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1.
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
2.
Avian Pathol ; 49(4): 325-334, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32208867

RESUMO

Waterfowl parvoviruses (WPVs) including goose parvovirus (GPV), novel GPV-related virus (NGPV) and Muscovy duck parvovirus (MDPV) cause significant economic losses and epizootic threat to the waterfowl industries, and little is known about the B-cell epitopes of WPVs. In this study, a monoclonal antibody (mAb) 5B5 against the VP3 protein of NGPV was used to identify the possible epitope in the three kinds of WPVs. The mAb 5B5 had neutralizing activities to the three viruses, and reacted with the conserved linear B-cell epitopes of 438LHNPPP443 in VP3 protein of GPV, NGPV and MDPV. To the authors' best knowledge, this is the first report on identification of the common conserved neutralizing linear B-cell epitope on VP3 protein of three different WPVs, which would facilitate the development of a novel immunodiagnostic assay for rapid detection of WPV infection.


Assuntos
Epitopos de Linfócito B/genética , Gansos/virologia , Infecções por Parvoviridae/veterinária , Parvovirinae/imunologia , Doenças das Aves Domésticas/virologia , Animais , Anticorpos Monoclonais/imunologia , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/virologia , Parvovirinae/genética , Parvovirinae/isolamento & purificação , Doenças das Aves Domésticas/diagnóstico , Proteínas Virais/genética
3.
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
4.
J Virol ; 93(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30333169

RESUMO

Adeno-associated virus serotype 5 (AAV5) is being developed as a gene delivery vector for several diseases, including hemophilia and Huntington's disease, and has a demonstrated efficient transduction in liver, lung, skeletal muscle, and the central nervous system. One limitation of AAV gene delivery is preexisting neutralizing antibodies, which present a significant challenge for vector effectiveness in therapeutic applications. Here, we report the cryo-electron microscopy (cryo-EM) and image-reconstructed structure of AAV5 in complex with a newly generated monoclonal antibody, HL2476, at 3.1-Å resolution. Unlike other available anti-AAV5 capsid antibodies, ADK5a and ADK5b, with epitopes surrounding the 5-fold channel of the capsid, HL2476 binds to the 3-fold protrusions. To elucidate the capsid-antibody interactions, the heavy and light chains were sequenced and their coordinates, along with the AAV5 viral protein, assigned to the density map. The high resolution of the complex enabled the identification of interacting residues at the 3-fold protrusions of the capsid, including R483, which forms two hydrogen bonds with the light chain of HL2476. A panel of AAV5 variants was generated and analyzed by native dot immunoblot and transduction assays. This identified variants with antibody escape phenotypes that maintain infectivity.IMPORTANCE Biologics based on recombinant AAVs (rAAVs) are increasingly becoming attractive human gene delivery vehicles, especially after the approval of Glybera in Europe and Luxturna in the United States. However, preexisting neutralizing antibodies against the AAV capsids in a large percentage of the human population limit wide-spread utilization of these vectors. To circumvent this problem, stealth vectors must be generated that are undetectable by these antibodies. This study details the high-resolution characterization of a new antigenic region on AAV5, a vector being developed for numerous delivery applications. The structure of AAV5 complexed with HL2476, a novel antibody, was determined by cryo-EM to 3.1-Å resolution. The resolution of the density map enabled the identification of interacting residues between capsid and antibody and the determinants of neutralization. Thus, the information obtained from this study can facilitate the generation of host immune escape vectors.


Assuntos
Anticorpos Monoclonais/metabolismo , Capsídeo/química , Epitopos/imunologia , Parvovirinae/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Capsídeo/imunologia , Microscopia Crioeletrônica , Dependovirus , Feminino , Células HEK293 , Humanos , Ligação de Hidrogênio , Camundongos , Parvovirinae/química , Engenharia de Proteínas
5.
J Virol ; 93(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30541847

