Assessment of replicate numbers for titrating avian influenza virus using dose-response models

Embryonating chicken eggs (ECEs) are among the most sensitive laboratory host systems for avian influenza virus (AIV) titration, but ECEs are expensive and require space for storage and incubation. Therefore, reducing ECE use would conserve resources. We utilized statistical modeling to evaluate the accuracy and precision of AIV titration with 3 instead of 5 ECEs for each dilution by the Reed–Muench method for 50% endpoint calculation. Beta-Poisson and exponential dose-response models were used in a simulation study to evaluate observations from actual titration data from 18 AIV isolates. The reproducibility among replicates of a titration was evaluated with one AIV isolate titrated in 3 replicates with the beta-Poisson, exponential, and Weibull dose-response models. The standard deviation (SD) of the error between input and estimated virus titers was estimated with Monte Carlo simulations using the fitted dose-response models. Good fit was observed with all models that were utilized. Reducing the number of ECEs per dilution from 5 to 3 resulted in the width of the 95% confidence interval increasing from ±0.64 to ±0.75 log1050% ECE infectious doses (EID50) and the SD of the error increased by 0.03 log10EID50. Our study suggests that using fewer ECEs per dilution is a viable approach that will allow laboratories to reduce costs and improve efficiency.

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Avian Influenza Virus Isolation, Propagation, and Titration in Embryonated Chicken Eggs

Methods in Molecular Biology - Animal Influenza Virus ◽

10.1007/978-1-0716-0346-8_12 ◽

2020 ◽

pp. 149-164 ◽

Cited By ~ 2

Author(s):

Erica Spackman ◽

Mary Lea Killian

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Virus Isolation ◽

Chicken Eggs ◽

Embryonated Chicken

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Isolation of Avian Influenza Virus (Subtype H5N2) from Chicken Eggs during a Natural Outbreak

Avian Diseases ◽

10.2307/1590473 ◽

1985 ◽

Vol 29 (4) ◽

pp. 1195 ◽

Cited By ~ 41

Author(s):

D. T. Cappucci ◽

D. C. Johnson ◽

M. Brugh ◽

T. M. Smith ◽

C. F. Jackson ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Chicken Eggs ◽

Avian Influenza Virus Subtype ◽

Natural Outbreak ◽

Virus Subtype

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Natural Reassortants of Potentially Zoonotic Avian Influenza Viruses H5N1 and H9N2 from Egypt Display Distinct Pathogenic Phenotypes in Experimentally Infected Chickens and Ferrets

Journal of Virology ◽

10.1128/jvi.01300-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 13

Author(s):

Mahmoud M. Naguib ◽

Reiner Ulrich ◽

Elisa Kasbohm ◽

Christine L. P. Eng ◽

Donata Hoffmann ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Highly Pathogenic Avian Influenza ◽

Parent Virus ◽

Highly Pathogenic ◽

Pathogenic Avian Influenza Virus ◽

Chicken Eggs ◽

Embryonated Chicken

ABSTRACT The cocirculation of zoonotic highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 and avian influenza virus (AIV) of subtype H9N2 among poultry in Egypt for at least 6 years should render that country a hypothetical hot spot for the emergence of reassortant, phenotypically altered viruses, yet no reassortants have been detected in Egypt. The present investigations proved that reassortants of the Egyptian H5N1 clade 2.2.1.2 virus and H9N2 virus of the G1-B lineage can be generated by coamplification in embryonated chicken eggs. Reassortants were restricted to the H5N1 subtype and acquired between two and all six of the internal segments of the H9N2 virus. Five selected plaque-purified reassortant clones expressed a broad phenotypic spectrum both in vitro and in vivo. Two groups of reassortants were characterized to have retarded growth characteristics in vitro compared to the H5N1 parent virus. One clone provoked reduced mortality in inoculated chickens, although the characteristics of a highly pathogenic phenotype were retained. Enhanced zoonotic properties were not predicted for any of these clones, and this prediction was confirmed by ferret inoculation experiments: neither the H5N1 parent virus nor two selected clones induced severe clinical symptoms or were transmitted to sentinel ferrets by contact. While the emergence of reassortants of Egyptian HPAIV of subtype H5N1 with internal gene segments of cocirculating H9N2 viruses is possible in principle, the spread of such viruses is expected to be governed by their fitness to outcompete the parental viruses in the field. The eventual spread of attenuated phenotypes, however, would negatively impact syndrome surveillance on poultry farms and might foster enzootic virus circulation. IMPORTANCE Despite almost 6 years of the continuous cocirculation of highly pathogenic avian influenza virus H5N1 and avian influenza virus H9N2 in poultry in Egypt, no reassortants of the two subtypes have been reported. Here, the principal compatibility of the two subtypes is shown by forcing the reassortment between copassaged H5N1 und H9N2 viruses in embryonated chicken eggs. The resulting reassortant viruses displayed a wide range of pathogenicity including attenuated phenotypes in chickens, but did not show enhanced zoonotic propensities in the ferret model.

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