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VP3 and 3’-UTR Affect Viral Replication of Infectious Bursal Disease Virus

Author: WangYongQiang
Tutor: WangXiaoMei
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: infectious bursal disease virus reverse-genetic system real-time RT-PCR 990 amino acid 3’-UTR viral replication
CLC: S852.65
Type: Master's thesis
Year: 2009
Downloads: 89
Quote: 1
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Abstract

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is a highly contagious immunosuppressive disease in young chickens. It induces vaccination failure and secondary infections within the flock. In addition, the emergence of variant strain and very virulent infectious bursal disease virus (vvIBDV) is causing new problems in the control of IBD. Besides, it has prompted extensive research efforts to find out the molecular determinations of the virulence, cell tropism and viral replication of IBDV. Recently, the vvIBDV has become the dominant strain gradually. So it is critical to detect the virus at an early stage.A real-time RT-PCR method was developed for the detection of infectious bursal disease virus (IBDV). The VP5 gene of IBDV was chosen firstly as the target binding region for a specific TaqMan probe. Results showed that viral genomic copy number could be quantified accurately ranging from 108 copies/μL to 101 copies/μL. No positive signal was detected for other avian pathogens (AIV, NDV, IBV, MDV, ARV and CAV) in the specificity test. This assay was highly sensitive and could detect as little as 30 copies of viral RNA. Both the coefficients of variation (CVs) of inter- and intra-assay reproducibility were less than 2%. Growth curves of the IBDV Gt strain in chicken embryo fibroblasts (CEF) and DF-1 cells were evaluated by the real-time RT-PCR. Data showed that cytopathic effects of the virus in CEF and DF-1 cells were similar. However, higher viral titers were detected in the DF-1 cell line. This study indicated that the real-time RT-PCR approach provided a powerful diagnostic tool with high sensitivity and specificity for identification and quantitation of IBDV. DF-1 cell line might be a more suitable continuous cell line for the propagation of IBDV compared to CEF.vvIBDV Gx strain causes over 60% mortality in chickens, but cannot replicate in CEF cultures. The attenuated Gt strain, however, has no virulence to chickens and replicates well in CEF cultures. Sequence comparison of the two strains showed a 6 amino acids difference in VP4, 4 amino acids difference in VP3 and 3 nucleotides in 3’-UTR. To determine whether VP4, VP3 or 3’-UTR are involved in the replication of IBDV, three chimeric viruses, of which the VP4/VP3/3’UTR, VP3/3’UTR or VP4 region of Gt were replaced by the corresponding region of Gx, were rescued and subsequently characterized in vitro and in vivo. Results showed that neither VP4 nor VP3 had effects on the virulence, and VP4 was not involved in the viral replication. However, the replication kinetics curves in CEF cultures indicated that the region of VP3/3’UTR affected viral replication in CEF cultures. Through site-directed mutagenesis, three rescued viruses with a single amino acid substitution in the C-terminus of VP3 of Gt strain (L981P, A990V and T1005A, respectively) were characterized in a similar manner. Results indicated that amino acid substitution at position 990 greatly affected viral replication in CEF cultures. Besides, the 3’-UTR region of Gt was replaced by the corresponding region of Gx based on the fusion PCR. One chimeric virus (rGt-Gx3’UTR) was rescued and characterized in the same way. Results indicated that 3’-UTR affected viral replication too. And the RNA dependent RNA polymerase of Gt strain not only recognized the 3’-UTR of Gt, but also recognized the 3’-UTR of Gx. The viral loads in bursa of fabricius of rGt-GxVP3/3’UTR, rGt-Gx3’UTR, rGtA-A990V and rmGt were evaluated by the real-time RT-PCR. In summary, data indicated that C-terminus of VP3 affected viral replication, to our knowledge, this is the first report that amino acid at position 990 plays a critical role in the replication of IBDV both in vitro and in vivo. Additionally, the 3’-UTR influenced the viral replication too.

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