RESUMEN
Human adenovirus 36 (HAdV-D36) is presently the sole virus identified to be associated with an elevated risk of obesity in both humans and animals. However, its impact on embryonated chicken eggs (ECEs) remains unexplored. This study endeavoured to examine the influence of HAdV-D36 on embryonic development by utilizing embryonated chicken eggs as a dynamic model. To simulate various infection routes, the allantoic cavity and the yolk sac of ECEs were inoculated with HAdV-D36. Subsequently, embryos from both the experimental (inoculated with virus) and control (inoculated with PBS) groups were weighed and subjected to daily histological examination. The daily embryo weights were assessed and compared between groups using the Shapiro-Wilk test. Histopathological changes in tissues were examined and compared between the tested and control groups to ascertain physiological alterations induced by the virus. Our study confirmed a significant increase in the body weight of ECEs. However, this phenomenon was not attributable to adipose tissue development; rather, it was characterized by an augmented number of cells in all observed tissues compared to control subjects. We posit that HAdV-D36 may impact developing organisms through mechanisms other than enhanced adipose tissue development. Specifically, our findings indicate an increased number of cells in all tissues, a phenomenon that occurs through an as-yet-unexplored pathway.
Asunto(s)
Adenovirus Humanos , Pollos , Animales , Humanos , Peso Corporal , ObesidadRESUMEN
Camels are susceptible to a variety of infectious diseases, such as trypanosomiasis, anthrax, hemorrhagic septicemia, brucellosis, mange, and pox, which can also affect other farm animal species. Camelpox is one of the most infectious skin diseases, which is caused by the Camelpox virus (CPV), a member of the Orthopoxvirus genus in the Poxviridae family. This study mainly aimed to detect and isolate CPV affecting camels in Wasit province, Iraq. Initially, the study focused principally on clinical manifestations of the disease in affected animals. Afterward, 110 skin samples were collected from infected animals under strict aseptic conditions. They were to be subjected to inoculation into the local embryonated chicken eggs to isolate the virus from the chicken embryo fibroblast cell culture. Finally, the isolates were confirmed using the molecular polymerase chain reaction (PCR) assay. Camelpox virus isolates appeared as several necrotic pock lesions on the Chorioallantoic Membrane (CAM) with cell clustering and sloughing or detachment from the monolayer. Molecular testing was conducted using the PCR assay by targeting the ATIP gene at 881bp. The findings demonstrated that all the investigated isolates were poxvirus positive. In addition, it was found that Camelpox disease is significantly endemic in Wasit province, Iraq. The visualization of the characteristic features of the virus also revealed it can easily adapt to replication in the CAM and cell culture.
Asunto(s)
Orthopoxvirus , Infecciones por Poxviridae , Embrión de Pollo , Animales , Orthopoxvirus/genética , Camelus/genética , Irak/epidemiología , Infecciones por Poxviridae/epidemiología , Infecciones por Poxviridae/veterinaria , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
Avian influenza virus and some mammalian influenza A viruses can be isolated, propagated, and titrated in embryonated chicken eggs (ECEs). Most sample types can be accommodated in ECE culture with appropriate processing. Isolation may also be accomplished in cell culture, and if a mammalian lineage influenza A is expected to be in an avian sample, for example swine influenza in turkey specimens, mammalian cell may be preferable. Culture in ECEs is highly sensitive but is not specific for influenza A, which may be an advantage because a sample may be screened for several agents at once. Once an agent is isolated in culture, the presence of influenza viruses can be confirmed by hemagglutination inhibition assay, antigen immunoassay, agar gel immunodiffusion assay, or RT-PCR. Finally, ECEs may be used to propagate and titrate an avian influenza virus.
