The nervous necrosis virus (NNV) mainly attacks the central nervous system of fish to cause viral nervous necrosis, which is an acute and serious prevalent disease in fish. Among different genotypes of NNV, red-spotted grouper nervous necrosis virus (RGNNV) is the most widely reported, with the highest number of susceptible species. To better understand the pathogenicity of RGNNV, we first developed a reverse genetic system for recombinant RGNNV rescue using B7GG and striped snakehead (SSN-1) cells. Furthermore, we constructed attenuated RGNNV strains rRGNNV-B2-M1 and rRGNNV-B2-M2 with the loss of B2 protein expression, which grew slower and induced less Mx1 expression than that of wild-type RGNNV. Moreover, rRGNNV-B2-M1 and rRGNNV-B2-M2 were less virulent than the wild-type RGNNV. Our study provides a potential tool for further research on the viral protein function, virulence pathogenesis, and vaccine development of RGNNV, which is also a template for the rescue of other fish viruses.

The larval zebrafish is a vertebrate model for in vivo monitoring and manipulation of whole-brain neuronal activity. Tracing its neural circuits remains challenging. Here, we report an applicable methodology tailored for larval zebrafish to achieve efficient retrograde trans-monosynaptic tracing from genetically defined neurons via EnvA-pseudotyped glycoprotein-deleted rabies viruses. By combinatorially optimizing multiple factors involved, we identified the CVS strain trans-complemented with advanced expression of N2cG at 36 °C as the optimal combination. It yielded a tracing efficiency of up to 20 inputs per starter cell. Its low cytotoxicity enabled the viable labeling and calcium imaging of infected neurons 10 days post-infection, spanning larval ages commonly used for functional examination. Cre-dependent labeling was further developed to enable cell-type-specific input tracing and circuit reconstruction. We mapped cerebellar circuits and uncovered the ipsilateral preference and subtype specificity of granule cell-to-Purkinje cell connections. Our method offers an efficient way for tracing neural circuits in larval zebrafish.

The larval zebrafish is a vertebrate model for in vivo monitoring and manipulation of whole-brain neuronal activity. Tracing its neural circuits remains challenging. Here, we report an applicable methodology tailored for larval zebrafish to achieve efficient retrograde trans-monosynaptic tracing from genetically defined neurons via EnvA-pseudotyped glycoprotein-deleted rabies viruses. By combinatorially optimizing multiple factors involved, we identified the CVS strain trans-complemented with advanced expression of N2cG at 36 °C as the optimal combination. It yielded a tracing efficiency of up to 20 inputs per starter cell. Its low cytotoxicity enabled the viable labeling and calcium imaging of infected neurons 10 days post-infection, spanning larval ages commonly used for functional examination. Cre-dependent labeling was further developed to enable cell-type-specific input tracing and circuit reconstruction. We mapped cerebellar circuits and uncovered the ipsilateral preference and subtype specificity of granule cell-to-Purkinje cell connections. Our method offers an efficient way for tracing neural circuits in larval zebrafish.