Tracking arboviruses, their transmission vectors and potential hosts by nanopore sequencing of mosquitoes

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.</p

Real-time RT-PCR for Venezuelan equine encephalitis complex, Madariaga and Eastern equine encephalitis viruses: application in human and mosquito public health surveillance in Panama

Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), and Venezuelan equine encephalitis virus complex (VEEV) are New World alphaviruses transmitted by mosquitoes. They cause febrile and sometimes severe neurological disease in human and equine hosts. Detecting them during the acute phase is hindered by nonspecific symptoms and limited diagnostic tools. We designed and clinically assessed reverse transcription polymerase chain reaction assays (rRT-PCRs) for VEEV complex, MADV, and EEEV using whole-genome sequences. Validation involved 15 retrospective serum samples from 2015-2017 outbreaks, 150 mosquito pools from 2015, and 118 prospective samples from 2021-2022 surveillance in Panama. The rRT-PCRs detected VEEV complex RNA in 10 samples (66.7%) from outbreaks, with one having both VEEV complex and MADV RNAs. VEEV complex RNA was found in 5 suspected dengue cases from disease surveillance. The rRT-PCR assays identified VEEV complex RNA in 3 Culex (Melanoconion) vomerifer pools, leading to VEEV isolates in 2. Phylogenetic analysis revealed the VEEV ID subtype in positive samples. Notably, 11.9% of dengue-like disease patients showed VEEV infections. Together, our rRT-PCR validation in human and mosquito samples suggests this method can be incorporated into mosquito and human encephalitic alphavirus surveillance programs in endemic regions

Zika virus infection among symptomatic patients from two healthcare centers in Sao Paulo State, Brazil: prevalence, clinical characteristics, viral detection in body fluids and serodynamics.

Zika virus (ZIKV) clinical presentation and frequency/duration of shedding need further clarification. Symptomatic ZIKV-infected individuals identified in two hospitals in Sao Paulo State, Brazil, were investigated regarding clinical characteristics, shedding in body fluids, and serodynamics. Ninety-four of 235 symptomatic patients (Site A: 58%; Site B: 16%) had Real-Time PCR-confirmed ZIKV infection; fever, headache and gastrointestinal symptoms were less frequent, and rash was more frequent compared to ZIKV-negative patients. Real-Time PCR in serum had worse performance compared to plasma, while urine had the highest sensitivity. Shedding in genital fluids and saliva was rare. IgM positivity was the highest 28 days (24%); IgG positivity increased >14 days (96%) remaining positive in 94% of patients >28 days. ZIKV prevalence varied importantly in two neighboring cities during the same transmission season. Urine Real-Time PCR can improve diagnostic sensitivity; serum testing is less useful. Accurate serological tests are needed to improve diagnosis and surveillance

Zika virus infection among symptomatic patients from two healthcare centers in Sao Paulo State, Brazil: prevalence, clinical characteristics, viral detection in body fluids and serodynamics

Zika virus (ZIKV) clinical presentation and frequency/duration of shedding need further clarification. Symptomatic ZIKV-infected individuals identified in two hospitals in Sao Paulo State, Brazil, were investigated regarding clinical characteristics, shedding in body fluids, and serodynamics. Ninety-four of 235 symptomatic patients (Site A: 58%; Site B: 16%) had Real-Time PCR-confirmed ZIKV infection; fever, headache and gastrointestinal symptoms were less frequent, and rash was more frequent compared to ZIKV-negative patients. Real-Time PCR in serum had worse performance compared to plasma, while urine had the highest sensitivity. Shedding in genital fluids and saliva was rare. IgM positivity was the highest &lt;14 days after the symptoms onset (86%), decreasing &gt;28 days (24%); IgG positivity increased &gt;14 days (96%) remaining positive in 94% of patients &gt;28 days. ZIKV prevalence varied importantly in two neighboring cities during the same transmission season. Urine Real-Time PCR can improve diagnostic sensitivity; serum testing is less useful. Accurate serological tests are needed to improve diagnosis and surveillance

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

Multiplex qPCR Discriminates Variants of Concern to Enhance Global Surveillance of SARS-CoV-2

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, 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 United Kingdom, could be serendipitously detected by the Thermo Fisher 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 (VOC) that lack spike Δ69-70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all 3 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 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1-2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. The protocol can be used to sequence other viral genomes using the online Primal Scheme primer designer software. It is suitable for sequencing either RNA or DNA viruses in the field during outbreaks or as an inexpensive, convenient method for use in the lab

Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil

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

Genomic Surveillance of Yellow Fever Virus Epizootic in São Paulo, Brazil, 2016 – 2018

