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    Recognition of dengue virus (DENV) serotypes by non-structural protein 3 (NS3)–specific monoclonal antibody (MAb) E1D8. A, C6/36 cells were infected with the indicated DENV serotypes at a multiplicity of infection of 0.1, and cell lysates were collected 72 hours later. Equal amounts of protein from lysates of uninfected cells and cells infected with each DENV serotype were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed by Western blot with MAb E1D8. B, Western blot was performed as in A on lysates of the indicated tissues from uninfected mice or DENV2-infected C6/36 cells.

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    Dengue virus (DENV) infection in mouse tissues. Mice were infected with 105 plaque-forming units of DENV2 PL046, and tissues were collected on day 3–4 post-infection. A–L, Immunohistochemical analysis of formalin-fixed, paraffin-embedded tissues. Tissue sections from uninfected mice were stained with monoclonal antibody (MAb) E1D8 to DENV nonstructural protein 3 (NS3) (A–D), and sections from DENV2-infected mice were stained with an IgG2a isotype control antibody (E–H) or MAb E1D8 (I–L). Tissues are spleen (A, E, and I), lymph node (B, F, and J), liver (C, G, and K), and bone marrow (D, H, and L ). In spleen and lymph node, infected cells displayed either macrophage morphology (arrowheads) or long processes typical of dendritic cells (arrows). In liver, rare positive hepatocytes were detected (arrow). Infected cells were detected in bone marrow (arrows), but morphology was not sufficient to identify cell types. M and N, Infected cell types in bone marrow were identified by dual-staining for NS3 (red) and the myeloid marker F4/80 (green) in bone marrow aspirates, with 4′-6-diamidino-2-phenylindole counter-stain (blue). Staining of NS3 was diffusely cytoplasmic in some cells (M), or present in a punctate perinuclear pattern in others (N). Dual staining for NS3 and B220 (B cells), TER-119 (erythroid progenitors and erythrocytes), or CD41 (megakaryocytes/platelets) did not detect any double-stained cells. Magnifications 400× (A–L) or 1,000× (M and N). This figure appears in color at www.ajtmh.org.

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    Dengue virus nonstructural protein 3 (NS3) in human lymphoid organs. A–D, Serial sections of tissues from fatal human dengue cases were stained with hematoxylin and eosin (right column), NS3 immunohistochemical (IHC) analysis (middle column), or IHC with an IgG2a isotype control antibody (left column). NS3 IHC staining is red-brown to brown, with blue hematoxylin counterstain. Brown pigments in hematoxylin and eosin–stained sections are hemosiderin deposits caused by erythrocyte necrosis. Samples shown are A, lymph node; B, splenic red pulp; C, splenic artery; and D, splenic sinusoidal endothelium. Intercellular spaces in lymph node are artifacts of the ethanol storage and paraffin-embedding process. All images were taken at 400× magnification. This figure appears in color at www.ajtmh.org.

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    Dengue virus nonstructural protein 3 (NS3) in human lung, liver, and brain. Tissues from fatal human DENV infections were stained with hematoxylin and eosin (right column), NS3 immunohistochemical (IHC) analysis (middle column), or IHC with an IgG2a isotype control antibody (left column). Tissues shown are A, lung; B, liver; and C, cerebrum. NS3 IHC staining is red-brown to brown, with blue hematoxylin counter-stain. Arrows indicate staining of NS3 in lung and brain. Punctate brown pigment in hematoxylin and eosin–stained lung is hemosiderin deposits caused by erythrocyte necrosis, and punctate dark pigment in liver hematoxylin and eosin–stained sections is caused by bile deposits secondary to cholestasis. Spaces surrounding cerebral blood vessels are artifacts of the paraffin embedding and the sectioning process. All images were taken at 400× magnification. This figure appears in color at www.ajtmh.org.

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Tropism of Dengue Virus in Mice and Humans Defined by Viral Nonstructural Protein 3-Specific Immunostaining

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  • 1 Division of Infectious Diseases, School of Public Health, and Department of Molecular and Cell Biology, University of California, Berkeley, California; Hospital de Infectología, Ministerio de Salud, Guayaquil, Ecuador; Instituto Nacional de Higiene, Ministerio de Salud, Guayaquil, Ecuador; Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, California

Previous attempts to define dengue virus (DENV) tropism in human autopsy tissues have detected DENV antigens that are abundant in circulation during severe dengue, and thus may be present in uninfected cells. To better define DENV tropism, we performed immunostaining for the DENV2 nonstructural protein 3 (NS3) in humans and in a mouse model of DENV infection. In mice, NS3 was detected in phagocytes of the spleen and lymph node, hepatocytes in liver, and myeloid cells in bone marrow. In human autopsy tissues, NS3 was present in phagocytes in lymph node and spleen, alveolar macrophages in lung, and perivascular cells in brain. This protein was also found in hepatocytes in liver and endothelial cells in spleen, although NS3 was not present in endothelium in any other tissue. Thus, NS3-specific immunostaining supports roles for infected phagocytes, hepatocytes, and, to a limited degree, endothelial cells in the pathogenesis of severe dengue.

