The HIV-1 virus is the etiologic agent of AIDS (Acquired Immunodeficiency Syndrome) and a life-long infection results in the destruction of lymphocytes, rendering the host immunocompromised 12. The development of AIDS in HIV-1 infected individuals has been defined as a result of a combination of two different types of infections characterized by an acute phase where the virus can rapidly deplete CD4+ T cells and a chronic phase where the damaged immune system gradually loses all functionality 345. Though the primary target is CD4+ T cells, the HIV-1 virus can also infect both monocytes/macrophages and dendritic cells (DCs), however, cellular tropism of the virus is determined by the expression of the cell surface receptor CD4 and the coreceptors CCR5 and CXCR4. Genetic variability in the expression of these cell surface markers can lead to differences in susceptibility by so-called R5 viruses which recognize CCR5, R5X4 viruses which recognize both CCR5 and CXCR4, and X4 viruses which recognize only CXCR4 678. The activity and longevity of the integrated HIV-1 provirus can be directly correlated to both the activation state as well as the survival of the cell. This phenomenon results in dramatically different viral pathogenicity in activated as compared to both resting and quiescent CD4+ T cells 3910. Primary HIV-1 infection is asymptomatic during the first two weeks after exposure to the virus; however, acute HIV-1 infection is evident by a dramatic burst of viral replication correlating with infection of activated T cells. This initial infection and high viral replication efficiency result in a high titer of virus present in the plasma of infected individuals that gradually drops off as the infection induces a cytopathic effect on the T cells after approximately nine weeks post infection. This acute viremia is also correlated with an active host immune response against the infection in the form of cytotoxic T lymphocyte (CTLs) CD8+ cells that recognize HIV-1 infected cells and induce cell death 111213. This CD8+ CTL response correlates with the production of HIV-1 neutralizing antibodies or seroconversion of the patient. An additional population of CD4+ T cells can be classified as resting or permissive where cellular replication is restricted at several different steps; however, there exists enough stimulatory signals to push the cell into the G₁ phase of the cell cycle. In HIV-1 positive individuals, the resting CD4+ T cells contain HIV-1 DNA in a linear form (in the cytoplasm of the cell) representing an inducible viral population that can be properly integrated upon the correct stimulation. Despite the cytoplasmic localization of the majority of viral DNA, low levels of integrated HIV-1 can be isolated from a small subset of the resting T-cell population which is most likely due to infected, activated CD4+ T cells that have reverted back to a resting state, a commonly seen phenomenon important for the establishment of immunologic memory 1415. Similarly, infected quiescent or refractory CD4+ T cells also exhibit viral replication restrictions where the provirus exists integrated in the genome in a silent or latent state 15161718. The establishment of transcriptionally silent provirus does not occur only in this subset of T cells; indeed, actively dividing T cells can contain viral reservoirs as latency can be an intrinsic property of the virus 19. It is assumed that the provirus is established in these cells during normal progression through the cell cycle and in response to the infection to avoid cytopathicity and immune clearance. After the reverse transcription step has been completed, the cell establishes itself at G₀, blocking further progression 31518. This establishment of a latent population of cells containing integrated provirus signifies the clinical latency period of infection, where the maintenance of T cell homeostasis and low viral loads occur until the terminal stages of infection and progression to disease 15182021.

The fidelity of the HIV-1 RT as well as the rapid viral replication rate contribute to the diversity of the viral progeny. In an active infection 10⁹-10¹⁰ virions are produced per day, and during each viral replication cycle there is a mutation rate of approximately 3 × 10^-5 nucleotides due primarily to a "slippery" RT 2223. The introduction of multiple point mutations in the viral genome results in many different strains of virus within an infected individual, as well as the possibility of one cell being infected by different strains, leading to recombination events. Additionally, the genomic variability leads to differences in protein sequence and structure, resulting in difficulties in developing antiretroviral drugs against the viral integrase, protease, and RT. This results in the appearance of drug-resistant HIV-1 variants in the face of antiretroviral therapies. This necessitates a cocktail of antiretroviral drugs known as HAART (highly active antiretroviral therapy) as the primary treatment for HIV-1 infected individuals who need to be constantly evaluated for treatment effectiveness against the viral strains present 242526272829.

