Differential effects of simian immunodeficiency virus infection on immune inductive and effector sites in the rectal mucosa of rhesus macaques

The rectal mucosa, a region involved in human immunodeficiency virus/simian immunodeficiency virus (SIV) infection and transmission, contains immune inductive sites, rectal lymphoid nodules (RLN), and effector sites, the lamina propria (LP). This study was designed to evaluate cell populations involved in rectal mucosal immune function in both RLN and LP, by immunocytochemical analysis of rectal mucosa from 11 SIV-infected (2 to 21 months postinfection) and five naive rhesus macaques. In the rectum, as previously observed in other intestinal regions, CD4(+) cells were dramatically reduced in the LP of SIV-infected macaques, but high numbers of CD4(+) cells remained in RLN indicating maintenance of T cell help in inductive sites. Cells expressing the mucosal homing receptor alpha4beta7 were dramatically decreased in the RLN and LP of most SIV-infected macaques. The RLN of both naive and SIV-infected macaques contained high numbers of CD68 + MHC-II+ macrophages and cells expressing the co-stimulatory molecules B7-2 and CD40, as well as IgM + MHCII+ and IgM + CD40+ B cells, indicating maintenance of antigen presentation capacity. The LP of all three macaques SIV-infected for 2 months contained many B7-2+ cells, suggesting increased activation of antigen-presenting cells. LP of SIV-infected rectal mucosa contained increased numbers of IgM+ cells, confirming previous observations in small intestine and colon. The data suggest that antigen-presentation capacity is maintained in inductive sites of SIV-infected rectal mucosa, but immune effector functions may be altered.     

Establishment and characterization of Indian muntjak cell lines transformed with simian virus 40.

Kidney cells of an Indian muntjak were transformed with simian virus 40 (SV40). The transformation efficiency of the tertiary cultures was very high when estimated by the agar suspension culture method. The efficiency was about 0.015% when infected at an input multiplicity of 0.4 p.f.u./cell. Clonal cell lines were established from the colonies in soft agar medium. Most of the cell lines and their subclones produced a small amount of infectious SV40. The SV40 virion antigen-positive cells in a clone increased from 0.2% to about 40% by the treatment with mitomycin C. More than 70% of the cells in two cell lines were normal in G- and C-banded karyotypes, indicating that chromosomal change is not a necessary step in the process of transformation of the Indian muntjak cells with SV40.     

Characterization of a simian human immunodeficiency virus encoding the envelope gene from the CCR5-tropic HIV-1 Ba-L

The tat, rev, vpu, and env genes from the monocytotropic CCR5-dependent HIV-1 Ba-L isolate were substituted for homologous simian immunodeficiency virus (SIV) sequences in the SIV genome. The resultant SHIV (SHIV Ba-L) replicated in CCR5-positive PM-1 cells but not in CCR5-negative CEMX174 cells. Infection of HOS cells expressing different co-receptors showed SHIV Ba-L to be strictly CCR5-dependent. Infection of PM-1 cells and rhesus peripheral blood mononuclear cells (PBMCs) was highly sensitive to RANTES but not to SDF-1. Although SHIV Ba-L infected rhesus and pigtail macaques intravenously or rectally, plasma viremia was controlled after 3 weeks. After serial passage through 4 pigtails by blood and bone marrow transfer, virus from pigtail PBMCs had higher in vitro infectious titers on rhesus PBMCs and was efficiently transmitted vaginally in rhesus and cynomolgus macaques. Plasma viremia generally persisted longer than after infection with unpassaged virus but was eventually controlled with no significant decrease in CD4+ T-cell counts in peripheral blood. The envelope gene of SHIV Ba-L revealed a very little genetic drift during in vivo passage. SHIV Ba-L provides a potentially useful model for R5 HIV-1 infection of humans.     

Monocyte adhesion to endothelium in simian immunodeficiency virus-induced AIDS encephalitis is mediated by vascular cell adhesion molecule-1/alpha 4 beta 1 integrin interactions.

