Molecular epidemiology and histopathological analysis of largemouth bass ranavirus

Micropterus salmoides is a high-quality freshwater fish among the main aquaculture species in my country. In recent years, diseases of M. salmoides have occurred frequently, which has caused a great impact on my country's aquaculture industry. Largemouth bass ranavirus (LMBV) is a pathogen that seriously harms the M. salmoides farming industry. In order to explore the pathological changes of M. salmoides after infection with LMBV, and the relationship between the dynamic changes of virus content in the body and the occurrence of diseaseases. The typical symptom of diseaseased fish is muscle necrosis, commonly known as rotting. In this study, 723 diseased M. salmoides samples collected from 2019 to 2021 in Guangdong Province and surrounding areas were classified and sorted, and their liver, spleen and kidney tissues were collected for quantitative PCR detection.. The results showed that the rate of positive LMBV was 63.62%. In addition, the incidence and detection rate are higher in the summer high temperature season. LMBV positive samples were selected to inoculate CPB cells, and a total of 93 strains of LMBV were obtained. Through PCR amplification and sequencing analysis of the isolates MCP, ATPase, DNA polymerase and methyltransferase, the results showed that these genes are highly conserved in LMBV isolates. The identity of the MCP and ATPase gene is 100.0%, and the methyltransferase gene identity is 99.7%-100.0%, and the DNA polymerase gene identity is 99.8%-100.0%, indicating that the variation between LMBV strains is minimal. The representative strain was selected to inject 100 μL 3.72×10⁶ TCID₅₀/mL LMBV into the pectoral fins of M. salmoides to artificially infect the fish with LMBV. The samples showed similar symptoms to those infected with LMBV in the natural state, including darkening of the fish body, surface ulceration, and liver atrophy, etc. Four fish were randomly selected at 0 h 4 h, 8 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d after infection, and liver, spleen, kidney, stomach, intestine, brain, heart and gill tissues were taken to make paraffin sections with HE staining for histopathological changes analysis. qPCR was used to detect the tissue distribution and kinetics of the virus. The results showed that the viral load of LMBV gradually increaseased within 5 days after infection, and the virus was distributed in the heart, liver, spleen, kidney, stomach, intestine, brain and other tissues, and the highest viral load in the heart was 9.5×10⁵ copies/mg. The minimum viral load in brain tissue was 2.9×10² copies/mg. The histopathological results after virus infection showed that LMBV can cause a variety of tissue necrosis of M. salmoides, among which liver, kidney and heart diseases become more serious. Typical pathological features include disorder of liver cell arrangement, dilatation of liver sinusoids, nucleus evacuation, renal tissue cell evacuation, Macrophage rupture, necrosis of ventricular membrane fibers, etc.