Journal of General Virology

Background: Klebsiella pneumoniae is a key causative agent of multidrug resistant nosocomial infections. Bacteriophages offer a promising alternative treatment, but the diversity of Klebsiella species requires the characterisation of a broad range of phages, particularly the poorly characterised jumbo phages. Materials and Methods: Phage BMCPR_005026 was isolated from sewage against Klebsiella pneumoniae. Whole genome sequencing, transmission electron microscopy and host range analysis against a diverse set of 40 clinically relevant Klebsiella strains were used to characterise the phage. Results: The virion had a myovirus morphology and genomic analysis placed it within an undefined family in Caudoviricetes where it forms a new species within the genus Alcyoneusvirus. BMCPR_005026 could form plaques on 3 of the 40 Klebsiella strains tested (7.5%) and formed halos. Conclusion: BMCPR_005026 represents a new species of phage within the genus Alcyoneusvirus with activity against 3 Klebsiella KL-types.

Urban waterfowl are important natural reservoirs of viruses that can affect wildlife, livestock and human health, yet the diversity and spatial distribution of these viral communities remain poorly understood. Here, we characterised the faecal viromes of urban waterfowl sampled from 13 locations across the length of Aotearoa (New Zealand). We identified 96 avian viruses (50 species) spanning nine viral families, including 33 previously undescribed viruses. Picornaviruses were the most widespread viral group, while several viruses were closely related to those previously identified in Australian waterfowl, indicating that some viral lineages are likely distributed across the broader Australasian region. Despite sampling locations spanning more than 1,500 km, virome composition exhibited little evidence of geographic structure, with little difference in viral diversity or community composition between sampling locations. Network analyses similarly demonstrated extensive sharing of virus species among sampling locations, indicating little geographic structuring of virome composition among urban waterfowl. In addition, we recovered the complete genome of a low-pathogenic A(H3N8) influenza A virus from mallards (Anas platyrhynchos) in the most southern region of Aotearoa. These findings reveal extensive viral diversity and widespread connectivity among urban waterfowl, providing new insights into viral ecology and the potential for widespread virus distribution across Aotearoa.

Protein kinase R (PKR) is an antiviral sensor that induces translational shutdown and stress granule (SG) formation through the phosphorylation of eIF2. Although previous studies have proposed that hepatitis C virus (HCV) non-structural protein 5A (NS5A) inhibits PKR dimerization, other studies consistently observe PKR activation and SG formation during HCV infection. To address this discrepancy, we interrogated the ability of NS5A from different genotypes and strains to regulate canonical PKR-eIF2 activation in a variety of cell culture models. Regardless of the genotype or strain tested, HCV NS5A failed to inhibit PKR dimerization or dsRNA-induced PKR activation, nor did it rescue downstream steps in the PKR-eIF2 pathway, such as translation halt and SG formation, in contrast to known viral PKR antagonists. Similarly, NS5A did not antagonise PKR activation when expressed in the context of other HCV non-structural proteins. We also tested the role of the host factor cyclophilin A (CypA), which interacts with both NS5A and PKR. However, the absence of CypA did not affect downstream PKR signaling or the activity of viral PKR antagonists or NS5A. Collectively, these findings indicate that NS5A does not effectively antagonise the PKR-eIF2 pathway and further support previous findings that HCV replication occurs in the presence of an active PKR response.

Since 2020, highly pathogenic avian influenza (HPAI) A(H5Nx) clade 2.3.4.4b viruses have spread globally, causing extensive outbreaks in domestic and wild birds. Increased circulation has resulted in frequent spillover to mammals, and occasional mammal-to-mammal transmission. Although human infections remain rare, the zoonotic potential of these viruses continues to be a public health concern. We investigated six cases of HPAI in red foxes (Vulpes vulpes) in Norway during 2022-2024 and identified infections with H5N1 and H5N5 viruses. Pathological and virological investigations demonstrated systemic infection, with prominent lesions in the brain and/or lungs. Phylogenetic analyses showed high similarity between viruses detected in foxes and those concurrently circulating in wild birds. The mammalian adaptation marker PB2:E627K was identified in one H5N1 virus and as a minority variant in a second H5N1 virus.

