Genomic epidemiology reveals multiple introductions of Zika virus into the United States.

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States; since then, hundreds of locally acquired infections have been reported in Florida. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least 4 introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission is likely to have started in the spring of 2016-several months before its initial detection. By analysing surveillance and genetic data, we show that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions were linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions.

Local Mosquito-Borne Transmission of Zika Virus - Miami-Dade and Broward Counties, Florida, June-August 2016.

During the first 6 months of 2016, large outbreaks of Zika virus disease caused by local mosquito-borne transmission occurred in Puerto Rico and other U.S. territories, but local mosquito-borne transmission was not identified in the continental United States (1,2). As of July 22, 2016, the Florida Department of Health had identified 321 Zika virus disease cases among Florida residents and visitors, all occurring in either travelers from other countries or territories with ongoing Zika virus transmission or sexual contacts of recent travelers.* During standard case investigation of persons with compatible illness and laboratory evidence of recent Zika virus infection (i.e., a specimen positive by real-time reverse transcription-polymerase chain reaction [rRT-PCR], or positive Zika immunoglobulin M [IgM] with supporting dengue serology [negative for dengue IgM antibodies and positive for dengue IgG antibodies], or confirmation of Zika virus neutralizing antibodies by plaque reduction neutralization testing [PRNT]) (3), four persons were identified in Broward and Miami-Dade counties whose infections were attributed to likely local mosquito-borne transmission. Two of these persons worked within 120 meters (131 yards) of each other but had no other epidemiologic connections, suggesting the possibility of a local community-based outbreak. Further epidemiologic and laboratory investigations of the worksites and surrounding neighborhood identified a total of 29 persons with laboratory evidence of recent Zika virus infection and likely exposure during late June to early August, most within an approximate 6-block area. In response to limited impact on the population of Aedes aegypti mosquito vectors from initial ground-based mosquito control efforts, aerial ultralow volume spraying with the organophosphate insecticide naled was applied over a 10 square-mile area beginning in early August and alternated with aerial larviciding with Bacillus thuringiensis subspecies israelensis (Bti), a group biologic control agent, in a central 2 square-mile area. No additional cases were identified after implementation of this mosquito control strategy. No increases in emergency department (ED) patient visits associated with aerial spraying were reported, including visits for asthma, reactive airway disease, wheezing, shortness of breath, nausea, vomiting, or diarrhea. Local and state health departments serving communities where Ae. aegypti, the primary vector of Zika virus, is found should continue to actively monitor for local transmission of the virus.(†).

Prolonged Detection of Zika Virus RNA in Pregnant Women.

OBJECTIVE: Zika virus infection during pregnancy is a cause of microcephaly and other fetal brain abnormalities. Reports indicate that the duration of detectable viral RNA in serum after symptom onset is brief. In a recent case report involving a severely affected fetus, Zika virus RNA was detected in maternal serum 10 weeks after symptom onset, longer than the duration of RNA detection in serum previously reported. This report summarizes the clinical and laboratory characteristics of pregnant women with prolonged detection of Zika virus RNA in serum that were reported to the U.S. Zika Pregnancy Registry. METHODS: Data were obtained from the U.S. Zika Pregnancy Registry, an enhanced surveillance system of pregnant women with laboratory evidence of confirmed or possible Zika virus infection. For this case series, we defined prolonged detection of Zika virus RNA as Zika virus RNA detection in serum by real-time reverse transcription-polymerase chain reaction (RT-PCR) 14 or more days after symptom onset or, for women not reporting signs or symptoms consistent with Zika virus disease (asymptomatic), 21 or more days after last possible exposure to Zika virus. RESULTS: Prolonged Zika virus RNA detection in serum was identified in four symptomatic pregnant women up to 46 days after symptom onset and in one asymptomatic pregnant woman 53 days postexposure. Among the five pregnancies, one pregnancy had evidence of fetal Zika virus infection confirmed by histopathologic examination of fetal tissue, three pregnancies resulted in live births of apparently healthy neonates with no reported abnormalities, and one pregnancy is ongoing. CONCLUSION: Zika virus RNA was detected in the serum of five pregnant women beyond the previously estimated timeframe. Additional real-time RT-PCR testing of pregnant women might provide more data about prolonged detection of Zika virus RNA and the possible diagnostic, epidemiologic, and clinical implications for pregnant women.

