柔嫩艾美耳球虫病毒的鉴定

Identification of Eimeria tenella virus

鸡球虫病是由艾美耳属(Eimeria)的一种单细胞寄生性原虫引起的严重危害养禽业发展的重要疾病之一,遍及世界各地。感染鸡的艾美耳属球虫中,国外已报道在巨型艾美耳球虫(E.maxima),堆型艾美耳球虫(E.acervu-lina),毒害艾美耳球虫(E.necatrix)和布氏艾美耳球虫(E.brunetti)中发现了病毒粒子或病毒RNA。但是作为危害最严重的柔嫩艾美耳球虫是否有病毒感染,国内外迄今尚无报道。本研究首次在柔嫩艾美耳球虫中发现病毒并对其进行了鉴定。通过核酸分析、RNA依赖的RNA聚合酶(RDRP)活性和电镜形态观察对病毒进行鉴定。核酸酶的敏感性试验结果表明在柔嫩艾美耳球虫总核酸电泳图谱上观察到的大小分别为1.4、2.4和3.6kb的3条病毒带均不能被DNA酶(100mg/L)降解,但可被RNA酶(1.0mg/L)降解,表明这些核酸为RNA。另外,这些核酸不能被高盐浓度(0.3mol/L NaCl)RNase A(10mg/L)降解,但可被低盐浓度(0.015mol/L NaCl)RNase A(10mg/L)降解,并且采用α-32P标记的UTP掺入法测得该病毒样核酸具有RNA依赖RNA聚合酶(RDRP)活性,表明这些核酸为双链RNA(dsRNA)。利用蔗糖梯度离心和透射电镜技术对柔嫩艾美耳球虫病毒进行了分离和鉴定。电镜负染观察到柔嫩艾美耳球虫病毒粒子外观呈球形,二十面体,无囊膜,直径38nm。

Coccidiosis is a worldwide disease in poultry caused by members attributed to Eimeira genus which can lead to serious economical losses.E.tenella is one of the most important and harmful species causing coccidiosis in chickens.Up to now parasitic protozoa viruses or RNA segments（RNAs） associated with viruses have been described in Eimeria species including E.stiedae,E.nieschulzi,E.necatrix,E.maxima,E.acervulina,and E.brunette.However,virus-like RNAs and virus-like particles（VLPs） have not been reported in E.tenella with similar assays.In the present report,for the first time in our knowledge,E.tenella virus was discovered and identified in E.tenella sporulated oocysts isolated from Changchun,China.To identify E.tenella viruses,total nucleic acids associated with RNAs were analyzed and virus morphology was observed under an electron microscopy.RNAs was analyzed by nuclease sensitivity assay,RNase-protection assay,and RNA-dependent RNA polymerase（RDRP） activity assay.The results of nuclease sensitivity analysis showed that three bands with sizes of 1.4,2.4 and 3.6 kb described above were DNase-resistant（100 mg/L）,but were digested by RNase A（1.0 mg/L）.These results suggested that the three bands were RNAs in nature.Moreover,the RNA bands were resistant to RNase A digestion at high salt concentration（0.3 mol/L NaCl）,but not at low salt concentration（0.015 mol/L NaCl）.These RNAs possessed RDRP activity as suggested by the α-32P-UTP incorporated assay.The results indicated that they were double-stranded RNAs（dsRNAs）.E.tenella virus was separated by sucrose gradient centrifugation and examined under electron microscopy.The results showed that they have a homogeneous icosahedral shape with a diameter of approximately 38 nm,without an envelope.