Untargeted LC-MS metabolomics reveals the metabolic responses in olive flounder subjected to hirame rhabdovirus infection.

Hirame novirhabdovirus (HIRRV), which mainly infects the olive flounder (Paralichthys olivaceus), is considered to be one of the most serious viral pathogens threatening the global fish culture industry. However, little is known about the mechanism of host-pathogen interactions at the metabolomic level. In this study, in order to explore the metabolic response of olive flounder to HIRRV infection, liquid chromatography mass spectrometry (LC-MS) was used to detect the changes of endogenous compounds of the olive flounder after HIRRV infection. A total of 954 unique masses were obtained, including 495 metabolites and 459 lipids. Among them, 7 and 173 qualified differential metabolites were identified at 2 days and 7 days post-infection, respectively. Distinct metabolic profiles were observed along with viral infection. At the early stage of infection, only a few metabolites were perturbed. Among them, the level of inosine and carnosine were increased and the potential antiviral ability of these two metabolites was further confirmed by exogenous addition experiment. At the late stage of HIRRV infection, the metabolic profiles changed remarkably. The changes in amino acids and nucleotides especially the 7-methylguanine also accelerated the amplification of viral particles. And the down-regulation of glutathione (GSH) implied an elevated level of ROS (reactive oxygen species) that attenuated the immune system of flounders. HIRRV also induced the accumulation of purine and reduction of pyrimidine, and elevated LPC and LPE levels. The unbalanced purine/pyrimidine and altered lipid profile may be beneficial for the replication and infection of HIRRV at the late stage of infection. These findings provide new insights into the pathogenic mechanism of HIRRV infection in olive flounder.

Expression characteristics of non-virion protein of Hirame novirhabdovirus and its transfection induced response in hirame natural embryo cells.

The non-virion (NV) protein is the signature of genus Novirhabdovirus, which has been of considerable concern due to its potential role in viral pathogenicity. However, its expression characteristics and induced immune response remain limited. In the present work, it was demonstrated that Hirame novirhabdovirus (HIRRV) NV protein was only detected in the viral infected hirame natural embryo (HINAE) cells, but absent in the purified virions. Results showed that the transcription of NV gene could be stably detected in HIRRV-infected HINAE cells at 12 h post infection (hpi) and then reached the peak at 72 hpi. A similar expression trend of NV gene was also found in HIRRV-infected flounders. Subcellular localization analysis further exhibited that HIRRV-NV protein was predominantly localized in the cytoplasm. To elucidate the biological function of HIRRV-NV protein, NV eukaryotic plasmid was transfected into HINAE cells for RNA-seq. Compared to empty plasmid group, some key genes in RLR signaling pathway were significantly downregulated in NV-overexpressed HINAE cells, indicating that RLR signaling pathway was inhibited by HIRRV-NV protein. The interferon-associated genes were also significantly suppressed upon transfection of NV gene. This research would improve our understanding of expression characteristics and biological function of NV protein during HIRRV infection process.

Genome characterization of Hirame novirhabdovirus (HIRRV) isolate CNPo2015 and transcriptome analysis of Hirame natural embryo (HINAE) cells infected with CNPo2015.

Hirame novirhabdovirus (HIRRV) is a fish rhabdovirus belonging to family Rhabdoviridae, genus Novirhabdovirus, which is highly contagious and virulent, and causes hemorrhagic disease in many fish species. In the present work, the whole genome sequence of HIRRV strain CNPo2015 that previously isolated from cultured flounders was obtained using high-throughput sequencing. It consists of 10,998 nucleotides and encodes six viral proteins arranged in order of 3'-N-P-M-G-NV-L-5'. Among Novirhabdovirus, L protein of CNPo2015 possessed the lowest amino acid sequence divergence with HIRRV isolate CA 9703 and HIRRV 080113, and the highest with Snakehead rhabdovirus. Furthermore, the immune response of Hirame natural embryo (HINAE) cell line to HIRRV infection was characterized by RNA-seq, and the results showed that 1976 differentially expressed genes (DEGs) including 1219 up-regulated and 727 down-regulated genes were identified in the HINAE cells infected with HIRRV at 48 h post infection (hpi). Several KEGG pathways were significantly enriched in the viral infected cells, such as cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, cell cycle, apoptosis, RIG-I-like receptors signaling pathway and P13K-AKT signaling pathway. Post viral infection, the flow cytometric Annexin V/PI assay found that apoptotic rate of HINAE cells showed a slight increase within 3 days and then the early and late apoptotic rate were significantly increased to 41 ± 2.65% and 12.37 ± 2.61% at day 4, respectively. Meanwhile, qRT-PCR results also showed that six apoptosis-related genes (BCL2L1, CASPASE 3, CASPASE 10, FAS, AKT and CDK1) were significantly upregulated. This investigation has not only enriched our knowledge of sequence difference characteristics between CNPo2015 and other Novirhabdoviruses, but also provided a data basis for deeper understanding of immune responses in flounder cells post viral infection.