Monkeypox virus (MPXV) is the causative agent of mpox disease in humans. The virus comprises two clades, Central African clade I and West African clade II, with case fatality rates of ~11 and ~4%, respectively. Since the discovery of mpox disease in 1970, the virus has been restricted to Africa. However, in 2022, a previously unrecognized subclade IIb caused the largest global outbreak of mpox disease with a case fatality rate of ~0.2%. The difference in virulence of MPXV subclades in human infection warrants further investigation; however, one critical limitation is the lack of susceptible small animal models. In this study, we investigated the susceptibility of four murine models, including CAST-EiJ and three immunocompromised models (C57BL/6 Ifnar-/-, C57BL/6 Ifngr-/-, and C57BL/6 Ifnar-/-/Ifngr-/-) to MPXV clade IIa (WR 7-61 and US-2003) and IIb (MA-2022) isolates. All four mouse models were susceptible to clade IIa infection, leading to severe disease marked by decreased body temperature, weight loss, and lethality. In contrast, clade IIb infection produced minimal to mild disease at similar doses in all four murine models. The clade IIb isolate produced severe disease (40% lethality) at only the highest dose (8.0 log10 PFU) in the most susceptible immunocompromised mouse model, C57BL/6 Ifnar-/-/Ifngr-/-. This is the first demonstration of lethal disease with clade IIb in a murine model. In addition, these data show that clade IIa is ~100 to 100,000-fold more virulent than clade IIb and provides three additional murine models for investigating MPXV infection and pathogenesis.

GC content-the proportion of guanine and cytosine nucleotides-varies widely among organisms and viruses. Although GC content is recognized to have biological significance, its functional role in viruses remains poorly understood. Here, we examined the impact of GC-content bias using three positive-sense single-stranded RNA viruses with distinct GC profiles: severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), Japanese encephalitis virus (JEV), and hepatitis C virus (HCV). Variants of the NanoLuc (Nluc) reporter gene, engineered with synonymous mutations to alter GC content, were inserted into each viral genome. Serial passaging experiments revealed strong effects on genome stability and viral fitness. In SARS-CoV-2, which has low GC content, the introduction of a GC-rich Nluc gene disrupted genome stability, leading to frequent deletions of Nluc. In both SARS-CoV-2 and HCV, Nluc with mismatched GC levels accumulated substitutions that optimized GC content toward that of the viral genome, predominantly at the third codon position. tRNA-seq analysis revealed a shift in the host tRNA pool toward a more GC-rich composition during HCV infection, consistent with this substitution bias. In contrast, JEV (intermediate GC content) maintained the Nluc variants across serial passages, suggesting reduced selective pressure. Taken together, these findings demonstrate that mismatches between viral genome GC content and inserted sequences profoundly affect genetic stability, suggesting evolutionary constraints that may shape RNA virus composition more broadly. This study provides mechanistic insight into how GC content influences viral genome maintenance and offers a framework for designing genetically stable recombinant reporter viruses.IMPORTANCEGC content is a fundamental genomic feature that influences gene expression, genome architecture, and adaptation. Although its role in cellular genomes has been extensively studied, the functional significance of GC content in viral genomes remains poorly understood. In this study, we show that incompatibility in GC content between a viral genome and an inserted transgene destabilizes RNA secondary structures, compromising viral genome integrity, and is accompanied by changes in the host tRNA pool consistent with this compositional bias. These findings identify GC content as a previously underappreciated determinant of virus-host compatibility and viral fitness. Importantly, our results provide mechanistic insight into how the GC content of a viral genome can be matched to its host cellular environment. By demonstrating that GC content matching is critical for the stability of recombinant viruses, this study provides a basis for the rational design of reporter viruses, vaccine platforms, and antiviral screening tools.