Because the mechanisms associated with recruitment of monocytes to brain in AIDS encephalitis are unknown, we used tissues from rhesus monkeys infected with simian immunodeficiency virus (SIV) to examine the relative contributions of various adhesion pathways in mediating monocyte adhesion to endothelium from encephalitic brain. Using a modified Stamper and Woodruff tissue adhesion assay, we found that the human monocytic cell lines, THP-1 and U937, and the B cell line, Ramos, preferentially bound to brain vessels from monkeys with AIDS encephalitis. Using a combined tissue adhesion/immunohistochemistry approach, these cells only bound to vessels expressing vascular cell adhesion molecule-1 (VCAM-1). Furthermore, pretreatment of tissues with antibodies to VCAM-1 or cell lines with antibodies to VLA-4 (CD49d) inhibited adhesion by more than 70%. Intercellular adhesion molecule-1 (ICAM-1)/beta 2 integrin interactions were not significant in mediating cell adhesion to the vasculature in encephalitic simian brain using a cell line (JY) capable of binding rhesus monkey ICAM-1. In addition, selectin-mediated interactions did not significantly contribute to cell binding to encephalitic brain as there was no immunohistochemical expression of E-selectin and P-selectin in either normal or encephalitic brain, nor was there a demonstrable adhesive effect from L-selectin using L-selectin-transfected 300.19 cells on simian encephalitic brain. These results demonstrate that using the tissue adhesion assay, THP-1, U937, and Ramos cells bind to vessels in brain from animals with AIDS encephalitis using VCAM-1/alpha 4 beta 1 integrin interactions and suggest that VCAM-1 and VLA-4 may be integral for monocyte recruitment to the central nervous system during the development of AIDS encephalitis.     

Foamy virus serotypes 1 and 2 in rhesus monkey tissues

Summary The isolation and serologic identification by cross neutralization tests showed the presence of simian foamy virus serotypes 1 and 2 in primary rhesus monkey brain, spleen, and kidney cell cultures. The frequency of isolation of foamy viruses from brain and spleen cell cultures was 86% and from kidney cell cultures it was 36%. In primary spleen cell cultures 50% of the foamy virus isolates were type 2. Of the 14 animals from which foamy virus was isolated, only 4 demonstrated serum neutralizing antibody to the homologous serotype. The cytopathology of foamy viruses in the primary cell cultures is described.     

Simian foamy virus type 1 (SFV-1) induces apoptosis

Foamy virus infection causes cytopathology in several cell types from different species. The mechanism of cell killing by foamy viruses is not known. In this report, the mechanism of cell death induced by simian foamy virus type 1 (SFV-1) infection was investigated in fibroblast and lymphoid derived cells lines. Infected L-929 (fibroblast) and Raji (B cell) cells showed chromatin condensation, chromatin cleavage into nucleosome oligomers, and ultrastructural changes consistent with apoptosis. These data suggest that SFV-1 induced apoptotic cell death in different cell lines from different species. The degree of apoptotic cell death in both L-929 and Raji cell lines correlated with increased virus replication. Apoptosis, therefore, is one mechanism by which SFV-1 causes cell death.     

Vaccination of rhesus macaques with a vif-deleted simian immunodeficiency virus proviral DNA vaccine

