Adaptation and Molecular Characterization of Two Malaysian Very Virulent Infectious Bursal Disease Virus Isolates Adapted in BGM-70 Cell Line

Two Malaysian very virulent infectious bursal disease virus (vvIBDV) strains UPM0081 and UPM190 (also known as UPMB00/81 and UPM04/190, respectively) isolated from local IBD outbreaks were serially passaged 12 times (EP12) in specific pathogen free (SPF) chicken embryonated eggs (CEE) by chorioallantoic membrane (CAM) route. The EP12 isolate was further adapted and serially propagated in BGM-70 cell line up to 20 passages (P20). Characteristic cytopathic effects (CPEs) were subtly observed at P1 in both isolates 72 hours postinoculation (pi). The CPE became prominent at P5 with cell rounding, cytoplasmic vacuoles, granulation, and detachment from flask starting from day 3 pi, up to 7 days pi with titers of 109.50 TCID50/mL andlog109.80 TCID50/mL for UPM0081 and UPM190, respectively. The CPE became subtle at P17 and disappeared by P18 and P19 for UPM0081 and UPM190, respectively. However, the presence of IBDV was confirmed by immunoperoxidase, immunofluorescence, and RT-PCR techniques. Phylogenetic analysis showed that these two isolates were of the vvIBDV. It appears that a single mutation of UPM190 and UPM0081 IBDV isolates at D279N could facilitate vvIBDV strain adaptability in CEE and BGM-70 cultures.

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

Journal of Pathogens ◽

10.1155/2018/1068758 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Nafi’u Lawal ◽

Mohd Hair-Bejo ◽

Siti Suri Arshad ◽

Abdul Rahman Omar ◽

Aini Ideris

Keyword(s):

Cell Line ◽

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Chorioallantoic Membrane ◽

Infectious Bursal Disease ◽

Phylogenetic Tree Analysis ◽

Cytopathic Effects ◽

Bursal Disease ◽

Specific Pathogen ◽

Syringe Filter

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.

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Growth and Replication of Infectious Bursal Disease Virus in the DF-1 Cell Line and Chicken Embryo Fibroblasts

BioMed Research International ◽

10.1155/2014/494835 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 12

Author(s):

Kaliyaperumal Rekha ◽

Chandran Sivasubramanian ◽

Ill-Min Chung ◽

Muthu Thiruvengadam

Keyword(s):

Cell Line ◽

Disease Virus ◽

Chicken Embryo ◽

Infectious Bursal Disease Virus ◽

Infectious Bursal Disease ◽

Population Doubling ◽

Cytopathic Effects ◽

Bursal Disease ◽

Embryo Fibroblasts ◽

Chicken Embryo Fibroblasts

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. To determine a suitable cell line for IBDV infection, replication, and growth kinetics of the virus, DF-1 cells and chicken embryo fibroblasts (CEF) were used. The population doubling per day (Pd/D) was found to be higher in DF-1 as compared to CEF cells. A suitable time of infection (TOI) was established for increased production of virus and greater infectivity titers. The DF-1 and CEF cells were found to be susceptible to infection by producing marked cytopathic effects (CPEs), and the growth curves of IBDV in DF-1 and CEF cells were evaluated by infectivity assay using tissue culture infectious dose (TCID50). The cytopathic effects of the virus in DF-1 and CEF cells were found to be similar, but higher viral titers were detected in the DF-1 cells as compared to CEF. Thus the DF-1 cell line had a higher growth potential and infectivity, which will be of advantage in vaccine production.

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