Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 ( C y HV ‐3) and possible latency of C y HV ‐3 by temperature shift in the cells

Abstract

A new cell line named CCF ‐ K 104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 ( C y HV ‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from C y HV ‐3‐infected CCF ‐ K 104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious C y HV ‐3 was produced stably in CCF ‐ K 104 cells over 30 viral passages. Our findings showed that CCF ‐ K 104 is a useful cell line for isolation and productive replication of C y HV ‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that C y HV ‐3 was present with low viral DNA loads, suggesting that C y HV ‐3 may establish latent infection in CCF ‐ K 104 cells. Amplification of the left and right terminal repeat sequences of the C y HV ‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of C y HV ‐3.

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