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We used nanopore sequencing and phylogenetic analyses to identify a cosmopolitan genotype of dengue virus serotype 2 that was isolated from a 56-year-old male patient from the state of Goiás in Brazil. The emergence of a cosmopolitan genotype in Brazil will require risk assessment and surveillance to reduce epidemic potential.

Dengue is an ongoing health risk for Peace Corps Volunteers (PCVs) working in the tropics. On May 2019, the Peace Corps Office of Health Services notified the Centers for Disease Control and Prevention (CDC) of a dengue outbreak among PCVs in Timor-Leste. The purpose of this investigation was to identify the clinical, demographic, and epidemiological characteristics of PCVs with dengue and recommend dengue preventive measures. To identify PCVs with dengue and describe disease severity, the medical records of PCVs reporting fever during September 2018–June 2019 were reviewed. To identify factors associated with dengue virus (DENV) infection, we administered a questionnaire on demographics, travel history, and mosquito avoidance behaviors and collected blood specimens to detect the anti-DENV IgM antibody to diagnose recent infection. Of 35 PCVs in-country, 11 (31%) tested positive for dengue (NS1, IgM, PCR), eight requiring hospitalization and medical evacuation. Among 27 (77%) PCVs who participated in the investigation, all reported having been recently bitten by mosquitoes and 56% reported being bitten most often at home; only 16 (59%) reported having screens on bedroom windows. Nearly all (93%) PCVs reported using a bed net every night; fewer (70%) reported using mosquito repellent at least once a day. No behaviors were significantly associated with DENV infection. Raising awareness of dengue risk among PCVs and continuing to encourage mosquito avoidance behavior to prevent dengue is critical. Access to and use of measures to avoid mosquito bites should be improved or implemented. Peace Corps medical officers should continue to receive an annual refresher training on dengue clinical management.

Investigation of a dengue case in a laboratory worker in North Carolina, USA, revealed that the case-patient prepared high-titer dengue virus stocks soon before illness onset. Improper doffing of gloves with an open finger wound likely resulted in cutaneous exposure. This case reinforces recommendations for enhanced precautions when working with high-titer dengue virus.

A dengue outbreak occurred on Hawaii Island between September 2015 and March 2016. Entomological investigations were undertaken between December 2015 and February 2016 to determine which Aedes mosquito species were responsible for the outbreak. A total of 3,259 mosquitoes were collected using a combination of CDC autocidal gravid ovitraps, Biogents BG-Sentinel traps, and hand-nets; immature mosquitoes were collected during environmental surveys. The composition of species was Aedes albopictus (58%), Aedes aegypti (25%), Wyeomyia mitchelli (7%), Aedes vexans (5%), Culex quinquefasciatus (4%), and Aedes japonicus (1%). Adult mosquitoes were analyzed by real-time reverse transcription polymerase chain reaction (PCR) for the presence of dengue virus (DENV) RNA. Of the 185 pools of female mosquitoes tested, 15 containing Ae. albopictus were positive for the presence of DENV type 1 RNA. No virus was detected in pools of the remaining species. Phylogenetic analysis showed the virus strain belonged to genotype I and was closely related to strains that were circulating in the Pacific between 2008 and 2014. This is the first report of detection of DENV in Ae. albopictus from Hawaii.

Fatal Dengue Acquired in Florida A woman presented with acute cholecystitis to a hospital in Miami. She subsequently died from complications of locally acquired dengue virus infection.

Zika virus (ZIKV) is a member of the Flaviviridae family, which includes other clinically notable viruses such as the 4 dengue virus serotypes (DENV-1–4). Distinguishing DENVs from ZIKV using the established serologic assays widely used for monitoring DENV transmission is difficult because of antibody cross-reactivity between these closely related flaviviruses. We describe a modified and improved recombinant envelope domain III–based serologic assay for detecting ZIKV type-specific antibodies in regions with endemic DENV transmission. When the assay was used to measure ZIKV seroprevalence in 2017 among children 9–14 years of age living in a region of the Philippines with endemic DENV transmission, we observed a ZIKV seroprevalence of 18%. Investigators should consider using the ZIKV envelope domain III–based assay, which is simple and readily adaptable for use in standard clinical and public health laboratories, to assess ZIKV seroprevalence in areas with endemic DENV transmission.

The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 103 genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases., The Trioplex real-time RT-PCR assay was developed for detection of Zika virus infections in areas with dengue and chikungunya transmission. Here, Santiago et al. describe the optimization and clinical performance of the assay, showing high sensitivity for detection and differentiation of the three viruses.

To evaluate potential enhancement of Zika virus (ZIKV) infection among patients with prior dengue virus (DENV) infection, we compared loads of viral RNA among patients infected with ZIKV (n = 1070), DENV-2 (n = 312), or DENV-3 (n = 260). Compared to patients without prior DENV infection, patients with prior DENV infection had significantly higher mean loads of viral RNA if infected with DENV-2 (10.6 vs 11.6 log10 GCE/mL, respectively; t test, P < .0001) or DENV-3 (10.3 vs 10.9 log10 GCE/mL; P < .0001), but not ZIKV (4.7 vs 4.7 log10 GCE/mL; P = .959). These findings provide evidence against in vivo enhancement of ZIKV by anti-DENV antibodies.

The global expansion of dengue viruses (DENV-1 to DENV-4) has contributed to the divergence, transmission, and establishment of genetic lineages of epidemiological concern; however, tracking the phylogenetic relationships of these virus is not always possible due to the inability of standardized sequencing procedures in resource-limited public health laboratories. Consequently, public genomic data banks contain inadequate representation of geographical regions and historical periods. In order to improve detection of the DENV-1 to DENV-4 lineages, we report the development of a serotype-specific Sanger-based method standardized to sequence DENV-1 to DENV-4 directly from clinical samples using universal primers that detect most DENV genotypes. The resulting envelope protein coding sequences are analyzed for genotyping with phylogenetic methods. We evaluated the performance of this method by detecting, amplifying, and sequencing 54 contemporary DENV isolates, including 29 clinical samples, representing a variety of genotypes of epidemiological importance and global presence. All specimens were sequenced successfully and phylogenetic reconstructions resulted in the expected genotype classification. To further improve genomic surveillance in regions where dengue is endemic, this method was transferred to 16 public health laboratories in 13 Latin American countries, to date. Our objective is to provide an accessible method that facilitates the integration of genomics with dengue surveillance.

Dengue is an acute febrile illness caused by any of four dengue virus types (DENV-1-4). DENVs are transmitted by mosquitos of the genus Aedes (1) and are endemic throughout the tropics (2). In 2010, an estimated 390 million DENV infections occurred worldwide (2). During 2007-2013, a total of three to 10 dengue cases were reported annually in Arizona and all were travel-associated. During September-December 2014, coincident with a dengue outbreak in Sonora, Mexico, 93 travel-associated dengue cases were reported in Arizona residents; 70 (75%) cases were among residents of Yuma County, which borders San Luis Río Colorado, Sonora, Mexico. San Luis Río Colorado reported its first case of locally acquired dengue in September 2014. To investigate the temporal relationship of the dengue outbreaks in Yuma County and San Luis Río Colorado and compare patient characteristics and signs and symptoms, passive surveillance data from both locations were analyzed. In addition, household-based cluster investigations were conducted near the residences of reported dengue cases in Yuma County to identify unreported cases and assess risk for local transmission. Surveillance data identified 52 locally acquired cases (21% hospitalized) in San Luis Río Colorado and 70 travel-associated cases (66% hospitalized) in Yuma County with illness onset during September-December 2014. Among 194 persons who participated in the cluster investigations in Yuma County, 152 (78%) traveled to Mexico at least monthly during the preceding 3 months. Four (2%) of 161 Yuma County residents who provided serum samples for diagnostic testing during cluster investigations had detectable DENV immunoglobulin M (IgM); one reported a recent febrile illness, and all four had traveled to Mexico during the preceding 3 months. Entomologic assessments among 105 households revealed 24 water containers per 100 houses colonized by Ae. aegypti. Frequent travel to Mexico and Ae. aegypti colonization indicate risk for local transmission of DENV in Yuma County. Public health officials in Sonora and Arizona should continue to collaborate on dengue surveillance and educate the public regarding mosquito abatement and avoidance practices. Clinicians evaluating patients from the U.S.-Mexico border region should consider dengue in patients with acute febrile illness and report suspected cases to public health authorities.

