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Next-generation sequencing technologies, exponential increases in the availability of virus genomic data, and ongoing advances in phylogenomic methods have made genomic epidemiology an increasingly powerful tool for public health response to a range of mosquito-borne virus outbreaks. In this review, we offer a brief primer on the scope and methods of phylogenomic analyses that can answer key epidemiological questions during mosquito-borne virus public health emergencies. We then focus on case examples of outbreaks, including those caused by dengue, Zika, yellow fever, West Nile, and chikungunya viruses, to demonstrate the utility of genomic epidemiology to support the prevention and control of mosquito-borne virus threats. We extend these case studies with operational perspectives on how to best incorporate genomic epidemiology into structured surveillance and response programs for mosquito-borne virus control. Many tools for genomic epidemiology already exist, but so do technical and nontechnical challenges to advancing their use. Frameworks to support the rapid sharing of multidimensional data and increased cross-sector partnerships, networks, and collaborations can support advancement on all scales, from research and development to implementation by public health agencies.

The emergence of Zika virus in the Americas in 2015 and its association with birth defects and other adverse health outcomes triggered an unprecedented public health response and a demand for testing. In 2016, when Florida exceeded state public health laboratory capacity for diagnostic testing, the state formed partnerships with federal and commercial laboratories. Eighty-two percent of the testing (n = 33 802 of 41 008 specimens) by the laboratory partners, including Florida’s Bureau of Public Health Laboratories (BPHL; n = 13 074), a commercial laboratory (n = 19 214), and the Centers for Disease Control and Prevention (CDC; n = 1514), occurred from July through November 2016, encompassing the peak period of local transmission. These partnerships allowed BPHL to maintain acceptable test turnaround times of 1 to 4 days for nucleic acid testing and 3 to 7 days for serologic testing. Lessons learned from this response to inform future outbreaks included the need for early planning to establish outside partnerships, adding specimen triage strategies to surge plans, and integrating state and CDC information systems.

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.(†).

Data on the duration of detectable Zika virus–specific IgM in infected persons are limited. Neutralizing antibody cross-reactivity occurs between Zika virus and related flaviviruses, but the degree to which this confounds diagnosis is uncertain. We tested serum specimens collected 12–19 months after illness onset from patients with confirmed Zika virus disease for Zika virus IgM and Zika virus and dengue virus neutralizing antibodies. Among 62 participants, 45 (73%) had detectable Zika virus IgM and 12 (19%) had an equivocal result. Although all patients tested had Zika virus neutralizing antibodies, 39 (63%) also had neutralizing antibodies against dengue virus; of those, 12 (19%) had 4-fold higher than Zika virus titer. Prolonged detection of IgM and neutralizing antibody cross-reactivity make it difficult to determine the timing of Zika virus infection and differentiate between related flaviviruses.

The ongoing Zika epidemic in the Americas has challenged public health surveillance, response, and control systems. Even as the epidemic appears to be near its end in the Americas, it is unclear whether substantial Zika virus transmission may still be ongoing. This issue is exacerbated by large discrepancies in local case reporting and significant delays in detecting outbreaks due to surveillance gaps. To uncover locations with lingering outbreaks in the Americas, we investigated travel-associated Zika cases diagnosed in the United States and Europe to identify signatures of transmission dynamics that were not captured by local reporting. We found that a large and unreported Zika outbreak occurred in Cuba during 2017, a year after peak transmission in neighboring countries, with cases still appearing in 2018. By sequencing Zika virus from infected travelers, we show that the 2017 outbreak in Cuba was sparked by long-lived lineages of Zika virus introduced from multiple places in the Americas a year prior. Our data suggest that while aggressive mosquito control in Cuba may initially have been effective at mitigating Zika virus transmission, in the absence of vaccines, herd immunity, or strong international coordination, such control measures may need to be maintained to be effective. Our study highlights how Zika virus may still be ‘silently’ spreading in the Americas and provides a framework for more accurately understanding outbreak dynamics.

BACKGROUND AND OBJECTIVE: In July 2016, local transmission of Zika virus (ZIKV) was announced in Miami-Dade County, Florida. In this report, we describe the epidemiology of pediatric ZIKV infections in locally acquired and travel-associated cases. METHODS: All children aged 1 to 17 years tested for ZIKV between October 1, 2015, and March 29, 2017, were included. SAS 9.4 was used to analyze age, sex, race and/or ethnicity, origin of exposure, onset date, affiliation with a household cluster, clinical symptoms, hospitalizations, viremia, viruria, and antibody detection in specimens. RESULTS: Among 478 confirmed ZIKV cases in Miami-Dade County, 33 (6.9%) occurred in children (1–17 years). Twenty-seven (82.3%) cases were travel-associated. The median age of a pediatric Zika case patient was 11 years. Seventeen (51.5%) case patients were boys, and 23 (69.9%) were Hispanic. Among 31 symptomatic cases, all reported having rash, 25 (80.6%) reported fever, 9 (29.0%) reported conjunctivitis, and 7 (22.6%) reported arthralgia. Sixteen (48.5%) cases reported 2 of 4 and 8 (24.2%) reported 3 of 4 main symptoms. CONCLUSIONS: This report found that the majority of children identified during the 2016 ZIKV outbreak only presented with 2 of the 4 main symptoms. In addition, pediatric ZIKV cases were frequently associated with symptomatic household members.

