First isolation of West Nile virus in Brazil

Serological evidence of West Nile virus (WNV) infection has been reported in different regions of Brazil from equine and human hosts but the virus had never been isolated in the country.

OBJECTIVES

We sought to identify the viral etiology of equine encephalitis in Espírito Santo state.

METHODS

We performed viral culture in C6/36 cells, molecular detection of WNV genome, histopathology and immunohistochemistry from horse cerebral tissue. We also carried out sequencing, phylogenetic analysis and molecular clock.

FINDINGS

Histopathologic analysis from horse cerebral tissue showed injury related to encephalitis and WNV infection was confirmed by immunohistochemistry. The virus was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) from brain tissue and subsequently isolated in C6/36 cells. WNV full-length genome was sequenced showing the isolated strain belongs to lineage 1a. The molecular clock indicated that Brazilian WNV strain share the same common ancestor that were circulating in US during 2002-2005.

MAIN CONCLUSIONS

Here we report the first isolation of WNV in Brazil from a horse with neurologic disease, which was clustered into lineage 1a with others US WNV strains isolated in beginning of 2000’s decade.

Key words:
West Nile virus; horses; Brazil

Authorship

Lívia Caricio Martins

Instituto Evandro Chagas, Seção de Arbovirologia e Febres Hemorrágicas, Ananindeua, PA, BrasilInstituto Evandro ChagasBrasilAnanindeua, PA, BrasilInstituto Evandro Chagas, Seção de Arbovirologia e Febres Hemorrágicas, Ananindeua, PA, Brasil

Eliana Vieira Pinto da Silva

Livia Medeiros Neves Casseb

Sandro Patroca da Silva

Ana Cecília Ribeiro Cruz

Jamilla Augusta de Sousa Pantoja

Daniele Barbosa de Almeida Medeiros

Arnaldo Jorge Martins Filho

Instituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, BrasilInstituto Evandro ChagasBrasilAnanindeua, PA, BrasilInstituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, Brasil

Ermelinda do Rosário Moutinho da Cruz

Instituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, BrasilInstituto Evandro ChagasBrasilAnanindeua, PA, BrasilInstituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, Brasil

Marialva Tereza Ferreira de Araújo

Instituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, BrasilInstituto Evandro ChagasBrasilAnanindeua, PA, BrasilInstituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, Brasil

Jedson Ferreira Cardoso

Instituto Evandro Chagas, Centro de Inovação tecnológica, Ananindeua, PA, BrasilInstituto Evandro ChagasBrasilAnanindeua, PA, BrasilInstituto Evandro Chagas, Centro de Inovação tecnológica, Ananindeua, PA, Brasil

Marcos Antônio Correia Rodrigues da Cunha

Secretaria de Estado da Saúde do Espírito Santo, Vitória, ES, BrasilSecretaria de Estado da Saúde do Espírito SantoBrasilVitória, ES, BrasilSecretaria de Estado da Saúde do Espírito Santo, Vitória, ES, Brasil

Gilton Luiz Almada

Alessandro Pecego Martins Romano

Ministério da Saúde, Secretaria de Vigilância em Saúde, Brasília, DF, BrasilMinistério da SaúdeBrazilBrasília, DF, BrazilMinistério da Saúde, Secretaria de Vigilância em Saúde, Brasília, DF, Brasil

Maria Guadalupe Dias Pestana Santos

Secretaria de Saúde do Município de Venécia, Venécia, ES, BrasilSecretaria de Saúde do Município de VenéciaBrazilVenécia, ES, BrazilSecretaria de Saúde do Município de Venécia, Venécia, ES, Brasil

Gilsa Aparecida Pimenta Rodrigues

Jannifer Oliveira Chiang

Juarez Antonio Simões Quaresma

Instituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, BrasilInstituto Evandro ChagasBrasilAnanindeua, PA, BrasilInstituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, Brasil

Valéria Lima Carvalho ⁺+ Corresponding author: valeriacarvalho@iec.gov.br

http://orcid.org/0000-0001-8385-8253

Pedro Fernando da Costa Vasconcelos

+ Corresponding author: valeriacarvalho@iec.gov.br

NLCM, PFCV and JOC - Supervision; VLC, EVPS and ACRC - writing, original draft; APMR, LMNC, MACRC, GLA, MGDPS and GAPR - investigation; SPS, JASP, AJMF, ERMC, MTFA and JASQ - methodology; JFC and DBAM - molecular clock. The author declares no conflicts of interest.

SCIMAGO INSTITUTIONS RANKINGS

Instituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, BrasilInstituto Evandro ChagasBrasilAnanindeua, PA, BrasilInstituto Evandro Chagas, Seção de Patologia, Ananindeua, PA, Brasil

Figures

Figures (4)

Thumbnail

Fig. 1:
detection of West Nile virus (WNV) antigen in C6/36 cells by indirect immunofluorescence assay (IFA). (A) WNV antigen (green) in infected cells detected by IFA using flavivirus group-reactive hyperimmune mouse ascitic fluid. (B) Uninfected cell control by IFA using the same hyperimmune mouse ascitic fluid.

