CAMILA ALVES MAIA DA SILVA FONZAGHI

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LIM/46 - Laboratório de Parasitologia Médica, Hospital das Clínicas, Faculdade de Medicina

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  • article 0 Citação(ões) na Scopus
    Phylogenetics, Epidemiology and Temporal Patterns of Dengue Virus in Araraquara, São Paulo State
    (2024) SOUZA, Caio Santos de; CALEIRO, Giovana Santos; CLARO, Ingra Morales; JESUS, Jaqueline Goes de; COLETTI, Thais Moura; SILVA, Camila Alves Maia da; COSTA, Angela Aparecida; INENAMI, Marta; RIBEIRO, Andreia C.; FELIX, Alvina Clara; PAULA, Anderson Vicente de; FIGUEIREDO, Walter M.; LUNA, Expedito Jose de Albuquerque; SABINO, Ester C.; ROMANO, Camila M.
    Dengue virus (DENV) is a prominent arbovirus with global spread, causing approximately 390 million infections each year. In Brazil, yearly epidemics follow a well-documented pattern of serotype replacement every three to four years on average. Araraquara, located in the state of Sao Paulo, has faced significant impacts from DENV epidemics since the emergence of DENV-1 in 2010. The municipality then transitioned from low to moderate endemicity in less than 10 years. Yet, there remains an insufficient understanding of virus circulation dynamics, particularly concerning DENV-1, in the region, as well as the genetic characteristics of the virus. To address this, we sequenced 37 complete or partial DENV-1 genomes sampled from 2015 to 2022 in Araraquara. Then, using also Brazilian and worldwide DENV-1 sequences we reconstructed the evolutionary history of DENV-1 in Araraquara and estimated the time to the most recent common ancestor (tMRCA) for serotype 1, for genotype V and its main lineages. Within the last ten years, there have been at least three introductions of genotype V in Araraquara, distributed in two main lineages (L Ia and L Ib, and L II). The tMRCA for the first sampled lineage (2015/2016 epidemics) was approximately 15 years ago (in 2008). Crucially, our analysis challenges existing assumptions regarding the emergence time of the DENV-1 genotypes, suggesting that genotype V might have diverged more recently than previously described. The presence of the two lineages of genotype V in the municipality might have contributed to the extended persistence of DENV-1 in the region.
  • article 5 Citação(ões) na Scopus
    Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing
    (2023) CLARO, I. M.; RAMUNDO, M. S.; COLETTI, T. M.; SILVA, C. A. M. da; VALENCA, I. N.; CANDIDO, D. S.; SALES, F. C. S.; MANULI, E. R.; JESUS, J. G. de; PAULA, A. de; FELIX, A. C.; ANDRADE, P. D. S.; PINHO, M. C.; SOUZA, W. M.; AMORIM, M. R.; PROENCA-MODENA, J. L.; KALLAS, E. G.; LEVI, J. E.; FARIA, N. R.; SABINO, E. C.; LOMAN, N. J.; QUICK, J.
    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.