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1.
PLoS Pathog ; 16(1): e1008262, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31971979

RESUMO

Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic.


Assuntos
Rim/virologia , Infecções por Parvoviridae/veterinária , Infecções por Parvoviridae/virologia , Parvovirinae/fisiologia , Doenças dos Roedores/virologia , Proteínas Virais/metabolismo , Tropismo Viral , Animais , Humanos , Camundongos , Parvovirinae/genética , Proteínas Virais/genética
2.
Nucleic Acids Res ; 48(1): 36-54, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31745548

RESUMO

Huntington disease (HD) is a fatal neurodegenerative disease caused by a pathogenic expansion of a CAG repeat in the huntingtin (HTT) gene. There are no disease-modifying therapies for HD. Artificial microRNAs targeting HTT transcripts for degradation have shown preclinical promise and will soon enter human clinical trials. Here, we examine the tolerability and efficacy of non-selective HTT lowering with an AAV5 encoded miRNA targeting human HTT (AAV5-miHTT) in the humanized Hu128/21 mouse model of HD. We show that intrastriatal administration of AAV5-miHTT results in potent and sustained HTT suppression for at least 7 months post-injection. Importantly, non-selective suppression of huntingtin was generally tolerated, however high dose AAV5-miHTT did induce astrogliosis. We observed an improvement of select behavioural and modest neuropathological HD-like phenotypes in Hu128/21 mice, suggesting a potential therapeutic benefit of miRNA-mediated non-selective HTT lowering. Finally, we also observed that potent reduction of wild type HTT (wtHTT) in Hu21 control mice was tolerated up to 7 months post-injection but may induce impairment of motor coordination and striatal atrophy. Taken together, our data suggests that in the context of HD, the therapeutic benefits of mHTT reduction may outweigh the potentially detrimental effects of wtHTT loss following non-selective HTT lowering.


Assuntos
Proteína Huntingtina/genética , Doença de Huntington/terapia , MicroRNAs/genética , Terapia de Alvo Molecular/métodos , Parvovirinae/genética , RNA Mensageiro/genética , Animais , Animais Geneticamente Modificados , Astrócitos/metabolismo , Astrócitos/patologia , Sequência de Bases , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dosagem de Genes , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteína Huntingtina/antagonistas & inibidores , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Parvovirinae/metabolismo , Desempenho Psicomotor , Estabilidade de RNA , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Repetições de Trinucleotídeos
3.
Gene ; 724: 144157, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629820

RESUMO

Cellular microRNAs are known to modulate the life-cycle of different viruses. Surprisingly, very little data exists on AAV-induced changes to the cellular microRNAome in general and in hepatic and retinal cells, in particular. We reasoned that inducible microRNA in response to recombinant AAV infection may regulate immediate and long-lived cellular responses necessary for the cell's own survival as well as its ability to control several aspects of viral life-cycle. To study this, we performed a global small RNA sequencing analysis in Adeno-associated virus (AAV) serotypes 2 and 3 infected hepatic and retinal cell models. This screen identified multiple differentially expressed microRNAs, in AAV infected Huh-7 and ARPE-19 cells. Among these, one microRNA (miR-4488) was found to be significantly down regulated (-2.24 fold for AAV2 and -3.32 fold for ARPE-19) in AAV infected cells. An enrichment and pathway analysis of miR-4488 predicted its possible effects on gene targets involved in multiple biological processes including cell-cycle regulation, endoplasmic reticulum stress response and lipid-signalling pathways. Moreover, validation studies in miR-4488 mimic or sponge transfected cells revealed modulation of these target pathways in a cell-specific manner. Further studies demonstrated that overexpression of miR-4488, modestly increased gene expression (126-128%) from AAV2 and AAV3 vectors in Huh-7 cells whereas miR-4488 inhibition in ARPE-19 cells had a similar increase (142-158%) on AAV2 or AAV3 transduction. Our results highlight that recombinant AAV mediated microRNA expression is cell-type and serotype-specific and can target specific host cellular biological pathways.


