The Mechanism Underlying Glutamine Regulating Snakeheadfish Vesiculovirus (SHVV) Replication

Rhabdoviridae consists of a group of viruses with single negative-sense RNA and envelope.Rhabdoviruses can infect broad host ranges,including both plants and animals.Rhabdoviruses are also important fish pathogens.Snakehead fish vesiculovirus(SHVV)belongs to the genus Perhabdovirus of Rhabdoviridae.SHVV infection has caused serious disease and great economic loss of snakehead fish culture.However,there is no effective way to prevent and control the disease.Understanding the mechanism of the SHVV replication will shed a new light on the prevention and control of SHVV disease of snakehead fish.Therefore,in this dissertation,we focused our studies on the relationship between viral replication and glutamine,meanwhile,the host range of SHVV was also investigated.The results were as follows:1.Effects of glutamine on SHVV replication.We observed that the lack of glucose but not glutamine could significantly inhibit the growth of SSN-1 cells.Therefore,we further investigated the role of glutamine on SHVV infection.The results showed that the lack of glutamine could significantly inhibit the replication of SHVV in SSN-1 cells.Furthermore,both L-and D-type glutamine had similar effects on the viral replication,indicating that chirality did not affect the effect of glutamine.2.Transcriptomic profiling of SHVV infected SSN-1 cells cultured in medium without glutamine.In order to explore the replication mechanism of the SHVV in SSN-1 cells,we conducted transcriptome sequencing of SHVV-infected and non-infected SSN-1 cells with glutamine deprivation.The results showed that there were 1441 differential expression gene(DEGs)with 1215 up-regulated and 226down-regulated ones.Enrichment analysis of DEGs through KEGG pathway found that the DEGs were mostly enriched in tricarboxylic acid circulating pathway,MAPK,apoptosis,RIG-1-like and Toll-like receptor signaling pathways and so on.The q RTPCR results of 8 random DEGSs showed similar expression patterns with the results of the transcriptomic analysis,indicating that transcriptomic analysis was reliable.3.Study on the relationship between viral replication and TCA cycle.Basing on the results of transcriptomic analysis,we further investigated the relationship between viral replication and TCA cycle.Supplementation of TCA cycle intermediates,such asα-ketoglutarate(α-KG),pyruvate and oxaloacetate(OAA),could significantly rescue the viral replication in SSN-1 cultured in the medium without glutamine,suggesting that glutamine was replenished in TCA cycling to increase the viral replication.Glutaminase(GLS)is a rate-limiting enzyme for the production of glutamate from glutamine,which is important for the entry of glutamine into the TCA cycle.We have cloned,expressed the GLS gene,and polyclonal antibody against GLS was generated in rabbit.Overexpression of GLS or inhibition of GLS activity by BPTES showed that the replication of the virus was enhanced or decreased,respectively,indicating that GLS regulated SHVV replication via dehydration of glutamine into glutamate,so as it could enter into TCA cycle.4.Study on the relationship between viral replication and cell autophagy.The transcriptomic results showed that the expression of autophagy related gene 4(ATG4)was increased in SHVV infected SSN-1 cells.Furthermore,we observed that SHVV infection could induce autophagy of SSN-1 by transmission electron microscopy.Therefore,we investigated the relationship between viral replication and cell autophagy.It is well known that glutamine is the precursor material for the synthesis of glutathione(GSH).We showed that the addition of GSH or glutamine could inhibit autophagy,the addition of glutathione synthesis inhibitor BSO could reduce GSH,resulting in the autophagy.We further showed that the lack of glutamine induced autophagy through oxidative stress-ATG4,resulting in the inhibition of SHVV replication at the levels of viral m RNA,protein and titer.5.SHVV pathogenicity on rice field eel(Monopterus albus).Monopterus albus rhabdovirus(Mo ARV)is a kind of Rhabdovirus which can cause serious loss of rice field eel culture.We determined the whole genome sequencing of SHVV,and the results showed that the genome similarity between SHVV and Mo ARV was more than94%.Therefore,we studied the susceptibility of rice field eel to SHVV.The results showed that rice field eel was susceptible to SHVV by either intraperitoneal injection or immersion.Severe hemorrhage was observed on the skin and internal organs(liver,spleen and heart)of the infected rice field eel.Histopathological examination revealed that the liver,kidney and heart tissue of the infected fish had vacuoles.Viral specific RNA or protein could be detected by reverse transcription polymerase chain reaction(RT-PCR),in situ hybridization(ISH)or immunohistochemistry(IHC).These results showed that rice field eel was susceptible to SHVV.