INTRODUCTION

Dengue virus (DENV) infections cause approximately 50 million cases of dengue fever and 250,000–500,000 cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) annually. 1,2 Several studies have examined DENV tropism in human autopsy tissues by immunohistochemical (IHC) analysis with antibodies against viral structural proteins310 or by in situ hybridization to positive-strand viral RNA.8 However, because virions containing structural proteins and positive-strand RNA circulate at high levels in DENV patients, 1113 these techniques may label infected cells and uninfected cells that have taken up virions by endocytosis or phagocytosis. We recently determined by flow cytometry analysis that 25–50% of DENV structural protein–positive peripheral blood mononuclear cells in patients in the acute phase of dengue are negative for nonstructural protein 3 (NS3), and thus likely represent DENV antigen uptake in the absence of infection. 14

Studies of DENV infection and pathogenesis have also been limited by the lack of a mouse model with demonstrated relevant cellular tropism. In interferon receptor–deficient (AG129) mice, DENV infects phagocytes in spleen and lymph nodes, 15 but the cellular tropism of DENV in other AG129 mouse tissues has not been determined, and the extent to which AG129 mice model human DENV infection would be better understood if tropism in mice and humans were directly compared.

Consequently, we developed an NS3-specific staining technique to improve our ability to identify DENV-infected cells in vivo. Although viral capsid, premembrane, envelope, and NS1 proteins are secreted, the NS3 protease/helicase is cytoplasmically localized and does not enter the secretory pathway. 16 Thus, NS3-specific IHC analysis should label DENV-infected cells but not uninfected cells that bind viral particles. We generated an NS3-specific monoclonal antibody (MAb), E1D8, which detects NS3 of all four DENV serotypes and detects NS3 in paraffin-embedded tissues. The MAb was used to characterize the cellular tropism of DENV in mice and in fatal human cases of dengue.

MATERIALS AND METHODS

Generation of MAb.

The DENV2 virus NS3 gene (1,853 basepairs) was obtained by reverse transcription–polymerase chain reaction (RT-PCR) amplification of RNA extracted from a first-passage culture supernatant of the Nicaraguan DENV2 strain N1098 by using oligonucleotides Bam HI-NS3Fwd 5′-CGGGATCCGCTGGAGTATTGTGGGAC GT-3′ and PSTI-NS3Rev 5′-AACTGCAGCTTTCTTCCAG CTGCAAATTCCT-3′, with 5 ng of RNA as template. The RT-PCR was performed with 1.25 units of Rav2 reverse transcriptase (Amersham Biosciences, Arlington Heights, IL) and 2.5 units of Vent polymerase (New England Biolabs, Ipswich, MA) under the following conditions: 42°C for 1 hour; 30 cycles at 94°C for 1 minute, 57°C for 1 minute, and 72°C for 3 minutes; and a final extension at 72°C for 10 minutes. Amplified product was subcloned into TA vector (Invitrogen, Carlsbad, CA), screened by restriction enzyme digestion, and sequenced. Selected clones were then digested with Bam HI/Pst I and ligated in-frame into the pQE30 vector (Qiagen, Valencia, CA) immediately downstream of the hexahistidine tag. Expression of the recombinant 75-kD protein in transformed Escherichia coli M15 cells was induced with 1 mM iso-propyl-d-thiogalactoside in a 50-mL of Luria-Bertani culture and assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).

Nonstructural protein 3 was purified from large-scale cultures by nickel affinity purification with Talon resin following the manufacturer’s recommendations (Clontech, Mountain View, CA). Purified protein was tested for reactivity by Western blot and enzyme-linked immunosorbent assay (ELISA) by using dengue immune polyclonal sera. BALB/c mice were injected intraperitoneally with 10 μg of NS3 in Ribi adjuvant eight weeks, four weeks, and four days prior to fusion of splenocytes with P3X63-Ag8.653 cells. Hybridomas were selected in hypoxanthine-aminopterin-thymidine medium for two weeks, followed by ELISA screening using DENV2-infected Aedes albopictus C6/36 cell lysates or uninfected lysates. Positive wells were re-screened using flow cytometry of permeabilized DENV2-infected C6/36 cells. Positive wells were cloned and retested by ELISA, flow cytometry, and Western blot. The E1D8 hybridoma was grown in serum-free medium in a CELLine flask (BD Biosciences, San Jose, CA) with supernatants collected once per week. The MAb was purified by protein G chromatography (Pierce, Rockford, IL), and purity was confirmed by SDS-PAGE. Purified E1D8 MAb and IgG2a isotype controls were conjugated to dinitrophenyl-biotin or Alexa594 using the FluoReporter Biotin/DNP and AlexaFluor 594 protein labeling kits (Invitrogen). Similar numbers of biotin-DNP and Alexa594 molecules per IgG molecule were present on E1D8 and control IgG2a preparations.

