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Public health responses to outbreaks of dengue, chikungunya, and Zika virus have been stymied by the inability to control the primary vector, Aedes aegypti mosquitos. Consequently, the need for novel approaches to Aedes vector control is urgent. Placement of three autocidal gravid ovitraps (AGO traps) in ~85% of homes in a community was previously shown to sustainably reduce the density of female Ae. aegypti by >80%. Following the introduction of chikungunya virus (CHIKV) to Puerto Rico, we conducted a seroprevalence survey to estimate the prevalence of CHIKV infection in communities with and without AGO traps and evaluate their effect on reducing CHIKV transmission. Multivariate models that calculated adjusted prevalence ratios (aPR) showed that among 175 and 152 residents of communities with and without AGO traps, respectively, an estimated 26.1% and 43.8% had been infected with CHIKV (aPR = 0.50, 95% CI: 0.37-0.91). After stratification by time spent in their community, protection from CHIKV infection was strongest among residents who reported spending many or all weekly daytime hours in their community:10.3% seropositive in communities with AGO traps vs. 48.7% in communities without (PR = 0.21, 95% CI: 0.11-0.41). The age-adjusted rate of fever with arthralgia attributable to CHIKV infection was 58% (95% CI: 46-66%). The monthly number of CHIKV-infected mosquitos and symptomatic residents were diminished in communities with AGO traps compared to those without. These findings indicate that AGO traps are an effective tool that protects humans from infection with a virus transmitted by Ae. aegypti mosquitos. Future studies should evaluate their protective effectiveness in large, urban communities.

Dengue is a mosquito-borne viral illness that causes a variety of health outcomes, from a mild acute febrile illness to potentially fatal severe dengue. Between 2005 and 2010, the annual number of suspected dengue cases reported to the Passive Dengue Surveillance System (PDSS) in Puerto Rico ranged from 2,346 in 2006 to 22,496 in 2010. Like other passive surveillance systems, PDSS is subject to under-reporting. To estimate the degree of under-reporting in Puerto Rico, we built separate inpatient and outpatient probability-based multiplier models, using data from two different surveillance systems-PDSS and the enhanced dengue surveillance system (EDSS). We adjusted reported cases to account for sensitivity of diagnostic tests, specimens with indeterminate results, and differences between PDSS and EDSS in numbers of reported dengue cases. In addition, for outpatients, we adjusted for the fact that less than 100% of medical providers submit diagnostic specimens from suspected cases. We estimated that a multiplication factor of between 5 (for 2010 data) to 9 (for 2006 data) must be used to correct for the under-reporting of the number of laboratory-positive dengue inpatients. Multiplication factors of between 21 (for 2010 data) to 115 (for 2008 data) must be used to correct for the under-reporting of laboratory-positive dengue outpatients. We also estimated that, after correcting for underreporting, the mean annual rate, for 2005-2010, of medically attended dengue in Puerto Rico to be between 2.1 (for dengue inpatients) to 7.8 (for dengue outpatients) per 1,000 population. These estimated rates compare to the reported rates of 0.4 (dengue outpatients) to 0.1 (dengue inpatients) per 1,000 population. The multipliers, while subject to limitations, will help public health officials correct for underreporting of dengue cases, and thus better evaluate the cost-and-benefits of possible interventions.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal disorder characterized by fever, pancytopenia, hepatosplenomegaly, and increased serum ferritin. HLH is being increasingly reported as a complication of dengue, a common tropical acute febrile illness. After a cluster of pediatric dengue-associated HLH patients was identified during the 2012-2013 dengue epidemic in Puerto Rico, active surveillance and a case-control investigation was conducted at four referral hospitals to determine the incidence of HLH in children and identify risk factors for HLH following dengue. Patients with dengue-associated HLH (cases) were matched by month of illness onset and admission hospital to dengue patients that did not develop HLH (controls). During 2008-2013, a total of 33 HLH patients were identified, of which 22 (67%) were associated with dengue and 1 died (dengue-associated HLH case-fatality rate: 4.5%). Two patients with dengue-associated HLH had illness onset in 2009, none had illness onset during the 2010 dengue epidemic, and 20 had illness onset during the 2012-2013 epidemic. Frequency of infection with either dengue virus (DENV)-1 or DENV-4 did not differ between cases and controls. Cases were younger than controls (median age: 1 vs. 13 years, p < 0.01), were hospitalized longer (18 vs. 5 days, p < 0.01), and were admitted more frequently to pediatric intensive care units (100% vs. 16%, p < 0.01). Cases had co-infection (18.2% vs. 4.5%, p = 0.04), recent influenza-like illness (54.5% vs. 25.0%, p = 0.01), and longer duration of fever (7 vs. 5 days; p < 0.01). Cases were more likely to have lymphadenopathy, hepatomegaly, splenomegaly, anemia, and elevated liver transaminases (p ≤ 0.02). During this cluster of dengue-associated HLH cases that was temporally associated with the 2012-2013 epidemic, most patients with dengue-associated HLH were infants and had higher morbidity than dengue inpatients. Physicians throughout the tropics should be aware of HLH as a potential complication of dengue, particularly in patients with anemia and severe liver injury.

