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1.
Avian Pathol ; 50(1): 61-77, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33034512

RESUMO

Inoculation of embryonated chicken eggs has been widely used during the past decades; however, inoculation success rates have not been investigated systematically. In this study named success rates were assessed in brown eggs incubated between 5 and 19 days, which were inoculated with 0.2 ml methylene blue per egg. Inoculations were performed in a simple and fully standardized way. Five embryonic compartments were targeted blindly (amniotic cavity, embryo, allantoic cavity, albumen and yolk) with needles of four different lengths; albumen and yolk were targeted with eggs in upside down position. Three compartments were inoculated within sight (air chamber, chorioallantoic membrane and blood vessel). Twenty embryos were used per incubation day, intended deposition site and needle length. Success rates were assessed by visual inspection after breaking the eggs. The inoculations targeting albumen, yolk, amniotic cavity and embryo yielded low scores. Magnetic resonance imaging was performed to elucidate the reason(s) for these low success rates: needles used were of appropriate length, but embryo and amniotic cavity had variable positions in the eggs, while albumen and yolk rapidly changed position after turning the eggs upside down. The latter led to adjustment of the inoculation method for albumen and yolk. Failures to inoculate compartments within sight were immediately visible; therefore, these eggs could be discarded. Except for the amniotic cavity, full scores (20/20) were obtained for all compartments although not always on every day of incubation. In conclusion, the present study may serve as a guide to more accurately inoculate the various chicken embryo compartments. RESEARCH HIGHLIGHTS Blind inoculation of embryonated egg compartments was successful, except for the amniotic cavity. MRI showed rapid position change of albumen and yolk after turning eggs upside down. In ovo vaccination against Marek's disease might be improved by using 38 mm needles.


Assuntos
Doença de Marek/virologia , Óvulo/ultraestrutura , Alantoide/ultraestrutura , Alantoide/virologia , Âmnio/ultraestrutura , Âmnio/virologia , Animais , Embrião de Galinha , Membrana Corioalantoide/ultraestrutura , Membrana Corioalantoide/virologia , Feminino , Injeções , Imageamento por Ressonância Magnética/veterinária , Masculino , Azul de Metileno , Óvulo/virologia
2.
Methods Mol Biol ; 2203: 107-117, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833208

RESUMO

The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, and yolk). The developing embryo and its membranes provide a diversity of cell types that allow for the successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). IBV replicates well in the embryonated chicken egg, regardless of inoculation route; however, the allantoic route is favored as the virus replicates well in epithelium lining the chorioallantoic membrane, with high virus titers found in these membranes and associated allantoic fluids. TCoV replicates only in epithelium lining the embryo intestines and bursa of Fabricius; thus, amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for detection, propagation, and characterization of other, novel coronaviruses.


Assuntos
Embrião de Galinha/virologia , Coronavirus do Peru/isolamento & purificação , Vírus da Bronquite Infecciosa/isolamento & purificação , Alantoide/virologia , Âmnio/virologia , Animais , Embrião de Galinha/citologia , Coronavirus do Peru/fisiologia , Vírus da Bronquite Infecciosa/fisiologia , Tropismo Viral
3.
Avian Pathol ; 47(4): 418-426, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29712443

RESUMO

Sequence variability in the S1 gene determines the genotype of infectious bronchitis virus (IBV) strains. A single RT-PCR assay was developed to amplify and sequence the full S1 gene for six classical and variant IBVs (M41, D274, 793B, IS/885/00, IS/1494/06 and Q1) enriched in allantoic fluid (AF) or the same AF inoculated onto Flinders Technology Association (FTA) cards. Representative strains from each genotype were grown in specific-pathogen-free eggs and RNA was extracted from AF. Full S1 gene amplification was achieved using primer A and primer 22.51. Products were sequenced using primers A, 1050+, 1380+ and SX3+ to obtain short sequences covering the full gene. Following serial dilutions of AF, detection limits of the partial assay were higher than those of the full S1 gene. Partial S1 sequences exhibited higher-than-average nucleotide similarity percentages (79%; 352 bp) compared to full S1 sequences (77%; 1756 bp), suggesting that full S1 analysis allows greater strain differentiation. For IBV detection from AF-inoculated FTA cards, four serotypes were incubated for up to 21 days at three temperatures, 4°C, room temperature (approximately 24°C) and 40°C. RNA was extracted and tested with partial and full S1 protocols. Through partial sequencing, all IBVs were successfully detected at all sampling points and storage temperatures. In contrast, using full S1 sequencing it was not possible to amplify the gene beyond 14 days or when stored at 40°C. Data presented show that for full S1 sequencing, a substantial amount of RNA is needed. Field samples collected onto FTA cards are unlikely to yield such quantity or quality. ABBREVIATIONS: AF: allantoic fluid; CD50: ciliostatic dose 50; FTA: Flinders Technology Association; IB: infectious bronchitis; IBV: infectious bronchitis virus.


