Evaluation and Attempted Optimization of Avian Embryos and Cell Culture Methods for Efficient Isolation and Propagation of Low Pathogenicity Avian Influenza Viruses

Moresco, Kira A.; Stallknecht, David E.; Swayne, David E.

doi:10.1637/9030-883709-digest.1

Cited by 7 publications

(9 citation statements)

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“…Their observations, however, occurred over a period of days, whereas we are suggesting Daphnia ingestion and inactivation of AIV can be rapid. Alternatively, negative culture results may be a consequence of method sensitivity (22,44). Inoculation of AIV into embryonated chicken eggs may lead to higher rates of successful virus isolation than cell lines (45), and future use of this method may be warranted.…”

Section: Discussionmentioning

confidence: 99%

Accumulation and Inactivation of Avian Influenza Virus by the Filter-Feeding Invertebrate Daphnia magna

Meixell

Borchardt

Spencer

2013

Appl Environ Microbiol

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The principal mode of avian influenza A virus (AIV) transmission among wild birds is thought to occur via an indirect fecal-oral route, whereby individuals are exposed to virus from the environment through contact with virus-contaminated water. AIV can remain viable for an extended time in water; however, little is known regarding the influence of the biotic community (i.e., aquatic invertebrates) on virus persistence and infectivity in aquatic environments. We conducted laboratory experiments to investigate the ability of an aquatic filter-feeding invertebrate, Daphnia magna, to accumulate virus from AIV-dosed water under the hypothesis that they represent a potential vector of AIV to waterfowl hosts. We placed live daphnids in test tubes dosed with low-pathogenicity AIV (H3N8 subtype isolated from a wild duck) and sampled Daphnia tissue and the surrounding water using reverse transcription-quantitative PCR (RT-qPCR) at 3-to 120-min intervals for up to 960 min following dosing. Concentrations of viral RNA averaged 3 times higher in Daphnia tissue than the surrounding water shortly after viral exposure, but concentrations decreased exponentially through time for both. Extracts from Daphnia tissue were negative for AIV by cell culture, whereas AIV remained viable in water without Daphnia present. Our results suggest daphnids can accumulate AIV RNA and effectively remove virus particles from water. Although concentrations of viral RNA were consistently higher in Daphnia tissue than the water, additional research is needed on the time scale of AIV inactivation after Daphnia ingestion to fully elucidate Daphnia's role as a potential vector of AIV infection to aquatic birds.

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Section: Discussionmentioning

confidence: 99%

Accumulation and Inactivation of Avian Influenza Virus by the Filter-Feeding Invertebrate Daphnia magna

Meixell

Borchardt

Spencer

2013

Appl Environ Microbiol

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“…We show that the protease is able to activate the monobasic cleavage site of the H9N2 virus and support its infection in A549 cells. The A549 cells are permissive to the H9N2 virus growth, but fibroblastic cells that lack the enzymes, require additional trypsin for virus replication (14,25).…”

Section: Discussionmentioning

confidence: 99%

Replication Efficiency of Influenza A Virus H9N2: A Comparative Analysis Between Different Origin Cell Types

Lebas

Shahsavandi

Mohammadi

et al. 2013

Jundishapur J Microbiol

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Background: Efficient isolation and detection of low pathogenic avian influenza viruses from surveillance samples continues to be a high priority. Currently, the new cell lines are considered for supporting the replication to high virus strains titers. Objectives: The replication efficiency of a low pathogenic avian influenza virus in different origin cells was evaluated under different conditions. Materials and Methods: Chicken embryo fibroblast (CEF) cell and human alveolar epithelial cell line (A549) were infected with H9N2 at a multiplicity of infection of 0.1. The amount of infectious virus released into the cell culture supernatants at various post-infection time intervals were tittered by tissue culture infectious dose (TCID50) assay. The impact of these cells adaptation was investigated by determination the virus genes nucleotide sequences. Results: The influenza virus infectivity was not significant difference in these cells in the presence of trypsin. The results of fusion assay and determination of cellular protease confirmed that A549 cells support virus entry with or without supplemental trypsin. However, the H9N2 virus showed lower titer and infectivity in the trypsin-free infected A549 cells within longer time. The comparative sequence analysis indicated several simultaneously nucleotide substitutions were occurred in NA of the virus replicated in A549 cells resulted in two fixed amino acid changes at positions G320 to A and G414 to A up to the fifth passage. Conclusions: After seven consecutive passages of both cell cultures, the H9N2 virus showed similar antigenicity, also no change on viral titer level and virus replication behavior in adaptation was found. The results highlighted the use of A549 cells for efficient virus isolation.