This work analyzed 30 drug resistance mutations (DRMs) in the surface antigen of hepatitis B virus (HBV) (i.e., HBsAg) selected by several FDA-approved anti-HBV nucleos(t)ide analogs. All DRM-bearing HBsAg variants supported the formation and secretion of hepatitis delta virus (HDV), and only 13/30 DRMs considerably decreased the yield of secreted HDV virions. However, the majority of DRMs (23/30) greatly reduced HDV infectivity, and four of them even caused the complete loss of infectivity, which was determined by analyzing the levels of HDV RNA genomes in the infected cells, and the fraction of HDV-infected cells. The ability to inhibit HDV infectivity was observed for DRMs located in the large cytosolic loop (LCL), major antigenic loop (MAL), HDV-binding site (HDV-BS), and the transmembrane domains II, III, and IV of HBsAg, which suggested that DRMs mediate their inhibitory influence by both direct and indirect mechanisms that could alter the attachment/entry, post-entry trafficking, and/or subsequent uncoating of the virions. This report also established the transmembrane domains II, III, and IV as critical regulators of the overall infectivity of HDV virions. It became apparent that in the settings of natural HBV/HDV infection, DRMs can therefore greatly reduce the spread/superinfection of HDV and diminish the size of the HDV reservoir in chronically infected livers. Overall, our findings advanced the understanding of how anti-HBV drug treatments influence the HDV life cycle, the roles of HBV envelope proteins in facilitating the assembly and infectivity of HDV, and the mechanism of complex HBV-HDV interactions.

With the broad spread of the chikungunya virus (CHIKV), there is an increasing demand for more effective and protective vaccines. In this study, we first developed a Newcastle disease virus (NDV) vector vaccine candidate expressing the complete envelope (E3-E1) protein of CHIKV. The immunoelectron microscopy confirmed the presence of E2 protein in the rNDV-CHIKV virion, and the virological results showed that the expression of E1 and E2 protein was stable after 10 serial passages in embryonated chicken eggs. Moreover, we assessed its safety and immunogenicity following intramuscular (i.m.) or subcutaneous (s.c.) administration in C57BL/6 mice. The immunogenicity analyses demonstrated that a single-dose immunization elicits high titers of CHIKV E2-specific IgG antibodies, potent neutralizing activity, and T-cell immune responses. Importantly, both routes of immunization conferred complete protection against wild-type CHIKV challenge in C57BL/6 mice. These findings present a promising novel vector-based vaccine candidate with significant potential for effective CHIKV prevention.IMPORTANCEChikungunya virus (CHIKV) imposes a substantial global health burden, characterized by arthralgia that can persist for years, neurological complications, and potentially fatal outcomes in vulnerable populations. Currently, two vaccines against CHIKV have been approved: a live-attenuated vaccine (Ixchiq) and a virus-like particle (VLP) vaccine (Vimkunya). However, Ixchiq has been withdrawn from clinical use due to significant side effects, highlighting the urgent need for the development of novel vaccine strategies. Our research has successfully developed a novel vaccine candidate that utilizes Newcastle disease virus as a vector to express CHIKV antigens. This innovative strategy offers multiple advantages: cost-effectiveness, genetic stability of the inserted gene, and robust immunogenicity with effective immune protection. This approach offers a contribution in addressing the emerging threat posed by CHIKV.

Superinfection, where a person with HIV acquires a second phylogenetically distinct strain, provides an opportunity to study reservoir evolutionary dynamics because the two strains can be tracked over time. We hereby characterize such a case that originally went undetected by clinical monitoring. The participant, who expressed the protective HLA-B*57:03 allele, initially controlled his subtype B infection but lost control after superinfection with a unique recombinant form (URF), which came to dominate in plasma without displacing the original B strain. Five years after initiating therapy, replication-competent proviruses from both strains persisted in blood, though URF proviruses dominated (80%). Despite their unequal representation within the reservoir, both strains rapidly rebounded upon treatment interruption, though most rebounding sequences belonged to a URF subclade that predated ART initiation. During the treatment interruption, genetically distinct viral lineages continually appeared in plasma, consistent with sequential "waves" of reactivation of diverse reservoir clones. Notably, immune escape variants emerged from the reservoir in a later "wave," displacing initial susceptible variants and underscoring the important role of immune responses in shaping rebound dynamics. Sequence analysis revealed that the URF transmitted/founder virus already harbored key HLA-B*57:03-associated escape mutations, likely explaining the loss of viral control after superinfection. Our results reveal that superinfection can remain undetected despite routine HIV clinical monitoring, pre-adaptation of the superinfecting strain to host HLA can undermine immune control, immune-escaped virus can re-emerge from reservoirs during treatment interruption, and immune responses can actively shape viral rebound dynamics.IMPORTANCEThis case demonstrates that HIV superinfection can go undetected by routine clinical monitoring, underscoring the importance of assays that capture HIV diversity-particularly those used to screen participants for cure trials. It also illustrates how pre-adaptation of a superinfecting strain to host HLA-restricted immune responses can evade existing immunity, with implications for understanding natural immune control, and underscoring the challenge of viral diversity for vaccine and immunotherapy design. The sequential emergence of genetically diverse HIV lineages during treatment interruption, including minority immune-escape variants that emerge from the reservoir to displace the initial susceptible population, highlights the difficulty of predicting rebound viral composition and underscores the key role of immune responses in shaping rebound dynamics. Critically, escaped rebound variants can then reseed the reservoir, thereby enriching it in escaped forms, highlighting a specific vulnerability for cure approaches that harness natural immune responses to eliminate reservoir cells.