Assuntos
Galinhas/virologia , Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/genética , Doenças das Aves Domésticas/virologia , Glicoproteína da Espícula de Coronavírus/genética , Alantoide/virologia , Animais , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Líquido Extracelular/virologia , Feminino , Genótipo , Óvulo/virologia , Filogenia , Doenças das Aves Domésticas/diagnóstico , Sensibilidade e Especificidade , Análise de Sequência de DNA/veterinária , Organismos Livres de Patógenos Específicos , Manejo de Espécimes/veterinária , Temperatura
4.
J Virol Methods ; 251: 1-6, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28969954

RESUMO

Inactivation of Newcastle disease virus (NDV) has been routinely achieved with heat, ß-propiolactone, binary ethylenimine, ultraviolet light and formalin. However, these strategies have not been tested for cell surface ligand or receptor phenotype in viral-infected chicken immune cells. To study the capacity of fixation buffers to preserve surface markers while inactivating NDV, a primary splenocyte culture was infected with NDV and incubated with a commercial intracellular fixation buffer (ICB), formulated with 4% formaldehyde. Splenocytes were fixed with a 1:2 dilution of ICB in phosphate buffered saline (PBS) for 45min at 23°C or 4°C and inactivation of NDV was tested in addition to recognition of antigens by antibodies in fixed and non-fixed splenocytes via flow cytometric analysis. The binding and percentage of splenic CD4+ and CD8+ cells were not affected. In addition, NDV titers as high as 109.5 and 107.6 EID50 in allantoic fluid (AF) and macrophages, respectively, were successfully inactivated after 45min at 23°C and 4°C, confirming the ICB's effectiveness in inactivating high concentrations of NDV. In conclusion, high concentrations of NDV in AF, chicken splenocytes, and macrophages can be inactivated using ICB. Additionally, this method did not compromise cell phenotyping of enriched chicken splenocytes.


Assuntos
Desinfetantes/farmacologia , Fixadores/farmacologia , Leucócitos/virologia , Viabilidade Microbiana/efeitos dos fármacos , Vírus da Doença de Newcastle/efeitos dos fármacos , Inativação de Vírus , Alantoide/virologia , Animais , Células Cultivadas , Galinhas , Vírus da Doença de Newcastle/fisiologia , Baço/virologia
5.
J Virol Methods ; 221: 62-7, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25929989

RESUMO

RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commercial lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTqPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing materials, including diluted virus positive allantoic fluid or cell culture supernatant, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material.


Assuntos
DNA Complementar/metabolismo , Orthomyxoviridae/isolamento & purificação , RNA Viral/isolamento & purificação , Manejo de Espécimes/métodos , Alantoide/virologia , Animais , Aves , Cloaca/virologia , DNA Complementar/genética , Fezes/virologia , Orofaringe/virologia , Orthomyxoviridae/genética , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Suínos
6.
J Gen Virol ; 96(12): 3499-3506, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27257648

RESUMO

Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavirus infectious bronchitis virus (IBV). It was thought that coronavirus virions were composed of three major viral structural proteins until investigations of other coronaviruses showed that the virions also include viral non-structural and genus-specific accessory proteins as well as host-cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs, purified by sucrose-gradient ultracentrifugation and analysed by mass spectrometry. Analysis of three preparations of purified IBV yielded the three expected structural proteins plus 35 additional virion-associated host proteins. The virion-associated host proteins had a diverse range of functional attributions, being involved in cytoskeleton formation, RNA binding and protein folding pathways. Some of these proteins were unique to this study, while others were found to be orthologous to proteins identified in severe acute respiratory syndrome coronavirus virions and also virions from a number of other RNA and DNA viruses.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Vírus da Bronquite Infecciosa/metabolismo , Proteínas Virais/metabolismo , Vírion/metabolismo , Alantoide/virologia , Animais , Embrião de Galinha , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/ultraestrutura , Espectrometria de Massas , Proteoma , Organismos Livres de Patógenos Específicos , Proteínas Virais/genética , Vírion/genética , Vírion/ultraestrutura
7.
J Virol Methods ; 210: 22-5, 2014 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-25261812