RESUMO

A key aspect to consider for vaccinal protection is the induction of a local line of defense consisting of nonrecirculating tissue-resident memory T cells (TRM), in parallel to the generation of systemic memory CD8+ T cell responses. The potential to induce TRM has now been demonstrated for a number of pathogens and viral vectors. This potential, however, has never been tested for recombinant adeno-associated virus (rAAV) vectors, which are weakly inflammatory and poor transducer of dendritic cells. Using a model rAAV2/1-based vaccine, we determined that a single intradermal immunization with rAAV2/1 vectors in mice induces fully functional TRM at the local site of immunization. The optimal differentiation of rAAV-induced transgene-specific skin TRM was dependent on local transgene expression and additional CD4+ T cell help. Transgene expression in dendritic cells, however, appeared to be dispensable for the priming of transgene-specific skin TRM, suggesting that this process solely depends on the cross-presentation of transgene products. Overall, this study provides needed information to properly assess rAAV vectors as T cell-inducing vaccine carriers.IMPORTANCE rAAVs display numerous characteristics that could make them extremely attractive as vaccine carriers, including an excellent safety profile in humans and great flexibility regarding serotypes and choice of target tissue. Studies addressing the ability of rAAV to induce protective T cell responses, however, are scarce. Notably, the potential to induce a tissue-resident memory T cell response has never been described for rAAV vectors, strongly limiting further interest for their use as vaccine carriers. Using a model rAAV2/1 vaccine delivered to the skin, our study demonstrated that rAAV vectors can induce bona fide skin resident TRM and provides additional clues regarding the cellular mechanisms underlying this process. These results will help widen the field of rAAV applications.


Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Parvovirinae/imunologia , Animais , Células Dendríticas/imunologia , Dependovirus , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Parvovirinae/genética , Pele/citologia , Pele/imunologia , Transgenes/genética , Transgenes/imunologia , Vacinação , Vacinas Virais/imunologia
6.
Acta Virol ; 62(4): 415-423, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30472872

RESUMO

Goose parvovirus (GPV) is a highly contagious and lethal disease in goslings and Muscovy ducklings, and is of concern to the waterfowl industry. With the aim of comparing the cellular immunogenicity of three capsid proteins of GPV, plasmids of pcDNA3.1(+)-VP1, pcDNA3.1(+)-VP2, and pcDNA3.1(+)-VP3 were constructed, and the recombinant protein VPs were expressed using an eukaryotic expression system. We detected the levels of immune-related genes (CD4, CD8α, IL-1ß, IL-6, IFNα, IFNγ, and IFNλ) in both goose embryo fibroblasts (GEF) and goose peripheral blood mononuclear cells (PBMCs) cellular models. The immune response conferred by a VP2 DNA vaccine in vivo was observed in a time course. Our data suggested that the cellular immune response to VP2 and VP3 was stronger than that to VP1, while VP2 and VP3 shared similar cellular immune reactivity. In addition, vaccination with VP2 plasmid can induce high level of IgY antibody that continued to increase through 28 days post vaccination. Therefore, our findings shed light on the host cellular immune response against GPV capsid proteins. Keywords: GPV; capsid proteins; cellular immune response; humoral immunity.


Assuntos
Proteínas do Capsídeo , Infecções por Parvoviridae , Vacinas Virais , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Leucócitos Mononucleares/imunologia , Infecções por Parvoviridae/imunologia , Parvovirinae/genética , Parvovirinae/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia
7.
Gene Ther ; 25(3): 205-219, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29785047

RESUMO

The successful application of adeno-associated virus (AAV) gene delivery vectors as a therapeutic paradigm will require efficient gene delivery to the appropriate cells in affected organs. In this study, we utilized a rational design approach to introduce modifications to the AAV2 and AAVrh8R capsids and the resulting variants were evaluated for transduction activity in the retina and brain. The modifications disrupted either capsid/receptor binding or altered capsid surface charge. Specifically, we mutated AAV2 amino acids R585A and R588A, which are required for binding to its receptor, heparan sulfate proteoglycans, to generate a variant referred to as AAV2-HBKO. In contrast to parental AAV2, the AAV2-HBKO vector displayed low-transduction activity following intravitreal delivery to the mouse eye; however, following its subretinal delivery, AAV2-HBKO resulted in significantly greater photoreceptor transduction. Intrastriatal delivery of AAV2-HBKO to mice facilitated widespread striatal and cortical expression, in contrast to the restricted transduction pattern of the parental AAV2 vector. Furthermore, we found that altering the surface charge on the AAVrh8R capsid by modifying the number of arginine residues on the capsid surface had a profound impact on subretinal transduction. The data further validate the potential of capsid engineering to improve AAV gene therapy vectors for clinical applications.