Highly pathogenic avian influenza A(H5N1) (HPAI H5N1) viruses of clade 2.3.4.4b have recently been detected in U.S. dairy cattle following multiple spillover events from avian reservoirs. In December 2025, HPAI H5N1 virus was identified in a dairy herd in Wisconsin through the National Milk Testing Strategy. Here, we report the isolation of a clade 2.3.4.4b, genotype D1.1 H5N1 virus, A/dairy cow/Wisconsin/25G05743-001/2025 (WI5743-H5N1), from bulk milk associated with the affected herd, describe its phylogenetic relationships, and assess its pathogenicity in mice. Infectious virus was recovered following blind passage in embryonated chicken eggs. Phylogenetic analysis demonstrated that WI5743-H5N1 is distinct from previously reported D1.1 viruses detected in dairy cattle in Nevada and Arizona, supporting an independent introduction into cattle, and indicating a likely local avian source. Compared with closely related avian viruses, WI5743-H5N1 encoded the mammalian-adapting substitution PB2-E627K and additional amino acid differences in HA, PB1-F2, and NS1. In mice, WI5743-H5N1 replicated efficiently in respiratory tissues and was detectable in the brain but exhibited lower lethality relative to other recent clade 2.3.4.4b, genotype B3.13 viruses. Together, these findings highlight the genetic and phenotypic diversity of HPAI H5N1 viruses infecting dairy cattle and underscore the importance of continued surveillance and functional characterization of emerging strains.IMPORTANCEHighly pathogenic avian influenza A(H5N1) viruses have recently entered U.S. dairy cattle through multiple spillover events from avian reservoirs, creating new opportunities for viral adaptation in mammals. Here, we describe the isolation and characterization of a clade 2.3.4.4b, genotype D1.1 H5N1 virus from bulk milk collected during a spillover event in Wisconsin in December 2025. Phylogenetic analyses demonstrated that this virus represents an independent introduction into dairy cattle distinct from previously reported D1.1 viruses identified in Nevada and Arizona. Although the virus encoded the mammalian-adapting PB2-E627K substitution, it exhibited comparatively low lethality in mice, highlighting the complexity of mammalian adaptation and pathogenicity in H5N1 viruses. These findings expand current understanding of the genetic and phenotypic diversity of H5N1 viruses infecting dairy cattle and emphasize the importance of continued surveillance and functional characterization of emerging strains.

Influenza B virus (IBV) exhibits antigenic diversity and significantly contributes to the annual burden of influenza-related disease, posing substantial impacts on global public health and the economy. In this study, we focused on the neuraminidase (NA) of IBV, immunized an alpaca with tetrameric IBV NA, and identified a series of nanobodies with a common feature of the "DYR" motif in CDR3, exhibiting broad inhibitory activity against diverse NAs of IBV. Among these, the most potent VHH5 broadly inhibits NA from multiple representative strains across ancestral, B/Yamagata/16/88-like, and B/Victoria/2/87-like lineages. Moreover, fusing VHH5 to a human immunoglobulin G1 Fc domain significantly increased its antiviral potency in vitro, with the 50% inhibitory concentration in the low nanomolar range. Structural modeling and molecular dynamics simulations revealed that the "DYR" motif within the elongated CDR3 loop of VHH5 penetrates the catalytic pocket and engages the highly conserved catalytic site of IBV NA. Experimental mutagenesis analysis of the predicted interface further supported this binding model. Furthermore, the VHH5-Fc protects mice from the lethal challenge with a high dose of the IBV-2024 virus in both prophylactic and therapeutic settings. Our findings highlight the potential of the Fc-fused VHH5 antibody in controlling influenza B virus infections.

