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LIM/46 - Laboratório de Parasitologia Médica, Hospital das Clínicas, Faculdade de Medicina
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- 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.
- Local Transmission of SARS-CoV-2 Lineage B.1.1.7, Brazil, December 2020(2021) CLARO, Ingra Morales; SALES, Flavia Cristina da Silva; RAMUNDO, Mariana Severo; CANDIDO, Darlan S.; SILVA, Camila A. M.; JESUS, Jaqueline Goes de; MANULI, Erika R.; OLIVEIRA, Cristina Mendes de; SCARPELLI, Luciano; CAMPANA, Gustavo; PYBUS, Oliver G.; SABINO, Ester Cerdeira; FARIA, Nuno Rodrigues; LEVI, Jose EduardoIn December 2020, research surveillance detected the B.1.1.7 lineage of severe acute respiratory syndrome coronavirus 2 in Sao Paulo, Brazil. Rapid genomic sequencing and phylogenetic analysis revealed 2 distinct introductions of the lineage. One patient reported no international travel. There may be more infections with this lineage in Brazil than reported.
- Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil(2021) FARIA, Nuno R.; MELLAN, Thomas A.; WHITTAKER, Charles; CLARO, Ingra M.; CANDIDO, Darlan da S.; MISHRA, Swapnil; CRISPIM, Myuki A. E.; SALES, Flavia C.; HAWRYLUK, Iwona; MCCRONE, John T.; HULSWIT, Ruben J. G.; FRANCO, Lucas A. M.; RAMUNDO, Mariana S.; JESUS, Jaqueline G. de; ANDRADE, Pamela S.; COLETTI, Thais M.; FERREIRA, Giulia M.; SILVA, Camila A. M.; MANULI, Erika R.; PEREIRA, Rafael H. M.; PEIXOTO, Pedro S.; KRAEMER, Moritz U.; GABURO JR., Nelson; CAMILO, Cecilia da C.; HOELTGEBAUM, Henrique; SOUZA, William M.; ROCHA, Esmenia C.; SOUZA, Leandro M. de; PINHO, Mariana C. de; ARAUJO, Leonardo J. T.; V, Frederico S. Malta; LIMA, Aline B. de; SILVA, Joice do P.; ZAULI, Danielle A. G.; FERREIRA, Alessandro C. de S.; SCHNEKENBERG, Ricardo P.; LAYDON, Daniel J.; WALKER, Patrick G. T.; SCHLUETER, Hannah M.; SANTOS, Ana L. P. dos; VIDAL, Maria S.; CARO, Valentina S. Del; FILHO, Rosinaldo M. F.; SANTOS, Helem M. dos; AGUIAR, Renato S.; PROENCA-MODENA, Jose L. P.; NELSON, Bruce; HAY, James A.; MONOD, Melodie; MISCOURIDOU, Xenia; COUPLAND, Helen; SONABEND, Raphael; VOLLMER, Michaela; GANDY, Axel; PRETE JR., Carlos A.; NASCIMENTO, Vitor H.; SUCHARD, Marc A.; BOWDEN, Thomas A.; POND, Sergei L. K.; WU, Chieh-Hsi; RATMANN, Oliver; FERGUSON, Neil M.; DYE, Christopher; LOMAN, Nick J.; LEMEY, Philippe; RAMBAUT, Andrew; FRAIJI, Nelson A.; CARVALHO, Maria do P. S. S.; PYBUS, Oliver G.; FLAXMAN, Seth; BHATT, Samir; SABINO, Ester C.Cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Manaus, Brazil, resurged in late 2020 despite previously high levels of infection. Genome sequencing of viruses sampled in Manaus between November 2020 and January 2021 revealed the emergence and circulation of a novel SARS-CoV-2 variant of concern. Lineage P.1 acquired 17 mutations, including a trio in the spike protein (K417T, E484K, and N501Y) associated with increased binding to the human ACE2 (angiotensin-converting enzyme 2) receptor. Molecular clock analysis shows that P.1 emergence occurred around mid-November 2020 and was preceded by a period of faster molecular evolution. Using a two-category dynamical model that integrates genomic and mortality data, we estimate that P.1 may be 1.7- to 2.4-fold more transmissible and that previous (non-P.1) infection provides 54 to 79% of the protection against infection with P.1 that it provides against non-P.1 lineages. Enhanced global genomic surveillance of variants of concern, which may exhibit increased transmissibility and/or immune evasion, is critical to accelerate pandemic responsiveness.
- 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.
