Dengue virus tropism in humanized mice recapitulates human dengue fever

Animal models of dengue virus disease have been very difficult to develop because of the virus' specificity for infection and replication in certain human cells. We developed a model of dengue fever in immunodeficient mice transplanted with human stem cells from umbilical cord blood. These mice show measurable signs of dengue disease as in humans (fever, viremia, erythema and thrombocytopenia), and after infection with the most virulent strain of dengue serotype 2, humanized mice showed infection in human cells in bone marrow, spleen and blood. Cytokines and chemokines were secreted by these human cells into the mouse bloodstream. We demonstrated that the pathology of dengue virus infection in these mice follows that reported in human patients, making this the first valid and relevant model for studying dengue fever pathogenesis in humans.     

Multiploid CD61+ Cells Are the Pre-Dominant Cell Lineage Infected during Acute Dengue Virus Infection in Bone Marrow

Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM). Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV) and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM) and humans were utilized to identify the cell lineage(s) that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.     

Humanized Mice Show Clinical Signs of Dengue Fever according to Infecting Virus Genotype

We demonstrated that the infection of humanized NOD-scid IL2rγ ^null mice with different strains (representing the four genotypes) of dengue virus serotype 2 (DEN-2) can induce the development of human-like disease, including fever, viremia, erythema, and thrombocytopenia. Newborn mice were irradiated and received transplants by intrahepatic inoculation of human cord blood-derived hematopoietic progenitor cells (CD34⁺). After 6 weeks, mouse peripheral blood was tested by flow cytometry to determine levels of human lymphocytes (CD45⁺ cells); rates of reconstitution ranged from 16 to 80% (median, 52%). Infection (with approximately 10⁶ PFU, the equivalent of a mosquito bite) of these humanized mice with eight low-passage-number strains produced a high viremia extending to days 12 to 18 postinfection. We observed a significant decrease in platelets at day 10 in most of the mice and an increase in body temperature (fever) and erythema (rash) in comparison with humanized mice inoculated with cell culture medium only. Comparison of Southeast (SE) Asian and other genotype viruses (American, Indian, and West African) in this model showed significant differences in magnitude and duration of viremia and rash, with the SE Asian viruses always being highest. Indian genotype viruses produced lower viremias and less thrombocytopenia than the others, and West African (sylvatic) viruses produced the shortest periods of viremia and the lowest rash measurements. These results correlate with virulence and transmission differences described previously for primary human target cells and whole mosquitoes and may correlate with epidemiologic observations around the world. These characteristics make this mouse model ideal for the study of dengue pathogenesis and the evaluation of vaccine attenuation and antivirals.Dengue viruses, which cause the disease dengue fever (DF) and its more severe form, dengue hemorrhagic fever (DHF), in humans, have been spreading to more areas of the world along with their mosquito (Aedes aegypti and Aedes albopictus) vectors. Now over 100 countries are affected, including some areas of the United States (Texas and Hawaii) (5, 26). Due to the fact that only humans show clinical signs and symptoms of disease, it has been difficult to directly test the mechanisms of pathogenesis of these viruses (4). Through decades of research, including clinical, epidemiologic, and laboratory studies, the factors involved in producing disease, whether it be DF or DHF, have remained unproved. However, there are many indications that both the virus and the host contribute to the occurrence and severity of disease: there are genetic differences in the virus and host immune response that can be measured in vitro, and these factors seem to lead to immunopathology in addition to the damage done by virus replication. Because there are four antigenically distinct dengue viruses (serotypes 1 to 4), humans can theoretically have dengue virus infections leading to clinical disease up to four times, and the immunity to the first virus enhances the probability of developing severe dengue after a subsequent infection. Thus, the development of vaccines has been hampered by the unknown effects of inoculating with a tetravalent preparation that might cause immunopathology or severe disease, and there are no appropriate animal models in which to test vaccine attenuation and efficacy for human applications.In 2005 we reported the development of humanized NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice that produced signs of DF upon infection with one strain of dengue virus (3). The mice were humanized by giving them transplants of purified hematopoietic stem cells from human umbilical cord blood (CB) samples taken from normal births. After subcutaneous infection with a low dose of a Southeast (SE) Asian virus, the viremia, rash, and thrombocytopenia were significantly higher, longer lasting, and more like human disease than in any other animal model described at the time. We concluded that this model could be used to test antiviral treatments, since these mice did not produce measurable human antibodies. Since then, many other immunodeficient mouse strains have been produced that can have enhanced human engraftment levels, and they develop functional human immune system cells, including some level of adaptive immunity (20). It has been reported that some of these mouse strains develop immunoglobulins specific for human immunodeficiency virus and dengue virus, albeit at low levels (14, 25).Here we present results of dengue virus pathogenesis studies in a new mouse strain, NOD-scid IL2rγ ^null, that has a much higher degree of human lymphocyte development (median of 52%, versus 14% previously). The comparison of viruses from different genetic subgroups of dengue serotype 2 has led us to conclude that this model is reflective of actual human dengue pathogenesis, and this development might bring us to a new era in testing the factors that contribute to dengue disease.     

Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice

Background

The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research.

Methodology/Principal Findings

We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor γ-chain knockout (NOD-scid IL2rγ^null) mice engrafted with human hematopoietic stem cells. Human CD45⁺ cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rγ^null mice with HLA-A2⁺ human cord blood hematopoietic stem cells, were able to secrete IFN-γ, IL-2 and TNF-α in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353_(111–119), NS4b 2423_(181–189), and NS4a 2148_(56–64).

Conclusions/Significance

This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections.     

HIV-1 tracing method of systemic viremia in vivo using an artificially mutated virus pool

The appearance of human immunodeficiency virus type 1 (HIV-1) plasma viremia is associated with progression to symptomatic disease and CD4⁺ T cell depletion. To locate the source of systemic viremia, this study employed a novel method to trace HIV-1 infection in vivo. We created JRCSFξnef, a pool of infectious HIV-1 (strain JR-CSF) with highly mutated nef gene regions by random mutagenesis PCR and infected this mutated virus pool into both Jurkat-CCR5 cells and hematopoietic stem cell-transplanted humanized mice. Infection resulted in systemic plasma viremia in humanized mice and viral RNA sequencing helped us to identify multiple lymphoid organs such as spleen, lymph nodes, and bone marrow but not peripheral blood cells as the source of systemic viremia. Our data suggest that this method could be useful for the tracing of viral trafficking in vivo.     

Mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice

Dengue viruses (DENV) are transmitted to humans by the bite of Aedes aegypti or Aedes albopictus mosquitoes, with millions of infections annually in over 100 countries. The diseases they produce, which occur exclusively in humans, are dengue fever (DF) and dengue hemorrhagic fever (DHF). We previously developed a humanized mouse model of DF in which mice transplanted with human hematopoietic stem cells produced signs of DENV disease after injection with low-passage, wild-type isolates. Using these mice, but now allowing infected A. aegypti to transmit dengue virus during feeding, we observed signs of more severe disease (higher and more sustained viremia, erythema, and thrombocytopenia). Infected mice mounted innate (gamma interferon [IFN-γ] and soluble interleukin 2 receptor alpha [sIL-2Rα]) and adaptive (anti-DENV antibodies) immune responses that failed to clear viremia until day 56, while a mosquito bite alone induced strong immunomodulators (tumor necrosis factor alpha [TNF-α], IL-4, and IL-10) and thrombocytopenia. This is the first animal model that allows an evaluation of human immunity to DENV infection after mosquito inoculation.     

Analysis of Murine CD8+ T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2K^d-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8⁺ CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.     

Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice

The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγ^null mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.     