Evaluation of the Honey-Card Technique for Detection of Transmission of Arboviruses in Florida and Comparison With Sentinel Chicken Seroconversion.

Zoonotic mosquito-borne viruses, such as the West Nile virus (WNV) and eastern equine encephalitis virus (EEEV), are major public health threats in the United States. Early detection of virus transmission and targeted vector management are critical to protect humans against these pathogens. Sentinel chickens and pool screening of mosquitoes, the most widely used methods of arbovirus early detection, have technical time-lags that compromise their early-detection value. The exploitation of sugar-feeding by trapped mosquitoes for arbovirus surveillance may represent a viable alternative to other methods. Here we compared effectiveness of sugar-impregnated nucleic-acid preserving substrates (SIPS) and sentinel chicken program for detecting WNV, EEEV, and St. Louis encephalitis virus in gravid traps, CO2-baited light traps, and resting traps at 10 locations in two Florida counties. In St. Johns County, comparable numbers of EEEV detections were made by SIPS traps (18) and sentinel chickens (22), but fewer WNV detections were made using SIPS (1) than sentinel chickens (13). In Volusia County, seven arbovirus detections were made via the sentinel chicken program (one EEEV and six WNV), whereas only one arbovirus detection (WNV) was made using SIPS. CO2-baited light traps captured >90% of total mosquitoes, yet yielded <30% of arbovirus detections. Resting traps and gravid traps captured a fraction of total mosquitoes, yet yielded roughly equivalent numbers of arbovirus detections, as did light traps. Challenges to successful deployment of SIPS include optimization of traps for collecting all vector species, increasing sugar-feeding rates of trapped vectors, and developing tractable methods for arbovirus detection.

Comparison of Test Results for Zika Virus RNA in Urine, Serum, and Saliva Specimens from Persons with Travel-Associated Zika Virus Disease - Florida, 2016.

In May 2015, Zika virus was reported to be circulating in Brazil. This was the first identified introduction of the virus in the Region of the Americas. Since that time, Zika virus has rapidly spread throughout the region. As of April 20, 2016, the Florida Department of Health Bureau of Public Health Laboratories (BPHL) has tested specimens from 913 persons who met state criteria for Zika virus testing. Among these 913 persons, 91 met confirmed or probable Zika virus disease case criteria and all cases were travel-associated (1). On the basis of previous small case studies reporting real time reverse-transcription polymerase chain reaction (RT-PCR) detection of Zika virus RNA in urine, saliva, and semen (2-6), the Florida Department of Health collected multiple specimen types from persons with suspected Zika virus disease. Test results were evaluated by specimen type and number of days after symptom onset to determine the most sensitive and efficient testing algorithm for acute Zika virus disease. Urine specimens were collected from 70 patients with suspected Zika virus disease from zero to 20 days after symptom onset. Of these, 65 (93%) tested positive for Zika virus RNA by RT-PCR. Results for 95% (52/55) of urine specimens collected from persons within 5 days of symptom onset tested positive by RT-PCR; only 56% (31/55) of serum specimens collected on the same date tested positive by RT-PCR. Results for 82% (9/11) of urine specimens collected >5 days after symptom onset tested positive by RT-PCR; none of the RT-PCR tests for serum specimens were positive. No cases had results that were exclusively positive by RT-PCR testing of saliva. BPHL testing results suggest urine might be the preferred specimen type to identify acute Zika virus disease.

Vector Competence and Capacity of Culex erraticus (Diptera: Culicidae) for Eastern Equine Encephalitis Virus in the Southeastern United States.

