| SHIV-89.6 is a nonpathogenic chimeric simian-human immunodeficiency virus (SHIV) that contains both SIVmac239 genome backbone and type B HIV-1 env gene of a cytopathic primary patient isolate 89.6 of HIV-1. SHIV-89.6P is a pathogenic SHIV variant derived from SHIV-89.6. And its molecular clone, KB9, hereafter termed SHIV-KB9. As a successful chimeric SHIV, SHIV-KB9 has been applied in AIDS research overseas. Because of the high similarity between the B subtype and the B' subtype that is a main popular subtype in our country at present. The SHIV-KB9 was introduced for assessing efficacy of candidate AIDS vaccines, which is targeting specific Chinese major viral strains. To titrate viral concentration of SHIV-KB9 to infect Chinese rhesus macaques, and establish a SHIV/Chinese rhesus macaques' model, we performed to titrate the SHIV-KB9 in Chinese rhesus monkeys.In total of ten rhesus monkeys free from BV, SIV, SRV and STLV viruses' infection were used for the 2 batches experiments in vivo. The first 6 monkeys in batch 1 were inoculated i. v. with 1ml of SHIV-KB9 virus stock solution at concentration of 10-0, 10-1, 10-2, 10-3, 10-4, 10-5 dilution, respectively. To test whether the first 6 animals were infected or not with the SHIV virus stock, blood samples were collected at different time points according to the time schedule. Proviral DNA from PBMCs and RNA from EDTA anti-coagulated plasma were extracted and detected with nested PCR and RT-PCR respectively, and also virus isolation by co-culture the PBMCs from normal monkeys with infected monkeys and CD4+/CD8+ ratio test were performed. The results strongly supported that the three from six rhesus monkeys were infected. On a basis of first batch of experiment, we have carried out second batch of experiment; the 4 monkeys in batch 2 were inoculated i. v. with lml of SHIV-KB9 virus stock solution at concentration of 10-1, 10-2, 10-3 and 10-4 dilution, respectively. Results of experiment revealed that the SHIV-KB9 is a highly pathogenicity strain, it could infect the Chinese-origin rhesus monkeys. The monkey that was infected even with titer of 4.8×105 copies/ml SHIV-KB9 appears to be high viremia. A419 (4.8×106) and A425 (4.8×105) appeared AIDS symptom. SHIV-KB9 successfully infected the Chinese-original rhesus monkeys and could be possibly used in the evaluation of the candidate AIDS vaccines.The cell mediated immunity is the critical for animal infection with viruses, especially in vaccine assessment. To measure much more accurately, the IFN-γ, ELISPOT to assess the cell mediated immunity that induce by virus or HIV vaccine were developed.In the study, Four Chinese-origin rhesus macaques were used. First, all the monkeys were screened by serological test to ensure that they were free of BV, SIV, SRV, and STLV viruses. And then they were infected with 100 MID50 SIVmac239. We analyzed results of Chinese-origin rhesus macaques by FACS, DNA-PCR, RT-PCR and ELISPOT detections.The result revealed the IFN-γELISPOT method could effectively test experiment monkey's cellular immunity situation, be clear about the basic tendency and the scope of CTL of Chinese rhesus monkey infect with SIVmac239. Our finding provided the new basic set point for SIV/Chinese-origin rhesus macaques' model, and it has provided the foundation condition for used this model appraisal medicine or vaccine.In conclusion, the research demonstrated that Chinese rhesus monkeys could be infected successfully with challenges of SHIV-KB9 virus, and the effective concentration, virus and immunology measurements were determinated. The successfully created SHIV-KB9/SAIDS rhesus monkey model could be possibly used in evaluation of HIV vaccines in China.IFN-γELISPOT technique has been tested to be sensitive and specific in cellular immunity studies of rhesus macaques infected with SIV in this study, and the IFN-γELISPOT test will play an important role in present available SIV/SHIV/SAIDS rhesus monkey models for the AIDS pathogenesy, infection immunity studies, and also the medicine screening and vaccine evaluating. |
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