Viruses.

DENV2 PL046 was obtained from H.-Y. Lei (National Cheng Kung University, Tainan, Taiwan, Republic of China). DENV1 448 (Indonesia) and DENV4 664 (Thailand) were gifts of S. Kliks (Pediatric Dengue Vaccine Initiative, Berkeley, CA). DENV3 3009 (Sri Lanka) was a gift of A. DeSilva (University of North Carolina, Chapel Hill, NC). All viruses were propagated in C6/36 cells and titered on baby hamster kidney (BHK21, clone 15) cells. 17

Mouse infections.

The 129/Pas mice lacking interferon-α/β and -γ receptors (AG129) were bred in the University of California, Berkeley animal facility. All experimental procedures were performed according to the Institutional Animal Care and Use Committee guidelines. DENV2 PL046 was diluted in phosphate-buffered saline to a concentration of 5 × 105 plaque-forming units/mL, and mice were injected subcutaneously with 200 μL under the skin of the ventral hindlimbs. Tissues were collected 2–4 days post-infection, placed into cold 10% buffered formalin phosphate, fixed for 48 hours, and embedded in paraffin. To collect bone marrow aspirates, femurs were flushed with Hanks’ balanced salt solution containing 3.5 mM EDTA and 0.5% bovine serum albumin, and aspirates were centrifuged onto glass slides using a Cytofuge2 cytocentrifuge (StatSpin; IRIS International, Inc., Westwood, MA), air-dried, fixed for 2 minutes in methanol at −20°C, air-dried, and stored at 4°C.

Human tissue samples.

As per Ecuadorian law, persons who are deceased at the public hospitals (e.g., the Hospital de Infectología in Guayaquil) suspected of having DHF/DSS undergo autopsy within 24 hours of death. As per hospital policy and protocol, the families of all cases in this study consented for research use of autopsy specimens. An institutional review board exemption was obtained from the University of California Berkeley Committee for the Protection of Human Subjects, CPHS #2004-8-15. Specimens (1 cm3) of each tissue were collected into 10% formalin, fixed for 2–7 days, and stored in 70% ethanol at 4°C until transport to the University of California, Berkeley. Paraffin embedding was performed by a commercial laboratory (AML Laboratories, Rosedale, MD), and 5-μm sections were prepared.

Western blot.

The C6/36 cells were infected at a multiplicity of infection of 0.1 with each DENV serotype, or mock-infected. Three days later, cells were dislodged and centrifuged. Cell pellets were frozen at −80°C, then thawed in lysis buffer. To prepare mouse tissue lysates, mouse spleen, liver, and lymph node samples were ground in lysis buffer and centrifuged. Protein concentrations were determined by the Bradford assay (Pierce), and equal protein amounts of lysates were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Immobilon; Millipore. Billerica, MA), which was probed with 4 μg/mL of MAb E1D8, followed by a 1:5,000 dilution of horseradish peroxidase–conjugated goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA), and visualized with Supersignal West Femto substrate (Pierce) using a Bio-Rad (Hercules, CA) ChemiDocEQ imager. Quantification of band intensities was performed with Bio-Rad Quantity One software.

Immunostaining.

Tissue sections were deparaffinized in xylenes and rehydrated through a graded series of ethanol. Endogenous peroxidase was quenched for 10 minutes in 3% H2O2 in methanol. Antigens were unmasked by boiling slides in 10 mM sodium citrate, pH 6.0, for 20 minutes (NS3 stain) or by incubating in 0.25% trypsin for 60 minutes (von Willebrand Factor [vWF] stain). Blocking solutions were as follows: for NS3, phosphate-buffered saline plus 5% normal mouse serum, 5% normal rabbit serum, and 5% normal human serum; for vWF, 5% normal goat serum. Primary antibodies were applied for 1 hour in blocking solution plus 5% nonfat dry milk. Primary antibodies were for NS3, 20 μg/mL of E1D8-dinitrophenyl-biotin; for isotype control, 20 μg/mL of IgG2a-dinitrophenyl-biotin; for vWF, a 1:100 dilution of rabbit anti-vWF (Zymed, South San Francisco, CA). Secondary antibodies were biotinylated rabbit anti-dinitrophenyl (Invitrogen) for NS3 and isotype control stains and biotinylated goat anti-rabbit IgG (Jackson Immunoresearch) for vWF. ABC amplification (Vector Laboratories, Burlingame, CA) was used, and stains were developed using ImmPACT DAB or NovaRED (Vector Laboratories). Slides were counterstained with Hematoxylin QS (Vector Laboratories), dehydrated in graded ethanols into xylenes, and mounted with Cytoseal XYL (Richard-Allan Scientific, Kalamazoo, MI).