Background Anti-dengue virus (DENV) immunoglobulin M (IgM) seroconversion has been the reference standard for dengue diagnosis. However, paired specimens are rarely obtained, and the interval for this testing negates its usefulness in guiding clinical case management. The presence of DENV viremia and appearance of IgM during the febrile phase of dengue provides the framework for dengue laboratory diagnosis by using a single serum specimen. Methods Archived paired serum specimens (n = 1234) from patients with laboratory-confirmed dengue from 2005 through 2011 were used to determine the diagnostic performance of real-time reverse transcription polymerase chain reaction (RT-PCR), for detection of DENV serotypes 1-4, and enzyme-linked immunosorbent assays (ELISAs), for detection of DENV nonstructural protein 1 (NS1) antigen and anti-DENV IgM. Results During 1-3 days after illness onset, real-time RT-PCR and NS1 antigen testing detected 82%-69% and 90%-84% of cases, respectively, as viremia levels declined, while anti-DENV IgM ELISA detected 5%-41% of cases as antibody appeared. Over the 10-day period of the febrile phase of dengue, the cumulative effect of using these 3 types of tests in a diagnostic algorithm confirmed ≥90% of dengue cases. Conclusions The use of molecular or NS1 antigen tests to detect DENV and one to detect anti-DENV IgM in a single serum specimen collected during the first 10 days of illness accurately identified ≥90% of dengue primary and secondary cases.

Dengue virus (DENV) is estimated to cause 390 million infections per year worldwide. A quarter of these infections manifest clinically and are associated with a morbidity and mortality that put a significant burden on the affected regions. Reports of increased frequency, intensity, and extended geographical range of outbreaks highlight the virus’s ongoing global spread. Persistent transmission in endemic areas and the emergence in territories formerly devoid of transmission have shaped DENV’s current genetic diversity and divergence. This genetic layout is hierarchically organized in serotypes, genotypes, and sub-genotypic clades. While serotypes are well defined, the genotype nomenclature and classification system lack consistency, which complicates a broader analysis of their clinical and epidemiological characteristics. We identify five key challenges: (1) Currently, there is no formal definition of a DENV genotype; (2) Two different nomenclature systems are used in parallel, which causes significant confusion; (3) A standardized classification procedure is lacking so far; (4) No formal definition of sub-genotypic clades is in place; (5) There is no consensus on how to report antigenic diversity. Therefore, we believe that the time is right to re-evaluate DENV genetic diversity in an essential effort to provide harmonization across DENV studies., SCOPUS: ar.j, info:eu-repo/semantics/published

BACKGROUND In the absence of active blood donation screening, dengue viruses (DENV) have been implicated in only a limited number of transfusion transmissions worldwide. This study attempted to identify if blood from donors testing negative by an NS1-antigen (Ag) enzyme-linked immunosorbent assay (ELISA) but confirmed positive for DENV RNA caused DENV-related disease in recipients during the epidemic years of 2010 to 2012 in Puerto Rico. STUDY DESIGN AND METHODS Donation aliquots testing negative by an investigational NS1-Ag ELISA were stored frozen and retested retrospectively using a research transcription-mediated amplification assay (TMA) detecting DENV RNA. All RNA-reactive donations were subject to confirmatory RNA and antibody testing. Recipient tracing was conducted for all components manufactured from TMA-reactive components. Medical chart review, recipient interview, and follow-up sampling occurred for 42 recipients transfused with TMA-reactive components. RESULTS Six of 42 recipients developed new-onset fever in the 2 weeks posttransfusion; three (50%) received RNA confirmed-positive, NS1-Ag–negative red blood cell (RBC) units. One recipient of a high-titer unit (7 × 107 DENV-4 RNA copies/mL) developed severe dengue, and a second recipient had only fever recorded but had a negative sepsis work-up. New fever attributable to DENV infection in a third recipient was confounded by fever potentially attributable to posttransfusion sepsis. CONCLUSIONS In our retrospective study, NS1-Ag detected 20% of all RNA confirmed-positive donations demonstrating limitations of NS1-Ag ELISA for blood donation screening. We identified one recipient with a clinical syndrome compatible with severe dengue who had received an NS1-Ag–negative but RNA confirmed-positive RBC unit. This investigation illustrates the difficulty in confirming transfusion transmission in dengue-endemic areas among severely ill transfusion recipients.

Case detection should be improved by instituting routine laboratory-based surveillance for acute febrile illnesses in Africa. Dengue in Angola, During the 2013 dengue epidemic in Luanda, Angola, 811 dengue rapid diagnostic test–positive cases were reported to the Ministry of Health. To better understand the magnitude of the epidemic and identify risk factors for dengue virus (DENV) infection, we conducted cluster surveys around households of case-patients and randomly selected households 6 weeks after the peak of the epidemic. Of 173 case cluster participants, 16 (9%) exhibited evidence of recent DENV infection. Of 247 random cluster participants, 25 (10%) had evidence of recent DENV infection. Of 13 recently infected participants who had a recent febrile illness, 7 (54%) had sought medical care, and 1 (14%) was hospitalized with symptoms consistent with severe dengue; however, none received a diagnosis of dengue. Behavior associated with protection from DENV infection included recent use of mosquito repellent or a bed net. These findings suggest that the 2013 dengue epidemic was larger than indicated by passive surveillance data.

Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses that cause widespread, acute febrile illnesses, notably microcephaly for fetuses of infected pregnant women. Detecting the viral cause of these illnesses is paramount to determine risks to patients, counsel pregnant women, and help fight outbreaks. A combined diagnostic algorithm for ZIKV and DENV requires Reverse transcription polymerase chain reaction (RT-PCR) and IgM antibody detection. RT-PCR differentiates between DENV and ZIKV infections during the acute phases of infection, but differentiation based on IgM antibodies is currently nearly impossible in endemic areas. We have developed a ZIKV/DENV protein array and tested it with serum samples collected from ZIKV- and DENV-infected patients and healthy subjects in Puerto Rico. Our analyses reveal a biomarker panel that are capable of discriminating ZIKV and DENV infections with high accuracy, including Capsid protein from African ZIKV strain MR766, and other 5 pair of proteins (NS1, NS2A, NS3, NS4B and NS5) from ZIKV and DENV respectively. Both sensitivity and specificity of the test for ZIKV from DENV are around 90%. We propose that the ZIKV/DENV protein array will be used in future studies to discriminate patients infected with ZIKV from DENV.

Cross-reactivity within flavivirus antibody assays, produced by shared epitopes in the envelope proteins, can complicate the serological diagnosis of Zika virus (ZIKAV) infection. We assessed the utility of the plaque reduction neutralization test (PRNT) to confirm recent ZIKAV infections and rule out misleading positive immunoglobulin M (IgM) results in areas with various levels of past dengue virus (DENV) infection incidence. We reviewed PRNT results of sera collected for diagnosis of ZIKAV infection from 1 January through 31 August 2016 with positive ZIKAV IgM results, and ZIKAV and DENV PRNTs were performed. PRNT result interpretations included ZIKAV, unspecified flavivirus, DENV infection, or negative. For this analysis, ZIKAV IgM was considered false positive for samples interpreted as a DENV infection or negative. In U.S. states, 208 (27%) of 759 IgM-positive results were confirmed to be ZIKAV compared to 11 (21%) of 52 in the U.S. Virgin Islands (USVI), 15 (15%) of 103 in American Samoa, and 13 (11%) of 123 in Puerto Rico. In American Samoa and Puerto Rico, more than 80% of IgM-positive results were unspecified flavivirus infections. The false-positivity rate was 27% in U.S. states, 18% in the USVI, 2% in American Samoa, and 6% in Puerto Rico. In U.S. states, the PRNT provided a virus-specific diagnosis or ruled out infection in the majority of IgM-positive samples. Almost a third of ZIKAV IgM-positive results were not confirmed; therefore, providers and patients must understand that IgM results are preliminary. In territories with historically higher rates of DENV transmission, the PRNT usually could not differentiate between ZIKAV and DENV infections.

