Nef genes of SIV

Molecular clones of SIVmac were constructed that differed only in sequences within the nef gene. DEAE-transfection of viral DNA containing an open from of nef yielded virus that replicated with similar kinetics and to a similar extent in macaque peripheral blood lymphocyte (PBL) cultures as virus with a deletion or stop codon within nef. Rhesus monkeys that received each kind of molecularly cloned virus became infected. Our results additionally suggest that mutant forms of virus are selected in vitro while open, functional forms are selected in vivo. In animals infected with virus containing a stop codon within nef, reversion of the stop codon to a coding codon was demonstrated in five of five clones analyzed. These results indicate that nef is playing some role crucial to the virus life cycle in vivo.     

Early helper T-cell dysfunction in simian immunodeficiency virus but not in human immunodeficiency virus type-2-infected macaques

Both naive and vaccinated macaques acquired a virus-specific proliferative helper T-cell reactivity in response to infection with the nonpathogenic human immunodeficiency virus type 2 (HIV-2). In contrast, macaques infected with the pathogenic simian immunodeficiency virus of the macaque strain (SIV_mac) did not develop a helper T-cell response. Furthermore, a vaccine-induced preexisting T-cell reactivity was abrogated after SIV_mac infection in vaccine failures. These differences may reflect the different pathogenicity of the two closely related viruses.     

Detection of simian immunodeficiency virus DNA in macrophages from infected rhesus macaques.

We have examined the frequency of infection of monocyte-derived and alveolar macrophages isolated from rhesus macaques inoculated with simian immunodeficiency virus (SIVmac) utilizing a semiquantitative PCR methodology. Animals were inoculated with either pathogenic (SIVmac239) or nonpathogenic (SIVmac1A11) molecularly cloned viruses of SIVmac, or with uncloned pathogenic SIVmacBIOL. The frequency of SIV DNA in macrophages was highest early after infection and at terminal stages of disease, whereas during the asymptomatic period, SIV DNA was present at very low levels in macrophages.     

Immunological responses and viral modulatory effects of vaccination with recombinant modified vaccinia virus Ankara (rMVA) expressing structural and regulatory transgenes of simian immunodeficiency virus (SIVmac32H/J5M)

Background The immunogenicity and protective efficacy of recombinant modified vaccinia virus Ankara (rMVA) vectors expressing structural (gag/pol, env) and regulatory (tat, rev, nef) genes of SIVmac251/32H‐J5 (rMVA‐J5) were assessed. Methods Immunization with rMVA constructs (2.5 × 10⁷ IU) 32, 20 and 8 weeks pre‐challenge was compared with 32 and 20 weeks but with a final boost 8 weeks pre‐challenge with 2 × 10⁶ fixed‐inactivated HSC‐F4 cells infected with SIVmac32H. Controls received rMVA vectors expressing an irrelevant transgene or were naïve challenge controls. All received 10 MID₅₀ SIVmac32H/J5 intravenously. Results Vaccinates immunized with rMVA‐J5 exhibited significant, albeit transient, control of peak primary viraemia despite inconsistent and variable immune responses elicted by vaccination. Humoral and cellular responses to Env were most consistent, with lower responses to Nef, Rev and Tat. Increasing titres of anti‐vaccinia neutralizing antibodies reflected the number and dose of rMVA inoculations. Conclusions Improved combinations of viral vectors are required to elicit appropriate immune responses to control viral replication.     

Thenef gene of SIVmac239 is necessary for efficient growth in H9 cells

AIDS viruses require an intact functionalnef gene in order to inducedisease. The nonpathogenic molecular cloned virus SIVmac239nef-deletion encodes a truncatednef gene. This attenuated reading frame is expressed both in vitro and in a virus-infected animal in vivo. Encoding the first 58 amino acids of Nef, the reading frame retained its ability to down-modulate CD4 from the surface of T cells. CD4-down-modulated stable cell lines expressing full-length and truncatednef genes were significantly less infected by SIV. SIV-mac239nef-open and SIVmacnef-deletion encoding a truncatednef clearly differed in replication kinetics in H9 cells and H9-derived cell lines. SIV-mac239nef-deletion replication was delayed in H9.     

