The transmission pattern of mpox has shifted from sporadic zoonotic outbreaks to sustained human-to-human spread. Epidemiological data indicate sexual contact as a crucial driver for efficient transmission and the associated devastating mpox outbreaks in recent years. However, our understanding of exact driving factors and transmission determinants is still limited. Here, we investigated MPXV clade Ia virus pathogenicity, shedding kinetics, and transmission potential in a prairie dog model (Cynomys ludovicianus). All tested mucosal inoculation routes (penile/preputial, vaginal, rectal, intranasal) resulted in a productive, systemic infection. Inoculation via urogenital routes generated the highest virus shedding and most severe clinical disease. A simulated sexual contact transmission resulted in 100% transmission efficiency with high virus shedding in sentinels on day 1, even before the onset of clinical signs. Our findings provide critical insights into mpox transmission, emphasizing the role of anogenital mucosal surfaces in facilitating rapid spread. These results advocate for a stronger focus on mucosal infection when evaluating countermeasures.

Abstract The recent MPXV epidemic across Africa revealed extensive viral diversity and complex transmission dynamics, prompting a continent-wide genomic investigation. We analysed 3,450 high-quality MPXV virus whole genomes from 24 African Union Member States, revealing the complex and concurrent circulation of Sub-clades Ia, Ib, IIa, and IIb. Subclade Ia showed high levels of virus diversity in reservoir hosts in Central Africa, detected through zoonotic transmission and some sustained human outbreak lastly detected. In contrast, Clade Ib exhibited signatures of sustained human-to-human transmission across Eastern and Southern Africa. Clade IIa remains largely zoonotic in West Africa. Like Ia, IIb shows continued zoonotic transmission, and sustained human outbreak linked to lineage G1 and G2 circulation. Phylogeographic analyses revealed frequent cross-border transmission and interconnectedness, which was aligned with both human mobility corridors and international boundaries. For instance, the Democratic Republic of the Congo or Sierra Leone seems to emerge as a source of regional exportation, while the Cameroon–Nigeria, CAR-Cameroon or CAR-DRC interfaces reflected ongoing cross-border zoonotic spillovers. These findings underscore the need for harmonised genomic surveillance, APOBEC3-aware triage, and integrated One Health strategies to prevent local outbreaks from escalating into regional epidemics and to inform vaccine deployment and public health preparedness.

The ongoing outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in the U.S. poses a significant public health threat. To date, 70 human cases have been confirmed in the United States, including two severe cases and one fatality. While suitable animal models are crucial for predicting the potential pandemic risk of newly emerging pathogens in humans, studies investigating contemporary HPAIV H5N1 transmission dynamics remain limited. Here, we investigate the pathogenicity and transmission efficiency of recent clade 2.3.4.4b H5N1 viruses isolated from a bovine, mountain lion, and a human case using Syrian hamsters. Intranasal inoculation results in productive virus replication in the respiratory tract and shedding for all three isolates. Transmission studies demonstrate limited efficiency via direct contact and airborne routes for all isolates. Although overall transmission is inefficient, the human H5N1 isolate demonstrates relatively greater contact transmissibility than the bovine and mountain lion isolates. Taken together, our findings demonstrate that the Syrian hamster model complements existing animal models for influenza A virus research and expands the resources available for investigating the pathogenicity, transmissibility, and efficacy of countermeasures against HPAIV H5N1.

In 2022, monkeypox virus (MPXV) clade IIb emerged resulting in a global epidemic driven by human-to-human transmission, mostly through sexual contact mainly among the population of men who have sex with men. To date, published data on the circulation of MPXV clade IIb in the central African region are absent. Here we describe a case of laboratory-confirmed mpox clade IIb lineage A2.2 in Pointe-Noire, the second largest city of the Republic of the Congo. Whole-genome phylogenetic analysis placed the MPXV in clade IIb, lineage A.2.2 currently emerging in West Africa, in particular Sierra Leone. An additional 16 cases of clade Ia, 32 cases of clade Ib and one additional introduction of clade IIb were identified by passive surveillance in the Republic of the Congo in 2025. The detection of clade IIb mpox marks the third distinct MPXV clade and lineage co-circulating in the human population, together with clade Ia and Ib. This underscores the need for improved surveillance and diagnostic strategies to identify the respective clade and lineage circulating in the human population. Strengthening of regional capacity for case detection, contact-tracing, public health measures and affordable vaccines are urgently needed to reduce the global risk for both clade I and clade II MPXV.

