Phylogenetics, Epidemiology and Temporal Patterns of Dengue Virus in Araraquara, São Paulo State.

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 São 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.

Lessons from a Multilaboratorial Task Force for Diagnosis of a Fatal Toxoplasmosis Outbreak in Captive Primates in Brazil.

Toxoplasmosis is an important zoonotic disease caused by the parasite Toxoplasma gondii and is especially fatal for neotropical primates. In Brazil, the Ministry of Health is responsible for national epizootic surveillance, but some diseases are still neglected. Here, we present an integrated investigation of an outbreak that occurred during the first year of the COVID-19 pandemic among eleven neotropical primates housed at a primatology center in Brazil. After presenting non-specific clinical signs, all animals died within four days. A wide range of pathogens were evaluated, and we successfully identified T. gondii as the causative agent within four days after necropsies. The liver was the most affected organ, presenting hemorrhage and hepatocellular necrosis. Tachyzoites and bradyzoite cysts were observed in histological examinations and immunohistochemistry in different organs; in addition, parasitic DNA was detected through PCR in blood samples from all specimens evaluated. A high prevalence of Escherichia coli was also observed, indicating sepsis. This case highlights some of the obstacles faced by the current Brazilian surveillance system. A diagnosis was obtained through the integrated action of researchers since investigation for toxoplasmosis is currently absent in national guidelines. An interdisciplinary investigation could be a possible model for future epizootic investigations in animals.

Genetic differences of dengue virus 2 in patients with distinct clinical outcome.

The genetic diversity of the dengue virus is characterized by four circulating serotypes, several genotypes, and an increasing number of existing lineages that may have differences in the potential to cause epidemics and disease severity. Accurate identification of the genetic variability of the virus is essential to identify lineages responsible for an epidemic and understanding the processes of virus spread and virulence. Here, we characterize, using portable nanopore genomic sequencing, different lineages of dengue virus 2 (DENV-2) detected in 22 serum samples from patients with and without dengue warning signs attended at Hospital de Base of São José do Rio Preto (SJRP) in 2019, during a DENV-2 outbreak. Demographic, epidemiological, and clinical data were also analyzed. The phylogenetic reconstruction and the clinical data showed that two lineages belonging to the American/Asian genotype of DENV-2-BR3 and BR4 (BR4L1 and BR4L2)-were co-circulating in SJRP. Although preliminary, these results indicate no specific association between clinical form and phylogenetic clustering at the virus consensus sequence level. Studies with larger sample sizes and which explore single nucleotide variants are needed. Therefore, we showed that portable nanopore genome sequencing could generate quick and reliable sequences for genomic surveillance to monitor viral diversity and its association with disease severity as an epidemic unfolds.

Dynamics of Early Establishment of SARS-CoV-2 VOC Omicron Lineages in Minas Gerais, Brazil.

Brazil is one of the nations most affected by Coronavirus disease 2019 (COVID-19). The introduction and establishment of new virus variants can be related to an increase in cases and fatalities. The emergence of Omicron, the most modified SARS-CoV-2 variant, caused alarm for the public health of Brazil. In this study, we examined the effects of the Omicron introduction in Minas Gerais (MG), the second-most populous state of Brazil. A total of 430 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) samples from November 2021 to June 2022 from Belo Horizonte (BH) city were sequenced. These newly sequenced genomes comprise 72% of all previously available SARS-CoV-2 genomes for the city. Evolutionary analysis of novel viral genomes reveals that a great diversity of Omicron sublineages have circulated in BH, a pattern in-keeping with observations across Brazil more generally. Bayesian phylogeographic reconstructions indicate that this diversity is a product of a large number of international and national importations. As observed previously, São Paulo state is shown as a significant hub for viral spread throughout the country, contributing to around 70% of all viral Omicron introductions detected in MG.

Characterization of insect-specific Culex flavivirus (Flaviviridae) nucleotide sequences in mosquitoes from urban parks in São Paulo, Brazil.