Sylvatic dengue viruses (DENV) are both evolutionarily and ecologically distinct from human DENV and are maintained in an enzootic transmission cycle. Evidence of sylvatic human infections from West Africa and Southeast Asia suggests that sylvatic DENV come into regular contact with humans. Thus, this potential of emergence into the human transmission cycle could limit the potential for eradicating this cycle with vaccines currently in late stages of development. We assessed the likelihood of sylvatic DENV-4 emergence in the face of natural immunity to current human strains and vaccination with two DENV-4 vaccine candidates. Our data indicate homotypic neutralization of sylvatic and human DENV-4 strains by human primary convalescent and vaccinee sera but limited heterotypic immunity. These results suggest that emergence of sylvatic strains into the human cycle would be limited by homotypic immunity mediated by virus neutralizing antibodies produced by natural infection or vaccination.

Background Dengue is a leading cause of morbidity throughout the tropics; however, accurate population-based estimates of mortality rates are not available. Methods/Principal Findings We established the Enhanced Fatal Acute Febrile Illness Surveillance System (EFASS) to estimate dengue mortality rates in Puerto Rico. Healthcare professionals submitted serum and tissue specimens from patients who died from a dengue-like acute febrile illness, and death certificates were reviewed to identify additional cases. Specimens were tested for markers of dengue virus (DENV) infection by molecular, immunologic, and immunohistochemical methods, and were also tested for West Nile virus, Leptospira spp., and other pathogens based on histopathologic findings. Medical records were reviewed and clinical data abstracted. A total of 311 deaths were identified, of which 58 (19%) were DENV laboratory-positive. Dengue mortality rates were 1.05 per 100,000 population in 2010, 0.16 in 2011 and 0.36 in 2012. Dengue mortality was highest among adults 19–64 years and seniors ≥65 years (1.17 and 1.66 deaths per 100,000, respectively). Other pathogens identified included 34 Leptospira spp. cases and one case of Burkholderia pseudomallei and Neisseria meningitidis. Conclusions/Significance EFASS showed that dengue mortality rates among adults were higher than reported for influenza, and identified a leptospirosis outbreak and index cases of melioidosis and meningitis., Author Summary Dengue is a major public health problem in the tropics. Despite its global importance, population-based mortality rates attributable to dengue are largely unknown. Dengue vaccines are now in late stage clinical trials and one vaccine has been licensed in several countries. Evidence-based decisions regarding the future use of dengue vaccines will depend on robust estimates of disease burden which should include mortality. To estimate mortality due to dengue in Puerto Rico, where dengue is endemic, we developed an enhanced surveillance system to detect fatalities due to a preceeding dengue-like acute febrile illness using more sensitive case identification and laboratory methods than the previous passive method. This surveillance system found the dengue mortality rate was 1.05 per 100,000 Puerto Rico residents in 2010, the highest rate ever detected. Among adults aged 19–64 years, mortality from dengue (1.17 deaths per 100,000) was higher than from other infectious diseases, including influenza. The utility of this enhanced surveillance system was further proven through the identification of an outbreak of leptospirosis as well as detection of other diseases of public health importance, including melioidosis and meningitis.

Zika virus is a flavivirus transmitted primarily by Aedes aegypti and Aedes albopictus mosquitoes, and infection can be asymptomatic or result in an acute febrile illness with rash (1). Zika virus infection during pregnancy is a cause of microcephaly and other severe birth defects (2). Infection has also been associated with Guillain-Barre syndrome (GBS) (3) and severe thrombocytopenia (4,5). In December 2015, the Puerto Rico Department of Health (PRDH) reported the first locally acquired case of Zika virus infection. This report provides an update to the epidemiology of and public health response to ongoing Zika virus transmission in Puerto Rico (6,7). A confirmed case of Zika virus infection is defined as a positive result for Zika virus testing by reverse transcription-polymerase chain reaction (RT-PCR) for Zika virus in a blood or urine specimen. A presumptive case is defined as a positive result by Zika virus immunoglobulin M (IgM) enzyme-linked immunosorbent assay (MAC-ELISA)* and a negative result by dengue virus IgM ELISA, or a positive test result by Zika IgM MAC-ELISA in a pregnant woman. An unspecified flavivirus case is defined as positive or equivocal results for both Zika and dengue virus by IgM ELISA. During November 1, 2015-July 7, 2016, a total of 23,487 persons were evaluated by PRDH and CDC Dengue Branch for Zika virus infection, including asymptomatic pregnant women and persons with signs or symptoms consistent with Zika virus disease or suspected GBS; 5,582 (24%) confirmed and presumptive Zika virus cases were identified. Persons with Zika virus infection were residents of 77 (99%) of Puerto Rico's 78 municipalities. During 2016, the percentage of positive Zika virus infection cases among symptomatic males and nonpregnant females who were tested increased from 14% in February to 64% in June. Among 9,343 pregnant women tested, 672 had confirmed or presumptive Zika virus infection, including 441 (66%) symptomatic women and 231 (34%) asymptomatic women. One patient died after developing severe thrombocytopenia (4). Evidence of Zika virus infection or recent unspecified flavivirus infection was detected in 21 patients with confirmed GBS. The widespread outbreak and accelerating increase in the number of cases in Puerto Rico warrants intensified vector control and personal protective behaviors to prevent new infections, particularly among pregnant women.

Chikungunya spread throughout the Dominican Republic (DR) after the first identified laboratory-confirmed cases were reported in April 2014. In June 2014, a U.S.-based service organization operating in the DR reported chikungunya-like illnesses among several staff. We assessed the incidence of chikungunya virus (CHIKV) and dengue virus (DENV) infection and illnesses and evaluated adherence to mosquito avoidance measures among volunteers/staff deployed in the DR who returned to the United States during July-August 2014. Investigation participants completed a questionnaire that collected information on demographics, medical history, self-reported illnesses, and mosquito exposures and avoidance behaviors and provided serum for CHIKV and DENV diagnostic testing by reverse transcription polymerase chain reaction and IgM enzyme-linked immunosorbent assay. Of 102 participants, 42 (41%) had evidence of recent CHIKV infection and two (2%) had evidence of recent DENV infection. Of the 41 participants with evidence of recent CHIKV infection only, 39 (95%) reported fever, 37 (90%) reported rash, and 37 (90%) reported joint pain during their assignment. All attended the organization's health trainings, and 89 (87%) sought a pretravel health consultation. Most (∼95%) used insect repellent; however, only 30% applied it multiple times daily and < 5% stayed in housing with window/door screens. In sum, CHIKV infections were common among these volunteers during the 2014 chikungunya epidemic in the DR. Despite high levels of preparation, reported adherence to mosquito avoidance measures were inconsistent. Clinicians should discuss chikungunya with travelers visiting areas with ongoing CHIKV outbreaks and should consider chikungunya when diagnosing febrile illnesses in travelers returning from affected areas.