Monkeypox virus (MPXV) clade IIb caused an outbreak of >100,000 cases globally, with highest disease burden in people living with advanced human immunodeficiency virus (HIV), those with CD4 count <200 cells/mm3. We performed an extensive autopsy on a fatal case of mpox in a person living with advanced HIV-1, who achieved HIV suppression but poor immune reconstitution despite > 6 months antiretroviral therapy and had progressive necrotizing mucocutaneous mpox disease despite multiple prolonged mpox-targeted therapies. We quantified MPXV DNA across 76 tissues and observed a high burden in skin and mucosal tissue and a lower burden in all central nervous system (CNS) tissues tested. However, we detected replication-competent MPXV in multiple CNS tissues and confirmed neuronal and glial cell infection using RNA-scope in situ hybridization. MPVX sequencing from body and brain tissues revealed widespread tecovirimat resistance-associated variants. This case proves that replication-competent MPXV and tecovirimat resistance-associated isolates can infect and persist in the CNS of people with advanced HIV thus informing future therapeutic design.

Hendra virus (HeV) was discovered in 1994 in Australia. Limited genomic data have hindered comprehensive understanding of HeV's evolutionary dynamics. Here we recovered 48 HeV genomes from bats and 9 from horses from Australia between 2016 and 2020, revealing four distinct clades. Each clade was distributed over a large spatial area with multiple clades co-circulating within a single bat roost on the same day and over consecutive years. The diversity and temporal stability of co-circulating clades suggest that viral dynamics are driven by episodic shedding of existing lineages maintained at the population level, rather than immune-driven strain-replacement dynamics. HeV isolates of different clades displayed variation in phenotypic properties but minimal antigenic differences. We provide an overview of evolutionary dynamics, phenotypic properties and assessment of countermeasures for HeV, and provide insights into the processes that maintain virus diversity in bats and influence the potential for viral emergence.

Monkeypox virus (MPXV), a zoonotic Orthopox virus endemic to West and Central Africa, causes mpox disease. Although Ghana had no confirmed human cases before 2022, the 2003 U.S. mpox outbreak was traced to rodents exported from Ghana, suggesting potential undetected exposure in the local population. This study assessed mpox exposure prior to the emergence of Clade IIb in humans. We tested 457 serum samples collected across 14 regions of Ghana using a commercial anti-MPXV IgG ELISA. These samples comprised 365 archived sera from 2021 SARS-CoV-2 surveillance and 92 sera from suspected mpox cases during the 2022 outbreak. Multivariable logistic regression was performed to examine associations between MPXV seropositivity and demographic factors, including age, sex, region, urban/rural status and inferred smallpox vaccination status. Overall MPXV seroprevalence was 6.6%. Participants from the Western Region had significantly increased odds of seropositivity (aOR = 6.70, 95% CI: 1.75–25.62, p = 0.005), whereas those from Greater Accra had decreased odds (aOR = 0.28, 95% CI: 0.09–0.90, p = 0.033). The findings suggest localized MPXV circulation or repeated zoonotic spillover may have occurred undetected, challenging the prevailing assumption that Ghana was unaffected by human mpox prior to 2022, underscoring the importance of strengthened surveillance and preparedness in Ghana.

Highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b emerged in dairy cows in the United States in early 2024. Since then, this clade simultaneously circulates in wild birds, cattle and poultry with ongoing transmission into several mammalian species. Given the historical role of swine in influenza ecology, susceptibility of pigs to this virus is critical for animal and public health. To address this concern, Sinclair nanopigs were infected with a bovine clade 2.3.4.4b HPAIV H5N1 isolate by combined intranasal, intratracheal and oral administration mimicking possible natural exposure routes. Pigs were productively infected developing either subclinical or mild disease with seroconversion. Virus replication occurred mainly in respiratory tissues resulting in shedding from upper respiratory tract mucosae. Limited transmission to naïve contact cage mates was documented in a subset of transmission pairs. The combination of subclinical clade 2.3.4.4b HPAIV H5N1 replication and limited transmission draws an alarming scenario for One Health considering pigs are a favorable influenza mixing vessel enabling mammalian adaptation.

Since early 2022, highly pathogenic avian influenza (HPAI) H5N1 virus infections have been reported in wild aquatic birds and poultry throughout the USA with spillover into several mammalian species1-6. In March 2024, HPAIV H5N1 clade 2.3.4.4b was first detected in dairy cows in Texas, USA, and continues to circulate on dairy farms in many states7,8. Milk production and quality are diminished in infected dairy cows, with high virus titres in milk raising concerns of exposure to mammals including humans through consumption9-12. Here we investigated routes of infection with bovine HPAIV H5N1 clade 2.3.4.4b in cynomolgus macaques, a surrogate model for human infection13. We show that intranasal or intratracheal inoculation of macaques could cause systemic infection resulting in mild and severe respiratory disease, respectively. By contrast, infection by the orogastric route resulted in limited infection and seroconversion of macaques that remained subclinical.

We compared clinical disease, virus shedding and dissemination following genital MPXV clade Ia and Ib infections in Mastomys natalensis. MPXV clade Ib resulted in a milder clinical disease but a similar shedding profile to clade Ia. These findings indicate a prolonged pre-symptomatic or prodromal shedding period in clade Ib infections. ### Competing Interest Statement The authors have declared no competing interest. Intramural Research Program of the National Institutes of Health

The emergence of the Omicron lineage represented a major genetic drift in SARS-CoV-2 evolution. This was associated with phenotypic changes including evasion of pre-existing immunity and decreased disease severity. Continuous evolution within the Omicron lineage raised concerns of potential increased transmissibility and/or disease severity. To address this, we evaluate the fitness and pathogenesis of contemporary Omicron variants XBB.1.5, XBB.1.16, EG.5.1, and JN.1 in the upper (URT) and lower respiratory tract (LRT). We compare in vivo infection in Syrian hamsters with infection in primary human nasal and lung epithelium cells and assess differences in transmissibility, antigenicity, and innate immune activation. Omicron variants replicate efficiently in the URT but display limited pathology in the lungs compared to previous variants and fail to replicate in human lung organoids. JN.1 is attenuated in both URT and LRT compared to other Omicron variants and fails to transmit in the male hamster model. Our data demonstrate that Omicron lineage evolution has favored increased fitness in the URT.

In 2022, mpox virus (MPXV) clade IIb emerged resulting in a global epidemic driven by human-to-human transmission mostly through sexual contact in the MSM population. To date, published data on the circulation of the MPXV clade IIb in the Central African region are absent. Here, we describe the first case of laboratory-confirmed mpox with MPXV clade IIb lineage A2.2 in Pointe-Noire, the second largest city of the Republic of the Congo (RoC). Whole genome phylogenetic analysis placed the MPXV in clade IIb, lineage A.2.2. currently emerging in West Africa, in particular Sierra Leone. The detection of clade IIb mpox, marks the third distinct MPXV clade and lineage co-circulating in the human population, together with clade Ia and clade Ib. This underscores the need for improved surveillance and diagnostic strategies to identify the respective clade and lineage circulating in the human population. Strengthening of the regional capacity for case detection, contact tracing, public health measures, and affordable vaccines are urgently needed to reduce the global risk for both clades I and Clade II MPXV.