In addition to the primary infection of susceptible populations of CD4+ T cells and monocytes/macrophages DCs can also support the integration of proviral DNA 330. Tissue macrophages are infected primarily through the CCR5 coreceptor, and individuals that lack CCR5 are highly resistant to infection, irrespective of CD4+ T cell infection 31323334. Infection of tissue macrophages assists in the progressive infection of CD4+ T cells due to interactions of the HIV-1 viral protein Nef through stimulation of the CD40 receptor and activation of the NF-κB pathway 35. Subsequent secreted proteins increase the expression of stimulatory receptors on B cells, which then interact with corresponding ligands on CD4+ T cells, allowing for either viral entry and the expression of viral proteins or the productive infection of susceptible CD4+ T cells 35.

The loss of CD4+ T cells in HIV-1 infected individuals leaves the host susceptible to opportunistic infections, many of which are normally blocked through mucosal barriers and innate immunity. The infection of the gut-associated lymphoid tissue (GALT) of the HIV-1 infected gastrointestinal (GI) tract and the pathogenesis surrounding this manifestation are termed HIV enteropathy 3637383940. Viral replication within the GALT tissue is compartmentalized with different anatomical areas of the gut exhibiting higher levels of infected cells in one site than others, i.e. esophagus, stomach, duodenum and colorectum 41. This is due largely to the wide range of distribution and composition of lymphoid tissues in the gut, including Peyer's patches in the small intestine, lymphoid follicles in the large intestine and rectum, and a majority of CD8+ T cells in the intraepithelium of the small intestine 41. This situation allows for the selection of various HIV-1 susceptible cell types within different areas of the GALT. The HIV-1 induced local activation and inflammation of the GI immune system result in the recruitment and infiltration of CD4+ T cells and CD8+ T cells to the mucosal tissues 38. Indeed in HIV-1 infected individuals, there is an increase in the proinflammatory lymphocyte response as well as an absence of CCR5+ CD4+ T cells within the GI tract during the acute stage of infection. Rapid elimination of CD4+T cells associated with structural damage of the gut is thought to cause leakage of bacterial pathogens/products into the blood stream resulting in hyperimmune activation, the hallmark of immunopathogenesis of HIV disease 42. CD4+ T cells in the GI tract are 10-fold more likely to be infected by HIV-1 than those in the peripheral blood; however, the predominance of HIV-1 specific CD8+ T cells in the GI tract is comparable to the CD4+ levels observed in peripheral blood 434445. The induction of a mucosal humoral immune response through activation of a functional HIV-1 specific T-cell response may help to control viral replication and inhibit viral spread within the GI tract.

The identification of HIV-1 as the causative agent of AIDS was followed only a year later by the recruitment of chimpanzees for the purpose of in vivo research into the disease and its associated pathogenesis, treatment, and prevention 46. Chimpanzees represented a logical and ideal starting animal model because of their documented DNA homology with humans; the two species share between 97 and 98% of their genomes. However, on a practical level, this animal was also recognized as an endangered species in certain areas; and despite genetic similarities, there are also many differences that affect immune responses and clinical manifestations of infection with human viruses, such as HIV-1 4647.