Asunto(s)
Pollos/virología , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Óvulo/virología , Virología/métodos , Animales , Embrión de Pollo , Membrana Corioalantoides/metabolismo , Manejo de Especímenes , Saco Vitelino/metabolismoRESUMEN
Influenza virus isolation is a procedure to obtain a live and infectious virus that can be used for antigenic characterization, pathogenesis investigation, vaccine production, and so on. Embryonated chicken egg inoculation is traditionally considered the "gold standard" method for influenza virus isolation and propagation. However, many primary cells and continuous cell lines have also been examined or developed for influenza virus isolation and replication. Specifically, influenza A virus in swine (IAV-S) isolation and propagation has been attempted and compared in embryonated chicken eggs, some primary porcine cells, and a number of continuous cell lines. Currently, Madin-Darby canine kidney (MDCK) cells remain the most commonly used cell line for the isolation, propagation, and titration of IAV-S. Virus isolation in embryonated chicken eggs or in different cell lines offers alternative approaches when IAV-S isolation in MDCK cells is unsuccessful. Optimal specimens for IAV-S isolation includes nasal swabs, nasopharyngeal swabs, oral fluids, bronchoalveolar lavage, lung tissues, and so on. In this chapter, we describe the procedures of sample processing, IAV-S isolation in MDCK cells and in embryonated chicken eggs, as well as the methods used for confirming the virus isolation results.
Asunto(s)
Pollos/virología , Virus de la Influenza A/aislamiento & purificación , Óvulo/virología , Porcinos/virología , Cultivo de Virus/métodos , Animales , Embrión de Pollo , Perros , Células de Riñón Canino Madin Darby , Especificidad de Órganos , Manejo de EspecímenesRESUMEN
Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9-11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12 h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24 h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-α, IFN-ß, IFN-γ, IL-1ß, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16 h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-γ, IL-1ß and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.
Asunto(s)
Virus de la Bronquitis Infecciosa/inmunología , Receptores Toll-Like/agonistas , Adyuvantes Inmunológicos/farmacología , Animales , Antivirales/farmacología , Proteínas Aviares/agonistas , Proteínas Aviares/inmunología , Embrión de Pollo , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Citocinas/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Inmunidad Innata , Virus de la Bronquitis Infecciosa/patogenicidad , Virus de la Bronquitis Infecciosa/fisiología , Ligandos , Lipopéptidos/farmacología , Lipopolisacáridos/farmacología , Oligodesoxirribonucleótidos/farmacología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/virología , Receptores Toll-Like/inmunología , Carga Viral/efectos de los fármacos , Carga Viral/inmunología , Replicación Viral/efectos de los fármacos , Replicación Viral/inmunologíaRESUMEN
Background & objectives: The susceptibility of influenza viruses to neuraminidase inhibitors (NAIs) is studied using enzyme-based assays, sequence analysis and in vitro and in vivo studies. Oseltamivir carboxylate (OC) is the active prodrug of the NAI oseltamivir. There is lack of information on the use of embryonated chicken eggs for studying susceptibility of highly pathogenic avian influenza (HPAI) H5N1 viruses to antiviral drugs. The aim of the present study was to assess the use of 10 day old embryonated chicken eggs for studying antiviral susceptibility of HPAI H5N1 viruses. Methods: Two HPAI H5N1 viruses isolated from India were used in the study. Fluorescence-based NAI assay was performed to determine antiviral susceptibility of these viruses. In ovo antiviral assays were carried out using 10 day old embryonated chicken eggs. The virus dilutions were incubated with 14 µg/ml of OC and inoculated in the allantoic cavity. In the eggs, 50 per cent egg infectious dose (EID50) titres as well as mortality were quantitated. Results: The two viruses used were susceptible to OC in the NAI assay. It was found that there was a significant drop in EID50titres; however, no significant protection from mortality after OC treatment was observed. Interpretation & conclusions: By measuring viral titres, the egg model was suitable to study the susceptibility of HPAI viruses to antiviral drugs along with NAI assay. The present study highlights the use of eggs as a model to study susceptibility of HPAI viruses to OC.