RESUMO

Isolation of viruses using chick embryos is a classical virological method. Inoculation of the allantoic cavity and use of allantoic fluid is a common method of passaging isolated avian influenza viruses. In the present study, 2490 fresh fecal samples and 4967 old fecal samples were investigated and subjected to conventional passaging (allantoic fluid method). Two newly developed methods-the allantochorion and allantoic fluid mixed method and the chick embryo and allantoic fluid mixed method-were also examined. The rates of influenza virus isolation for these three methods were compared. There appeared to be little difference among these methods when fresh fecal samples were used. However, for the old fecal samples, isolation rates for influenza virus were significantly higher for the chick embryo and allantoic fluid mixed method compared with the conventional allantoic fluid method. All viruses isolated using the conventional allantoic fluid method were isolated successfully using the two newly developed methods. These results suggest that using chick embryos in conjunction with allantoic fluid is effective for early virus isolation, especially for fecal samples that are not fresh. Additionally, practical chick embryo passage methods are described that improve significantly the rate of isolation of influenza viruses from fecal samples of migratory birds in a complex wild ecological environment.


Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Alantoide/virologia , Animais , Aves , Embrião de Galinha , Fezes/virologia
8.
Cancer Gene Ther ; 21(11): 463-71, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25257305

RESUMO

Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic potential. Detailed preclinical information regarding the safety of oncolytic NDV is scarce. In this study, we evaluated the toxicity, biodistribution and shedding of intravenously injected oncolytic NDVs in non-human primates (Macaca fascicularis). Two animals were injected with escalating doses of a non-recombinant vaccine strain, a recombinant lentogenic strain or a recombinant mesogenic strain. To study transmission, naive animals were co-housed with the injected animals. Injection with NDV did not lead to severe illness in the animals or abnormalities in hematologic or biochemistry measurements. Injected animals shed low amounts of virus, but this did not lead to seroconversion of the contact animals. Postmortem evaluation demonstrated no pathological changes or evidence of virus replication. This study demonstrates that NDV generated in embryonated chicken eggs is safe for intravenous administration to non-human primates. In addition, our study confirmed results from a previous report that naïve primate and human sera are able to neutralize egg-generated NDV. We discuss the implications of these results for our study and the use of NDV for virotherapy.


Assuntos
Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Vírus da Doença de Newcastle/genética , Terapia Viral Oncolítica/métodos , Alantoide/virologia , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular , Embrião de Galinha , DNA Complementar/genética , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Injeções Intravenosas , Macaca fascicularis , Masculino , Mutagênese Sítio-Dirigida , Testes de Neutralização , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas Virais/administração & dosagem , Eliminação de Partículas Virais
9.
J Virol Methods ; 182(1-2): 76-81, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22465103

RESUMO

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of duck hepatitis virus type-1 (DHV-1) was established. Using primers specific to the highly conserved 3D gene of DHV-1, the developed RT-LAMP assay detected the viral RNA of DHV-1 extracted from both allantoic fluid and liver samples of infected ducks. The assay is as sensitive as RT-PCR, and shows no cross-reaction with other common avian viral and bacterial pathogens. In addition to detection via ethidium bromide staining following gel electrophoresis, naked-eye observation after staining with SYBR Green I dye can be used to detect RT-LAMP products; this enables field application of this assay. The findings demonstrate that RT-LAMP can serve as a helpful tool for the detection and surveillance of DHV-1 in the poultry industry.


Assuntos
Vírus da Hepatite do Pato/isolamento & purificação , Hepatite Viral Animal/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções por Picornaviridae/veterinária , Transcrição Reversa , Virologia/métodos , Alantoide/virologia , Animais , Benzotiazóis , Reações Cruzadas , Primers do DNA/genética , Diaminas , Patos , Eletroforese em Gel de Ágar , Vírus da Hepatite do Pato/genética , Fígado/virologia , Compostos Orgânicos , Infecções por Picornaviridae/virologia , Quinolinas , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
10.
Vopr Virusol ; 56(3): 24-8, 2011.
Artigo em Russo | MEDLINE | ID: mdl-21786623

RESUMO

Infectivity of pandemic influenza virus A(H1N1) infectivity is shown to be activated through proteolytic cleavage of hemagglutinin HA0 --> HA1 + HA2 during virus propagation in the human intestinal cell line Caco-2 and chicken embryonated eggs. Injection of aprotinin, a natural serine protease inhibitor, into the liquid culture or allantoic cavity of chicken embryos inhibited the proteolysis of the viral HA0 and suppressed the proteolytic activation of the synthesized virus and its multicycle replication. These data allow aprotinin to be recommended as an antiviral drug for the treatment of swine influenza in humans.


