Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing.

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters  available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

Epidemiological and clinical characteristics of the COVID-19 epidemic in Brazil.

The first case of COVID-19 was detected in Brazil on 25 February 2020. We report and contextualize epidemiological, demographic and clinical findings for COVID-19 cases during the first 3 months of the epidemic. By 31 May 2020, 514,200 COVID-19 cases, including 29,314 deaths, had been reported in 75.3% (4,196 of 5,570) of municipalities across all five administrative regions of Brazil. The R0 value for Brazil was estimated at 3.1 (95% Bayesian credible interval = 2.4-5.5), with a higher median but overlapping credible intervals compared with some other seriously affected countries. A positive association between higher per-capita income and COVID-19 diagnosis was identified. Furthermore, the severe acute respiratory infection cases with unknown aetiology were associated with lower per-capita income. Co-circulation of six respiratory viruses was detected but at very low levels. These findings provide a comprehensive description of the ongoing COVID-19 epidemic in Brazil and may help to guide subsequent measures to control virus transmission.

Silent circulation of dengue virus in Aedes albopictus (Diptera: Culicidae) resulting from natural vertical transmission.

Vertical transmission in Aedes aegypti and Aedes albopictus is considered a maintenance mechanism for dengue virus (DENV) during unfavorable conditions and may be implicated in dengue outbreaks. Since DENV infection dynamics vary among wild-type viruses and vector populations, vertical transmission rates can also vary between regions. However, even though São Paulo is the most populous city in the Americas and has experienced major dengue epidemics, natural vertical transmission had never been detected in this area before. Here we confirm and describe for the first time natural vertical transmission of DENV-3 in two pools of male Ae. albopictus from the city of São Paulo. The detection of DENV-3 in years when no human autochthonous cases of this serotype were recorded suggests that silent circulation of DENV-3 is occurring and indicates that green areas may be maintaining serotypes that are not circulating in the human population, possibly by a vertical transmission mechanism.

Gonotrophic discordance in Culex quinquefasciatus (Diptera: Culicidae) in the city of São Paulo, Brazil

Abstract INTRODUCTION: This study aimed to assess the occurrence of gonotrophic discordance in females of Culex quinquefasciatus in São Paulo, Brazil. METHODS: Resting females were collected monthly for 8 months. Females of Cx. quinquefasciatus were identified, and their midgut and ovaries were dissected. RESULTS: Two hundred females were dissected, out of which, 27.5% were nulliparous and 57% were parous. Most females had no blood in the midgut, but gonotrophic discordance was found in 21% females. CONCLUSIONS: Females of Cx. quinquefasciatus showed a high parity rate and gonotrophic discordance, which could favor the vector capacity of this species.

Gonotrophic discordance in Culex quinquefasciatus (Diptera: Culicidae) in the city of São Paulo, Brazil.

INTRODUCTION: This study aimed to assess the occurrence of gonotrophic discordance in females of Culex quinquefasciatus in São Paulo, Brazil. METHODS: Resting females were collected monthly for 8 months. Females of Cx. quinquefasciatus were identified, and their midgut and ovaries were dissected. RESULTS: Two hundred females were dissected, out of which, 27.5% were nulliparous and 57% were parous. Most females had no blood in the midgut, but gonotrophic discordance was found in 21% females. CONCLUSIONS: Females of Cx. quinquefasciatus showed a high parity rate and gonotrophic discordance, which could favor the vector capacity of this species.