Asunto(s)
Antivirales/farmacología , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Aviar/tratamiento farmacológico , Oseltamivir/farmacología , Animales , Pollos , Farmacorresistencia Viral/genética , Huevos/virología , Inhibidores Enzimáticos/farmacología , India/epidemiología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Aviar/epidemiología , Gripe Aviar/virologíaRESUMEN
Fowl adenovirus 9 (FAdV-9) has one of the largest genomes (45 kb) so far sequenced from all adenoviruses studied. Genus-specific genes located within the early (E) regions at the right and left ends of the viral genome have unknown functions except for ORF8 (Gam-1 gene), ORF22 and ORF1 (dUTPase gene). ORF19, located at the right end of the genome (nts 34,220-36,443), is predicted to encode a lipase protein and its homologs are also found in all FAdV genomes so far sequenced. The role of ORF19 in virus replication and virulence is unknown. To study ORF19 and explore its potential as a locus for foreign gene insertion, we generated one ORF19-deleted mutant virus (rFAdV-9Δ19-SwaI) and three FAdV-9Δ19-based recombinant viruses replacing ORF19 as follows: rFAdV-9Δ19-CAT and enhanced-green fluorescent protein (EGFP) cassette (CMV promoter-EGFP-poly A) in a rightward (rFAdV-9Δ19-EGFP-R) and leftward orientation (rFAdV-9Δ19-EGFP-L). All recombinant viruses were stable after three passages. In chicken hepatoma cells, rFAdV-9Δ19-SwaI, rFAdV-9Δ19-CAT and rFAdV-9Δ19-EGFP-R replicated at titers similar to that of the wild-type virus, whilst rFAdV-9Δ19-EGFP-L replicated at a much lower titer. Interestingly, FAdV-9Δ19-SwaI replicated at higher titers in cells and in embryonated eggs, respectively than those of wild-type and recombinant viruses. These observations suggest ORF19 is nonessential for replication and can be used as a novel cloning site for engineering FAdV-9-based recombinant viruses and rFAdV-9Δ19-SwaI could be used to determine its role for virus replication in vivo.
Asunto(s)
Aviadenovirus/fisiología , Expresión Génica , Proteínas Recombinantes/biosíntesis , Proteínas Virales/metabolismo , Replicación Viral , Animales , Aviadenovirus/genética , Línea Celular , Pollos , Eliminación de Gen , Vectores Genéticos , Inestabilidad Genómica , Proteínas Recombinantes/genética , Carga Viral , Proteínas Virales/genéticaRESUMEN
Hazara virus (HAZV) is a member of the genus Orthonairovirus of the family Nairoviridae. HAZV is closely related to Crimean-Congo hemorrhagic fever virus but differs in that it is non-pathogenic to humans. To establish an infection model system, we tested whether embryonated chicken eggs, which are classically used for evaluating viral pathogenicity, are susceptible to HAZV infection. We demonstrated that HAZV replicates well in embryonated chicken eggs and kills 100% of the embryos. This can be a valuable tool to evaluate the lethality of nairoviruses in a biosafety level 2 laboratory.
Asunto(s)
Embrión de Pollo/patología , Embrión de Pollo/virología , Virus de la Fiebre Hemorrágica de Crimea-Congo/patogenicidad , Animales , Análisis de Supervivencia , Cultivo de Virus , Replicación ViralRESUMEN
Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of 4 zoospore concentrations into the egg allantoic cavity at 3 embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 hours after inoculation, and 95% embryos died within 72 hours. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only 1 liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis.