Assuntos
Dependovirus/genética , MicroRNAs/genética , Infecções por Parvoviridae/genética , Epitélio Pigmentado da Retina/virologia , Transdução Genética/métodos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Parvovirinae/genética , Reprodutibilidade dos Testes , Epitélio Pigmentado da Retina/citologia , Transgenes
4.
Avian Dis ; 63(4): 731-736, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31865690

RESUMO

Goose astrovirus is a novel and distinct astrovirus that causes fatal visceral gout in 4- to 21-day-old goslings. Goose parvovirus is the etiologic agent of Derzsy disease, an acute, contagious, and fatal disease that affects mainly young goslings. This paper describes the clinical signs and gross and histopathologic features of co-infection with astrovirus and goose parvovirus. Clinical signs and history included increased mortality, depression, anorexia, enteritis, joint swelling, and paralysis. Postmortem examination showed a considerable amount of urate covering the internal organs, especially the heart, liver, and kidney. Some goslings had swollen duodenum and ileum. Histologic lesions in the kidney, liver, spleen, lung, proventriculus, and brain included hemorrhage, congestion, edema, cell necrosis, inflammatory cell infiltration, and an eosinophilic protein-like substance in renal tubules. The extensive infiltration of heterophil myelocytes into the kidney, spleen, liver, lung, bursa of Fabricius, and pancreas is a new finding.


Assuntos
Infecções por Astroviridae/veterinária , Coinfecção/veterinária , Gansos , Infecções por Parvoviridae/veterinária , Doenças das Aves Domésticas/diagnóstico , Animais , Astroviridae/isolamento & purificação , Infecções por Astroviridae/diagnóstico , Infecções por Astroviridae/virologia , China , Coinfecção/diagnóstico , Coinfecção/virologia , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/virologia , Parvovirinae/isolamento & purificação , Doenças das Aves Domésticas/virologia
5.
Virol J ; 16(1): 136, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727103

RESUMO

BACKGROUND: Goose parvovirus (GPV) is the etiological agent of Derzsy's disease and is fatal for gosling. Research on the molecular basis of GPV pathogenicity has been hampered by the lack of a reliable reverse genetics system. At present, the GPV infectious clone has been rescued by transfection in the goose embryo, but the growth character of it is unclear in vitro. METHODS: In this study, we identified the full-length genome of GPV RC16 from the clinical sample, which was cloned into the pACYC177, generating the pIRC16. The recombinant virus (rGPV RC16) was rescued by the transfection of pIRC16 into goose embryo fibroblasts (GEFs). The rescued virus was characterized by whole genome sequencing, indirect immunofluorescence assays (IFA) and western blot (WB) using rabbit anti-GPV Rep polyclonal antibody as the primary antibody. Previously, we found the 164 K, 165 K, and 167 K residues in the 160YPVVKKPKLTEE171 are required for the nuclear import of VP1 (Chen S, Liu P, He Y, et al. Virology 519:17-22). According to that, the GPV infectious clones with mutated K164A, K165A, or K167A in VP1 were constructed, rescued and passaged. RESULTS: The rGPV RC16 has been successfully rescued by transfection of pIRC16 into the GEFs and can proliferate in vitro. Furthermore, the progeny virus produced by pIRC16 transfected cells was infectious in GEFs. Moreover, mutagenesis experiments showed that the rGPV RC16 with mutated 164 K, 165 K and 167 K in VP1 could not proliferate in GEFs based on the data of IFA and WB in parental virus and progeny virus. CONCLUSIONS: The rGPV RC16 containing genetic maker and the progeny virus are infectious in GEFs. The 164 K, 165 K, and 167 K of VP1 are vital for the proliferation of rGPV RC16 in vitro.


Assuntos
Proteínas do Capsídeo/genética , Fibroblastos/virologia , Infecções por Parvoviridae/virologia , Parvovirinae/fisiologia , Animais , Proteínas do Capsídeo/química , Núcleo Celular/virologia , Células Cultivadas , Gansos , Genoma Viral/genética , Mutação , Sinais de Localização Nuclear/genética , Parvovirinae/classificação , Parvovirinae/genética , Filogenia , Doenças das Aves Domésticas/virologia , Coelhos , Genética Reversa , Replicação Viral/genética
6.
Invest Ophthalmol Vis Sci ; 60(14): 4849-4857, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31747684

