3株樱桃谷肉鸭源和2株番鸭源新型鸭呼肠孤病毒的分离与鉴定

The isolation and identification of novel duck reovirus from Cherry Valley ducks (3 strains) and Muscovy ducks (2 strains)

[目的]本文旨在对临床表现为肝、脾坏死的病死鸭进行鸭呼肠孤病毒(NDRV)分离与鉴定。[方法]通过接种10日龄鸭胚进行病毒的初步分离培养,结合PCR检测及σC基因序列分析进行分子生物学鉴定。将NDRV分离毒株接种鸡肝癌细胞(LMH)、鸡胚成纤维细胞(CEF)和鸡胚肝细胞(CEL),经绒毛尿囊膜接种11日龄樱桃谷肉鸭胚,经腿部肌肉接种4日龄雏樱桃谷肉鸭进行生物学特性鉴定。通过测定NDRV分离毒株对氯仿和胰蛋白酶的敏感性进行理化特性鉴定。[结果]分离到5株新型鸭呼肠孤病毒(NDRV),分别为RPVA1311、RPVA1312、RPVA1313、RPVA1314和RPVA1315。NDRV分离毒株σC基因序列与新型鸭呼肠孤病毒的核苷酸同源性为94%～98%,与禽呼肠孤病毒(ARV)和番鸭呼肠孤病毒(MDRV)相似性较低,分别为38%～41%和40%～50%; NDRV分离毒株能在LMH、CEF和CEL中增殖并产生细胞融合,形成合胞体的细胞病变,2株番鸭源分离病毒的LMH细胞毒滴度(lg[TCID_(50)]:6.32、6.63)明显高于3株樱桃谷肉鸭源病毒的滴度(lg[TCID_(50)]:5.83、5.68、5.63); NDRV分离毒株能100%致死11日龄樱桃谷肉鸭胚,且2株番鸭源分离病毒的鸭胚组织毒滴度(lg[ELD_(50)]:5.50、5.63)稍高于3株樱桃谷肉鸭源病毒的滴度(lg[ELD_(50)]:5.32、5.17、5.00),主要病变表现为全身性出血; 4日龄雏樱桃谷肉鸭接种NDRV分离毒株后,出现与自然发病鸭相似的病症,发病率为100%,同时能从病死鸭的肝、脾中再次分离到原病毒;病死鸭主要病变为脾脏肿大、坏死,组织病理学观察可见脾脏淋巴细胞数量减少、组织细胞坏死,肝细胞变性、炎性细胞在汇管区周围浸润;理化性质测定结果显示,NDRV分离毒株对氯仿不敏感,对胰蛋白酶具有不同程度的敏感性。[结论]分离的5株病毒是属于呼肠孤病毒科正呼肠孤病毒属的一种新型鸭呼肠孤病毒。

[Objectives] The paper aims to isolate and identify duck reovirus from the dead ducks with clinical manifestations characterized by necrosis of liver and spleen.[Methods] The virus was isolated and propagated by inoculation of 10 days old duck embryos and further molecular biology identification was operated with PCR detection and the analysis of σC gene sequence.The detection of the biological characteristics included the infection of Leghorn male chicken hepatocellular carcinoma(LMH),chicken embryo fibroblast(CEF)and chicken embryo liver cell(CEL),the chorioallantoic membrane inoculation of 11 days old Cherry Valley duck embryos,and leg muscle injection of 4 days old Cherry Valley ducks.The physicochemical characteristics were determined by the detection of the sensitivity of the isolated NDRV to chloroform and trypsin.[Results] 5 novel duck reoviruses(NDRV)were identified,RPVA1311,RPVA1312,RPVA1313,RPVA1314 and RPVA1315,respectively.Compared with novel duck reovirus,avian reovirus(ARV)and Muscovy duck reovirus(MDRV),the nucleotide homology of σC gene sequence of the isolated NDRV were 94%-98%,38%-41% and 40%-50%,respectively.The isolated NDRV could proliferate in LMH,CEF and CEL,and induce cytopathy characterized by cell fusion and the formation of syncytium,and the LMH viral titers of two virus isolated from Muscovy ducks(lg [TCID50 ]:6.32,6.63)were significantly higher than those of three virus isolated from Cherry Valley ducks(5.83,5.68,5.63).All of 11 days old Cherry Valley duck embryo inoculated with the isolated NDRV died,and the main lesion of dead embryo was systemic bleeding points and ELD50 was titrated to be higher than 10^5.0.The virus titers of the duck embryos infected with the virus isolated from Muscovy ducks(lg[ELD50 ]:5.50,5.63)were a little higher than those of the duck embryos infected with the virus isolated from Cherry Valley ducks(5.32,5.12,5.00).The study also found that 4 days old ducks injected with the isolated NDRV were able to show the same symptoms as the natural DRV cases,and the morbidity was 100%.Besides,the identical viruses could be isolated again from the livers and spleens of dead ducks.As for necropsy,spleen was obviously swelling with necrotic spots.In the histopathological examination,necrosis and notable lymphocyte decrease in spleen,and hepatocyte degeneration and infiltration of inflammatory cells around the portal area in the liver were observed.The tests of physicochemical characteristics indicated that the isolated NDRV strains were insensitive to chloroform and sensitive to trypsin of various degree.[Conclusions] The five isolated virus are a new-type duck reovirus of Reoviridae Orthoreovirus.