Migration can increase population connectivity and gene flow but may also facilitate pathogen transmission among hosts and habitats. The way life-history strategies shape viral diversity in closely related freshwater fishes remains poorly understood. Here, we used metatranscriptomics to compare the viromes of two fish in the family Galaxiidae with contrasting life-history strategies: amphidromous [i]nanga (Galaxias maculatus) and river-resident Gollum galaxias (Galaxias gollumoides). We analysed 54 pooled libraries derived from 416 individuals collected across 27 sites spanning multiple catchments and fish-passage barriers in southern New Zealand. A total of 13 novel fish-associated viruses were identified from five viral families (Astroviridae, Circoviridae, Coronaviridae, Hepeviridae, and Picornaviridae). Virome composition differed markedly between species, with only a single virus-positive pool detected among [i]nanga, whereas Gollum galaxias harboured 11 novel viruses, with circoviruses dominating the virome. No fish-associated viruses were detected in post-larval juveniles of either species, and alpha diversity did not differ significantly among catchments, sites or positions relative to fish-passage barriers. However, beta diversity analyses revealed significant spatial structuring of viral communities among sites and catchments, indicating localised variation in virome composition. Contrary to our predictions, the migratory species exhibited substantially lower viral diversity than the river-resident species. The absence of detectable viruses in juveniles, combined with the greater viral richness of the longer-lived Gollum galaxias, suggests that host longevity and cumulative environmental exposure may be important drivers of virome assembly. These findings demonstrate that host life-history strategy influences virome composition and reveal unexpectedly low viral diversity in a globally widespread migratory galaxiid, providing new insights into virus-host ecology and connectivity in freshwater fishes.

Oysters are vital to global aquaculture and marine ecosystems, yet, their associated RNA viruses remain largely unexplored. Here, we analysed ~1,000 publicly available metatranscriptomic datasets from 13 oyster species across 14 countries and 5 continents to characterize the oyster RNA virome (ORV). We identified 1,515 distinct RNA viruses putatively representing 17 viral orders and 766 species-level viral operational taxonomic units (vOTUs), with ~62% being previously undocumented putative species. These viruses not only populate extant branches of RNA virus evolution, but also represent previously unrecognized families and genera, and include viruses that putatively infect bacteria, fungi, protists, plants, invertebrates, and vertebrates. Based on sequence similarity, we found many calicivirus-like, orthoreovirus-like, astrovirus-like, and picornavirus-like viruses that do not infect invertebrates or microbes; thus, oysters accumulate diverse viruses from the surrounding water, and can potentially be interrogated as reporters of viruses circulating in the environment.

In resource-limited settings plasma is usually transported to centralized laboratories for HIV RNA quantification; often by motor vehicles using dry ice. Any decay in plasma HIV RNA when near the threshold used to define virologic failure could impact clinical management. However, maintaining a cold chain during transport can be costly and limit the frequency of shipments, which can delay clinical assessments and also negatively impact patient care. Transporting plasma at ambient temperatures could facilitate timely measurements of plasma HIV RNA viral loads (VL). We conducted a stability study of HIV RNA in plasma held for 1, 3, 5, and 7 days at a range of temperatures (4{degrees}C, 30{degrees}C, and 37{degrees}C) to simulate the durations and temperatures specimens could experience during transport. Thirteen large volume plasma specimens with VL ranging from 2.7-5.53 log10 copies/mL were evaluated. Mean changes in VL between Day-0 to Day-1, Day-3, Day-5 or Day-7 were calculated, with decays >0.5 log10 c/mL considered clinically relevant. Compared to Day 0, the VL of plasma stored at 4{degrees}C and 30{degrees}C remained stable across the Days tested. The VL in plasma stored at 37{degrees}C had a progressive decline at Day-1, Day-3, Day-5 and Day-7 (means of -0.04, -0.19, -0.33. -0.34 log10 c/mL, respectively), with a decay of >0.5 log10 observed in 3/13 (23%) specimens at Day-7. A linear regression analysis showed that plasma specimens stored at 37{degrees}C significantly decayed over time (slope -0.054, 95% CI -0.079, -0.029; p=0.01). Any decay observed among specimens stored at 4{degrees}C and 30{degrees}C was not clinically relevant nor statistically significant. Our observations suggest that HIV RNA levels in plasma should remain stable if transported to centralized laboratories at ambient temperatures of [≤]30{degrees}C or chilled by cold packs over a period [≤]7 days. Transporting specimens with cold packs instead of dry ice could reduce costs and facilitate the timely monitoring of VL used to assess and guide antiretroviral therapy.

