vvIBDVVery Virulent Infectious Bursal Disease Virus
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The confluent BGM-70 cells growing on the microcarrier were infected with vvIBDV UPM190BGMP15 with CPE development at 72 hours pi at which time, the virus was harvested and designated as UPM190EP12BGMP15BP1.
RT-PCR of the Bioreactor Propagated vvIBDV. The PCR products upon electrophoresis revealed a 643 bp fragments (Figure 3) and sequencing results obtained were analyzed using MEGA version 7 [26] and Bioedit version 7 bioinformatic software [29] as shown below (Figures 4 and 5).
The main traditional means of producing vvIBDV seed virus for vaccine production has been using CEE from SPF chicken flocks [24].
The present study demonstrated the successful growth of vvIBDV in BGM-70 cell line on cytodex 1 microcarriers using stirring parameter of 12rpm/5 minutes for the first 24 hours followed by 14rpm/5 minutes for the rest of the culture period until harvest.
Caption: Figure 2: vvIBDV infected BGM-70 cells detaching from cytodex.
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).
When the nucleotide sequences of the preadaptation, CEE, BGM-70 adapted, and reference isolates were translated to amino acids, the putative motifs identifying vvIBDV pathotypes were seen revealing isoleucine (I) at positions 242, 256, and 294 and serine (S) at position 299 (Figure 8).
Phylogenetic analysis of the nucleotide sequences revealed that our isolates cluster with the sequences of the vvIBDV isolates from Europe, Asia, Middle East, and Africa deposited in GenBank included in the analysis (Figure 9).
Outbreaks of vvIBDV in chickens as confirmed through RTPCR and restriction fragment length polymorphism (RFLP) techniques were reported in Malaysia [22] and other parts of the world.
The presence of VP2 antigen as demonstrated by immunoperoxidase and immunofluorescence within the cytoplasm of infected cells 48 hours pi indicated the ability of BGM-70 cell line to support the growth and replication of these two vvIBDV isolates, because the virus was reported to replicate within the cytoplasm of infected cells with generation of viral capsid proteins (VP2 and VP3) within 48 hours pi [30, 49, 50].
The molecular analyses using RT-PCR, sequence, and sequence analysis revealed that the two isolates are phylogenetically related to the vvIBDV found in Asia, Europe, Middle East, and Africa due to the presence of A222 [51,52] and I242, I256, I294, and S299 [51] in their VP2 hypervariable amino acid composition compared to the caIBDV or vaIBDV that possessed V222, VA, or E256 or the serotype 2 viruses that possessed P222, V242, I or S256, N or F294 and T or P299 [51] in the same region.
Phylogenetically, all the isolates fall within the vvIBDV clades by clustering with other published vvIBDV reference sequences deposited in GeneBank, indicating that the BGM-70 passaging and the mutations observed did not lead to a change in the pathotypes of the isolates.