Assuntos
Aprotinina/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/tratamento farmacológico , Vírus Reordenados/fisiologia , Replicação Viral/efeitos dos fármacos , Alantoide/virologia , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Aprotinina/uso terapêutico , Células CACO-2 , Embrião de Galinha , Cães , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H1N1/química , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Pandemias/prevenção & controle , Vírus Reordenados/química , Inibidores de Serina Proteinase/farmacologia , Inibidores de Serina Proteinase/uso terapêutico
11.
Arch Virol ; 156(4): 557-63, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21203786

RESUMO

The avian influenza vaccine strain A/duck/Hokkaido/Vac-1/2004 (H5N1) (Vac-1) was found to be pathogenic in chicken embryos (CEs). In order to decrease the pathogenicity of Vac-1 in CEs, a series of reassortant viruses was generated between Vac-1 and A/Puerto Rico/8/1934 (H1N1) (PR8), and their pathogenicity and growth potential were compared in CEs. The results indicated that either the PB1 or PA protein was responsible for the pathogenicity of Vac-1 in CEs. The HA titers of the allantoic fluids of CEs inoculated with the recombinant H5N1 viruses, of which pathogenicity was lower than that of the recombinant Vac-1 prepared by reverse genetics in CEs, were equivalent to those of CEs inoculated with the recombinant Vac-1. One of the reassortant viruses, rg-PR8-PA/Vac-1 (H5N1), in which the PA gene was replaced with the corresponding gene of PR8, yielded allantoic fluids with the same HA titer as that of Vac-1, indicating that this reassortant should be a good candidate as an improved vaccine strain.


Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/efeitos adversos , Vírus Reordenados/patogenicidade , Alantoide/virologia , Animais , Embrião de Galinha , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Carga Viral
12.
Poult Sci ; 89(8): 1647-50, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20634520

RESUMO

The survival rate of Korean H5N1 highly pathogenic avian influenza (HPAI) viruses was investigated at different temperatures under the laboratory conditions. The estimated survival days for a starting viral concentration of 10(6.5) 50% egg infectious dose/0.1 mL were 930, 1,042, and 3,213 d at 4 degrees C; 226, 232, and 293 d at 20 degrees C; and 51, 55, and 58 d at 30 degrees C for A/chicken/Korea/ES/03, A/chicken/Korea/IS/06, and A/chicken/Korea/Gimje/08 (Gimje/08) viruses, respectively. The stability of the Gimje/08 virus was statistically significant compared with the other 2 viruses except for the data between Gimje/08 and A/chicken/Korea/IS/06 virus at 30 degrees C. This result indicated that the survival rate of 3 Korean HPAI viruses is different at various temperatures, which might have partially influenced the large scale of HPAI outbreak in Korea in 2008.


Assuntos
Virus da Influenza A Subtipo H5N1/fisiologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/epidemiologia , Alantoide/virologia , Animais , Galinhas/virologia , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Influenza Aviária/virologia , Análise de Regressão , Organismos Livres de Patógenos Específicos , Temperatura
13.
J Virol Methods ; 168(1-2): 72-7, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20435070

RESUMO

Avian influenza viruses (AIVs) of the H9N2 subtype are a major economic problem in the poultry industry in Israel. Most field isolates from the last decade differ significantly from H9N2 isolates from Europe and the USA, rendering published detection methods inadequate. This study aimed to develop a real-time TaqMan((R)) RT-PCR assay, based on a conserved region in the HA9 gene. The assay was validated with viruses representing different genetic subtypes and other common avian pathogens, and was found specific to H9N2. The real-time RT-PCR assay was compared to RT-PCR, which is in routine diagnostic use. Real-time RT-PCR was found to be more sensitive than RT-PCR by 1.5-2.5 orders of magnitude when testing tracheal swabs directly and by 2-3 orders of magnitude allantoic fluid after AIV propagation in embryonated eggs. Sensitivity was quantified by using 10-fold dilutions of the H9-gene amplification fragment, and real-time RT-PCR was found to be 10(4)-fold more sensitive than RT-PCR. Clinical samples, which included tracheal and cloacal swabs, as well as allantoic fluid, were tested by both methods. By real-time RT-PCR 20% more positive H9N2 samples were detected than by RT-PCR. The real-time RT-PCR assay was found suitable for detection and epidemiological survey not only of Israeli H9N2 viruses, but also for isolates from other parts of the world.