Early experiments in the 1980s utilizing chimpanzees demonstrated a series of important insights into HIV-1 infection, including the ability to be transmitted through blood and vaginal secretions 46. These investigations were able to establish an HIV-1 infection of HIV-1 in chimpanzees with successful viral entry, expression, subsequent productive viral replication and even IgG immune response mimicking human conditions. However, important differences in cell-mediated immune responses began to emerge, especially in the case of the studies by Zarling et al. where they observed that CTLs that developed in humans and played an important role in pathogenesis were not present in chimpanzees 48. Chimpanzees were also not developing the same markers of disease as humans, such as increases in β2 microglobulin, TNF-α, and IL-6. Attention shifted to other options including the use of HIV-2 and Simian Immunodeficiency Virus (SIV) as infection models. HIV-2 proved successful in infecting cynomologus macaques while SIV was useful for investigating clinical progression, particularly in juvenile macaques, of immunodeficiency as it compared to the disease in humans 495051. However both of these systems have limitations including differences in the natural progression of disease as well as challenges in accurately targeting therapeutic interventions, in addition to the high cost of animals. The combination of the HIV-1 envelope gene with the naturally occurring lentivirus in primates, SIV, produced a chimeric virus known as SHIV 52. SHIV models in rhesus and pigtail macaques have provided some success as surrogates for HIV-1 infection in humans. However, a major difference remains, the development of AIDS, occurring in this primate model within about 2–6 months period as opposed to the often longer latency observed in humans. Therefore this SHIV model is considered a useful representation of acute infection that progresses rapidly but is not necessarily an accurate reflection of the insidious HIV-1 infection and disease course. Some SIV strains such as SIVmac251 do in fact demonstrate more of a chronic infection and have found some success in efforts aimed at vaccine development, though some differences with HIV-1 still exist with regard to pathogenesis. Recent data show that chimpanzees infected with SIVcpz are able to develop an immunopathology similar to human AIDS 53 suggesting that this model holds further utility.

Despite the usefulness of non-human primates for investigations of human retroviruses, the difficulties encountered with respect to ethical, financial, and immunological challenges have led quickly to the exploration of smaller animal models (Table 1). One such model utilizing feline immunodeficiency virus (FIV) infection has provided limited insight for comparison to human disease, though this model has shown some promise vaccine development efforts and also in relevance for to human neuropathy related to HIV infection 5455. Rats have also been utilized for pharmacological research as well as HIV-1 associated dementia 47. Transgenic animals, both rat and mouse, have also demonstrated value especially for investigations concerning entry or the effects of viral integration on specific tissues 47. However, transgenic animals are limited in the ability to study therapeutics or vaccines since viral replication and proliferation are not fully achieved in these models 47. In particular, the major impairment in the transgenic rat models occurs at the level of viral gene expression and maturation of viral particles 5657. While it is possible to infect these animal models with HIV-1, problems arise in the later stages of the viral life cycle resulting in an inability to sustain viral production. Although these transgenic models could mimic the early events in viral replication, a significant block is encountered at the point of integration, ultimately creating a limited picture of productive systemic infection 58. Recent developments have shown that murine models (e.g. humanized mice) have become increasingly desirable for retroviral infection studies. Mice represent an ideal research option not only for their relatively low cost and ease of access, but also because of the ever increasing ability to manipulate the mouse genome in order to more accurately reflect what is happening in human infection at both the molecular and clinical levels 47. These murine models are continuing to evolve, and new approaches are being developed for establishing an accurate picture of human retroviral infection and for allowing relevant investigation of therapeutic and preventive options.

An inherent problem associated with the engraftment of any foreign tissue into another host is the risk of incompatibility, either rejection of the graft by the host or graft vs. host disease (GVHD). GVHD is an interesting and especially relevant syndrome that is often observed in organ and bone marrow transplants when functional immune cells in the transplanted tissue or fluid recognize the host cells and tissue as foreign and subsequently initiate an immunologic response against the host. This response quickly spreads to become an established systemic attack and results in the death of the host. In the context of xenografted small animals, how is it that these humanized mice can support and establish a functioning human immune system without exhibiting any GVHD symptoms? One possible answer is found in the Thy/Liv model which has proven particularly useful in preventing GVHD due to the complete exclusion of mature CD3+ T cells, a phenomenon that can be mimicked clinically with some success. Additionally, the presence of the fetal thy/liv organ allows for innate maturation of human CD4+ and CD8+ T cells in the context of the animal's own immune system.