RESUMO

Purpose: We reported previously that retinas of mice with inherited retinal degeneration make less protein than retinas of normal mice. Despite recent studies suggesting that diminished protein synthesis rates may contribute to neurologic disorders, a direct link between protein synthesis rates and the progression of neurodegeneration has not been established. Moreover, it remains unclear whether reduced protein synthesis could be involved in retinal pathogenesis. Dysregulation of AKT/mTOR signaling has been reported in the retina during retinal degeneration, but to what extent this signaling contributes to translational attenuation in these mice remains uncertain. Methods: C57BL/6J and rd16 mice were subcutaneously injected with anisomycin to chronically inhibit protein synthesis rates. An AAV2 construct encoding constitutively active 4ebp1 was subretinally delivered in wildtype animals to lower protein synthesis rates. 4ebp1/2 were knocked out in rd16 mice. Results: Anisomycin treatment lowered retinal translation rates, accelerated retinal degeneration in rd16 mice, and initiated cell death in the retinas of C57BL/6J mice. AAV-mediated transfer of constitutively active 4ebp1-4A into the subretinal space of wildtype animals inhibited protein synthesis, and led to reduced electroretinography amplitudes and fewer ONL nuclei. Finally, we report that restoring protein synthesis rates by knocking out 4ebp1/2 was associated with an approximately 2-fold increase in rhodopsin levels and a delay in retinal degeneration in rd16 mice. Conclusions: Our study indicates that protein synthesis inhibition is likely not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but may be harmful to degenerating retinas, and that restoring protein synthesis may have therapeutic potential in delaying the progression of retinal degeneration.


Assuntos
Biossíntese de Proteínas/fisiologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Anisomicina/farmacologia , Proteínas de Ciclo Celular/genética , Morte Celular , Eletrorretinografia , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica/fisiologia , Marcação In Situ das Extremidades Cortadas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parvovirinae/genética , Inibidores da Síntese de Proteínas/farmacologia , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Transfecção
7.
Arch Med Res ; 50(6): 384-392, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31678897

RESUMO

BACKGROUND: T helper 2 (Th2) lymphocytes and associated interleukin (IL) 4 and IL-13 play crucial roles in asthma pathogenesis. In this study, we explored an adeno-associated virus 5 (AAV5) based gene therapy by delivering truncated IL-4 protein to antagonize IL-4 receptor α chain and interrupt asthmatic signal pathway. RESULTS: A recombinant adeno-associated virus 5 (AAV5) vector harboring a truncated mouse IL-4 gene (AAV5-mIL-4ΔC22) was prepared. Western blotting showed that the IL-4 mutant protein lacking the C-terminal 22 amino acids was expressed well in AAV5-mIL-4ΔC22 infected 16HBE and BEAS-2B cells. AAV5-drivn green fluorescent protein (AAV5-GFP) served as a control. The biodistribution of vector DNA after AAV5 vector aerosol inhalation was examined by PCR and the result showed that foreign DNA was detectable in the lungs but not in other organs including gonads. The aerosol inhalation-mediated delivery of AAV5-expressed antagonistic IL-4 mutant protein improved the lung function of ovalbumin-induced asthma mice. CONCLUSIONS: The inhalation of aerosolized AAV5-mIL-4ΔC22 significantly improved the lung function and modulated the immune cell infiltration and associated cytokine expression in the bronchoalveolar lavage fluid (BALF) of ovalbumin-induced asthma mice.


Assuntos
Asma/terapia , Terapia Genética/métodos , Subunidade alfa de Receptor de Interleucina-4/antagonistas & inibidores , Interleucina-4/genética , Administração por Inalação , Animais , Asma/induzido quimicamente , Líquido da Lavagem Broncoalveolar/química , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interleucina-4/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/toxicidade , Parvovirinae/genética , Distribuição Tecidual
8.
BMC Vet Res ; 15(1): 389, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676004

RESUMO

BACKGROUND: Classic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV. RESULTS: After genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/µl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A., S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings. CONCLUSIONS: We established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.


Assuntos
Infecções por Parvoviridae/veterinária , Parvovirinae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Doenças das Aves Domésticas/virologia , Animais , DNA Bacteriano/genética , DNA Complementar/genética , DNA Viral/genética , Patos , Especificidade de Hospedeiro , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/virologia , Doenças das Aves Domésticas/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
9.
Invest Ophthalmol Vis Sci ; 60(13): 4479-4488, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31661548