BACKGROUND: Despite their worldwide occurrence, the distribution and role of insect-specific flaviviruses (ISFs) remain unclear. METHODS: We evaluated the presence of ISFs in mosquitoes collected in São Paulo, Brazil, using reverse transcription and semi-nested polymerase chain reaction (PCR). Some of the positive samples were subjected to nanopore sequencing. RESULTS: Twelve mosquito pools (2.8%) tested positive for flavivirus infection. Nanopore sequencing was successfully performed on six samples. Phylogenetic analysis grouped these sequences into genotype 2 of Culex flavivirus (CxFV). CONCLUSIONS: The identification of CxFV genotype 2 at new locations in São Paulo highlights the importance of understanding the role of ISFs in mosquito vector competence.

Molecular characterization and sequecing analysis of SARS-CoV-2 genome in Minas Gerais, Brazil.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in Wuhan, China, is the causative agent of the coronavirus disease 2019 (COVID-19). Since its first notification in São Paulo state (SP) on 26th February 2020, more than 22,300,000 cases and 619,000 deaths were reported in Brazil. In early pandemic, SARS-CoV-2 spread locally, however, over time, this virus was disseminated to other regions of the country. Herein, we performed genomic sequencing and phylogenetic analysis of SARS-CoV-2 using 20 clinical samples of COVID-19 confirmed cases from 9 cities of Minas Gerais state (MG), in order to evaluate the molecular properties of circulating viral strains in this locality from March to May 2020. Our analyses demonstrated the circulation of B.1 lineage isolates in the investigated locations and nucleotide substitutions were observed into the genomic regions related to important viral structures. Additionally, sequences generated in this study clustered with isolates from SP, suggesting a dissemination route between these two states. Alternatively, monophyletic groups of sequences from MG and other states or country were observed, indicating independent events of virus introduction. These results reinforce the need of genomic surveillance for understand the ongoing spread of emerging viral pathogens.

Retrospective Genomic Surveillance of Chikungunya Transmission in Minas Gerais State, Southeast Brazil.

Brazil accounted for a total number of 1,276,194 reported cases of chikungunya fever between 2014 and 2022. Additionally, since 2015, the country has experienced an increasing death toll, in which the Northeast and Southeast regions appear to report the worst scenarios. Although the CHIKV transmission dynamics have been studied in many parts of the country since its introduction in 2014, little is still known about chikungunya virus (CHIKV) transmission and genetic diversity in the state of Minas Gerais, located in southeast Brazil. Moreover, no studies have been published characterizing CHIKV genomic surveillance in this state. Thus, to retrospectively explore the CHIKV epidemic in Minas Gerais, we generated 40 genomes from clinical samples using Nanopore sequencing. Phylogenetic analysis indicated that multiple introductions of CHIKV occurred, likely from the northeastern Brazilian states, with the most recent common ancestral strain dating to early March 2016, which is in agreement with local epidemiological reports. Additionally, epidemiological data reveals a decline in the number of reported cases from 2017 to 2021, indicating that population immunity or changes in vector activity may have contributed to the decreasing waves of CHIKV infection. Together, our results shed light on the dispersion dynamics of CHIKV and show that infections decreased from March 2017 to January 2021 despite multiple introductions into Minas Gerais State. In conclusion, our study highlights the importance of combining genomic and epidemiological data in order to assist public health laboratories in monitoring and understanding the patterns and diversity of mosquito-borne viral epidemics. IMPORTANCE Arbovirus infections in Brazil, including chikungunya, dengue, yellow fever, and Zika, result in considerable morbidity and mortality and are pressing public health concerns. However, our understanding of these outbreaks is hampered by the limited availability of genomic data. In this study, we combine epidemiological analysis and portable genome sequencing to retrospectively describe the CHIKV epidemic in Minas Gerais between 2017 and 2021. Our results indicate that the East/Central/South African (ECSA) CHIKV lineage was introduced into Minas Gerais by three distinct events, likely from the North and Northeast regions of Brazil. Our study provides an understanding of how CHIKV initiates transmission in the region and illustrates that genomics in the field can augment traditional approaches to infectious disease surveillance and control.

Characterization of insect-specific Culex flavivirus (Flaviviridae) nucleotide sequences in mosquitoes from urban parks in São Paulo, Brazil

ABSTRACT Background: Despite their worldwide occurrence, the distribution and role of insect-specific flaviviruses (ISFs) remain unclear. Methods: We evaluated the presence of ISFs in mosquitoes collected in São Paulo, Brazil, using reverse transcription and semi-nested polymerase chain reaction (PCR). Some of the positive samples were subjected to nanopore sequencing. Results: Twelve mosquito pools (2.8%) tested positive for flavivirus infection. Nanopore sequencing was successfully performed on six samples. Phylogenetic analysis grouped these sequences into genotype 2 of Culex flavivirus (CxFV). Conclusions: The identification of CxFV genotype 2 at new locations in São Paulo highlights the importance of understanding the role of ISFs in mosquito vector competence.

