Investigation of neurotropic arboviruses in wild and domestic animals in Amazon region, 2023-2024

Hayashi, Daniel Jun; Nunes, Bruno Tardelli Diniz; Silva, Sandro Patroca da; Cunha, Tânia Cristina Alves da Silveira da; Paiva, Francisco Amilton dos Santos; Prazeres, Ivy Tsuya Essashika; Santos, Alessandra da Conceição Miranda; Barros, Landeson Junior Leopoldino; Ribeiro, Ana Cláudia da Silva; Sobral, Felipe Baraldi; Fernandes, Luiz Mário; Coelho, Taciana Fernandes Souza Barbosa; Carvalho, Valéria Lima; Cruz, Ana Cecilia Ribeiro; Medeiros, Daniele Barbosa de Almeida; Casseb, Livia Medeiros Neves

doi:10.1590/0074-02760250013

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RESEARCH ARTICLE • Mem. Inst. Oswaldo Cruz 121 • 2026 • https://doi.org/10.1590/0074-02760250013 linkcopy

Investigation of neurotropic arboviruses in wild and domestic animals in Amazon region, 2023-2024

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

BACKGROUND Arboviruses represent a potential threat to global public health due to their ability to infect various vertebrate hosts and vectors, as well as their adaptability to diverse ecosystems, allowing them to expand geographically across continents.

OBJECTIVE The present study aimed to describe the molecular epidemiology of encephalitic arboviruses, including Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Toscana virus (TOSV), and Rift Valley fever virus (RVFV) in nervous tissue samples from domestic and wild animals from the Northern region of Brazil, between February 2023 and June 2024.

METHODS Samples negative for rabies virus were analysed by reverse transcription real-time polymerase chain reaction (RT-qPCR) targeting Alphavirus, Orthoflavivirus and Phlebovirus species. Positive samples were subjected to viral isolation in cell culture and whole-genome sequencing using next-generation sequencing. Phylogenetic and molecular clock analyses were performed to characterise viral lineages and evolutionary relationships.

FINDINGS Two samples tested positive for arboviruses by RT-qPCR: one SLEV sample from the state of Amazonas, which showed a low viral load, preventing virus isolation and sequencing, and one Madariaga virus (MADV) sample from the state of Pará, which could be isolated and sequenced. The isolated agent belongs to lineage III of EEEV, showing most similarity to strains from Guyana and Argentina.

MAIN CONCLUSIONS The present study detected two arboviruses in animals, suggesting its circulation in the study area.

Key words:
Encephalitis virus ; arboviruses; Alphavirus ; Flavivirus ; Orthoflavivirus ; Brazil

Virus	Target	Sequence
MADV/ EEEV	EEEV ALL F	5'-GTGGAACAACAACCARARCTTYTT-3'
	EEEV ALL R	5'-GGGATCCCCACCTTGTTCG-3'
	EEEV ALL Probe	5'-FAM-ATCACYGCT/ZEN/GTGTGYTCGTAYGCTGC-IBFQ-3'
WEEV¹⁰	WEEV 8220F	5'-AGGGATACCCCGAAGGTT-3'
	WEEV 8322R	5'-GTGAATAGCACACGGGTGGTT-3'
	WEEV 8274 Probe	5'- HEX-CTTTCGAAT/ZEN/GTCACGTTCCCATGCG-IBFQ-3'
VEEV	VEEV NSP1 41 F	5'- AAGTTCACGTTGAYATCGAG-3'
	VEEV NSP1 166R	5'-GCGAAAACGCUCURGC-3'
	VEEV NSP1 103 Probe	5'- Cy5-CAGTTTGAG/TAO/GTAGAAGCCAAGCAGGT-3IAbRQSp-3'
WNV¹¹	WNRTF	5'-CGGAAGTYGRGTAKACGGTGCTG-3'
	WNRTR	5'-CGGTWYTGAGGGCTTACRTGG-3'
	WNV MGB Probe	5'-FAM-WCCCCAGGWGGACTG-3MGBEq-3'
SLEV¹²	SLEV PrM 834F	5'-GAAAACTGGGTTCTGCGCA-3'
	SLEV PrM 905R	5'-GTTGCTGCCTAGCATCCATCC-3'
	SLEV PrM 857p	5'-HEX-TGGATATGC/ZEN/CCTAGTTGCGCTGGC-IBFQ-3'
RVFV¹³	RVFV F	5'-TGCCACGAGTYAGAGCCA-3'
	RVFV R	5'-GTGGGTCCGAGAGTYTGC-3'
	RVFV Probe	5'-FAM-TCCTTCTCC/ZEN/CAGTCAGCCCCAC-IBFQ-3'
TOSV	TOSVS 1631 F	5'-AAGGTTCAGCACAATCATCATCTTCAC-3'
	TOSVS 1808 R	5'-ATTGCTCTTGCTTTTCTTGATGAG-3'
	TOSVS 1773c Probe	5'-HEX-TGGGACCAT/ZEN/CAATGCATGGGTGAATGA GTTTGCTTA-IBFQ-3'
MS2⁹	MS2 F	5´-CATAAGTTAGATGGCCGTCTGT-3´
	MS2 R	5'- TAGAGACGACAACCATGCCAAAC -3'
	MS2 S Probe	5'-FAM-TCCAGACAA/ZEN/CGTGCAACATATCGCGACGTATCGTGATATGG-IBFQ-3'

Clock type	Tree prior	Length of chain	Path sampling	Stepping stone sampling
Strict clock	Constant size	5,00E+07	-28448,8718	-28448,5614
Strict clock	Constant size	5,00E+07	-28448,6177	-28448,4488
Strict clock	Constant size	5,00E+07	-28449,6215	-28449,4039
Strict clock	Bayesian skygrid	5,00E+07	-28447,6246	-28447,6141
Strict clock	Bayesian skygrid	5,00E+07	-28456,7644	-28456,6223
Strict clock	Bayesian skygrid	5,00E+07	-28448,0490	-28448,4519
Strict clock	GMRF Bayesian skyride	5,00E+07	-28449,8180	-28449,7006
Strict clock	GMRF Bayesian skyride	5,00E+07	-28450,2185	-28449,9547
Strict clock	GMRF Bayesian skyride	5,00E+07	-28449,7259	-28449,5267
Uncorrelated relaxed clock	Constant size	2,00E+08	-28424,9145	-28424,5184
Uncorrelated relaxed clock	Constant size	2,00E+08	-28424,5897	-28424,4118
Uncorrelated relaxed clock	Constant size	2,00E+08	-28424,9650	-28424,5068
Uncorrelated relaxed clock	Bayesian skygrid	2,00E+08	-28422,8338	-28422,5312
Uncorrelated relaxed clock	Bayesian skygrid	2,00E+08	-28426,1797	-28426,5661
Uncorrelated relaxed clock	Bayesian skygrid	2,00E+08	-28422,3869	-28422,2041
Uncorrelated relaxed clock	GMRF Bayesian skyride	2,00E+08	-28424,0788	-28423,5740
Uncorrelated relaxed clock	GMRF Bayesian skyride	2,00E+08	-28424,6073	-28424,4704
Uncorrelated relaxed clock	GMRF Bayesian skyride	2,00E+08	-28424,1785	-28423,9712

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Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
E-mail: memorias@fiocruz.br

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[TFN1] EEEV: Eastern equine encephalitis virus; MADV: Madariaga virus; RVFV: Rift Valley fever virus; SLEV: Saint Louis encephalitis virus; TOSV: Toscana virus; VEEV: Venezuelan equine encephalitis virus; WEEV: Western equine encephalitis virus; WNV: West Nile virus.