In general, the proliferation of human cells in these humanized mouse models is clearly evident; however, the functionality of the system is under scrutiny. Uittenbogaart et al. have shown that the maturation of engrafted human T cells occurs within the microenvironment of the SCID mouse; however, the possibility of phenotypic changes, especially on cell-surface markers is evident 74. It is possible that these animals may actually exhibit an atypical GVH reaction, where the xenografted human T cells become anergic within the mouse 75. The CD4+ and CD8+ populations of T cells in particular, exhibit anergy in that they are not activated to secrete cytokines after stimulation with CD3; however, when grown in vitro, the chimeric CD4+ cells were able to display anti-SCID mouse reactivity 75. These data suggest that although the SCID mouse is able to support a human T cell system the immune system may not always be properly functional. It has been proposed that up to three weeks post engraftment, a majority of the injected human cells will survive, proliferate, and mature; however, after this time, anti-mouse-reactive clones that are selected for and the engrafted immune system becomes nonfunctional 63. Finally, exploring the apparent contradictory lack of GVDH in these model systems, it is important to note that GVHD typically refers to events associated with allogenic grafts; the syndrome is not as well defined, understood, or quantified in xenogenic grafts.

The development of the NOD-SCID mouse model especially the CB17-prkdc^scid mice has been described as one of the most important breakthroughs in the humanized mouse model field. The NOD-SCID mouse was created by transferring the SCID mutation into a non-obese diabetic (NOD) mouse which is often used as a model for insulin-dependent diabetes 111. For more than a decade, NOD-SCID mice have been the "gold standard" for studies of human hematolymphoid engraftment in small animal models. The enhanced ability of NOD-SCID mice to engraft with human hematolymphoid tissues as compared with CB17-SCID mice was reported in 1995 by the Schultz group 67. Mice in the NOD genetic background exhibit deficiencies in NK cell activity, at least partially due to impairment of the activating receptor NKG2D 112. They are also impaired in complement activation due to C5 deficiency 113, and finally they lack LPS-induced production of IL-1 by macrophages 67. All these features contribute to these mice showing improved engraftment of human PBMCs and hematopoietic stem cells 6466114115 (Table 2). A downside to the NOD-SCID model is the tendency of the mice to develop thymic lymphomas which can compromise the life-span of the animals 111116.

Koyanagi et al. described NOD-SCID as a novel immunodeficient mouse strain based its genetic background 117. In particular, the authors described the NOD-SCID hu-PBL mouse where engraftment of human PBLs resulted in defective T, B and NK cell populations which can model a high level of HIV-1 infectable human cells. Upon infection with HIV-1, these mice exhibited high levels of viremia, as well as detectable viral RNA in infected cells, and free virions in the blood stream. This model also exhibited HIV-1 infection in vital organs such as the liver, lungs, and brain. The uniqueness of this model is derived from its lack of NK cells; therefore, the lack of innate immunity allows for the presentation of a susceptible model for the development of HIV-1 viremia as well as for multiple organ pathogenesis 117.

In the bone marrow/liver/thymus, or "BLT" mouse model, NOD-SCID mice are implanted with fetal thymic and liver organs, similar to the SCID hu Thy/Liv model 118. The mice are then sublethally irradiated and transplanted with fetal liver tissue-derived CD34⁺ stem cell suspension. In this model, the mice essentially undergo a bone marrow transplant to complement the human fetal thymus/liver implants 118. This mouse model results in a large number of reconstituted human mature T and B lymphocytes, monocytes, macrophages, and DCs in lymphoid organs 118. This model also exhibits systemic populations of a large number of human B cells, monocytes, macrophages, and DCs, in addition to the infiltration of the liver, lung, and GI tract with human immune cells (Table 2). The humanized BLT mouse is an attractive scaffold for HIV-1 research in that the robust systemic reconstitution of the mouse with human cells is possible due to the education of human T cells within the engrafted thymus, as well as the maturation of human hematopoietic cells. This system has shown functional immune responses in the form of immunoglobulin production, T cell receptor expression, and cytokine production in response to various toxins and to the xenografting itself 78. The BLT mouse in particular contains HIV-1 susceptible populations of human cells within the GI tract as well as in the vaginal and rectal tissues 119 (Table 3). Human mucosal cells within the BLT mice are targets for mimicking HIV-1 induced CD4+ T cell depletion seen in human GALT 78119. In particular, the reconstituted DCs found in the gut epithelium are lineage negative, HLA-DR^bright CD11c⁺ cells that are also found within the human vagina, ectocervix, endocervix, uterus, and lungs 78. The reconstitution of the female genital tract in the BLT mice specifically provides an ideal model for the investigation of vaginal HIV-1 transmission; an infection which results in systemic dissemination of the virus in the animal.