Assuntos
Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/diagnóstico , Influenza Aviária/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virologia/métodos , Alantoide/virologia , Animais , Galinhas , Cloaca/virologia , Hemaglutininas Virais/genética , Israel , Sensibilidade e Especificidade , Traqueia/virologia
14.
J Gen Virol ; 91(Pt 1): 51-8, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19759237

RESUMO

The regions at the left and right ends of fowl adenovirus (FAdV) genomes are not well-characterized in comparison to those of human adenoviruses. Using a series of deletion mutants, we analysed a 2.4 kb region near the left end of the FAdV-9 genome (nt 400-2782) that contains packaging-signal motifs VI and VII and open reading frames (ORFs) 0, 1, 1A, 1B, 1C and 2. Viable viruses with specific deletions in this region had wild-type characteristics in vitro, as measured by cytopathic effect, plaque morphology, virus titres and growth kinetics. However, one mutant (FAdV-9Delta4), which lacked these ORFs and retained the packaging motifs, did not replicate at wild-type levels in vivo, as judged in infected eggs by virus titres in allantoic fluid and in infected chickens by antibody responses, virus titres in faeces and virus genome copy numbers in tissues. These findings indicate that some of the ORFs in this region, although dispensable in vitro, are important for in vivo replication of FAdV-9.


Assuntos
Aviadenovirus/fisiologia , DNA Viral/genética , Genoma Viral , Alantoide/virologia , Animais , Aviadenovirus/genética , Aviadenovirus/crescimento & desenvolvimento , Aviadenovirus/patogenicidade , Embrião de Galinha , Galinhas , Efeito Citopatogênico Viral , Ovos/virologia , Fezes/virologia , Viabilidade Microbiana , Deleção de Sequência , Carga Viral , Ensaio de Placa Viral , Virulência
15.
J Vet Med Sci ; 72(4): 519-23, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20032626

RESUMO

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an established gene amplification method for rapid diagnosis of various infectious diseases. In order to detect avian influenza viruses, particularly in field specimens, specific primers targeting the matrix gene were designed. Thirty-four virus samples, including isolates from wild and domestic avian hosts belonging to various geographical areas, were used to confirm the validity of the primers. All samples were confirmed to be positive in less than 1 hr. The RT-LAMP assay was also able to detect avian influenza virus in the various field samples, such as swabs, tissues, and feces. These results indicate that the developed RT-LAMP assay with uniquely designed primers is potentially useful in comprehensive avian influenza surveillance.


Assuntos
Aves/virologia , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Alantoide/virologia , Animais , Sequência de Bases , Encéfalo/virologia , Primers do DNA , DNA Viral/genética , Fezes/virologia , Amplificação de Genes , Músculo Esquelético/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Transcrição Reversa , Traqueia/virologia
16.
J Virol Methods ; 153(2): 218-22, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18652846

RESUMO

Influenza is a serious global public health problem and an economic burden. With the continual emergence of new influenza A virus strains, new antiviral drugs are needed urgently. In this study, an improved embryonated chicken egg model for evaluating antiviral activity against Influenza A virus was developed. In the model, the influenza A virus was injected into the allantoic cavity and ribavirin was injected into the albumen of the egg. The levels of influenza A virus in the allantoic fluid was titrated by the hemagglutination test after incubation for 72 h at 35.5 degrees C and 12 h at 4 degrees C. Ribavirin treatment at a dose of 25 mg/kg to 100 mg/kg decreased significantly the hemagglutination titers both of Influenza virus A/FM1, H1N1 (IVA1) (p < 0.01) and influenza virus A/Wuhan/359/95, H3N2 (IVA3) (p < 0.01). In a time-dependent drug addition assay, significant efficacy of ribavirin against both IVA1 and IVA3 was observed when the drug was administered before and shortly after viral inoculation (p < 0.01 or p < 0.05). In conclusion, ribavirin treatment showed significant antiviral activity against IVA1 and IVA3 in this model, suggesting that the improved model would be useful for evaluating the anti-influenza virus activity of potential inhibitors.


Assuntos
Alantoide/virologia , Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Ribavirina/farmacologia , Animais , Antivirais/administração & dosagem , Linhagem Celular , Embrião de Galinha , Testes de Hemaglutinação , Humanos , Modelos Animais , Ribavirina/administração & dosagem
17.
J Clin Microbiol ; 46(8): 2561-7, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18562585

RESUMO

Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA(0) is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay targeting the HA(0) cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA(0) cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluid samples and 11 diagnostic swab samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR assay--followed by cycle sequencing--can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.


