Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods

Diseases of Aquatic Organisms

Peter Hostnik¹  Vlasta Jencic¹  Marjeta Strancar¹  Ivan Toplak¹  Joze Grom¹  Darja Barlic-Maganja¹ 

关键词: Infectious hematopoietic necrosis virus;    IHNV;    Diagnosis;    Virus isolation;    RT-PCR;    Storage;   

DOI  :  10.3354/dao052179

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenatesand cell culture supernatants were incubated at 4 and 25°C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezingand thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4°C but, only on the first day ofincubation at 25°C. Viral RNA could be amplified during the incubation period of 35 d at 4°C but only during 8 d of incubation at 25°C. In IHNV-infected cell culture supernatant stored at 4°C, it was possible to detect virus for 36 and 16 d in supernatantstored at 25°C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while thevirus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of differentdetection methods are very important for efficient IHNV diagnosis.

【 授权许可】

Unknown