Background. The dengue virus serotype 3 (DENV-3) Indian subcontinent strain emerged in Puerto Rico in 1998 after a 21-year absence. The rapid expansion of DENV-3 on the island correlated with the withdrawal of the other serotypes for 7 years. The DENV-3 prevalence declined in 2008 and remains undetected. Methods. We sequenced complete genomes of 92 DENV-3 clinical isolates to characterize the molecular evolution and phylogeography throughout 10 years of continued sampling (1998–2007). Results. We documented 8 distinct lineages that emerged simultaneously and evolved independently. Two of the 8 lineages were highly associated with transient introductions of foreign viruses, and 2 of the 3 endemic lineages covered the entire study period. We found evidence of temporal-geographical clustering only within the 3 endemic lineages. The phylogeography analysis combined with serotype-specific incidence data showed that transmission of a DENV serotype in a given location and time is usually correlated with the absence of the other serotype. Conclusions. Our study shows the cotransmission of DENV-3 lineages through a complex dissemination pattern dissimilar to the evolutionary dynamics of the other serotypes in the island. High virus genetic diversity and a large naive population were underlying factors in the expansion and collapse of DENV-3 in Puerto Rico.

BACKGROUND: In 2007, a total of 10,508 suspected dengue cases were reported in Puerto Rico. Blood donations were tested for dengue virus (DENV) RNA and recipients of RNA-positive donations traced to assess transfusion transmission. STUDY DESIGN AND METHODS: Blood donation samples from 2007 were maintained in a repository and tested individually for DENV RNA by transcription-mediated amplification (TMA); a subset was further tested by an enhanced TMA (eTMA) assay. TMA-reactive samples were considered confirmed if TMA (including eTMA) was repeat reactive (RR). All TMA-RR samples were tested by quantitative, DENV type–specific reverse transcriptase–polymerase chain reaction (RT-PCR) and for anti-DENV immunoglobulin (Ig)M by enzyme-linked immunosorbent assay. Samples positive by RT-PCR were further tested for infectivity in mosquito cell culture. Patients receiving components from TMA-RR donations were followed. RESULTS: Of 15,350 donation samples tested, 29 were TMA-RR for a prevalence of 1 per 529 (0.19%). DENV Types 1, 2, and 3 with viral titers of 105 to 109 copies/mL were detected by RT-PCR in 12 samples of which all were infectious in mosquito culture. Six TMA-RR samples were IgM positive. Three of the 29 recipients receiving TMA-RR donations were tested. One recipient in Puerto Rico transfused with red blood cells containing 108 copies/mL DENV-2 became febrile 3 days posttransfusion and developed dengue hemorrhagic fever. The recipient was DENV-2 RNA positive by RT-PCR; both the donor and the recipient viruses had identical envelope sequences. CONCLUSIONS: High rates of viremia were detected in blood donors in Puerto Rico coupled with the first documented transfusion transmission of severe dengue disease, suggesting that further research on interventions is needed.

Epidemic dengue fever (DF) and the emergence of dengue hemorrhagic fever (DHF) in the Americas are associated with increased endemicity and cocirculation of the 4 dengue virus (DENV) serotypes, 1–4 (1). These increases have been particularly evident in Puerto Rico, where transmission increased during the past 25 years (2–4). The first DHF epidemics in the Americas occurred in the 1980s and were caused by the Asian/American genotype of DENV-2, then new to the region, which rapidly replaced the American genotype (5–7). This replacement has been linked to a potential to cause higher viremia and severe illness (8–10). Introduction of DENV-3 in the mid 1990s and increased human population and travel further fostered larger and more frequent DF and DHF epidemics in the region (11–13). Although all 4 DENV serotypes circulate on the island, DENV-2 circulated continuously for 25 years. Previously, a partial sequence analysis from 74 DENV-2 isolates collected in Puerto Rico during 7 years throughout a 14-year period (1987–2001) showed a DENV-2 lineage evolving through a series of turnover events (14). A lineage replacement in 1994 appeared to be associated with a foreign virus but only 3 other reintroductions were found, all linked to the 1998 epidemic, the largest in Puerto Rico history (14). This was a turning point in the epidemiology of dengue, with DENV-2 (and DENV-1 and -4) rapidly declining during the expansion of DENV-3. However, transmission of DENV-2 persisted at low levels during 1999–2003 and increased thereafter. This serotype turnover offers new opportunities to study the evolution of DENV-2. Our analysis illustrates the genetic composition and population diversity of DENV-2 throughout 22 consecutive years of sampling in Puerto Rico and may explain the evolutionary resilience and long-term establishment of this virus.

Dengue is a mosquito-borne viral disease that affects 40% of the world’s population. Nearly four million U.S. citizens live in dengue-endemic areas; the most affected population resides in Puerto Rico. Data from a dengue surveillance system were used to describe all suspected cases reported in Puerto Rico in 2007. Rates of infection per 10,000 residents were calculated by age, sex, and residence. Rates and clinical outcomes were compared with those from outbreaks in 1994–1995 and 1998. In 2007, 10,508 suspected cases were reported; 52.5% persons were hospitalized, 31.8% reported hemorrhage, 2.2% had dengue hemorrhage fever, and 44 died. A total of 3,293 (33.0%) of processed specimens were laboratory positive for dengue virus (DENV); DENV-3 (1,342, 61.7%) and DENV-2 (677, 31.1%) were detected most often. The overall incidence of laboratory-positive dengue was 8.6 infections per 10,000 population. Rates were highest among persons 10–14 years of age (19.0), followed by persons 15–19 years of age (17.9) and infants (10.9). Higher rates of hospitalization and hemorrhage were reported in 2007 than in 1994–1995 or 1998. United States citizens residing in Puerto Rico are at risk of acquiring dengue. Data suggest that the severity is worsening, and persons 10–19 years of age and infants continue to be most affected.

Early diagnosis of dengue is challenging because the initial symptoms are often non-specific, viraemia may be below detectable levels and serological tests confirm dengue late in the course of illness. Identifying dengue early in the clinical course could be useful in reducing dengue virus transmission in a community. This study analyzed data from 145 laboratory-positive and 293 laboratory-negative dengue cases in Puerto Rico to define the early clinical features of dengue infection in children and adults and to identify the clinical features that predict a laboratory-positive dengue infection. Among children, rash and age were independently associated with laboratory-positive dengue infection. Rash in the absence of cough had a positive predictive value of 100% and a negative predictive value of 82.4% as a paediatric dengue screen. Among adults, eye pain, diarrhoea and absence of upper respiratory symptoms were independently associated with laboratory-positive dengue infection. No useful early predictors of dengue infection among adults were found. Using clinical features may promote earlier identification of a subset of paediatric dengue patients in Puerto Rico. Laboratory confirmation is still necessary for the accurate diagnosis of dengue infection.

Dengue virus (DENV) is a major cause of febrile illness and hemorrhagic fever in tropical and subtropical regions. Typically, patients presenting with acute dengue disease are viremic but may not have yet developed detectable titers of antibody. Therefore, early diagnosis depends mostly on detection of viral components, such as the RNA. To define the potential use of transcription-mediated amplification (TMA) DENV RNA as a diagnostic tool, we first compared its analytic sensitivity using a routine real-time reverse transcription (RT)-PCR and found that TMA is approximately 10 to 100 times more sensitive. In addition, we tested acute-phase serum samples (

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

BACKGROUND: A single instance of transfusion-transmitted dengue infection has been reported. The high incidence of dengue in endemic countries, the high proportion of asymptomatic infection, and the median 5-day viremia, however, suggest that transfusion-associated dengue transmission may be more widespread than documented. STUDY DESIGN AND METHODS: The prevalence of dengue virus (DENV) RNA was determined in all blood donations to the American Red Cross in Puerto Rico from September 20 to December 4, 2005, using a specific type of nucleic acid amplification test called transcription-mediated amplification (TMA). TMA-positive donations were defined as those having two repeatedly reactive TMA results. TMA-positive donations were tested by enzyme-linked immunosorbent assay for immunoglobulin M (IgM) antibodies, by reverse transcription–polymerase chain reaction (RT-PCR), and by viral culture. RESULTS: Twelve (0.07%) of 16,521 blood donations tested were TMA-positive. Four were positive by RT-PCR (DENV serotypes 2 and 3). Virus was cultured from 3 of 4 RT-PCR–positive donations. One of the 12 TMA-positive donations was IgM-positive. Only 5 donations remained TMA-positive when diluted 1:16, as is done for routine minipool screening for other transfusion-transmissible viral infections (hepatitis C, human immunodeficiency, West Nile viruses [WNVs]). CONCLUSION: Nearly 1 in 1000 blood donations contained DENV RNA, and virus could be cultured from TMA-positive donations, suggesting a transfusion transmission risk similar to that which existed in the United States for WNV before universal donation screening. Similar to WNV, IgM antibody screening is likely to be ineffective, and some potentially infectious donations will be missed by minipool screening. Transfusion transmission should be considered in patients with dengue after blood transfusion.

