Infectious Hematopoietic Necrosis Virus (IHNV) Diagnostic Protocol

Infectious Hematopoietic Necrosis Virus (IHNV) is a highly pathogenic virus that seriously threatens the health of fish, especially causing significant economic losses in aquaculture. Establishing a scientific and efficient diagnostic protocol is crucial to control the spread of this virus. Below is a draft of an IHNV diagnostic protocol based on current best practices:

I. Preliminary Diagnosis

1. Clinical Observation

• Symptom identification: Observe if the fish population shows symptoms such as acute mortality, darkening of body color, exophthalmia, abdominal swelling, and gill filament hemorrhage.
• Epidemiological investigation: Collect data on the geographical location, species, age, stocking density, water temperature of affected fish populations, analyze potential sources of infection, and transmission routes.

2. Pathological Examination

• Tissue sampling: Collect samples of kidney, spleen, brain, and other tissues from moribund or recently dead fish.
• Histopathology: Use a microscope to examine tissue sections, identify typical cellular pathological features such as lymphocyte infiltration, and hematopoietic tissue necrosis.

II. Laboratory Confirmation

1. Virus Isolation and Culture

• Cell culture: Use suitable cell lines (such as FHM, CHSE-214) for virus isolation, observe cytopathic effects (CPE).
• Identification: Confirm the virus type of the isolated material using immunofluorescence staining or PCR techniques.

2. Molecular Biology Detection

• Polymerase Chain Reaction (PCR): Design specific primers, amplify IHNV gene fragments, and conduct virus nucleic acid detection through agarose gel electrophoresis or real-time quantitative PCR.
• Sequence analysis: Determine the sequence of PCR products, compare them with known IHNV strains to identify the viral genotype.

3. Serological Testing

• Neutralization test (NT): Use known IHNV antiserum to detect neutralizing antibodies in the test samples, evaluate the population's immune status.
• Enzyme-linked immunosorbent assay (ELISA): Use specific antibodies to detect IHNV antigens or antibodies in the samples.

III. Result Interpretation and Reporting

Based on laboratory test results, combined with clinical symptoms and epidemiological data, provide a comprehensive diagnostic report. The report should include:

• Case description: Detailed record of the disease progression, clinical symptoms, pathological changes, etc.
• Laboratory test results: List specific data and conclusions for each test.
• Diagnostic conclusion: Clearly indicate whether it is an IHNV infection and possible strain type.
• Recommendations: Provide targeted prevention and control suggestions, including isolation treatment, disinfection measures, vaccination, etc.

IV. Follow-up Monitoring and Management

• Regular monitoring: Continuously monitor suspected infection areas to promptly detect new outbreaks.
• Health management: Strengthen aquaculture environment management, enhance fish immunity, and reduce the risk of viral infections.
• Information sharing: Collaborate with relevant research institutions and government departments to share epidemic information and collectively address the threat of IHNV.

Please note that this diagnostic protocol serves as a basic framework, and adjustments and optimizations should be made based on specific circumstances and local regulations. Furthermore, all procedures involving live animals must adhere to relevant ethical and animal welfare standards.