Highly pathogenic avian influenza H5N1 viruses of clade 2.3.4.4b have caused widespread avian mortality and sporadic mammalian infections, raising concerns about their potential for efficient replication in the human population. Efficient replication in the human upper respiratory tract is considered a key barrier to transmission. Here, we demonstrate that an H5N1 virus isolated from bovine milk in Texas in 2024 (H5N1Tex/24) replicates as efficiently as the 2009 pandemic H1N1 virus (H1N1HH4/09) in well-differentiated human nasal epithelial cells. These cells express both avian- and human-type influenza receptors, indicating receptor adaptation is unnecessary for entry. H5N1Tex/24 replicates effectively at 33 degrees Celsius, reflecting nasal cavity temperature, whereas earlier avian H5N1 strains require 37 degrees Celsius, suggesting that H5N1Tex/24 has acquired another key adaptive feature to the human upper respiratory tract. H5N1Tex/24 remains sensitive to interferon-λ (IFN-λ) despite inducing low cytokine levels. Notably, no known mammalian-adaptive mutations such as PB2-E627K were detected. These findings suggest that H5N1Tex/24 possesses intrinsic traits enabling efficient replication in the human upper airways, a critical step toward potential airborne transmission, underscoring the need for vigilant surveillance. ### Competing Interest Statement The authors have declared no competing interest. Federal Food Safety and Veterinary Office, https://ror.org/01hwpsz06, 1.24.m

African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic pigs and wild boars, which poses severe threats to the global pork industry. Despite the promise of live attenuated vaccines (LAVs), their narrow margin between efficacy and residual virulence presents major safety challenges. This study bridges a critical knowledge gap in ASF vaccinology by identifying innate and adaptive correlates of protection. This was achieved by using an established model with two groups of pigs differing in baseline immunological status (farm and specific pathogen-free [SPF]). The animals were immunized with an attenuated ASFV strain and subsequently challenged with a related, highly virulent genotype II strain. By applying a systems immunology approach, we correlated kinetic data, including serum cytokines, blood transcription modules (BTMs), T-cell responses, and antibody levels, with clinical outcomes to track protective and detrimental immune responses to the virus over time. Key innate correlates of protection included early and sustained IFN-α response, activation of antigen presentation BTMs, and controlled IL-8 levels during immunization. Lower baseline immune activation observed in SPF pigs in steady state was linked to increased protection. Adaptive correlates encompassed cell cycle, plasma cell, and T-cell BTM responses lasting until day 15 post-immunization. Consequently, an effective response from ASFV-specific Th cells prior to challenge indicated protection. After the challenge, an early IFN-α response, along with low levels of pro-inflammatory cytokines and a strong induction of memory Th and Tc cells, correlated with improved clinical outcomes. The model highlights the critical role of host-specific factors in vaccine efficacy and provides a valuable framework for optimizing ASFV vaccine design while distinguishing between protective and detrimental immune responses.

Histopathology and immunohistochemistry (IHC) are central to COVID-19 tissue evaluation. However, conventional manual scoring is limited by certain subjectivity and its semiquantitative nature. In this retrospective study of experimentally infected mice, we implemented a deep learning-based digital pathology workflow using com-mercially available software to quantitatively assess SARS-CoV-2 antigen burden in lung (n=135) and brain (n=67) tissues. The performance of digital quantification was evaluated against conventional manual scoring, and its biological relevance was as-sessed by correlation with established virological and pathological parameters across different stages of disease progression. Digital IHC quantification demonstrated near-perfect agreement with manual scoring in both lung [R=0.94, p< 0.0001, concord-ance correlation coefficient (CCC)=0.969] and brain (R=0.98, p< 0.0001; CCC=0.98) in-dicating high reproducibility and accuracy. In addition, digital antigen quantification showed significant positive correlations with viral RNA levels, infectious viral titers, and histopathological scores, indicating that it provides biologically meaningful readout of SARS-CoV2 infection. Although computational image analysis requires ad-ditional infrastructure, technical expertise, and increased analysis time, these invest-ments provide a more objective and reproducible alternative to the traditional manual gold standard while generating quantitative data that enable a more precise assess-ment of SARS-CoV-2–associated disease.

