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Evidence for cell apoptosis suppressing white spot syndrome virus replication in Procambarus clarkii at high
Xiao-Guo Wu1, Hai-Tao Xiong, Yi-Zhen Wang
1Zhejiang University, Hangzhou, People's Republic of China.
Abstract:
In shrimp, higher water temperatures (~32°C) can suppress the ability of white spot syndrome virus (WSSV) to replicate and cause mortality, but the mechanisms remain unclear. To investigate whether cell apoptosis might be involved, a Tdt-mediated dUTP nick-end label (TUNEL) method was used to assess levels of chromosomal DNA fragmentation in hepatopancreas and gill cells of Procambarus clarkii crayfish infected with WSSV and maintained at either 32 ± 1°C or 24 ± 1°C. Based on relative cell numbers with yellow-green colored TUNEL-positive nuclei, the apoptotic index was elevated in WSSV-infected crayfish maintained at 32°C. In gill tissue sections examined by transmission electron microscope, cells with nuclei displaying apoptotic bodies or marginated, condensed and fragmented chromatin without concurrent cell cytoplasm damage were also more prevalent. Flow cytometry sorting of annexin-stained cells showed apoptosis to be most prevalent in granular haemocytes, and assays for caspase-3 activity showed it to be most elevated in hepatopancreas tissue. Despite these indicators of cell apoptosis but consistent with WSSV replication being restricted at elevated temperatures, no increases in transcription of the viral anti-apoptosis genes ORF390 and ORF222 were detected by RT-PCR in shrimp maintained at 32°C, possibly due to the elevated levels of cellular apoptosis.
Insights
Elevated temperatures induce apoptosis in crayfish infected with white spot syndrome virus (WSSV), potentially explaining restricted viral replication. This cellular response may be key to understanding WSSV disease progression.
Area of Science:
- Aquatic animal health
- Virology
- Cellular biology
Background:
- White spot syndrome virus (WSSV) causes significant mortality in shrimp.
- Higher water temperatures (around 32°C) are known to suppress WSSV replication and reduce mortality, but the underlying mechanisms are not fully understood.
- Cell apoptosis, or programmed cell death, is a potential factor influencing viral replication and host response.
Purpose of the Study:
- To investigate the role of cell apoptosis in WSSV-infected crayfish at different temperatures.
- To determine if elevated temperatures trigger apoptotic responses in WSSV-infected crustaceans.
- To explore the relationship between temperature, apoptosis, and WSSV replication.
Main Methods:
- Utilized the Tdt-mediated dUTP nick-end label (TUNEL) assay to detect DNA fragmentation in crayfish tissues.
- Examined gill tissue using transmission electron microscopy to identify cellular signs of apoptosis.
- Employed flow cytometry with annexin staining to identify apoptotic cells in haemolymph.
- Assessed caspase-3 activity in hepatopancreas tissue.
- Used RT-PCR to analyze the transcription of viral anti-apoptosis genes (ORF390 and ORF222).
Main Results:
- The apoptotic index was significantly higher in WSSV-infected crayfish maintained at 32°C compared to those at 24°C.
- Transmission electron microscopy revealed more cells with apoptotic features (e.g., apoptotic bodies, chromatin condensation) in gill tissues at 32°C.
- Flow cytometry indicated that granular haemocytes were the primary cells undergoing apoptosis.
- Caspase-3 activity, a marker of apoptosis, was highest in the hepatopancreas.
- Despite increased cellular apoptosis at 32°C, transcription of viral anti-apoptosis genes (ORF390, ORF222) did not increase, suggesting a potential suppression mechanism.
Conclusions:
- Elevated water temperatures (32°C) induce significant cellular apoptosis in WSSV-infected Procambarus clarkii crayfish.
- The observed increase in apoptosis at higher temperatures correlates with restricted WSSV replication.
- Cellular apoptosis, rather than viral gene regulation, may be the primary mechanism by which elevated temperatures suppress WSSV.
- These findings provide insights into the host-pathogen interaction and temperature-dependent disease dynamics in WSSV infections.
