Wu Ching Ching, Rubinelli Peter, Lin Tsang Long
Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, 406 South University Street, West Lafayette, IN 47907-2065, USA.
Avian Dis. 2007 Jun;51(2):515-26. doi: 10.1637/0005-2086(2007)51[515:MDADOI]2.0.CO;2.
Vaccination of hens, with the subsequent maternal immunity imparted to chicks, is the primary means of controlling infectious bursal disease virus (IBDV). Effective vaccination depends on rapid and accurate diagnosis of the subtype present in a flock because vaccines based on the classic subtype of IBDV can fail to protect against challenge with a variant subtype. This review describes the various methods available to detect and differentiate between IBDV subtypes. Serotype 1 IBDV causes economically significant immunosuppressive disease in young chickens. Within serotype 1, two subtypes, classic and variant, can be differentiated by the virus neutralization assay. Antigen capture enzyme-linked immunosorbent assay (AC-ELISA) with MAbs has been successful at differentiating the very virulent IBDV phenotype (vvIBDV) from less pathogenic types. More rapid and sensitive molecular diagnostic methods based on reverse transcription-polymerase chain reaction (RT-PCR) for amplification of the IBDV VP2 gene have been a major focus of investigation in recent years. Conventional RT-PCR has been useful in detecting IBDV serotypes and, to a lesser extent, differentiating IBDV subtypes. One of the approaches has been the use of SspI and NgoM IV restriction enzymes, for restriction endonuclease (RE) analysis of RT-PCR products (RT-PCR-RE) and BstNI and MboI for restriction fragment length polymorphism (RFLP) analysis (RT-PCR-RFLP) to find unique banding patterns associated with antigenic variation within the variable region of the IBDV VP2 protein. However, these approaches were ultimately found to be unreliable because subtypes could not be consistently distinguished with restriction enzymes. These limitations led to studies in differentiating subtypes by detection of single nucleotide differences in sequence through real-time RT-PCR or DNA sequencing of RT-PCR products. Conventional RT-PCR, amplifying the VP2 hypervariable region, in combination with DNA sequencing of the PCR product, can differentiate classic, variant, and vvIBDV strains because variant and vvIBDV have characteristic nucleotide and amino acid substitutions. Real-time RT-PCR, targeting different regions of the IBDV genome, including VP1, VP2, and VP4 genes, in conjunction with melting-curve analysis is being investigated as a promising tool for molecular diagnosis of IBDV infection. These methods potentially allow for more rapid, sensitive, and specific detection and differentiation of IBDV classic, very virulent, and variant subtypes.
给母鸡接种疫苗，随后将母源免疫力传递给雏鸡，是控制传染性法氏囊病病毒（IBDV）的主要手段。有效的疫苗接种取决于对鸡群中存在的病毒亚型进行快速准确的诊断，因为基于IBDV经典亚型的疫苗可能无法保护鸡群免受变异亚型的攻击。本综述描述了用于检测和区分IBDV亚型的各种方法。血清1型IBDV可在雏鸡中引起具有经济重要性的免疫抑制性疾病。在血清1型中，经典型和变异型这两种亚型可通过病毒中和试验进行区分。使用单克隆抗体的抗原捕获酶联免疫吸附测定（AC-ELISA）已成功区分超强毒力IBDV表型（vvIBDV）和致病性较低的类型。近年来，基于逆转录-聚合酶链反应（RT-PCR）扩增IBDV VP2基因的更快速、灵敏的分子诊断方法一直是研究的重点。常规RT-PCR在检测IBDV血清型方面很有用，在区分IBDV亚型方面作用较小。其中一种方法是使用SspI和NgoM IV限制性内切酶，对RT-PCR产物进行限制性内切酶（RE）分析（RT-PCR-RE），以及使用BstNI和MboI进行限制性片段长度多态性（RFLP）分析（RT-PCR-RFLP），以找到与IBDV VP2蛋白可变区内抗原变异相关的独特条带模式。然而，这些方法最终被发现不可靠，因为无法用限制性内切酶一致地区分亚型。这些局限性促使人们通过实时RT-PCR或RT-PCR产物的DNA测序检测序列中的单核苷酸差异来区分亚型。扩增VP2高变区的常规RT-PCR与PCR产物的DNA测序相结合，可以区分经典型、变异型和vvIBDV毒株，因为变异型和vvIBDV具有特征性的核苷酸和氨基酸替换。针对IBDV基因组不同区域（包括VP1、VP2和VP4基因）的实时RT-PCR结合熔解曲线分析，正作为一种有前景的IBDV感染分子诊断工具进行研究。这些方法有可能实现对IBDV经典型、超强毒力型和变异型亚型更快速、灵敏和特异的检测与区分。
