Dengue Viral RNA Levels in Peripheral Blood Mononuclear Cells are Associated with Disease Severity and Preexisting Dengue Immune Status

Background Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF). The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known. Method The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR. Results Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells. Conclusions B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC). Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis

Evaluation of Cardiac Involvement in Children with Dengue by Serial Echocardiographic Studies

Background: Infection with dengue virus results in a wide range of clinical manifestations from dengue fever (DF), a self-limited febrile illness, to dengue hemorrhagic fever (DHF) which is characterized by plasma leakage and bleeding tendency. Although cardiac involvement has been reported in dengue, the incidence and the extent of cardiac involvement are not well defined. Methods and Principal Findings: We characterized the incidence and changes in cardiac function in a prospective in-patient cohort of suspected dengue cases by serial echocardiography. Plasma leakage was detected by serial chest and abdominal ultrasonography. Daily cardiac troponin-T levels were measured. One hundred and eighty one dengue cases were enrolled. On the day of enrollment, dengue cases that already developed plasma leakage had lower cardiac index (2695 (127) vs 3188 (75) (L/min/m2), p = .003) and higher left ventricular myocardial performance index (.413 (.021) vs .328 (.026), p = .021) and systemic vascular resistance (2478 (184) vs 1820 (133) (dynes·s/cm5), p = .005) compared to those without plasma leakage. Early diastolic wall motion of the left ventricle was decreased in dengue cases with plasma leakage compared to those without. Decreased left ventricular wall motility was more common in dengue patients compared to non-dengue cases particularly in cases with plasma leakage. Differences in cardiac function between DF and DHF were most pronounced around the time of plasma leakage. Cardiac dysfunction was transient and did not require treatment. Transient elevated troponin-T levels were more common in DHF cases compared to DF (14.5% vs 5%, p = 0.028). Conclusions: Transient left ventricular systolic and diastolic dysfunction was common in children hospitalized with dengue and related to severity of plasma leakage. The functional abnormality spontaneously resolved without specific treatment. Cardiac structural changes including myocarditis were uncommon. Author Summary: Dengue is a viral infection with a wide range of symptoms from a self-limiting fever called dengue fever (DF) to dengue hemorrhagic fever (DHF) which is characterized by leaky blood vessels and bleeding that can lead to shock in severe cases. Abnormal heart function has been reported but the frequencies and the progression of heart involvement are not well defined. In this study children with dengue had serial evaluation of their heart function during the course of the illness. Patients with DHF had comparatively low blood volume at the time of fever resolution and had decreased blood flow into the left lower heart chamber compared to DF cases. Relaxation and contraction of the left side of the heart were also relatively decreased in DHF. These abnormalities may contribute to the clinical response and complications of fluid replacement in dengue

Epidemiology of Infant Dengue Cases Illuminates Serotype-Specificity in the Interaction between Immunity and Disease, and Changes in Transmission Dynamics

BACKGROUND: Infants born to dengue immune mothers acquire maternal antibodies to dengue. These antibodies, though initially protective, decline during the first year of life to levels thought to be disease enhancing, before reaching undetectable levels. Infants have long been studied to understand the interaction between infection and disease on an individual level. METHODS/FINDINGS: Considering infants (cases \u3c 1 year old) as a unique group, we analyzed serotype specific dengue case data from patients admitted to a pediatric hospital in Bangkok, Thailand. We show differences in the propensity of serotypes to cause disease in individuals with dengue antibodies (infants and post-primary cases) and in individuals without dengue antibodies (primary cases). The mean age of infant cases differed among serotypes, consistent with previously observed differential waning of maternal antibody titers by serotype. We show that trends over time in epidemiology of infant cases are consistent with those observed in the whole population, and therefore with trends in the force of infection. CONCLUSIONS/SIGNIFICANCE: Infants with dengue are informative about the interaction between antibody and the dengue serotypes, confirming that in this population DENV-2 and DENV-4 almost exclusively cause disease in the presence of dengue antibody despite infections occurring in others. We also observe differences between the serotypes in the mean age in infant cases, informative about the interaction between waning immunity and disease for the different serotypes in infants. In addition, we show that the mean age of infant cases over time is informative about transmission in the whole population. Therefore, ongoing surveillance for dengue in infants could provide useful insights into dengue epidemiology, particularly after the introduction of a dengue vaccine targeting adults and older children

