key: cord-0033670-5ffp71nd authors: Xia, Yu-chen; Hu, Zhi-hong; Qiu, Zhi-juan; Ma, Zhong-bin; Wang, Hua-lin; Deng, Fei title: An improved culture system for virus isolation and detection date: 2008-10-10 journal: Virol Sin DOI: 10.1007/s12250-008-2968-1 sha: d800dfd11a8200915ebe5144b41eae3116adbe5a doc_id: 33670 cord_uid: 5ffp71nd Cell culture plays an important role in virology. It provides a platform for the detection and isolation of viruses as well as for the biochemistry and molecular biology based studies of viruses. In the present work, a new system that could permits multiple (different) cell lines to be simultaneously cultured in one dish was developed. In the system, each cell line was cultured in an isolated zone in the same dish or well and the system is therefore called an isolated co-culture system. The usefulness of this novel approach for virus isolation was demonstrated using a model system based on adenovirus. lines for viral infection. This approach becomes less effective when there is a limited supply of specimen and a number of different cell lines have to be used for isolation of novel or unknown viruses. To this end, we have developed a new isolated co-culture cell system which allows virus isolation in multiple cell lines using a small amount of starting materials. The BHK cell line, which could not support the propagation of adenovirus, was used as a control. A strain of type III adenovirus was kindly provided by CCGVCC (China Centre General Viruses Culture Collection). Anti-adenovirus human serum was kindly provided by Professor Lin Chen from Guangzhou Institute of Biomedicine and Health. A 35mm TC-Treated culture dish (Corning, United State) was divided into seven areas by marker as shown in Fig. 1A . Each of the area was seeded with 10 4 cells of one of the above cell lines. After 4 h incubation in incubator with CO 2 , the media were carefully removed, and the dish was washed twice with phosphate buffered saline (PBS). Type III adenovirus was chosen to test the isolated co-culture cell system because of its wide host-range and low risk. An aliquot of 1.5 mL virus stock was gently loaded to each dish covering the whole surface so that all seven cells received equal exposure of the specimen, and the dish was incubated for one hour at 37 in an incubator with CO ℃ 2 . Then the viral medium was discarded, the cells were washed by PBS and 1.5 mL maintenance medium (DMEM+2%FBS) was As all the cells need to be cultured in the same and BHK cells in comparison to the un-infected controls. (Fig.2) The supernatant of infected co-cultured cells was used as the template for PCR identification. Using adenovirus specific primers hex1885 and hex1913, a PCR product with a size of 300 bp could be detected (Fig. 3) . The expected size of the PCR product of the adenovirus was 301 bp, therefore the PCR result indicated that the adenovirus was replicated in the cocultured cells. Immunofluorescence assay was used to detect the (Fig.4) . The development of new research fields frequently depends on the development of new techniques. It was when the specimen quantity is limited, the success rate is often low. In this study, an isolated co-cultured cell system that could support several different cell lines to be maintained in the same container was developed. The cells were seeded in isolated zones of the container and were co-cultured with the same medium. It increases the opportunity of the virus to encounter its favoured cells and reduces the use of specimens. (3, 16) . It is shown in this study that it can be coupled with the co-culture system for pathogen identification. In fact the immunofluorescence assay made the co-culture system more sensitive, as the LLC-MK2 cells which did not show CPE could be detected by immunofluorescence assay. In summary, in this study an isolated co-culture system was developed which has the potential to be applied torapid isolation of unknown virus when the starting material is limited. Future studies will examine the feasibility of increasing the number of different cell lines in the same dish to further enhance the success rate. Facing with the increasing number of emerging viruses in recent times, it is hoped that this novel virus isolation system will play a important role in rapid isolation and identification of new pathogens in future disease investigation. 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