Human Breast Milk and Antiretrovirals Dramatically Reduce Oral HIV-1 Transmission in BLT Humanized Mice

Currently, over 15% of new HIV infections occur in children. Breastfeeding is a major contributor to HIV infections in infants. This represents a major paradox in the field because in vitro, breast milk has been shown to have a strong inhibitory effect on HIV infectivity. However, this inhibitory effect has never been demonstrated in vivo. Here, we address this important paradox using the first humanized mouse model of oral HIV transmission. We established that reconstitution of the oral cavity and upper gastrointestinal (GI) tract of humanized bone marrow/liver/thymus (BLT) mice with human leukocytes, including the human cell types important for mucosal HIV transmission (i.e. dendritic cells, macrophages and CD4⁺ T cells), renders them susceptible to oral transmission of cell-free and cell-associated HIV. Oral transmission of HIV resulted in systemic infection of lymphoid and non-lymphoid tissues that is characterized by the presence of HIV RNA in plasma and a gradual decline of CD4⁺ T cells in peripheral blood. Consistent with infection of the oral cavity, we observed virus shedding into saliva. We then evaluated the role of human breast milk on oral HIV transmission. Our in vivo results demonstrate that breast milk has a strong inhibitory effect on oral transmission of both cell-free and cell-associated HIV. Finally, we evaluated the effect of antiretrovirals on oral transmission of HIV. Our results show that systemic antiretrovirals administered prior to exposure can efficiently prevent oral HIV transmission in BLT mice.     

Role of the chemokine receptors CCR1, CCR2 and CCR4 in the pathogenesis of experimental dengue infection in mice

Dengue virus (DENV), a mosquito-borne flavivirus, is a public health problem in many tropical countries. Recent clinical data have shown an association between levels of different chemokines in plasma and severity of dengue. We evaluated the role of CC chemokine receptors CCR1, CCR2 and CCR4 in an experimental model of DENV-2 infection in mice. Infection of mice induced evident clinical disease and tissue damage, including thrombocytopenia, hemoconcentration, lymphopenia, increased levels of transaminases and pro-inflammatory cytokines, and lethality in WT mice. Importantly, infected WT mice presented increased levels of chemokines CCL2/JE, CCL3/MIP-1α and CCL5/RANTES in spleen and liver. CCR1^-/- mice had a mild phenotype with disease presentation and lethality similar to those of WT mice. In CCR2^-/- mice, lethality, liver damage, levels of IL-6 and IFN-γ, and leukocyte activation were attenuated. However, thrombocytopenia, hemoconcentration and systemic TNF-α levels were similar to infected WT mice. Infection enhanced levels of CCL17/TARC, a CCR4 ligand. In CCR4^-/- mice, lethality, tissue injury and systemic inflammation were markedly decreased. Despite differences in disease presentation in CCR-deficient mice, there was no significant difference in viral load. In conclusion, activation of chemokine receptors has discrete roles in the pathogenesis of dengue infection. These studies suggest that the chemokine storm that follows severe primary dengue infection associates mostly to development of disease rather than protection.     

Immunopathogenesis of dengue virus infection

Dengue virus infection causes dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), whose pathogeneses are not clearly understood. Current hypotheses of antibody-dependent enhancement, virus virulence, and IFN-gamma/TNFalpha-mediated immunopathogenesis are insufficient to explain clinical manifestations of DHF/DSS such as thrombocytopenia and hemoconcentration. Dengue virus infection induces transient immune aberrant activation of CD4/CD8 ratio inversion and cytokine overproduction, and infection of endothelial cells and hepatocytes causes apoptosis and dysfunction of these cells. The coagulation and fibrinolysis systems are also activated after dengue virus infection. We propose a new hypothesis for the immunopathogenesis for dengue virus infection. The aberrant immune responses not only impair the immune response to clear the virus, but also result in overproduction of cytokines that affect monocytes, endothelial cells, and hepatocytes. Platelets are destroyed by crossreactive anti-platelet autoantibodies. Dengue-virus-induced vasculopathy and coagulopathy must be involved in the pathogenesis of hemorrhage, and the unbalance between coagulation and fibrinolysis activation increases the likelihood of severe hemorrhage in DHF/DSS. Hemostasis is maintained unless the dysregulation of coagulation and fibrinolysis persists. The overproduced IL-6 might play a crucial role in the enhanced production of anti-platelet or anti-endothelial cell autoantibodies, elevated levels of tPA, as well as a deficiency in coagulation. Capillary leakage is triggered by the dengue virus itself or by antibodies to its antigens. This immunopathogenesis of DHF/DSS can account for specific characteristics of clinical, pathologic, and epidemiological observations in dengue virus infection.     

