Effects Of Glutamine On Red-spotted Grouper Nervous Necrosis Virus (RGNNV) Replication

Virus needs to hijack cellular metabolic machinery during its replication inside the host.Glucose and glutamine are two main sources for carbon metabolic pathways which have been altered by some viruses to maintain their life cycle.Nervous necrosis virus(NNV)can infect many economically important fish and has caused great economic loss in aquaculture industry worldwide.Despite its high pathogenicity,little is known about the relationship between the host metabolic pathways and its replication.Here we studied the effects of glucose and glutamine on red spotted grouper nervous necrosis virus(RGNNV)replication in GF-1,Grouper fin cell line and SSN-1 cells,striped snakehead fish cell line,using qRT-PCR to find mRNA yields,Western blotting and Immune fluorescence Assay(IFA)for protein expression.The results showed that glucose but not glutamine was essential for the viability of both GF-1 and SSN-1 cells.During RGNNV infection,in both GF-1 and SSN-1 cells,cultured in the medium without glutamine,we didn't observe efficient production of NNV virion.However,the yield of virion could be rescued with the addition of several intermediates of glutamine in Tri carboxylic acid(TCA)cycle,i.e.pyruvate,oxaloacetic acid(OAA),and dimethyl a-ketoglutarate(a-KG).We also performed western blotting and immune fluorescence assay and noticed that these intermediates of TCA cycle also rescued glutamine deprivation at protein level.Furthermore,addition of bis-2-(5-phenylacetamido-1,3,4-thiadiazo 1-2-ethyl sulfide(BPTES),a glutaminolysis inhibitor,was able to inhibit virus multiplication in GF-las well as SSN-1 cells cultured in complete medium having no BPTES.However it is also rescued by the supplementation of dimethyl a-ketoglutarate(a-KG).We also examined the transcription levels of RdRp and capsid genes of RGNNV at different time points which also showed that glutamine is necessary for RGNNV production and no virion production was observed at either time points in glutamine free medium,and again a-KG rescued glutamine deficiency at either time points.Taken together,these results revealed that glutamine could regulate RGNNV replication via TCA cycle and it lead to find a new route to target RGNNV.