Sylvatic dengue viruses (DENVs) are transmitted between non-human primates and arboreal Aedes spp. mosquitoes in Southeast Asia and west Africa. Recent evidence suggests that the risk for re-emergence of sylvatic DENV into the urban endemic/epidemic cycle may be high, which could limit the potential for eradicating the human transmission cycle with vaccines now under development. We assessed the likelihood of sylvatic DENV re-emergence in the face of immunity to current endemic strains or vaccines by evaluating the neutralization capacity of sera from DENV vaccinees and convalescent patients after primary infection with DENV-2 and DENV-3 serotypes. Our data indicate robust homotypic cross-immunity between human sera and sylvatic DENV strains, but limited heterotypic neutralization. Should a licensed vaccine lead to the eradication of the urban transmission cycle in the future, re-emergence of sylvatic strains into the urban cycle would be limited by homotypic immunity mediated by virus-neutralizing antibodies.

Dengue has emerged globally as a major human health problem since the 1950s and is now the most important arboviral disease of humans, infecting nearly 400 million people annually. While some cases are asymptomatic, others can develop a febrile illness (dengue fever) or even progress to severe and fatal dengue. Dengue is caused by any of four closely related but distinct viruses, known as Dengue virus serotype 1 to 4 (DENV-1 to DENV-4) which are maintained in endemic transmission to humans in large urban centers of the tropics by Aedes mosquitoes. Since the early 1960s, Puerto Rico, a major metropolitan center in the Caribbean, has experienced increasingly larger and clinically more severe epidemics following the introduction of all four dengue serotypes. The first dengue hemorrhagic fever epidemic in 1986, and a particularly severe outbreak in 1998 were dominated by novel DENV-4 strains that evolved in Puerto Rico, replacing earlier strains and spreading throughout the region. Sequence characterization of 54 complete DENV-4 genomes and their comparative evolution against 74 previously published viral sequences from the region over several decades shows that DENV-4 strains from these periods were genetically distinct based on unique changes in the envelope and non-structural genes. Their replacement of earlier strains in Puerto Rico progressed rapidly, suggesting that strong natural selection played a role in their fixation. This study confirms that DENVs evolve through rapid lineage turnover driven in part by natural selection and genetic drift.

The devil in the dengue details Along with their mosquito vectors, dengue viruses are spreading worldwide to infect millions of people. For a few, subsequent infection results in lethal hemorrhagic disease. Katzelnick et al. used antibody-binding data to map structural divergence and antigenic variation among dengue viruses. Comparing results in monkeys and humans, the viruses approximately clustered into the four known groups. However, the four virus groups showed as much antigenic distance within a group as between groups. This finding helps explain why immune responses to dengue are highly variable, and it has complex implications for epidemiology, disease, and vaccine deployment. Science , this issue p. 1338

Background/Objectives In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/β and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/β signaling. Methodology/Principal Findings In order to determine the relative inhibition of IFN-α/β signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/β signaling. Most of the strains were able to inhibit IFN-α/β to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells. Conclusions The ability of DENVs to inhibit IFN-α/β signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines., Author Summary Dengue is a viral illness acquired through the bite of an infected mosquito. This flu-like illness, which in rare instances can be fatal, threatens more than half of the world’s population. Both in vitro and clinical studies looking at how the virus operates have consistently found that the interferon response is modulated by the virus during infection. We looked at the ability of dengue virus (DENV) strains to inhibit phosphorylated signal transducer and activator of transcription (pSTAT1) after IFN-β stimulation and observed that contrary to earlier published reports; all DENVs are capable of inhibiting IFN-α/β signaling. Strains from the DENV-2 sylvatic genotype, which mainly infect non-human primates (NHP), displayed an increased ability to inhibit pSTAT1 compared to the Asian strain 16681. To our surprise, DENVs were only capable of inhibiting pSTAT1 in human cell lines, but not in NHP cell lines. Inhibition of pSTAT1 is observed in both human and NHP primary dendritic cells. These results have important implications in the use of NHP cell lines for studies of IFN-α/β inhibition by DENV in vitro and may be a relevant consideration when using NHPs for DENV pre-clinical studies.

Dengue is an acute febrile illness caused by any of four mosquito-transmitted dengue virus (DENV) types. Dengue is endemic in Jamaica, where an epidemic occurred in 2012. An investigation was conducted by multiple agencies for 66 missionaries traveling from nine US states to Jamaica after 1 missionary from the group was confirmed to have dengue. Travelers were offered diagnostic testing, and a survey was administered to assess knowledge, behaviors, and illness. Of 42 survey respondents, 9 (21%) respondents reported an acute febrile illness during or after travel to Jamaica. Of 15 travelers that provided serum specimens, 4 (27%) travelers had detectable anti-DENV immunoglobulin M antibody, and 1 traveler also had DENV-1 detected by reverse transcriptase polymerase chain reaction. Recent or past infection with a DENV was evident in 93% (13 of 14) missionaries with available sera. No behavioral or demographic factors were significantly associated with DENV infection. This investigation shows that even trips of short duration to endemic areas present a risk of acquiring dengue.

