Broiler stunting diseases have long frustrated veterinary virologists. Runting-stunting syndrome, often abbreviated as RSS, is associated with malabsorption and typically presents in young chicks as stunted growth, poor feathering and abnormal droppings. A related but distinct condition, transmissible viral proventriculitis or TVP, involves maldigestion linked to inflammation of the proventriculus, the glandular stomach of the bird. Viral infections have repeatedly been proposed as contributors to both syndromes, and numerous candidate agents have been put forward over the decades. Yet the aetiological roles of specific viruses remain obscure. Conventional virome studies, which typically sequence viral genetic material from homogenised whole tissues or faecal samples, lack spatial resolution. They cannot determine which viruses are physically localised to lesion sites and which are merely passing through the gut lumen, a limitation that severely constrains inference about direct viral involvement in pathogenesis.

The team’s methodological innovation was to couple laser-capture microdissection of formalin-fixed paraffin-embedded tissues with metatranscriptomic sequencing. Formalin-fixed paraffin-embedded, or FFPE, tissues are the standard material of diagnostic histopathology, preserving cellular architecture so that pathologists can score lesions and identify affected regions. Using a laser microdissection system, the researchers excised histologically defined lesion areas from four regions of the gastrointestinal tract, the duodenum, jejunum, proventriculus and gizzard, alongside matching unaffected non-lesion regions from the same tissue sections. This design meant that viral communities within lesions could be compared directly with those in adjacent healthy tissue within the same birds and the same flocks, effectively using each animal as its own experimental control and filtering out background viral noise from feed, environment and commensal carriage.

The sequencing generated a rich catalogue of viral operational taxonomic units across the sampled tissues. In the RSS-derived laser-microdissected samples, which totalled 71 distinct lesion and non-lesion preparations, three enteric viruses emerged as consistently enriched within gastrointestinal lesion tissues relative to non-lesion tissues. The first was Gallivirus spp., detected as multiple strains, a genus of small RNA viruses that has circulated in poultry for years without a clearly defined disease role. The second was Avastrovirus galli, an avian astrovirus species known to infect the intestinal tract. The third was Rotavirus deltagastroenteritidis, a member of the rotavirus species that infects the gastrointestinal systems of birds. All three belong to virus groups long suspected of involvement in enteric disease, but this study provided the spatial evidence that conventional approaches could not deliver: their genetic material concentrates precisely where the tissue damage is.

Importantly, these candidate viruses were not confined to a single outbreak. The researchers detected them across multiple affected farms, and in individual lesion samples the viruses co-occurred, suggesting that co-infections may contribute to the development or severity of stunting disease. This pattern of co-occurrence raises questions familiar from other enteric disease systems, where combinations of viruses, rather than any single agent, may overwhelm the gut’s absorptive capacity or immune defences. The consistency of enrichment across farms strengthens the case that these three viruses are meaningfully associated with RSS pathology rather than being incidental findings, although the authors are careful to frame them as candidate agents whose causal roles still require further investigation.

A particularly revealing component of the study came from the comparative analysis with transmissible viral proventriculitis. The team analysed 49 laser-microdissected samples derived from TVP cases and found that the enteric virome compositions of the two syndromes were clearly distinct. Differential enrichment of vaccine-like strains was observed in the TVP samples, indicating that live attenuated vaccine viruses may occupy a different ecological or pathological niche in this maldigestion-associated condition. The contrast between the RSS and TVP viral signatures confirmed that the spatially informed approach can discriminate disease-specific viral profiles, a proof of principle that extends beyond this single question. Where two clinically related but pathologically distinct syndromes could previously be interrogated only with blunt whole-tissue sequencing, the lesion-anchored method now separates their viromes with histological precision.

Suspicions raised by sequencing were then tested against independent material. All three RSS candidate viruses were confirmed in matched fresh-frozen tissue homogenates from the same birds using reverse transcription PCR, quantitative RT-PCR and amplicon sequencing, verifying that the microdissection findings were not artefacts of FFPE processing or sequencing bias. The researchers went a step further and attempted virus isolation, successfully recovering chicken astrovirus in cell culture. This chain of evidence, from histological lesion to spatially resolved viral sequence to independent molecular confirmation in frozen tissue to live virus in culture, represents a rigorous workflow for implicating candidate pathogens in a disease complex that has resisted definitive attribution for decades.

The technical achievement rests on careful handling of a notoriously difficult sample type. FFPE tissue is chemically preserved and fragmented, which complicates RNA extraction and library preparation, and laser-capture microdissection demands expert histological guidance to distinguish lesion from non-lesion tissue reliably. The authors acknowledge the histological laboratory of the Agri-Food and Biosciences Institute at Stormont for preparing tissue samples, and Professor Imre Lengyel and Dr Jamie Mitchell of Queen’s University Belfast’s Wellcome Wolfson Institute for Experimental Medicine for training and laboratory support in the microdissection experiments. The work was funded by the UK Biotechnology and Biological Sciences Research Council under grant number BB/X01732X/1, with additional collaboration from the Animal and Plant Health Agency and St. David’s poultry team in clinical diagnosis, post-mortem investigation and tissue sample preparation.