RESUMO

Purpose: Glutamate excitotoxicity seems to contribute to retinal ganglion cell (RGC) death in various eye diseases, but the underlying molecular mechanisms are not fully understood. We studied the roles of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, known as cellular stress-related senescence markers, in N-methyl-d-aspartate (NMDA)-induced RGC death. Methods: Gene expression was analyzed using quantitative reverse transcription (qRT)-PCR, in situ hybridization, and immunochemistry. Cdkn2a and Cdkn2b gain- and loss-of-function experiments were performed using the adeno-associated virus type 2 (AAV2)-mediated gene delivery system. AAV2-CRISPR-Cas9-mediated knockout of Cdkn2a or Cdkn2b was validated using cultured cells by T7 endonuclease I assay and Western blot analysis. The effects of altered expression of Cdkn2a and Cdkn2b on NMDA-induced RGC death were evaluated by quantification of RNA binding protein with multiple splicing (Rbpms)-immunoreactive RGCs. Results: Intravitreal NMDA injection resulted in upregulation of Cdkn2a and Cdkn2b expression in RGCs of the mouse retina. AAV2-mediated overexpression of Cdkn2b led to increased expression of Cdkn2a in RGCs, but not vice versa. Overexpression of Cdkn2b, but not Cdkn2a, resulted in a further reduction in RGC viability in NMDA-injected retinas. However, excessive levels of Cdkn2a or Cdkn2b had no effect on RGC viability in healthy mice. AAV2-CRISPR-Cas9-mediated knockout of either Cdkn2a or Cdkn2b attenuated NMDA-induced RGC death. Conclusions: Cdkn2a and Cdkn2b have pivotal roles in the regulation of excitotoxic RGC degeneration under NMDA-induced pathologic conditions. Our findings imply that Cdkn2a and Cdkn2b are novel therapeutic targets for ocular diseases displaying excitotoxicity-induced neuronal degeneration.


Assuntos
Inibidor de Quinase Dependente de Ciclina p15/fisiologia , Agonistas de Aminoácidos Excitatórios/toxicidade , N-Metilaspartato/toxicidade , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Western Blotting , Sistemas CRISPR-Cas , Morte Celular , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Imunoquímica , Hibridização In Situ , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parvovirinae/genética , Células Ganglionares da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
10.
Arch Virol ; 164(11): 2837-2841, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31494776

RESUMO

Since January 2019, abnormal molting has been observed frequently in approximately 40-day-old Pekin ducks in China. To investigate the possible involvement of a virus, we tested the prevalence of duck circovirus (DuCV), goose hemorrhagic polyomavirus (GHPyV), and goose parvovirus (GPV) in 11 molt cases in two provinces. GPV was detected in all cases, particularly in all samples collected from the feather area. The complete genome sequences of three GPV strains were determined and found to have 52 nucleotide changes relative to GPVs associated with short beak and dwarfism syndrome of Pekin ducks. These data will enhance our understanding of GPV diversity and outcomes of GPV infection in Pekin ducks.


Assuntos
Patos/virologia , Gansos/virologia , Muda/fisiologia , Parvovirinae/isolamento & purificação , Doenças das Aves Domésticas/virologia , Animais , China/epidemiologia , Circovirus/genética , Circovirus/isolamento & purificação , Genoma Viral/genética , Parvovirinae/genética , Polyomavirus/genética , Polyomavirus/isolamento & purificação , Doenças das Aves Domésticas/epidemiologia
11.
Clin Exp Nephrol ; 23(12): 1345-1356, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31482255

RESUMO

BACKGROUND: Previous studies showed that microRNA-29b (miR-29b) inhibits renal fibrosis. Therefore, miR-29b replacement therapy represents a promising approach for treating renal fibrosis. However, an efficient method of kidney-targeted miRNA delivery has yet to be established. Recombinant adeno-associated virus (rAAV) vectors have great potential for clinical application. For kidney-targeted gene delivery, the most suitable AAV serotype has yet to be established. Here, we identified the most suitable AAV serotype for kidney-targeted gene delivery and determined that AAV-mediated miR-29b delivery can suppress renal fibrosis in vivo. METHOD: To determine which AAV serotype is suitable for kidney cells, GFP-positive cells were identified by flow cytometry after the infection of rAAV serotype 1-9 vectors containing the EGFP gene. Next, we injected rAAV vectors into the renal pelvis to determine transduction efficiency in vivo. GFP expression was measured seven days after injecting rAAV serotype 1-9 vectors carrying the EGFP gene. Finally, we investigated whether rAAV6-mediated miR-29b delivery can suppress renal fibrosis in UUO mouse model. RESULTS: We found that rAAV6 vector is the most suitable for targeting kidney cells regardless of animal species in vitro and rAAV6 is the most suitable vector for kidney-targeted in vivo gene delivery in mice. Intra-renal pelvic injection of rAAV vectors can transduce genes into kidney TECs. Furthermore, rAAV6-mediated miR-29b delivery attenuated renal fibrosis in UUO model by suppressing Snail1 expression. CONCLUSION: Our study has revealed that rAAV6 is the most suitable serotype for kidney-targeted gene delivery and rAAV6-mediated miR-29b delivery into kidney TECs can suppress established renal fibrosis.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Nefropatias/prevenção & controle , Túbulos Renais Proximais/metabolismo , MicroRNAs/genética , Parvovirinae/genética , Obstrução Ureteral/terapia , Animais , Linhagem Celular , Modelos Animais de Doenças , Fibrose , Humanos , Nefropatias/diagnóstico , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Parvovirinae/metabolismo , Ratos , Fator de Crescimento Transformador beta1/toxicidade , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
12.
BMC Vet Res ; 15(1): 274, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370852