Wesselsbron virus (WSLV) disease is an important neglected cause of hepatitis in ruminants with potential for zoonotic transmission, yet its histological lesions have been scarcely studied. We performed a thorough machine learning-driven, pathologist-led digital histopathological assessment of WSLV-induced hepatitis in ewes and lambs infected with clade I (rSA999) (n = 6) or clade II (SAH117) (n = 8) strains and a mock group (n = 6). The analysis was performed on immunohistochemical (IHC) staining for T cells (CD3), B cells (PAX5), histiocytes (Iba1), the WSLV nonstructural protein 1 (NS1), and a double stain for arginase 1 and Ki67 to assess the hepatocyte proliferation index (PI). WSLV-infected animals exhibited significantly higher lymphohistiocytic infiltration and higher hepatocyte PI compared to the mock group. The T cell density was 10 folds higher than the B cell density and was more pronounced in the rSA999 group than in the SAH177 group. Digitally quantified parameters positively correlated with WSLV reverse transcription quantitative PCR (RT-qPCR) results and hepatic injury markers (aspartate transferase [AST], bilirubin, and adenosine deaminase [ADA]), indicating that digital histopathology reliably detects liver damage and disease severity. Among the parameters assessed, the positive correlation between the density of Iba1+ staining and the WSLV viral load in the liver was the strongest, underscoring the prominent involvement of histiocytes in WSLV-induced hepatitis. This study demonstrates the value of digital histopathological analysis in viral-induced hepatitis using formalin-fixed paraffin-embedded (FFPE) tissue, leveraging whole-slide imaging and deep learning (DL) to objectively characterize key hepatic alterations caused by the viral infection.

Highly pathogenic avian influenza (H5N1) viruses of clade 2.3.4.4b have caused significant losses in bird populations worldwide and repeatedly infected mammals, including humans, without sustained human to human transmission. Here we show that an H5N1 virus (H5N1Tex/24) isolated from bovine milk in Texas in 2024 replicates just as efficiently in differentiated human nasal epithelial cells as a pandemic H1N1 virus strain from 2009 (H1N1HH4/09), at both 37 °C and 33 °C. The adaptive mutations PB2 M631L and PA K497R promoted replication at 33 °C but had no effect on replication at 37 °C. An H5N1 virus (H5N1BE/22) isolated from a pelican in 2022, which lacked these mutations, replicated efficiently at 37 °C but poorly at 33 °C, and this limitation was not overcome by the introduction of the PB2 M631L and PA K497R mutations. The differentiated nasal epithelial cell cultures expressed receptors for both human and avian influenza viruses. Accordingly, no HA mutations associated with altered receptor specificity were detected. H5N1Tex/24 was able to effectively suppress the production of interferon-λ, yet remained sensitive to the antiviral effects of this cytokine. These findings suggest that H5N1Tex/24 possesses intrinsic traits supporting efficient replication in differentiated human upper airway cell cultures.