The NOD/SCID model also served as the basis for the development of another breakthrough animal model. This time the target for mutation was the interleukin 2 receptor common gamma chain (IL2rγ^-/-) since a defect here is responsible for the human manifestation of X-linked SCID. This mutation resulted in a significant reduction in both the innate and the adaptive immune functions and has been utilized in several different strains for the purposes of investigating the benefits of humanization 69. In particular, the NOD/Shi-scid IL2rγ^-/- or NOG mouse was developed in 2000 and Ito et al. demonstrated its success with efficient engraftment of human hematopoietic stem cells 71. Shultz et al. used a similar approach to establish the NOD/LtSz-scid IL2rγ^-/- mouse model 72. These two models differ in their use of distinct NOD substrains as well as the choice of the IL2rγ^-/- mouse 68. The NOG animal is the product of a cross between the NOD/Shi-scid mouse with an IL2rγ^-/- mouse that has a defect in exon 7. Conversely, Shultz's model is the result of the NOD/LtSz-scid animal in combination with an IL2rγ^-/- mouse that has a defect in exon 1 68. Thus far no significant differences in engraftment efficiency have been observed between the two animals, and they are considered to be comparable choices for use in investigations requiring a humanized model 68 (Table 2).

These mouse models have served as excellent tools for conducting various HIV-1 studies. This model was first shown to support human hematopoiesis by Ishikawa et al. who transplanted newborn NOD/SCID IL2rγ^-/- mice via a facial vein with purified human CD34+ cord blood cells 70. The cells were readily reconstituted and differentiated into mature myelomonocytes, DCs, erythrocytes, platelets, and lymphocytes. This humanized model was improved upon, and it was shown that CD4+T cells in the peripheral blood, spleen, and bone marrow expressed both CXCR4 and CCR5 antigens and showed a long-lasting viremia after infection with HIV-1 viral isolates specific for both receptors 120. The infected animals also produced both anti-HIV Env and anti-HIV Gag specific antibodies indicating a high sustained rate of viral infection. The engraftment and infection procedures employed by these studies resulted in an infection lasting only 43 days, after which the animals died; however, when the CD34+ cells were transplanted without myeloablation methods, the mice were able to survive for longer than 300 days 121 (Table 3). The establishment of a stable HIV-1 infection and a steady decline in CD4+ T cell counts resulted in one of the most efficient humanized mouse models of HIV-1 infection to date.

The humanized NOD-SCID models are based on the SCID mutation which can result in a leakiness marked by low level production of mouse immunoglobulins and T-cell receptors over time. Additionally, these mice have a significantly decreased viability due to the development of lethal thymic lymphomas in as little as 5 months and susceptibility to GVHD. Pertaining to HIV-1 infection, inadequate sustained hematopoietic cell populations in these mice allows for only the study of acute HIV-1 infection rather than the chronic, latent infection observed in HIV-1 infected individuals. Therefore, the development of a more stable humanized mouse model, exhibiting a functional human immune system, was needed to address the shortcomings of the hu-SCID models. This was accomplished through the development of the Rag2^-/-γ_c^-/- mice which are completely devoid of all T, B, and NK cells 122123. These mutant mice were created by crossing homozygous recombinase activating gene 2 (Rag2) knockout mice with homozygous common cytokine receptor γ chain (γc) knockouts 122123. The Rag2 mutation results in the lack of maturation of thymus-derived T cells and peripheral B cells where the γc mutation results in the lack of the functional subunit of the interleukin-2 (IL-2), IL-4, IL-7, IL-9 and IL-15 receptors, preventing the development of lymphocytes and NK cells 122123 (Table 2). The Rag2 knockout is not a leaky mutation; it does not result in spontaneously forming tumors; nor does it confer radiation-sensitivity to the mice as the SCID mutation does. Therefore, the Rag2^-/-γ_c^-/- mouse may be an ideal scaffold for repopulation of the animal with human hematopoietic cells.

