The contribution of T cells to the host response to dengue virus (DENV) infection is not well understood. We previously demonstrated a protective role for CD8+ T cells during primary DENV infection using a mouse-passaged DENV strain and IFN-α/βR−/− C57BL/6 mice, which are susceptible to DENV infection. In this study, we examine the role of CD4+ T cells during primary DENV infection. Four I-Ab–restricted epitopes derived from three of the nonstructural DENV proteins were identified. CD4+ T cells expanded and were activated after DENV infection, with peak activation occurring on day 7. The DENV-specific CD4+ T cells expressed intracellular IFN-γ, TNF, IL-2, and CD40L, and killed peptide-pulsed target cells in vivo. Surprisingly, depletion of CD4+ T cells before DENV infection had no effect on viral loads. Consistent with this observation, CD4+ T cell depletion did not affect the DENV-specific IgG or IgM Ab titers or their neutralizing activity, or the DENV-specific CD8+ T cell response. However, immunization with the CD4+ T cell epitopes before infection resulted in significantly lower viral loads. Thus, we conclude that whereas CD4+ T cells are not required for controlling primary DENV infection, their induction by immunization can contribute to viral clearance. These findings suggest inducing anti-DENV CD4+ T cell responses by vaccination may be beneficial.

T he four serotypes of dengue virus (DENV1 to -4) belong to the Flaviviridae family, which also includes West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV). More than 2.5 billion people live in areas where DENV is endemic, and approximately 50 million people are infected each year (17). DENV generally manifests as an acute systemic infection that is cleared within 14 days (46). In most cases, infected individuals develop a febrile illness (dengue fever [DF]) characterized by flu-like symptoms, often accompanied by retro-orbital pain, ostealgia, and maculopapular rash. In nearly 500,000 cases per year, patients develop severe vascular leakage, which may result in hypovolemic shock (dengue hemorrhagic fever/dengue shock syndrome [DHF/ DSS]), ultimately leading to fatality rates as high as 10 to 15% in some countries or more than 25,000 deaths worldwide each year (15,17). In a subset of both DF and DHF/DSS cases (16), DENV infection was linked to encephalitis and signs of encephalopathy, including lethargy, confusion, seizure, and coma, as well as delayed neurological symptoms, such as paralysis of extremities, loss of sensation, and psychosis (2,21,23,30,36,38,43). DENVinduced neurological disease is being increasingly recognized as an important component of dengue disease in humans independent of DF and DHF/DSS (24). The prevalence of DENV-induced neurological disease has been estimated to be 4.2% of DENV cases (44) and as high as 18% of undiagnosed suspected viral central nervous system (CNS) infections in regions where DENV is endemic (21, 23).In the last decade, DENV has increasingly been linked to neurological disease in both the presence and absence of DF. Viruses of the Flaviviridae family generally cluster in a phylogenetic pattern that mirrors the typical disease manifestations they cause (13); for example, encephalitic flaviviruses cluster together. DENV, however, usually causes hemorrhagic illness but is genetically more homologous to the encephalitic viruses, including WNV, SLEV, and JEV, than it is to other hemorrhagic flaviviruses, such as YFV, based on several analyses of the nonstructural (NS) proteins NS3 and NS5 (11, 13). The close relationship of DENV to encephalitic flaviviruses may help explain the capacity of DENV to cause neurological disease and, possibly, infection of the CNS in humans (6, 37). Additionally, DENV-induced paralysis has been reported in numerous mouse models (4, 49), but only a few studies have investigated this phenomenon (1,18,20). Presently, whether DENV infects the CNS in natural human infections continues to be a subject of debate (25,30,32).Mice lacking the alpha/beta interferon and gamma interferon receptors (IFN-␣/␤R and IFN-␥R) in the 129/Sv genetic background (AG129) are highly susceptible to disease resembling human DHF/DSS, as well as paralysis, following infection with mouse-passaged variants of DENV2 clinical isolate PL046 (5,35,40,51). In contrast, congenic mice lacking only IFN-␣/␤R (A12...

Human postmortem studies of natural dengue virus (DENV) infection have reported systemically distributed viral antigen. Although it is widely accepted that DENV infects mononuclear phagocytes, the sequence in which specific tissues and cell types are targeted remains uncharacterized. We previously reported that mice lacking alpha/beta and gamma interferon receptors permit high levels of DENV replication and show signs of systemic disease (T. R. Prestwood et al., J. Virol. 82:8411-8421, 2008). Here we demonstrate that within 6 h, DENV traffics to and replicates in both CD169؉ and SIGN-R1 ؉ macrophages of the splenic marginal zone or draining lymph node, respectively, following intravenous or intrafootpad inoculation. Subsequently, high levels of replication are detected in F4/80 ؉ splenic red pulp macrophages and in the bone marrow, lymph nodes, and Peyer's patches. Intravenously inoculated mice begin to succumb to dengue disease 72 h after infection, at which time viral replication occurs systemically, except in lymphoid tissues. In particular, high levels of replication occur in CD68؉ macrophages of the kidneys, heart, thymus, and gastrointestinal tract. Over the course of infection, proportionately large quantities of DENV traffic to the liver and spleen. However, late during infection, viral trafficking to the spleen decreases, while trafficking to the liver, thymus, and kidneys increases. The present study demonstrates that macrophage populations, initially in the spleen and other lymphoid tissues and later in nonlymphoid tissues, are major targets of DENV infection in vivo.T he four serotypes of dengue virus (DENV1 to DENV4) belong to the Flaviviridae family and cause a mosquito-borne febrile illness in more than 50 million people per year. In nearly 500,000 cases per year, individuals develop severe hemorrhage and sometimes shock, resulting in more than 25,000 deaths annually (12). The 10.7-kb positive-stranded RNA genome of DENV encodes a polyprotein that is cleaved into 3 structural proteins, the envelope (E), the premembrane/membrane (prM/M), and the capsid (C), and 7 nonstructural (NS) proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. During replication, all DENV proteins and both positive-and negative-sense DENV RNAs are produced intracellularly, while only positive-sense RNA, the structural proteins, and NS1 are known to be secreted at high levels. Thus, based on current knowledge, NS proteins, with the exception of NS1, are restricted predominantly to cells in which DENV replicates.The cellular tropism of DENV has been investigated in humans (2, 5, 6, 8, 13, 15, 16, 25, 29) and mice (3, 9, 20, 38). In human studies, DENV antigen has been reported in the skin, liver, brain, kidney, spleen, lymph nodes, lungs, stomach, and intestines (2,5,6,8,13,15,16,25,29). These studies have mainly relied upon detection of secreted DENV antigens, which may actually localize elsewhere, complicating the interpretation of results. Recently two studies detected DENV NS antigen in the lymph nodes, spleen, liver, and ...