Since emerging in 2020, clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) has disseminated globally, causing substantial infection and mortality in wild birds and poultry. Additionally, numerous spill-over events into mammals have occurred, including mass mortalities and sporadic human infections. Such events can drive the acquisition of mammalian-adaptive mutations, which further increase the risk to humans; the most well characterised mutation is the glutamate to lysine change at amino acid position 627 in the polymerase basic 2 protein (PB2-627K). Across avian influenza viruses, PB2-627K enhances polymerase activity and replication in mammalian cells, increasing zoonotic and pandemic potential. However, bioinformatic analyses indicate that PB2-627K remains rare among clade 2.3.4.4b H5N1 viruses circulating in avian populations. Here, we investigated the impact of PB2-627K on viral fitness in avian hosts to assess the potential for mammalian-adapted viruses to re-establish in birds, mimicking infection of avian species following scavenging on mammalian species where PB2-627K has already emerged. Using a representative H5N1 virus (European genotype AB), PB2-627K increased polymerase activity and replication in human cells without compromising replication in avian cells. A mammalian-derived PB2-627K H5N1 isolate from a fox exhibited comparable replication kinetics and transmission efficiency in chickens and ducks to a closely related PB2-627E virus. Notably, PB2-627K remained genetically stable at consensus level following infection and transmission in both avian hosts. These findings demonstrate that mammalian-adaptive mutations acquired during spill-over can be maintained in avian populations, potentially elevating zoonotic risk and underscoring the importance of surveillance for such mutations in birds.

TThe discovery of virophages and polinton-like viruses (PLVs) sparked studies of their hyperparasitic relationships with their associated giant viruses and hosts. To date, every cultured virophage and PLV depends on an Imitervirales helper virus. The freshwater alga Chrysochromulina parva is infected by viruses in the Imitervirales and Algavirales, and recent work showed that the PLV, CpV-PLV Moe, replicates via co-infection with the Imitervirales host CpV-BQ3. The other known PLVs, CpV PLV Larry and CpV PLV Curly, were subsequently isolated alongside CpV-BQ1, a putative Algavirales virus, raising the possibility that they depend on an Algavirales helper. To determine the nature of Larry and Curlys hyperparasitic relationships, infection experiments were conducted with each PLV and either CpV-BQ3 or CpV-BQ1. These experiments demonstrated that that neither Larry nor Curly replicated when introduced to C. parva alone, nor during mixed infections that included the Imitervirales virus BQ3. In contrast, the Algavirales virus CpV-BQ1 supported replication of both PLVs, with each increasing roughly 1000-fold over the course of infection while CpV-BQ1 replication was simultaneously reduced up to 100-fold. Together, these findings provide the first culture based evidence that PLV replication can be supported by an Algavirales helper and demonstrate that C. parva supports at least two distinct hyperparasitic relationships spanning different viral orders within the class Megaviricetes.

Background and Aims: Genetic diversity within the hepatitis B virus (HBV) surface antigen (HBsAg) drives the emergence of immune escape variants, but studying this diversity in vivo has been limited by the difficulty of establishing infection from patient isolates. We aimed to perform unbiased in vivo screening of variants in the HBs antigenic loop for antibody escape and fitness, using synthetic DNA launch of HBV in human hepatocyte chimeric mice. Approach and Results: Direct intrahepatic injection (IHI) of barcoded, recombinant cccDNA established infection from up to 32,000 independent launch events of a genotype A strain and successfully launched clinical consensus sequences across the major genotypes. Using IHI, we performed deep mutational scanning of 4,608 codon variants across the HBs antigenic loop. Under control antibody treatment, diverse variants expanded, revealing differences in variant fitness in the absence of adaptive immune pressure. Anti-HBs monoclonal antibody treatment suppressed wild-type viremia while permitting breakthrough of resistant variants from the library. Sequencing identified strong enrichment for substitutions at G145 (G145Q, G145H, G145R). Notably, G145Q, rather than the clinically dominant G145R, showed the greatest fitness advantage, indicating that G145R's clinical prevalence does not reflect superior intrinsic fitness. Conclusions: Unbiased in vivo screening reveals that clinically observed HBV escape mutations reflect which substitutions are genetically accessible by single nucleotide change and compatible with the overlapping polymerase reading frame, rather than which confer optimal fitness. This approach establishes a platform for high-resolution mapping of viral variant fitness under antiviral selection pressure.