Zika virus (ZIKV) is currently causing an unprecedented pandemic linked to severe congenital syndromes1,2. In July 2016, mosquito-borne ZIKV transmission was first reported in the continental United States and since then, hundreds of locally acquired infections have been described3. To gain insights into the timing, source, and likely route(s) of introduction into the United States, we tracked the virus from its first detection in Miami, Florida by direct sequencing of ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We detected at least four distinct ZIKV introductions and estimate that 11-52 introductions contributed to the outbreak in Florida. Furthermore, our data suggests that ZIKV transmission likely started in the spring of 2016 - several months before initial detection. By analyzing epidemiological, surveillance, and genetic data, we discovered that several spatially distinct ZIKV transmission zones were likely portions of the same outbreak, rather than isolated events. Our analyses show that most introductions are linked to the Caribbean, which is supported by the high incidence rates and traffic, especially via cruises, from the region into Miami. By comparing mosquito abundance and travel capacity across the United States, we find that southern Florida is especially vulnerable to ZIKV introductions and at risk of repeat occurrences. By tracking the virus from its initial introduction into the United States, we provide a deeper understanding of how ZIKV initiates and sustains transmission in new regions.

We assessed IgM detection in Zika patients from the 2016 outbreak in Miami-Dade County, Florida, USA. Of those with positive or equivocal IgM after 12-19 months, 87% (26/30) had IgM 6 months later. In a survival analysis, ≈76% had IgM at 25 months. Zika virus IgM persists for years, complicating serologic diagnosis.

Summary The Zika epidemic in the Americas has challenged surveillance and control. As the epidemic appears to be waning, it is unclear whether transmission is still ongoing, which is exacerbated by discrepancies in reporting. To uncover locations with lingering outbreaks, we investigated travel-associated Zika cases to identify transmission not captured by reporting. We uncovered an unreported outbreak in Cuba during 2017, a year after peak transmission in neighboring islands. By sequencing Zika virus, we show that the establishment of the virus was delayed by a year and that the ensuing outbreak was sparked by long-lived lineages of Zika virus from other Caribbean islands. Our data suggest that, although mosquito control in Cuba may initially have been effective at mitigating Zika virus transmission, such measures need to be maintained to be effective. Our study highlights how Zika virus may still be “silently” spreading and provides a framework for understanding outbreak dynamics. Video Abstract, Graphical Abstract, Highlights • Travel surveillance and genomics uncovered hidden Zika transmission • An unreported and 1-year delayed Zika outbreak was detected in Cuba • Mosquito control may delay, not prevent, Zika virus establishment • A surveillance framework to detect hidden outbreaks was created, A combination of travel surveillance and clinical virus genomic sequencing of infected travelers provides a framework for detecting hidden outbreaks, such as an unreported Zika outbreak in Cuba during 2017.

Objective The Centers for Disease Control and Prevention and the Association of Public Health Laboratories have proposed a new HIV-1/2 Diagnostic Algorithm: a fourth-generation HIV-1/2 Ag/Ab immunoassay (IA) followed, when repeatedly reactive, by an HIV-1/HIV-2 antibody differentiation test, and if that is non-reactive, HIV-1 nucleic acid amplification testing (NAT). The objective of the study was to evaluate performance of the new algorithm after five months of utilization in our high volume, high HIV-1 seroprevalence public health population. Methods Algorithm sensitivity and specificity was evaluated on 51,953 prospective serum or plasma specimens from individuals self-referring for HIV serostatus determination. Specimens were tested on the day of receipt or maintained at 4 °C until the next testing opportunity. If the initial HIV-1/2 Ag/Ab IA (Abbott Combo) was nonreactive, a negative lab interpretation report would follow. If the initial IA was reactive, repeat screening in duplicate was immediately performed. Repeatedly reactive specimens were tested with an HIV-1/HIV-2 differentiation assay (Multispot [MS] HIV-1/HIV-2 Rapid Test) on the same or next workday. If the Abbott Combo-MS assays were discordant, HIV-1 NAT (APTIMA® HIV-1 RNA) was performed. In addition to the algorithm performance, we also evaluated the laboratory “specimen receipt to reporting” turnaround time (TAT). Results The sensitivity and specificity of the new HIV Diagnostic Algorithm with serum and plasma specimens over the initial 5 month period was 100% (922/922) and 99.99% (51,030/51,031), respectively. Two algorithm-defined acute HIV-1 infections (AHI) were detected. In addition only 3 of the 992 MS secondary tests performed were interpreted as HIV-1 Indeterminate (HIV-1 recombinant gp41 reactivity only). Of these, 2 were HIV-1 NAT reactive, defined in-house as an early HIV infection (EHI) and one was HIV-1 NAT nonreactive, indicating a false positive initial screening result. Laboratory TAT for reporting concordant reactive Abbott Combo-MS results in ≤2 workdays was 96%, compared to 22% for reporting concordant reactive 3rd generation IA-Western blot results. Conclusions In our public health testing population, results from the new HIV Diagnostic Algorithm exceeded those of the 3rd generation IA-WB algorithm with respect to HIV-1 sensitivity. The identification of two algorithm-defined AHIs provided the opportunity to inform these individuals of their HIV status and link them to medical care earlier than the scheduled posttest counseling appointment.