Use of infectious molecular clones of simian immunodeficiency virus for pathogenesis studies

Three infectious molecular clones of SIVmac and one of HIV-2 exhibit remarkable variation in their biological properties despite similarities in genome organization and sequence relatedness. Cloned viruses differed in their ability to grow in various cultured cells, in their ability to infect macaques, and in the location of the env stop codon. Sequences from the 3' end predict that at least three of the four clones do not have an intact, functional nef gene. All four cloned viruses yield infectious virus in HUT-78 and all four cloned viruses are cytopathic.     

猴免疫缺陷病毒（SIV）感染恒河猴血液学和血液生化的动态变化

目的探讨SIVmac239感染中国恒河猴的血液学和血清生物化学的变化,研究艾滋病的发生机制。方法 20只恒河猴感染SIVmac239后分别在感染前和感染后2、6、9、12、15和18个月在猴空腹状态用盐酸氯胺酮麻醉下静脉釆血。抗凝血用全自动血液细胞分析仪检测血液常规,血清用全自动生化分析仪检测生化指标。结果SIV感染猴红白细胞计数呈逐渐的减少。淋巴细胞计数有较明显减/增的波动。血清酶类ALT和AST没有明显的上升,但在15月时均有所下降P〈0.05～P〈0.01。LDH持续降低,原因未明。CK则逐月上升,至12月时达高峰,后又有所下降。血尿素氮逐月上升,并在6～18个月时P〈0.01。总蛋白量上升,白蛋白逐月降低,球蛋白升高,白蛋白/球蛋白比率倒置。SIV感染猴2个月后总胆固醇、甘油三脂、低密度脂蛋白和高密度脂蛋白均呈上升,至12月时略有下降,但到18月又上升。血糖（GLU）在SIV感染后逐月上升,后期上升更为明显,这可能与胰岛的损害有关。结论 SIVmac239感染猴后,通过SIV侵入相关的器官病变和体液调节失衡导致血液学和血液生物化学产生异常,从而提供有关猴艾滋病的一些基础资料,这将对研究人的艾滋病也许有所帮助。     

Molecular changes associated with replication of simian immunodeficiency virus in human cells

The SIVmac239 infectious clone does not have a premature stop codon in its transmembrane protein (TMP) region and it produces full-length (41 kilodalton, kDa) TMP in macaque peripheral blood lymphocytes (PBL) in vitro and in vivo. However, viruses with truncated forms of TMP (28kDa) are selected during propagation in human cell types; truncated forms arise from point mutations, CAG (glutamine) to TAG (stop), in the viral genome. These results document molecular changes associated with adaptation of SIVmac for growth in human cells.     

SIVMAC vaccine studies using whole inactivated virus antigen sequentially depleted of viral proteins

Groups of four rhesus monkeys were immunised at 0, 1, 2, and 13 months with whole inactivated SIV_mac32H, SIV_mac depleted of the outer envelope glycoprotein gp130, virus cores depleted of the lipid membrane (and hence transmembrane glycoproteins), or purified gag protein. These macaques plus controls were challenged with either the homologous SIV_mac251–32H. grown in human cells or the same virus passed once through monkey cells. None of those challenged with monkey-grown virus were protected, whereas all in the whole and gp130-depleted virus groups, and one in the core group resisted challenge with human-grown virus. As the only difference between the challenge viruses was a single in vitro passage in monkey cells it can be concluded that protection was solely due to human cell components. Finally, passive transfer of high titer IgG from monkeys infected with the homologous challenge virus failed to protect monkeys from infection despite the presence of circulating neutralising antibodies.     

两株SIV_(mac)的滴定和特性

SIVmac251的MID100为32TCID,而SIVmac239的MID100高于320TCID。体内滴定感染成功的5只猴（SIVmac2513只,SIVmac2392只）和用ZMID100SIVmac251感染的7只猴感染后有全身淋巴结肿大,并出现规律性的血浆病毒血症和抗体反应。SIVmac251感染的7只恒河猴和2只食蟹猴的淋巴结和脾脏的病理组织学检查,显现规律的SIVmac感染后的组织学变化。上述结果表明两株SIVmac均能诱发SIVmac感染猴的系列表现和变化,可应用于抗艾滋病药物猴体疗效的评价。