- Neutralisation of SARS-CoV-2 lineage P.1 by antibodies elicited through natural SARS-CoV-2 infection or vaccination with an inactivated SARS-CoV-2 vaccine: an immunological study(2021) SOUZA, William M.; AMORIM, Mariene R.; SESTI-COSTA, Renata; COIMBRA, Lais D.; BRUNETTI, Natalia S.; TOLEDO-TEIXEIRA, Daniel A.; SOUZA, Gabriela F. de; MURARO, Stefanie P.; PARISE, Pierina L.; BARBOSA, Priscilla P.; BISPO-DOS-SANTOS, Karina; MOFATTO, Luciana S.; SIMEONI, Camila L.; CLARO, Ingra M.; DUARTE, Adriana S. S.; COLETTI, Thais M.; ZANGIROLAMI, Audrey B.; COSTA-LIMA, Carolina; GOMES, Arilson B. S. P.; I, Lucas Buscaratti; SALES, Flavia C.; COSTA, Vitor A.; FRANCO, Lucas A. M.; CANDIDO, Darlan S.; PYBUS, Oliver G.; JESUS, Jaqueline G. de; SILVA, Camila A. M.; RAMUNDO, Mariana S.; FERREIRA, Giulia M.; PINHO, Mariana C.; SOUZA, Leandro M.; ROCHA, Esmenia C.; ANDRADE, Pamela S.; CRISPIM, Myuki A. E.; MAKTURA, Grazielle C.; MANULI, Erika R.; SANTOS, Magnun N. N.; CAMILO, Cecilia C.; ANGERAMI, Rodrigo N.; MORETTI, Maria L.; SPILKI, Fernando R.; ARNS, Clarice W.; ADDAS-CARVALHO, Marcelo; BENITES, Bruno D.; VINOLO, Marco A. R.; MORI, Marcelo A. S.; GABURO, Nelson; DYE, Christopher; MARQUES-SOUZA, Henrique; MARQUES, Rafael E.; FARIAS, Alessandro S.; DIAMOND, Michael S.; FARIA, Nuno R.; SABINO, Ester C.; GRANJA, Fabiana; PROENCA-MODENA, Jose LuizBackground Mutations accrued by SARS-CoV-2 lineage P.1-first detected in Brazil in early January, 2021-include amino acid changes in the receptor-binding domain of the viral spike protein that also are reported in other variants of concern, including B.1.1.7 and B.1.351. We aimed to investigate whether isolates of wild-type P.1 lineage SARS-CoV-2 can escape from neutralising antibodies generated by a polyclonal immune response. Methods We did an immunological study to assess the neutralising effects of antibodies on lineage P.1 and lineage B isolates of SARS-CoV-2, using plasma samples from patients previously infected with or vaccinated against SARS-CoV-2. Two specimens (P.1/28 and P.1/30) containing SARS-CoV-2 lineage P.1 (as confirmed by viral genome sequencing) were obtained from nasopharyngeal and bronchoalveolar lavage samples collected from patients in Manaus, Brazil, and compared against an isolate of SARS-CoV-2 lineage B (SARS.CoV2/SP02.2020) recovered from a patient in Brazil in February, 2020. Isolates were incubated with plasma samples from 21 blood donors who had previously had COVID-19 and from a total of 53 recipients of the chemically inactivated SARS-CoV-2 vaccine CoronaVac: 18 individuals after receipt of a single dose and an additional 20 individuals (38 in total) after receipt of two doses (collected 17-38 days after the most recent dose); and 15 individuals who received two doses during the phase 3 trial of the vaccine (collected 134-230 days after the second dose). Antibody neutralisation of P.1/28, P.1/30, and B isolates by plasma samples were compared in terms of median virus neutralisation titre (VNT50, defined as the reciprocal value of the sample dilution that showed 50% protection against cytopathic effects). Findings In terms of VNT50, plasma from individuals previously infected with SARS-CoV-2 had an 8.6 times lower neutralising capacity against the P.1 isolates (median VNT50 30 [IQR <20-45] for P.1/28 and 30 [<20-40] for P.1/30) than against the lineage B isolate (260 [160-400]), with a binominal model showing significant reductions in lineage P.1 isolates compared with the lineage B isolate (p <= 0.0001). Efficient neutralisation of P.1 isolates was not seen with plasma samples collected from individuals vaccinated with a first dose of CoronaVac 20-23 days earlier (VNT(50)s below the limit of detection [<20] for most plasma samples), a second dose 17-38 days earlier (median VNT50 24 [IQR <20-25] for P.1/28 and 28 [<20-25] for P.1/30), or a second dose 134-260 days earlier (all VNT(50)s below limit of detection). Median VNT(50)s against the lineage B isolate were 20 (IQR 20-30) after a first dose of CoronaVac 20-23 days earlier, 75 (<20-263) after a second dose 17-38 days earlier, and 20 (<20-30) after a second dose 134-260 days earlier. In plasma collected 17-38 days after a second dose of CoronaVac, neutralising capacity against both P.1 isolates was significantly decreased (p=0.0051 for P.1/28 and p=0.0336 for P.1/30) compared with that against the lineage B isolate. All data were corroborated by results obtained through plaque reduction neutralisation tests. Interpretation SARS-CoV-2 lineage P.1 might escape neutralisation by antibodies generated in response to polyclonal stimulation against previously circulating variants of SARS-CoV-2. Continuous genomic surveillance of SARS-CoV-2 combined with antibody neutralisation assays could help to guide national immunisation programmes.