Avian Diseases Section, Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka, India.
Microbiol Spectr. 2021 Dec 22;9(3):e0098921. doi: 10.1128/spectrum.00989-21. Epub 2021 Dec 8.
We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I "F gene" class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research. The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.
我们提供了一种新颖的单限制酶（RE；BsaHI）消化方法，用于检测新城疫病毒（NDV）的不同致病型。在 NCBI 数据库中扫描了 4000 个 F 基因核苷酸序列后，我们在切割位点发现了一个单一的 RE（BsaHI）消化位点。使用 APMV-I“F 基因”II 类特异性引物基于逆转录 PCR 扩增了一个 535bp 的片段，然后用 RE（BsaHI）对禽源 NDV 田间分离株和鸽副黏病毒-1 分离株进行致病型分析。弱毒（禽源和中间毒力株）分别产生 189bp 和 346bp 的片段，但在强毒力株中仍保持未消化的 535bp 片段。此外，还使用了 45 个田间 NDV 分离株和 8 个疫苗株来验证该方法。基于序列的分析也与使用单一 RE（BsaHI）消化方法获得的数据一致。该技术有可能在短时间内区分弱毒和强毒株，因此在 NDV 监测和研究中具有重要价值。NDV 疫苗株的广泛使用和野鸟中存在的弱毒 NDV 株使得难以诊断新城疫病毒（NDV）。确定强毒 NDV 所需的脑内致病指数（ICPI）和/或基于测序的鉴定是耗时、昂贵、困难和残酷的技术。我们评估了 4000 个 F 基因核苷酸序列，并发现了一种在短时间内检测 NDV 和疫苗致病型的限制酶（RE；BsaHI）消化技术，该技术具有成本效益，对现场病例以及大规模 NDV 监测和监测都很有用。使用单一 RE BsaHI 消化技术获得的数据与基于序列的分析一致。