Assuntos
Terapia Genética/métodos , Parvovirinae/crescimento & desenvolvimento , Parvovirinae/imunologia , Animais , Encéfalo/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Dependovirus/imunologia , Técnicas de Transferência de Genes , Vetores Genéticos , Células HeLa , Heparitina Sulfato , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Transdução Genética/métodos
8.
Virology ; 518: 369-376, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29604478

RESUMO

Adeno-associated viruses (AAVs) are being developed as vectors for the treatment of genetic disorders. However, pre-existing antibodies present a significant limitation to achieving optimal efficacy for the AAV gene delivery system. Efforts aimed at engineering vectors with the ability to evade the immune response include identification of residues on the virus capsid important for these interactions and changing them. Here K531 is identified as the determinant of monoclonal antibody ADK6 recognition by AAV6, and not the closely related AAV1. The AAV6-ADK6 complex structure was determined by cryo-electron microscopy and the footprint confirmed by cell-based assays. The ADK6 footprint overlaps previously identified AAV antigenic regions and neutralizes by blocking essential cell surface glycan attachment sites. This study thus expands the available repertoire of AAV-antibody information that can guide the design of host immune escaping AAV vectors able to maintain capsid functionality.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Parvovirinae/imunologia , Anticorpos Monoclonais/ultraestrutura , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/ultraestrutura , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Dependovirus , Parvovirinae/ultraestrutura , Ligação Proteica
9.
Trop Anim Health Prod ; 49(5): 1085-1088, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28470582

RESUMO

The prevention of Ungulate protoparvovirus 1 (UPV1) infection and consequently the reproductive losses is based on vaccination of all pigs intended for breeding. As maternally derived antibodies (MDA) can interfere with the development of immunity following vaccination, it is important to know the duration of anti-UPV1 MDA to determine the optimal age for the best vaccination efficacy. To elucidate the association between dam and piglet antibody levels against UPV1 and to estimate the decrease rate of MDA, sera and colostrum of 127 gilts (before the first vaccination against UPV1; 15 days after the second vaccine dose; at farrowing; and during the second pregnancy) and sera of 276 piglets (prior to initial colostrum intake; at 7, 21, 57, 87, and 128 days-old) were tested by ELISA. Most gilts (85.8%) had anti-UPV1 antibodies before vaccination and after vaccination all were positive. At 7 days old, the majority of the piglets had anti-UPV1 antibodies, but around 57 days old, only 35.3% were positive and at 87 days old, all were negative. The estimated average half-life of MDA was 29.8 (28.8-30.9) days. A strong correlation was determined between piglet serum at 7 days old with gilt serum at farrowing time (r = 0.77, n = 248, P < 0.001) and with piglet serum at 7 days old with colostrum (r = 0.73, n = 248, P < 0.001). The MDA decreased earlier and the antibody half-life was a little longer than previously reported. Based on these findings, UPV1 vaccination can be performed earlier than usual.


Assuntos
Imunidade Materno-Adquirida , Infecções por Parvoviridae/veterinária , Parvovirinae/imunologia , Doenças dos Suínos/imunologia , Vacinação/veterinária , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Colostro/imunologia , Feminino , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/virologia , Sus scrofa , Suínos , Doenças dos Suínos/virologia
10.
Sci Rep ; 6: 25545, 2016 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-27150912

RESUMO

Both Tembusu virus (TMUV) and goose parvovirus (GPV) are causative agents of goose disease. However, the host immune response of the goose against these two different categories of virus has not been well documented. Here, we compared the clinical symptoms and pathological characteristics, antigen distribution and intensity, and expression of immune-related genes in TMUV- and GPV- infected goose. The immunohistochemistry analysis demonstrated that GPV was primarily located in the liver, lung, small intestine, and rectum, while TMUV was situated in the liver, brain, spleen, and small intestine. The induction of IFNγ and proinflammatory cytokines is highly associated with the distribution profiles of antigen and CD8α+ molecules. The effector function of CD8 T cells may be accomplished by the secretion of IFNγ together with high expression of proinflammatory cytokines such as IL1 and IL6. Remarkably, significant increases in the transcription of immune genes were observed after infection, which suggested that both GPV and TMUV can effectively induce immune response in goose PMBCs. This study will provide fundamental information for goose molecular immunology in defending against pandemic viruses.


