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1.
Avian Dis ; 65(1): 122-131, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-34339131

RESUMO

A Korean field strain of fowl adenovirus (FAdV) 8b was isolated from chickens showing high mortality. Isolated FAdV-8b strains with the hexon and fiber genes were genetically analyzed. The Korean FAdV-8b (K194/19) strain isolated in 2019 showed higher sequence identity with the FAdV-8b strain isolated in China but lower sequence identity with the Korean FAdV-8b (K187/08) strain isolated in 2008. The K194/19 strain formed a distinct subcluster within the FAdV-8b cluster in a phylogenetic tree based on hexon and fiber genes. FAdV can infect day-old chicks through vertical transmission, and so blood samples were obtained from 54-, 60-, and 63-wk-old parent chickens. FAdV-specific antibody levels were investigated with ELISA and virus neutralization (VN) tests with the K194/19 and K187/08 strains as antigens. In VN tests, all sera neutralized the K187/08 strain. However, the K194/19 strain was neutralized by sera collected from 60- and 63-wk-old chickens but not sera obtained from 54-wk-old chickens, indicating natural infection. Finally, to determine the pathogenicity of the K194/19 strain, 1-day-old and 4-wk-old specific-pathogen-free birds were infected with the K194/19 and K187/08 strains. No significant difference in pathogenicity was observed between the two strains. Although the K194/19 strain showed similar pathogenicity with the K187/08 strain, differences in nucleotide and amino acid sequences of the hexon and fiber genes may determine the evasion ability of the K187/08 neutralizing antibody, indicating the need for development of a novel FAdV vaccine.


Assuntos
Infecções por Adenoviridae/veterinária , Galinhas , Adenovirus A das Aves/genética , Adenovirus A das Aves/patogenicidade , Doenças das Aves Domésticas/virologia , Infecções por Adenoviridae/virologia , Animais , República da Coreia , Organismos Livres de Patógenos Específicos , Virulência
2.
Avian Dis ; 65(1): 177-187, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-34339138

RESUMO

Currently, the poultry industry worldwide is facing an emerging trend of fowl adenovirus (FAdV)-associated diseases with a significant economic impact, especially in meat-type chickens. Vertical transmission is an important feature of all FAdVs; hence, preventive measures mostly revolve around breeding stocks. However, knowledge about temporal development of FAdV infections in modern commercial settings is rare or even nonexistent. In the present study, longitudinal monitoring for FAdV was conducted in broiler breeder flocks located in a confined geographical region with intensive poultry production in Iran. For this, the antibody status of birds from 4 to 32 wk of age was monitored with a commercial FAdV-ELISA and virus neutralization test (VNT). In parallel, fecal shedding of FAdV was determined at the peak of egg production with real-time PCR and virus isolation. Overall, the commercial ELISA showed seroconversion of flocks before onset of production. VNT resolved in detail infection patterns of individual serotypes with a primordial FAdV-D (FAdV-2/-11) infection, frequently followed by FAdV-E (FAdV-8a, -8b) superinfection. FAdV-A (FAdV-1) was traced in half of the investigated flocks, while no evidence of infection with FAdV-C (FAdV-4, -10) was noted. Common serological profiles between different houses of the same farm indicate an overarching biosecurity. Serological profiles coupled with virological findings at the peak of egg production indicated that higher antibody levels, determined by ELISA, correlated with lower amounts of viral DNA in fecal excretion. Simultaneously, the number of isolated FAdVs belonging to distinct serotypes declined in accordance with a rise of neutralizing antibodies in birds, underlining the significance of serotype-specific antibodies in the epidemiology of FAdV in breeders. Investigations in breeders were complemented with screening of FAdV-associated diseases in local broilers over a 3-yr period; 26 cases of inclusion body hepatitis with dominant involvement of FAdV-11/FAdV-8b, one outbreak of adenoviral gizzard erosion related to FAdV-1, and no evidence of hepatitis-hydropericardium syndrome suggest that identical serotypes are maintained in the local poultry industry.