São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species

Real-time, rapid detection and sequencing of arboviruses in Brazil

Os vírus emergentes e reemergentes transmitidos por artrópodes (arbovírus) sempre foram uma preocupação global para a saúde humana. O Brasil é um grande país tropical que fornece condições ideais para a existência de muitos arbovírus que são mantidos em uma ampla variedade de ciclos zoonóticos. Nos últimos anos, o Brasil foi afetado por uma onda de graves epidemias, vírus sobrepostos, principalmente causados pelos vírus Zika (ZIKV), dengue (DENV), imunodeficiência humana (HIV), Ebola (EBOV), febre amarela (YFV), e Chikungunya (CHIKV). As epidemias resultantes causaram alta morbidade, mortalidade e custos econômicos. Além disso, há no Brasil a co-circulação de outras arboviroses negligenciadas ou que são pouco discutidas na literatura médica que vêm sendo responsáveis por causar pequenos surtos ou casos esporádicos em diferentes regiões do país. No entanto, a nossa compreensão destes surtos é dificultada pelo desafio do diagnóstico laboratorial (muitas vezes possui baixa especificidade e/ou requer o conhecimento a priori dos vírus a serem analisados) e diagnóstico clínico (geralmente febre, dor de cabeça, dores nas articulações, erupções cutâneas), que por si só, se sobrepõem àquelas causadas pela co-circulação dos vírus transmitidos por artrópodes, resultando em uma disponibilidade limitada de dados de vigilância epidemiológica. Portanto, há uma necessidade urgente de melhorar nossa capacidade de realizar a vigilância de arbovírus a partir de amostras clínicas através de melhores diagnósticos moleculares. Ferramentas de sequenciamento são abordagens que podem, por sua vez, serem usadas para detectar e monitorar a diversidade de arbovírus, identificar linhagens emergentes e controlar o potencial de introdução de novos vírus, sendo capaz de preparar ou até mesmo prevenir novos surtos. Em 2016, o projeto ZiBRA (Zika no Brasil Real Time Analysis) foi pioneiro de uma nova abordagem para a vigilância de arbovírus usando uma tecnologia de sequenciamento portátil baseada em nanopores para obtenção de genomas completos do vírus Zika em tempo real - chamada \"epidemiologia genômica\". Sendo uma abordagem ainda complexa e limitada pelo número de vírus que podem ser sequenciados simultaneamente (uma reação para cada vírus), o que aumenta muito o custo e não permite a identificação de outros vírus que não estão sendo testados na reação, esta opção ainda é de difícil adoção como protocolo e tecnologia de instrumento de vigilância de rotina em laboratórios de pesquisa e saúde pública. Neste trabalho buscamos gerar novas metodologias que possam ser utilizadas para a vigilância completa de arbovírus, incluindo um painel de detecção e sequenciamento do genoma completo dos principais arbovírus de importância pública e duas abordagens de sequenciamento por metagenômica para rastreio e potencial descoberta de novos vírus. Além disso, buscamos realizar a redução do custo de sequenciamento de arbovírus a US$10 por amostra e padronizar uma melhoria no fluxo de trabalho o que acarretará na adoção desta tecnologia por pesquisadores, laboratórios de diagnóstico e laboratórios de vigilância pública resultando em uma maior precisão na vigilância de arboviroses com potencial de introdução e disseminação no Brasil e em outros países e acelerar o ritmo das pesquisas sobre genética e epidemiologia de infecções por arbovírusEmerging and re-emerging viruses transmitted by arthropods (arboviruses) have always been a global concern for human health. Brazil is a largely tropical country that provides ideal conditions for the existence of many arboviruses that are maintained in a wide variety of zoonotic cycles. In recent years, Brazil has been affected by a wave of serious epidemics, overlapping viruses, mainly caused by the Zika virus (ZIKV), dengue virus (DENV), human immunodeficiency virus (HIV), Ebola virus (EBOV), yellow fever virus (YFV), and Chikungunya virus (CHIKV). The resulting epidemics caused high morbidity, mortality, and economic costs. In addition, there is in Brazil the co-circulation of other arboviruses that are neglected or that are little discussed in the medical literature, which have been responsible for causing small outbreaks or sporadic cases in different regions of the country. However, our understanding of these outbreaks is hampered by the challenge of laboratory diagnosis (often has low specificity and/or requires a priori knowledge of the viruses to be analyzed) and clinical diagnosis (generally fever, headache, joint pain, rashes), which by themselves overlap with those caused by the co-circulation of arthropod-borne viruses, resulting in limited availability of epidemiological surveillance data. Therefore, there is an urgent need to improve our ability to conduct arbovirus surveillance from clinical specimens through better molecular diagnostics. Sequencing tools are approaches that can, in turn, be used to detect and monitor arbovirus diversity, identify emerging strains and control the potential for the introduction of new viruses, being able to prepare or even prevent new outbreaks. In 2016, the ZiBRA (Zika in Brazil Real-Time Analysis) project pioneered a new approach to arbovirus surveillance using portable nanopore-based sequencing technology to obtain complete genomes of the Zika virus in real time - called \"genomic epidemiology \". Being an approach that is still complex and limited by the number of viruses that can be sequenced simultaneously (one reaction for each virus), which greatly increases the cost and does not allow the identification of other viruses that are not being tested in the reaction, this option is still difficult to be adopted as protocol and technology as a routine surveillance tool in research and public health laboratories. In this work, we seek to generate new methodologies that can be used for complete arbovirus surveillance, including a full genome detection and sequencing panel of the main arboviruses of public importance and two metagenomic sequencing approaches for screening and potential discovery of new viruses. In addition, we seek to reduce the cost of arbovirus sequencing to US$ 10 per sample and standardize an improvement in the workflow which will lead to the adoption of this technology by researchers, diagnostic laboratories, and public surveillance laboratories resulting in greater accuracy in the surveillance of arboviruses with the potential for introduction and spread in Brazil and other countries, and accelerate the pace of research on the genetics and epidemiology of arbovirus infection