The herpesvirus DNA replication machinery comprises a battery of viral enzymes that orchestrate viral genome synthesis. In herpes simplex virus type 1 (HSV-1), the machinery consists of seven essential components, including the origin-binding protein UL9, the single-stranded DNA (ssDNA)-binding protein ICP8, the heterodimeric DNA polymerase complex UL30-UL42, and the heterotrimeric helicase-primase complex UL5-UL8-UL52. UL9, a superfamily 2 (SF2) helicase, functions as a dimer that specifically recognizes replication origins and unwinds duplex DNA to initiate replication. Furthermore, UL9 recruits the replication machinery through interactions with viral components and engages cellular proteins that regulate its function. However, the molecular mechanisms underlying the multifunctionality of UL9 remain incompletely understood due to the lack of structural information. Here, we present cryo-electron microscopy structures of UL9 in both apo and DNA-bound states. Together with biochemical and enzymatic assays, we elucidate the molecular basis of UL9 dimerization, origin recognition and allosteric regulation by ICP8.IMPORTANCEHerpes simplex virus 1 (HSV-1) is a widespread virus that causes lifelong infections, leading to periodic outbreaks ranging from common cold sores to life-threatening encephalitis, and no current treatment can eradicate the dormant virus. To multiply, HSV-1 relies on a protein-based molecular machine to replicate its genome, where the unwinding of double-stranded DNA at specific replication origins is coordinated by the viral origin-binding protein UL9. Here, we present the high-resolution structures of UL9, both alone and bound to DNA, revealing how it forms a stable homodimer to grab onto the origin. Combined with precise biochemical experiments, we further show how UL9 collaborates with another viral helper protein, ICP8, to unwind DNA efficiently. These discoveries solve a long-standing puzzle in herpesvirus biology and offer a vital structural blueprint for designing new antiviral drugs that can block viral replication at its very earliest stage.

The accumulation of subgenomic flavivirus RNAs (sfRNAs) modulates viral fitness and pathogenicity in culture and in vivo. These noncoding RNAs are produced by incomplete digestion of the flavivirus genome by the cellular 5'-3' exoribonuclease (XRN1). Diverse flaviviruses have conserved RNA structural elements (RSEs) that map to their 3'-untranslated region (3'-UTR): Xrn-resistant RNA structures, dumbbell structures, and a 3'-stem loop (3'-SL). Despite the importance of the 3'-UTR RSEs for flavivirus replication, the structural dynamics of sfRNA during flavivirus infection are understudied. Here, we use digital droplet PCR to quantify sfRNA levels during infection for a panel of mosquito-borne flaviviruses, including dengue virus serotypes 1 (DENV1), 2 (DENV2), and 4 (DENV4), and Zika virus. We then used SHAPE-MaP on XRN1-digested, in vitro-transcribed sfRNAs from each virus to determine their secondary structures compared to the corresponding sfRNAs obtained from flavivirus-infected A549 cells. Results seen in-cell and in vitro were largely similar; however, motifs within the dumbbell, the small hairpin directly upstream of the 3'-SL, and 3'-SL regions showed significant differences in the extent of nucleotide reactivity. These differences were consistent among the four flaviviruses examined and may indicate regions of sfRNA that are shielded by interaction with proteins or other nucleic acids during infection. However, strong protection indicative of sustained interaction was not apparent. Our findings suggest that sfRNA interactions with viral and host factors within the cell are few, occur via base-paired regions, or are highly transient.IMPORTANCEFlaviviruses are highly prevalent human pathogens. The flavivirus genome contains RNA structural elements (RSEs), including those encoded in the 3'-UTR, that are necessary for viral replication. Subgenomic flavivirus RNAs (sfRNAs) are produced by incomplete digestion of flavivirus genomic RNA due to the cellular exoribonuclease XRN1 encountering 3'-UTR RSEs that promote XRN1 stalling and disassociation. Viruses unable to produce sfRNAs are highly attenuated, underlining their biological importance. sfRNA secondary structure has been investigated previously, but little information is available on sfRNA secondary structure dynamics in infected cells. By comparing SHAPE-MaP reactivities in vitro and in cells, we determined that previously inferred structures are likely maintained within infected cells. We also identified differences in the extent of SHAPE reactivity between in vitro and in-cell environments that were common to multiple mosquito-borne flaviviruses. These differences suggest that sfRNAs may engage in transient interactions within the cell that may be important for their function.

