| White spot syndrome virus (WSSV) is an non-occluded, rod-shaped, enveloped and double-stranded DNA virus. It is also the main pathogen of shrimp. In the present study,we investigated the effects of water temperature(8-33℃)on WSSV invasion, genome replication and gene transcription differences in Procambarus clarkii.According to the detection results of WSSV genome through quantitative PCR, 27℃is the optimum temperature for WSSV proliferation in vivo; the virus proliferation was difficult below 12℃; at 32℃, the proliferation was inhibited at a certain level, and survival time of crayfish was extended. From the transfer experiment, we found that crayfish infected at 27℃and transferred to 12°C, or infected at 33℃and transferred to 27℃could slightly.delay WSSV proliferation process in crayfish, following 2-3 days extension of surival time. This result showed that low temperature delayed rather than reduced original viral replication; hight temperature could still inhibit WSSV-CN proliferation for a short time after crayfish were transferd to low water temperature. but crayfish could not survive with the sustaining WSSV infection.We labelled intact virion using DAPI, and then injected high doses of this virion directly into blood circulatory system of crayfish kept at 12℃and 33℃. Fluorescence miscrope observation showed that WSSV was able to invade hemocyte even at 12℃, but the infected hemocyte number significantly decreased compared with 33℃or 27℃. This indicated that low temperature could only reduce the efficiency of invasion, but unable to prevent entry of virion into hemocyte. We assumed that viral particles incubated in WSSV-infected crayfish without being eliminated at low or high temperature.Crayfish kept at different water temperature were challenged by 109 copies of virions injection. We found that at 32℃the proliferation process was only two days later than 27℃; but at 12℃the viral replication level was inhibited at 105 for about 20 days when crayfish were still alive. This indicated that during outbreak infection of WSSV, low temperature still reduced WSSV proliferation when high temperature had less efficiency. These results also suggest mechanism differences in inhibiting WSSV proliferation between low and high temperature.Temporal analyses of the transcription of immediate early gene (IE1) and late gene VP28 showed that, at 12℃the two genes transcription was all postponed after 12 hpi(hours post infection); at 32℃their transcription was detected dramatically early by 2 hpi and 4hpi and then maintained at a constant level until the end of the analysis. We concluded that low temperature inhibited not only the replication of WSSV genome, but also the packaging and the formation of complete virus particles, thus making the process of WSSV infection slowed down. Transcription of two genes were induced by hyperthermia may due to some enzymes of signal transducer were activated in crayfish at high temperature. The differences in gene transcription and expression level of those regulate genes or related enzymes at different temperature need to be explored further in order to better understand the negative effect of water temperature on gene replication of WSSV.
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