SARS-CoV-2 reinfection caused by the P.1 lineage in Araraquara city, Sao Paulo State, Brazil.

Reinfection by the severe acute respiratory syndrome coronavirus type 2 (SARS-COV-2) has been reported in many countries, suggesting that the virus may continue to circulate among humans despite the possibility of local herd immunity due to massive previous infections. The emergence of variants of concern (VOC) that are more transmissible than the previous circulating ones has raised particular concerns on the vaccines effectiveness and reinfection rates. The P.1 lineage was first identified in December 2020 in Manaus city and is now globally spread. We report the first case of reinfection of SARS-CoV-2 caused by the P.1 variant outside of Manaus. The potential of these new variants to escape naturally and vaccine- induced immunity highlights the need for a global vigilance.

Epidemiologic History and Genetic Diversity Origins of Chikungunya and Dengue Viruses, Paraguay.

Paraguay has been severely affected by emergent Zika and chikungunya viruses, and dengue virus is endemic. To learn more about the origins of genetic diversity and epidemiologic history of these viruses in Paraguay, we deployed portable sequencing technologies to strengthen genomic surveillance and determine the evolutionary and epidemic history of arthropod-borne viruses (arboviruses). Samples stored at the Paraguay National Central Laboratory were sequenced and subjected to phylogenetic analysis. Among 33 virus genomes generated, we identified 2 genotypes of chikungunya and 2 serotypes of dengue virus that circulated in Paraguay during 2014-2018; the main source of these virus lineages was estimated to be Brazil. The evolutionary history inferred by our analyses precisely matched the available travel history of the patients. The genomic surveillance approach used was valuable for describing the epidemiologic history of arboviruses and can be used to determine the origins and evolution of future arbovirus outbreaks.

PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern.

With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses 1-3 , there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant first detected in the UK 4,5 could be serendipitously detected by the ThermoFisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern that lack spike Δ69-70, such as B.1.351 (also 501Y.V2) detected in South Africa 6 and P.1 (also 501Y.V3) recently detected in Brazil 7 . We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all three variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open source PCR assay to detect SARS-CoV-2 variants of concern 8 . Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence spread of B.1.1.7, B.1.351, and P.1.

Local Transmission of SARS-CoV-2 Lineage B.1.1.7, Brazil, December 2020.

In December 2020, research surveillance detected the B.1.1.7 lineage of severe acute respiratory syndrome coronavirus 2 in São 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.

First case of SARS-COV-2 sequencing in cerebrospinal fluid of a patient with suspected demyelinating disease.

The association between coronaviruses and central nervous system (CNS) demyelinating lesions has been previously shown. However, no case has been described of an association between the novel coronavirus (SARS-COV-2) and CNS demyelinating disease so far. SARS-COV-2 was previously detected in cerebrospinal fluid (CSF) sample of a patient with encephalitis. However, the virus identity was not confirmed by deep sequencing of SARS-COV-2 detected in the CSF. Here, we report a case of a patient with mild respiratory symptoms and neurological manifestations compatible with clinically isolated syndrome. The viral genome of SARS-COV-2 was detected and sequenced in CSF with 99.74-100% similarity between the patient virus and worldwide sequences. This report suggests a possible association of SARS-COV-2 infection with neurological symptoms of demyelinating disease, even in the absence of relevant upper respiratory tract infection signs.

Importation and early local transmission of COVID-19 in Brazil, 2020.

We conducted the genome sequencing and analysis of the first confirmed COVID-19 infections in Brazil. Rapid sequencing coupled with phylogenetic analyses in the context of travel history corroborate multiple independent importations from Italy and local spread during the initial stage of COVID-19 transmission in Brazil.

Genomic detection of a virus lineage replacement event of dengue virus serotype 2 in Brazil, 2019.