The American Society for Virology (ASV) 2025 Annual Meeting, held from 14 to 17 July at the Palais des congrès de Montréal and hosted by McGill University, featured a dedicated Plant Virology Satellite Symposium entitled "Pioneering Plant Virology Research for a Healthier Future." This symposium emphasized the inclusion of topics of significance to early-career scientists. The program highlighted advances across diverse areas of plant virology, including mechanisms of transgenerational virus transmission, the economic and biological impacts of grapevine viruses on viticulture, emerging plant virus vectors, and innovative applications of plant viruses as nanomaterials for cancer immunotherapy. Additionally, emerging research on small functional proteins encoded by plant viruses and plant defense mechanisms underscored the complexity of viral genomes and host interactions. The symposium showcased cutting-edge approaches and strategies aimed at addressing global challenges in plant health and food security.

The global rise of antimicrobial resistance (AMR) demands innovative strategies to limit the spread of multidrug-resistant bacteria. Conjugative plasmids, particularly those in the incompatibility group P (IncP), play a central role in disseminating resistance genes across bacterial species via their encoded type IV secretion system (T4SS). Here, we characterize the single-stranded RNA (ssRNA) bacteriophage (ssRNA phage) PRR1, which selectively targets bacteria carrying the IncP plasmid RP4, including many Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli (ESKAPEE) pathogens, and assess its ability to inhibit conjugation. Using cryo-electron microscopy, we resolved the mature PRR1 virion at 3.45 Å resolution, revealing two phage maturation protein (Mat)-RNA interactions within the 3' untranslated region: a conserved interaction (Mat-U1) and a novel interaction (Mat-V1) for ssRNA phages. To characterize the PRR1-RP4 pilus interaction, we performed alanine-scanning mutagenesis and pinpointed four critical TrbC pilin residues (S12, W13, S72, and R77) for infection. Computational modeling revealed that these residues are located near the termini of the pilin at the phage-pilus interface. Notably, native and non-infectious, UV-cross-linked PRR1 was sufficient to block RP4 transfer, indicating conjugation inhibition does not require a complete infection cycle. Finally, combining PRR1 and antibiotic treatment yielded nine unique phage-resistant mutants within T4SS-associated genes on the RP4 plasmid. Eight of these mutants nearly abolished conjugation, while the trbE frameshift mutant retained ~30% of wild-type efficiency, which is pivotal to clarifying the relationship between phage infection and pilus function. Collectively, these results establish ssRNA phages as specific T4SS plasmid-targeting agents and underscore their potential to limit horizontal gene transfer in AMR pathogens.IMPORTANCEAntimicrobial resistance (AMR) spreads rapidly through horizontal gene transfer, largely driven by conjugative plasmids. Despite their central role, few strategies exist to directly block plasmid transfer. Here, we show that the IncP plasmid-dependent ssRNA phage PRR1 can inhibit the spread of antibiotic resistance genes by targeting the RP4 T4SS pilus. Structural and mutational analyses reveal previously unrecognized RNA packaging interactions and identify four pilin residues critical for infection. Remarkably, non-infectious PRR1 particles alone are sufficient to block conjugation, offering inhibition without the selective pressure from phage replication. Almost all PRR1-resistant RP4 mutants lost or had severely reduced plasmid transfer, while the remaining mutant is critical for studying the link between T4SS function and phage infection. These results highlight ssRNA phages as precise agents for limiting AMR gene dissemination.

Post-acute sequelae of COVID-19 (PASC) poses a major health burden after SARS-CoV-2 infection. Although type 2 diabetes (T2D) is associated with PASC, the mechanism of T2D-mediated PASC in the lung remains elusive. Here, we found that people with T2D (PWT2D) exhibited significantly upregulated fibrosis-related genes in monocytes, which positively correlated with pulmonary fibrosis-related biomarkers up to 3 months after acute SARS-CoV-2 infection. Using db/db mice to model human T2D, we found consistently that SARS-CoV-2 infection resulted in upregulation of fibrosis-related genes in lung macrophages and persistent pulmonary fibrosis. Moreover, the macrophage-depletion demonstrated that pro-inflammatory macrophages in db/db mice were determinants for inducing pulmonary fibrosis post-infection. Importantly, the anti-T2D glucagon-like peptide-1 receptor agonist (GLP1-RA) reprogramed macrophage responses to SARS-CoV-2 by normalizing fibrosis-related genes, significantly reducing the pulmonary fibrosis in a glucose-independent manner. These findings demonstrated that SARS-CoV-2-induced proinflammatory macrophages are detrimental factors in T2D-mediated PASC, which can be prevented by GLP1-RA.

