Propagation and Molecular Characterization of Bioreactor Adapted Very Virulent Infectious Bursal Disease Virus Isolates of Malaysia

Abstract

Two Malaysian very virulent infectious bursal disease virus (vvIBDV) strains UPM0081 (also known as B00/81) and UPM190 (also known as UPM04/190) isolated from local IBD outbreaks in 2000 and 2004, respectively, were separately passaged for 12 consecutive times in 11-day-old specific pathogen free (SPF) chicken embryonated eggs (CEE) via the chorioallantoic membrane (CAM) route. The CEE passage 8 (EP8) isolates were passaged once in BGM-70 cell line yielding UPM0081EP8BGMP1 and UPM190EP8BGMP1, while the EP12 isolates were passaged 15 times in BGM-70 cell line yielding UPM0081EP12BGMP15 and UPM190EP12BGMP15 using T25 tissue culture flask. These isolates were all propagated once in bioreactor using cytodex 1 as microcarrier at 3 g per liter (3 g/L) yielding UPM0081EP8BGMP1BP1, UPM190EP8BGMP1BP1, UPM0081EP12BGMP15BP1, and UPM190EP12BGMP15BP1 isolates. The viruses were harvested at 3 days after inoculation, following the appearance of cytopathic effects (CPE) characterized by detachment from the microcarrier using standard protocol and filtered using 0.2 μm syringe filter. The filtrates were positive for IBDV by RT-PCR and immunofluorescence. Sequence and phylogenetic tree analysis indicated that the isolates were of the vvIBDV strains and were not different from the flask propagated parental viruses.

Figure 1

BGM-70 cells attaching to cytodex microcarrier 1 in a bioreactor. (a) BGM-70 cells attaching to cytodex at 6 hours ps. (b) Confluent cells on cytodex at 24 hours ps. Bar=100 μm.

Figure 2

vvIBDV infected BGM-70 cells detaching from cytodex. (a) Detachment of BGM-70 cells from microcarriers at 72 hours pi with UPM0081EP8BGMP1BP1. (b) Detachment of BGM-70 cells from microcarriers at 72 hours pi with UPM190EP8BGMP1BP1. (c) Detachment of BGM-70 cells from microcarriers at 72 hours pi with UPM0081EP12BGMP15BP1. (d) Detachment of BGM-70 cells from microcarriers at 72 hours pi with UPM190EP12BGMP15BP1. Bar=100 μm.

Figure 3

Gel electrophoresis of bioreactor passaged viruses. RT-PCR product showing 643 bp fragment of bioreactor passaged vvIBDV UPM0081EP12BGMP15BP1 (lane 2), UPM0081EP8BGMP1BP1 (lane 3), UPM190EP12BGMP15BP1 (lane 4), UMP190EP8BGMP1BP1 (lane 5), and original BGM-70 and egg passaged isolates UPM0081BGMP15 (lane 6), UPM190BGMP15 (lane 7), UPM0081EP8 (lane 8), UPM190EP8 (lane 9), UPM0081EP12 (lanes 10 and 11), UPM190EP12 (lanes 12 and 13), positive controls (lanes 14, 15, and 16), and negative control (lane 17). A 1000 bp DNA ladder (lanes 1 and 15) (MBI Fermentas, Lithuania) was used to flank the PCR products.

Figure 4

Comparison of nucleotide sequences of bioreactor, conventional flask, and CAM propagated UPM0081 and UPM190. Nucleotide alignment of bioreactor, BGM-70, and SPF egg UPM190 and UPM0081 isolates with 30 reference sequences showing no nucleotide changes between the original parent and the bioreactor passaged isolates.

Figure 5

Comparison of amino acid sequences of bioreactor and BGM-70 propagated UPM0081 and UPM190. Protein alignment of bioreactor, BGM-70, and SPF egg passaged UPM190 and UPM0081 isolates with 30 reference sequences showing no amino acid changes between the original parental isolates and the bioreactor passaged isolates.

Figure 6

Phylogenetic analysis of bioreactor, CAM adapted, and conventional flask propagated UPM0081 and UPM190. Phylogenetic relationship of taxa between bioreactor, BGM-70, and CEE passaged UPM190 and UPM0081 isolates with reference sequences to determine their phylogenetic relationship. The evolutionary history was inferred using the Neighbor-Joining method with bootstrap (1000 replicates). The evolutionary distances were computed using Poisson correction method. The analysis involved 30 amino acid sequences with a total of 80 positions. The analyses were conducted in MEGA7. Note that the UPM strains formed 2 distinct clusters within the vvIBDV branches from the reference sequences indicating their close relationship.

Figure 7

The indirect immunofluorescence test on the bioreactor propagated UPM0081EP12BGMP15BP1 and UPM190EP12BGMP15BP1 showing high amount of IBDV antigen within the cytoplasm of BGM-70 cells at 72 hours after inoculation. ((a) to (c)) BGM-70 cells infected with bioreactor propagated UPM0081EP12BGMP15BP1 and ((d) to (f)) UPM190EP12BGMP15BP1 showing positive signals for VP2 antigen (green) when stained with FITC-conjugated anti-chicken antibody raised against chicken anti-VP2 antibody. ((g) to (i)) Uninoculated control. Bar=50μm.

Figure 8

The indirect immunofluorescence test on the flask propagated UPM0081BGMP15 and UPM190BGMP15 showing low amount of IBDV antigen within the cytoplasm of infected BGM-70 cells at 72 hours after inoculation. ((a) to (c)) BGM-70 cells infected with flask propagated UPM0081BGMP15 and ((d) to (f)) UPM190BGMP15 showing positive signals for VP2 antigen (green) when stained with FITC-conjugated anti-chicken antibody raised against chicken anti-VP2 antibody. Bar=50 μm.