Study On Biomarkers In Body Fluids Of Rhesus Monkey With AIDS Encephalopathy

AIDS is acquired immunodeficiency syndrome(AIDS),which is caused by human immunodeficiency virus(HIV).AIDS patients will suffer from organ and tissue related diseases,such as gastrointestinal diseases、HIV-associated neurocognitive disorders(HAND)etc.HAND can be divided into three types according to the severity of the disease:asymptomatic neurocognitive disorder(ANI),mild neurocognitive disorder(MND),and HIV-associated dementia(HAD).Although Highly active antiretroviral therapy(HAART)has reduced the incidence of HAD,the proportion of virus-induced ANI and MND has increased.At present,the diagnosis of the disease is mainly aided by imaging and pathological examinations.Although these methods can accurately determine whether a patient has AIDS encephalopathy,there are certain limitations.For example,it is mainly aimed at advanced patients,and application is limited in animal models.In order to find relevant biomarkers to predict the occurrence and development of encephalopathy,especially in the early stages of the disease,we conducted the following two parts of research.Part I:Changes in expression of biomarkers in the plasma and cerebrospinal fluid of SIVmac251-21755-BG infected monkeys.Clinically,the use of HAART reduces the proportion of HAD,but the incidence of AN1 and MND is still relatively high.Patients with AIDS encephalopathy still have poor prognosis and reduced quality of life.How to achieve early prediction in order to implement the intervention as soon as possible become the focus of HAND research.In recent years,studies have found that the expression of tau protein and neurofilament light chain NFL in the blood and cerebrospinal fluid of patients with AIDS encephalopathy change significantly.Therefore,scientists hope to use biomarkers to predict the progress of encephalopathy.In this study,we tracked the changes in the biomarkers of AIDS encephalopathy animals in the early stage of infection,in order to find biomarkers that can play a predictive role in the early stage of the disease.In this study,we used the neurotropic virus SIVmac251-21755-BG to infect 10 Chinese rhesus monkeys and detected the expression of biomarkers in the plasma and cerebrospinal fluid of infected monkeys.The results showed that in the early stage of infection,animals with AIDS encephalopathy had no obvious clinical symptoms and imaging changes.There were no obvious changes in plasma YKL-40 and neopterin,and NFL was too low to be detected in the plasma.Only sCD163 showed significant small fluctuations in most animals in the first three months after infection.On the contrary,NFL,neopterin and YKL-40 in the cerebrospinal fluid of infected animals all have a significant change in the acute phase.Both neopterin and YKL-40 have a transient increase in the acute phase.Although the duration is very short,the increase is very large,which is significantly different from that of SIVmac239 and SHIVSF162P3 infected monkeys.Not only does NFL rise rapidly after infection,but also lasts for about 150 days,showing the great potential as a biomarker to predict the progress of encephalopathy,which prompting us that the early stage of encephalopathy have obvious and lasting neuronal damage which is the clinical basis of the cognitive disorder.Part II:The establishment and application of absolute quantitative method for SIV DNA by droplet digital PCR.After SIV infects rhesus monkeys,monkeys can develop persistent infections and form virus reservoirs,which is very similar to the process of HIV infection in humans.Strategies for clearing virus reservoirs are a hot topic in current AIDS research,so it is very important to accurately assess the size of virus reservoirs.The current commonly used method for quantitative detection of virus reservoirs is Real Time Polymerase Chain Reaction,or RT-PCR.This method calculates the copies number of viral DNA in a sample by using a standard curve to achieve"absolute quantification" in a relative sense.Digital PCR(dPCR)is a new technology developed in recent years.Compared with qPCR quantification,it doesn’t need to rely on standards,it can directly detect the copy number in the sample,and achieve absolute quantification in the true sense.In this part of the study,in order to establish droplet digital PCR(ddPCR)as a key indicator,the precise detection of SIV DNA load in cells and tissues of AIDS animal models can be used for real-time dynamic assessment of hidden virus in the virus reservoirs.According to the mature qPCR method,the reaction conditions of droplet dPCR(ddPCR)were optimized to determine the optimal annealing temperature.The detection range of ddPCR was determined by testing the 10-fold serial dilution of pGEM-SIVgag477 plasmid standard.The stability of the detection method was judged by multiple tests on several DNA samples.The results show that the annealing temperature of ddPCR is 60℃;the detection range is 0.3～3.96 ×104 copies/μL;the droplet dPCR has a good correlation with qPCR,with a correlation coefficient of r=0.9981.That is,this study established an absolute quantification method of SIV viral DNA based on droplet dPCR,which can effectively perform dynamic absolute quantification of viral DNA load in virus repository samples in cells and tissues,greatly improving the accuracy of model application evaluation degree.