Temperature-dependent viral replication and antiviral apoptotic response in viral haemorrhagic septicaemia virus (VHSV)-infected olive flounder (Paralichthys olivaceus).

摘要

The olive flounder (Paralichthys olivaceus) shows a high rate of mortality to viral haemorrhagic septicaemia virus (VHSV) in the winter and spring but has zero mortality over 20 degrees C. In this experiment, we studied the effect of rearing temperature on viral replication, viral transcription and antiviral apoptotic immune response in VHSV-infected olive flounder by real-time polymerase chain reaction. Olive flounder were given intra-peritoneal injections of VHSV (107.8 TCID₅₀/ml) and were reared at 15 degrees C or 20 degrees C. Five fish were randomly sampled for head kidney at 3, 6 and 12 h post-infection (hpi) and 1, 2, 4 and 7 days post-infection (dpi). Total RNA extracted from the tissue was reverse transcribed and used as template for real-time PCR. In the 15 degrees C group, the number of viral gRNA copies peaked after 2 dpi and remained high through 7 dpi, while in the 20 degrees C group, the copy number was at the highest at 1 dpi but drastically declined at later stages. Viral mRNA levels in the 15 degrees C group gradually increased starting at 3 hpi to reach their maximum value at 12 hpi and remained high until 2 dpi, whereas the other group showed much lower copy numbers that were undetectably low at 4 and 7 dpi. Type II IFN expression increased as the viral copies increased and the 20 degrees C group showed quicker and stronger expression than the 15 degrees C group. The MHC class I and CD8 expression was high in both the groups at early stage of infection (3-6 hpi) but at later stages (2-7 dpi) in 15 degrees C group expression reduced below control levels, while they expressed higher to control in 20 degrees C group. The expression of granzyme in 15 degrees C fish showed a single peak at 2 dpi, but was consistently expressing in 20 degrees C fish. Individuals expressed very high levels of perforin expressed very high levels of caspase 3. In 15 degrees C fish, TNF alpha , FasL and p53 expressed significantly higher than 20 degrees C only at initial stages of infection (3-6 hpi). Caspase 3 expression found to be low in 15 degrees C fish whereas it was significantly elevated in 20 degrees C group. Interestingly individual fish with high caspase 3 expression contained very low viral RNA. Thus, from our experiment, we can conclude that an effective apoptotic immune response in VHSV-infected olive flounder plays a crucial role in the survival of the host at higher temperatures.