Several pathogenic viruses benefit plants under specific circumstances, e.g., by inducing drought resistance. It is hypothesised that RNA viruses in the Endornaviridae and Partitiviridae families, sometimes called persistent since they are exclusively seed-transmitted at ~100% efficiency, have followed evolutionary trajectories towards mutualism. However, evidence comes from crossing non-identical host backgrounds making it impossible to distinguish definitively between the effects of viral versus host genotypes. We used virus-induced gene silencing (VIGS) to cure pepper plants of bell pepper endornavirus (BPEV) and the partitiviruses pepper cryptic virus 1 (PCV1) and PCV2 and compared these with genetically identical plants retaining these viruses. We found that PCV1, PCV2 and BPEV can significantly affect aerial and root growth and fruit/seed characteristics in ways that could benefit host fitness. VIGS-mediated curing allows unambiguous hypothesis-testing of potential benefits of persistent viruses and exploration of how viral and host genotypes shape persistent virus-host relationships.

BackgroundHuman Mastadenovirus B (HAdV-B) causes ocular and respiratory infections and is capable of causing outbreaks in healthcare settings. HAdV-B114, a recombinant genotype only formally recognised in 2023, has been reported predominantly from Germany, the USA, China and Japan; genomic data from UK clinical settings remain limited. This study analysed 68 HAdV-B whole genomes from ocular and respiratory-associated samples collected at a Nottingham hospital across the pre- and post-COVID-19 restriction periods (2017-2023). MethodsClinical samples typed positive for HAdV-B by partial hexon Sanger sequencing were selected for whole-genome sequencing using a novel overlapping PCR amplicon strategy, sequenced on the Oxford Nanopore Technologies PromethION platform. Phylogenetic and pairwise difference analyses were used to characterise genotype composition and genomic relatedness. SimPlot and amino-acid comparison characterised divergence between sub-lineages. ResultsWhole-genome phylogenetics confirmed that two species B genotypes were detected in Nottingham University Hospitals between 2017-23: HAdV-B114 (n=56) and HAdV-B7 (n=12). HAdV-B114 was detected before and after COVID-19 restrictions, with an increase in 2022-2023, consistent with post-restriction expansion of a pre-existing lineage. Pairwise difference analysis identified 24 genomically identical B114 sequences; associated metadata argued against a single nosocomial transmission chain interpretation, with the pattern more consistent with repeated detection of a widely circulating community strain, with the possibility of localised onward transmission. Within the B114 clade, multiple monophyletic groups were identified by whole-genome phylogenetics. ConclusionsThis study adds whole-genome resolution to the existing UK HAdV-B114 data, demonstrates the utility of WGS for clinical adenovirus surveillance and shows the benefits of combining epidemiological data with genomic analysis.

Powassan virus (POWV) is an emerging tick-borne orthoflavivirus that causes severe encephalitis, long-term neurologic sequelae, and death, yet no approved antiviral therapies exist. Here, we evaluated several previously characterized nucleoside analogs as candidate countermeasures for POWV encephalitis. Favipiravir, molnupiravir, 4'-fluorouridine (4'-FlU), and GS-441524 inhibited POWV replication in vitro and demonstrated comparable activity against West Nile virus (WNV). We established C3H/HeNCrl mice as a stringent POWV disease model, showing increased susceptibility, higher day 7 brain titers, and more pronounced neurologic signs resembling severe human disease, compared with C57BL/6 mice. In prophylactic and therapeutic studies, 4'-fluorouridine was the most efficacious compound tested, significantly reducing brain viral burden even when treatment was delayed. Direct comparison with molnupiravir, favipiravir, and GS-441524 in mice confirmed superior survival benefit and antiviral activity for 4'-FlU against both POWV and WNV. These data identify 4'-fluorouridine as a promising therapeutic candidate for neurotropic flavivirus disease.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis, with an estimated 20 million human infections annually. Its diverse host range presents a significant zoonotic threat, yet the molecular mechanisms underpinning HEV-host interactions remain poorly characterised. Here, we used transposon-based insertion screening of the HEV pORF1 polyprotein to identify a novel region within the viral PCP domain that permits HA-epitope insertion, enabling the first epitope tagging of this domain. Combined with V5 tagging of the hypervariable region (HVR), this approach enabled the generation of a replication-competent stable cell line expressing dual-tagged ORF1. Co-immunoprecipitation and TMT-based LC-MS proteomics revealed that pORF1 interacts with host proteins involved in endoplasmic reticulum-associated degradation (ERAD) and the 26S proteasome. Pharmacological inhibition demonstrated that both pathways are required for efficient HEV replication. Treatment with the FDA-approved proteasome inhibitor bortezomib altered pORF1 precursor abundance and led to loss of full-length pORF1 through enhanced proteolysis. Importantly, the kinetics of ORF1 degradation following proteasome inhibition correlated with immediate loss of HEV replication, demonstrating a functional link between pORF1 proteostasis and viral replication. Collectively, our findings identify the ERAD network as a previously unrecognised requirement for HEV replication and establish a role for the 26S proteasome in maintaining pORF1 proteostasis.

