Suínos , Doenças dos Suínos/virologia , Vietnã/epidemiologia
15.
Nucleic Acids Res ; 49(2): 969-985, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398341

RESUMO

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.


Assuntos
Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , Edição de Genes , Vetores Genéticos/genética , Parvovirinae/genética , Reparo de DNA por Recombinação , Ribonucleoproteínas/metabolismo , Adulto , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Células HEK293 , Humanos , Ácidos Hidroxâmicos/farmacologia , Mutação INDEL , Células-Tronco Pluripotentes Induzidas , Cinética , RNA Guia/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Linfócitos T , Transdução Genética
16.
Transbound Emerg Dis ; 68(3): 1062-1068, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-32815299

RESUMO

Carnivore protoparvovirus 1 is one of the most important pathogens affecting both wild and domestic carnivores. Here, we reported the genetic characterization of canine parvovirus (CPV-2) strains from a rescued guiña (Leopardus guigna) and domestic dogs from Chile. Guiña strain was classified as CPV-2c, and phylogenetic analysis of the complete coding genome showed that the guiña CPV-2c strain shares a recent common ancestor with Chilean domestic dogs' strains. These viruses showed >99% identity and exhibited three changes in the NS1 protein (V596A, E661K and L582F). This is the first detection and genetic characterization of CPV-2c infection in guiña worldwide, and one of the few comparative studies that show the source of infection was domestic dogs. The current findings highlight the fact that guiña is a susceptible species to protoparvovirus infection and that domestic dogs represent an important threat to its conservation. The CPV-2 cross-species transmission between domestic dogs and guiña should be taken into account for protection programmes of this endangered species.


Assuntos
Doenças do Cão/transmissão , Felidae , Infecções por Parvoviridae/veterinária , Parvovirinae/isolamento & purificação , Animais , Chile , Doenças do Cão/virologia , Cães , Infecções por Parvoviridae/virologia
17.
Exp Eye Res ; 203: 108388, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33333046

RESUMO

PURPOSE: To explore the role of nucleotide-binding oligomerization domain-like receptors (NLRs) family caspase-activation and the recruitment domain containing 4 (NLRC4) inflammasome in retinal ganglion cell (RGC) injury induced by an acute glaucoma mouse model. METHOD: A mouse model of acute ocular hypertension, which can lead to retinal ischemia-reperfusion (I/R) injury, was established. The expression level of NLRC4 was detected by polymerase chain reaction and western blotting. Localized expression of NLRC4 was detected by examining immunofluorescence in eyeball sections. Intravitreal adeno-associated virus 2(AAV2) administration was used to knockdown retinal Nlrc4. Fluoro-Gold labeled RGCs and TdT-mediated dUTP nick end labeling were used to evaluate the survival and apoptosis of RGCs. Tlr4-/- mice were utilized to explore whether NLRC4 inflammasome is influenced by Toll-like receptor4 (TLR4). RESULTS: NLRC4, expressed in RGCs and microglial cells, was actively involved in mouse retinal I/R injury. Knockdown of Nlrc4 using an AAV2 vector caused an obvious reduction in the generation of IL-1ß led by the rapidly elevated intraocular pressure, and thereby improved the RGC survival. In addition, activation of the NLRC4 inflammasome could influence the phosphorylation of p38 and Jun N-terminal kinase, which was largely dependent on TLR4 signaling. CONCLUSION: Our study demonstrated the role of NLRC4 inflammasome in promoting RGC damage in mouse retinal I/R injury. Inhibition of NLRC4 might be leveraged as a potential therapeutic target in glaucomatous retinopathy.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Morte Celular/fisiologia , Glaucoma/patologia , Inflamassomos/metabolismo , Células Ganglionares da Retina/patologia , Doença Aguda , Animais , Western Blotting , Modelos Animais de Doenças , Glaucoma/metabolismo , Marcação In Situ das Extremidades Cortadas , Pressão Intraocular , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Hipertensão Ocular/metabolismo , Hipertensão Ocular/patologia , Parvovirinae/genética , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Traumatismo por Reperfusão/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
PLoS One ; 15(12): e0243359, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33320893