Assuntos
Infecções por Adenoviridae/veterinária , Galinhas , Ensaio de Imunoadsorção Enzimática/veterinária , Adenovirus A das Aves/isolamento & purificação , Doenças das Aves Domésticas/epidemiologia , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/virologia , Animais , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Irã (Geográfico)/epidemiologia , Doenças das Aves Domésticas/virologia , Prevalência
3.
J Virol Methods ; 294: 114172, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33915232

RESUMO

The CMV immediate early promoter from the EGFP expression plasmid pEGFP-N1 was replaced with the very left end of the fowl adenovirus 9 (FAdV-9) genome (ntds 73-574) to demonstrate and delineate the promoter function of this sequence. Expression of an EGFP ORF which replaced ORF1 and ORF2 demonstrated that the native promoter can drive down stream foreign gene expression. Replacement of ORF1 and ORF2 with a bicistronic cassette, incorporating a 493 bp IRES from an Ontario strain of avian encephalomyelitis virus (AEV) separating an EGFP ORF and mCherry ORF allowed for expression of both ORFs from a recombinant FAdV. These results provide an additional platform for multivalent vaccines development based on a native FAdV-9 promoter and an avian virus IRES.


Assuntos
Infecções por Adenoviridae , Aviadenovirus , Adenovirus A das Aves , Doenças das Aves Domésticas , Animais , Aviadenovirus/genética , Galinhas , Adenovirus A das Aves/genética , Expressão Gênica , Fases de Leitura Aberta , Plasmídeos , Regiões Promotoras Genéticas
4.
Virulence ; 12(1): 754-765, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33616472

RESUMO

The outbreaks of hepatitis-hydropericardium syndrome (HPS) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) have caused a huge economic loss to the poultry industry globally since 2013. Although the Fiber-2 has been identified as a key virulent related factor for FAdV-4, little is known about its molecular basis. In this study, we identified the efficient interaction of the Fiber-2 with the karyopherin alpha 3/4 (KPNA3/4) protein via its N-terminus of 1-40aa. The analysis of the overexpression and knockout of KPNA3/4 showed that KPNA3/4 could efficiently assist the replication of FAdV-4. Moreover, a fiber-2-edited virus FAV-4_Del with a deletion of 7-40aa in Fiber-2 was rescued through the CRISPR-Cas9 technique. In comparison with the wild type FAdV-4, FAV-4_Del was highly attenuated in vitro and in vivo. Notably, the inoculation of FAV-4_Del in chickens could provide full protection against the lethal challenge with the wild type FAdV-4. All these findings not only give novel insights into the molecular basis for the pathogenesis of Fiber-2 but also provide efficient targets for developing antiviral strategies and live-attenuated vaccine candidates against the highly pathogenic FAdV-4.


Assuntos
Adenovirus A das Aves/genética , Adenovirus A das Aves/patogenicidade , Proteínas Virais/genética , Replicação Viral , alfa Carioferinas/metabolismo , Animais , Anticorpos Antivirais/sangue , Linhagem Celular Tumoral , Galinhas/virologia , Adenovirus A das Aves/classificação , Adenovirus A das Aves/fisiologia , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , Sorogrupo , Virulência , alfa Carioferinas/genética
5.
Avian Pathol ; 50(1): 2-5, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32795192

RESUMO

Fowl adenovirus (FAdV) infections in chickens have undergone substantial changes in recent decades, driven by host and pathogen factors. Based on the pathogenesis of inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS), modern broilers are much more inclined to have difficulties keeping the metabolic homeostasis, whereas adenoviral gizzard erosion (AGE) is noticed equally in broilers and egg-layers. Defining the importance of certain serotypes for specific FAdV diseases is a major achievement of recent years but the isolation of viruses from clinically healthy birds remains unexplained, as virulence factors are hardly known and continue to be a "black box". Together with further studies on pathogenesis of FAdV-induced diseases, such knowledge on virulence factors would help to improve protection strategies, which presently mainly concentrate on autogenous vaccines of breeders to prevent vertical transmission.


Assuntos
Infecções por Adenoviridae/veterinária , Galinhas/virologia , Adenovirus A das Aves/fisiologia , Interações Hospedeiro-Patógeno , Doenças das Aves Domésticas/patologia , Vacinas Virais/imunologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/prevenção & controle , Infecções por Adenoviridae/virologia , Animais , Autovacinas/imunologia , Feminino , Adenovirus A das Aves/imunologia , Adenovirus A das Aves/patogenicidade , Moela das Aves/patologia , Moela das Aves/virologia , Masculino , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Sorogrupo , Virulência , Fatores de Virulência
6.
Vet Res ; 51(1): 143, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33267862