Pathogenesis of the delayed phase of Rauscher virus-induced thrombocytopenia

BALB/c (H-2d) mice infected with Rauscher murine leukemia virus (RMuLV) developed two phases of thrombocytopenia: an acute phase, probably due to direct virus-platelet interactions, and a delayed phase, starting 2 to 3 wk after virus injection, which was associated with the infection of megakaryocytes by RMuLV and with the expression of RMuLV gp70 and p30 antigens on platelet membranes. This study was concerned with the pathogenesis of this second phase of thrombocytopenia. During this period, the number of marrow megakaryocytes was increased. A peripheral platelet destruction was further indicated by reduced platelet life span. It was shown that radiolabeled platelets, either normal or infected, were submitted to a more rapid clearance in infected recipients than in normal recipients. This might be due to the splenomegaly observed in infected recipients. However, the immediate clearance of gp70+ platelets was more accelerated in infected recipients with high titers of serum anti-gp70 antibodies than in infected recipients without detectable serum anti-gp70 antibodies. In addition, the passive transfer of anti-RMuLV serum to normal BALB/c mice induced a rapid and specific clearance of previously injected radiolabeled platelets expressing RMuLV antigens. In H-2d mice, viral gp70 antigen expression on platelets correlated with the development of delayed thrombocytopenia; but H-2k strains of mice, although susceptible to RMuLV and expressing RMuLV-related antigens on their platelets, did not develop any anti-RMuLV antibodies nor any delayed thrombocytopenia. These results suggest that specific clearance of gp70+ platelets in the presence of significant amounts of serum antiviral antibodies and nonspecific hypersplenism play a role in the development of delayed thrombocytopenia in RMuLV-infected mice.     

Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus.

Seven calves between 1 week and 2 months of age were infected with a noncytopathic field isolate of bovine viral diarrhea virus (BDV) in order to evaluate the effect of BDV infection on the concentration of circulating platelets in the blood. All calves were determined to be free of BDV and neutralizing antibodies to BDV before infection. Platelet counts were performed on a daily basis over a 30-day period beginning at the time of infection. By 2 weeks postinfection, all calves showed a significant drop in the number of circulating platelets and a marked hyperplasia of megakaryocytes in the bone marrow. In three of the seven calves, thrombocytopenia was severe (less than or equal to 5,000/microliters) for 1 to 6 days. In two of these three animals, extensive petechial and ecchymotic hemorrhages were observed on all mucosal surfaces and on various internal organs during the period of severe thrombocytopenia. BDV was consistently isolated from the platelets during the early phases of the infection, and viral antigen was occasionally detected on platelets by a fluorescent-antibody assay. The results demonstrate that BDV infection is associated with decreases in platelet numbers and suggest that platelets may serve as carriers of circulating virus.     

Suppressive Effects of Anthrax Lethal Toxin on Megakaryopoiesis

Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. LT challenge suppresses platelet counts and platelet function in mice, however, the mechanism responsible for thrombocytopenia remains unclear. LT inhibits cellular mitogen-activated protein kinases (MAPKs), which are vital pathways responsible for cell survival, differentiation, and maturation. One of the MAPKs, the MEK1/2-extracellular signal-regulated kinase pathway, is particularly important in megakaryopoiesis. This study evaluates the hypothesis that LT may suppress the progenitor cells of platelets, thereby inducing thrombocytopenic responses. Using cord blood-derived CD34⁺ cells and mouse bone marrow mononuclear cells to perform in vitro differentiation, this work shows that LT suppresses megakaryopoiesis by reducing the survival of megakaryocytes. Thrombopoietin treatments can reduce thrombocytopenia, megakaryocytic suppression, and the quick onset of lethality in LT-challenged mice. These results suggest that megakaryocytic suppression is one of the mechanisms by which LT induces thrombocytopenia. These findings may provide new insights for developing feasible approaches against anthrax.     