BACKGROUND Despite efforts to mitigate the impact of dengue virus (DENV) epidemics, the virus remains a public health problem in tropical and subtropical regions around the world. Most DENV cases in the Americas between January and July 2019 were reported in Brazil. São Paulo State in the southeast of Brazil has reported nearly half of all DENV infections in the country. OBJECTIVES To understand the origin and dynamics of the 2019 DENV outbreak. METHODS Here using portable nanopore sequencing we generated20 new DENV genome sequences from viremic patients with suspected dengue infection residing in two of the most-affected municipalities of São Paulo State, Araraquara and São José do Rio Preto. We conducted a comprehensive phylogenetic analysis with 1,630 global DENV strains to better understand the evolutionary history of the DENV lineages that currently circulate in the region. FINDINGS The new outbreak strains were classified as DENV2 genotype III (American/Asian genotype). Our analysis shows that the 2019 outbreak is the result of a novel DENV lineage that was recently introduced to Brazil from the Caribbean region. Dating phylogeographic analysis suggests that DENV2-III BR-4 was introduced to Brazil in or around early 2014, possibly from the Caribbean region. MAIN CONCLUSIONS Our study describes the early detection of a newly introduced and rapidly-expanding DENV2 virus lineage in Brazil.

Author Correction: Nanopore native RNA sequencing of a human poly(A) transcriptome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic detection of a virus lineage replacement event of dengue virus serotype 2 in Brazil, 2019

BACKGROUND Despite efforts to mitigate the impact of dengue virus (DENV) epidemics, the virus remains a public health problem in tropical and subtropical regions around the world. Most DENV cases in the Americas between January and July 2019 were reported in Brazil. São Paulo State in the southeast of Brazil has reported nearly half of all DENV infections in the country. OBJECTIVES To understand the origin and dynamics of the 2019 DENV outbreak. METHODS Here using portable nanopore sequencing we generated20 new DENV genome sequences from viremic patients with suspected dengue infection residing in two of the most-affected municipalities of São Paulo State, Araraquara and São José do Rio Preto. We conducted a comprehensive phylogenetic analysis with 1,630 global DENV strains to better understand the evolutionary history of the DENV lineages that currently circulate in the region. FINDINGS The new outbreak strains were classified as DENV2 genotype III (American/Asian genotype). Our analysis shows that the 2019 outbreak is the result of a novel DENV lineage that was recently introduced to Brazil from the Caribbean region. Dating phylogeographic analysis suggests that DENV2-III BR-4 was introduced to Brazil in or around early 2014, possibly from the Caribbean region. MAIN CONCLUSIONS Our study describes the early detection of a newly introduced and rapidly-expanding DENV2 virus lineage in Brazil.

Importation and early local transmission of COVID-19 in Brazil, 2020

We conducted the genome sequencing and analysis of the first confirmed COVID-19 infections in Brazil. Rapid sequencing coupled with phylogenetic analyses in the context of travel history corroborate multiple independent importations from Italy and local spread during the initial stage of COVID-19 transmission in Brazil. (AU)

Genomic detection of a virus lineage replacement event of dengue virus serotype 2 in Brazil, 2019

BACKGROUND Despite efforts to mitigate the impact of dengue virus (DENV) epidemics, the virus remains a public health problem in tropical and subtropical regions around the world. Most DENV cases in the Americas between January and July 2019 were reported in Brazil. São Paulo State in the southeast of Brazil has reported nearly half of all DENV infections in the country. OBJECTIVES To understand the origin and dynamics of the 2019 DENV outbreak. METHODS Here using portable nanopore sequencing we generated20 new DENV genome sequences from viremic patients with suspected dengue infection residing in two of the most-affected municipalities of São Paulo State, Araraquara and São José do Rio Preto. We conducted a comprehensive phylogenetic analysis with 1,630 global DENV strains to better understand the evolutionary history of the DENV lineages that currently circulate in the region. FINDINGS The new outbreak strains were classified as DENV2 genotype III (American/Asian genotype). Our analysis shows that the 2019 outbreak is the result of a novel DENV lineage that was recently introduced to Brazil from the Caribbean region. Dating phylogeographic analysis suggests that DENV2-III BR-4 was introduced to Brazil in or around early 2014, possibly from the Caribbean region. MAIN CONCLUSIONS Our study describes the early detection of a newly introduced and rapidly-expanding DENV2 virus lineage in Brazil.

Nanopore native RNA sequencing of a human poly(A) transcriptome.

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.