Kaposi's Sarcoma-associated Herpesvirus (KSHV) Infection And Molecular Epidemiology Research In China, Evaluation And Screening Of Anti-KSHV Drugs In Vitro

ZHU biao,CHEN yagang,WU nanping et al.The institute of infectious disease,the 1^staffiliated hospital of medical collage,Zhejiang University,Hangzhou China 310006Kaposi's sarcoma-associated herpesvirus(KSHV),also known as human herpesvirus 8(HHV-8), was first identified in 1994 by Columbian pathologist Chang et al.They identified it in biopsy tissue from an Kaposi's sarcoma patient by using representational different analysis.There were many important advancements in the pathology and epidemiology and molecular biology pathogenesis of KSHV infection.It found out that KSHV is etiologically linked to Kaposi's sarcoma(KS);multicentric Castleman disease(MCD),and primary effusion lymphoma.(PEL),and immunodeficiency subjects are easy to get the three kinds of disease.KSHV infection has highly variablity from different nationality and geography.The epidemiological survey,in particular,the strain features of KSHV infection in China are very limited.The report about KSHV infection state and it's epidemic strains in immunodeficiency subjects of China are more rare.Many drugs could inhibit the KSHV lytic replication,but there are no drug acting on latent KSHV infection.This research consists of four parts,we study KSHV infection and features in different groups of Chinese people,especially those immunodeficiency patients and the epidemic strains,we also explore the drugs which could control or eradicate latent KSHV.Part A The expression and purification of KSHV ORF65 recombinant proteinAccording to Genbank GI:87196820 sequence,we syntheses 258 bp fragment of KSHV ORF65, corresponding to 86 aa-170aa amino acid.The fragment insert into EcoRV site of PUC57 plasmid. Digested with restriction endonuclease EcoRⅠand HindⅢand recombinant it into PET44a prokaryotic expression plasmid.Transform DE3 Escherichia coli.,express recombinant protein and commissioned Pharmacia CO.purificated recombinant protein.We got 0.7ug/ul purity KSHV ORF65 recombinant protein.Western blot confirmed that this recombinant protein can recongnized the serum of KSHV infection patients.The sensitivity and specificity meet the test requirements.The molecular weight of KSHV ORF65 recombinant protein is about 75 KD.This recombinant protein provides an effective method for KSHV serological study in ChinaPartB Establishment of KSHV ELISA and KSHV infection state and characteristics of portion population in ChinaRecombination protein KSHV ORF65 were diluted in carbonate buffer(pH 9.6)to a final concentration of 1μg/ml;100μl of the solution was incubated in each well of ELISA plates.The detection plasma and positive and negtive control sera were diluted 1:100 with PBS-T,100μl of dilution samples were added to each well of ELISA plates.Horseradish peroxidase-conjugate goat anti-human immunoglobulin G was second antibody.The color reaction was developed with tetramethylbenzidine.Plates were read at 450 nm with an ELISA plate reader.The cutoff point was 3-fold STD(Standard Deviation)added average OD450 of KSHV negative control.We design the nested PCR primers and Taq man probe in the region of KSHV ORF26,perform nested PCR and real-time PCR to detect KSHV-DNA in different population of China.Including the blood spread high-risk population:intravenous drug users,renal transplant recipients,primarily blood borne hepatitis patients;high-risk groups sexually transmitted through;non-intravenous drug users, organ transplant recipients(renal transplantation,liver transplantation);HIV infection (antiretroviral treatment,without antiretroviral treatment);general population.We further analyzed KSHV infection characteristics in chronic hepatitis B patients and the interference relationship between HBV/KSHV replication and treatment-related factors.Results suggest that KSHV infection exists in general population in China.The KSHV infection rates is relatively high in intraveneous drug users,blood borne hepatitis patients and renal transplant recipients.Blood transmission is possibly the main route of KSHV infection in China. KSHV infection and active KSHV replication is higher in the immunodeficiency patients;The tendency for KSHV infection increased with age in the different age groups of chronic hepatitis B patients.KSHV infection may promote HBV replication;licorice preparations in the body possibly suppress the replication of KSHV.PartC The endemic strains and cladograms of KSHV from Zhejiang immunodeficient patients in ChinaBased on KSHV ORF26 gene subtype analysis,the KSHV ORF26 was amplified using nested PCR.Purified PCR products were directly sequenced bilaterally using a BigDye sequencing kit and an ABI 377 automatic sequencer.The partial KSHV ORF26 sequences were obtained and aligned using Contig Express sequence splicing software.Gene phylogeny was analyzed using the Clustal 1.81 software program and compared with 10 KSHV DNA sequences deposited in GenBank(AY043000,AY042999,AY219458,AY219457,AY707887,AY707886,AY735096, AY735095,AY736237,AY736236).The phylogenetic tree was obtained using TreeView software. We obtained 13 sequences after assembling DNA sequence contigs.Gene diversity and cladograms of the 13 sequences were compared with ten internationally predominant strains.The KSHV ORF26 sequence diversity and cladogram analyses suggested that KSHV strains from the Zhejiang province of China were closest to the Hungary AY707887 strain and belonged to the A3 subtype.Numbers of GenBank are EU026382;EU089809-EU089818.These are the first time that China upload the data of KSHV subtypes.Endemic KSHV A3 subtype may provide a possible explanation for the low incidence of KS in immunodeficient subjects and HIV/AIDS patients from this area in China.Part D Evaluation and screening of anti-KSHV drugs in vitroThe representation of current clinical anti-herpesvirus chemical drugs is the nucleoside analogues, which inhibits herpesvirus replication by inhibition of DNA polymerase.By far,an effective drug for anti-latent KSHV infection is lack.In my Part B research,the clinical case studies suggest that licorice preparations may be have the role of anti-KSHV in the body.In this Part,We emplyed BC-3 cells for cell model and used real-time fluorescence quantitative PCR of KSHV-DNA for core technology.TPA(phorbol ester)stimulate BC-3 cell to promote latent KSHV enter into lytic replication to produce KSHV virion.The nucleoside analogues GCV(ganciciovir)for the control, the KSHV inhibition of glycyrrhizic acid,Allicin and EGCG(epigallocatechin gallate)were evaluated.The results suggest that GA and ALLICIN could inhibit circular KSHV DNA and linar KSHV DNA in vitro.GCV and EGCG only inhibited KSHV viral particles DNA and no obvious effect on circular KSHV DNA.The results show that this part,we establish a drug evaluation system can be successfully used in the anti-KSHV drugs screening in vitro.Glycyrrhizin and garlic may be have a role of anti-latent KSHV infection in-vitro.