Aminopeptidase N (APN, EC 3.4.11.2) is an ectopeptidase expressed in lung at the apical surface of alveolar type II epithelial cells. Its expression is upregulated during fetal lung development. To begin to understand the regulation of APN expression during lung development, we used the rapid modification of cDNA ends (RACE) to clone the 5′ end of the major APN transcript in rat lung and alveolar type II cells. The cloned sequence revealed a unique 135 bp untranslated exon which genomic cloning and restriction mapping indicated was located more than 14 kb upstream from the coding sequence. A 172 bp genomic fragment flanking the untranslated exon produced a high level of expression of a reporter gene in transient transfection assays using a human lung adenocarcinoma cell line. The DNA fragment includes elements known to be important for expression of lung specific proteins, including the surfactant-associated proteins A, B, and C and the Clara cell specific protein. Comparison of the APN genomic sequences and gene structure from human and rat suggests that the exon present in the rat lung transcript may result from the use of a previously uncharacterized APN promoter.

Genome sequencing of Zika virus samples from infected patients and Aedes aegypti mosquitoes in Florida shows that the virus was probably introduced into the United States on multiple occasions, and that the Caribbean is the most likely source. Three papers in this issue present a wealth of new Zika virus (ZIKV) genome sequences and further insights into the genetic epidemiology of ZIKV. Nathan Grubaugh et al. provide 39 new ZIKV genome sequences from infected patients and Aedes aegypti mosquitoes in Florida. Phylogenetic analysis suggests that the virus has been introduced on multiple separate occasions, probably linked to travel from the Caribbean. They find a low probability of long-term persistence of ZIKV transmission chains within Florida, suggesting that the potential for future ZIKV outbreaks there will depend on transmission dynamics in the Americas. Nuno Faria et al. and Hayden Metsky et al. reconstruct the spread of ZIKV in Brazil and the Americas. Faria et al. provide 54 new ZIKV genomes, several sequenced in real time in a mobile genomics laboratory. They trace the spatial origins and spread of ZIKV in Brazil and the Americas and date the timing of the international spread of ZIKV from Brazil. They find that northeast Brazil had a crucial role in the establishment of the epidemic and the spread of the virus within Brazil and the Americas. Metsky et al. generate 110 ZIKV genomes from clinical and mosquito samples from ten regions. They also see rapid expansion of the epidemic within Brazil and multiple introductions to other geographic areas. In agreement with Faria et al., they find that ZIKV circulated unobserved for many months before transmission was detected. Metsky et al. additionally describe ZIKV evolution and discuss how the accumulation of mutations might affect the performance of diagnostic tests in the future. Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities1,2. 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 Florida3,4. 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.

Simian virus 40 (SV40)-induced sarcomas and adenovirus 7-induced sarcomas (Adv-7) exhibit both specific tumor-specific transplantation antigens (TSTA) and cross-protective embryonic antigens at the cell surface in the LAK:LVG(SYR) strain of Syrian golden hamsters. Specific SV40 TSTA could be released from the surfaces of living hamster sarcoma cells in a 2.5% crude 1-butanol extract (CBE) and served as immunogen to protect syngeneic recipients against subsequent homologous but not heterologous tumor cell challenge. The CBE-extracted SV40-induced TSTA (tumor-specific) was observed to be free of detectable, cross-protective embryonic antigens (EA) by tumor transplantation assays. The induction of cytotoxic lymphocyte-mediated immunity with the CBE-released TSTA was dependent on the administration of a single sensitizing injection of 12-20 micrograms antigen protein. Higher concentrations (50-1,000 micrograms) of the CBE tumor cell extract, given in a single injection, enhanced tumor growth as did two injections of 12.5 micrograms CBE-extracted SV40-induced TSTA at 1-week intervals. A cross-protective antigen(s), not detected in the CBE tumor extracts, was retained in the intact, 1-butanol-extracted SV40 and Adv-7-induced tumor cell lines after completion of the CBE extraction procedure and in similarly extracted 10-day hamster fetal cells. Some alterations in the normal immunogenicity of EA extracted with CBE followed by KCI from SV40-induced sarcoma cells could be detected in the transplantation assays and lymphocyte transformation assays, whereas EA extracted from CBE-KCI-treated Adv-7 cells or 10-day hamster fetal cells retained normal immunogenicity in vivo and in vitro. These procedures provide a means for successful separation of immunogenic SV40- and Adv-7-induced TSTA from detectable, biologically active, cross-protective EA from the surfaces of these sarcoma cells.