| BackgroundThe genus Flavivirus is a group of RNA virus transmitted by mosquitoes.As the southern part of China is located in the tropical and subtropical regions,the annual average temperature is high and the climate is hot and humid.It is especially suitable for the propagation of various arbovirus pathogens and becomes the natural epidemic area and place of acute arbovirus infectious diseases,especially In Guangdong Province,the flow of inbound and outbound people is high,so the risk of local input and popularity is extremely high.ZIKV is a member of the Flavivirus family and causes only mild symptoms after ZIKV infection,so it has received little attention since its first separation in 1947 and before 2007.After the first outbreak of the Pacific Island in 2007,The prevalence of ZIKV has been reported intermittently in the subtropical region.With the outbreak of the epidemic,the incidence of neurological complications is increasing.In the outbreak of South America and Central America at the end of 2015,Brazil reported thousands of cases of suspected neonatal small head disease.As of the end of 2017,more than 80 countries and regions reported evidence of mosquito-borne ZIKV infection,and there are risks of further expansion and cross-border.In China,in February 2016,after the first case of Zika was reported,as of May 2017,there were25 cases of ZIKV infection,and the provinces imported into China were mainly in Guangdong Province,with 15 cases imported,accounting for 60.0%of the total number of imported cases.With the expansion of the epidemic and the characteristics of its long-term serious impact on the nervous system,With the expansion of the epidemic and its characteristics of invading the nervous system leading to long-term adverse effects,China as a potential natural epidemic area should reserve appropriate treatment and response measures,especially early rapid diagnosis that can distinguish other flaviviruses.In the early diagnosis,we can refer to dengue virus,one of the important members of the genus Flavivirus.Because there are many natural foci in the south of China,the diagnostic method of DENV is more mature than ZIKV.In addition to nucleic acid diagnosis,rapid diagnosis of DENV immunochromatography has become the first choice in clinical screening,including rapid detection of IgM/IgG and NS1 gold immunochromatography assay(GICA).In particular,the viral NS1antigen,which is secreted as a soluble hexamer from DENV-infected cells,circulates in the bloodstream of infected patients.NS1 is detectable simultaneously in the viremia phase and is detectable after the viremia phase,providing a longer diagnostic window than viral RNA and is therefore often used as one of the diagnostic targets.Fast and accurate diagnosis is critical to rapid clinical management at the outbreak.Therefore,whether in ZIKV or DENV,joint early rapid detection,multi-strategy diagnosis to control the epidemic is undoubtedly a gain.Part 1 Establishment of early rapid antigen detection method for Zika virus infection ObjectiveTo establish a colloid gold-immunochromatography assay(GICA)for detecting of ZIKV NS1 antigen for early diagnosis of ZIKV infection.MethodsThe 5 monoclonal antibodies obtained from human anti-ZIKV NS1 were subjected to antibody kinetic assay and competitive inhibition experiments by biofilm layer optical interferometry,and combined with pairwise random combination pairing results to screen out the best capture antibody andcoated antibody.The selected paired antibodies were finally prepared into colloidal gold test papers.The specificity and sensitivity were examined by recombinant NS1 protein,infected virus cell culture supernatant,and serum/plasma infected with DENV patients.Among them,the virus was quantified by the Focus-Forming Assay(FFA)method.Results1.Pairing of antibodies and preparation of NS1 antigen colloidal gold test paperIn the kinetic assay,5 antibodies showed high affinity for ZIKV NS1.Through the competition inhibition experiments,the antigen binding sites of ZKns3G2 and ZKns4B8 antibodies were similarly classified into one class,and the antigen binding sites of the other three antibodies ZKns4F10,ZKns14G5,and ZKns2E11 were similarly classified into another class.Through the principle of random pairing,ZKns2E11 was used as the coating antibody,and ZKns3G2 labeled with colloidal gold was used as the detection antibody.Finally,the colloidal gold test paper was successfully constructed.2.Sensitivity and specificity identification of colloidal gold test paperIn the sensitivity analysis of the test paper,the NS1 protein released from the cells infected with the ZIKV virus amount of 2×10~4 FFU/ml and 31.25ng/ml recombinant NS1 protein were detected,and the test strip was able to read the positive result within the effective