The significance of the framework extends beyond the broiler house. Spatially informed viromics, as the authors describe it, moves beyond conventional virome surveys by linking viral abundance patterns to histopathological context, converting a list of detected viruses into a map of where each virus resides relative to tissue injury. The same logic applies to any disease in which a virome has been catalogued but causation remains contested, from respiratory infections in livestock to chronic inflammatory conditions in humans where archived biopsy material is abundant. By demonstrating that lesion-anchored analysis can separate disease-specific viral signatures between two syndromes in the same host species, the study establishes a template for turning diagnostic pathology archives into high-resolution virological evidence.

For the poultry industry, the immediate value lies in the candidate agents themselves. Gallivirus, avian astrovirus and the avian rotavirus now have concrete spatial and molecular evidence tying them to RSS lesions across multiple farms, giving researchers specific targets for experimental infection studies, vaccine development and diagnostic assay design. The open question the authors highlight is whether these viruses function as primary pathogens or as co-factors that act alongside other infectious or husbandry-related stresses to produce stunting disease. Answering that question will require controlled challenge experiments and longitudinal field work, but for the first time those experiments can be aimed at a shortlist of candidates selected not merely by abundance in a sequencing library but by their physical presence within the lesions where the damage is done.

Subject of Research: Spatial viromics of broiler stunting disease lesions to identify candidate viral pathogens

Article Title: Lesion-anchored viromics identifies candidate viral agents of broiler stunting diseases

Article References: Hsieh, S.-Y., Creevey, C. J., Sheridan, P. M., Poulos, C. G., Smyth, V. J., & Kyriazakis, I. (2026). Lesion-anchored viromics identifies candidate viral agents of broiler stunting diseases. Microbiome. https://doi.org/10.1186/s40168-026-02556-w

Image Credits: AI Generated

DOI: 10.1186/s40168-026-02556-w

Keywords: broiler, runting-stunting syndrome, transmissible viral proventriculitis, viromics, metatranscriptomics, laser-capture microdissection, astrovirus, rotavirus, gallivirus, poultry health, viral enteritis, histopathology

The target of the investigation was a group of microorganisms known as formatotrophs—organisms capable of consuming formate, a simple one-carbon compound that serves as a critical metabolic currency in anaerobic digesters. Formate shuttles carbon and reducing power between different members of the microbial consortium, and the microbes that consume it occupy a pivotal position in the food web that ultimately yields methane. Because these organisms are metabolically active only when formate is available, conventional sequencing approaches struggle to distinguish their viruses from the vast background of viral genetic material produced by dormant or inactive community members. The research team needed a way to tag the active players before going hunting for their viruses.

Their solution was to feed laboratory microcosms—small, controlled batch anaerobic digestion reactors—with formate labeled with carbon-13, a heavy isotope of carbon. Any microorganism that consumed the labeled formate would incorporate the heavy carbon into its own DNA, making that DNA measurably denser than the DNA of organisms relying on other carbon sources. By spinning the extracted DNA in a density gradient, the researchers could separate the heavy, isotope-enriched fractions from the lighter ones, effectively isolating the genetic material of the active formatotrophs. This technique, known as stable isotope probing or SIP, has been used before to study active bacteria, but coupling it with viral metagenomics—sequencing of viromes, the collective viral genomes in a sample—is what made the new approach distinctive.

The enrichment experiment revealed that two primary guilds of formate consumers dominated the labeled fractions. The first consisted of hydrogenotrophic methanogens, archaea belonging to the order Methanobacteriales, with the species Methanobacterium subterraneum predominating. These archaea consume formate and release methane directly, placing them at the very end of the anaerobic food chain. The second guild comprised acetogenic bacteria from the family Natronincolaceae, specifically the genus Andreesenella, which convert formate and other substrates into acetate and other products that feed the methanogens. Together, these two groups formed the metabolic backbone of the formate-driven community, and any viruses found associated with their isotope-labeled DNA could be confidently assigned to these active hosts.

From the cross-assembly of six viromes and six isotope-labeled microbiome fractions, the team assembled a catalogue of 2,368 viral operational taxonomic units, or vOTUs—genetic clusters that roughly correspond to viral species. Of these, 261 were selected for detailed analysis. The results delivered a striking demonstration of the method’s power. Viruses associated with methanogenic archaeal hosts were dramatically enriched in the heavy, carbon-13-labeled fractions of the microbiome, yet they were nearly absent from the total viromes sequenced from the same samples. In other words, the viruses infecting the active methanogens were hiding in plain sight, invisible to conventional virome sequencing but clearly revealed once the isotope label was used to filter for activity. This contrast shows that DNA-based stable isotope probing can resolve virus-host associations at the level of entire viral communities, not just individual pairs.