RESUMO

BACKGROUND: In Poland, the leader in goose production in Europe, goose parovirus infection, or Derzsy's disease (DD), must be reported to the veterinary administration due to the serious economic and epizootic threat to waterfowl production. Prophylactic treatment for DD includes attenuated live or inactivated vaccines. Moreover, the control of DD includes the monitoring of maternal derived antibody (MDA) levels in the offspring and antibody titers in the parent flock after vaccination. The aim of this study was to develop an ELISA for the detection of goose parvovirus (GPV) antibodies. RESULTS: Two recombinant protein fragments derived from VP3 (viral protein 3) GPV, namely VP3ep6 and VP3ep4-6 with a mass of 20.9 and 32.3 kDa, respectively, were produced using an Escherichia coli expression system. These proteins were purified by one-step nickel-affinity chromatography, which yielded protein preparations with a purity above 95%. These recombinant proteins were useful in the detection of serum anti-GPV antibodies, and this was confirmed by Western blotting. However, recombinant VP3ep4-6 protein showed a greater ability to correctly identify sera from infected geese. In the next stage of the project, a pool of 166 goose sera samples, previously examined by a virus neutralization test (VN), was tested. For further studies, one recombinant protein (VP3ep4-6) was selected for optimization of the test conditions. After optimization, the newly developed ELISA was compared to other serological tests, and demonstrated high sensitivity and specificity. CONCLUSION: In conclusion, the VP3ep4-6 ELISA method described here can be used for the detection of antibodies to GPV in serum.


Assuntos
Anticorpos Antivirais/sangue , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Parvoviridae/veterinária , Parvovirinae/imunologia , Doenças das Aves Domésticas/diagnóstico , Animais , Ensaio de Imunoadsorção Enzimática/normas , Infecções por Parvoviridae/sangue , Infecções por Parvoviridae/diagnóstico , Doenças das Aves Domésticas/sangue , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade
13.
Invest Ophthalmol Vis Sci ; 60(10): 3644-3651, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31469404

RESUMO

Purpose: Previously we showed that AAV5-mediated expression of either human M- or L-opsin promoted regrowth of cone outer segments and rescued M-cone function in the treated M-opsin knockout (Opn1mw-/-) dorsal retina. In this study, we determined cone viability and window of treatability in aged Opn1mw-/- mice. Methods: Cone viability was assessed with antibody against cone arrestin and peanut agglutinin (PNA) staining. The rate of cone degeneration in Opn1mw-/- mice was quantified by PNA staining. AAV5 vector expressing human L-opsin was injected subretinally into one eye of Opn1mw-/- mice at 1, 7, and 15 months old, while the contralateral eyes served as controls. M-cone-mediated retinal function was analyzed 2 and 13 months postinjection by full-field ERG. L-opsin transgene expression and cone outer segment structure were examined by immunohistochemistry. Results: We showed that dorsal M-opsin dominant cones exhibit outer segment degeneration at an early age in Opn1mw-/- mice, whereas ventral S-opsin dominant cones were normal. The remaining M-opsin dominant cones remained viable for at least 15 months, albeit having shortened or no outer segments. We also showed that AAV5-mediated expression of human L-opsin was still able to rescue function and outer segment structure in the remaining M-opsin dominant cones when treatment was initiated at 15 months of age. Conclusions: Our results showing that the remaining M-opsin dominant cones in aged Opn1mw-/- mice can still be rescued by gene therapy is helpful for establishing the window of treatability in future blue cone monochromacy clinical trials.