For detection of DENV infection in human tissues, tissues were considered positive for DENV NS3 if at least 10 cells per slide exhibited clear cytoplasmic staining with MAb E1D8, and similar staining was not observed with isotype control antibody staining of the same tissue, or in MAb E1D8 staining of non–DENV-infected tissue.

For immunofluorescence, slides were blocked in 5% normal mouse serum, then probed with 20 μg/mL of E1D8-Alexa594 and one of the following antibodies: 20 μg/mL of Alexa488-IgG2a control, a 1:50 dilution of fluorescein isothiocyanate [FITC]–conjugated anti-erythroid antigen (TER-119), a 1:25 dilution of Alexa488-anti-CD45R/B220 (eBioscience, San Diego, CA), a 1:2 dilution of Alexa488-anti-F4/80 (Serotec, Raleigh, NC), or a 1:50 dilution of FITC-conjugated anti-CD41 (Pharmingen, San Diego, CA). Slides were mounted in Vectashield with 4′-6-diamidino-2-phenylindole (Vector Laboratories).

PCR analysis of RNA in paraffin-embedded tissues.

RNA was extracted from three 20-μm sections of paraffin-embedded human liver, lung, and/or spleen samples from each of the cases in the study using the RecoverAll RNA extraction kit (Ambion, Austin, TX). Extracted RNA was then tested for DENV RNA by serotype-specific RT-PCR as previously described. 18

RESULTS

Recognition of NS3 from all DENV serotypes by MAb E1D8.

The MAbs were generated by immunizing mice with recombinant DENV2 NS3 protein and creating hybridomas from spleen cells of immunized mice, which were screened for reactivity by ELISA, flow cytometry, and Western blot with DENV-infected cell lysates or infected cells. The MAb E1D8 recognized a band of the expected molecular weight of NS3 (70 kD) on Western blots of DENV-infected mosquito cell lysates (Figure 1A) and reacted with DENV2-infected C6/36 cells, but not uninfected cells, in ELISA and flow cytometry assays. Notably, MAb E1D8 recognized NS3 from all four DENV serotypes by Western blot (Figure 1A) and immunofluorescence staining of infected cells (data not shown). Several lower-molecular weight bands that are visible on the Western blot appear to be NS3 breakdown products because they are present only in infected cells (Figure 1A). Further screening for non-specific reactivity of MAb E1D8 was performed by Western blot of uninfected mouse tissue lysates with DENV2-infected C6/36 cells as an NS3 positive control (Figure 1B). The staining of NS3 was robust, and uninfected mouse tissue lysates exhibited little reactivity with MAb E1D8; the strongest cross-reactive band in uninfected tissue lanes was a weak band at of approximately 50 kD in liver, which stained with 5% of the intensity of staining of NS3. These results show that strong staining with MAb E1D8 in Western blots occurs only when NS3 is present.

Localization of DENV NS3 in mice.

To determine if MAb E1D8 could detect NS3 in paraffin-embedded tissues, AG129 mice were infected with DENV2. Because spleen, liver, lymph node, and bone marrow were previously identified as sites of viral infection, 15,19 these tissues were collected 2–4 days post-infection and processed into paraffin sections. Lung, small and large intestines, and brain were collected as negative controls because these tissues contain little or no DENV at early time points by plaque assay. 15 Slides were stained in parallel using MAb E1D8 or an IgG2a isotype control antibody; both primary antibodies were conjugated to a dinitrophenyl-biotin moiety to enable sensitive detection and avoid use of anti-mouse secondary antibodies on mouse tissues.

The IHC analysis with MAb E1D8 did not label any cells in uninfected mouse tissues (Figure 2A–D) or in infected mouse tissues probed with the isotype control antibody (Figure 2E–H). Staining was also absent from lung, intestines, and brain from infected mice. In contrast, MAb E1D8 labeled the cytoplasm of many cells in infected mouse spleen (Figure 2I). Cells positive for NS3 occurred primarily in red pulp areas, with some stained cells located at the periphery of the white pulp. The NS3-positive cells were identified by morphology and location as macrophages with large cytoplasm and rounded shape, and dendritic cells with distinctive elongated processes. Staining in lymph nodes was similar to that in the spleen (Figure 2J). These results are consistent with those of a previous study, in which negative-strand DENV RNA was detected in macrophages and dendritic cells in infected mouse spleen and lymph node. 15

In liver, NS3-positive hepatocytes were observed (Figure 2K), although rarely. Interestingly, NS3-positive hepatocytes, when present, were invariably adjacent to the lobule central vein. The NS3-positive cells in bone marrow exhibited variable morphology that was not sufficient to identify the infected cell types (Figure 2L). Consequently, bone marrow aspirates from infected mice were dual-stained by immunofluorescence for NS3 and cellular markers for myeloid cells (F4/80), B cells (B220), erythroid progenitors (TER-119), or megakaryocytes and platelets (CD41). Staining of NS3 was observed only in F4/80 + cells, indicating that DENV exclusively infects myeloid-lineage cells in mouse bone marrow (Figure 2M and N). Interestingly, NS3 immunofluorescence showed two patterns of staining of NS3: a broad distribution throughout the cytoplasm (Figure 2M), or a punctuate perinuclear pattern (Figure 2N). No NS3 signal was present in uninfected bone marrow aspirates stained with MAb E1D8.