RESUMO

A recombinant fowl adenovirus (FAdV) fiber protein, derived from a FAdV-8a strain, was tested for its efficacy to protect chickens against inclusion body hepatitis (IBH). FAdV-E field isolates belonging to both a homotypic (FAdV-8a) and heterotypic (-8b) serotype were used as challenge. Mechanisms underlying fiber-induced protective immunity were investigated by fiber-based ELISA, virus neutralization assays and flow cytometry of peripheral blood mononuclear cells, monitoring the temporal developments of humoral and cellular responses after vaccination and challenge exposure. Birds were clinically protected from the homologous challenge and showed a significant reduction of viral load in investigated target organs, whereas fiber-based immunity failed to counteract the heterologous serotype infection. These findings were supported in vitro by the strictly type-specific neutralizing activity of fiber immune sera. In protected birds, fiber vaccination prevented a post-challenge drop of peripheral B cells in blood. Furthermore, fiber immunization stimulated CD4+ T lymphocyte proliferation while moderating the CD8α+ T cell response and prevented challenge-induced changes in systemic monocytes/macrophages and γδ+ T cell subpopulations. Both vaccinated and adjuvant-only injected birds experienced a priming of systemic B cells and TCRγδ+ T lymphocytes, which masked possible pre-challenge effects due to the antigen. In conclusion, within FAdV-E, recombinant fiber represents a vaccine candidate to control the adverse effects of homotypic infection by eliciting an effective humoral immunity and regulating B and T cell response, whereas the failure of heterotypic protection suggests a primordial role of humoral immunity for this vaccine.


Assuntos
Galinhas , Adenovirus A das Aves/metabolismo , Hepatite Viral Animal/prevenção & controle , Imunidade Celular/imunologia , Vacinas contra Hepatite Viral/imunologia , Proteínas Virais/imunologia , Animais , Linfócitos B/classificação , Linfócitos B/metabolismo , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Organismos Livres de Patógenos Específicos , Linfócitos T/classificação , Linfócitos T/metabolismo
7.
Avian Dis ; 64(1): 16-22, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32267121

RESUMO

Hydropericardium syndrome (HPS) is caused by fowl adenovirus serotype 4 (FAdV-4). HPS has caused outbreaks in Chinese populations of broiler chickens since 2015. However, little is known about the molecular mechanisms underlying HPS. In this study, we used transcriptomic analysis to screen differentially expressed genes (DEGs) in the livers of FAdV-4-infected and noninfected chicks. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the gene network associated with the arginine metabolism pathway was enriched in livers infected by FAdV-4; 10 genes were downregulated and 8 genes were upregulated in these livers when compared to noninfected livers. The DEGs identified in livers were reanalyzed by real-time fluorescence quantitative PCR (qPCR); results indicated that the mRNA levels of the DEGs concurred with the data derived from KEGG analysis. Next, we used qPCR to detect the DEGs of the arginine metabolism pathway in a hepatocellular carcinoma cell line (LMH) after infection with FAdV-4 for 24 hr; this also indicated that the mRNA levels of the DEGs concurred with that seen in the liver. We also used si-RNA oligonucleotides to knock down the mRNA levels of iNOS in LMH cells infected with FAdV-4 and found that the viral load of FAdV-4 was increased. Further investigation revealed that the addition of 240 µg/ml of arginine into the culture medium of LMH cells infected with FAdV-4 for 24 hr led to a significant increase in the mRNA levels of iNOS but a significant reduction in the viral load of FAdV-4. Therefore, our data indicated that when broiler chickens become infected with FAdV-4, the arginine metabolic pathway in the liver becomes dysfunctional and the iNOS mRNA level decreases. This will add benefit to the replication of FAdV-4 but can be inhibited by the addition of an appropriate amount of arginine.


Assuntos
Infecções por Adenoviridae/veterinária , Arginina/metabolismo , Galinhas , Adenovirus A das Aves/fisiologia , Doenças das Aves Domésticas/virologia , Replicação Viral , Infecções por Adenoviridae/virologia , Animais , Feminino , Adenovirus A das Aves/classificação , Fígado/virologia , Sorogrupo , Carga Viral
8.
Vaccine ; 38(2): 143-149, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31672334