Assuntos
Defeitos da Visão Cromática/terapia , Terapia Genética/métodos , Células Fotorreceptoras Retinianas Cones/fisiologia , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/fisiologia , Envelhecimento/fisiologia , Animais , Arrestinas/genética , Defeitos da Visão Cromática/genética , Defeitos da Visão Cromática/fisiopatologia , Modelos Animais de Doenças , Eletrorretinografia , Regulação da Expressão Gênica/fisiologia , Vetores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parvovirinae/genética , Retina/fisiopatologia
14.
Mol Cell Probes ; 47: 101439, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31445110

RESUMO

Both Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) can cause high mortality and morbidity in Muscovy ducklings. MDPVs and GPVs share high nucleotide identity, which can cause errors during differential diagnosis. In this study, the NS genes of both MDPVs and GPVs were chosen for the design of specific primers after genetic comparison. Only three primers (GF1, MF1 and MGR1) were designed for the duplex PCR assay: GF1 is specific for GPV only; MF1 is specific for MDPV only; and MGR1 is highly conserved for both MDPV and GPV. After a series of optimization experiments, the duplex PCR assay amplified a 161-bp fragment specifically for GPV, a 1197-bp fragment specifically for MDPV, and two fragments (161-bp and 1197-bp) for both GPV and MDPV. The lowest detection limit was 103 copies/µl. No amplification was obtained using nucleic acids from other pathogens (including DAdV-A, DuCV, DEV, GHPV, R.A., E. coli., P.M. and S.S.) occurring in Muscovy ducks. Application of the duplex PCR assay in field samples showed that even one-day-old Muscovy ducklings were both MDPV-positive and GPV-positive. In conclusion, a duplex PCR assay for the simultaneous detection and differentiation of MDPV and GPV was established using only three highly specific primers. Our finding suggested that country-wide vaccination with MDPV and GPV vaccines in waterfowls are necessary.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Parvoviridae/veterinária , Parvovirus/classificação , Doenças das Aves Domésticas/virologia , Proteínas não Estruturais Virais/genética , Animais , Diagnóstico Diferencial , Patos , Gansos , Limite de Detecção , Parvovirinae , Parvovirus/genética , Parvovirus/isolamento & purificação , Filogenia , Especificidade da Espécie
15.
Res Vet Sci ; 125: 212-217, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31271953

RESUMO

Goose parvovirus (GPV) is the etiological agent of Derzsy's disease, with a natural reservoir consisting only of geese and Muscovy ducks. However, the pathological changes in the immune organs of ducklings experimentally infected with GPV remain unknown. In this study, 2-day-old Cherry Valley ducklings were intramuscularly injected with GPV. Immune organs (e.g., thymus, bursa of Fabricius, spleen, Harderian gland, cecal tonsil, bone marrow, and peripheral blood lymphocytes [PBLs]) were collected 1, 3, 5, 7, 10, and 14 days post-infection (dpi). Pathological lesions were assessed by histology and the viral load was concurrently assessed using quantitative real-time polymerase chain reaction. GPV antigen was detected via immunofluorescence staining and immunohistochemistry. No clinical symptoms or death were observed in the infected ducklings from 1 to 14 dpi; however, lesions with different degrees of hemorrhage and hyperemia were observed in the thymus, spleen and Harderian gland. Lymphocyte necrosis was identified in the thymus and spleen. In the immune organs, the highest viral loads were found in the spleen at 7 dpi, followed by the bone marrow, PBLs, and cecal tonsil at 3 dpi, and the bursa, Harderian gland, and thymus at 1 dpi. GPV antigen was primarily expressed in the cecal tonsil, spleen, and Harderian gland at 5 dpi, as well as in the PBLs and bone marrow at 3 dpi. Our findings indicate widespread GPV replication and dissemination in the immune organs of Cherry Valley ducklings.


Assuntos
Bolsa de Fabricius/patologia , Patos , Infecções por Parvoviridae/veterinária , Parvovirinae , Doenças das Aves Domésticas/virologia , Baço/patologia , Animais , Bolsa de Fabricius/virologia , Gansos , Infecções por Parvoviridae/patologia , Parvovirus/genética , Doenças das Aves Domésticas/patologia , Reação em Cadeia da Polimerase em Tempo Real , Baço/virologia , Carga Viral
16.
Proc Natl Acad Sci U S A ; 116(29): 14755-14760, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31262807