In this study, we investigated differences in tissue tropism of two HPAI H5N1 strains, the isolate A/Vietnam/1203/2004 (VN1203) isolated from a fatal human case in 2004 and the bovine isolate A/Bovine/Ohio/B24osu-342/2024 (Bov342) isolated in 2024, in C57BL/6J mice. Infection via aerosols was uniformly lethal in mice. However, tissue tropism differed significantly: while VN1203 replication was largely restricted to the respiratory tract, Bov342 successfully replicated in the respiratory tract as well as various regions of the brain. Correspondingly, cytokine profiles in the brain differed significantly between the isolates. Notably, in addition to abundant evidence of CNS infection in Bov342-challenged mice via immunohistochemistry, sporadic intranuclear and intracytoplasmic immunoreactivity was observed in other tissues in the head, including the choroid plexus, retina, and inner ear. This study demonstrates that while both HPAI H5N1 isolates are uniformly lethal in C57BL/6J mice upon aerosol exposure, significant differences exist in tissue tropism.

The detection of high-consequence viral pathogens is essential for spillover prevention and reduction in transmission but is limited by the low sensitivity of next-generation sequencing technology. Low-titer field samples from a variety of hosts are primarily composed of non-viral genomic material, reducing the probability of obtaining usable sequence data. Targeted enrichment, such as VirCapSeq-VERT, removes background genomic material to improve virus detection but is mainly used for sequencing clinical samples. We customized the VirCapSeq-VERT probe system to aid in the detection of zoonotic viruses of interest and adapted it for use on the Oxford Nanopore sequencing platform. We validated the method on a variety of samples, including a mock virome consisting of seven RNA viruses, samples from an animal laboratory study, and a set of animal field samples. We also developed Nanite, a lightweight bioinformatics pipeline, to perform bioinformatic analyses. Results indicated that the optimized enrichment protocol improved sequencing by enhancing the detection of viruses, increasing read lengths, and, in some cases, improving genomic coverage. Most importantly, the sequencing of zoonotic viruses was improved in field samples with low titers, suggesting that this protocol is a useful tool for increasing the efficacy of Oxford Nanopore sequencing for field-oriented applications.

Rapid influenza diagnostic tests (RIDTs) could be useful in the current bovine H5N1 outbreak. Here, we evaluated three RIDTs with H5N1. The RDITs showed comparable sensitivity with H5N1 compared to seasonal influenza A virus H3N2, and no difference was observed in sensitivity between raw milk and the PBS control.

Sequencing and bioinformatic analysis of mpox virus (MPXV) remain challenging in resource-limited settings. We developed and validated a PCR-based sequencing assay that targets a 12.5 kilobase (kb) region that is phylogenetically representative of the whole ∼ 200 kb MPXV genome. We combined this sequencing assay with a lightweight, downloadable, on-and-off-grid-bioinformatics pipeline for rapid phylogenetic analysis. Our findings demonstrate that this simplified sequencing method, and the associated bioinformatics pipeline accurately distinguished clades, subclades, and clusters of MPXV. Therefore, this assay will provide rapid sequence information for understanding transmission patterns and sources of outbreaks in resource-limited settings. In addition, this assay provides a unique opportunity to decentralize mpox molecular surveillance capacities that are needed to contain the ongoing outbreak.

Rotaviruses belong to genotype VP4-P[8] are a significant cause of severe loose diarrhea in infants and young children. In the present study, we characterised the complete genome of three of the Pakistani P[8]b RVA strains by Illumina HiSeq sequencing technology to determine the complete genotype constellation providing insight into the evolutionary dynamics of their genes using maximum likelihood analysis. The maximum genomic sequences of our study strains were similar to more recent human Wa-Like G1P[8]a, G3P[8]a, G4P[6], G4P[8], G9P[4], G9P[8]a, G11P[25],G12P[8]a and G12P[6] strains circulating around the world. Therefore, strains PAK274, PAK439 and PAK624 carry natively distinctive VP4 gene with universally common human Wa-Like genetic backbone. Comparing our study P[8]b strains with vaccines strains RotarixTM and RotaTeqTM, multiple amino acid differences were examined between vaccine virus antigenic epitopes and Pakistani isolates. Over time, these differences may result in the selection for strains that will escape the vaccine-induced RVA-neutralizing-antibody effect.