Cross-Reactivity and Expansion of Dengue-Specific T cells During Acute Primary and Secondary Infections in Humans

Serotype-cross-reactive memory T cells responding to secondary dengue virus (DENV) infection are thought to contribute to disease. However, epitope-specific T cell responses have not been thoroughly compared between subjects with primary versus secondary DENV infection. We studied CD8+ T cells specific for the HLA-A*1101-restricted NS3133 epitope in a cohort of A11+ DENV-infected patients throughout acute illness and convalescence. We compared the expansion, serotype-cross-reactivity, and activation of these cells in PBMC from patients experiencing primary or secondary infection and mild or severe disease by flow cytometry. Our results show expansion and activation of DENV-specific CD8+ T cells during acute infection, which are predominantly serotype-cross-reactive regardless of DENV infection history. These data confirm marked T cell activation and serotype-cross-reactivity during the febrile phase of dengue; however, A11-NS3133-specific responses did not correlate with prior antigenic exposure or current disease severity

Longitudinal Analysis of Memory B and T Cell Responses to Dengue Virus in a 5-Year Prospective Cohort Study in Thailand

Prior exposure to dengue virus (DENV) has a profound impact on the outcome of infection, which varies according to the interval between infections. Antibodies secreted by B cells and cytokines secreted by T cells are thought to contribute both to protective immunity against DENV and the pathogenesis of dengue disease. We analyzed peripheral blood mononuclear cells (PBMC) collected from Thai children over a 5-year prospective cohort study to define the dynamics of DENV-specific memory B and T cell responses and the impact of symptomatic or subclinical DENV infections. To measure B cell responses, PBMC were stimulated with IL-2 plus R848 and culture supernatants were tested for DENV-binding antibodies by ELISA. To measure T cell responses, PBMC were stimulated in dual-color ELISPOT assays with overlapping peptide pools of structural and non-structural proteins from the four DENV types. B cell responses were low to one or more DENV types prior to symptomatic infection and increased with reactivity to all four types after infection. Subjects who had a subclinical infection or who did not experience a DENV infection during the study period showed strong memory B cell responses to all four DENV types. T cell responses to DENV peptides demonstrated a cytokine hierarchy of IFN-γ \u3e IL-2 \u3e IFN-γ/IL-2. T cell responses were low or absent prior to secondary infections. The trends in T cell responses to DENV peptides over 3 year post-infection were highly variable, but subjects who had experienced a secondary DENV1 infection showed higher cytokine responses compared to subjects who had experienced a secondary DENV2 or subclinical infection. The longitudinal nature of our study demonstrates persistent memory B cell responses over years and a lasting but variable impact of secondary DENV infection on DENV-specific T cell responses

Eliminating a Region of Respiratory Syncytial Virus Attachment Protein Allows Induction of Protective Immunity without Vaccine-enhanced Lung Eosinophilia

In a murine model of respiratory syncytial virus disease, prior sensitization to the attachment glycoprotein (G) leads to pulmonary eosinophilia and enhanced illness. Three different approaches were taken to dissect the region of G responsible for enhanced disease and protection against challenge. First, mutant viruses, containing frameshifts that altered the COOH terminus of the G protein, were used to challenge mice sensitized by scarification with recombinant vaccinia virus (rVV) expressing wild-type G. Second, cDNA expressing these mutated G proteins were expressed by rVV and used to vaccinate mice before challenge with wild-type respiratory syncytial virus (RSV). These studies identified residues 193–205 to be responsible for G-induced weight loss and lung eosinophilia and showed that this region was not was not necessary for induction of protective immunity. Third, mice were sensitized using an rVV that expressed only amino acids 124–203 of the G protein. Upon RSV challenge, mice sensitized with this rVV developed enhanced weight loss and eosinophilia. This is the first time that a region within RSV (amino acids 193–203) has been shown to be responsible for induction of lung eosinophilia and disease enhancement. Moreover, we now show that it is possible to induce protective immunity with an altered G protein without inducing a pathological response

