Metagenomic screening of faeces from apparently healthy Magellanic penguins (Spheniscus magellanicus) at Magdalena Island, Chile, uncovered four highly divergent novel picornaviruses — Sphenifaro virus, Sphenigellan virus, Sphenimaju virus, and Sphenilena virus — each likely representing a new genus in the family Picornaviridae, with over 20% of the sampled penguins shedding at least one virus and frequent co-infections. Nothing is known about the natural history of these viruses: their host range beyond Magellanic penguins, their transmission route, the drivers of their high prevalence and co-infection, their pathogenic potential, their connectivity among penguin populations across South America, and the broader picornavirus diversity of penguins (Sphenisciformes) all remain open. No published study has yet re-detected or further characterized these four viruses, while related work on other penguin picornaviruses leaves the broader questions unresolved.

Background

The family Picornaviridae comprises small, non-enveloped, positive-sense single-stranded RNA viruses whose genomes (~7–10 kb) encode a single polyprotein that is cleaved into structural capsid proteins (region P1) and non-structural proteins (regions P2 and P3, including helicase and RNA-dependent RNA polymerase). Several picornaviruses are major veterinary pathogens of poultry — for example duck hepatitis A virus (genus Avihepatovirus) and avian encephalomyelitis virus (genus Tremovirus), while the genus Hepatovirus contains hepatitis A virus of humans. Under International Committee on Taxonomy of Virus (ICTV) conventions, picornavirus genera are demarcated by low amino-acid identity in the P1 capsid region (below roughly 40%) together with phylogenetic placement. With the advent of viral metagenomics (unbiased high-throughput sequencing of total viral nucleic acid from samples such as faeces, without culturing or prior sequence knowledge), many picornaviruses have been discovered in apparently healthy wild birds, raising the question of whether such viruses are benign commensals or cryptic pathogens.

Penguins (order Sphenisciformes) have become a productive system for this question. Metagenomic surveys of Antarctic penguins have revealed diverse RNA viromes, including unassigned picornaviruses; Pingu virus, found in 12.9% of gentoo penguins (Pygoscelis papua) on King George Island but not in sympatric Adélie or chinstrap penguins (de Souza et al. 2019); and penguin megrivirus from an Adélie penguin (Yinda et al. 2019). A 15-year serological survey in Argentine Magellanic penguins (Uhart et al. 2020) detected only trace antibody exposure to Tremovirus A, so the Hepatovirus/Tremovirus lineage was essentially undocumented in this host. The Magellanic penguin breeds in large colonies along the Atlantic and Pacific coasts of southern South America and migrates north along both coasts; it is classified as Near Threatened, which makes the disease ecology of its viruses a conservation concern.

Against this background, Hayer et al. (2021) (study first posted as bioRxiv 10.1101/2020.10.26.356485) sequenced faecal samples from apparently healthy Magellanic penguins at Magdalena Island, Chile, and discovered four novel picornaviruses, named Sphenifaro virus, Sphenigellan virus, Sphenimaju virus, and Sphenilena virus (GenBank genome assemblies LR897978, LR897979, LR897980 and LR897982). Each shares less than 40% P1 amino-acid identity with any known picornavirus and carries exceptionally long phylogenetic branches within the avian Hepatovirus/Tremovirus lineage (distantly related to the genera Hepatovirus, Tremovirus, Gruhelivirus, and Crahelvirus), so each likely constitutes a new genus. Reverse-transcription PCR (RT-PCR, which converts RNA to DNA and amplifies target sequences) screening of the 107 faecal samples — collected from 72 Magellanic penguins, one rockhopper penguin and three kelp gulls — detected at least one of the four viruses in 28 of the 107 samples (25%; 95% CI 18–34%) and in 22 of the 72 individual Magellanic penguins (individual-level prevalence 21%; 95% CI 14–30%), all positives being from Magellanic penguins; the study's Discussion summarises this as over 20% of the sampled penguins shedding at least one virus. Seven individuals carried two or three of the viruses, and amplicon sequencing showed within-virus pairwise nucleotide identities of only 91–98%. The authors' own discussion states that several important questions remain unresolved: the host range of these viruses beyond Magellanic penguins (one rockhopper penguin and three kelp gulls screened were negative, a sample far too small to establish host restriction); the transmission route, plausibly faecal–oral but purely speculative; the ecological and evolutionary drivers of the high prevalence and co-infection rates; the pathogenic potential of apparently asymptomatic infections for wild birds; the connectivity of these viruses among Magellanic penguin populations across South America and along migratory flyways; and, more broadly, the vast undersampling of picornavirus diversity in Sphenisciformes, where the authors expect many more Hepatovirus- and Tremovirus-like viruses to be found.

Problem Statement

Establish the natural history — the ecology, transmission, and significance — of the four novel Magellanic penguin picornaviruses (Sphenifaro virus, Sphenigellan virus, Sphenimaju virus, and Sphenilena virus) discovered by Hayer et al. (2021), whose joint characterization determines where this novel picornavirus radiation lies between a host-restricted, colony-restricted, and benign component of the Magellanic penguin virome and a generalist, mobile, and potentially pathogenic one. Concretely, as posed by the source study: (1) Host range — do these viruses infect other penguin species (e.g., rockhopper, African, little blue, Galápagos penguins) or birds sharing their habitat such as kelp gulls, beyond the tiny negative screen already reported? (2) Transmission — what is the actual route (the faecal–oral route is plausible but unconfirmed), and what are the duration and environmental persistence of shedding? (3) Ecology and pathogenesis — what ecological and evolutionary factors drive the observed high prevalence (over 20% of sampled birds) and frequent co-infections, and do these apparently asymptomatic infections cause subclinical effects, reduced fitness, or disease under stress in wild birds? (4) Connectivity — how are these viruses structured among Magellanic penguin populations across South America and potentially along migratory flyways, given that sampling so far covers a single colony (Magdalena Island, Chile)? (5) Broader diversity — given that picornavirus diversity in Sphenisciformes is considered vastly undersampled, how much related picornavirus diversity exists across penguin species, and what is the evolutionary history of this lineage? The five components define one objective — a coherent characterization of this newly discovered virus group.

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