Field studies of the ecology of eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) in the southeastern United States have demonstrated that Culex erraticus (Dyar and Knab) is the most common mosquito at many enzootic sites and is often infected with the virus. However, the competence of Cx. erraticus for EEEV has not been explored in detail. Culex erraticus females were collected from the field and fed upon EEEV-infected chicks. The infected mosquitoes were provided honey for nutrition and to monitor for time to infectiveness. Of the mosquitoes that survived the 14-d postfeeding period, 89% were infected and 84% had evidence of a disseminated infection, though titers were generally low. EEEV was first detected in honey 6 d postinfection and was detected in samples collected from 94% of the mosquitoes with a disseminated infection overall. These data and others were then employed to estimate the relative vectorial capacity of Cx. erraticus at an EEEV enzootic site in Alabama. The vectorial capacity of Cx. erraticus at this site was 44% of Culiseta melanura (Coquillett), the accepted enzootic vector, suggesting Cx. erraticus may play a role in transmitting EEEV in areas where it is abundant and Cs. melanura rare.

Ecology of Culiseta Melanura and Other Mosquitoes (Diptera: Culicidae) from Walton County, FL, During Winter Period 2013-2014.

Winter ecology of putative vectors of eastern equine encephalomyelitis virus (EEEV) in northern Florida was investigated at field locations with evidence of historic EEEV winter transmission. Light traps and resting shelters were used to sample the mosquito community in the vicinity of eight sentinel flocks throughout the winter period (November-April) of 2013 and 2014 in Walton County, FL. Overall mosquito activity was relatively low, although mosquitoes were captured during each week of the study period. Mosquito activity was linked to morning temperature, and females were captured when ambient morning temperatures were quite low (1-5°C). Anopheles crucians Wiedemann, Culex erraticus (Dyar and Knab), Culex territans Walker, and Culiseta melanura (Coquillett) were the most commonly collected mosquito species (of 20 total species). Analysis of blood-engorged mosquitoes revealed a number of mosquito species feeding upon chickens, other birds, amphibians, and domestic and wild mammals. Cs. melanura fed primarily upon chickens and songbirds (Passeriformes), suggesting that this mosquito species is the likely winter vector of EEEV to sentinel chickens in northern Florida. Both resident and nonresident songbird species were fed upon, constituting 63.9 and 36.1% of total songbird meals, respectively. Our results suggest important roles for Cs. melanura and songbird hosts for the winter transmission of EEEV in northern Florida.

Field investigations of winter transmission of eastern equine encephalitis virus in Florida.

Studies investigating winter transmission of Eastern equine encephalitis virus (EEEV) were conducted in Hillsborough County, Florida. The virus was detected in Culiseta melanura and Anopheles quadrimaculatus in February 2012 and 2013, respectively. During the winter months, herons were the most important avian hosts for all mosquito species encountered. In collections carried out in the summer of 2011, blood meals taken from herons were still common, but less frequently encountered than in winter, with an increased frequency of mammalian- and reptile-derived meals observed in the summer. Four wading bird species (Black-crowned Night Heron [Nycticorax nycticorax], Yellow-crowned Night Heron [Nyctanassa violacea], Anhinga [Anhinga anhinga], and Great Blue Heron [Ardea herodias]) were most frequently fed upon by Cs. melanura and Culex erraticus, suggesting that these species may participate in maintaining EEEV during the winter in Florida.

Innate preference or opportunism: mosquitoes feeding on birds of prey at the Southeastern Raptor Center.

The amplification of mosquito-borne pathogens is driven by patterns of host use by vectors. While each mosquito species is innately adapted to feed upon a particular group of hosts, this "preference" is difficult to assess in field-based studies, because factors such as host defenses and spatial and temporal overlap of mosquitoes and hosts affect which host animals actually get bitten. Here we examined patterns of host use by mosquitoes feeding on caged raptors at a rehabilitation and education center for birds of prey in Alabama, U.S.A. PCR-based techniques were used to determine the host species fed upon. Of 19 raptor species at the facility, seven were found to be fed upon by mosquitoes. Feeding indices and linear regression indicated that no species or family of raptor were significantly preferred over another (R(2)=0.46). Relative abundance adjusted for bird size explained a statistically significant amount of the variation in relative host use (R(2)=0.71), suggesting that bird size is an important component of host selection by mosquitoes. These findings support the hypothesis that traits of host animals drive patterns of host use by mosquitoes in nature, an interaction that leads to amplification of mosquito-borne viruses.