time.The test paper has good specificity,whether it is detecting recombinant protein,infected virus cell culture supernatant or clinical sample,and does not cross with DENV.Conclusions1.The 5 antibodies isolated from the recovery period of infected ZIKV patients have strong binding activity to ZIKV NS1 protein;2.Antibodies can be divided into two types according to binding sites,one is ZKns3G2 and ZKns4B8,and the other is ZKns4F10,ZKns2E11,ZKns14G5;3.The test strip prepared by using ZKns2E11 as the coated antibody and ZKns3G2labeled colloidal gold as the capture antibody has good sensitivity and can detect31.25ng/ml ZIKV recombinant NS1 protein,and does not cross with DENV,showing good specificity.It can be used as a supplement to clinical nucleic acid diagnosis of Zika virus disease,providing a technical reserve for ZIKV and DENV co-infected areas.Part 2 Influence of host immune status on the diagnosis of NS1 colloidal gold ObjectiveTo analyze the effects of viral antigenemia,host immune status and type of infectious virus strain on early diagnosis in patients with dengue virus infection clinically occurring in July and August 2018.MethodsA simple analysis was performed on the laboratory data of 15 patients who were hospitalized in Guangzhou Eighth People's Hospital in July and August 2018 and confirmed DENV-2 infection by RT-PCR.Serum/plasma samples were collected from 7 patients during hospitalization(serum/plasma was collected from misdiagnosed patient on day 5,6,and 32 of the disease onset),and NS1,IgM/IgG ELISA tests were performed to analyze the NS1 antigen and antibody secretion levels in the patient.The above samples were subjected to viral RNA extraction,amplified and constructed plasmids to obtain the full-length sequences of E and NS1.Through amino acid sequence alignment and the drawing of evolutionary tree,gene mutation sites and genotyping of infected virus strains were obtained,so as to further analyze its influence on early diagnosis.Results1.Collection and organization of clinical dataIn the 15 laboratory data collected,11 of the 12 individuals with rapid NS1 testing showed weak positive or positive,which was consistent with their RT-PCR results.As for IgM/IgG rapid detection,most cases(8/10)were negative by day 6after onset.2.NS1 and IgM/IgG ELISA assay OD values of serum/plasma samples at day 5 and day 6 in Misdiagnosed case were0.21 and 0.32 respectively in the NS1 ELISA method(critical value=0.16).and were negative in both IgM and IgG ELISA assays.Only on day 32,plasma samples were positive in the IgM ELISA assay,while in the IgG assay,plasma collected on days 5,6,and 32 were negative.Among the plasma samples collected by other patients,5 patients in the IgM ELISA test were positive but only one was weakly positive in the rapid test.In the IgG ELISA test,two patients had positive serum/plasma samples and negative rapid tests.3.E,NS1 gene sequence acquisition and analysisThe E gene sequence was successfully amplified from 5 samples,and the NS1 gene sequence amplified from 4 samples,and the above sequence was uploaded to the Genbank database.In the alignment of the E and NS1 sequence,the amino acid sequence of the E and NS1 protein were very similar between the virus strains isolated in 2018,with only a few amino acid differences.Among the obtained amino acid sequences of E and NS1 protein,there were samples that were completely consistent with pt3.The obtained strains are all DENV-2 global genotype,and the obtained strains in the molecular evolution tree(based on the E gene)can be divided into three branches,The strains obtained in the molecular evolutionary tree can be divided into three branches,of which the denv-2 virus strain isolated from pt3belongs to a branch that is similar to the epidemic strain in India in 2013(MH822956),and the other two branches are respectively closely related to the epidemic strain in Kenya in 2017(MG779202)and the epidemic strain in Malaysia in 2013(KJ806783).In the molecular phylogenetic tree based on the NS1 gene,it can be divided into two branches according to the obtained sequence,one of which branches(from pt3,pt2,and pt6,respectively)is similar to India in 2013(MH822956),and the other branch is similar to Indonesia in 2008(KC762671).Conclusions1.The immune status of the host can affect the sensitivity of rapid detection of the antigen;2.Analysis of E protein sequence showed that there were two strains of DENV-2 in2018,all of which were cosmopolitan,and the cases in this study were all imported cases,mainly from Southeast Asia;3.The low NS1 antigenemia in DENV-2 infected patients in this study can be related to the host,suggesting that multiple strategies should be adopted for highly suspected cases to assist in diagnosis. |
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