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Herpesviruses are significant pathogens of ruminants. In water buffaloes (Bubalus bubalis), however, herpesviruses have not been thoroughly studied. Although bubaline alphaherpesvirus 1 (BuAHV1) and bovine alphaherpesvirus 1 (BoAHV1) have already been recovered from water buffaloes, to date, no reports on the occurrence of bovine alphaherpesvirus 5 (BoAHV5) in these animals have been published. Therefore, the aim of this study was to search for BuAHV1, BoAHV1, and BoAHV5 in palatine tonsils of apparently healthy water buffaloes from the Pará state, Northern Brazil. Tissue samples of tonsils (n = 293) were screened by a nested PCR (nPCR) targeting a region of UL44 (gC coding gene), followed by sequencing, to detect and differentiate between the viral types. Viral genome segments were detected in 18 out of 293 (6.1%) of the palatine tonsil samples. Two animals carried genomes of BoAHV1 only, eleven animals carried BoAHV5 genomes only, and four animals carried BuAHV1 only. Another animal had both BoAHV1 and BoAHV5 genomes in its tonsils. No infectious virus could be recovered from any of the samples. The BuAHV1 sequences identified here were more closely related to BuAHV1 genomes identified in India. Phylogenetic analyses suggested a closer relationship between the recovered BoAHV5 and BuAHV1 genomes. Therefore, evidence is provided here to confirm that not only BoAHV1 and BuAHV1, but also BoAHV5, can infect water buffaloes. This report highlights (i) the first detection of BoAHV5 in water buffaloes and (ii) the occurrence of coinfections with BoAHV1 and BoAHV5 in that species. Such findings and the similarity of BoAHV5 to Indian herpesvirus genomes suggest that the origin of type 5 may be linked to recombinations between bovine and bubaline herpesviruses within bubalines, since the scenario for generation of recombinants in buffaloes is potentially present. [ABSTRACT FROM AUTHOR]