Co-infections of different pathogenic microorganisms usually cause complex effects, and receive more attention. Red-grouper nervous necrosis virus (RGNNV) and Vibrio are the common viral and bacterial pathogens of fish, and are often detected simultaneously in diseased fish. However, the understanding of co-infection of RGNNV and Vibrio is still unclear. In this study, we have established a grouper (Epinephelus coioides) model of the co-infection of RGNNV and Vibrio harveyi (V. harveyi). Compared with single pathogen infection, co-infection of RGNNV and V. harveyi significantly caused more severe pathologic changes with higher mortality (P <
0.05), and promoted the proliferation of the pathogens by RNA-FISH and qRT-PCR (P <
0.05), demonstrating a synergistic effect of RGNNV and V. harveyi in grouper. Furthermore, we found that V. harveyi inhibited the induction and migration of neutrophils by RGNNV, resulting the obviously reduced neutrophils of co-infection groups (P <
0.05). In addition, transcriptome analysis showed that differentially expressed genes (DEGs) of brain tissues of different experimental groups were enriched in immune signaling pathways, such as JAK-STAT signaling, NF-κB signaling and TNF signaling pathways. For the liver and spleen tissues, the DEGs of different experimental groups were enriched in metabolism-related pathways, such as glycolysis/gluconeogenesis and glycerolipid metabolism. Further analysis of the selected DEGs, co-infection of RGNNV and V. harveyi significantly suppressed the host immune response and up-regulated host glucose and lipid metabolism, compared with single-pathogen infection. Taken together, the RGNNV and V. harveyi make synergic reaction in grouper, possibly due to the down regulation of host immune response and up regulation of metabolism to facilitate the replication of both pathogens. These results provide new insights into the pathogenesis of multiple pathogens, and contribute to develop new therapies.