Sex-biased avian host use by arbovirus vectors.

Prevalence of arthropod-borne parasites often differs drastically between host sexes. This sex-related disparity may be related to physiological (primarily hormonal) differences that facilitate or suppress replication of the pathogen in host tissues. Alternately, differences in pathogen prevalence between host sexes may be owing to differential exposure to infected vectors. Here, we report on the use of PCR-based assays recognizing bird sex chromosomes to investigate sex-related patterns of avian host use from field-collected female mosquitoes from Florida, USA. Mosquitoes took more bloodmeals from male birds (64.0% of 308 sexed samples) than female birds (36.0%), deviating significantly from a hypothetical 1:1 sex ratio. In addition, male-biased host use was consistent across mosquito species (Culex erraticus (64.4%); Culex nigripalpus (61.0%) and Culiseta melanura (64.9%)). Our findings support the hypothesis that sex-biased exposure to vector-borne pathogens contributes to disparities in parasite/pathogen prevalence between the sexes. While few studies have yet to investigate sex-biased host use by mosquitoes, the methods used here could be applied to a variety of mosquito-borne disease systems, including those that affect health of humans, domestic animals and wildlife. Understanding the mechanisms that drive sex-based disparities in host use may lead to novel strategies for interrupting pathogen/parasite transmission.

Detection of eastern equine encephalomyelitis virus RNA in North American snakes.

The role of non-avian vertebrates in the ecology of eastern equine encephalomyelitis virus (EEEV) is unresolved, but mounting evidence supports a potential role for snakes in the EEEV transmission cycle, especially as over-wintering hosts. To determine rates of exposure and infection, we examined serum samples from wild snakes at a focus of EEEV in Alabama for viral RNA using quantitative reverse transcription polymerase chain reaction. Two species of vipers, the copperhead (Agkistrodon contortrix) and the cottonmouth (Agkistrodon piscivorus), were found to be positive for EEEV RNA using this assay. Prevalence of EEEV RNA was more frequent in seropositive snakes than seronegative snakes. Positivity for the quantitative reverse transcription polymerase chain reaction in cottonmouths peaked in April and September. Body size and sex ratios were not significantly different between infected and uninfected snakes. These results support the hypothesis that snakes are involved in the ecology of EEEV in North America, possibly as over-wintering hosts for the virus.

Vertebrate hosts of Aedes aegypti and Aedes mediovittatus (Diptera: Culicidae) in rural Puerto Rico.

The distribution of Aedes (Stegomyia) aegypti (L.), the main vector of dengue viruses (DENV) worldwide, overlaps with Aedes (Gymnometopa) mediovittatus (Coquillett), the Caribbean treehole mosquito, in urban, suburban, and rural areas. Ae. mediovittatus is a competent vector of DENV with high rates of vertical DENV transmission in the laboratory. This study determined whether Ae. mediovittatus feeds on humans and compared its feeding patterns with co-occurring Ae. aegypti in two rural communities of Puerto Rico. Adult mosquitoes were captured for three consecutive days every week from July 2009 to May 2010 using BG-Sentinel traps with skin lures that were placed in the front yard of houses in both communities. Three methods were used to identify the 756 bloodmeals obtained in this study: a multiplex polymerase chain reaction (PCR) for humans and dogs targeting cytochrome b; a PCR targeting the 16S rRNA; and a nested PCR targeting cytochrome b. Ae. mediovittatus fed mostly on humans (45-52%) and dogs (28-32%) but also on cats, cows, horses, rats, pigs, goats, sheep, and chickens. Ae. aegypti fed mostly on humans (76-79%) and dogs (18-21%) but also on cats, horses, and chickens. Our results indicate that Ae. mediovittatus may have a relatively high rate of vector-human contact, which might facilitate virus transmission or harborage in rural areas of Puerto Rico.