Human tissue samples.

Patients with suspected cases of dengue who came to the Hospital de Infectología in Guayaquil, Ecuador, in 2006 and 2007 with hemorrhagic manifestations, thrombocytopenia, and/or shock were considered for inclusion in the study. Upon review of available clinical and laboratory data for each case, five patients (1–5) who died of hypovolemic shock and tested positive for IgM antibodies to DENV by ELISA and/or DENV RNA by RT-PCR were included as confirmed fatal DENV cases (Table 1). Another patient (case 6) exhibited gastrointestinal hemorrhage, petechiae, and thrombocytopenia, but did not exhibit hypovolemic shock characteristic of DSS and was negative for IgM antibodies to DENV and for DENV RNA. The cause of death for this patient was reported as hepatitis of unknown etiology. This patient was included as a probable non-DENV control.

The RT-PCR amplification of RNA extracted from paraffin blocks confirmed DENV infection in cases 1, 4, and 5 and identified the infecting serotype in cases 4 and 5 as DENV2. DENV RNA in the remaining cases could not be detected; this may be a result of RNA instability during storage in aqueous ethanol while samples awaited transport to the United States.

Immunohistochemical analysis of NS3 in human tissues.

Paraffin sections of tissues from all cases were stained for NS3. Isotype control staining was performed on serial sections to identify nonspecific staining. All dengue cases exhibited MAb E1D8–specific staining in at least one tissue, and the probable non-dengue control case was negative for NS3 in all tissues, demonstrating that the MAb E1D8 does not produce spurious staining in human tissues (Table 2). The tissues tested by NS3 IHC analysis and the NS3-positive cell types detected are shown in Table 2. Nonstructural protein 3 was detected in lymph node, spleen, lung, cerebrum, and liver, but not in cerebellum, stomach, small or large intestine, pancreas, adrenal gland, skin, heart, or bone marrow.

In lymph node, NS3 was observed in macrophages in reactive germinal centers, as identified by cell morphology (Figure 3A). Sections from two spleens exhibited staining of NS3. The NS3-positive cells were abundant in red pulp, although some positive cells were also found near the edges of white pulp regions. Cell morphology identified the infected cells as mononuclear phagocytes, which could be macrophages and/or dendritic cells, but no infected lymphocytes were seen (Figure 3B). Nonstructural protein 3 was also present in splenic endothelial cells of central arteries in two cases (Figure 3C) and in sinusoidal endothelium in one case (Figure 3D). Staining for vWF confirmed that NS3-positive cells in vascular tissue were endothelial cells (data not shown). Infected spleens exhibited extensive vascular congestion, hemolysis, hemosiderin deposition, and vasculitis (Figure 3B–D).

Nonstructural protein 3 was detected in the lungs of three patients, where NS3 was present in alveolar macrophages (Figure 4A) but not in other cell types. In two cases, substantial pathologic changes were present, as demonstrated by alveolar spaces filled by erythrocytes, edematous fluid, and macrophages or lymphocytes (Figure 4A).

In liver sections, NS3 was detected in hepatocytes in four of six patients. The NS3-positive hepatocytes were invariably proximal to the lobule central vein; NS3 was not detected in hepatocytes near the portal triads, and NS3 was not found in endothelial or Kupffer cells (Figure 4B). Infected livers displayed variable histopathologic changes: steatosis and cholestasis were present in all infected livers, and midzonal necrosis was found in two cases. In necrotic areas, hepatocytes exhibited fragmented nuclei indicative of apoptosis, as previously reported for DENV infection,5 and Kupffer cells were abundant. Although NS3 was common in hepatocytes adjacent to necrotic areas, staining of NS3 was not observed within necrotic areas.

In cerebrum, NS3 was present in cells adjacent to capillaries (Figure 4C), but not in endothelial cells, neurons, or glial cells in other locations. Based upon morphology and location, the infected cells may be perivascular astrocytes or peripheral blood monocyte/macrophages extravasating into the brain; DENV antigen has previously been reported in both of these cell types. 3,9

Similar to our observations in mice, two distinctive patterns of NS3 localization were seen in human cells. Phagocytic cells contained either diffuse or punctate staining (compare Figure 3A and B), and endothelium exhibited diffuse staining of NS3 throughout the cytoplasm (compare Figure 3C and D), and staining of NS3 in hepatocytes was sharply punctate and concentrated near the nucleus (Figure 4B). The significance of this cell type-specific variation in NS3 localization is unclear; the different staining patterns may represent cell type-specific differences in the subcellular localization of NS3 or simply different stages of an infectious cycle that progresses at different rates in distinct cell types.