RESUMO

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the loss of upper and lower motor neurons. Transgenic mice that overexpress mutant SOD1 develop paralysis and accumulate misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and endoplasmic reticulum (ER). Recently, macrophage migration inhibitory factor (MIF) was shown to directly inhibit mutant SOD1 misfolding and binding to intracellular membranes. In addition, complete elimination of endogenous MIF accelerated disease onset and late disease progression, as well as shortened the lifespan of mutant SOD1 mice with higher amounts of misfolded SOD1 detected within the spinal cord. Based on these findings, we used adeno-associated viral (AAV) vectors to overexpress MIF in the spinal cord of mutant SOD1G93A and loxSOD1G37R mice. Our data show that MIF mRNA and protein levels were increased in the spinal cords of AAV2/9-MIF-injected mice. Furthermore, mutant SOD1G93A and loxSOD1G37R mice injected with AAV2/9-MIF demonstrated a significant delay in disease onset and prolonged survival compared with their AAV2/9-GFP-injected or noninjected littermates. Moreover, these mice accumulated reduced amounts of misfolded SOD1 in their spinal cords, with no observed effect on glial overactivation as a result of MIF up-regulation. Our findings indicate that MIF plays a significant role in SOD1 folding and misfolding mechanisms and strengthen the hypothesis that MIF acts as a chaperone for misfolded SOD1 in vivo and may have further implications regarding the therapeutic potential role of up-regulation of MIF in modulating the specific accumulation of misfolded SOD1.


Assuntos
Esclerose Amiotrófica Lateral/terapia , Terapia Genética/métodos , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Medula Espinal/patologia , Superóxido Dismutase-1/metabolismo , Idade de Início , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/mortalidade , Animais , Células Cultivadas , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Injeções Espinhais , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Mutação , Parvovirinae/genética , Cultura Primária de Células , Agregados Proteicos , Dobramento de Proteína , Medula Espinal/citologia , Superóxido Dismutase-1/genética , Fatores de Tempo , Transdução Genética/métodos , Resultado do Tratamento
17.
Arch Virol ; 164(9): 2315-2320, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31168750

RESUMO

Novel protoparvoviruses genetically related to human and non-human primate bufaviruses (BuVs) have been detected recently in respiratory and enteric specimens collected from dogs and cats. In this study, by molecular screening of archival collections of faecal samples from wolves and foxes, we detected BuVs with a rate of 17.1% (7/41) and 10.5% (9/86), respectively. Sequence analysis of a portion of the ORF2 gene region of nine positive samples showed that the viruses in these samples were closely related to BuVs (97.5-99.0% nucleotide sequence identity) found in domestic carnivores.


Assuntos
Animais Selvagens/virologia , Raposas/virologia , Infecções por Parvoviridae/veterinária , Parvovirinae/genética , Parvovirinae/isolamento & purificação , Lobos/virologia , Animais , Animais Domésticos/virologia , Carnívoros/virologia , Cães , Fases de Leitura Aberta , Infecções por Parvoviridae/virologia , Parvovirinae/classificação , Filogenia
18.
Transbound Emerg Dis ; 66(5): 1834-1839, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31237413

RESUMO

Recently, short beak and dwarfism syndrome (SBDS) had a sudden outbreak in Cherry Valley duck flocks, followed by Pekin ducks and mule ducks in various regions of mainland China. This widely spreading infectious disease was characterized by growth retardation, smaller beak and tarsus with high morbidity and low mortality rate. In this study, we identified and characterized virus from domestic Linwu sheldrakes (namely as HuN18) with SBDS. HuN18 isolates shared high nucleotide identity with novel goose parvovirus (N-GPV). A 5110-nucleotide full-length genome sequence of HuN18 was found with no deletion in ITR region. Alignment studies of HuN18 showed 96.8%-99.0% identity with other N-GPVs and 92.9%-96.3% identity with classic GPV. According to the recombination analysis, HuN18 showed the potential major parent was the N-GPV sdlc01 strain, the potential minor parent was the classical GPV Y strain, and the secondary potential minor parent was the SYG61v strain. To the best of our knowledge, this is the first report of N-GPV in domestic Linwu sheldrakes with SBDS; these data provide evidence that attenuated live viruses are involved in genetic recombination with prevailing wild parvoviruses, which contributes to the novel emerging variants of waterfowl parvoviruses.


Assuntos
Surtos de Doenças/veterinária , Nanismo/veterinária , Genoma Viral/genética , Infecções por Parvoviridae/veterinária , Parvovirinae/isolamento & purificação , Doenças das Aves Domésticas/virologia , Animais , Bico/virologia , China/epidemiologia , Patos , Gansos , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Parvovirinae/genética , Filogenia , Doenças das Aves Domésticas/epidemiologia
19.
Invest Ophthalmol Vis Sci ; 60(7): 2494-2502, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31185088