Dengue virus selectively induces human mast cell chemokine production

Severe dengue virus infections usually occur in individuals who have preexisting anti-dengue virus antibodies. Mast cells are known to play an important role in host defense against several pathogens, but their role in viral infection has not yet been elucidated. The effects of dengue virus infection on the production of chemokines by human mast cells were examined. Elevated levels of secreted RANTES, MIP-1alpha, and MIP-1beta, but not IL-8 or ENA-78, were observed following infection of KU812 or HMC-1 human mast cell-basophil lines. In some cases a >200-fold increase in RANTES production was observed. Cord blood-derived cultured human mast cells treated with dengue virus in the presence of subneutralizing concentrations of dengue virus-specific antibody also demonstrated significantly (P < 0.05) increased RANTES production, under conditions which did not induce significant degranulation. Chemokine responses were not observed when mast cells were treated with UV-inactivated dengue virus in the presence or absence of human dengue virus-specific antibody. Neither antibody-enhanced dengue virus infection of the highly permissive U937 monocytic cell line nor adenovirus infection of mast cells induced a RANTES, MIP-1alpha, or MIP-1beta response, demonstrating a selective mast cell response to dengue virus. These results suggest a role for mast cells in the initiation of chemokine-dependent host responses to dengue virus infection.     

Both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage

Hemorrhage is a common clinical manifestation in dengue patients. However, the pathogenic mechanism of dengue virus (DV)-induced hemorrhage awaits clarification. We established a mouse model of DV hemorrhage using immunocompetent C57BL/6 mice by injecting DV serotype 2 strain 16681 intradermally. While inoculation of 3 x 10(9) PFU of DV induced systemic hemorrhage in all of the mice by day 3 of infection, one out of three of those injected with 4 x 10(7) to 8 x 10(7) PFU developed hemorrhage in the subcutaneous tissues. The mice that were inoculated with 4 x 10(7) to 8 x 10(7) PFU but that did not develop hemorrhage were used as a basis for comparison to explore the pathogenic mechanism of dengue hemorrhage. The results showed that mice with severe thrombocytopenia manifested signs of vascular leakage and hemorrhage. We observed that high viral titer, macrophage infiltration, and tumor necrosis factor alpha (TNF-alpha) production in the local tissues are three important events that lead to hemorrhage. Immunofluorescence staining revealed that DV targeted both endothelial cells and macrophages. In addition, the production of high levels of TNF-alpha in tissues correlated with endothelial cell apoptosis and hemorrhage. By comparing TNF-alpha(-/-) to IgH(-/-), C5(-/-), and wild-type mice, we found that TNF-alpha was important for the development of hemorrhage. In vitro studies showed that mouse primary microvascular endothelial cells were susceptible to DV but that TNF-alpha enhanced DV-induced apoptosis. Our mouse model illustrated that intradermal inoculation of high titers of DV predisposes endothelial cells to be susceptible to TNF-alpha-induced cell death, which leads to endothelium damage and hemorrhage development. This finding highlights the contribution of the innate immune response to dengue hemorrhage.     

Immune Response to Dengue Virus Infection in Pediatric Patients in New Delhi,India—Association of Viremia,Inflammatory Mediators and Monocytes with Disease Severity

Dengue virus, a mosquito-borne flavivirus, is a causative agent for dengue infection, which manifests with symptoms ranging from mild fever to fatal dengue shock syndrome. The presence of four serotypes, against which immune cross-protection is short-lived and serotype cross-reactive antibodies that might enhance infection, pose a challenge to further investigate the role of virus and immune response in pathogenesis. We evaluated the viral and immunological factors that correlate with severe dengue disease in a cohort of pediatric dengue patients in New Delhi. Severe dengue disease was observed in both primary and secondary infections. Viral load had no association with disease severity but high viral load correlated with prolonged thrombocytopenia and delayed recovery. Severe dengue cases had low Th1 cytokines and a concurrent increase in the inflammatory mediators such as IL-6, IL-8 and IL-10. A transient increase in CD14⁺CD16⁺ intermediate monocytes was observed early in infection. Sorting of monocytes from dengue patient peripheral blood mononuclear cells revealed that it is the CD14+ cells, but not the CD16+ or the T or B cells, that were infected with dengue virus and were major producers of IL-10. Using the Boruta algorithm, reduced interferon-α levels and enhanced aforementioned pro-inflammatory cytokines were identified as some of the distinctive markers of severe dengue. Furthermore, the reduction in the levels of IL-8 and IL-10 were identified as the most significant markers of recovery from severe disease. Our results provide further insights into the immune response of children to primary and secondary dengue infection and help us to understand the complex interplay between the intrinsic factors in dengue pathogenesis.     

