Dengue Virus in Platelets from Dengue Patients

Though thrombocytopenia or dysfunction of platelets is common in dengue virus infection, the role of platelets has not been established. We enrolled 33 hospitalized children with serologically confirmed dengue virus infection. Blood specimens were collected during hospitalization. Platelets and plasma were isolated from the whole blood. Detection of dengue virus in plasma and platelets was carried out by RT-PCR with primers that can differentiate different dengue serotypes simultaneously, and by electron transmission microscopy (EM). Dengue viral RNA was detected in the platelets and plasma by conventional RT-PCR. A significantly higher percentage of dengue viral RNA was detected in platelets than in plasma (p=0.03). Platelets isolated 5 days after onset of fever were most likely positive for viral RNA. Concurrent infection or co-circulation with multiple dengue serotypes was observed in 12% of patients. Infrequently, negative-stranded dengue viral RNA was detected in platelets and in plasma. Importantly, EM confirmed the presence of dengue viral-like particles inside platelets prepared from dengue patients. Our findings suggest the presence of dengue virus in platelets may be associated with the dysfunction of platelets observed in dengue patients. bility and mortality in urban tropical areas. Changing climate, such as global warming and the effects of El Nino, unplanned urbanization, increased international travel, and the lack of effective vector-control programs are predominant causes of the increased threat of dengue infection (Barclay, 2008). The National Institute of Allergy and Infectious Diseases (NIAID) has listed dengue virus as a Category A priority bio-threat pathogen (NIAID, 2005). Over 100 million people living in tropical and subtropical areas are at risk of infection with the dengue INTRODUCTION Infection with dengue virus (DENV) is considered a major public heath issue internationally (Mackenzie et al, 2004). Dengue virus infection is the most important mosquito borne human disease in terms of moSOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 254 Vol 40 No. 2 March 2009 virus. An estimated 50 million cases of dengue infection are documented globally per year. The death rate due to dengue infection is approximately 2 to 5%, and occurs predominantly in children under 15 years of age (WHO, 2004). The recent outbreak of dengue infection in Brazil highlights the potential for the virus spread to the Americas (Morens and Fauci, 2007). Dengue infection is a potential threat to public health in the US. Currently, there is no known vaccine or effective chemotherapeutic treatment. Dengue virus is a single-stranded, positive sense RNA virus whose genome is 11 kb in length. The virus is spherical in shape, 40-50 nm in diameter. A single large protein is translated from the positive RNA and is digested into viral components by viral or host proteases. There are four serologically distinct but geneticcally related dengue viruses: DENV-1, DENV-2, DENV-3, and DENV-4. The Aedes aegypti mosquito is responsible for most of the transmission (WHO, 2004). The spectrum of dengue disease includes asymptomatic cases, dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) (Anderson et al, 2007). DENV infections can be life threatening, usually when caused by secondary infection with heterologous virus serotype (WHO, 2004). The incubation period of DENV infection varies from 5 to 10 days. Viremia occurs roughly 2 days prior to clinical manifestations and lasts for 5-6 days (Halstead, 2007). One of the clinical hallmarks in dengue virus infection is thrombocytopenia or dysfunction of platelets. There are three mechanisms that can lead to thrombocytopenia: decreased production, increased consumption, or immune-complex lysis (Srichaikul and Nimmannitya, 2000; Schexneider and Reedy, 2005; Oishi et al, 2007). However, the precise mechanism for the development of thrombocytopenia or dysfunction of platelets in dengue infection remains elusive. Platelets are anuclear cells derived from megakaryocytes. Although the size of platelets is small, platelets carry out biological functions, including protein synthesis and protein modification; they possess receptors on their surface for signal transduction. Importantly, several platelet surface receptors linked to the entry of the dengue virus, such as DC-SIGN or enhancement of the receptor FcγII have been found (Michelson, 2007). With the era of microarray and proteomic technology, the understanding of platelet function has expanded in recent years, including the understanding that platelets act as immune cells (von Hundelshausen and Weber, 2007), cross-talking with lymphocytes (Li, 2008) and being actively involved in shaping the immune response upon encountering infectious agents (Elzey et al, 2003). In dengue infection, studies have found viral antigen on the surface of platelets, immune-complex containing platelets on skin biopsy and an association between dengue virus and platelets in vitro (Boonpucknavig et al, 1979a,b; Saito et al, 2004; Oishi et al, 2007). However, no active dengue virus products have been demonstrated in platelets from infected patients. The exact role of platelets in the pathogenesis of dengue virus infection and its association with thrombocytopenia and dysfunction of platelets is still unknown. We enrolled 33 clinically and serologically confirmed dengue fever patients into this study to investigate the relationship between platelets and dengue virus, and the potential association with clinical outcome. MATERIALS AND METHODS Patients and samples The patients enrolled in the study were children hospitalized at Siriraj Hospital, DENGUE VIRUS IN PLATELETS FROM DENGUE PATIENTS Vol 40 No. 2 March 2009 255 Mahidol University, Bangkok, Thailand with a clinical diagnosis of DF or DHF according to WHO criteria from November 2006 to September 2007. Two to 3 milliliters of blood was collected in an EDTA tube (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) from each patient following hospital admission after informed written consent was given in accordance with a protocol approved by the Siriraj Hospital Ethics Committee. Thirtythree patients with serological confirmation of acute dengue infection were studied. Isolation of human platelets and plasma Human platelets and plasma were isolated from collected blood using OptiPrep (Axis-Shield, Monroe, NC). Briefly, 5 volumes of OptiPrep were diluted with 22 volumes of 0.85% (w/v) NaCl, 1 mM EDTA, 20 mM Herpes-NaOH, pH 7.4 to produce a 1.063 g/ml solution. In a 15-ml centrifuge tube, 2-3 ml of blood was layered over 5 ml of the 1.063 g/ml solution and centrifuged at 350g for 15 minutes at 22oC in a swinging bucket rotor with no break during deceleration. The clear plasma layer was removed and thereafter the broad turbid band containing platelets, which extended into the density barrier from just above the interface, was harvested. The collected plasma and platelets were aliquoted, and subjected to RNA extraction and EM study.