Detection Of The Immediately Early Genes Of Cyprinid Herpesvirus Ⅱ And The Effect Of Viral Infection On Reactive Oxygen-related Signal Pathways

China is a big carp breeding country.Since the outbreak of Cyprinidherpesvirus(CYHV-2)in China,Cyprinidherpesvirus(CYHV-2)has caused great harm to crucian carp breeding industry,so it is urgent to further study the virus.Cyprinus herpesvirusⅡ type is a double stranded DNA encapsulated virus.The genome of the virus has obvious expression sequence in the amplification process.Among them,the genes that can be expressed in the absence of new protein synthesis by utilizing proteases such as RNA polymerase existing in the host are the very early genes of the virus.The very early viral genes and the very early proteins they express can affect the expression of viral genes and host genes,regulate the rate of viral amplification and the expression of host proteins to create conditions for viral amplification,and are the key regulatory genes in the process of viral infection.As the first gene expressed in the viral genome,very early genes are very potential targets for drug screening for prevention and control.At the same time,the very early genes can be used as representative genes to monitor the replication and amplification of the virus.Its promoter is also a good candidate for an efficient promoter.The very early CYHV-2 gene screened can provide accurate data for the subsequent study of the virus.According to existing studies,viral infection can cause intracellular ROS changes.The change of ROS is an important change indicator in the process of viral infection,which increases with the advancement of the pathological process of viral diseases.In order to maintain the intracellular oxidative balance,cells will activate the expression of anti-oxidative stress related genes and regulate the immune system to resist viral infection.So far,no studies have been conducted on the changes of ROS pathway in the host after CYHV-2 infection.In order to further understand the changes of the oxidative stress system in the process of virus infection,detection and exploration were carried out.Through the experiment,we obtained the following results:1、An extremely early gene screening model of CYHV-2 was established using 12μg/m L actinomycone and 300 μg/m L cytarabine.Through high-throughput sequencing and verification,ORF54,ORF121,ORF141,ORF147,ORF155,five CYHV-2extremely early stages were screened and 34 early genes and 38 late genes.2、By detecting intracellular ROS,it was found that CYHV-2 infection would cause the increase of ROS in the host,and the change trend was firstly increased and then decreased.It was also found that CYHV-2 could up-regulate the expression of NOX4-related genes in the oxidase subunit of NADHP and promote the production of ROS.At the same time,persistent CYHV-2 infection can cause intracellular ROS accumulation,activate the expression of genes related to Nrf2/ARE antioxidant response elements,and remove intracellular excessive accumulation of ROS.3、In order to explore the interaction between CYHV-2 infection and host ROS-related genes,Ryuf-2 cells were treated with anti-oxidants EGCG and BBH and then infected with CYHV-2 virus.Furthermore,changes in genes related to oxidative stress pathway and changes in ROS were detected.It was found that BBH and EGCG could effectively inhibit the transcription and amplification of CYHV-2 in cells.Meanwhile,the expression of NAPDH related genes up-regulated by CYHV-2infection and the production of ROS were inhibited.With the increase of infection time,BBH and EGCG could significantly up-regulate the expression of Nrf2 gene and regulate the Nrf2/ARE antioxidant elements to inhibit the increase of intracellular ROS.The results showed that BBH and EGCG could inhibit the amplification of CYHV-2 by inhibiting ROS production,which also indicated that ROS was an important signal molecule for CYHV-2 amplification.