OVID-19 is a severe acute respiratory infection (SARI) that emerged in early December 2019 in Wuhan, China 1. The outbreak was declared a public health emergency of international concern by the World Health Organization on 30 January 2020. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped, single-stranded positive-sense RNA virus that belongs to the Betacoronavirus genus and Coronaviridae family 2. SARS-CoV-2 is closely related genetically to bat-derived SARS-like coronaviruses 3. Human-to-human transmission occurs primarily via respiratory droplets and direct contact, similar to human influenza viruses, SARS-CoV and Middle East respiratory syndrome coronavirus 4. The most commonly reported clinical symptoms are fever, dry cough, fatigue, dyspnoea, anosmia, ageusia, or some combination of these 1,4,5. As of 16 June 2020, more than 7.9 million cases have been confirmed worldwide, resulting in 434,796 deaths 6. Brazil declared COVID-19 a national public health emergency on 3 February 2020 7. After the development of a national emergency plan and the early establishment of molecular diagnostic facilities across Brazil's network of public health laboratories, the country reported its first confirmed COVID-19 case on 25 February 2020, in a traveller returning to São Paulo from northern Italy 8. São Paulo is the largest city in South America and no other Brazilian city receives a greater proportion of international flights 9. Currently, Brazil has one of the fastest-growing COVID-19 epidemics in the world, now accounting for 1,864,681 cases and 72,100 deaths, comprising over 55% of the total number of reported cases in Latin America and the Caribbean (as of 14 July 2020) 6. About 21% of Latin American and Caribbean populations are estimated to be at risk of severe COVID-19 illness 10. The region has been experiencing large outbreaks, with growing epidemics in Brazil,
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.
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 method 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, ‘SMART-9N’, and a version compatible with barcoded PCR primers available from Oxford Nanopore Technologies, ‘Rapid SMART-9N’, for the detection, characterization, and whole-genome sequencing of RNA viruses. 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 6e00 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.
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 6e00 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.
The objective of this study was to assess the parity, presence of blood in the stomach, and the gonotrophic discordance of females of Aedes aegypti and Aedes albopictus captured in two areas of the city of São Paulo. The captures were undertaken monthly, by aspiration, in the period from January, 2015 to August, 2017. All the females of the two species had their midguts and ovaries dissected to determine the presence of blood and the parity/stage of maturation. With regard to parity, 27% and 34% of the females of Ae. aegypti and Ae. albopictus, respectively, were parous or were in advanced stages of the development of their ovaries (33% and 27%, respectively). The larger part of the females of Ae. aegypti and Ae. albopictus contained blood in their stomachs (77% and 60%, respectively), beyond which 36% and 27% of the females of Ae. aegypti and Ae. albopictus, respectively, were in gonotrophic discordance. Our results indicate favorable conditions in the study areas because of the presence of parous females. Moreover, this frequent and multiple contact of Ae. aegypti and Ae. albopictus females with vertebrate hosts, such as humans, increases the possibility of transmitting the viruses they may be carrying.
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.
Aos meus pais Vanda e Cristiano, as minhas irmãs Tâmara e Taciane, que sempre me apoiaram nas minhas escolhas, e ao meu pequeno sobrinho curioso Pedro Henrique. Ao meu namorado que sempre foi o meu maior incentivador e companheiro, sempre acreditou em mim, mais do que eu mesma. Ao meu cunhado Cláudio, meus primos Anderson e Adisson, a minha querida tia Lúcia, e todos os familiares que me apoiaram nesta etapa. À minhas amigas especiais Gisele Alves e Gislaine Alves, e toda sua família que aprendi a amar. Aos grandes professores Mauro e Julia do ensino médio, me fizeram ter certeza que a biologia era o caminho certo a seguir. A todos os professores da minha graduação em biologia, que me deram base e ensinos preciosos que me transformaram. À professora Drª. Carmen Taipe Lagos, foi minha professora de zoologia dos invertebrados, e indicou a Faculdade de Saúde Pública da USP, para a continuação dos meus estudos, ótimo conselho. Aos meus bons e grandes amigos da graduação Juliana Borges, Juliana
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