RESUMO

Recently, outbreaks of adenoviral gizzard erosion (AGE) have been documented in pullets and layers housed free range and in enriched cage systems characterized by increased mortality and a negative impact on egg production. In the present study the pathogenicity of a fowl adenovirus serotype 1 (FAdV-1) field strain as well as the aetiological role of a FAdV-8a strain, both isolated from AGE affected pullets, were investigated in vivo in 20-week-old specific-pathogen-free (SPF) layer-type chickens. Furthermore, the efficacy of a single (week 17) and double (week 14 and 17) application of a live vaccine consisting of an apathogenic FAdV-1 (CELO strain) against challenge with virulent FAdV-1 was investigated. For the first time, AGE was successfully reproduced in adult birds after oral infection of 20-week-old SPF birds with a virulent FAdV-1 field isolate, characterized by pathological changes of the gizzard from 7 days post challenge onwards. In addition, a negative impact of the FAdV-1 infection on the development of the reproductive tract was observed. Thus, confirming the pathogenicity and aetiological role of FAdV-1 in the development of AGE and economic losses due to AGE in layers. In contrast, no pathological changes were observed in birds infected with FAdV-8a. Independent of a single or double application of the live FAdV-1 vaccine strain CELO, no gross pathological changes were observed in gizzards post challenge with the virulent FAdV-1, indicating that complete protection of layers against horizontal induction of AGE was achieved. Nonetheless, virulent FAdV-1 was detected in cloacal swabs and gizzards in both vaccinated groups post challenge determined by the application of an amplification refractory mutation system quantitative PCR used to differentiate between vaccine and challenge strains.


Assuntos
Adenovirus A das Aves/genética , Moela das Aves/virologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas Virais/administração & dosagem , Adenoviridae/genética , Infecções por Adenoviridae/prevenção & controle , Infecções por Adenoviridae/veterinária , Animais , Aviadenovirus/genética , Galinhas/virologia , Feminino , Masculino , Doenças das Aves Domésticas/virologia , Sorogrupo , Organismos Livres de Patógenos Específicos , Vacinação , Vacinas Virais/imunologia
9.
Avian Dis ; 63(4): 568-576, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31865670

RESUMO

Gizzard erosions have been noticed in slaughtered broiler chickens during inspection at a processing plant in Iran. The condition was detected in piled gizzards derived from seven commercial broiler farms brought to slaughter on the same day. In total, 48 gizzards with lesions underwent thorough pathologic and virologic investigation. Perforation, roughening, and discoloration of the koilin layer as well as inflammation of the mucosa were observed macroscopically. Histologic examination showed dissociation of and cellular debris in the koilin layer accompanied by a loss and degeneration of glandular epithelium with mild to marked infiltration of inflammatory cells in the mucosa, submucosa, and muscular layer. Fowl adenovirus serotypes 1 (FAdV-1), 11 (FAdV-11), and 8a (FAdV-8a) were found in 13, 12, and 1 gizzard(s), respectively. Therein included were two gizzards that showed mixed infections with FAdV-1 and FAdV-11. Detailed analysis of the hexon gene revealed that the Iranian FAdV-1 isolates could be divided into two subclusters, more closely related to either the European (CELO) or the Asian (Ote) FAdV-1 reference strains. The present study, for the first time, describes not only the appearance of gizzard erosion but also the isolation of FAdV-1 and FAdV-8a from broilers in Iran and offers insights on the epidemiology of FAdV infection in Iranian flocks.


Assuntos
Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/veterinária , Galinhas , Adenovirus A das Aves/fisiologia , Moela das Aves/patologia , Doenças das Aves Domésticas/patologia , Infecções por Adenoviridae/virologia , Animais , Moela das Aves/virologia , Irã (Geográfico) , Doenças das Aves Domésticas/virologia
10.
Avian Dis ; 63(4): 619-624, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31865676

RESUMO

Fowl adenovirus (FAdV) infection is an emerging problem in the world poultry industry, especially in broilers, as the causal agent of inclusion body hepatitis or hepatitis-hydropericardium syndrome. From December 2017 to January 2019, we recorded 116 cases of suspected hepatitis-hydropericardium syndrome in chicken farms throughout Indonesia. Necropsy was done on each farm site with three to five freshly dead birds per farm. Tissue samples were collected in virus transport medium and frozen at -20 C. The virus was cultivated in 9-day-old fertilized specific-pathogenic-free chicken eggs. FAdV was detected using polymerase chain reaction with a published primer set. The polymerase chain reaction products were sequenced and subjected to a BLAST search. The phylogeny was inferred using the neighbor-joining method and tested using the bootstrap test. FadV-D and -E are present in Indonesia and confirmed in 40 of 116 suspected cases. The affected chicken ages were 27.27 ± 8.94 days. Most affected farms were raising broiler chickens. The only typical clinical sign was unusual daily mortality of >1%, while the three most frequent pathologic lesions were swelling and hemorrhage of kidney and liver, as well as hydropericardium. To reduce economic loss, a vaccine should be developed immediately.


