一株新型鸭细小病毒的分离鉴定和遗传演化特征分析

Isolation, identification, and phylogenetic characterization of a strain of novel duck parvovirus

摘要:

【背景】2015年以来，我国东部沿海地区陆续暴发樱桃谷鸭等商品肉鸭以短喙、舌外伸肿大、生长发育不良、腿骨易折等为主要临床症状的疾病，并逐步向内陆蔓延，研究发现引起该病的病原为新型鸭细小病毒(novel duck parvovirus, NDPV)。【目的】开展流行病学调查，研究NDPV的遗传进化特征，为了解NDPV遗传演化规律，完善NDPV流行病学数据及致病机制的研究提供科学依据。【方法】取发病雏鸭的肝脏研磨、离心后取上清，使用SPF鸭胚成功分离病毒并测其含量，对分离的病毒进行PCR鉴定及外源病毒检测，扩增病毒全基因组并进行序列同源性分析，对VP1蛋白进行分子特征和遗传进化分析。【结果】从山东省某鸭场疑似短喙侏儒综合征(short beak and dwarfism syndrome, SBDS)的病料中分离到一株NDPV，命名为SDGT0628。该分离株在SPF鸭胚上培养可导致鸭胚出现特异性死亡，测得鸭胚半数致死量(DELD₅₀)为10^−4.5/0.2 mL。全基因组核苷酸序列和VP1蛋白氨基酸序列同源性分析结果发现：分离株SDGT0628与2023年分离株TX2302全基因组核苷酸序列相似性最高为99.8%，与SD0101和LYG23的核苷酸同源性次之，为99.7%，VP1蛋白氨基酸序列与2018年分离株SD0101相似性为99.9%；SDGT0628与鹅细小病毒(goose parvovirus, GPV)的同源性较番鸭细小病毒(muscovy duck parvovirus, MDPV)高。全基因组核苷酸序列系统发育树表明，SDGT0628与NDPV形成一个独立的小分支，证明GPV在不断地进化。与GPV VP1蛋白氨基酸序列比对结果发现，13株NDPV (含SDGT0628株)有Q89L、D142E、S450N这3个氨基酸位点的共同变异；分离株SDGT0628在497位氨基酸位点产生突变(W→R)，其他NDPV和GPV未产生此差异。本实验室新分离出5株NDPV，与SDGT0628株VP1氨基酸序列突变位点进行比较，5株NDPV均存在89、142、450位点突变。【结论】从山东省某鸭场中分离得到了一株NDPV，命名为SDGT0628株(GenBank登录号PQ316314)。VP1蛋白的氨基酸序列分析发现，NDPV存在3个氨基酸位点的突变，SDGT0628在497位氨基酸位点产生了新的突变。

Abstract:

[Background] Since 2015, disease outbreaks have occurred in Cherry Valley ducks and other commercial meat ducks in the eastern coastal areas of China and have gradually spread inland. The diseased ducks present short beaks, enlarged tongues, stunted growth, and easy fractures of leg bones. The pathogenic agent of this disease was identified as novel duck parvovirus (NDPV). [Objective] To reveal the genetic evolution of NDPV by epidemiological investigation, so as to provide a basis for understanding the genetic evolution laws, enriching the epidemiological data, and deciphering the pathogenic mechanism of NDPV. [Methods] The liver of a diseased duckling was grinded and the supernatant was obtained by centrifugation of the homogenate. The SPF-grade duck embryo was used for virus isolation and the virus titer was measured. PCR was employed to identify the isolated virus and detect exogenous viruses. The whole genome of the isolated virus was amplified and analyzed for the sequence homology. Molecular characteristics and genetic evolution of the VP1 protein were analyzed. [Results] A NDPV strain, SDGT0628, was isolated from a duck farm with suspected short beak and dwarfism syndrome (SBDS) in Shandong Province. The strain was cultured with SPF-grade duck embryos, with the median lethal dose in duck embryos (DELD₅₀) being 10^−4.5/0.2 mL. SDGT0628 showed the genome-wide nucleotide sequence homology of 99.8% with the strain TX2302 isolated in 2023 and 99.7% with strains SD0101 and LYG23. SDGT0628 showed the VP1 amino acid sequence homology of 99.9% with the strain SD0101 isolated in 2018. SDGT0628 had higher homology with goose parvovirus (GPV) than with muscovy duck parvovirus (MDPV). The phylogenetic tree built based on genome-wide nucleotide sequences showed that SDGT0628 formed a small independent branch with NDPV, demonstrating the continuous evolution of GPV. Compared with the amino acid sequence of GPV VP1 protein, those of 13 NDPV strains (including SDGT0628) had three common amino acid mutations, Q89L, D142E, and S450N. SDGT0628 produced a mutation W497R, which was not observed in other NDPV strains or GPV. Five strains of NDPV were newly isolated in our laboratory, and they were compared with SDGT0628 to reveal the amino acid mutations of the VP1 protein. The results showed that all the 5 strains of NDPV had mutations at positions 89, 142, and 450. [Conclusion] In this study, a strain of NDPV was isolated from a duck farm in Shandong Province and named SDGT0628 (GenBank accession number PQ316314). NDPV had 3 mutations in the VP1 protein, and SDGT0628 had a new mutation of W497R.