Analysis Of A Strain Of Virus HTLV-1Whole Genome Sequencing

Background and purposeHuman T-cell lymphotropic virus (HTLV) is the first retrovirus which was found.It has two subtypes, HTLV-1and HTLV-2. Gene homology of them is as high as70%. HTLV-2infection is mainly reported in eskimos. HTLV-1is a kind of exogenous retrovirus which can continue to infect humans. It infects10-20million people worldwide with endemic regions in Japan, equitorial Africa, the Caribbean and South America. Southeast coastal areas, such as Guangzhou, fujian and other places there is also a minor popular area in China. The virus is transmitted from mother to child, between sexual partners, by needle sharing and in contaminated blood products.Once infect people,the virus can exists for a long time in the humsn body, the incubation period can be as long as20years and can cause fatal or disabling diseases such as leukemia or paralysis of lower limb after many years. So far, effective treatments and vaccine of virus prevention has not been developed.As in the southeast coastal areas of China there are a certain number of infected people and the migration of people along with the development of the economy and characteristics of the spread of HTLV-1and the lack of screening programmes in transfusion of blood. Thus HTLV-1may cause more infections in traditional non-endemic domestic areas. In order to explore molecular features of HTLV-1virus infection gene in China’s traditional non-endemic areas, We do the whole genome sequencing and analysis to a strain of virus HTLV-1whole genome on the basis of large-scale investigation in Hubei, Henan.Methods1. ELISA detection of the sample:Take100healthy human and6serum samples of positive HTLV Antibody testing, using Double antigen sandwich enzyme-linked immunoassay to detection and reinspection.2. WB identification:6serum samples of positive HTLV Antibody testing, using WB identification reagents (MP Diagnostics (MPD) HTLV BLOT2.4of MP Biomedicals Asia Pacific Pte. Ltd co. Singapore)3. Fragment amplification cloning and sequencing of HTLV-1pro virus DNA: Extract the genome contains the proviral DNA from HTLV-1positive lymphocytes.using9pairs of primers of HTLV-1segmented amplification for PCR amplification respectively, getting a nine overlapping HTLV-1pleces, each pleces connected and recombination with T carrier. Identificate of restructuring using PCR amplification and NcoI and SalI double enzyme. Using sanger double deoxidizing termination method to identify the two-way (positive and negative) sequencing. Using sequencher software version3.1for DNA sequence Mosaic, getting a total length of HTLV-1proviral genome DNA sequence.4. The isolates HTLV-1gene and protein evolutionary tree analysis: Results1. Double antigen sandwich enzyme-linked immunoassay tests showed that100healthy serum specimens were all negative;5of6positive samples of serum in initial check.1is at the critical value.2. Using MP Diagnostics (MPD) HTLV BLOT2.4of MP Biomedicals Asia Pacific Pte. Ltd co. Singapore to confirm the inspection,getting1case of HTLV-1specimen. 3. HTLV provirus DNA fragment amplification results:Segmented amplification of HTLV-1, getting1150bp of the first fragment,1149bp of the second fragment,1087bp of the third fragment,1045bp of the fifth fragment,1050bp of the sixth fragment,1136bp of the seventh fragment,1148bp of the eighth fragment and960bp of the ninth fragment,each fragment is consistent with the predict.4. PCR amplification identification results showed that9fragments were all inserted into the positive clone.5.9recombinant clone can be cut by Ncol and SalI double enzyme, fragment length of the insertion sequences is also consistent with the design.6. Total length of HTLV-1provirus genome DNA sequence (Numbers for KC807984in GenBank database), total length of9034bp. Of the total length,"A",2088, take up23.11%,"C",3159,take up34.96%,"G",1707, take up18.89%,’T",2080, take up23.02%. Among them,1-756nt for the5’LTR area;803-2092nt for gag protein coding regions;2499-5188nt for pol protein coding regions;5125-7128nt for rex protein coding regions;5181-8360nt for tax protein coding regions;5181-6647nt for env protein coding regions;8279-9304nt for3’LTR area.7. HTLV-1isolates KC807984genome evolution analysis results show that it still belongs to the group A, closest relative is the chongqing isolates AF259264.Their genetic sequence homology is as high as99.72%; take second place homology with Japan isolates (U19949, J02029, AB513134); And Solomon islands isolates farest relatives, to serve central isolates.ConclusionHTLV-1isolates KC807984genome-wide9034bp, Gene subtypes belonging to group A, with a highly homologous with China isolates and Japan isolates.