N2a细胞中猪伪狂犬病毒潜伏感染和再激活模型的建立OA

As a neurotropic alphaherpesvirus,research on the latency and reactivation mechanisms of pseudorabies virus(PRV)has long been limited by the lack of stable,reproducible,and easily visualized neuronal cell models.This study aimed to establish and characterize a latent infection and reactivation model of PRV in mouse neuro-2a(N2a)cells.N2a cells were infected with a recombinant pseudorabies virus expressing enhanced green fluorescent protein(rPRV-EGFP).Latent infection was induced by heat stress at 42℃for 3 days,followed by transfer to 37℃for 7 days to induce reactivation.Multidimensional validation was performed through green fluorescent protein(GFP)signal tracking,real-time fluorescent quantitative polymerase chain reaction(qPCR)detection of glycoprotein B(gB)gene copy numbers in culture supernatants,qPCR analysis of the relative mRNA transcription levels of immediate-early protein 180(IE180)and latency-associated transcript(LAT),PCR detection of gB gene expression,determination of viral titers by the 50％tissue culture infectious dose(TCID50)assay,and Western blot(WB)analysis of viral proteins.The results showed that after 3 days of latent infection induction at 42℃,compared with the lytic infection group(37℃),the latent infection group(42℃)exhibited markedly weakened GFP signals in N2a cells,significantly reduced gB gene copy numbers(P<0.000 1),significantly decreased relative mRNA transcription levels of IE180 and LAT(P<0.000 1),and substantially reduced gB gene expression.Viral titers in the cell supernatants were below the detection limit,and the expression of IE180 and major capsid protein VP5 was absent.After transfer to 37℃,GFP signals recovered from day 1 and gradually increased.The gB gene copy number and the relative mRNA transcription levels of IE180 and LAT increased progressively and were significantly higher on day 7 than those observed after 3 days of latency induction(P<0.000 1).The expression level of the gB gene increased,viral titers continuously rose,and viral proteins including pseudorabies virus thymidine kinase(TK),serine/threonine protein kinase(US3),gB,VP5,and IE180 became detectable on day 3 after transfer,indicating successful controlled reactivation.This study achieved visualized tracking of PRV latency and reactivation and provides an efficient and reliable tool for investigating the molecular mechanisms underlying PRV latent infection and reactivation.