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As part of a study to investigate drivers of dengue virus (DENV) transmission dynamics, this qualitative study explored whether DENV-infected residents of Iquitos, Peru, considered it acceptable (1) to participate in direct mosquito feeding experiments (lab-reared Aedes aegypti mosquitoes fed directly on human volunteers) and (2) to provide blood meals indirectly (Ae. aegypti fed on blood drawn from participants by venipuncture). Twelve focus group discussions (FGDs; 94 participants: 82 females and 12 males) were conducted in January 2014 to explore six themes: (1) concerns and preferences regarding direct mosquito feeds and blood draws, (2) comprehension of and misconceptions about study procedures, (3) motivating factors for participation, (4) acceptability of children’s participation, (5) willingness to provide multiple samples over several days, and (6) preference for direct feedings in homes versus the study laboratory. Results of FGDs, including one with 5 of 53 past direct mosquito feed participants, indicated that mosquito feeding procedures are acceptable to Iquitos residents when they are provided with information and a few key messages are properly reinforced. FGD participants’ concerns focused primarily on safety issues rather than discomfort associated with mosquito bites. A video explaining the study dramatically increased comprehension of the study procedures. The majority of participants expressed a preference for mosquito feeding over venipuncture. Adults supported child participation if the children themselves assented. For most participants, home feedings were preferred over those in a laboratory. A major impetus for participation was the idea that results would contribute to an improved understanding of DENV transmission in Iquitos. Findings from our study will support future large-scale studies that employ direct mosquito feeding, a low-risk, non-invasive procedure that is experimentally superior to artificial mosquito feeding methods., Author summary Approximately half of the world’s population is at risk of contracting dengue virus (DENV). Ethical and logistical concerns with feeding lab-raised mosquitoes directly on naturally infected human subjects, and the lack of a relevant animal model for DENV experimental infection, are important obstacles to better understanding DENV transmission from humans to mosquitoes. Results from artificial infectious blood meals can bias estimates of mosquito infection and transmission rates. Based on 12 focus group discussions, we determined that the practice of feeding uninfected lab-raised mosquitoes on naturally infected human subjects is highly acceptable to people living in Iquitos, Peru, especially after common concerns are addressed. The majority of participants were willing to have mosquitoes feed on them directly and to give venous blood samples to feed to mosquitoes indirectly. Most participants stated a preference for direct feeding. This formative research, including recognition of and addressing common misconceptions, will help guide future development of protocols using biologically relevant direct mosquito feeding methods.

Background Transmission of dengue virus (DENV) from humans to mosquitoes represents a critical component of dengue epidemiology. Examinations of this process have generally been hampered by a lack of methods that adequately represent natural acquisition of DENV by mosquitoes from humans. In this study, we assessed artificial and natural blood feeding methods based on rates of DENV infection and dissemination within mosquitoes for use in a field-based epidemiological cohort study in Iquitos, Peru. Methodology/Principal findings Our study was implemented, stepwise, between 2011 and 2015. Participants who were 5 years and older with 5 or fewer days of fever were enrolled from ongoing clinic- and neighborhood-based studies on dengue in Iquitos. Wild type, laboratory-reared Aedes aegypti were fed directly on febrile individuals or on blood collected from participants that was either untreated or treated with EDTA. Mosquitoes were tested after approximately 14 days of extrinsic incubation for DENV infection and dissemination. A total of 58 participants, with viremias ranging from 1.3 × 102 to 2.9 × 106 focus-forming units per mL of serum, participated in one or more feeding methods. DENV infection and dissemination rates were not significantly different following direct and indirect-EDTA feeding; however, they were significantly lower for mosquitoes that fed indirectly on blood with no additive. Relative to direct feeding, infection rates showed greater variation following indirect-EDTA than indirect-no additive feeding. Dissemination rates were similar across all feeding methods. No differences were detected in DENV infection or dissemination rates in mosquitoes fed directly on participants with different dengue illness severity. Conclusions/Significance Our study demonstrates the feasibility of using direct and indirect feeding methods for field-based studies on vector competence. Direct mosquito feeding is preferable in terms of logistical ease, biosecurity, and reliability., Author summary In the context of ongoing clinic- and field-based epidemiological studies on dengue virus (DENV), we compared methods of feeding mosquitoes on blood from naturally infected humans. Participants chose to participate in direct (uninfected mosquitoes applied directly to skin) and/or indirect (uninfected mosquitoes fed on a membrane feeder in the laboratory with blood drawn from a participant) methods. Overall, rates of DENV infection and dissemination were lower in mosquitoes fed indirectly on blood with no additive than in mosquitoes fed directly on a participant. Rates of DENV infection and dissemination were similar between mosquitoes fed directly and indirectly when anticoagulant (EDTA) was added. The indirect-EDTA method resulted in more variable infection rates than the direct method. Our results demonstrate the feasibility of using either direct or indirect feeding methods to study DENV-human infectiousness to mosquito vectors. In our experience, however, direct feeding is preferable to indirect feeding in terms of logistical ease, biosecurity, and reliability.