DISCUSSION

Efforts to define the tropism of DENV have been hampered by the lack of a reagent able to distinguish virus-infected cells from those demonstrating endocytic or phagocytic uptake of viral antigen. Previous autopsy studies located DENV antigen and RNA in phagocytes in spleen, lymph node, lung, liver, and brain, although it has been unclear whether all or only some of these phagocytes are truly infected. 3,4,6,810 Dengue virus antigen has also been detected in lymphocytes, 3,8 hepatocytes, 3,5,7,9 endothelium,3,6,8,10 and cerebral neurons and astrocytes, 3,10 but other studies examining the same tissues have not found DENV antigen in these cell types. 6,8,9 This uncertainty over DENV tropism complicates development of relevant in vitro and in vivo models for DENV infection and clouds our understanding of dengue pathogenesis. We sought to examine DENV tropism using the anti-NS3 MAb E1D8, which is specific for a viral protein not secreted by infected cells, reacts with all DENV serotypes, and detects NS3 in paraffin-embedded tissues. Monoclonal antibody E1D8 was used to define DENV cellular tropism in a mouse model of infection and in autopsy tissues from fatal human dengue cases.

AG129 mice are susceptible to infection with clinical isolates of all four DENV serotypes, with virus replication occurring in peripheral tissues including spleen, lymph node, bone marrow, peripheral blood, and, to a lesser extent, liver. 15,17,1922 Using MAb E1D8, we detected NS3 in macrophages and dendritic cells of the spleen and lymph node in infected mice. We have previously shown that these cell types contain negative-strand viral RNA, which confirms the accuracy of the NS3-specific stain. 15 In liver, NS3 was detected in a limited number of hepatocytes, consistent with the low viral titers present in this tissue early after subcutaneous infection. The absence of DENV in mouse brain, alveolar macrophages, and endothelial cells and the low level of infection in the liver may reflect differences in tropism between mice and humans or may reflect the lower viremia and lack of overt disease present in mice early after infection compared with fatal human DHF cases late in infection. However, the cell types infected in mouse spleen, liver, and lymph node were also infected in humans, demonstrating that AG129 mice reproduce some aspects of human DENV tropism.

Consequently, we used the AG129 model to examine DENV tropism in bone marrow. Data from studies on DENV tropism in human bone marrow have been conflicting. One study found that myeloid cells are targets of DENV in long-term human bone marrow cultures, 23 another study identified erythroid progenitors as the major target cells, 24 and a third study found myeloid cells in bone marrow to be positive for viral antigen by IHC, but negative for viral RNA by in situ hybridization, leaving uncertainty as to whether bone marrow cells were truly infected.8 Furthermore, thrombocytopenia is common in DENV infections, raising the possibility that direct infection of platelet progenitor megakaryocytes could contribute to DENV pathogenesis. In mouse bone marrow, NS3 was present only in myeloid cells, but not in B cells, erythroid progenitors, megakaryocytes, or platelets, which supports the hypothesis that bone marrow myeloid cells are targets of DENV infection. However, because no NS3 was detected in our human bone marrow samples, staining of NS3 with a larger panel of human bone marrow samples will be required to confirm or refute this hypothesis.

Splenic phagocytes, lymph node macrophages, and alveolar macrophages were identified as DENV targets in humans by staining of NS3. These results confirm other studies that have implicated these same cell types as targets of DENV infection. 3,8,9 Although it is feasible that phagocytes could contain NS3 antigen after phagocytosis of other cells, this possibility is unlikely because of the absence of other infected cell types in lymph node and lung. Although infected spleens contained NS3-positive phagocytes and endothelium, infected endothelium did not exhibit signs of cell fragmentation or death, and NS3 positivity in phagocytes did not correlate with proximity to infected endothelium.

Although some studies have reported DENV infection or antigen in lymphocytes, 3,8,25 no NS3 was detected in lymphoctyes in any tissue, even when abundant infection was detected in other cell types. Thus, lymphocytes may be another example of a cell type that takes up viral antigen but does not become infected. This hypothesis is supported by the observations that DENV antigen–positive lymphocytes in human tissues do not contain detectable DENV RNA,8 and that primary human lymphocytes are refractory to DENV infection in vitro.26,27 Additionally, flow cytometric analysis of peripheral blood mononuclear cells from pediatric dengue cases during the acute phase of the disease identified activated monocytes, not lymphocytes, as the primary targets of DENV infection when MAbs were used to identify structural proteins and NS3. 14

Dengue virus infection of liver macrophages (Kupffer cells) has been controversial. Although some groups have detected DENV antigen in Kupffer cells, 3,9,28 others have not. 5,7 In primary cell culture, DENV enters Kupffer cells but fails to establish productive infection, 29 and one autopsy study found Kupffer cells to be positive for viral antigen but not RNA.8 In the present study, no NS3 was detected in Kupffer cells even when widespread infection of hepatocytes was present. Collectively, the available data suggest that Kupffer cells may not be susceptible to DENV infection, but can take up significant amounts of viral structural antigens by phagocytosis.