RESUMO

Osteoarthritis (OA) is the most common arthropathy, characterized by progressive degeneration of the articular cartilage. Currently, there are no disease-modifying approaches for OA treatment. Adeno-associated virus (AAV)-mediated gene therapy has recently become a potential treatment for OA due to its exceptional characteristics; however, the tropism and transduction efficiency of different AAV serotypes to articular joints and the safety profile of AAV applications are still unknown. The present study aims to screen an ideal AAV serotype to efficiently transfer genes to arthritic cartilage. AAV vectors of different serotypes expressing eGFP protein were injected into the knee joint cavities of mice, with all joint tissues collected 30 days after AAV injection. The transduction efficiency of AAVs was quantified by assessing the fluorescent intensities of eGFP in the cartilage of knee joints. Structural and morphological changes were analyzed by toluidine blue staining. Changes to ECM metabolism and pyroptosis of chondrocytes were determined by immunohistochemical staining. Fluorescence analysis of eGFP showed that eGFP was expressed in the cartilage of knee joints injected with each AAV vector. Quantification of eGFP intensity indicated that AAV2, 7 and 8 had the highest transduction efficiencies. Both toluidine blue staining and Mankin score showed that AAV6 aggravated cartilage degeneration. The analysis of key molecules in ECM metabolism suggested that AAV5 and 7 significantly reduced collagen type II, while AAV9 increased ADAMTS-4 but decreased MMP-19. In addition, transduction with AAV2, 5, 7 and 8 had no obvious effect on pyroptosis of chondrocytes. Comprehensive score analysis also showed that AAV2 had the highest score in intra-articular gene transfer. Collectively, our findings point to AAV2 as the best AAV serotype candidate for gene transfer on arthritic cartilage, resulting in minimal impact to ECM metabolism and pyroptosis of chondrocytes.


Assuntos
Artrite Experimental , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Terapia Genética , Vetores Genéticos , Articulação do Joelho/metabolismo , Parvovirinae , Proteína ADAMTS4/biossíntese , Proteína ADAMTS4/genética , Animais , Artrite Experimental/genética , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Experimental/terapia , Cartilagem Articular/patologia , Condrócitos/patologia , Colágeno Tipo II/biossíntese , Colágeno Tipo II/genética , Articulação do Joelho/patologia , Masculino , Metaloproteinases da Matriz Secretadas/biossíntese , Metaloproteinases da Matriz Secretadas/genética , Camundongos , Transdução Genética
19.
BMC Vet Res ; 16(1): 456, 2020 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-33228649

RESUMO

BACKGROUND: Goose parvoviruses (GPVs) spread globally and cause a huge economic loss to the poultry industry. Although the attenuated GPV vaccines play a key role in preventing the disease caused by GPV, the molecular basis for the attenuation of GPV is barely known. RESULTS: A highly attenuated GPV strain, GPV-CZM-142, was generated through blindly passaging of the highly pathogenic strain, GPV-CZM, in goose embryonic fibroblasts (GEF) for 142 generations. The GEF-adapted GPV strain's virulence was 10,000 times weaker than its wild type counterpart, GPV-CZM, based on the ELD50 (50% Embryo Lethal Dose). By comparing with the wild type strain, genome sequencing analysis identified adapted mutations either in ITR or in NS and VP1 of GPV-CZM-142. CONCLUSIONS: The highly attenuated GPV strain, GPV-CZM-142, provides a GPV vaccine candidate, and the identified virulence-related mutations give a novel insight into the molecular determinants of GPV virulence.


Assuntos
Infecções por Parvoviridae/veterinária , Parvovirinae/crescimento & desenvolvimento , Parvovirinae/genética , Doenças das Aves Domésticas/virologia , Animais , Células Cultivadas , Fibroblastos , Gansos , Infecções por Parvoviridae/embriologia , Infecções por Parvoviridae/virologia , Parvovirinae/imunologia , Doenças das Aves Domésticas/embriologia , Doenças das Aves Domésticas/imunologia , Análise de Sequência de DNA , Vacinas Atenuadas , Vacinas Virais , Virulência
20.
Trop Anim Health Prod ; 53(1): 36, 2020 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-33230580

RESUMO

Derzsy's disease, which is seen in goslings (Anser anser domestica) and Muscovy ducks (Cairina moschata), progresses to high mortality and causes significant yield losses. The disease agent is goose parvovirus (GPV), which is common in countries with waterfowl production. It has not previously been reported in Turkey. Using qPCR and sequencing of the VP3 protein-encoding gene, GPV is identified as the causative agent of high mortality among geese between 2018 and 2019. The VP3 sequences were also compared with the similar GenBank sequences phylogenetically. All the sequences were found to be most similar (98.90%) with Polish and Taiwan GPV strains. Phylogenetic analysis of the VP3 gene in strains in Turkey and comparison with strains from other countries demonstrated that the Turkish strains are native to the geography and circulated locally. This study detected the presence of the GPV gene for the first time in Turkey and demonstrated the importance of comparing the vaccine strain and wild type.


Assuntos
Patos/virologia , Gansos/virologia , Infecções por Parvoviridae/veterinária , Parvovirinae , Doenças das Aves Domésticas/virologia , Animais , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Filogenia , Doenças das Aves Domésticas/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Turquia/epidemiologia
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