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.</p

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

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 without isolation remains challenging for viruses such as Zika, where 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, optimised 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 starting with clinical samples in 1-2 days 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.</jats:p

Genomic epidemiology of the SARS-CoV-2 epidemic in Brazil

The high numbers of COVID-19 cases and deaths in Brazil have made Latin America an epicentre of the pandemic. SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, but important gaps remain in our understanding of virus transmission dynamics at a national scale. We use 17,135 near-complete genomes sampled from 27 Brazilian states and bordering country Paraguay. From March to November 2020, we detected co-circulation of multiple viral lineages that were linked to multiple importations (predominantly from Europe). After November 2020, we detected large, local transmission clusters within the country. In the absence of effective restriction measures, the epidemic progressed, and in January 2021 there was emergence and onward spread, both within and abroad, of variants of concern and variants under monitoring, including Gamma (P.1) and Zeta (P.2). We also characterized a genomic overview of the epidemic in Paraguay and detected evidence of importation of SARS-CoV-2 ancestor lineages and variants of concern from Brazil. Our findings show that genomic surveillance in Brazil enabled assessment of the real-time spread of emerging SARS-CoV-2 variants.Pan American Health OrganizationInternational Centre for Genetic Engineering and BiotechnologyCenter for Information TechnologyLaboratório de Flavivirus Fundacao Oswaldo CruzLaboratório de Genética Celular e Molecular Instituto de Ciências Biologicas Universidade Federal de Minas Gerais, Minas GeraisDepartment of Science and Technology for Humans and the Environment University of Campus Bio-Medico di RomaBlood Center of Ribeirão Preto Ribeirão Preto Medical School University of São Paulo Ribeirão PretoButantan InstitutePan American Health Organization (PAHO)/World Health Organization (WHO), Distrito FederalCentre for Epidemic Response and Innovation (CERI) School of Data Science and Computational Thinking Stellenbosch UniversityKwaZulu-Natal Research Innovation and Sequencing Platform (KRISP) School of Laboratory Medicine and Medical Sciences University of KwaZulu–NatalLaboratório Central de Saúde Pública do Estado de Minas Gerais (LACEN-MG) Fundação Ezequiel Dias, Minas GeraisLaboratório Central de Saúde Pública do Estado da Bahia (LACEN-BA), BahiaLaboratório Central de Salud PúblicaInstituto Regional de Investigación em Salud Universidad Nacional del CaaguazúLaboratório de Biología Molecular Hospital Regional de Coronel Oviedo Ministerio de Salud Pública y Bienestar SocialLaboratório Central de Saúde Pública do Estado de Mato Grosso (LACEN-MT), Mato GrossoLaboratório Central de Saúde Pública do Estado de Mato Grosso do Sul (LACEN-MS), Mato Grosso do SulUniversidade Federal do Mato Grosso do Sul, Mato Grosso do SulCentro de Genômica Funcional da ESALQ University of São Paulo, São PauloNGS Soluções Genômicas, São PauloMendelics Análise GenômicaInstituto de Biologia Molecular Laboratório AntonelloUniversidade Federal de Ciencias da Saúde de Porto Alegre (UFCSPA) Universidade Feevale Grupo Exame LaboratóriosVigilancia em Saúde do Estado de Mato Grosso, Mato GrossoSecretaria de Saude do Estado do Mato Grosso do Sul, Mato Grosso do SulLaboratório Central de Saúde Pública do Estado do Paraná (Lacen-PR), ParanáHospital de Clínicas da Universidade Federal do Paraná, ParanáLaboratório de Virologia Molecular Instituto Carlos Chagas/Fiocruz-PR, ParanáDepartment of Bioprocesses and Biotechnology School of Agricultural Sciences São Paulo State University (UNESP), São PauloDepartment of Veterinary Medicine School of Animal Science and Food Engineering University of São Paulo PirassunungaMedicine School of São José do Rio Preto (FAMERP), São PauloMolecular Biology Laboratory Applied Biotechnology Laboratory Clinical Hospital of the Botucatu Medical SchoolFundação Oswaldo Cruz Bio-Manguinhos Rio de JaneiroCoordenação Geral dos Laboratórios de Saúde Pública/Secretaria de Vigilância em Saúde Ministério da Saúde (CGLAB/SVS-MS), Distrito FederalCoordenação Geral das Arboviroses Secretaria de Vigilância em Saúde/Ministério da Saúde (CGARB/SVS-MS), Distrito FederalDepartamento de Imunização e Doenças Transmissíveisa/Secretaria de Vigilancia em Saude Ministerio da Saude, Distrito FederalDepartment of Zoology University of OxfordBiosystems and Integrative Sciences Institute Universidade de LisboaCentre for the AIDS Programme of Research in South Africa (CAPRISA)Department of Global Health University of WashingtonMarie Bashir Institute for Infectious Diseases and Biosecurity School of Life and Environmental Sciences and School of Medical Sciences University of SydneyDepartment of Bioprocesses and Biotechnology School of Agricultural Sciences São Paulo State University (UNESP), São PauloMolecular Biology Laboratory Applied Biotechnology Laboratory Clinical Hospital of the Botucatu Medical SchoolPan American Health Organization: APO21-00010098International Centre for Genetic Engineering and Biotechnology: CRP/BRA20-03Center for Information Technology: U01 AI15169