Paridade, desenvolvimento ovariano e transmissão vertical do vírus dengue em fêmeas de Aedes aegypti e Aedes albopictus (Diptera: Culicidae), na cidade de São Paulo / Parity ovarian development and vertical transmission of dengue virus in Aedes aegypti and Aedes albopictus (Diptera: Culicidae), in the city of São Paulo

No Brasil, a transmissão dos quatro sorotipos do vírus dengue ao homem ocorre através da picada da fêmea infectada de Aedes aegypti, também considerado vetor principal de outros arbovírus, como chikungunya e Zika. Além dessa espécie, o Aedes albopictus é considerado vetor potencial desses arbovírus. Populações brasileiras de Ae. aegypti e Ae. albopictus demonstraram a capacidade de realizar alguns repastos sanguíneos em diferentes hospedeiros dentro de um único ciclo de oviposição, o que chamamos de discordância gonotrófica. Paralelamente, a vigilância do DENV a partir de sua detecção em fêmeas de Ae. aegypti e Ae. albopictus que nunca se alimentaram de sangue é essencial para uma melhor compreensão da dinâmica de transmissão vertical desse agente etiológico. O objetivo desse estudo foi avaliar a paridade, a presença de sangue no estômago e o desenvolvimento ovariano de fêmeas de Ae. aegypti e de Ae. albopictus coletadas no Parque Esporte para Todos da Universidade de São Paulo - Cidade Universitária e na Faculdade de Saúde Pública da Universidade de São Paulo, bem como detectar DENV em fêmeas dessas espécies, oriundas de ovos coletados no Parque Municipal do Piqueri, São Paulo. Fêmeas de Ae. aegypti e Ae. albopictus capturadas tiveram o abdômen e ovários avaliados, através da técnica de dissecção. A investigação do RNA viral foi feita através de Transcrição Reversa seguida da Reação em Cadeia da Polimerase (RT-PCR). Foram encontrados 27% e 34% de fêmeas de Ae. aegypti e Ae. albopictus paridas, respectivamente, bem como 77% e 60% de fêmeas de Ae. aegypti e Ae. albopictus, respectivamente, com sangue no estômago. Foi constatado que 36% das fêmeas de Ae. aegypti estavam em discordância gonotrófica, enquanto essa taxa para Ae. albopictus foi de 27%. A transmissão vertical não foi detectada nas populações estudas. O campus da Faculdade de Saúde Pública e o Parque dos Esportes mostraram-se favoráveis para a manutenção e longevidade das duas espécies estudas. Apesar da transmissão vertical natural não ter sido detectada em nosso estudo, a investigação desse fenômeno é essencial para a compreensão da dinâmica de transmissão de arbovírus na natureza

In Brazil, the transmission of the four serotypes of the dengue virus to human is transmitted out through the bite of the infected female of Aedes aegypti, it is also considered a vector of the main etiological agents of other arboviruses, such as chikungunya and Zika. In addition, Aedes albopictus is considered a potential arbovirus vector. Brazilian populations of Ae. aegypti and Ae. albopictus have already demonstrated an ability to perform some blood supply in different hosts within a single oviposition cycle, which we call gonotrophic discordance. At the same time, surveillance of the DENV from its detection in Ae. aegypti and Ae. albopictus that has never fed on blood is essential for a better understanding of the dynamics of vertical transmission of etiologic agent. The objective was to evaluate the parity, the presence of blood in the stomach and the ovarian development of Ae. aegypti and Ae. albopictus collected in the Parque Esporte para todos of Universidade de São Paulo - Cidade Universitária and Faculdade de Saúde Pública of Universidade de São Paulo, as well as detecting DENV in females of these species, from eggs collected in the Parque Municipal do Piqueri, São Paulo. Females of Ae. aegypti and Ae. albopictus captured, from the results obtained, through the technique of dissection. The investigation of the virus was done through Reverse Transcription followed by Polymerase Chain Reaction. We found 27% and 34% of Ae. aegypti and Ae. albopictus parous, 77% and 60% of Ae. aegypti and Ae. albopictus, respectively, with blood in the stomach. It was found that 36% of Ae. aegypti was in gonotrophic discordance, while that rate for Ae. albopictus was 27%. Vertical transmission was not detected in the selections studied. The campus of the Faculdade de Saúde Pública and the Parque Esporte para todos were favorable to the maintenance and longevity of the two species studied. Vertical nature transmission was not detected in our study, an investigation is an essential event for understanding the transmission of arbovirus data in nature