Severe Acquired Idiopathic Thrombocytopenia in a Female Cynomolgus Macaque (Macaca fascicularis)

A 4-y-old female cynomolgus macaque presented for veterinary evaluation prior to placement in a preclinical study showed markedly low platelet counts that continued to decrease over time. Physical examination over the next several days showed areas of pale red discoloration in forelimbs, anterior thorax, and inguinal area and multifocal pinpoint areas of erythema or scabs. An area of dark red discoloration approximately 2 cm in diameter on the dorsal surface of the tongue was discovered on day 9. The macaque was euthanized, and histopathologic evaluation showed multifocal, ulcerative or erosive, hemorrhagic, lymphohistiocytic and neutrophilic glossitis and tonsillitis. The lesions on the tongue were associated with opportunistic fungi consistent with Candida albicans. The bone marrow showed megakaryocytic hyperplasia. There was no evidence of increased consumption of platelets, sequestration of platelets, or bone marrow suppression. The monkey was serologically negative for simian retrovirus, SIV, and simian T-lymphotropic virus. In light of cases reported in humans, immune-mediated destruction of platelets due to autoantibodies secondary to Candida albicans infection was considered. However, we were unable to detect antiplatelet antibodies on the platelet surface or in serum to support this etiology; therefore idiopathic thrombocytopenia was diagnosed. To our knowledge, this case represents the second reported observation of acquired thrombocytopenia in a nonhuman primate and the first reported observation in a cynomolgus macaque.Abbreviations: SVV, simian varicella virusDrug-induced thrombocytopenia is a serious but relatively common clinical condition, because numerous drugs have been associated with development of DIT. The diagnosis of drug-induced thrombocytopenia is usually made after the exclusion of all other possible causes, such as infectious agents and artifactual decreases due to clumping of platelets in vitro (pseudothrombocytopenia), and the establishment of a temporal relationship between the administration of a drug and the development of thrombocytopenia. Consequently, scientists involved with preclinical studies in NHP need to be aware of nondrug-related cases of unexplained marked thrombocytopenia like the one we describe in this case report because they may confound a drug-related effect.     

Human Herpesvirus 6A Infection and Immunopathogenesis in Humanized Rag2?/?γc?/? Mice

Although serious human diseases have been correlated with human herpesvirus 6A (HHV-6A) and HHV-6B, the lack of animal models has prevented studies which would more definitively link these viral infections to disease. HHV-6A and HHV-6B have recently been classified as two distinct viruses, and in this study we focused specifically on developing an in vivo model for HHV-6A. Here we show that Rag2^−/−γc^−/− mice humanized with cord blood-derived human hematopoietic stem cells produce human T cells that express the major HHV-6A receptor, CD46. Both cell-associated and cell-free viral transmission of HHV-6A into the peritoneal cavity resulted in detectable viral DNA in at least one of the samples (blood, bone marrow, etc.) analyzed from nearly all engrafted mice. Organs and cells positive for HHV-6A DNA were the plasma and cellular blood fractions, bone marrow, lymph node, and thymic samples; control mice had undetectable viral DNA. We also noted viral pathogenic effects on certain T cell populations. Specific thymocyte populations, including CD3⁻ CD4⁺ CD8⁻ and CD3⁺ CD4⁻ cells, were significantly modified in humanized mice infected by cell-associated transmission. In addition, we detected significantly increased proportions of CD4⁺ CD8⁺ cells in the blood of animals infected by cell-free transmission. These findings provide additional evidence that HHV-6A may play a role in human immunodeficiencies. These results indicate that humanized mice can be used to study HHV-6A in vivo infection and replication as well as aspects of viral pathogenesis.