RESUMO

Purpose: We determine whether intravitreal angiopoietin-1 combined with the short coiled-coil domain of cartilage oligomeric matrix protein by adeno-associated viral serotype 2 (AAV2.COMP-Ang1) delivery following the onset of vascular damage could rescue or repair damaged vascular beds and attenuate neuronal atrophy and dysfunction in the retinas of aged diabetic mice. Methods: AAV2.COMP-Ang1 was bilaterally injected into the vitreous of 6-month-old male Ins2Akita mice. Age-matched controls consisted of uninjected C57BL/6J and Ins2Akita males, and of Ins2Akita males injected with PBS or AAV2.REPORTER (AcGFP or LacZ). Retinal thickness and visual acuity were measured in vivo at baseline and at the 10.5-month endpoint. Ex vivo vascular parameters were measured from retinal flat mounts, and Western blot was used to detect protein expression. Results: All three Ins2Akita control groups showed significantly increased deep vascular density at 10.5 months compared to uninjected C57BL/6J retinas (as measured by vessel area, length, lacunarity, and number of junctions). In contrast, deep microvascular density of Ins2Akita retinas treated with AAV2.COMP-Ang1 was more similar to uninjected C57BL/6J retinas for all parameters. However, no significant improvement in retinal thinning or diabetic retinopathy-associated visual loss was found in treated diabetic retinas. Conclusions: Deep retinal microvasculature of diabetic Ins2Akita eyes shows late stage changes consistent with disorganized vascular proliferation. We show that intravitreally injected AAV2.COMP-Ang1 blocks this increase in deep microvascularity, even when administered subsequent to development of the first detectable vascular defects. However, improving vascular normalization did not attenuate neuroretinal degeneration or loss of visual acuity. Therefore, additional interventions are required to address neurodegenerative changes that are already underway.


Assuntos
Angiopoietina-1/administração & dosagem , Proteína de Matriz Oligomérica de Cartilagem/administração & dosagem , Retinopatia Diabética/prevenção & controle , Vetores Genéticos , Parvovirinae/genética , Neovascularização Retiniana/prevenção & controle , Vasos Retinianos/efeitos dos fármacos , Animais , Glicemia/metabolismo , Western Blotting , Capilares/efeitos dos fármacos , Diabetes Mellitus Tipo 1/complicações , Retinopatia Diabética/fisiopatologia , Portadores de Fármacos , Combinação de Medicamentos , Feminino , Terapia Genética , Insulina/genética , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retina/patologia , Neovascularização Retiniana/fisiopatologia , Vasos Retinianos/patologia , Acuidade Visual/fisiologia
20.
Hum Gene Ther Methods ; 30(3): 81-92, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31140323

RESUMO

Innate immune signals that promote B cell responses in gene transfer are generally ill-defined. In this study, we evaluate the effect of activating endosomal Toll-like receptors 7, 8, and 9 (TLR7, TLR7/8, and TLR9) on antibody formation during muscle-directed gene therapy with adeno-associated virus (AAV) vectors. We examined whether activation of endosomal TLRs, by adenine analog CL264 (TLR7 agonist), imidazolquinolone compound R848 (TLR7/8 agonist), or class B CpG oligodeoxynucleotides ODN1826 (TLR9 agonist), could augment antibody formation upon intramuscular administration of AAV1 expressing human clotting factor IX (AAV1-hFIX) in mice. The TLR9 agonist robustly enhanced antibody formation by the 1st week, thus initially eliminating systemic hFIX expression. By contrast, the TLR7 and TLR7/8 agonists did not markedly promote antibody formation, or significantly reduce circulating hFIX. We concurrently investigated the effects of these TLR agonists during muscle gene transfer on mature B cells and dendritic cells (DCs) in the draining lymph nodes including conventional DCs (CD11b+ or CD8α+ cDCs), monocyte-derived dendritic cells (moDCs), and plasmacytoid dendritic cells (pDCs). Only TLR9 stimulation caused a striking increase in the frequency of moDCs within 24 h. The TLR7/8 and TLR9 agonists activated pDCs, both subsets of cDCs, and mature B cells, whereas the TLR7 agonist had only mild effects on these cells. Thus, these TLR ligands have distinct effects on DCs and mature B cells, yet only the TLR9 agonist enhanced the humoral immune response against AAV-expressed hFIX. These new findings indicate a unique ability of certain TLR9 agonists to stimulate B cell responses in muscle gene transfer through enrichment of moDCs.


Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Células Dendríticas/imunologia , Fator IX/imunologia , Parvovirinae/genética , Músculo Quadríceps/imunologia , Receptor Toll-Like 9/agonistas , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Fator IX/genética , Terapia Genética , Imidazóis/farmacologia , Masculino , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos , Músculo Quadríceps/metabolismo , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia
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