A significant advance in the humanized mouse model field was marked by the successful xenotransplantation of immunodeficient mice with human CD34+ hematopoietic stem cells (HSC). Reconstitution of human immune cells in the Rag2^-/-γ_c^-/- model and the development of human adaptive immunity has been shown by Traggiai et al. 73. BALB/c Rag2^-/-γ_c^-/- neonates were sublethally irradiated, injected intrahepatically (i.h.) with CD34+ human cord blood stem cells 4–12 hours post irradiation, and allowed to reconstitute for a period of 26 weeks. Transplanted mice exhibited lymph node development at 8 weeks of age as well as the presentation of CD45+ human hematopoietic cells. The transplanted mice also developed human DC, T, and B cells, and engrafted human cells were found in the bone marrow and spleen. The investigators also showed that the engraftment was sufficient to stimulate a human immune response when exposed to tetanus toxins and Epstein-Barr virus. This was the first humanized mouse model to show any kind of normal human cytotoxic immune response. Gimeno et al. utilized the same mouse strain and a similar set of experiments to model the knockdown of tumor suppressor genes (i.e. p53) and monitor the development of hematopoietic cells in vivo 124. Here, Rag2^/-γ_c^-/- neonates were sublethally irradiated, injected i.p. with CD34+ human cells isolated from fetal liver and allowed to reconstitute 124. The authors also investigated the age-dependence of engraftment in these mice and found that neonates can form 80% human cells, while one-week old animals can form 30% human cells, and two-week old animals can form 10% human cells at 8 weeks post implantation 116124. The preference for using neonates when reconstituting human cells is most likely due to a lesser developed murine thymus as compared to older mice or due to macrophages or neutrophils being less developed and conferring less resistance in newborns 116124. Using newborn Rag2^-/-γ_c^-/- animals, this study showed greater than 60% human cell engraftment in peripheral blood leukocytes and liver, and greater than 50% human cell engraftment in spleen and bone marrow 116124. This significant improvement in xenotransplantation in the Rag2^-/-γ_c^-/- model compared to the hu-SCID model provides a suitable environment to study infectious diseases and other maladies in a reliable small animal model.

The humanized Rag2^-/-γ_c^-/- scaffold is an ideal system to study HIV-1 pathogenesis due to the presence of an intact human immune system and its ability to support multi-lineage hematopoiesis. Two groups published the first evidence that this humanized mouse model can support a sustained HIV-1 infection 125126. The Baenziger et al. study utilized the Traggiai method of xenotransplantation into Rag2^-/-γ_c^-/- animals, and at 10–28 weeks of age the animals were infected i.p. with CCR5-tropic YU-2 or CXCR4-tropic NL4-3 HIV-1 viral strains 73125. Both HIV-1 strains were able to produce a chronic infection of up to 190 days as well as an initial acute burst phase of viral replication as detected by plasma viral RNA 125. This group observed some strain-specificity in terms of CD4 T cell depletion and thymic infection. The CXCR4-tropic infected mice exhibited a marked depletion in CD4 T cell levels in the blood as compared to the CCR5-tropic strain, whereas the latter strain was able to infect the thymus of these animals almost exclusively. The Berges et al. study, which was focused on testing the permissiveness of this model to HIV-1 infection, was also performed using the xenotransplantation method of Traggiai et al. into conditioned neonatal BALB/c Rag2^-/-γ_c^-/- animals 126. At 16 weeks post engraftment, thymic, splenic, and lymphoid tissue samples were taken from an animal and successfully infected with an X4-tropic NL4-3 HIV-1 reporter virus ex-vivo as measured by p24 ELISA. As the engrafted human cells were infectable, an in vivo infection was subsequently performed i.p. with HIV-1 X4-tropic NL4-3 or R5-tropic BaL viruses and sufficient levels of viral DNA was detected in the blood at up to 30 weeks post infection. This infection model also exhibited CD4 T cell depletion in the animals, a characteristic of chronic HIV-1 infections in humans. This study proved that the Rag2^-/-γ_c^-/- humanized mouse model can support an active infection in vivo and provide characteristic symptoms of viremia as seen in humans. These two studies were confirmed by Zhang et al. who reported that CCR5 and CXCR4 are both expressed on the reconstituted human T cells and peripheral lymphoid organs of this humanized mouse model 127. They also reported that the HIV-1 infection in these mice persists for at least 19 weeks and that this model can serve to recapitulate HIV-1 immunopathogenesis.