Assuntos
Infecções por Adenoviridae/veterinária , Galinhas , Epidemias/veterinária , Adenovirus A das Aves/fisiologia , Doenças das Aves Domésticas , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/patologia , Animais , Adenovirus A das Aves/classificação , Indonésia/epidemiologia , Filogenia , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/patologia , Organismos Livres de Patógenos Específicos
11.
Viruses ; 11(8)2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408986

RESUMO

Since 2015, severe outbreaks of hepatitis-hydropericardium syndrome (HHS), caused by hypervirulent fowl adenovirus serotype 4 (FAdV-4), have emerged in several provinces in China, posing a great threat to poultry industry. So far, factors contributing to the pathogenesis of hypervirulent FAdV-4 have not been fully uncovered. Elucidation of the pathogenesis of FAdV-4 will facilitate the development of effective FAdV-4 vaccine candidates for the control of HHS and vaccine vector. The interaction between pathogen and host defense system determines the pathogenicity of the pathogen. Therefore, the present review highlights the knowledge of both viral and host factors contributing to the pathogenesis of hypervirulent FAdV-4 strains to facilitate the related further studies.


Assuntos
Infecções por Adenoviridae/veterinária , Suscetibilidade a Doenças , Adenovirus A das Aves/fisiologia , Doenças das Aves Domésticas/virologia , Animais , Adenovirus A das Aves/classificação , Adenovirus A das Aves/patogenicidade , Interações Hospedeiro-Patógeno , Doenças das Aves Domésticas/metabolismo , Sorogrupo , Proteínas Virais/metabolismo , Virulência , Fatores de Virulência
12.
Viruses ; 11(6)2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31195615

RESUMO

Fowl adenovirus serotype 4 (FAdV-4) is the pathogenic agent of hydropericardium hepatitis syndrome (HHS) in chickens and ducks, which has caused huge economic losses for the Chinese poultry industry since 2015. In order to objectively determine the prevalence and co-infection status of the virus in Shandong province in China, we analyzed a total of 679 clinical cases of chickens and ducks from 36 farms in the province. The results showed that the FAdV-4 infection rate was 65.2% (443/679), and the rate in breeder ducks was almost two-fold higher than that in breeder chickens (68.57% vs. 34.30%). Notably, co-infection by H9N2 avian influenza virus, infectious bursal disease virus, and/or chicken infectious anemia virus was very common in the 443 FAdV-4-positive cases. Furthermore, phylogenetic analysis of the hexon genes of four Shandong FAdV-4 isolates revealed that these strains clustered into Indian reference strains, indicating that the Shandong FAdV-4 strains might have originated in India. These findings provide the first data on the prevalence and co-infection status of FAdV-4 in Shandong province, which may serve as a foundation for the prevention of FAdV-4 in the field.


Assuntos
Infecções por Adenoviridae/veterinária , Coinfecção/veterinária , Adenovirus A das Aves , Doenças das Aves Domésticas/epidemiologia , Adenoviridae/genética , Adenoviridae/imunologia , Adenoviridae/isolamento & purificação , Infecções por Adenoviridae/epidemiologia , Animais , Proteínas do Capsídeo/genética , Vírus da Anemia da Galinha/isolamento & purificação , Galinhas/virologia , China/epidemiologia , Coinfecção/epidemiologia , Coinfecção/virologia , Patos/virologia , Adenovirus A das Aves/genética , Adenovirus A das Aves/imunologia , Adenovirus A das Aves/isolamento & purificação , Genes Virais , Hepatite Animal/epidemiologia , Hepatite Animal/virologia , Vírus da Doença Infecciosa da Bursa/isolamento & purificação , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Filogenia , Filogeografia , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , Prevalência , Sorogrupo
13.
Poult Sci ; 98(9): 3514-3522, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30993349