Studies in non-human primates, with simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) have demonstrated that live-attenuated viral vaccines are highly effective; however these vaccine viruses maintain a low level of pathogenicity. Lentivirus attenuation associated with deletion of the viral vif gene carries a significantly reduced risk for pathogenicity, while retaining the potential for virus replication of low magnitude in the host. This report describes a vif-deleted simian immunodeficiency virus (SIV)mac239 provirus that was tested as an attenuated proviral DNA vaccine by inoculation of female rhesus macaques. SIV-specific interferon-gamma enzyme-linked immunospot responses of low magnitude were observed after immunization with plasmid containing the vif-deleted SIV provirus. However, vaccinated animals displayed strong sustained virus-specific T cell proliferative responses and increasing antiviral antibody titers. These immune responses suggested either persistent vaccine plasmid expression or low level replication of vif-deleted SIV in the host. Immunized and unvaccinated macaques received a single high dose vaginal challenge with pathogenic SIVmac251. A transient suppression of challenge virus load and a greater median survival time was observed for vaccinated animals. However, virus loads for vaccinated and unvaccinated macaques were comparable by twenty weeks after challenge and overall survival curves for the two groups were not significantly different. Thus, a vif-deleted SIVmac239 proviral DNA vaccine is immunogenic and capable of inducing a transient suppression of pathogenic challenge virus, despite severe attenuation of the vaccine virus.     

Intracellular state of Epstein-Barr virus DNA in producer cell lines.

The physical state of the Epstein-Barr virus (EBV) DNA in three cell lines which spontaneously produce virus has been characterized. Circular EBV DNA molecules have been found in P3HR-I, B95-8 and M8I cells. The size of the intracellular M8I circular EBV DNA molecules is comparable with the linear virus genome isolated from virus particles but the circular P3HR-I and B95-8 DNA molecules are shorter than the virion DNA . In addition to the circular form, some EBV DNA with physical properties indicative of integrated sequences was found in all three producer cell lines. There was no marked change in the amount of either the circular or integrated forms of EBV DNA when these producer cell lines were grown in the presence of phosphonacetic acid to suppress the spontaneous virus production which occurs in a small percentage of the cells in untreated cultures.     

Factors affecting the growth and titration by immunofluorescence of simian foamy virus

Summary This paper presents some observations concerned with the growth of simian foamy virus and some modifications which should be introduced to the fluorescence assay of foamy virus. The modified procedure is the most sensitive method described for the titration of foamy virus.Examination of the optimal conditions for the growth and titration by fluorescence assay of simian foamy virus showed that the virus was particularly sensitive to changes in virus and cell concentration. At the low cell concentrations employed previously a saturation-type response was obtained with high titre virus and virus adsorption efficiency was decreased as input virus was diluted. Maximum virus production was obtained with high cell concentrations at input multiplicities of 5 and 10. At high multiplicities of infection more than 90 per cent of the cells adsorbed virus but only 45 per cent became infected, this appeared to be related to cell DNA synthesis.With 3 FiguresThis work formed part of a thesis for the degree of Doctor of Philosophy by Dr. Janis Hartley (formerly Janis Samuels).     

Localization of simian immunodeficiency virus in the central nervous system of rhesus monkeys.

Simian immunodeficiency virus (SIV), like the human immunodeficiency virus (HIV), is a lentivirus that is both immunosuppressive and neurovirulent. Rhesus macaques (Macaca mulatta) inoculated with SIV often develop a giant cell encephalitis similar to that seen in humans infected with HIV. The authors examined SIV expression by immunohistochemistry and RNA in situ hybridization in the cerebrum, cerebellum, choroid plexus, and spinal cord from five macaques with and two macaques without giant cell encephalitis. Selected portions of the central nervous system (CNS) also were examined by electron microscopy. Simian immunodeficiency virus was detected in the CNS of all seven monkeys whether or not they had giant cell encephalitis. Both SIV antigen and RNA were present in all levels of the CNS examined. Macrophage/giant cell lesions always contained viral RNA and antigen and were the only sites where viral particles were detected by electron microscopy. However, SIV antigen and RNA also were commonly associated with small vessels, the choroid plexus, and meninges; these were the only locations where virus was detected in animals without giant cell encephalitis. Immunophenotyping showed that the cellular infiltrates consisted primarily of monocyte/macrophages and occasional CD8-positive T cells. Macrophages and T cells also were present in the stroma of the choroid plexus and were intimately associated with vessels in the CNS of SIV-infected but not uninfected macaques. Simian immunodeficiency virus infection of the macaque CNS provides an excellent model for studying the pathogenesis, treatment, and prevention of HIV-1-encephalitis.     

