1. Introduction

Metagenomic analyses have revealed that viruses with single-stranded DNA (ssDNA) genomes are ubiquitous, remarkably diverse, and infect cellular organisms across the tree of life (Rosario et al. 2012, Krupovic 2013, Zhao et al. 2019). One of the most diverse groups of ssDNA viruses is represented by members of the phylum Cressdnaviricota (Krupovic et al. 2020a), formerly known as the circular, Rep-encoding single-stranded DNA (CRESS DNA) viruses (Rosario et al. 2012). These viruses share the characteristic two-domain rolling circle replication (RCR) initiation protein (Rep) comprising the N-terminal HUH superfamily endonuclease domain and the C-terminal superfamily 3 helicase domain (Kazlauskas et al. 2019). All cressdnaviricots characterized thus far build nonenveloped, icosahedral capsids and encode CPs with the jelly-roll fold (antiparallel 8-strand β-barrel) (Krupovic et al. 2020a, Kulshrestha et al. 2020). Whereas the Rep is universally encoded across Cressdnaviricota, the capsid protein (CP) genes of these viruses display considerable sequence divergence and in some lineages have been demonstrated to undergo gene replacement with nonorthologous but structurally related CPs from other viruses (Diemer and Stedman 2012, Roux et al. 2013, Kazlauskas et al. 2017, Munke et al. 2022). Thus, the phylogenetic analysis of the Rep proteins is used as a framework for the classification of cressdnaviricots into families, orders, and classes.

Currently, Cressdnaviricota comprises two classes (Arfiviricetes and Repensiviricetes), 13 orders, and 24 families (Simmonds et al. 2025). All cressdnaviricots for which the hosts have been experimentally determined infect eukaryotes, including plants (e.g. Nanoviridae, Geminiviridae, Amesuviridae, Metaxyviridae), fungi (Genomoviridae [also shown to replicate in insects]), animals (e.g. Circoviridae, Redondoviridae), algae (Bacilladnaviridae), and protists (e.g. Naryaviridae, Nenyaviridae, Vilyaviridae, Oomyviridae) (Krupovic et al. 2020a,b, Varsani and Krupovic 2024, Varsani et al. 2024). However, hosts of cressdnaviricot viruses representing a considerable fraction of families are currently unknown. Furthermore, many ssDNA viruses, especially those discovered through metagenomics, remain unclassified. Thus, the extent of cressdnaviricot diversity and their distribution in eukaryotes remain to be fully appreciated.

Several ssDNA viruses have been described in economically important plant pathogenic filamentous fungi, including Sclerotinia sclerotiorum (infected by Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, SsHADV1), Fusarium graminearum (Fusarium graminearum gemytripvirus 1, FgGMTV1), Diaporthe sojae (Diaporthe sojae circular DNA virus 1, DsCDV1), and Botrytis cinerea (Botrytis cinerea ssDNA virus 1, BcssDV1) (Yu et al. 2010, Li et al. 2020, Hao et al. 2021, Ruiz-Padilla et al. 2023, Wang et al. 2024). No ssDNA viruses infecting oomycetes have been isolated but metagenomic studies have uncovered the existence of multiple endogenous viral elements (EVEs), encompassing the Rep and CP genes typical of ssDNA viruses, in the genomes of oomycetes (Sabanadzovic et al. 2025). In particular, EVEs were found in chromosome 5 of Phytophthora plurivora, P. parasitica (synonym P. nicotianae), P. fragariae, Aphanomyces astaci, and Pythium insidiosum. Furthermore, transcripts corresponding to ssDNA virus genes were detected in the transcriptomes of Plasmopara halstedii and A. invadans, suggesting the presence of transcriptionally active viral elements and raising the possibility of ongoing or recent viral replication rather than solely degenerated genomic remnants (Sabanadzovic et al. 2025). Environmental metatranscriptomic analyses have further identified diverse cressdnaviricot sequences in various habitats known to harbour fungi and oomycetes, including soil, riverbed sediments, plant samples, and animal faeces (Eaglesham and Hewson 2013, Reavy et al. 2015, Rosario et al. 2018, Fehér et al. 2021, Varsani and Krupovic 2021, Zhao et al. 2021). Therefore, ssDNA viruses of fungi and oomycetes may play important, yet underappreciated roles in ecosystem structuring by controlling the behaviour and dynamics of their hosts (Kondo et al. 2022). Indeed, several SsHADV1-like ssDNA viruses (family Genomoviridae) have been shown to induce hypovirulence of their phytopathogenic fungal hosts (Yu et al. 2010, Li et al. 2020).

In marine ecosystems, viral infections are observed in all organisms from bacteria to whales, and the presence of these viruses influences marine biological community composition and serves as a potential driving force in biogeochemical cycles (Suttle 2005). Although dsDNA phages infecting bacteria are the most abundant viruses in marine ecosystems, metagenomic and metatranscriptomic studies have uncovered a substantial contribution to the global marine virome of ssDNA and RNA viruses that infect protists, including unicellular algae, fungi, and fungus-like oomycetes (Coy et al. 2018). RNA viruses have been identified from various host fungi infecting Posidonia oceanica (seagrass) and Holothuria poli, and oomycetes, including Phytophthora condilina and Halophytophthora frigida (Nerva et al. 2016, 2019, Botella et al. 2020, Botella and Jung 2021). However, no DNA viruses infecting marine fungi or oomycetes have been identified to date.

The genus Halophytophthora constitutes a sister genus of the well-known plant pathogenic oomycete genus Phytophthora, comprising species with similar morphology and life cycles, almost exclusively inhabiting brackish and saltwater environments (Sullivan et al. 2018). While some species, such as H. lateralis (previously Halophytophthora sp. Zostera), have been demonstrated to be pathogenic to eelgrass (Zostera marina) (Govers et al. 2016), the genus Halophytophthora has been primarily considered as decomposers in mangrove ecosystems (Newell and Fell 1992, 1997). The only report of viral infection in this genus is the co-infection of eight bunya-like viruses in H. frigida collected from estuaries in southern Portugal, and the effects of viral infection on the host remain unclear (Botella et al. 2020).

To expand the limited knowledge on viruses in Halophytophthora, we focused our viral discovery and characterization efforts on H. thermoambigua, a species inhabiting marine and brackish-water environments (Maia et al. 2022). Halophytophthora thermoambigua emerged as a compelling candidate for virus screening due to two notable biological features: its self-sterile breeding system, with no observed oogonia, oospores, or antheridia, and its unusual phenotypic variability in colony morphology and optimal growth temperature among isolates (20°C, 25°C, or 27.5°C). Here, we report the discovery of the first DNA virus identified in an oomycete. We demonstrate its mitochondrial localization, confirm its extrachromosomal replicative form, and provide evidence of stable vertical transmission. The virus is associated with reduced host growth and altered temperature-dependent performance.