RESUMO

Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium syndrome. To clarify the effects of FAdV-4 on immune organs in birds, we conducted a detailed examination of dynamic morphology and damage mechanisms in chickens randomly divided into 4 groups (FAdV-4, vaccination, FAdV-4 plus vaccination, and control). FAdV-4 caused the depletion of lymphocytes and subsequent growth impairment in the thymus and bursa. Chickens infected with FAdV-4 and subjected to vaccination experienced greater inhibition of antibody responses to inactivated vaccines against Newcastle disease and avian influenza virus subtype H9 than uninfected and vaccinated chickens. The mechanisms underlying adenovirus-mediated lymphoid organ damage were further investigated via transferase-mediated dUTP nick-end labeling and apoptotic genes transcription analyses. Notably, lymphocytes apoptosis in lymphoid organs and expression of specific gene transcripts was significantly upregulated after infection (P < 0.05). Furthermore, increased expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α mRNA was observed (P < 0.05), compared to the control group. Our collective findings suggested that FAdV-4 caused structural and functional damage of immune organs via apoptosis along with induction of a severe inflammatory response.


Assuntos
Infecções por Adenoviridae/veterinária , Galinhas , Adenovirus A das Aves/fisiologia , Tolerância Imunológica/imunologia , Imunidade Humoral/imunologia , Doenças das Aves Domésticas/imunologia , Tropismo Viral/imunologia , Infecções por Adenoviridae/imunologia , Animais , Apoptose , Adenovirus A das Aves/imunologia , Inflamação , Distribuição Aleatória , Sorogrupo
14.
BMC Vet Res ; 15(1): 103, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30935399

RESUMO

BACKGROUND: Major viruses, including duck-origin avian influenza virus, duck-origin Newcastle disease virus, novel duck parvovirus, duck hepatitis A virus, duck Tembusu virus, fowl adenovirus, and duck enteritis virus, pose great harm to ducks and cause enormous economic losses to duck industry. This study aims to establish a multiplex polymerase chain reaction (m-PCR) method for simultaneous detection of these seven viruses. RESULTS: Specific primers were designed and synthesized according to the conserved region of seven viral gene sequences. Then, seven recombinant plasmids, as the positive controls, were reconstructed in this study. Within the study, D-optimal design was adopted to optimize PCR parameters. The optimum parameters for m-PCR were annealing temperature at 57 °C, Mg2+ concentration at 4 mM, Taq DNA polymerase concentration at 0.05 U/µL, and dNTP concentration at 0.32 mM. With these optimal parameters, the m-PCR method produced neither cross-reactions among these seven viruses nor nonspecific reactions with other common waterfowl pathogens. The detection limit of m-PCR for each virus was 1 × 104 viral DNA copies/µL. In addition, the m-PCR method could detect a combination of several random viruses in co-infection analysis. Finally, the m-PCR method was successfully applied to clinical samples, and the detection results were consistent with uniplex PCR. CONCLUSION: Given its rapidity, specificity, sensitivity, and convenience, the established m-PCR method is feasible for simultaneous detection of seven duck-infecting viruses and can be applied to clinical diagnosis of viral infection in ducks.


Assuntos
Patos/virologia , Reação em Cadeia da Polimerase Multiplex/veterinária , Doenças das Aves Domésticas/virologia , Animais , Coinfecção/diagnóstico , Coinfecção/veterinária , Coinfecção/virologia , Flavivirus , Adenovirus A das Aves , Vírus da Hepatite do Pato , Reação em Cadeia da Polimerase Multiplex/métodos , Vírus da Doença de Newcastle , Orthomyxoviridae , Parvovirinae , Doenças das Aves Domésticas/diagnóstico , Sensibilidade e Especificidade
15.
Avian Pathol ; 48(3): 278-283, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30663340

RESUMO

Extinct from nature, captive young Alagoas curassows (Pauxi mitu) were found agonizing or dead with respiratory disease. Intranuclear inclusion bodies were found in the epithelia of the trachea, associated with marked necrotic tracheitis. An Aviadenovirus was isolated in chicken eggs and characterized genetically with 99% identity to the fowl Aviadenovirus A, as based on the hexon protein gene. This is the first report of respiratory disease caused by Aviadenovirus in any cracid species in Brazil, recommending for stricter biosecurity in the conservation premises. RESEARCH HIGHLIGHTS Fatal tracheitis in curassows extinct from nature was associated with Aviadenovirus A. Seven-month-old Alagoas curassows (Aves: Cracidae) died with haemorrhagic tracheitis. Aviadenovirus A with 99% identity to fowl adenovirus 1 was detected in dead curassows. Fatal tracheitis by Aviadenovirus was described in Pauxi mitu (Aves: Cracidae).


