Abstract
ABSTRACTWe 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.IMPORTANCE 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.
Highlights
We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV)
ND is divided into five pathotypes based on clinical signs and pathological lesions: (i) viscerotropic velogenic Newcastle disease (VVND) with digestive tract hemorrhagic lesions, (ii) neurotropic velogenic Newcastle disease (NVND) with respiratory and neurological signs, (iii) mesogenic pathotype, (iv) lentogenic pathotype with a mild or inapparent respiratory infection, and (v) asymptomatic with no obvious disease
Various fast techniques, such as Reverse transcription (RT)-PCR followed by restriction enzyme (RE) analysis, became available with the development of molecular tools for discriminating between avirulent and virulent NDV isolates [15,16,17,18], and low-virulence lentogenic field and vaccine strains were distinguished from mesogenic and velogenic field strains by RE digestion with BglI [19]
Summary
We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). 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. Due to widespread use of the NDV vaccine strain and the presence of avirulent NDV strains in wild birds, sequence analysis of the RT-PCR result is not suitable for all field instances Various fast techniques, such as RT-PCR followed by restriction enzyme (RE) analysis, became available with the development of molecular tools for discriminating between avirulent and virulent NDV isolates [15,16,17,18], and low-virulence lentogenic field and vaccine strains were distinguished from mesogenic and velogenic field strains by RE digestion with BglI [19]. Probe hybridization [23, 24] and TaqMan fluorogenic probe hybridization [25] are additional techniques to differentiate NDV pathotypes; these methods may not be fit for routine laboratory diagnosis with limited facilities and could possibly increase the cost and time of the diagnosis
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