Evidence of reassortment of avian influenza A (H2) viruses in Brazilian shorebirds

Abstract

Influenza A viruses of the H2 subtype represent a zoonotic and pandemic threat to humans due to a lack of widespread specific immunity. Although A(H2) viruses that circulate in wild bird reservoirs are distinct from the 1957 pandemic A(H2N2) viruses, there is concern that they could impact animal and public health. There is limited information on AIVs in Latin America, and next to nothing about H2 subtypes in Brazil. In the present study, we report the occurrence and genomic sequences of two influenza A viruses isolated from wild-caught white-rumped sandpipers (Calidris fuscicollis). One virus, identified as A(H2N1), was isolated from a bird captured in Restinga de Jurubatiba National Park (PNRJ, Rio de Janeiro), while the other, identified as A(H2N2), was isolated from a bird captured in Lagoa do Peixe National Park (PNLP, Rio Grande do Sul). DNA sequencing and phylogenetic analysis of the obtained sequences revealed that each virus belonged to distinct subtypes. Furthermore, the phylogenetic analysis indicated that the genomic sequence of the A(H2N1) virus isolated from PNRJ was most closely related to other A(H2N1) viruses isolated from North American birds. On the other hand, the A(H2N2) virus genome recovered from the PNLP-captured bird exhibited a more diverse origin, with some sequences closely related to viruses from Iceland and North America, and others showing similarity to virus sequences recovered from birds in South America. Viral genes of diverse origins were identified in one of the viruses, indicating local reassortment. This suggests that the extreme South of Brazil may serve as an environment conducive to reassortment between avian influenza virus lineages from North and South America, potentially contributing to an increase in overall viral diversity.

Fig 1. Main avian migratory routes are described, with emphasis on the Atlantic route that passes through the collection sites marked on the map.

Lagoa do Peixe National Park (PNLP) in Rio Grande do Sul and Restinga de Jurubatiba National Park (PNRJ) in Rio de Janeiro. Republished from MapChart under a CC BY license, with permission from Minas Giannekas, original copyright CC BY-SA 4.0 Legal Code, 2024.

Fig 2. Phylogenetic analysis of the PNLP and PNRJ influenza isolates hemagglutinin (HA) gene.

Brazilian samples in this study are written in a bold font. The clusters of high similarly sequences related are indicated by branches colored red (Human HA2, green (Eurasia and Oceania), blue (North America), gold (Iceland) and purple (South America). A total of 780 complete HA2 sequences available in NCBI Influenza Virus Database were used for this phylogenetic analysis. The tree was constructed using the GTR+F+I substitution model as selected by IQ-TREE Model Finder in a 1777 nt length alignment. The scale bar represents the number of substitutions per site. Bootstraps values greater than 50% were obtained in the analysis of 1000 replicates and are presented at the branching points. The tree was rooted with the A/goose/Guangdong/1/1996H5N1) HA sequence (NC_007362.1) as the outgroup.

Fig 3. Phylogenetic analysis of the PNRJ influenza isolate neuraminidase (NA-N1) gene.

The PNRJ sample is written in a bold font. North America branches are colored blue and South America are colored purple. The GTR+F+I substitution model was used as selected by IQ-TREE Model Finder, in a 1410 nt length alignment. The scale bar represents the number of substitutions per site. Bootstraps values greater than 50% were obtained in the analysis of 1000 replicates and are presented at the branching points. The tree was rooted with the A/District_Of_Columbia/50/2022(H1N1) NA sequence (OQ203115) as the outgroup.

Fig 4. Phylogenetic analysis of PNLP influenza isolate neuraminidase (NA-N2) gene.

The PNRJ sample is written in a bold font. North America branches are colored blue, Eurasia and Oceania are colored green and South America are colored purple. The scale bar represents the number of substitutions per site. The GTR+F+I substitution model was used as selected by IQ-TREE Model Finder, in a 1410 nt length alignment. Bootstraps values greater than 50% were obtained in the analysis of 1000 replicates and are presented at the branching points. The tree was rooted with the A/Korea/426/1968(H2N2) NA2 sequence (NC_007382) as the outgroup.