Assuntos
Aviadenovirus/classificação , Galliformes/virologia , Doenças das Aves Domésticas/diagnóstico , Traqueíte/veterinária , Animais , Aviadenovirus/genética , Aviadenovirus/isolamento & purificação , Brasil , Evolução Fatal , Adenovirus A das Aves/genética , Corpos de Inclusão Viral/virologia , Corpos de Inclusão Intranuclear/virologia , Necrose/veterinária , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Traqueia/patologia , Traqueia/virologia , Traqueíte/diagnóstico , Traqueíte/patologia , Traqueíte/virologia
16.
PLoS One ; 14(12): e0225863, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31891571

RESUMO

Fowl adenovirus (FAdV) is the causative agent of inclusion body hepatitis (IBH) in chickens with significant economic losses due to high mortality and poor production. It was objectives of the study to attenuate and determine the molecular characteristic of FAdV isolate (UPM1137) of Malaysia passages in primary chicken embryo liver (CEL) cells. The cytopathic effect (CPE) was recorded and the present of the virus was detected by polymerase chain reaction (PCR). Nucleotide and amino acid changes were determined and a phylogenetic tree was constructed. The pathogenicity and immunogenicity of the virus at passage 35 (CEL35) with virus titre of 106.7TCID50/mL was determined in day old specific pathogen free (SPF) chicks via oral or subcutaneous route of inoculation. The study demonstrated that the FAdV isolate was successfully propagated and attenuated in CEL cells up to 35th consecutive passages (CEL35) with delayed of CPE formation within 48 to 72 post inoculation (pi) from CEL20 onwards. The virus caused typical CPE with basophilic intranuclear inclusion bodies, refractile and clumping of cells. The virus is belong to serotype 8b with substitution of amino acid at position 44, 133 and 185 in L1 loop of hexon gene and in knob of fiber gene at position 348 and 360 at CEL35. It is non-pathogenic, but immunogenic in SPF chickens. It was concluded that the FAdV isolate was successfully attenuated in CEL cells with molecular changes in major capsid proteins which affect its infectivity in cell culture and SPF chickens.


Assuntos
Infecções por Adenoviridae , Galinhas , Adenovirus A das Aves , Hepatite Viral Animal , Fígado , Doenças das Aves Domésticas , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/veterinária , Animais , Células Cultivadas , Embrião de Galinha , Galinhas/imunologia , Galinhas/virologia , Adenovirus A das Aves/imunologia , Adenovirus A das Aves/isolamento & purificação , Adenovirus A das Aves/patogenicidade , Hepatite Viral Animal/imunologia , Hepatite Viral Animal/patologia , Hepatite Viral Animal/virologia , Fígado/imunologia , Fígado/virologia , Malásia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia
17.
Artigo em Inglês | MEDLINE | ID: mdl-30245048

RESUMO

Fowl adenovirus-4 (FAdV-4) causes hydropericardium syndrome (HPS) in poultry worldwide. An understanding of viral structural protein composition is important for developing novel immunodiagnostics and immunoprophylactics. Here we report isolation, culture, molecular and protein profile of FAdV-4 isolates recovered from HPS outbreaks in chicken in the states of Himachal Pradesh and Tamil Nadu in India. We performed a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting-based protein profiling of FAdV-4 isolates against a reference FAdV-1 or Chicken Embryo Lethal Orphan (CELO) virus. SDS-PAGE analysis showed that seven protein bands in FAdV-4 isolates were similar to CELO expect an additional band of 110 kDa in CELO virus. On Western blotting, two protein fractions of 43 kDa and 78 kDa size were observed in FAdV-4 isolates. Overall, results show that FAdV-4 isolates recovered from different regions of the country had similar protein profile and possibly a common source of origin.


Assuntos
Infecções por Adenoviridae/veterinária , Aviadenovirus/química , Galinhas , Doenças das Aves Domésticas/virologia , Proteínas Virais/análise , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/genética , Aviadenovirus/crescimento & desenvolvimento , Aviadenovirus/isolamento & purificação , Western Blotting/métodos , DNA Viral/química , Surtos de Doenças/veterinária , Eletroforese em Gel de Poliacrilamida/métodos , Adenovirus A das Aves/química , Soros Imunes/imunologia , Índia/epidemiologia , Masculino , Filogenia , Reação em Cadeia da Polimerase , Aves Domésticas , Doenças das Aves Domésticas/epidemiologia , Coelhos , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
18.
J Vet Sci ; 19(6): 759-770, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30173491

RESUMO

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 108.7TCID50/mL (TCID50, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity.