To the Editor: Leptospirosis, caused by Leptospira spp. bacteria, and dengue, caused by dengue viruses (DENVs), are potentially fatal acute febrile illnesses (AFI) endemic to the tropics (1,2). Because their clinical manifestations are similar (3), leptospirosis may be misidentified as dengue (4). We report a fatal case of co-infection with Leptospira spp. and DENV-1 in a man in Puerto Rico. On May 23, 2010, a 42-year-old unemployed male carpenter came to an outpatient clinic in Puerto Rico reporting a 4-day history of fever, headache, generalized myalgia, anorexia, nausea, and vomiting. He was being treated for chronic hypertension and had been released from jail 2 weeks before illness onset. On evaluation, he was febrile, hypertensive, and tachycardic; laboratory results showed thrombocytopenia and leukocytosis with a predominance of neutrophils. Viral syndrome was diagnosed, and the patient was given acetaminophen, solumedrol, and ketoprofen. The patient returned to the clinic on May 25 with continued fever, myalgia, worsening headache, and bilateral calf pain; he was afebrile and tachycardic and appeared acutely ill. He had no rash, jaundice, icteric sclera, cardiac murmurs, or organomegaly, and his lungs were clear on auscultation. He was given intravenous (IV) saline, and results of laboratory tests performed afterward showed leukocytosis with a predominance of neutrophils, thrombocytopenia, increased blood urea nitrogen (BUN)–to-creatinine ratio, hyponatremia, hyperglycemia, and elevated aspartate aminotransferase. He was given IV ampicillin, meperidine, and promethazine and was transferred to a local hospital for admission, with a presumptive diagnosis of pre–renal azotemia and leptospirosis. On arrival at the emergency department on the same day, the patient was febrile, tachycardic, and hypotensive, with cold, clammy skin. Results of an electrocardiogram showed sinus tachycardia; cardiac enzymes were not elevated. He was given repeat IV saline and piperacillin/tazobactam. New laboratory findings included anemia, prolonged prothrombin time, elevated creatinine kinase, hematuria, and a further increase in BUN-to-creatine ratio. Chest radiograph showed cardiomegaly with increased pulmonary vascularity and perihilar alveolar densities. Arterial blood gas (ABG) results showed compensated metabolic acidosis, with low oxygen partial pressure (pO2). He was given IV saline again, and vancomycin and ceftriaxone were added to his medication regimen. On admission to the intensive care unit, the patient continued to be hypotensive and was again given IV saline. Although ABG results on the morning of May 26 were somewhat improved, the patient was started on respiratory treatments for new-onset cough and increasing respiratory rate. Laboratory test results showed a large drop in hematocrit, worsening thrombocytopenia and leukocytosis, hypocalcemia, and hypoalbuminemia; he was given an infusion of 25% albumin. The patient’s condition continued to worsen, with ABG results showing further decline in pO2. Severe respiratory distress developed, and he was placed on mechanical ventilation and given IV saline. Repeat ABG results showed severe respiratory acidosis and metabolic acidosis. Soon after, generalized edema developed, and the patient became cyanotic, with no measurable pulse; despite aggressive resuscitation efforts, he died on March 26. All results of bacterial cultures were negative, as was detection of anti-Leptospira IgM. Postmortem examination showed rash and pleural effusion, and blood and tissue specimens were taken for diagnostic testing. Liver sections showed bile stasis, dilated sinusoidal space, and pericentral hepatocellular necrosis (Figure, panel A); lung sections showed intraalveolar hemorrhage, edema, and focal inflammatory infiltrates (Figure, panel B). Heart sections showed perivascular edema, and kidney sections showed evidence of interstitial inflammatory infiltrates and acute tubular necrosis (Figure, panel C). Immunohistochemical analysis of kidney (Figure, panel D), liver, lung, and heart sections showed Leptospira antigen. Dengue virus nonstructural (NS) protein 1 was detected in whole blood, and flavivirus NS5 gene was amplified from RNA extracted from the liver; sequencing showed 98% homology with DENV-1. Figure Histopathologic evaluation of tissue samples collected postmortem from a person co-infected with Leptospira spp. and dengue virus 1. Tissue specimens were taken from the liver (A), lung (B) and kidney (C and D) and stained with hemotoxylin-eosin (A, B, ... This case report demonstrates the need for antigen-based rapid diagnostic tests (RDT) for AFI patients. All available leptospirosis RDTs detect anti-Leptospira IgM (5), which was not detectable in this patient’s blood on the seventh day of illness, although Leptospira antigen was detected in postmortem analysis. Therefore, it is unlikely that any available leptospirosis RDT would have been clinically useful when leptospirosis signs first were recorded on the fourth day of illness. Because the incidence of both dengue and leptospirosis is increasing worldwide (6,7), physicians should have access to antigen-based RDT to make timely and thorough diagnoses. Nonetheless, even if leptospirosis had been diagnosed in this patient, dengue virus infection would likely still have been overlooked. Therefore, clinicians in areas where both Leptospira spp. and DENVs are endemic should include both pathogens in the differential diagnosis when evaluating AFI patients and should consider the possibility of co-infection. Early administration of doxycycline and penicillin G to treat mild and severe leptospirosis, respectively, may reduce the duration and severity of illness (8). For cases of severe dengue, packed red blood cells should be given in response to severe anemia. For patients with either dengue or leptospirosis, intravenous fluid administration should be closely monitored to prevent fluid overload.

Identifying etiologies of acute febrile illnesses (AFI) is challenging due to non-specific presentation and limited availability of diagnostics. Prospective AFI studies provide a methodology to describe the syndrome by age and etiology, findings that can be used to develop case definitions and multiplexed diagnostics to optimize management. We conducted a 3-year prospective AFI study in Puerto Rico. Patients with fever ≤7 days were offered enrollment, and clinical data and specimens were collected at enrollment and upon discharge or follow-up. Blood and oro-nasopharyngeal specimens were tested by RT-PCR and immunodiagnostic methods for infection with dengue viruses (DENV) 1–4, chikungunya virus (CHIKV), influenza A and B viruses (FLU A/B), 12 other respiratory viruses (ORV), enterovirus, Leptospira spp., and Burkholderia pseudomallei. Clinical presentation and laboratory findings of participants infected with DENV were compared to those infected with CHIKV, FLU A/B, and ORV. Clinical predictors of laboratory-positive dengue compared to all other AFI etiologies were determined by age and day post-illness onset (DPO) at presentation. Of 8,996 participants enrolled from May 7, 2012 through May 6, 2015, more than half (54.8%, 4,930) had a pathogen detected. Pathogens most frequently detected were CHIKV (1,635, 18.2%), FLU A/B (1,074, 11.9%), DENV 1–4 (970, 10.8%), and ORV (904, 10.3%). Participants with DENV infection presented later and a higher proportion were hospitalized than those with other diagnoses (46.7% versus 27.3% with ORV, 18.8% with FLU A/B, and 11.2% with CHIKV). Predictors of dengue in participants presenting, Author summary We conducted a prospective study of acute febrile illness (AFI) in Puerto Rico to better understand the etiology of AFI among all age groups in the tropics. Such findings could assist clinicians to identify disease-specific characteristics, which can then be used to initiate proper patient management. We enrolled 8,996 AFI patients and tested them for dengue viruses 1–4 (DENV 1–4) and 21 other pathogens. A pathogen was detected in 55% of patients, most frequently chikungunya virus (CHIKV, 18%), influenza A or B virus (FLU A/B, 12%), DENV 1–4 (11%), or another respiratory virus (ORV, 10%). Participants with dengue presented later after symptom onset and were hospitalized more often (47%) than patients with another etiology of AFI (27% with ORV, 19% with FLU A/B, and 11% with CHIKV). Predictors of patients with dengue differed by timing of presentation but included eye pain, nausea, and low white blood cell or platelet counts; negative predictors included symptoms of respiratory illness. By enrolling febrile patients at clinical presentation, we identified unbiased predictors of patients with dengue as compared to other common AFI. Findings can be used to diagnose dengue patients to provide early and appropriate clinical management.

The flaviviruses dengue, West Nile, and Japanese encephalitis represent three major mosquito-borne viruses worldwide. These pathogens impact the lives of millions of individuals and potentially could affect non-endemic areas already colonized by mosquito vectors. Unintentional transport of infected vectors (Aedes and Culex spp.), traveling within endemic areas, rapid adaptation of the insects into new geographic locations, climate change, and lack of medical surveillance have greatly contributed to the increase in flaviviral infections worldwide. The mechanisms by which flaviviruses alter the immune and the central nervous system have only recently been examined despite the alarming number of infections, related deaths, and increasing global distribution. In this review, we will discuss the expansion of the geographic areas affected by flaviviruses, the potential threats to previously unaffected countries, the mechanisms of pathogenesis, and the potential therapeutic interventions to limit the devastating consequences of these viruses.