Plant-derived flavonoids show measurable anti-dengue activity in cell culture, but their translational value is limited by a narrow therapeutic window: the concentrations that inhibit the virus approach or exceed those that are cytotoxic. Whether nanoparticle formulation can resolve this constraint, rather than simply add potency, remains untested for a chemically defined fraction. Here we show that biogenic silver nanoparticle (AgNP) formation using a flavonoid-enriched fraction of Carica papaya inverts an unusable selectivity profile into a viable one. The unformulated fraction was cytotoxic below the concentrations required for antiviral activity: its 50% cytotoxic concentration (CC50 = 134.9 ug/mL) lay below its 50% effective concentration against dengue virus serotype 2 (DENV-2; EC50 = 254.4 ug/mL), giving a Selectivity Index (SI) of 0.53. Using the same flavonoids as sole reducing and capping agents produced AgNPs (Z-average 128.4 nm; PDI 0.232; zeta potential -28.4 mV) that moved both parameters simultaneously. CC50 rose approximately 8.6-fold to 1,165.74 ug/mL while EC50 fell approximately 8.8-fold to 29.05 ug/mL, raising the SI to 40.12, an approximately 75-fold shift. Time-of-addition analysis localised the effect to the extracellular phase: inhibition was significant under pre-treatment and co-treatment but not after viral adsorption, identifying the AgNPs as entry inhibitors rather than replication inhibitors. Consistent with a serotype-independent physical mechanism, AgNP treatment at 30 ug/mL reduced viral RNA across all four serotypes, using inocula standardised against WHO-traceable NAAT reference reagents. These findings identify capping chemistry, rather than silver content alone, as a determinant of the therapeutic window in phytosynthesised nanoantivirals.

Porcine epidemic diarrhea virus (PEDV) was introduced into the United States (USA) in 2013 and has circulated ever since. PEDV causes significant losses in the swine industry, with the greatest impact observed during the first years following its introduction into the country. However, the evolutionary dynamics of the virus remain underexplored. Recently, analyses of the Spike protein subunit S1 suggested that the PEDV population currently circulating in the USA represents a fraction of its historical diversity and comprises only two clades restricted to specific geographic regions. These observations were based on a limited set of S1 sequences. Furthermore, our understanding of evolutionary dynamics at the genomic level is even more limited, as complete PEDV genomes have not been generated since 2017. Here, we characterized the evolutionary dynamics of PEDV populations in the USA using complete genomes and assessed the contribution of different evolutionary mechanisms to shaping population structure. We analyzed 338 complete genomes, of which 25 were obtained in this study, together with 623 Spike S1 sequences, of which 41 were obtained in this study. All sequences obtained in this study were assigned to the non-S-INDEL strain. No S-INDEL sequences were recovered in this study, although these viruses continue to circulate in the country. Our analyses revealed that most sequences sampled after 2023 formed a monophyletic clade divergent from sequences collected in previous years, suggesting an ongoing temporal population structuring. Population structure analyses further supported evidence of population differentiation over time. Recombination analyses indicated that sequences belonging to the contemporary clade shared a single recombination event, suggesting that this clade may have emerged through recombination. Time-scaled S1 phylogenies were consistent with previous findings, supporting the circulation of only two contemporary clades; however, one of these clades was detected for the first time outside its previously reported geographic range. Our findings document temporal genetic differentiation in US PEDV populations, consistent with continued viral evolution, and broaden the known geographic distribution of contemporary S1 clades. These results highlight the value of continued genomic surveillance to monitor changes in PEDV diversity and distribution and provide sequence data to inform vaccine research.

The bean thrips, Caliothrips phaseoli, is an emergent pest affecting peanut production. Thus, gaining a deeper understanding of the naturally occurring viral populations associated with this insect is of major importance, as such knowledge could support the design of novel pest management strategies that are both ecologically responsible and sustainable over the long term. In this study, we performed the first RNA high-throughput sequencing (HTS) analysis of C. phaseoli to unravel its virome, discovering a wide number of novel RNA viruses. Our HTS analysis revealed the presence of 31 viral segments, corresponding to 18 viruses, including double-stranded RNA viruses as well as positive- and negative-sense single-stranded RNA viruses. All these are novel viruses and cover multiple viral lineages belonging to diverse families. Overall, these results indicates that C. phaseoli hides a complex virome. The findings presented here provide an important framework for subsequent studies aimed at understanding the ecological significance, evolutionary patterns, and possible use of these viruses as components of biologically based management programs for this emerging peanut pest.