Assuntos
Infecções por Adenoviridae/veterinária , Proteínas do Capsídeo/genética , Adenovirus A das Aves , Infecções por Adenoviridae/virologia , Animais , Embrião de Galinha/virologia , Galinhas/virologia , Clonagem Molecular , DNA Forma Z/genética , Adenovirus A das Aves/genética , Adenovirus A das Aves/isolamento & purificação , Adenovirus A das Aves/patogenicidade , Fígado/patologia , Fígado/virologia , Malásia , Filogenia , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/virologia , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA , Organismos Livres de Patógenos Específicos
19.
Biologicals ; 54: 50-57, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29752158

RESUMO

Inclusion body hepatitis and hydropericardium-hepatitis syndrome caused by serotype 4 fowl adenovirus (FAdV-4) have emerged in China since 2013. FAdV is usually propagated in primary chicken embryonic liver cells or embryo yolk sac. The aim of this work was to develop an immortalized CEL cell line by stable expression of human mitochondrial ribosomal protein 18S-2, named CEL-hMRP18S-2 cells, for the propagation of FAdV-4. The maximum cell density of CEL-hMRP18S-2 cells could reach 2.65 × 106 cells/ml in four-days culture. According to the mRNA levels of cell-cycle related genes in CEL-hMRP18S-2 cells tested by qRT-PCR, we speculated that the transformation of hMRP18S-2 into CEL cells caused the functional inactivation of p53 and the significant down-regulation of p15INK4b might cause the hyperphosphorylated form of Rb, releasing E2F-1 factor and enhancing the E2F-dependent transcription for cell cycle progression. It was suspected that the up-regulated c-Myc mRNA level at the initial period of immortalization might prompt transformed cells through the G0-G1 checkpoint. The normal CPE was observed in CEL-hMRP18S-2 cells infected by FAdV-4 and microcarrier suspension culture performed for FAdV-4 propagation with 9.0 lgTCID50/ml suggested that CEL-hMRP18S-2 cells could be a useful continuous cell line for isolation of wild FAdV and production of FAdV-inactivated vaccine.


Assuntos
Infecções por Adenoviridae , Adenovirus A das Aves/crescimento & desenvolvimento , Expressão Gênica , Fígado , Proteínas Mitocondriais , Proteínas Ribossômicas , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/metabolismo , Infecções por Adenoviridae/patologia , Animais , Linhagem Celular Transformada , Embrião de Galinha , Galinhas , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética
20.
Vet Res ; 49(1): 29, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29523195

RESUMO

A recent outbreak of hepatitis-hydropericardium syndrome caused by serotype 4 fowl adenovirus (FAdV-4) has resulted in significant economic losses to the poultry industry worldwide. However, little is known about the molecular pathogenesis of FAdV-4. In this study, a novel monoclonal antibody (mAb) targeting the fiber-2 protein of FAdV-4 was generated, mAb 3C2. Indirect immunofluorescence assay showed that mAb 3C2 neither reacted with serotype 8 fowl adenovirus (FAdV-8) nor reacted with the fiber-1 protein of FAdV-4; it specifically reacted with the fiber-2 protein of FAdV-4. Notably, mAb 3C2 could efficiently immunoprecipitate the fiber-2 protein in chicken liver cells either infected with FAdV-4 or transfected with pcDNA3.1-Fiber2. Moreover, mAb 3C2 demonstrated marked neutralizing activity against FAdV-4 and could efficiently inhibit the infection of FAdV-4 in vitro. Using truncated fiber-2 constructs, the epitope recognized by mAb 3C2 was determined to be located between amino acids 416-448 at the C-terminus of fiber-2. Our data not only provide a foundation for the establishment of a rapid fiber-2 peptide-based diagnostic assay for FAdV-4 but also highlight the critical role of the fiber-2 protein in mediating infection by FAdV-4. Furthermore, the epitope recognized by 3C2 might serve as a novel target for the development of a vaccine targeting FAdV-4.


Assuntos
Infecções por Adenoviridae/veterinária , Anticorpos Antivirais/imunologia , Adenovirus A das Aves/fisiologia , Doenças das Aves Domésticas/prevenção & controle , Proteínas Virais/genética , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/prevenção & controle , Infecções por Adenoviridae/virologia , Animais , Anticorpos Monoclonais/imunologia , Galinhas , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Sorogrupo , Proteínas Virais/metabolismo
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