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. HuNoV replication studies have been hampered by the inability to grow the virus in cultured cells. The HuNoV genome is a positive-sense single-stranded RNA (ssRNA) molecule with three open reading frames (ORFs). We established a reverse genetics system driven by a mammalian promoter that functions without helper virus. The complete genome of the HuNoV genogroup II.3 U201 strain was cloned downstream of an elongation factor-1α (EF-1α) mammalian promoter. Cells transfected with plasmid containing the full-length genome (pHuNoV U₂₀₁F) expressed the ORF1 polyprotein, which was cleaved by the viral protease to produce the mature nonstructural viral proteins, and the capsid proteins. Progeny virus produced from the transfected cells contained the complete NoV genomic RNA (VP1, VP2, and VPg) and exhibited the same density in isopycnic cesium chloride gradients as native infectious NoV particles from a patient’s stool. This system also was applied to drive murine NoV RNA replication and produced infectious progeny virions. A GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA. RNA from virions containing the encapsidated GFP-genomic RNA was successfully transfected back into cells producing fluorescent puncta, indicating that the encapsidated RNA is replication-competent. The EF-1α mammalian promoter expression system provides the first reverse genetics system, to our knowledge, generalizable for human and animal NoVs that does not require a helper virus. Establishing a complete reverse genetics system expressed from cDNA for HuNoVs now allows the manipulation of the viral genome and production of reporter virions. Significance Human noroviruses are the predominant cause of acute gastroenteritis worldwide, but they remain noncultivatable. A tractable system is needed to understand the host restriction to cultivation. We established a reverse genetics system driven by a mammalian elongation factor-1α promoter without helper virus. This system supports genome replication, particle formation, and particles containing a GFP-marked genomic RNA. RNA from these particles is infectious. The system also produces infectious murine norovirus, confirming its broad applicability to other noroviruses.

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.

Emerging epidemics are challenging to track. Only a subset of cases is recognized and reported, as seen with the Zika virus (ZIKV) epidemic where large proportions of infection were asymptomatic. However, multiple imperfect indicators of infection provide an opportunity to estimate the underlying incidence of infection. We developed a modeling approach that integrates a generic Time-series Susceptible-Infected-Recovered epidemic model with assumptions about reporting biases in a Bayesian framework and applied it to the 2016 Zika epidemic in Puerto Rico using three indicators: suspected arboviral cases, suspected Zika-associated Guillain-Barré Syndrome cases, and blood bank data. Using this combination of surveillance data, we estimated the peak of the epidemic occurred during the week of August 15, 2016 (the 33 rd week of year), and 120 to 140 (50% credible interval [CrI], 95% CrI: 97 to 170) weekly infections per 10,000 population occurred at the peak. By the end of 2016, we estimated that approximately 890,000 (95% CrI: 660,000 to 1,100,000) individuals were infected in 2016 (26%, 95% CrI: 19% to 33%, of the population infected). Utilizing multiple indicators offers the opportunity for real-time and retrospective situational awareness to support epidemic preparedness and response.

Protein trafficking between the endoplasmic reticulum (ER) and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development.

Background After Zika virus (ZIKV) emerged in the Americas, laboratory-based surveillance for arboviral diseases in Puerto Rico was adapted to include ZIKV disease. Methods and findings Suspected cases of arboviral disease reported to Puerto Rico Department of Health were tested for evidence of infection with Zika, dengue, and chikungunya viruses by RT-PCR and IgM ELISA. To describe spatiotemporal trends among confirmed ZIKV disease cases, we analyzed the relationship between municipality-level socio-demographic, climatic, and spatial factors, and both time to detection of the first ZIKV disease case and the midpoint of the outbreak. During November 2015-December 2016, a total of 71,618 suspected arboviral disease cases were reported, of which 39,717 (55.5%; 1.1 cases per 100 residents) tested positive for ZIKV infection. The epidemic peaked in August 2016, when 71.5% of arboviral disease cases reported weekly tested positive for ZIKV infection. Incidence of ZIKV disease was highest among 20-29-year-olds (1.6 cases per 100 residents), and most (62.3%) cases were female. The most frequently reported symptoms were rash (83.0%), headache (64.6%), and myalgia (63.3%). Few patients were hospitalized (1.2%), and 13 (<0.1%) died. Early detection of ZIKV disease cases was associated with increased population size (log hazard ratio [HR]: -0.22 [95% confidence interval -0.29, -0.14]), eastern longitude (log HR: -1.04 [-1.17, -0.91]), and proximity to a city (spline estimated degrees of freedom [edf] = 2.0). Earlier midpoints of the outbreak were associated with northern latitude (log HR: -0.30 [-0.32, -0.29]), eastern longitude (spline edf = 6.5), and higher mean monthly temperature (log HR: -0.04 [-0.05, -0.03]). Higher incidence of ZIKV disease was associated with lower mean precipitation, but not socioeconomic factors. Conclusions During the ZIKV epidemic in Puerto Rico, 1% of residents were reported to public health authorities and had laboratory evidence of ZIKV disease. Transmission was first detected in urban areas of eastern Puerto Rico, where transmission also peaked earlier. These trends suggest that ZIKV was first introduced to Puerto Rico in the east before disseminating throughout the island.

Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality. During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1-3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001). Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis.

We reconstructed the 2016-2017 Zika virus epidemic in Puerto Rico by using complete genomes to uncover the epidemic's origin, spread, and evolutionary dynamics. Our study revealed that the epidemic was propelled by multiple introductions that spread across the island, intricate evolutionary patterns, and ≈10 months of cryptic transmission.