Evidence for current circulation of an ancient West Nile virus strain (NY99) in Brazil

We report the first case of WNV infection in a horse from Ceará state and the complete genome sequence of an isolate from Espírito Santo state. Both infections occurred in 2019.

METHODS:

WNV was isolated from the tissues of a horse with neurological signs in Espírito Santo and sequenced by MiSeq.

RESULTS:

Phylogenetic analysis revealed that the isolate belongs to lineage 1a, clustering with the NY99 strain, a strain that has not circulated in the USA since 2005.

CONCLUSIONS:

Our findings reinforce the hypothesis that WNV has been silently circulating in Brazil for many years.

Keywords:
West nile virus; NY99 strain; Horses

Authorship

Márcio Junio Lima Siconelli

Universidade São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Clínica Médica, Ribeirão Preto, SP, Brasil. Universidade São PauloBrasilRibeirão Preto, SP, Brasil Universidade São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Clínica Médica, Ribeirão Preto, SP, Brasil.

Daniel Macedo de Melo Jorge

Universidade São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular, Ribeirão Preto, SP, Brasil. Universidade São PauloBrasilRibeirão Preto, SP, Brasil Universidade São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular, Ribeirão Preto, SP, Brasil.

Luiza Antunes de Castro-Jorge

Antônio Augusto Fonseca-Júnior

Ministério da Agricultura, Pecuária e Abastecimento, Laboratório Federal de Defesa Agropecuária de Minas Gerais, Pedro Leopoldo, MG, Brasil. Ministério da Agricultura, Pecuária e AbastecimentoBrasilPedro Leopoldo, MG, Brasil Ministério da Agricultura, Pecuária e Abastecimento, Laboratório Federal de Defesa Agropecuária de Minas Gerais, Pedro Leopoldo, MG, Brasil.

Mateus Laguardia Nascimento

Vitor Gonçalves Floriano

Fransérgio Rocha de Souza

Instituto de Defesa Agropecuária e Florestal do Estado do Espírito Santo, Ibiraçu, ES, Brasil. Instituto de Defesa Agropecuária e Florestal do Estado do Espírito SantoBrasilIbiraçu, ES, Brasil Instituto de Defesa Agropecuária e Florestal do Estado do Espírito Santo, Ibiraçu, ES, Brasil.

Eudson Maia de Queiroz-Júnior

Agência de Defesa Agropecuária do Estado do Ceará, Boa Viagem, CE, Brasil. Agência de Defesa Agropecuária do Estado do CearáBrasilBoa Viagem, CE, Brasil Agência de Defesa Agropecuária do Estado do Ceará, Boa Viagem, CE, Brasil.

Marcelo Fernandes Camargos

Eliana Dea Lara Costa

Ministério da Agricultura, Pecuária e Abastecimento, Departamento de Saúde Animal, Brasília, DF, Brasil. Ministério da Agricultura, Pecuária e AbastecimentoBrasilBrasília, DF, Brasil Ministério da Agricultura, Pecuária e Abastecimento, Departamento de Saúde Animal, Brasília, DF, Brasil.

Adolorata Aparecida Bianco Carvalho

Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Departamento de Patologia, Reprodução e Saúde Única, Jaboticabal, SP, Brasil.Universidade Estadual PaulistaBrasilJaboticabal, SP, Brasil Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, Departamento de Patologia, Reprodução e Saúde Única, Jaboticabal, SP, Brasil.

Benedito Antonio Lopes da Fonseca

https://orcid.org/0000-0003-3159-5687

Authors’ contribution: MJLS: Conceptualization, Methodology, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration, Funding acquisition; DMMJ: Conceptualization, Software, Investigation, Resources, Writing - Original Draft, Writing - Review & Editing; LACJ: Visualization, Data Curation, Writing - Review & Editing; AAF-J, MLN, MFC and EDLC: Investigation, Resources; VGF, FRS and EMQ-J: Investigation; AABC: Supervision, Writing - Review & Editing; BALF: Conceptualization, Methodology, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. All authors read, reviewed and approved the final version of the manuscript.

Conflict of Interest: The authors declare that there is no conflict of interest.