Dengue is a potentially fatal acute febrile illness caused by four mosquito-transmitted dengue viruses (DENV-1–4). Although dengue outbreaks regularly occur in many regions of the Pacific, little is known about dengue in the Republic of the Marshall Islands (RMI). To better understand dengue in RMI, we investigated an explosive outbreak that began in October 2011. Suspected cases were reported to the Ministry of Health, serum specimens were tested with a dengue rapid diagnostic test (RDT), and confirmatory testing was performed using RT-PCR and IgM ELISA. Laboratory-positive cases were defined by detection of DENV nonstructural protein 1 by RDT, DENV nucleic acid by RT-PCR, or anti-DENV IgM antibody by RDT or ELISA. Secondary infection was defined by detection of anti-DENV IgG antibody by ELISA in a laboratory-positive acute specimen. During the four months of the outbreak, 1,603 suspected dengue cases (3% of the RMI population) were reported. Of 867 (54%) laboratory-positive cases, 209 (24%) had dengue with warning signs, six (0.7%) had severe dengue, and none died. Dengue incidence was highest in residents of Majuro and individuals aged 10–29 years, and ,95% of dengue cases were experiencing secondary infection. Only DENV-4 was detected by RT-PCR, which phylogenetic analysis demonstrated was most closely related to a virus previously identified in Southeast Asia. Cases of vertical DENV transmission, and DENV/Salmonella Typhi and DENV/Mycobacterium leprae co-infection were identified. Entomological surveys implicated water storage containers and discarded tires as the most important development sites for Aedes aegypti and Ae. albopictus, respectively. Although this is the first documented dengue outbreak in RMI, the age groups of cases and high prevalence of secondary infection demonstrate prior DENV circulation. Dengue surveillance should continue to be strengthened in RMI and throughout the Pacific to identify and rapidly respond to future outbreaks.

In June of 2007, West Nile virus (WNV) was detected in sentinel chickens and blood donors in Puerto Rico, where dengue virus (DENV) is hyperendemic. Enhanced human surveillance for acute febrile illness (AFI) began in eastern Puerto Rico on July 1, 2007. Healthcare providers submitted specimens from AFI cases for WNV and DENV virology and serology testing. Over 6 months, 385 specimens were received from 282 cases; 115 (41%) specimens were DENV laboratory-positive, 86 (31%) specimens were laboratory-indeterminate, and 32 (11%) specimens were laboratory-negative for WNV and DENV. One WNV infection was detected by anti-WNV immunoglobulin M (IgM) antibody and confirmed by a plaque reduction neutralization test. DENV and WNV infections could not be differentiated in 27 cases (10%). During a period of active WNV transmission, enhanced human surveillance identified one case of symptomatic WNV infection. Improved diagnostic methods are needed to allow differentiation of WNV and DENV in dengue-endemic regions.

Background Dengue is a potentially fatal acute febrile illness (AFI) caused by four mosquito-transmitted dengue viruses (DENV-1–4) that are endemic in Puerto Rico. In January 2010, the number of suspected dengue cases reported to the passive dengue surveillance system exceeded the epidemic threshold and an epidemic was declared soon after. Methodology/Principal Findings To characterize the epidemic, surveillance and laboratory diagnostic data were compiled. A suspected case was a dengue-like AFI in a person reported by a health care provider with or without a specimen submitted for diagnostic testing. Laboratory-positive cases had: (i) DENV nucleic acid detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in an acute serum specimen; (ii) anti-DENV IgM antibody detected by ELISA in any serum specimen; or (iii) DENV antigen or nucleic acid detected in an autopsy-tissue specimen. In 2010, a total of 26,766 suspected dengue cases (7.2 per 1,000 residents) were identified, of which 46.6% were laboratory-positive. Of 7,426 RT-PCR-positive specimens, DENV-1 (69.0%) and DENV-4 (23.6%) were detected more frequently than DENV-2 (7.3%) and DENV-3 (, Author Summary Dengue is a potentially fatal acute febrile illness that is endemic throughout the tropics and sub-tropics. Dengue has been endemic in Puerto Rico for several decades and recent epidemics occurred in 1994–5, 1998 and 2007. In January 2010, dengue surveillance indicated that an epidemic had begun. The epidemic peaked in early August and ended in December with a total of 26,766 suspected dengue cases identified, of which 128 were fatal. The 2010 epidemic was one of the longest in Puerto Rico history and resulted in the greatest number of cases and deaths ever detected. We analyzed the epidemiologic and immunologic characteristics of laboratory-confirmed dengue cases and age group-specific attack rates, and determined the frequency of first DENV infection and DENV-types among persons experiencing their first infection. This analysis indicated that 10–19 year-olds were most affected during the epidemic, and that DENV-1 was roughly four times more likely to be associated with clinically apparent illness upon first DENV infection than were DENV-2 or -4. The 2010 dengue epidemic demonstrated the heavy burden of illness due to dengue in Puerto Rico, re-emphasizing the critical need for effective primary prevention tools to reduce the morbidity and mortality due to dengue worldwide.

Dengue is a disease caused by infection with one of the four dengue virus serotypes (DENV-1, -2, -3, and -4). The virus is transmitted to humans by Aedes sp. mosquitoes. This enveloped virus contains a positive single-stranded RNA genome. Clinical manifestations of dengue can have a wide range of outcomes varying from a mild febrile illness to a life-threatening condition. New techniques have largely replaced the use of DENV isolation in disease diagnosis. However, virus isolation still serves as the gold standard for detection and serotyping of DENV and is common practice in research and reference laboratories where clinical isolates of the virus are characterized and sequenced, or used for a variety of research experiments. Isolation of DENV from clinical samples can be achieved in mammalian and mosquito cells or by inoculation of mosquitoes. The experimental methods presented here describe the most common procedures used for the isolation, serotyping, propagation, and quantification of DENV.

The objective of the study was to evaluate if the antibodies elicited after immunization with a tetravalent dengue vaccine, based on chimeric yellow fever 17D/dengue viruses, can neutralize a large range of dengue viruses (DENV). A panel of 82 DENVs was developed from viruses collected primarily during the last decade in 30 countries and included the four serotypes and the majority of existing genotypes. Viruses were isolated and minimally amplified before evaluation against a tetravalent polyclonal serum generated during vaccine preclinical evaluation in monkey, a model in which protection efficacy of this vaccine has been previously demonstrated (Guirakhoo et al., 2004). Neutralization was observed across all the DENV serotypes, genotypes, geographical origins and isolation years. These data indicate that antibodies elicited after immunization with this dengue vaccine candidate should widely protect against infection with contemporary DENV lineages circulating in endemic countries.

Dengue is an acute febrile illness caused by four mosquito-borne dengue viruses (DENV-1 to -4) that are endemic throughout the tropics. After returning from a 1-week missionary trip to Haiti in October of 2010, 5 of 28 (18%) travelers were hospitalized for dengue-like illness. All travelers were invited to submit serum specimens and complete questionnaires on pre-travel preparations, mosquito avoidance practices, and activities during travel. DENV infection was confirmed in seven (25%) travelers, including all travelers that were hospitalized. Viral sequencing revealed clos- est homology to a 2007 DENV-1 isolate from the Dominican Republic. Although most (88%) travelers had a pre-travel healthcare visit, only one-quarter knew that dengue is a risk in Haiti, and one-quarter regularly used insect repellent. This report confirms recent DENV transmission in Haiti. Travelers to DENV-endemic areas should receive dengue education during pre-travel health consultations, follow mosquito avoidance recommendations, and seek medical care for febrile ill- ness during or after travel.