Among the novel viruses identified were archaeal viruses infecting Methanobacteriales, including members of the family Anaerodiviridae and representatives of a possible entirely new viral family. On the bacterial side, the researchers discovered two viruses infecting Andreesenella that carry diversity-generating retroelements—genetic systems that allow viruses to rapidly shuffle and diversify the sequences of certain genes, potentially helping them adapt to changing host defenses. The team also detected a provirus, a viral genome integrated into a host chromosome, predicted to infect Methanothrix, a strictly acetoclastic methanogen that consumes acetate rather than formate. Because Methanothrix occupies a different trophic level from the directly labeled formatotrophs, its detection illustrates how the SIP-viromics approach can capture viral associations that ripple across multiple levels of the anaerobic food web.

Beyond simply cataloguing new viruses, the study examined the auxiliary metabolic genes carried within the viral genomes. These are genes of host origin that viruses maintain and express during infection, and they can meaningfully alter host metabolism. The analysis revealed numerous defense-associated genes, including dcm, metK, and queC, which may help viruses protect themselves or their hosts from competing genetic elements. More intriguingly, the researchers identified genuine metabolic auxiliary gene candidates, such as cysH, involved in assimilatory sulfate reduction, and a contiguous cluster of genes including uxe, galU, and serB, which participate in surface polysaccharide biosynthesis. If these genes are expressed during infection, they could influence sulfur cycling within the digester and modify the surface properties of host cells, with potential downstream consequences for how the microbial community functions and how efficiently biogas is produced.

The significance of these findings extends beyond anaerobic digestion itself. Methanogens and acetogens are central players in global carbon cycling, mediating the flow of carbon in wetlands, sediments, rice paddies, and the digestive tracts of animals. Viruses that infect these organisms have been almost impossible to study in situ because methanogenic archaea are difficult to culture and their viruses are even harder to isolate. By demonstrating that isotope labeling can pull the viruses of active methanogens out of a complex community without any need for cultivation, the study opens a path toward understanding viral regulation of methane emissions and carbon flux in a wide range of anoxic environments, not just engineered bioreactors.

For biotechnologists, the implications are equally compelling. Anaerobic digestion is a cornerstone of renewable energy and waste management, and process performance depends on the delicate balance of microbial guilds that hydrolyze polymers, ferment sugars, produce acetate and hydrogen, and finally generate methane. Viral infections can crash populations of key players, destabilize reactors, and shift metabolic pathways in unpredictable ways. Conversely, viruses carrying auxiliary metabolic genes might enhance process performance by supplementing host functions. The catalogue of 2,368 vOTUs and the detailed characterization of 261 representative viruses provide a foundation for monitoring viral dynamics in full-scale digesters and, eventually, for managing them—whether by suppressing harmful infections or exploiting beneficial ones.

The study, funded by the French National Research Agency through the VIRAME and VIRALSONG projects, also underscores the value of methodological integration in microbial ecology. Neither stable isotope probing nor viromics alone could have delivered these results: isotope labeling without sequencing would have revealed only that active hosts existed, while viromics alone would have missed the methanogen viruses entirely. By coupling the two, the researchers transformed a fundamental limitation of environmental virology—the difficulty of linking viral sequences to their hosts—into a solvable problem. As the approach is refined and applied to other isotope-labeled substrates and other anoxic ecosystems, the hidden viral diversity governing microbial carbon metabolism may finally come into focus, one labeled guild at a time.

Subject of Research: Viruses infecting active formatotrophic methanogens and acetogens during anaerobic digestion, identified by coupling stable isotope probing with metagenomics

Article Title: Coupling of stable isotope probing and metagenomics reveals the diversity of viruses infecting formatotrophs during anaerobic digestion process

Article References: Ngo, V. Q. H., Talleu, C., Enault, F., Midoux, C., Mariadassou, M., Bouchez, T., Krupovic, M., & Bize, A. (2026). Coupling of stable isotope probing and metagenomics reveals the diversity of viruses infecting formatotrophs during anaerobic digestion process. Microbiome. https://doi.org/10.1186/s40168-026-02509-3

Image Credits: AI Generated

DOI: 10.1186/s40168-026-02509-3

Keywords: anaerobic digestion, stable isotope probing, viromics, methanogens, formatotrophs, viral ecology, metagenomics, biogas, archaeal viruses, auxiliary metabolic genes, Andreesenella, Methanobacteriales