A Preliminary Study On The Mechanism Of GCRV Infection Mediated By HSP90 In Grass Car

Grass carp(Ctenopharyngodon idellus)is the largest freshwater fish breeding breed in China,and its breeding output accounts for about 1/5 of the total freshwater fish breeding in China,which is an important part of China’s fishery economic development.However,Grass carp reovirus(GCRV)infection has always been an "old,big and difficult" problem in the industry and academia while Grass carp culture pursues high efficiency and high yield.According to incomplete statistics,the annual economic loss caused by GCRV single pathogen accounts for about 40% of the total loss of grass carp diseases,which seriously restricts the healthy development of grass carp breeding industry.Practice and research show that the incidence of GCRV infection is closely related to environmental temperature,20 ℃-33 ℃ is the onset temperature of GCRV,28 ℃ is the high incidence temperature,and environmental temperature stress is easy to induce grass carp hemorrhagic disease.At present,the temperature-dependent pathogenic mechanism of GCRV is still unclear,which has become one of the bottleneck factors restricting the prevention and control of grass carp hemorrhagic disease.HSP90,a member of the Heat shock proteins(HSPs)family,is widely present in various prokaryotic and eukaryotic organisms,and is one of the most abundant proteins in organisms.It is easily induced by various environmental stress factors,such as physical,chemical and biological factors,and functions as dimers.At present,the association between HSP90 and grass carp hemorrhagic disease and the effect of HSP90 on GCRV proliferation remain unclear.In view of this,this study preliminarily explored the h SP90-mediated temperature-dependent pathogenesis of GCRV.The paper recorded the natural incidence of grass carp hemorrhagic disease in fish ponds in the experimental base in mid-September 2021,and found that the incidence of GCRV infection was closely related to temperature stress and HSP90.The ambient water temperature increased from 18 ℃ to 28 ℃ within 2 days,and a large number of grass carps became infected with GCRV and died.Subsequently,the water temperature gradually decreased from 28 ℃ to 18 ℃,and the onset of the grass carps slowed down greatly and stopped.Rt-pcr was used to detect GCRV genome proliferation and HSP90 expression levels in fish infected at 28 ℃ and asymptomatic fish at 18 ℃,and it was found that HSP90 was significantly positively correlated with GCRV proliferation.To explore the relationship between GCRV temperaturely-dependent pathogenesis and HSP90,we first analyzed the proliferation level of GCRV in grass carp kidney cell line(CIK)at different temperatures,and found that the proliferation rate of GCRV at onset temperature 28 ℃ was significantly higher than that at non-onset temperature 18 ℃,which was 50-100 times higher than that at18 ℃.In addition,GCRV infection with CIK induced HSP90 expression in a similar temperature-dependent manner,and infection at 28 ℃ significantly induced HSP90 expression compared with infection at 18 ℃.In order to explore the relationship between HSP90 expression and GCRV infection or temperature stress,we changed CIK cell lines from 18 ℃ incubator stress to 28 ℃ incubator,and transcriptional data analysis showed that 18 ℃-28 ℃ stress significantly induced the expression of several HSPs family genes including HSP90.Rt-pcr further verified that THE CIK cell line and the 18 ℃ to 28 ℃ stress treatment in gill and intestinal tissues of grass carp could significantly induce the expression of HSP90,suggesting that HSP90 may promote GCRV infection.To further explore the possible role of HSP90 in promoting GCRV proliferation,we overexpressed HSP90 in different grass carp cell lines(CIK and GCO)and infected GCRV 24 h later.Rt-pcr analysis showed that overexpression of HSP90 promoted GCRV proliferation in CIK and GCO cells.GCRV proliferation was dose-dependent on HSP90 level.In addition,CIK cells were treated with two types of HSP90 specific inhibitors(AUY922 and 17-AAG),and the proliferation of GCRV decreased after HSP90 was inhibited,which further confirmed that GCRV proliferation depended on the expression of HSP90,and HSP90 played a role in promoting GCRV.In conclusion,our data confirm that the onset of GCRV infection is closely related to temperature,and the temperature stress-related protein HSP90 plays a key role in promoting GCRV.The existing data provide data and theoretical basis for the systematic analysis of the temperature-dependent pathogenesis of GC...