What are viruses, if not nucleic acids surrounded by proteinaceous shells? While the energy-dependent viral packaging motors of tailed bacteriophages have been studied in great depth, our understanding of how eukaryotic double-stranded DNA (dsDNA) viruses package their genomes into nascent capsids remains incomplete. Herpesviruses are ubiquitous human pathogens that drive diverse clinical symptoms. The success of letermovir, a first-in-class human cytomegalovirus genome packaging inhibitor, suggests that this critical step in the virus life cycle can be targeted in other herpesvirus infections. Here, we integrate recent structural, biophysical, and genetic information from representative dsDNA viruses to define the overarching principles of viral genome packaging and highlight evolutionary distinctions. What lessons can we learn from bacteriophages to further our understanding of herpesvirus packaging, and can these insights generate new opportunities for antiherpesvirus therapeutics?

Highly pathogenic avian influenza (HPAI) virus clade 2.3.4.4b genotype B3.13 infected the mammary gland of dairy cattle; the new tissue tropism and host heightened concern about its ability to cross species barriers with zoonotic potential. Pigs play a key role in influenza A virus (IAV) adaptation, serving as a "mixing vessel" for the emergence of reassortants. The susceptibility of porcine mammary gland to HPAI infection remains unexplored. In this study, we profiled IAV receptors in porcine mammary gland as well as respiratory tract tissues. Additionally, we evaluated the binding capacity of IAVs to these tissues. Furthermore, we isolated primary cells from porcine mammary gland and respiratory tract, and immortalized them. We examined the growth potential of IAV isolates from bovine, avian, swine, and human on these cells. We showed that porcine mammary gland displays both SA-α2,3 and SA-α2,6, and that IAVs bind to mammary gland tissues with variable affinities. While bovine H5N1 virus replicates efficiently in mammary gland and respiratory tract cells, replication of other IAVs in mammary epithelial cells is moderate but is efficient in respiratory cells. These findings suggest that porcine mammary gland could support the infection by HPAI 2.3.4.4b genotype B3.13.

Human metapneumovirus (HMPV, genus Metapneumovirus) and respiratory syncytial virus (HRSV, genus Orthopneumovirus) are pediatric respiratory pathogens of the Pneumoviridae family. We previously developed live-intranasal HRSV vaccine candidates attenuated by genetically stabilized temperature sensitivity/attenuating (ts/att) mutations in the HRSV polymerase (L) ORF, including sites "1030s" (L codon changes S1313(AGC-to-TCA)/Y1321K(AAA), named after clone number 1030), and "Δ1313/I1314L," comprising a genetically stabilized deletion of L codon 1313. In pediatric vaccine studies, HRSV candidates with "1030s" or Δ1313/I1314L were attenuated, immunogenic, and genetically stable. Given the similarities between HRSV and HMPV, we introduced corresponding modifications into the HMPV L ORF, resulting in HMPV with deletion of codon 1238 (Δ1238), corresponding to HRSV Δ1313/I1314L, and HMPV with C1238S(TCA)/Y1246K(AAA) substitutions together (C1238S/Y1246K, corresponding to HRSV "1030s"), or individually (C1238S, Y1246K), serving as experimental controls. All versions replicated well in Vero cells at 32°C. At 37°C, replication of Δ1238, C1238S/Y1246K, and Y1246K was delayed, and peak titers were ~80-fold, 200-fold, and 25-fold lower than wild-type HMPV. As predicted, the C1238S control was not temperature-sensitive (shutoff temperature [TSH] > 40°C, similar to wild-type HMPV), while Y1246K and Δ1238 (TSH = 39°C) and C1238S/Y1246K (TSH = 38°C) had a ts phenotype. Thus, the addition of the C1238S(TCA) substitution increased the temperature sensitivity of Y1246K. The HMPV ts/att sites were genetically stable under temperature stress. In hamsters inoculated intranasally, C1238S/Y1246K and Δ1238 were restricted in replication, yet immunogenic and protective against HMPV challenge. Thus, inclusion in the HMPV L ORF of C1238S/Y1246K and Δ1238 mutations, corresponding to HRSV ts/att sites, provided genetically stable live-attenuated HMPV, suitable for clinical development.