Assuntos
Antígenos Virais/análise , Linfócitos T CD8-Positivos/imunologia , Infecções por Flavivirus/veterinária , Flavivirus/imunologia , Infecções por Parvoviridae/veterinária , Parvovirinae/imunologia , Estruturas Animais/virologia , Animais , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/patologia , Gansos , Imuno-Histoquímica , Interferon gama/metabolismo , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/patologia
11.
Virus Res ; 203: 77-83, 2015 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-25882914

RESUMO

Newcastle disease virus (NDV) and Goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting geese. In this study, we generated a recombinant rmNA-VP3, expressing GPV VP3 using a modified goose-origin NDV NA-1 by changing the multi-basic cleavage site motif RRQKR↓F of the F protein to the dibasic motif GRQGR↓L as that of the avirulent strain LaSota as a vaccine vector. Expression of the VP3 protein in rmNA-VP3 infected cells was detected by immunofluorescence and Western blot assay. The genetic stability was examined by serially passaging 10 times in 10-day-old embryonated SPF chicken eggs. Goslings were inoculated with rmNA-VP3 showed no apparent signs of disease and developed a strong GPV and NDV neutralizing antibodies response. This is the first study demonstrating that recombinant NDV has the potential to serve as bivalent live vaccine against Goose parvovirus and Newcastle disease virus infection in birds.


Assuntos
Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/imunologia , Infecções por Parvoviridae/veterinária , Parvovirinae/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas Virais/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Gansos , Instabilidade Genômica , Vírus da Doença de Newcastle/genética , Infecções por Parvoviridae/prevenção & controle , Parvovirinae/genética , Inoculações Seriadas , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
12.
Vet Q ; 34(4): 180-4, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25312356

RESUMO

BACKGROUND: Goose parvovirus (GPV) is a highly contagious and deadly disease for goslings and Muscovy ducklings. OBJECTIVES: To compare the differences in immune response of geese immunized with GPV-VP1 DNA-based and live attenuated vaccines. ANIMALS AND METHODS: Shitou geese were immunized once with either 20 µg pcDNA-GPV-VP1 DNA gene vaccine by gene gun bombardment via intramuscular injection, or 300 µg by i.m. injection, or 300 µL live attenuated vaccine by i.m. injection, whereas 300 µg pcDNA3.1 (+) i.m. or 300 µL saline i.m. were used as positive and negative controls, respectively. Each group comprised 28 animals. Peripheral blood samples were collected from 2-210 days after immunization and the proliferation of T lymphocytes, the number of CD4(+) and CD8(+) T cells and the level of IgG assessed. Statistical analysis was performed using a one-way analysis of variance with group multiple comparisons via Tukey's test. RESULTS: The pcDNA-GPV-VP1 DNA and attenuated vaccine induced cellular and humoral responses, and there were no differences between the 20 and 300 µg group in the responses of proliferation of T lymphocyte and the CD8(+) T-cell. However, as to CD4(+) T-cell response and humoral immunity, the 20 µg group performed better than the 300 µg group, which induced better cellular and humoral immunity than live attenuated vaccine. CONCLUSIONS: This study showed that it is possible to induce both cellular and humoral response using DNA-based vaccines and that the pcDNA-GPV-VP1 DNA gene vaccine induced better cellular and humoral immunity than live attenuated vaccine.


Assuntos
Gansos , Imunidade Celular , Imunidade Humoral , Infecções por Parvoviridae/veterinária , Parvovirinae/imunologia , Doenças das Aves Domésticas/imunologia , Vacinas Virais/imunologia , Animais , China , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/virologia , Doenças das Aves Domésticas/virologia , Vacinas Atenuadas/imunologia , Vacinas de DNA/imunologia
13.
Braz J Microbiol ; 44(2): 519-21, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24294250

RESUMO

Through bioinformatic prediction, between Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV), there were one epitope AA503-509 (RANEPKE) on non-structural protein and three epitopes AA426-430 (SQDLD), 540-544 (DPYRS), 685-691 (KENSKRW) on structural protein might cross-react with each other. Furthermore, the four epitops were expressed in Escherichia coli. All the four recombinant proteins could react with GPV-antisera and MDPV-antisera in Western blot.


Assuntos
Epitopos/imunologia , Parvovirinae/imunologia , Animais , Anticorpos Antivirais/sangue , Clonagem Molecular , Biologia Computacional/métodos , Reações Cruzadas , Patos , Epitopos/genética , Escherichia coli/genética , Expressão Gênica , Parvovirinae/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologia
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