Construction And Characterization Of A New SHIV Clone Carrying An Env Gene Derived From A CRF07_BC Strain

Human immunodeficiency virus(HIV) infection and AIDS continue to be a growing problem for the world's population.The need to develop a safe and efficacious vaccine against HIV is more pressing than ever.Human immunodeficiency virus(HIV) and simian immunodeficiency virus(SIV) are classified to Lentivirus of Retroviridae.The genome of HIV and SIV is highly homologous and most closely in Lentivirus group.SHIV,SIV/HIV chimeric virus,was constructed by replacing the corresponding region of SIV and HIV.A pathogenic R5 simian-human immunodeficiency virus(SHIV) encoding gene of the predominant prevalent HIV-1 B'/C Recombinant(CRF07_BC) strain in China was highly desirable to study the role of HIV-1 envelopes in transmission and pathogenesis as well as to evaluate candidate AIDS vaccines in nonhuman primates. SHIV-KB9,the clone of SHIV-89.6P was used as starting backbone for the construction of Chinese recombinant B'/C SHIV.The 2.1kb KpnI-BamHI fragment encoding the Chinese HIV envelope gene(covering N-terminal 50 to 780 amino acids) amplified from different HIV infections genomic DNA was replaced with the corresponding region of SHIV-KB9 individually.76 full-length SHIV clones were constructed and their infectious activity was tested in TZM,macaque and human PBMC.Among all full-length proviral clones derived from 7 different HIV-1 samples,only four clones were found to be infectious,they are SHIV-XJN0363A4,SHIV-XJN0363B8,SHIV-XJN0421A17 and SHIV-XJDC6431.One clone SHIV-XJDC6431 was chosed to be the most infectious activity due to dected infectivity in vitro,and was used to check the infectious charactization in vitro/in vivo.The virus particle of SHIV-XJDC6431 was packed in transfected 293T cells and could be observed under transmitted electron micrograph.The coreeeptor usage of SHIV-XJDC6431 was tested in GHOST cell lines,which showed that was CCR5-specific SHIVs.The infectivity of SHIV-XJDC6431 in human and rhesus monkey's PBMC were verified by DNA polymerase chain reaction(PCR),reverse transcriptase PCR(RT-PCR) and Simian immunodeficiency virus(SIV) p27 gag antigen titration assay.Also the distinct cytopathogenicity of rhesus monkey PBMC infected could be observed under optical microscope.To this end,SHIV-XJDC6431 was adapted by two passages in three Chinese-origin Rhesus Macaques,separately.Up to now,SHIV-XJDC6431 could replicate effectively in first passage 443,444 and second passage 445.It was revealed that the infectivity property of SHIV-XJDC6431 in first passage 443 and 444 was low replicated by measuring viral load and CD4/CD8 ratio analysis. Compared with SHIV-KB9 and SHIV-CN97001,the peak of viral load was low and the time point of deteced plasma viremia was delayed in two macaques post SHIV-XJDC6431 infection.But,the two animals infected had inclined setpoint viral load.After that,444 was passaged in vivo at day 65 post SHW-XJDC6431 infection. It was founded that the infectivity property of SHW-XJDC6431 enhanced during in vivo passage,the viral RNA load of second passage was higher.In conclusion,we constructed four R5-tropic and CRF07_BC SHIV strains which could establish persistent infection in human and macaque lymphocyte cells in vitro,and one of them SHIV-XJDC6431 could establish infection also in Chinese rhesus macaque in vivo.In addition,we observed that SHIV-XJDC6431 was capable of replicating more efficiently in macaque through animal passage,and hope for gaining more replication capacity and pathogenicity through continuous passaging in the rhesus macaque passage,which will entitle this strain as a useful tool in AIDS/HIV pathogenic and vaccine research.