University of Massachusetts Medical School. Worcester, MA, USA. University of Alabama at Birmingham. Birmingham, Al, USA. Biomed, Universidad de Carabobo. Maracay, Venezuela. Biomed, Universidad de Carabobo. Maracay, Venezuela. University of Massachusetts Medical School. Worcester, MA, USA. The Broad Institute of MIT and Harvard. Cambridge, MA, USA. University of Massachusetts Medical School. Worcester, MA, USA. Universidad Central de Venezuela. Caracas, Venezuela / Banco Municipal de Sangre del Distrito Capital. Caracas, Venezuela. University of Massachusetts Medical School. Worcester, MA, USA. Faculdade de Medicina de S?o Jos? do Rio Preto. Departamento de Doen?as Infecciosas e Parasit?rias. Laborat?rio de Pesquisas em Virologia. S?o Jos? do Rio Preto, SP, Brasil. Faculdade de Medicina de S?o Jos? do Rio Preto. Departamento de Doen?as Infecciosas e Parasit?rias. Laborat?rio de Pesquisas em Virologia. S?o Jos? do Rio Preto, SP, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Instituto Nacional de Ci?ncia e Tecnologia para Febres Hemorr?gicas. Ananindeua, PA, Brasil / Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Bioqu?mica e Imunologia. Instituto Nacional de Ci?ncia e Tecnologia em Dengue. Belo Horizonte, MG, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Instituto Nacional de Ci?ncia e Tecnologia para Febres Hemorr?gicas. Ananindeua, PA, Brasil / Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Bioqu?mica e Imunologia. Instituto Nacional de Ci?ncia e Tecnologia em Dengue. Belo Horizonte, MG, Brasil. Centers for Disease Control and Prevention. SanJuan, Puerto Rico, USA. Centers for Disease Control and Prevention. SanJuan, Puerto Rico, USA. Universidad Industrial de Santander. Santander, Colombia. Massachusetts Institute of Technology. Division of Heath Science and Technology. Cambridge, MA, USA. University of Alabama at Birmingham. Birmingham, Al, USA. The Broad Institute of MIT and Harvard. Cambridge, MA, USA. The Broad Institute of MIT and Harvard. Cambridge, MA, USA. University of Massachusetts Medical School. Worcester, MA, USA. Dengue virus currently causes 50?100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007?2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007?2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007?2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level.

Background The production of type I interferon alpha/beta (IFN-α/β) is crucial to viral clearance during dengue virus (DENV) infection; however, in vitro-infected dendritic cells (DCs) exhibit a decreased capacity to respond to IFN-α/β stimulation, and antigen-presenting cells (APCs) isolated from patients with acute DENV infection exhibit defects in T cell priming. Methods In order to ascertain the stimulatory capacity of primary human monocyte-derived DCs infected with wild-type DENV isolates, representing a range of genotypes and disease outcomes, we cocultured infected DCs with allogeneic-naive CD4(+) T cells. The gene expression patterns of IFN-α/β sensitive genes were quantitated to determine if the infected DCs displayed a blunted IFN-α/β response. Results DENV-infected DCs induced the initial proliferation of naive CD4(+) T cells but they remained nonpolarized in effector function. The expression of IFN-α/β-stimulated genes was downregulated, revealing that the inhibition of IFN-α/β signaling is conserved among endemic DENV serotype 2 strains. Conclusions The failure of naive CD4(+) T cells to differentiate into IFN gamma-producing effector T cells when primed by DENV-infected DCs cannot be explained solely by a block in IFN-α/β signaling, suggesting that the ability of DENV to evade the early host response is multifaceted.

Background Periodic outbreaks of dengue fever occur in the United States Virgin Islands. In June 2005, an outbreak of dengue virus (DENV) serotype-2 with cases of dengue hemorrhagic fever (DHF) was detected in St. Croix, US Virgin Islands. The objective of this report is to describe this outbreak of DENV-2 and the findings of a case-control study examining risk factors for DHF. Methodology/Principal Findings This is the largest dengue outbreak ever recorded in St. Croix, with 331 suspected dengue cases reported island-wide during 2005 (62.2 cases/10,000 population); 54% were hospitalized, 21% had at least one hemorrhagic manifestation, 28% had thrombocytopenia, 5% had DHF and 1 patient died. Eighty-nine laboratory-positive hospitalized patients were identified. Of these, there were 15 (17%) who met the WHO criteria for DHF (cases) and 74 (83%) who did not (controls). The only variable significantly associated with DHF on bivariate or multivariable analysis was age, with an adjusted odds ratio (95% confidence interval) of 1.033 (1.003,1.064). Conclusions/Significance During this outbreak of DENV-2, a high proportion of cases developed DHF and increasing age was significantly associated with DHF.

Dengue is an emerging tropical disease infecting tens of millions of people annually. A febrile illness with potentially severe hemorrhagic manifestations, dengue is caused by mosquito-borne viruses (DENV-1 to -4) that are maintained in endemic transmission in large urban centers of the tropics with periodic epidemic cycles at 3- to 5-year intervals. Puerto Rico (PR), a major population center in the Caribbean, has experienced increasingly severe epidemics since multiple dengue serotypes were introduced beginning in the late 1970s. We document the phylodynamics of DENV-4 between 1981 and 1998, a period of dramatic ecological expansion during which evolutionary change also occurs. The timescale of viral evolution is sufficiently short that viral transmission dynamics can be elucidated from genetic diversity data. Specifically, by combining virus sequence data with confirmed case counts in PR over these two decades, we show that the pattern of cyclic epidemics is strongly correlated with coalescent estimates of effective population size that have been estimated from sampled virus sequences using Bayesian Markov Chain Monte Carlo methods. Thus, we show that the observed epidemiologic dynamics are correlated with similar fluctuations in diversity, including severe interepidemic reductions in genetic diversity compatible with population bottlenecks that may greatly impact DENV evolutionary dynamics. Mean effective population sizes based on genetic data appear to increase prior to isolation counts, suggesting a potential bias in the latter and justifying more active surveillance of DENV activity. Our analysis explicitly integrates epidemiologic and sequence data in a joint model that could be used to further explore transmission models of infectious disease.

Background. As the incidence of dengue increases globally, US travelers to endemic areas may be at an increased risk of travel-associated dengue. Methods. Data from the US Centers for Disease Control and Prevention's laboratory-based Passive Dengue Surveillance System (PDSS) were used to describe trends in travel-associated dengue reported from January 1, 1996 to December 31, 2005. The PDSS relies on provider-initiated requests for diagnostic testing of serum samples via state health departments. A case of travel-associated dengue was defined as a laboratory-positive dengue infection in a resident of the 50 US states and the District of Columbia who had been in a dengue-endemic area within 14 days before symptom onset. Dengue infection was confirmed by serologic and virologic techniques. Results. One thousand one hundred and ninety-six suspected travel-associated dengue cases were reported—334 (28%) were laboratory-positive, 597 (50%) were laboratory-negative, and 265 (22%) were laboratory-indeterminate. The incidence of laboratory-positive cases varied from 1996 to 2005, but had an overall increase with no significant trend (53.5 to 121.3 per 108 US travelers, p = 0.36). The most commonly visited regions were the Caribbean, Mexico and Central America, and Asia. The median age of laboratory-positive cases was 37 years (range

Dengue virus is sensed in mammalian cells by Toll-like receptors and DExD/H box RNA helicases, triggering a Type 1 interferon response. Interferon acts upon infected and noninfected cells by stimulating the JAK/STAT signaling pathway resulting in the activation of interferon stimulated genes that lead cells toward the establishment of an antiviral response. The recognition of the importance of this rapid protective response should come with the realization that dengue virus would circumvent the interferon response to propagate in the host. There is recent, mounting evidence for mechanisms encoded by the dengue virus that weaken interferon signaling. Nonstructural proteins expressed separately or in replicon vectors block phosphorylation and down-regulate expression of major components of the JAK/STAT pathway, causing reduced activation of gene expression in response to IFNalpha/beta interferon. As our understanding of viral-host interaction increases, opportunities for improved biological models and therapeutics discovery arise.

Dengue virus is sensed in mammalian cells by Toll-like receptors and DExD/H box RNA helicases, triggering a Type 1 interferon response. Interferon acts upon infected and noninfected cells by stimulating the JAK/STAT signaling pathway resulting in the activation of interferon stimulated genes that lead cells toward the establishment of an antiviral response. The recognition of the importance of this rapid protective response should come with the realization that dengue virus would circumvent the interferon response to propagate in the host. There is recent, mounting evidence for mechanisms encoded by the dengue virus that weaken interferon signaling. Nonstructural proteins expressed separately or in replicon vectors block phosphorylation and down-regulate expression of major components of the JAK/STAT pathway, causing reduced activation of gene expression in response to IFNα/β interferon. As our understanding of viral-host interaction increases, opportunities for improved biological models and therapeutics discovery arise.