Studies have also implicated hepatocyte involvement in DENV pathogenesis. Increased levels of liver enzymes are common in dengue patients, 3032 DENV has been isolated from infected liver, 33 and fulminant DENV-associated hepatic failure has been reported. 34 Primary hepatocytes are permissive for DENV infection in vitro,35 but autopsy studies have differed over whether hepatocytes are infected by DENV in vivo.59 Immunohistochemical analysis of NS3 showed that infected hepatocytes were abundant in four of the five dengue cases. Infected hepatocytes were always located near the lobule central vein, necrosis was observed in the midzone, and steatosis was prominent throughout the tissue. This pattern of infection and pathologic changes has been reported for DENV 5,7,36 and mirrors the pathologic changes in the liver during yellow fever. 37,38 Our data suggest that direct infection of hepatocytes is responsible for the liver involvement observed in DENV infection.

There are multiple reports of central nervous system (CNS) involvement during DENV infection, 3,9,10,39,40 and DENV has been isolated from cerebrospinal fluid, 39,40 but the tropism of DENV in human CNS has been unclear. Two studies detected DENV in neurons, astrocytes, and microglia, but neither appears to have used isotype control stains to distinguish DENV-specific from background staining. 3,10 A third study found no DENV infection in the brain,8 and another study detected DENV antigen in infiltrating peripheral blood-derived monocyte/macrophages, but not in neurons, in three dengue cases with significant neuronal pathologic changes.9 Our study confirmed previous observations of DENV in perivascular cells, which may represent infected astrocytes or infiltrating monocytes. However, no infection of microglia or neurons was observed. Interestingly, none of the patients in our study presented with unusual signs of CNS involvement, demonstrating that DENV-infected cells may be present in the CNS even in patients who have with classic DHF/DSS signs and symptoms. Further investigation is required to determine if infection of neurons or microglia occurs in DHF/DSS or in dengue patients with pronounced neurologic symptoms.

Finally, endothelium has been an interesting but controversial proposed target of DENV. Primary human endothelial cells and human endothelial cell lines are permissive for DENV infection, 41,42 autopsy studies have found DENV antigen in the endothelium of various tissues, 3,8,28 and fatal dengue is characterized by systemic vascular dysfunction. However, destructive vascular lesions are generally absent in autopsy tissues, 28 and detection of viral structural antigens in or on endothelium may not indicate infection. We detected NS3 in splenic endothelium in sinusoidal endothelium and larger vessels. Notably, human splenic sinusoidal endothelium expresses mannose receptor, a known attachment receptor for DENV. 43,44 However, endothelial cells were not infected in any other tissue, even when other infected cell types and substantial pathologic changes were present. Thus, endothelial infection does not appear to be distributed widely enough to account for the systemic nature of DHF/DSS, and endothelial infection is not required for severe pathologic changes in individual tissues. Rather, DENV infection of endothelium may occur in a limited fashion and perhaps only in select tissue sites such as the spleen.

In conclusion, NS3-specific immunostaining can identify DENV-infected cells in human tissues. Phagocytes in lung, spleen, and lymph node, splenic endothelial cells, hepatocytes, and perivascular cells in cerebrum were found to be infected with DENV in humans in this study. Thus, these cell types are relevant for modeling DENV infection, and should be considered in efforts to understand dengue pathogenesis. To that end, AG129 mice reproduce several aspects of human DENV tropism and will continue to serve as a useful model of DENV infection. To more fully define DENV tropism, staining of NS3 must be performed on a larger panel of autopsy tissues, including tissues from multiple geographic locations and infected with each of the DENV serotypes.

Table 1

Autopsy details of patients who died of case of dengue*

Table 1
Table 2

Summary of staining for dengue virus nonstructural protein 3 (NS3)

Table 2
Figure 1.
Figure 1.

Recognition of dengue virus (DENV) serotypes by non-structural protein 3 (NS3)–specific monoclonal antibody (MAb) E1D8. A, C6/36 cells were infected with the indicated DENV serotypes at a multiplicity of infection of 0.1, and cell lysates were collected 72 hours later. Equal amounts of protein from lysates of uninfected cells and cells infected with each DENV serotype were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed by Western blot with MAb E1D8. B, Western blot was performed as in A on lysates of the indicated tissues from uninfected mice or DENV2-infected C6/36 cells.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 3; 10.4269/ajtmh.2009.80.416

Figure 2.
Figure 2.