State-of-the-art monitoring in treatment of dengue shock syndrome: a case series

BACKGROUND: Early recognition and treatment of circulatory volume loss is essential in the clinical management of dengue viral infection. We hypothesized that a novel computational algorithm, originally developed for noninvasive monitoring of blood loss in combat casualties, could: (1) indicate the central volume status of children with dengue during the early stages of shock ; and (2) track fluid resuscitation status. METHODS: Continuous noninvasive photoplethysmographic waveforms were collected over a 5-month period from three children of Thai ethnicity with clinical suspicion of dengue. Waveform data were processed by the algorithm to calculate each child\u27s Compensatory Reserve Index, where 1 represents supine normovolemia and 0 represents the circulatory volume at which hemodynamic decompensation occurs. Values between 1 and 0 indicate the proportion of reserve remaining before hemodynamic decompensation. RESULTS: This case report describes a 7-year-old Thai boy, another 7-year-old Thai boy, and a 9-year-old Thai boy who exhibited signs and symptoms of dengue shock syndrome; all the children had secondary dengue virus infections, documented by serology and reverse transcriptase polymerase chain reaction. The three boys experienced substantial plasma leakage demonstrated by pleural effusion index \u3e 25, ascites, and \u3e 20 % hemoconcentration. They received fluid administered intravenously; one received a blood transfusion. All three boys showed a significantly low initial Compensatory Reserve Index ( \u3e /=0.20), indicating a clinical diagnosis of near shock . Following 5 days with fluid resuscitation treatment, their Compensatory Reserve Index increased towards normovolemia (that is, Compensatory Reserve Index \u3e 0.75). CONCLUSIONS: The results from these cases demonstrate a new variation in the diagnostic capability to manage patients with dengue shock syndrome. The findings shed new light on a method that can avoid possible adverse effects of shock by noninvasive measurement of a patient\u27s compensatory reserve rather than standard vital signs or invasive diagnostic methods

Analysis of cell-associated DENV RNA by oligo(dT) primed 5’ capture scRNAseq

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5’ capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5’ to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5’ end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5’ capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naïve B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5’ capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells

The spatial dynamics of dengue virus in Kamphaeng Phet, Thailand.

BACKGROUND: Dengue is endemic to the rural province of Kamphaeng Phet, Northern Thailand. A decade of prospective cohort studies has provided important insights into the dengue viruses and their generated disease. However, as elsewhere, spatial dynamics of the pathogen remain poorly understood. In particular, the spatial scale of transmission and the scale of clustering are poorly characterized. This information is critical for effective deployment of spatially targeted interventions and for understanding the mechanisms that drive the dispersal of the virus. METHODOLOGY/PRINCIPAL FINDINGS: We geocoded the home locations of 4,768 confirmed dengue cases admitted to the main hospital in Kamphaeng Phet province between 1994 and 2008. We used the phi clustering statistic to characterize short-term spatial dependence between cases. Further, to see if clustering of cases led to similar temporal patterns of disease across villages, we calculated the correlation in the long-term epidemic curves between communities. We found that cases were 2.9 times (95% confidence interval 2.7-3.2) more likely to live in the same village and be infected within the same month than expected given the underlying spatial and temporal distribution of cases. This fell to 1.4 times (1.2-1.7) for individuals living in villages 1 km apart. Significant clustering was observed up to 5 km. We found a steadily decreasing trend in the correlation in epidemics curves by distance: communities separated by up to 5 km had a mean correlation of 0.28 falling to 0.16 for communities separated between 20 km and 25 km. A potential explanation for these patterns is a role for human movement in spreading the pathogen between communities. Gravity style models, which attempt to capture population movement, outperformed competing models in describing the observed correlations. CONCLUSIONS: There exists significant short-term clustering of cases within individual villages. Effective spatially and temporally targeted interventions deployed within villages may target ongoing transmission and reduce infection risk