Propagation and Molecular Characterization of Fowl Adenovirus Serotype 8b Isolates in Chicken Embryo Liver Cells Adapted on Cytodex™ 1 Microcarrier Using Stirred Tank Bioreactor

Chidozie C Ugwu,Abdul R Omar,Mohd Hair-Bejo,Mat I Nurulfiza,Aini Ideris

doi:10.3390/pr8091065

Chidozie C Ugwu, Abdul R Omar + Show 3 more

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https://doi.org/10.3390/pr8091065

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Abstract

Large volume production of vaccine virus is essential for prevention and control of viral diseases. The objectives of this study were to propagate Fowl adenovirus (FAdV) isolate (UPM08136) in chicken embryo liver (CEL) cells adapted to Cytodex™ 1 microcarriers using stirred tank bioreactor (STB) and molecularly characterize the virus. CEL cells were prepared and seeded onto prepared Cytodex™ 1 microcarriers and incubated first in stationary phase for 3 h and in STB at 37 °C, 5% CO2, and 20 rpm for 24 h. The CEL cells were infected with FAdV isolate (UPM08136) passage 5 (UPM08136CELP5) or passage 20 (UPM08136CELP20) and monitored until cell detachment. Immunofluorescence, TCID50, sequencing, alignment of hexon and fiber genes, and phylogenetic analysis were carried out. CEL cells were adapted well to Cytodex™ 1 microcarriers and successfully propagated the FAdV isolates in STB with virus titer of 107.5 (UPM08136CELP5B1) and 106.5 (UPM08136CELP20B1) TCID50/mL. These isolates clustered with the reference FAdV serotype 8b in the same evolutionary clade. The molecular characteristics remained unchanged, except for a point substitution at position 4 of the hexon gene of UPM08136CELP20B1, suggesting that propagation of the FAdV isolate in STB is stable and suitable for large volume production and could be a breakthrough in the scale-up process.

Highlights

Fowl adenovirus (FAdV) is associated with inclusion body hepatitis (IBH) in chickens which is responsible for heavy losses in poultry industries worldwide [1,2,3]
The chicken embryo liver (CEL) cells attached and adapted to the CytodexTM 1 microcarriers within 3 h of incubation (Figure 1a,b) and grew confluent on the microcarriers beads after 24 h incubation in the bioreactor (Figure 1c,d)
Propagation of FAdV in CEL Cells Adapted on CytodexTM 1 Microcarrier

Summary

Introduction

Fowl adenovirus (FAdV) is associated with inclusion body hepatitis (IBH) in chickens which is responsible for heavy losses in poultry industries worldwide [1,2,3]. Availability of vaccines in large volumes to meet the ever-growing production animal population could be easier through cell culture-based. The use of tissue culture flasks provided a solution to the issue of cell culture contaminations and improved cell proliferation, its limitation with regards to large volume production of vaccines has been inability to provide enough space [14]. A variety of microcarriers are available for vaccine development, but spherical bead type microcarriers such as CytodexTM 1 are suitable especially for stirred tank bioreactors [16]. It is a multipurpose microcarrier which can be used to grow a variety of cells. While culturing influenza virus vaccine in stirred tank bioreactor on Vero cells, CytodexTM 1 was used and optimized to the production capacity of 6000 L [16]

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