Human noroviruses (HuNoVs) are the most common cause of non-bacterial foodborne illnesses worldwide. HuNoVs are notoriously difficult to culture, and current cell culture systems, such as human intestinal enteroids have limitations in efficiency and scalability, which has limited their applications in food safety studies. Recently, it has been shown that transformed salivary gland cell lines (including NS-SV-TT-DC) allow for replication and passaging of norovirus GII.4. Herein, we examined whether we can independently observe viral replication in salivary gland cells and if other strains of human norovirus can replicate in these cells. For this purpose, we first evaluated the replication of several norovirus strains in NS-SV-TT-DC cells by either infecting them with vesicle-cloaked viruses or total viruses in stool filtrates. Low levels of viral replication was observed and vesicle isolation did not improve viral replication. Next, we screened for replication of 50 norovirus samples from 9 genotypes, and 14 isolates demonstrated over a two-fold viral replication. We observed that the initial viral load in the samples does not have a significant effect on replication efficiency. We were also able to passage several isolates. Subsequently, we examined whether interferon inhibitors such as Ruxolitinib, TPCA-1, and BX795 would improve viral replication and observed negligible effects. Finally we tested multiple supplements including MgCl2, bile salt, ceramide, and TAK-779. Most supplements had little or no effect on HuNoV replication in NS-SV-TT-DC cells. Bile salt and TAK-779 showed limited, genotype-specific benefits. Altogether these results indicate that salivary gland cells allow limited replication of certain HuNoV isolates.

SARS-CoV-2 continues to circulate globally at high levels and to evolve into new variants. Recently, a new saltation variant harbouring a highly mutated spike protein, PJ.2.1, emerged in North America. Here, we provide a brief epidemiological and virological characterization of PJ.2.1 and cocirculating variants. We report that PJ.2.1 is spreading in the USA, Canada, and Spain and mainly infects individuals aged 60 years and older, as do most other variants, with the exception of BA.3.2, which preferentially infects individuals aged 0-9 years. PJ.2.1 was less susceptible to inhibition by an antibody against ACE2 than were the other variants, indicating increased ACE2 binding. In contrast, PJ.2.1 did not show augmented evasion of antibodies from vaccinees who had received the LP.8.1-adapted vaccine compared with most other variants analysed. These results suggest that augmented ACE2 affinity rather than increased antibody evasion might drive the current, rapid spread of PJ.2.1.

Recent zoonotic coronavirus outbreaks have sparked an interest in understanding coronaviruses circulating in animal reservoirs. While bats are recognised as major reservoirs for these viruses, coronaviruses have also been detected in a wide range of terrestrial small mammals, particularly rodents. However, coronavirus diversity in these mammals, specifically in Europe, is still poorly characterised. In this study, we detected and characterised coronaviruses in terrestrial small mammals and bats from Belgium, France, Germany, and Ireland. We screened tissue samples from rodents and shrews and environmental bat guano using pan-coronavirus real-time and conventional PCRs, with positive results confirmed by sanger sequencing. Among 2667 bat and 1152 terrestrial mammal samples, we detected 59 positive samples, 36 from bats and 23 from rodents, from which we recovered 60 coronavirus sequences, spanning both Alphacoronavirus and Betacoronavirus lineages. Through next generation sequencing we identified one Pipistrellus pygmaeus bat guano sample carrying two distinct alphacoronaviruses, representing Nyctacovirus and Pedacovirus subgenera. Among rodents, most coronavirus-positive samples originated from a zoo in Belgium, where several CoV lineages co-circulated. The identified rodent coronaviruses clustered among previously described rodent-associated lineages, some of which also include human and other mammal-derived coronavirus sequences. Consistent with previous studies, we found sarbecoviruses in horseshoe bats (particularly Rhinolophus ferrumequinum and R. hipposideros) and merbecoviruses in Plecotus auritus bats, and these viruses are distantly related to the human coronaviruses of the respective families. Similar to bat coronaviruses, some rodent coronaviruses strains seemed to be association with specific rodent species. In conclusion, our study enhances the understanding of coronaviruses in rodents and bats, revealing potential hotspots for these viruses and their implication for human and animal health. These findings underscore the necessity for continued surveillance of coronaviruses in wildlife reservoirs to mitigate future zoonotic spillover risks.