Detection, Characterization and Sequencing of BTV Serotypes Circulating in Cuba in 2022

Abstract

In Cuba, despite a high sero-prevalence of bluetongue virus (BTV), circulating serotypes remain unknown. The aim of this study was to identify circulating BTV serotypes in farms throughout the western region of Cuba. Blood samples were collected from 200 young cattle and sheep between May and July 2022 for virological analyses (PCR, viral isolation and virus neutralization) and genome sequencing. The results confirmed viral circulation, with viro-prevalence of 25% for BTV. The virus was isolated from 18 blood samples and twelve BTV serotypes were identified by sequencing RT-PCR products targeting the segment 2 of the BTV genome (BTV-1, 2, 3, 6, 10, 12, 13, 17, 18, 19, 22 and 24). Finally, the full genome sequences of 17 Cuban BTV isolates were recovered using a Sequence Independent Single Primer Amplification (SISPA) approach combined to MinION Oxford Nanopore sequencing technology. All together, these results highlight the co-circulation of a wide diversity of BTV serotypes in a quite restricted area and emphasize the need for entomological and livestock surveillance, particularly in light of recent changes in the global distribution and nature of BTV infections.

Keywords: BTV; Nanopore sequencing; PCR; Sanger; isolation; serotypes.

Figure 1

Geographical location of animals sampled in Cuba between May and July 2022. Colored dots indicate the collection sites of the blood samples. The dot color determines the municipalities: in Habana Province—Guanabacoa (Vista Hermosa, represented by a yellow dot) and in Mayabeque Province—San José de las Lajas (CENSA and Guayabal, represented by a pink dot), Catalina de Güines (ICA, represented by orange dot), Jaruco (Tenería 1 Perú, Típica 14 Perú and Típica 4 Perú, represented by blue dot).

Figure 2

Phylogenetic analysis of S2 sequences of the Cuban BTV strains. Phylogenetic analysis of S2 sequences of BTV-1, 2, 3, 13, 18, 19 (A), 10, 12, 17, 22 and 24 (B) strains using the maximum likelihood method and Tamura–Nei model with 1000 bootstrap replicates in MEGA X. This analysis involved 39 (A) and 33 (B) nucleotide sequences, and there were a total of 2932 (A) and 2997 (B) positions in the final dataset. Bootstrap values appeared at the corresponding nodes. In the phylogenetic tree, accession number, serotype, country and year of sample collection are given. The Cuban sequences are marked in red. Brackets indicate nucleotypes clustering.

Figure 3

Phylogenetic analysis of S6 sequences of the Cuban BTV strains. Phylogenetic analysis of S6 sequences of BTV-1, 2, 3, 13, 18, 19 (A), 10, 12, 17, 22 and 24 (B) strains using the maximum likelihood method and Tamura–Nei model with 1000 bootstrap replicates in MEGA X. This analysis involved 51 (A) and 37 (B) nucleotide sequences, and there were a total of 1581 (A) and 1645 (B) positions in the final dataset. Bootstrap values appeared at the corresponding nodes. In the phylogenetic tree, accession number, serotype, country and year of sample collection are given. The Cuban sequences are marked in red. Brackets indicate nucleotypes clustering.