| Various chimeric SHIVs and their animal models have been widely used in AIDS research. However, up to now, there is no SHIV encoding genes of the predominant prevalent HIV-1 B'/C subtype strain in China and could cause AIDS-like symptoms in Chinese-original rhesus macaques. SHIV-XJ02170 constructed by Nankai University was generated in the background of highly pathogenic B subtype SHIV-KB9 gene, carrying a CRF07_BC HIV-1 env gene. It has been passaged in serial Chinese-original rhesus macaques, only in the second passaging animal A408 showed high viral loads, and isolates from A408 could be infectious to next monkeys. According to the sequence analysis of env gene, significant increase in the genetic distance during serial passaging, and env gene evolved forward along passaging.Based on all the previous research on SHIV-XJ02170, this study steps further to passage the mutated virus in serial Chinese-original rhesus macaques, which has already adapted in A408(numbered G0401V in this research) for a long time, and to passage after CD8+T cells deletion in vivo. According to the characteristic changes of SHIV-XJ02170 during serial passages in animals in late infection stage as well as sequence variation of env gene, the next step work is to construct infectious full-length clones and analyze their infectity. Our goal is to obtain a strong infectious SHIV virus that carries the gene of the predominant prevalent HIV-1 B'/C subtype strain in China, with high pathogenicity after infecting Chinese-original rhesus macaques to cause AIDS-like symptoms, and to explore the mechanism of enhanced virulence on molecular level.Viral bloods from macaque G0401V infected with SHIV-XJ02170 in late infection stage of 5 years were used for passaging 2 monkeys (G0401V→G0402V→G0403V) without CD8+T deletion and 1 monkey (G0401V→G0404V) with CD8+T deletion. Flow cytometry, viral load detecting and sequence alignment were used to analyze the change features during serial passages. The results suggested that SHIV-XJ02170 incubated in G0401V has showed stable infectivity during late infection for 5 years and its virulence was enhanced after passaging in vivo, especially after CD8+T cell deletion by injection of antibody CM-T807. G0402V showed a sharp decline of weight as well as CD4+T cell counts in peripheral blood, and dead suddenly on day 41 after infection. Monkeys G0403 and G0404 showed a viral load perk on day 14, then maintained low viral load level (104-105copies/mL) up to now after passaging (141d). Sequence alignment analysis indicated significant variation in env region and changes of N-glycosites in gp120 region.In order to have a further understanding about the influence on virulence caused by sequence variation, in the meantime to obtain the mutated virus with enhanced virulence, infectious clone construction and other following research was taken. First of all, pSHIV-KB9-pCI full-length clone was generated by inserting the whole genome of SHIV-KB9 into pCI-Sph I vector with the usage as clone backbone, and pSHIV-KB9-Eco47Ⅲ-3'half-length clone containing 3'half part of SHIV-KB9 genome was constructed for env gene replacing. Then,2.2kb DNA fragments belonging to env region were collected from samples of G0402V 13d and G0404V 7d, which represented different directions of sequence variation, and were used to replace the corresponding part of SHIV-KB9 genome. Finally 4 infectious full-length clones named pSHIV-TC containing various sequences were acquired. All the clones were transfected in 293T cells and the supernatant was collected to infect TZM-bl cells for the purpose of infectity analysis in vitro. The result indicated that SHIV-TC21 derived from G0402V had the strongest infectiveness, and on the contrary, SHIV-TC22 showed no biological activity. Infectity of both SHIV-TC41 and SHIV-TC42 was not as strong as SHIV-TC21.Because of its strongest infectity to cells compared with other pSHIV-TC clones, SHIV-TC21 was chosen for the next research on animal level. SHIV-TC21 could infect G0405V successfully and obtained enhanced virulence by passaging to G0406V. There were some obvious symptoms, such as a high level of viral replication during acute stage and detectable for several days, a severe reverse of CD4+/CD8+ratio near 0.2 from 52d post infection until now, with chronic diarrhea. Compared to SHIV-XJ02170, SHIV-TC21 has much more powerful virulence to animals.In conclusion, by the analysis of characteristic changes of SHIV-XJ02170 during serial passages in animals in late infection stage as well as sequence variation of env gene, we got a preliminary comprehension of the mechanism of enhanced virulence on molecular level; 4 SHIV-TC full-length clones with different env gene sequence were constructed and analyzed with their infectities on cell level; eventually we obtained an infectious full-length clone SHIV-TC21 with CCR5-specific tropic and relatively strong infectivity, which carries a CRF07_BC HIV-1 env gene. The animal model established by SHIV-TC21 infection was anticipated to become a useful tool in AIDS/HIV pathogenic and vaccine evaluation.
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