Dengue virus (DENV) infection in mouse tissues. Mice were infected with 105 plaque-forming units of DENV2 PL046, and tissues were collected on day 3–4 post-infection. A–L, Immunohistochemical analysis of formalin-fixed, paraffin-embedded tissues. Tissue sections from uninfected mice were stained with monoclonal antibody (MAb) E1D8 to DENV nonstructural protein 3 (NS3) (A–D), and sections from DENV2-infected mice were stained with an IgG2a isotype control antibody (E–H) or MAb E1D8 (I–L). Tissues are spleen (A, E, and I), lymph node (B, F, and J), liver (C, G, and K), and bone marrow (D, H, and L ). In spleen and lymph node, infected cells displayed either macrophage morphology (arrowheads) or long processes typical of dendritic cells (arrows). In liver, rare positive hepatocytes were detected (arrow). Infected cells were detected in bone marrow (arrows), but morphology was not sufficient to identify cell types. M and N, Infected cell types in bone marrow were identified by dual-staining for NS3 (red) and the myeloid marker F4/80 (green) in bone marrow aspirates, with 4′-6-diamidino-2-phenylindole counter-stain (blue). Staining of NS3 was diffusely cytoplasmic in some cells (M), or present in a punctate perinuclear pattern in others (N). Dual staining for NS3 and B220 (B cells), TER-119 (erythroid progenitors and erythrocytes), or CD41 (megakaryocytes/platelets) did not detect any double-stained cells. Magnifications 400× (A–L) or 1,000× (M and N). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 3; 10.4269/ajtmh.2009.80.416

Figure 3.
Figure 3.

Dengue virus nonstructural protein 3 (NS3) in human lymphoid organs. A–D, Serial sections of tissues from fatal human dengue cases were stained with hematoxylin and eosin (right column), NS3 immunohistochemical (IHC) analysis (middle column), or IHC with an IgG2a isotype control antibody (left column). NS3 IHC staining is red-brown to brown, with blue hematoxylin counterstain. Brown pigments in hematoxylin and eosin–stained sections are hemosiderin deposits caused by erythrocyte necrosis. Samples shown are A, lymph node; B, splenic red pulp; C, splenic artery; and D, splenic sinusoidal endothelium. Intercellular spaces in lymph node are artifacts of the ethanol storage and paraffin-embedding process. All images were taken at 400× magnification. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 3; 10.4269/ajtmh.2009.80.416

Figure 4.
Figure 4.

Dengue virus nonstructural protein 3 (NS3) in human lung, liver, and brain. Tissues from fatal human DENV infections were stained with hematoxylin and eosin (right column), NS3 immunohistochemical (IHC) analysis (middle column), or IHC with an IgG2a isotype control antibody (left column). Tissues shown are A, lung; B, liver; and C, cerebrum. NS3 IHC staining is red-brown to brown, with blue hematoxylin counter-stain. Arrows indicate staining of NS3 in lung and brain. Punctate brown pigment in hematoxylin and eosin–stained lung is hemosiderin deposits caused by erythrocyte necrosis, and punctate dark pigment in liver hematoxylin and eosin–stained sections is caused by bile deposits secondary to cholestasis. Spaces surrounding cerebral blood vessels are artifacts of the paraffin embedding and the sectioning process. All images were taken at 400× magnification. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 3; 10.4269/ajtmh.2009.80.416

*

Address correspondence to Eva Harris, Division of Infectious Diseases, School of Public Health, University of California Berkeley, 1 Barker Hall, Berkeley, CA 94720-7354. E-mail: eharris@berkeley.edu

Authors’ addresses: Scott J. Balsitis, Josefina Coloma, Diana Flores, and Eva Harris, Division of Infectious Diseases, School of Public Health, University of California Berkeley, 1 Barker Hall, Berkeley, CA 94720-7354. Glenda Castro, Microbiologia, Biologia Molecular, Infectious Diseases Hospital Jose Rodriguez Marideña, Julian Coronel y Esmeraldas, Postal 09-04-465P, Guayaquil, Ecuador. Aracely Alava, Division of Research and Microbiological Diagnostics, National Institute of Hygiene and Tropical Medicine Leopoldo Izquieta Perez, Julian Coronel y Esmeraldas 901-905, Postal 6224, Guayaquil, Ecuador. James H. McKerrow, Box 2550, University of California San Francisco, QB3, Mission Bay, San Francisco, CA 94158. P. Robert Beatty, Department of Molecular and Cell Biology, University of California Berkeley, 142 Life Sciences Addition, Berkeley, CA 94720-3200.

Acknowledgments: We thank Jennifer Kyle, Katherine Williams, Milena Gutierrez, Carolina Pérez, Fei Lin, and Dianna Edgil for their contributions to this work, and the staff of the University of California Berkeley Northwest Animal Facility for animal research support.

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