Goldfish Hemorrhagic Virus (CHV) Diagnostic Protocol

Purpose

Koi Herpesvirus, also known as Cyprinid Herpesvirus 3 (CHV3), is a virus that affects koi and other cyprinid fish species. It primarily targets the fish's hematopoietic organs, causing organ necrosis and a decline in immune function, which can lead to massive mortality in severe cases. This diagnostic protocol aims to provide a rapid and accurate diagnosis of goldfish suspected of being infected with the Goldfish Hemorrhagic Virus (CHV) through scientific methods, in order to implement appropriate prevention and control measures and reduce the risk of virus transmission.

Sample Collection

1. Collection target: Choose goldfish showing clinical symptoms (such as pale gills, difficulty breathing, skin ulcers, etc.) suspected of CHV infection.
2. Sample types: Collect blood, gill tissue, kidney tissue, spleen tissue, etc.
3. Collection method: Use sterile tools for live sampling, or perform necropsy sampling on recently deceased goldfish.
4. Sample preservation: Immediately place collected samples in sterile containers with appropriate preservation liquid, and transport them refrigerated to the laboratory.

Laboratory Testing

1. Histopathological examination

1. Fix, slice, and stain collected tissue samples.
2. Observe for pathological changes consistent with CHV infection in tissue slices, such as hemorrhagic organ necrosis, gill tissue inflammation, etc.

2. Polymerase Chain Reaction (PCR) testing

1. Extract sample DNA.
2. Design specific primers for PCR amplification targeting the CHV genome.
3. Confirm the presence of CHV DNA fragments through gel electrophoresis analysis of PCR products.

3. Real-time quantitative PCR (qPCR) testing

1. Monitor DNA amplification in real-time using fluorescence probe technology.
2. Quantitatively detect CHV DNA copy numbers in samples to assess viral load.

4. In Situ Hybridization (ISH) testing

1. Perform in situ hybridization on tissue slices using labeled CHV -specific probes.
2. Observe for probe signals under a microscope to determine the virus distribution in tissues.

Result Interpretation

1. If histopathological examination shows typical CHV lesions and PCR or qPCR testing results are positive, a diagnosis of CHV infection can be confirmed.
2. If there are only histopathological changes without molecular biology testing support, consider other possible causes.
3. Interpret qPCR results in conjunction with viral load to assess the degree of infection and potential infectivity.

Report Writing

1. Record sample information, collection time, testing methods, and results.
2. Provide clear diagnostic recommendations based on the test results.
3. Suggest follow-up actions such as isolation, disinfection, treatment, etc.

Precautions

1. Conduct all operations in a biosafety cabinet to prevent cross-contamination.
2. Use disposable sterile instruments to prevent sample contamination.
3. Strictly adhere to laboratory waste disposal regulations to ensure environmental safety.