Threat to Humans from Virus Infections of Non-human Primates

Several hundred distinct non human primate species are recognised, and they are likely to harbour a similar range of viruses to humans. Simians such as cynomolgus and rhesus macaques, African green monkeys, and marmosets are widely used for biomedical research, but despite this extensive close contact very few simian viruses have been shown to pose a threat of infection or illness to humans. Herpesvirus Simiae is the best recognised zoonotic hazard of simians. It is an alphaherpes virus of Asiatic macaques, which causes a mild or subclinical primary infection followed by latency in its natural host. It can be acquired by humans following a bite and causes an ascending meningoencephalitis. Less than 40 human cases have been described and the mortality rate in untreated human infections is 70%. The infection is treatable with acyclovir and extensive guidelines for managing simians and potential exposures have been developed. Ebola virus and Marburg virus have caused epizootics in cynomolgus macaques and vervet monkeys respectively, which have resulted in human infection and fatalities. However, non human primates are unlikely to be their natural host. More recently simian immunodeficiency virus and simian foamy virus have infected researchers, but infection has not been linked to illness. Simian viruses also pose a direct threat to humans through the use of primary monkey tissue cultures in laboratory work and vaccine manufacture, indeed a significant exposure of the human population occurred when cells contaminated with SV40 a polyomavirus of rhesus monkeys were used for polio vaccine production. New medical interventions such as xenotransplantation using primate organs pose a potential risk which requires careful assessment. © 1997 by John Wiley & Sons Ltd.     

云南苗族Bardet-Biedl综合征家系外周血B淋巴母细胞系的建立

目的 建立云南苗族巴德-毕氏综合征(Bardet-Biedl syndrome,BBS)家系外周血B淋巴母细胞系,为研究BBS的发病机制、诊断和治疗提供长期的标本来源.方法 取1个BBS核心家系成员新鲜肝素抗凝全血,采用EB病毒转化技术,并加入环孢霉素A(cyclosporine A,CyA)抑制T淋巴细胞,将B淋巴细胞转化成永生化淋巴母细胞系.结果 成功建立了苗族BBS核心家系中12个个体的永生细胞株.结论 EB病毒转化技术可有效保存原来个体完整的基因组,为以后的相关研究提供了长期的DNA资源.

Abstract：

Objective To establish immortalized lymphoblastoid cell lines of a Miao core pedigree with Bardet-Biedl syndrome (BBS), in order to provide a long-term source of material for research. Methods With Epstein-Barr virus transformation of B cells and addition of cyclosporine A to inhibit the activity of T cells, fresh anticoagulated blood samples with heparin were collected from 12 members of the core pedigree,and were used to establish the immortalized lymphoblastoid cell lines of B lymphocytes. Results Twelve immortalized lymphoblastoid cell lines of the core BBS pedigree were obtained successfully. Conclusion The immortalized B lymphoblastoid cell lines of the Miao pedigree with BBS can preserve the whole genome information and provide long-term research materials for BBS study.     

Direct measurement of CD8+ T cell responses in macaques infected with simian immunodeficiency virus

The simian immunodeficiency virus (SIV) macaque model system has been used extensively to study AIDS pathogenesis and to test candidate vaccines for their ability to protect against homologous or heterologous challenge with pathogenic SIV or SHIV. Recent studies suggest that stimulation of HIV-1-specific CTL responses is important for effective vaccination against HIV-1. While quantitative measurements of SIV-specific cytotoxic T lymphocyte (CTL) responses have been facilitated by the use of tetrameric peptide complexes, this technique is currently limited to the study of Mamu-A*01-positive rhesus macaques. Furthermore, very few SIV-specific CTL epitopes have been identified, and there is limited identification of other MHC alleles in macaques. In this study, cytokine flow cytometry (CFC) was used to quantify SIV-specific CD8+ antigen-reactive T cells in macaques infected with SIV. We found a strong correlation (r = 0.96, P < 0.001) between CD8+ antigen-reactive T cells stained with the Mamu-A*01 p11C, C-M tetramer and production of intracellular TNF-alpha in the CFC assay. Furthermore, the CFC assay was used to identify a novel SIV-specific CTL epitope in Envelope (SIV Env, a.a. 486-494, sequence AEVAELYRL). The use of the CFC assay facilitates the study of antigen-reactive T cell responses in SIV infection and vaccination.     