Once the HIV-1 humanized mouse model was established in Rag2^-/-γ_c^-/- animals, multiple studies have improved upon the model through increased robustness of infection and the generation of infectable human cells. These mice were found to display both the Treg phenotype and functions of regulatory CD4+CD25+ T cells in vivo 128. Specifically, it was found that the Treg cells and their interaction with the FoxP3 transcription factor (CD4+FoxP3+ cells) allow for the preferential infection by HIV-1 in the Rag2^-/-γ_c^-/- animals. Gorantla et al. developed an alternative irradiation dosing of neonatal mice at the time of xenotransplantation 129. They combined lower-dosage irradiation (400-cGy) with busulfan-mediated myeloablation (destruction of quiescent stem cells), to result in a stable chimerism. Additionally, they found that all components of the human immune system were present at 16 weeks of age; however, the maturation of the immune system was not functional until sometime between five and six months of age. In terms of functional HIV-1 infection and viremia in this study, a low dose of HIV-1 C1157 was able to sustain a stable infection for at least eight to ten weeks. Additionally, infection with HIV-1 ADA resulted in the expansion of CD8+ cells, activation of B cells, and physical changes in the lymph nodes, similar to what occurs in human HIV-1 patients. A study by An et al. recently described similar successes in infecting the Rag2^-/-γ_c^-/- model with a reconstituted human immune system with R5 HIV-1 isolates 130. Their results were in accordance with previous studies as they were able to detect virus in HIV-1 infected animals by co-culturing infected cells with non-infected cells in vitro as well as observing a decrease in the CD4+/CD8+ ratio as early as two weeks post-infection. With a low dose HIV-1 infection, this group detected B cell production of IgM and IgG, but were unable to detect an antibody response against HIV-1 antigens. Similarly, Van Duyne et al. showed successful infection of differentiated human CD45+ lymphocytes in their reconstituted Rag2^-/-γ_c^-/- model by ex-vivo infection with T-tropic or macrophage-tropic HIV-1 viruses 131 (Table 3). They also investigated the effect of inhibitors, i.e., AZT, Cyc202, and Tat peptide analogs on viral production in the Rag2^-/-γ_c^-/-model. In HIV-1 infected and treated animals, viral DNA was still observed; however, there was a marked decrease in Gag DNA/RNA as compared to untreated animals. Importantly, this model is now being investigated for the efficiency of RNAi gene therapy against HIV-1 infection. Ter Brake et al. recently evaluated the inhibitory effect of a shRNA against Nef protein in HIV-1 infected Rag2^-/-γ_c^-/- animals 132. The shRNA was transduced into the human hematopoietic stem cells prior to xenotransplantation, and the cells were allowed to differentiate into a normal percentage of cell subsets. Mature human CD4+ T cells were infected ex vivo with HIV-1, and a marked inhibition of viral replication was seen in the cells from the animals that received RNAi therapy. This study has very important implications for further experiments exploring RNAi as a therapeutic method.