Assuntos
Primers do DNA/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Alantoide/virologia , Animais , Animais Domésticos/virologia , Animais Selvagens/virologia , Aves/virologia , Vírus da Influenza A/isolamento & purificação , Dados de Sequência Molecular , Precursores de Proteínas/genética , RNA Viral/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA
18.
Jpn J Infect Dis ; 60(6): 342-6, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18032831

RESUMO

General theories on the inactivation of viruses in the presence of a concentrated protein, such as the allantoic fluid of chicken eggs, are not useful. That is, although sodium hypochlorite and sodium hydroxide are generally known as strong virucidal agents, they do not sufficiently inactivate viruses in allantoic fluid. We found that benzalkonium chloride (BC) is an effective virucidal agent against influenza, Newcastle disease, and avian infectious bronchitis viruses even in the presence of a concentrated protein. BC is easily biodegradable by activated sludge and is not very harmful to humans. We strongly recommend BC as a useful virucidal agent, especially in the manufacture of vaccines for these viruses.


Assuntos
Alantoide/virologia , Anti-Infecciosos/farmacologia , Vírus da Bronquite Infecciosa/efeitos dos fármacos , Vírus da Doença de Newcastle/efeitos dos fármacos , Orthomyxoviridae/efeitos dos fármacos , Inativação de Vírus/efeitos dos fármacos , Animais , Compostos de Benzalcônio/farmacologia , Embrião de Galinha , Etanol/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Hidróxido de Sódio/farmacologia , Hipoclorito de Sódio/farmacologia
19.
Vaccine ; 25(22): 4456-63, 2007 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-17418461

RESUMO

A yield enhancement technology for use in influenza vaccine manufacturing has been developed to maximize the recovery of influenza virus from allantoic fluid of virus-infected chick embryos; the standard raw material for influenza vaccine. Virus associated with amorphous debris in the allantoic fluid can be dissociated from the debris and recovered, thereby increasing viral yield. Dissociation can be achieved by subjecting the virus-debris complex to conditions of increased ionic strength at defined pH. Multifold increases in viral yield per ml of allantoic fluid were observed. The degree of yield enhancement is strain-specific, however, increases were observed in all type A and type B influenza strains tested. The heightened influenza virus recoveries can facilitate rapid vaccine manufacture, with increased numbers of doses produced, and may become essential at a time of influenza pandemic.


Assuntos
Alantoide/virologia , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Vacinas contra Influenza , Animais , Biotecnologia/métodos , Centrifugação com Gradiente de Concentração/métodos , Embrião de Galinha , Galinhas , Indústria Farmacêutica/métodos , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza A/classificação , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza B/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/virologia , Fatores de Tempo , Cultura de Vírus/métodos
20.
J Vet Diagn Invest ; 19(2): 155-60, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17402609

RESUMO

A rapid and simple latex agglutination test (LAT) for the detection of avian influenza virus (AIV) subtype H5N1 in chicken allantoic fluids, tracheal swabs, and tissues was developed. Monoclonal antibodies against the hemagglutinin glycoprotein of H5N1 were covalently coupled onto the surface of carboxylated latex bead using a water-soluble carbodiimide to obtain sensitized latex particles (SLP). These SLPs strongly agglutinated in the presence of allantoic fluid containing H5N1, but not fluids containing other AIV sub-types such as HIN1, H3N2, H4N6, and H9N2. Using this LAT, the virus was detectable in tracheal swabs 24 hours to 30 days after inoculating chickens with H5N1, with detection rates ranging from 45.5 to 79.2%. Much higher rates of detection were obtained from tissues collected postmortem from H5N1 experimentally infected chickens; lung tissue yielded the highest detection rate (96.7%), followed by kidney, spleen, brain, and liver tissues (90%). Lower detection rates were achieved with heart (41.7%) and cloacal tissues (26.8%). When the LAT was compared with other detection methods, the agreement with the viral isolation, H5 antigen immunochromatographic test,and H5 real-time RT-PCR test was 93.97, 95.18, and 87.95%, respectively. The test was highly specific for H5N1 in chickens and water fowls and had sensitivity comparable to other diagnostic tests evaluated.


Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/diagnóstico , Testes de Fixação do Látex/veterinária , Alantoide/virologia , Animais , Galinhas , Virus da Influenza A Subtipo H5N1/classificação , Traqueia/virologia
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