While human illness associated with hantavirus infection has been documented in many countries of South America, evidence for hantavirus transmission in Peru has been limited to the isolation of Rio Mamore virus from a pigmy mouse rat (Oligoryzomys microtis) in the Amazon city of Iquitos. To address the possibility of human hantavirus exposure in the region, we screened febrile patients reporting to health clinics in Iquitos from 2007 to 2010 for serological evidence of recent hantavirus infection. In addition, we conducted a serological survey for hantavirus-reactive IgG among healthy participants residing in Iquitos and rural areas surrounding the city. Through the febrile surveillance study, we identified 15 participants (0.3%; 15/5174) with IgM reactive to hantavirus (Andes virus) antigen, all with relatively mild, self-limited illness. From the cross-sectional serosurvey we found that 1.7% (36/2063) of residents of the Iquitos area had serum IgG reactive to one or more hantaviruses, with a higher prevalence in the urban population (2.2%, compared to 1.1% in rural areas). These results suggest that human infection with hantavirus has occurred in Peru.

Background Nearly half of the world’s population is at risk for dengue, yet no licensed vaccine or anti-viral drug is currently available. Dengue is caused by any of four dengue virus serotypes (DENV-1 through DENV-4), and infection by a DENV serotype is assumed to provide life-long protection against re-infection by that serotype. We investigated the validity of this fundamental assumption during a large dengue epidemic caused by DENV-2 in Iquitos, Peru, in 2010–2011, 15 years after the first outbreak of DENV-2 in the region. Methodology/Principal Findings We estimated the age-dependent prevalence of serotype-specific DENV antibodies from longitudinal cohort studies conducted between 1993 and 2010. During the 2010–2011 epidemic, active dengue cases were identified through active community- and clinic-based febrile surveillance studies, and acute inapparent DENV infections were identified through contact tracing studies. Based on the age-specific prevalence of DENV-2 neutralizing antibodies, the age distribution of DENV-2 cases was markedly older than expected. Homologous protection was estimated at 35.1% (95% confidence interval: 0%–65.2%). At the individual level, pre-existing DENV-2 antibodies were associated with an incomplete reduction in the frequency of symptoms. Among dengue cases, 43% (26/66) exhibited elevated DENV-2 neutralizing antibody titers for years prior to infection, compared with 76% (13/17) of inapparent infections (age-adjusted odds ratio: 4.2; 95% confidence interval: 1.1–17.7). Conclusions/Significance Our data indicate that protection from homologous DENV re-infection may be incomplete in some circumstances, which provides context for the limited vaccine efficacy against DENV-2 in recent trials. Further studies are warranted to confirm this phenomenon and to evaluate the potential role of incomplete homologous protection in DENV transmission dynamics., Author Summary Dengue is a mosquito-borne viral illness that imposes a tremendous public health burden on tropical and sub-tropical regions. An estimated 390 million infections occur globally each year, and up to 4 billion people are at risk. Dengue is caused by four dengue virus (DENV) serotypes (DENV-1 to DENV-4). Infection with any DENV can lead to a range of disease outcomes, from mild febrile illness to severe, hemorrhagic manifestations and death. Infection by one serotype has been assume to provide complete and lifelong protection against re-infection by the same serotype, and to our knowledge, instances of re-infection by the same serotype have not been rigorously documented. However, few long-term studies have been conducted in such a way that re-infection by the same serotype could be observed, if it did in fact occur. Our study provides evidence that re-infection may occur in certain circumstances. We draw from data collected during a 2010–2011 DENV-2 epidemic in northeastern Peru, 15 years after the initial DENV-2 outbreak in the region. This finding has significant implications for our understanding of dengue epidemiology and for dengue vaccine formulation, which may need to consider multiple genotypes of each serotype. Data from other long-term dengue epidemiology studies should be analyzed to determine if homologous re-infection is a more widespread phenomenon.