Thumbnail

Fig. 2:
photomicrograph of cerebral tissue of a horse with neurologic manifestation from Espírito Santo state with description of histopathological aspects and positive immunostaining for West Nile virus (WNV). (A) Inflammatory infiltrate predominantly constituted by mononuclear cells with perivascular cuffing (arrow) and neuronal necrosis (arrow heads). (B) Brain tissue showed neuronophagy (arrows) and associated gliosis (arrow head). (C) Perivascular infiltrate with congestion (arrows) and foci of neuronal necrosis of different intensities (arrow heads). (D) Immunohistochemistry of WNV antigen (reddish staining) in the cell cytoplasm in several foci of brain tissue (arrows).

Thumbnail

Fig. 3:
midpoint phylogenetic tree of nucleotide sequences using only polyprotein coding region of 1582 West Nile virus (WNV) strains representing different viral lineages. The analysis of those nucleotide sequences was performed using the maximum likelihood method based on the GTR matrix-based model. Different phylogenetic groups are assigned to previously defined lineages; highlighted in dark blue, strains included in lineage 1a from USA and in light blue strains included in lineage 1a from Europe, Asia and Africa. Strains from Brazil (red), Mexico (orange), Colombia (purple), and Argentina (green) were also identifies on the phylogenetic tree. Numbers over each main node of the tree correspond to bootstrap values (1000 replicates). Values < 70 are not supported by reproducible topologies. The bar represents nucleotide substitutions along of the branch.

Thumbnail

Fig. 4:
(A) Posterior probability densities and root-to-tip analysis based on maximum likelihood tree (Supplementary data II) demonstrated a good temporal correlation (R² = 0749), indicating that evolutionary clock models were appropriate for inferring the evolutionary origins of the West Nile virus (WNV) samples. It was used, with a relaxed uncorrelated log-normal molecular clock and a Bayesian skyline tree model. (B) Log marginal likelihood estimates, using distinct molecular clock and coalescent model combinations for temporal reconstruction using entire WNV open reading frames (ORFs) by path sampling model selection (PS) and stepping-stone model selection (SS) methods. The best-fitting model is highlighted in boldface. Coalescent models used were the parametric Constant models and the non-parametric was the Skygrid and Skyline models. The molecular clock models used were the strict molecular clock (Strict) and the uncorrelated relaxed lognormal molecular clock (UCLN). (C) Maximum clade credibility (MCC) tree of WNV genomes constructed using BEAST version 1.10.1 software. Colors of branches indicate geographic locations per the color key. Branch lengths correspond to lengths of time.

Fig. 1: detection of West Nile virus (WNV) antigen in C6/36 cells by indirect immunofluorescence assay (IFA). (A) WNV antigen (green) in infected cells detected by IFA using flavivirus group-reactive hyperimmune mouse ascitic fluid. (B) Uninfected cell control by IFA using the same hyperimmune mouse ascitic fluid.

Fig. 2: photomicrograph of cerebral tissue of a horse with neurologic manifestation from Espírito Santo state with description of histopathological aspects and positive immunostaining for West Nile virus (WNV). (A) Inflammatory infiltrate predominantly constituted by mononuclear cells with perivascular cuffing (arrow) and neuronal necrosis (arrow heads). (B) Brain tissue showed neuronophagy (arrows) and associated gliosis (arrow head). (C) Perivascular infiltrate with congestion (arrows) and foci of neuronal necrosis of different intensities (arrow heads). (D) Immunohistochemistry of WNV antigen (reddish staining) in the cell cytoplasm in several foci of brain tissue (arrows).

Fig. 3: midpoint phylogenetic tree of nucleotide sequences using only polyprotein coding region of 1582 West Nile virus (WNV) strains representing different viral lineages. The analysis of those nucleotide sequences was performed using the maximum likelihood method based on the GTR matrix-based model. Different phylogenetic groups are assigned to previously defined lineages; highlighted in dark blue, strains included in lineage 1a from USA and in light blue strains included in lineage 1a from Europe, Asia and Africa. Strains from Brazil (red), Mexico (orange), Colombia (purple), and Argentina (green) were also identifies on the phylogenetic tree. Numbers over each main node of the tree correspond to bootstrap values (1000 replicates). Values < 70 are not supported by reproducible topologies. The bar represents nucleotide substitutions along of the branch.

Fig. 4: (A) Posterior probability densities and root-to-tip analysis based on maximum likelihood tree (Supplementary data II) demonstrated a good temporal correlation (R² = 0749), indicating that evolutionary clock models were appropriate for inferring the evolutionary origins of the West Nile virus (WNV) samples. It was used, with a relaxed uncorrelated log-normal molecular clock and a Bayesian skyline tree model. (B) Log marginal likelihood estimates, using distinct molecular clock and coalescent model combinations for temporal reconstruction using entire WNV open reading frames (ORFs) by path sampling model selection (PS) and stepping-stone model selection (SS) methods. The best-fitting model is highlighted in boldface. Coalescent models used were the parametric Constant models and the non-parametric was the Skygrid and Skyline models. The molecular clock models used were the strict molecular clock (Strict) and the uncorrelated relaxed lognormal molecular clock (UCLN). (C) Maximum clade credibility (MCC) tree of WNV genomes constructed using BEAST version 1.10.1 software. Colors of branches indicate geographic locations per the color key. Branch lengths correspond to lengths of time.

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First isolation of West Nile virus in Brazil

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[1] + Corresponding author: valeriacarvalho@iec.gov.br