Asunto(s)
Modelos Animales de Enfermedad , Pitiosis/patología , Pythium/crecimiento & desarrollo , Pythium/patogenicidad , Animales , Embrión de Pollo , Histocitoquímica , Inmunohistoquímica , Técnicas Microbiológicas , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Análisis de Supervivencia , Factores de TiempoRESUMEN
Traditionally, embryonated chicken eggs (ECE) are considered the gold standard for Influenza virus isolation and vaccine production. Nowadays, different biological systems have been improved and performed, in order to evaluate a feasible alternative to ECE. In fact, in a previous study, mammalian and avian cell cultures were successfully used for avian influenza viruses primary isolation from target tissues and virus propagation. This research is focused on the investigation of adaptive mutations that occur after influenza A virus amplification in ECE and cell cultures. The results of the study shows that avian influenza viruses after multiple passages in different biological systems undergo mutations, in particular, the largest number of amino acid substitutions occurred in all biological substrates in the hemagglutinin.
Asunto(s)
Sustitución de Aminoácidos , Virus de la Influenza A/genética , Mutación , Proteínas Virales/genética , Adaptación Biológica , Virus de la Influenza A/metabolismo , Proteínas Virales/metabolismo , Cultivo de Virus/veterinariaRESUMEN
Avian influenza (AI) H9N2 viruses are endemic in many bird species, and human infections of H9N2 viruses have been reported. Oseltamivir phosphate (Tamiflu(®)) is the available antiviral drug for the treatment and prophylaxis of influenza. There are no reports of use of embryonated chicken egg as a model to study susceptibility of AI viruses to oseltamivir carboxylate (OC), the active metabolite. The present study was undertaken to explore the use of embryonated chicken eggs as a model for testing OC against the AI H9N2 viruses. A total of three AI H9N2 viruses, isolated in poultry in India, were used. Various virus dilutions were tested against 14µg/ml of OC. Three methods, namely (1) the in vitro virus-drug treatment, (2) drug delivery and virus challenge by allantoic route, and (3) drug delivery by albumen route and virus challenge by allantoic route were explored. The viruses were also tested using the fluorescence-based neuraminidase inhibitor (NAI) assay. There was significant inhibition (p<0.05) of the H9N2 viruses in presence of OC. The infectious virus titers as well as hemagglutination titers were significantly lower in presence of OC as compared to controls. The in vitro treatment of virus and drug; and drug and virus delivery at the same time by allantoic route showed significantly higher inhibition (p<0.05) of virus growth than that by the albumen route. In the NAI assay, the half maximal inhibitory concentration (IC50) values of the H9N2 viruses were within the standard range for known susceptible reference virus. In conclusion, the H9N2 viruses used in the study were susceptible to OC. Embryonated chicken egg could be used as a model to study susceptibility of AI viruses to antiviral drugs.
Asunto(s)
Antivirales/farmacología , Subtipo H9N2 del Virus de la Influenza A/efectos de los fármacos , Oseltamivir/análogos & derivados , Animales , Embrión de Pollo , India , Subtipo H9N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana/métodos , Oseltamivir/farmacología , Aves de Corral , Carga ViralRESUMEN
Gene families are common to all kingdoms of live and most likely derived from gene duplications with subsequent specification for the adaptation to environmental conditions. However, the exact contribution of single members to cellular physiology is difficult to predict. Here, we analysed a family of 3-ketoacyl-CoA thiolases composed of Pot1p, Fox3p and Pot13p from the dimorphic yeast Candida albicans and studied their contribution to fatty acid utilisation and virulence. The presence of three 3-ketoacyl-CoA thiolases in C. albicans contrasts the existence of only one single gene in closely related Saccharomycetales such as Saccharomyces cerevisiae. Phylogenetic analyses revealed that two of the thiolases, Pot1p and Fox3p, were closely related to the S. cerevisiae Pot1p. The third protein clustered with yet uncharacterised thiolases from filamentous fungi. Single, double and triple mutants were generated for phenotypic characterisations. While Pot1p was of general importance for utilisation of fatty acids, Fox3p partially contributed to fatty acid utilisation at elevated temperatures. No phenotype was detectable for pot13 deletions. When virulence of the different mutants was assessed in an embryonated chicken egg infection model, no significant attenuation was observed for any of the mutants, confirming previous assumptions that ß-oxidation is dispensable for C. albicans virulence.