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“…Moreover, ECE VI is widely accepted as the gold standard test with high Se and Sp values in chickens (7) and is considered to be the most efficient system for growth of wild-bird LPAIV isolates compared with various other embryo types and cell lines (18,19). In this study, ECE VI performed well, with 89% (95% CI: 72%-98%) of viruses isolated on first passage, which is comparable to the 87% isolation rate reported by others (19).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Avian Influenza Serologic and Virologic Diagnostic Methods in Wild Anseriformes and Charadriiformes

Curran

Robertson

Ellis³

et al. 2014

Avian Diseases

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SUMMARY. Evaluation of avian influenza virus (AIV) diagnostic methods, including a nucleoprotein (NP) competitive enzyme-linked immunosorbent assay (c-ELISA), hemagglutination inhibition (HI) test, type A real-time reverse transcription polymerase chain reaction (RRT-PCR), and embryonating chicken egg (ECE) virus isolation (VI), suggested validity of these tests in wild birds comparable to that reported in poultry. This was determined by analyzing the results from experimental inoculation of three species of wild birds with a low-pathogenicity AIV and from field surveillance data. The NP c-ELISA in a high-AIV prevalence setting had 100% diagnostic sensitivity (Se; 95% confidence interval [CI]: 81.5%-100%) and 91% diagnostic specificity (Sp; 95% CI: 70.8%-98.9%) in negative controls compared with the RRT-PCR. In low-AIV prevalence flocks using a .60% inhibition positivity threshold, relative to the HI test, c-ELISA performed with 90.5% Se (95% CI: 86.2%-93.8%) and 41.2% Sp (95% CI: 38.1%-44.5%). Assessment of HI suggests a titer $8 is a positive test result in wild-bird sera, and using this titer had 83.3% Se (95% CI: 58.6%-96.4%) in experimentally infected birds. The RRT-PCR diagnostic performance compared with VI in cloacal swabs varied over 2-6 days postinoculation, having high Se (83.3%-100%) and Sp (94.1%-100%) with substantial agreement (kappa 5 0.8). The cycle thresholds (C t ) for the RRT-PCR of C t , 37 for positivity and C t 5 37-40 as indeterminate were found to be valid for the species included in this study. In view of the interpretative diagnostic difficulties in heterogeneous populations of wild birds, this evaluation in three species of wild birds and in surveillance data should provide greater confidence in the application of these methods routinely used in poultry. RESUMEN. Evaluación de los métodos de diagnóstico serológicos y virológicos para influenza aviar en Anseriformes y Charadriiformes silvestres.La evaluación de los métodos de diagnóstico para la influenza aviar, incluyendo un ensayo por inmunoabsorción competitivo (C -ELISA) para la nucleoproteína (NP), una prueba de inhibición de la hemaglutinación (HI), un método de reacción en cadena de la polimerasa en tiempo real (RRT-PCR) para el tipo A y el aislamiento viral en embriones de pollo, sugirió que la validez de estas pruebas en las aves silvestres es comparable a la reportada con las aves comerciales. Esto se determinó mediante el análisis de los resultados de la inoculación experimental de tres especies de aves silvestres con un virus de la influenza aviar de baja patogenicidad y de los datos de vigilancia de campo. La prueba de ELISA para la nucleoproteína mostró una sensibilidad diagnóstica del 100% en un entorno de alta prevalencia de la influenza (Se; 95% intervalo de confianza: 81.5% a 100%) y una especificidad diagnóstica del 91% (Sp; 95% intervalo de confianza: 70.8%-98.9%) en controles negativos en comparación con la RRT-PCR. En parvadas de baja prevalencia de influenza aviar, utilizando un umbral de positividad por inhibici...

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Evaluation and Attempted Optimization of Avian Embryos and Cell Culture Methods for Efficient Isolation and Propagation of Low Pathogenicity Avian Influenza Viruses

Cited by 7 publications

References 0 publications

Accumulation and Inactivation of Avian Influenza Virus by the Filter-Feeding Invertebrate Daphnia magna

Accumulation and Inactivation of Avian Influenza Virus by the Filter-Feeding Invertebrate Daphnia magna

Replication Efficiency of Influenza A Virus H9N2: A Comparative Analysis Between Different Origin Cell Types

Evaluation of Avian Influenza Serologic and Virologic Diagnostic Methods in Wild Anseriformes and Charadriiformes

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