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Figures

Figures (2)

Thumbnail

FIGURE 1:
Map showing the states (in red) with molecular evidence of WNV circulation in horses with neurological disease. Black dots: municipalities of WNV occurrence (João Neiva in Espírito Santo (ES) and Boa Viagem in Ceará). Black star: municipality of São Mateus/ES where the first isolation and complete sequencing was done in 2018. Indirect immunofluorescence assay for the detection of West Nile virus antigens in VERO cells at 40X magnification. (A) Mock cells (uninfected cells were used as negative controls). (B) Infected cells after 48h post-inoculation with the 2nd passage of WNV isolate. Hyperimmune serum obtained from mice infected with WNV (NY99 reference strain) were used in both mock and infected cells. Blue: nuclei stained with DAPI Green: WNV antigen.

Thumbnail

FIGURE 2:
Maximum-likelihood (ML) phylogenetic reconstruction of WNV from 1953-2019. (A) a selection of 117 complete sequences from the global dataset were included in this analysis, presenting 0.1 substitutions per site. The GTR+F+I+G4 substitution model and support analysis of 10,000 replicates were used to obtain the phylogenetic tree; (B) dashed box highlighting South America (SA) strains, including the new isolate (MT905060), that cluster with NY99 strain, lineage 1a; only bootstrap values equal or bigger than 70% were shown; (C) root-to-tip analyses obtained with TempEst, using ML reconstruction obtained from 94 complete genomes from 1a and 1b lineages; graph correlation between time (years) and genetic divergence (substitutions per site) from the root of the tree (time to the Most Recent Common Ancestors, tMRCA) to the tips (sampled genomes) is shown; evolutionary slope rate: 4.146 x 10^-4 substitutions/site/year; black arrow outline = new isolate. All colored strains belong to South America: Brazil (red), Argentina (blue), and Colombia (green). Old world lineages include: 3 (Rabensburg), 4a, 4b (9), 7 (Koutango), and 8.

FIGURE 1: Map showing the states (in red) with molecular evidence of WNV circulation in horses with neurological disease. Black dots: municipalities of WNV occurrence (João Neiva in Espírito Santo (ES) and Boa Viagem in Ceará). Black star: municipality of São Mateus/ES where the first isolation and complete sequencing was done in 2018. Indirect immunofluorescence assay for the detection of West Nile virus antigens in VERO cells at 40X magnification. (A) Mock cells (uninfected cells were used as negative controls). (B) Infected cells after 48h post-inoculation with the 2nd passage of WNV isolate. Hyperimmune serum obtained from mice infected with WNV (NY99 reference strain) were used in both mock and infected cells. Blue: nuclei stained with DAPI Green: WNV antigen.

FIGURE 2: Maximum-likelihood (ML) phylogenetic reconstruction of WNV from 1953-2019. (A) a selection of 117 complete sequences from the global dataset were included in this analysis, presenting 0.1 substitutions per site. The GTR+F+I+G4 substitution model and support analysis of 10,000 replicates were used to obtain the phylogenetic tree; (B) dashed box highlighting South America (SA) strains, including the new isolate (MT905060), that cluster with NY99 strain, lineage 1a; only bootstrap values equal or bigger than 70% were shown; (C) root-to-tip analyses obtained with TempEst, using ML reconstruction obtained from 94 complete genomes from 1a and 1b lineages; graph correlation between time (years) and genetic divergence (substitutions per site) from the root of the tree (time to the Most Recent Common Ancestors, tMRCA) to the tips (sampled genomes) is shown; evolutionary slope rate: 4.146 x 10^-4 substitutions/site/year; black arrow outline = new isolate. All colored strains belong to South America: Brazil (red), Argentina (blue), and Colombia (green). Old world lineages include: 3 (Rabensburg), 4a, 4b (9), 7 (Koutango), and 8.

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Evidence for current circulation of an ancient West Nile virus strain (NY99) in Brazil

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[1] Authors’ contribution: MJLS: Conceptualization, Methodology, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration, Funding acquisition; DMMJ: Conceptualization, Software, Investigation, Resources, Writing - Original Draft, Writing - Review & Editing; LACJ: Visualization, Data Curation, Writing - Review & Editing; AAF-J, MLN, MFC and EDLC: Investigation, Resources; VGF, FRS and EMQ-J: Investigation; AABC: Supervision, Writing - Review & Editing; BALF: Conceptualization, Methodology, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. All authors read, reviewed and approved the final version of the manuscript.