DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus.

BACKGROUND: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. RESULTS: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. CONCLUSIONS: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Introduction and Spread of Dengue Virus 3, Florida, USA, May 2022-April 2023.

During May 2022-April 2023, dengue virus serotype 3 was identified among 601 travel-associated and 61 locally acquired dengue cases in Florida, USA. All 203 sequenced genomes belonged to the same genotype III lineage and revealed potential transmission chains in which most locally acquired cases occurred shortly after introduction, with little sustained transmission.

Oropouche Virus Disease Among U.S. Travelers - United States, 2024.

Beginning in late 2023, Oropouche virus was identified as the cause of large outbreaks in Amazon regions with known endemic transmission and in new areas in South America and the Caribbean. The virus is spread to humans by infected biting midges and some mosquito species. Although infection typically causes a self-limited febrile illness, reports of two deaths in patients with Oropouche virus infection and vertical transmission associated with adverse pregnancy outcomes have raised concerns about the threat of this virus to human health. In addition to approximately 8,000 locally acquired cases in the Americas, travel-associated Oropouche virus disease cases have recently been identified in European travelers returning from Cuba and Brazil. As of August 16, 2024, a total of 21 Oropouche virus disease cases were identified among U.S. travelers returning from Cuba. Most patients initially experienced fever, myalgia, and headache, often with other symptoms including arthralgia, diarrhea, nausea or vomiting, and rash. At least three patients had recurrent symptoms after the initial illness, a common characteristic of Oropouche virus disease. Clinicians and public health jurisdictions should be aware of the occurrence of Oropouche virus disease in U.S. travelers and request testing for suspected cases. Travelers should prevent insect bites when traveling, and pregnant persons should consider deferring travel to areas experiencing outbreaks of Oropouche virus disease.

Dengue Outbreak Response during COVID-19 Pandemic, Key Largo, Florida, USA, 2020.

We report a dengue outbreak in Key Largo, Florida, USA, from February through August 2020, during the COVID-19 pandemic. Successful community engagement resulted in 61% of case-patients self-reporting. We also describe COVID-19 pandemic effects on the dengue outbreak investigation and the need to increase clinician awareness of dengue testing recommendations.

Travel surveillance uncovers dengue virus dynamics and introductions in the Caribbean.

Dengue is the most prevalent mosquito-borne viral disease in humans, and cases are continuing to rise globally. In particular, islands in the Caribbean have experienced more frequent outbreaks, and all four dengue virus (DENV) serotypes have been reported in the region, leading to hyperendemicity and increased rates of severe disease. However, there is significant variability regarding virus surveillance and reporting between islands, making it difficult to obtain an accurate understanding of the epidemiological patterns in the Caribbean. To investigate this, we used travel surveillance and genomic epidemiology to reconstruct outbreak dynamics, DENV serotype turnover, and patterns of spread within the region from 2009-2022. We uncovered two recent DENV-3 introductions from Asia, one of which resulted in a large outbreak in Cuba, which was previously under-reported. We also show that while outbreaks can be synchronized between islands, they are often caused by different serotypes. Our study highlights the importance of surveillance of infected travelers to provide a snapshot of local introductions and transmission in areas with limited local surveillance and suggests that the recent DENV-3 introductions may pose a major public health threat in the region.

DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus.

Background: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. Results: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 101-102 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. Conclusions: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Travel surveillance uncovers dengue virus dynamics and introductions in the Caribbean.

Dengue is the most prevalent mosquito-borne viral disease in humans, and cases are continuing to rise globally. In particular, islands in the Caribbean have experienced more frequent outbreaks, and all four dengue virus (DENV) serotypes have been reported in the region, leading to hyperendemicity and increased rates of severe disease. However, there is significant variability regarding virus surveillance and reporting between islands, making it difficult to obtain an accurate understanding of the epidemiological patterns in the Caribbean. To investigate this, we used travel surveillance and genomic epidemiology to reconstruct outbreak dynamics, DENV serotype turnover, and patterns of spread within the region from 2009-2022. We uncovered two recent DENV-3 introductions from Asia, one of which resulted in a large outbreak in Cuba, which was previously under-reported. We also show that while outbreaks can be synchronized between islands, they are often caused by different serotypes. Our study highlights the importance of surveillance of infected travelers to provide a snapshot of local introductions and transmission in areas with limited local surveillance and suggests that the recent DENV-3 introductions may pose a major public health threat in the region.