Studies On Establishment Of Cell Lines From Clostera Anachoreta And Several Important Forest Insects And Replication Of C. Anachoreta Granulovirus In Vitro

Since Grace successfully established long-term cultures of insect cells, more than 800 continuous cell lines have been established from over 150 insect species. There are more than 260 lepidopteran cell lines today. These cell lines have attracted considerable attention because of their application in diverse areas of research, especially in medicine and agriculture. However, the availability of insect host cell lines is still a limiting factor for the study of some insect viruses. Moreover, cell lines originating from different insect speciecs tend to differ in their capacity to produce different viruses. Therefore, there is a need to develop more cell lines as substrates for replication of a variety of baculoviruses. We focus the study on how to establish cell lines of nine kinds of important forest insects and replication of baculovirus in vitro. The serum-free culture of the established cell lines was examined. The study is to furnish effective and valuable support for the large-scale production of the forest insect virus pesticides and Baclovirus Expression Vector System.The embryos, ovaries, testes, hemocytes and fat bodies from nine kinds of forest insects (Clostera anachoreta, Hyphantria cunea, Apocheima cinerarius, Sucra jujube, Ecotropis Obliqua, Anoplophora glabripennis, Apriona germari, Chinolyda flagellicornis, Arge pagana) were chose to initiate primary cultures. Following insect culture medium were chose: TNM-FH, IPL-41, Excell-405, Excell-420, TC-100. The cells of primary culture were obtained successfully from the insects. As a result, the 4-day old embryos were used as the best material in establishing insect cell lines.Two cell lines designated CAF-Clan I and CAF-Clan II have been established from embryos of Clostera anachoreta (Lepidoptera: Notodontidae) in TNM-FH medium containing 10% inactivated fetal bovine serum (FBS). Its optimum pH is 6.4. The two cell lines are the first cell lines ever generated from C. anachoreta. CAF-Clan I consist of a mixture of three cell types: spherical cells, spindle-shaped cells and giant cells. Most of the cultured cells formed a suspension in the medium and were subcultured 78 passages. CAF-Clan II mainly consists of spindle-shaped and spherical cells which attached to the culture surface and have undergone 57 passages. The cell population doubling time at 27℃of CAF-Clan I at passage 22 and CAF-Clan II at passage 24 was about 68.5h and 38.2h, respectively. The chromosome number of both cell lines at passage 15 varied from 62-100 in the majority of cells though a few cells exceeded 260 (n=30). DAF-PCR analysis confirmed that the origination of the two cell lines was C. anachoreta. The susceptibility of the cell lines to baculoviruses was tested. The results showed that CAF-Clan II was susceptible to infection of Clostera anachoreta Granulovirus(ClanGV), Autographa californica nucleopolyhedrovirus (AcMNPV) and Ecotropis Oblique nucleopolyhedrovirus (EoNPV). Occlusion bodies (OBs) production was 129±4 OBs/cell and 124±15 OBs/cell for AcMNPV and EoNPV, respectively. The TCID50 of ClanGV to CAF-Clan II was 6.5×10~5 TCID50/mL。CAF-Clan I was less susceptible to AcMNPV compared with CAF-Clan II, while nonpermissive to EoNPV, HcNPV and AciNPV. Both the new cell lines established in the paper were stored at -80℃and regenerated successfully. Single colony isolation was carried out with CAF-Clan I and the colonies were magnifying.The replication of ClanGV in CAF-ClanI and CAF-ClanII cultured at 15℃and 27℃was observed under the transmission electron microscope. The virogenic stroma, nucleocapsids, virions and occlusion bodies were observed. However, the single occluded virion in the occlusion bodies was not observed. The size of the OBs in vitro and in viva was same.Serum-free culture of the two cell lines was examined. CAF-Clan I and CAF-Clan II grow successfully in a modified insect medium and have been subcultured 5 passages. The cost of the new culture medium is equivalent to 31.8～47% of the commercial insect culture medium's.Four passages have been achieved in one of a primary culture from embryos of Hyphantria cunea and it can thus possibly to be a new cell line. The optimum medium for Hyphantria cunea and Apochema cinerarius is IPL-41+10%FBS. The primary culture of Apochema cinerarius, Anoplophora glabripennis, etc was still being done.