International audience; Three-quarters of the estimated 390 million dengue virus (DENV) infections each year are clinically inapparent. People with inapparent dengue virus infections are generally considered dead-end hosts for transmission because they do not reach sufficiently high viremia levels to infect mosquitoes. Here, we show that, despite their lower average level of viremia, asymptomatic people can be infectious to mosquitoes. Moreover, at a given level of viremia, DENV-infected people with no detectable symptoms or before the onset of symptoms are significantly more infectious to mosquitoes than people with symptomatic infections. Because DENV viremic people without clinical symptoms may be exposed to more mosquitoes through their undisrupted daily routines than sick people and represent the bulk of DENV infections, our data indicate that they have the potential to contribute significantly more to virus transmission to mosquitoes than previously recognized.

During 2010 and 2011, the Loreto region of Peru experienced a dengue outbreak of unprecedented magnitude and severity for the region. This outbreak coincided with the reappearance of dengue virus-2 (DENV-2) in Loreto after almost 8 years. Whole-genome sequence indicated that DENV-2 from the outbreak belonged to lineage II of the southeast Asian/American genotype and was most closely related to viruses circulating in Brazil during 2007 and 2008, whereas DENV-2 previously circulating in Loreto grouped with lineage I (DENV-2 strains circulating in South America since 1990). One amino acid substitution (NS5 A811V) in the 2010 and 2011 isolates resulted from positive selection. However, the 2010 and 2011 DENV-2 did not replicate to higher titers in monocyte-derived dendritic cells and did not infect or disseminate in a higher proportion of Aedes aegypti than DENV-2 isolates previously circulating in Loreto. These results suggest that factors other than enhanced viral replication played a role in the severity of this outbreak.

Infectious disease models play a key role in public health planning. These models rely on accurate estimates of key transmission parameters such as the force of infection (FoI), which is the per-capita risk of a susceptible person being infected. The FoI captures the fundamental dynamics of transmission and is crucial for gauging control efforts, such as identifying vaccination targets. Dengue virus (DENV) is a mosquito-borne, multiserotype pathogen that currently infects ∼390 million people a year. Existing estimates of the DENV FoI are inaccurate because they rely on the unrealistic assumption that risk is constant over time. Dengue models are thus unreliable for designing vaccine deployment strategies. Here, we present to our knowledge the first time-varying (daily), serotype-specific estimates of DENV FoIs using a spline-based fitting procedure designed to examine a 12-y, longitudinal DENV serological dataset from Iquitos, Peru (11,703 individuals, 38,416 samples, and 22,301 serotype-specific DENV infections from 1999 to 2010). The yearly DENV FoI varied markedly across time and serotypes (0–0.33), as did daily basic reproductive numbers (0.49–4.72). During specific time periods, the FoI fluctuations correlated across serotypes, indicating that different DENV serotypes shared common transmission drivers. The marked variation in transmission intensity that we detected indicates that intervention targets based on one-time estimates of the FoI could underestimate the level of effort needed to prevent disease. Our description of dengue virus transmission dynamics is unprecedented in detail, providing a basis for understanding the persistence of this rapidly emerging pathogen and improving disease prevention programs.

Viruses from the Alphavirus genus are responsible for numerous arboviral diseases impacting human health throughout the world. Confirmation of acute alphavirus infection is based on viral isolation, identification of viral RNA, or a fourfold or greater increase in antibody titers between acute and convalescent samples. In convalescence, the specificity of antibodies to an alphavirus may be confirmed by plaque reduction neutralization test. To identify the best method for alphavirus and neutralizing antibody recognition, the standard solid method using a cell monolayer overlay with 0.4% agarose and the semisolid method using a cell suspension overlay with 0.6% carboxymethyl cellulose (CMC) overlay were evaluated. Mayaro virus, Una virus, Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV) were selected to be tested by both methods. The results indicate that the solid method showed consistently greater sensitivity than the semisolid method. Also, a “semisolid-variant method” using a 0.6% CMC overlay on a cell monolayer was assayed for virus titration. This method provided the same sensitivity as the solid method for VEEV and also had greater sensitivity for WEEV titration. Modifications in plaque assay conditions affect significantly results and therefore evaluation of the performance of each new assay is needed.