The i.p. and i.v. methods of HIV-1 infection suffice for establishing a strong infection in these mouse models; however, they are not the natural routes of HIV-1 exposure in humans. Therefore, some recent studies have investigated the proficiency of these humanized mouse models in rectal and vaginal transmission. Berges et al. investigated the efficiency of transmission and infection of both R5 and X4 tropic HIV-1 viruses via both vaginal and rectal routes in the humanized Rag2^-/-γ_c^-/- mice 133. Interestingly, not only did these humanized animals contain susceptible human cells in both the rectal and vaginal mucosa, these animals were readily infected with HIV-1 through intravaginal or intrarectal exposures 133. A more efficient systemic infection was seen with the R5 mucosal infection as compared to the X4-HIV; however, more importantly, both viruses were able to infect the animals without mucosal abrasion or other means that are designed to make the animal more susceptible 133. Interestingly, an opposing study was recently published where it was determined that the Rag2^-/-γ_c^-/- animals are not suitable for rectal transmission of HIV-1 134. Humanized Rag2^-/-γ_c^-/- animals were exposed rectally with both cell-free and cell-associated HIV-1 but the resulting viral load was negative as compared to animals infected via the traditional i.p. route 134. Even upon various proinflammatory stimuli to increase the animals' susceptibility to HIV-1 infection, there was still a very low transmission rate due to low levels of human cellular engraftment in the gastro-intestinal associated tissues and cells 134.

The humanized mouse models described above are clearly valuable research tools for the study of many kinds of disease. Additionally, a true model of HIV-1 infection in humans should not rule out the possibility and probability of co-infections amongst individuals. For example, Kaposi's sarcoma-associated herpesvirus (KSHV) or HHV-8 has been shown to proliferate in the SCID-hu Thy/Liv model both in the presence and absence of a concurrent HIV-1 infection 161. Similarly, Human Herpesvirus 6 (HHV-6) is a herpesvirus that infects immunosupressed people as a result of HIV-1 infection. HHV-6 has been evaluated as a potential cofactor in the progression of HIV-1 when co-infected as modeled through the SCID-hu Thy/Liv system 162. Both HIV-1 and HHV-6 are able to replicate in the engrafted humanized thymus in this model, and further studies can be done to evaluate the interplay between these two viruses in vivo. Finally, a unique interaction exists between HIV-1 infected individuals who are also infected with the protozoan Toxoplasma gondii. In immunocompetent individuals, the parasite persists in the CNS as an asymptomatic chronic infection; however, in the presence of an HIV-1 infection, and the subsequent decrease in CD4+ cells, the T. gondii infection can reactivate and cause a disease known as Toxoplasma encephalitis 163. Alfonzo et al. investigated the co-infection of T. gondii in SCID mice humanized with PBMCs from HIV-1 infected patients who had been treated with HAART for at least one year 163. Mice humanized with blood from patients undergoing HAART were more resistant to parasitic infection than those mice without any antiretroviral treatment. This study concluded that there is a partial immune reconsistitution against parasitic infection in HIV-1 infected individuals undergoing HAART therapy. Future studies should also look at the impact of genital HSV-2 infection on the acquisition of HIV-1 in humanized mice since epidemiological human data suggest that prior HSV-2 infection significantly enhances sexual transmission of HIV in women.

Although still in its infancy, the field of humanized animal models holds enormous potential to grow as a primary research tool for human retroviral studies. Here we have reviewed the advances in this field over the past three decades, during which the technology and applications have grown exponentially. Future studies will most likely address how to increase the efficiency of mucosal infection in order to mimick the primary routes of HIV-1 transmission. The humanized mouse models also hold great promise for the development and testing of novel anti-retroviral therapies, bypassing the complications of larger animal (i.e., simian or human) studies. More generally, the humanized small animal model can benefit research in other human diseases such as cancers.

The authors declare that they have no competing interests.

Both RVD and CP contributed equally to this review.