Increased viral replication in simian immunodeficiency virus/simian-HIV-infected macaques with self-administering model of chronic alcohol consumption

Alcohol abuse constitutes a major cohort among HIV-infected individuals. The precise effect of alcohol addiction on HIV pathogenesis remains inconclusive, however. This study was designed to determine the effect of alcohol dependence on virus replication and CD4 profiles in simian immunodeficiency virus/simian-HIV-infected rhesus macaques. A group of 3 male Indian rhesus macaques was adapted to a self-drinking model of alcohol consumption, whereas another group of 3 macaques was provided a Nutrasweet solution. After 7 weeks of alcohol consumption, the alcohol-dependent animals along with controls were intravenously inoculated with a mixture of SHIV(KU), SHIV(89.6)P, and SIV/17E-Fr. These animals were followed for a period of 24 weeks for complete blood cell counts, CD4 cell profiles, and viral loads in the blood and cerebral compartments. The alcohol and control groups showed comparable peak viral loads in the blood. The plasma viral load in the alcohol group was 31- to 85-fold higher than that in the control group at weeks 18 through 24 after infection, however. The pattern of cerebrospinal fluid viral replication was also comparable during the acute phase; however, the virus continued to replicate in the brain of alcohol-dependent animals, whereas it became undetectable in the controls. The extent of CD4 cell loss in the alcohol group was significantly higher than that in the control animals at week 1 after infection.     

Cold agglutinins in rhesus macaques infected with simian immunodeficiency virus (SIV)

Cold agglutinins are uncommon autoimmune phenomena associated with mycoplasma pneumonia, viral infections, lymphoproliferative neoplasia and recently, acquired immunodeficiency syndrome (AIDS) in human beings. Six rhesus macaques infected with pathogenic isolates of simian immunodeficiency virus (SIV) developed cold agglutinins late in the course of viral infection in association with hyperproteinaemia, hyperglobulinaemia and thrombocytopenia. Cold agglutinin titres ranged from 1:1024 to 1:8192. The development of cold agglutinins in SIV-infected rhesus macaques may be another manifestation of immune dysfunction in this non-human primate model of AIDS.     

Longitudinal analysis of monocyte/macrophage infection in simian immunodeficiency virus-infected, CD8+ T-cell-depleted macaques that develop lentiviral encephalitis

The histopathological hallmark of lentiviral-associated encephalitis is an abundance of infected and activated macrophages. Why a subset of infected hosts develops lentiviral encephalitis and others do not is unknown. Using a CD8(+) T-cell depletion model of simian immunodeficiency virus (SIV)-infected rhesus macaques, we examined the relationship between peripheral SIV infection of monocytes/macrophages and the development of encephalitis. At the same time that cerebral spinal fluid viral load increased in macaques that developed encephalitis, we observed that monocyte-derived macrophages from these macaques produced more virus than those from macaques that did not develop encephalitis. However, during the course of infection, the number of blood monocyte-associated SIV DNA copies did not distinguish macaques that developed simian immunodeficiency virus encephalitis from macaques that did not develop encephalitis. Paradoxically, in this model, macaques that developed encephalitis had fewer SIV-infected macrophages in lungs and thymus at postmortem than macaques that did not develop encephalitis. These findings suggest that inherent differences in host monocyte viral production are related to development of encephalitis.