Interferons (IFNs) are secreted during virus infection and induce antiviral responses through receptor-mediated phosphorylation of signal transducer and activator of transcription (STAT) proteins, leading to IFN-stimulated gene expression with antiviral activity. We previously reported that the SARS-CoV-2 main protease, 3CLpro, is secreted from infected cells through gasdermin D/E pores and retains proteolytic activity in human serum against extracellular substrates. Here, we show that 3CLpro selectively cleaves glycosylated IFN-L1, IFN-L2, and a rare naturally occurring variant of IFN-γ (Arg160Gln), but does not cleave wild-type IFN-γ, IFN-L3, IFN-L4, IFN-alpha proteins or IFN-β. We identified sites of O-linked glycosylation of IFN-L1 at Thr137 and one or more threonines or serine in the sequence 24TSKPTTT30, and N-linked glycosylation at Asn65 that were indispensable for signaling. Unexpectedly, O-glycosylation was also required for the cleavage and inactivation of IFN-L1 by 3CLpro at two sites. Cleavage reduced STAT1 phosphorylation and impaired the induction of the IFN-stimulated proteins MX1, OAS2, and IFIT1. Although 3CLpro cleaved IFN-L2 proximal to its N-terminus at ARLH32↓GALP, cleavage neither disrupted signaling nor antiviral activity against SARS-CoV-2 and vesicular stomatitis virus. We further show that matrix metalloproteinases (MMPs) 2, 7, 8, and 12 degrade 3CLpro, whereas 3CLpro shows no activity against these MMPs.

Background: African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic pigs and wild boars. While live attenuated vaccines (LAVs) provide protection, their use raises safety concerns. Therefore, the aim of the present study was to identify viral B-cell antigens associated with protection and to test their potential using highly immunogenic vaccine delivery platforms. Methods: We employed a microarray of 169 ASFV proteins expressed in a cell-free prokaryotic system to identify immunodominant antigens using sera from immune pigs. Six structural proteins were selected and formulated into AP205 virus-like particles (VLPs). Additionally, replication-defective vesicular stomatitis virus (VSV)-based vaccine candidates expressing glycosylated CD2v and EP153R proteins were generated. Three groups of specific pathogen-free pigs were immunized with either VLP- or VSV-based vaccines and challenged with the virulent ASFV Georgia 2007 strain. Control groups included pigs immunized with the attenuated ASFV Estonia 2014 strain and a naïve group. Results: Most vaccine candidates induced detectable antibody responses against target ASFV proteins. However, neither VLP- nor VSV-based vaccines provided protection, as clinical scores, hematology, cytokine responses, and viremia levels were similar to those in the negative control group. In contrast, only the ASFV Estonia 2014 strain elicited a robust T-cell response and protective immunity. Conclusions: These findings highlight the challenges in identifying protective B-cell antigens of ASFV and emphasize the pivotal role of cellular immunity in mediating protection.

Deaths from viral hepatitis continue to rise around the world due to the lack of early biomarkers. We aimed here to evaluate the chemokine CXCL14, as a novel biomarker in acute viral hepatitis. We used a mouse model of acute hepatitis induced by murine hepatitis virus (MHV), a hepatotropic and lytic coronavirus, and showed that CXCL14 is overexpressed in the liver and sera of infected mice. Using primary cultures of murine and human hepatocytes, we showed that hepatocytes are the main source of CXCL14 after lytic hepatotropic virus infection and that CXCL14 expression is also induced by the pro-inflammatory cytokines IL-6 and TNFα. CXCL14 KO mice infected with MHV were partially protected and showed an attenuated antiviral immune response compared to wild-type mice. Finally, we show that CXCL14 is overexpressed in the sera of human patients infected with hepatitis viruses A, B, and E or herpes simplex virus. A positive correlation between CXCL14 and ALT levels in the sera of patients with acute herpetic hepatitis, as well as in mice models, suggests that hepatocyte lysis is necessary for the release of CXCL14. Overall, these data highlight that CXCL14 expression is associated with the occurrence of acute viral hepatitis and could be considered an alarmin and a new indicator of inflammation. CXCL14 serum levels are also associated with the severity of viral-induced liver injury.

African swine fever virus (ASFV) is a major threat for pig health and meat production in many countries. The development and commercialization of vaccine candidates are complicated by efficacy and safety concerns. Improved vaccine design requires further studies to identify factors that regulate immune responses to vaccines leading to protective immunity against a virulent challenge. In a previous study, we reported that infection with the moderately virulent ASFV field strain Estonia 2014 was less severe in specific pathogen-free (SPF) pigs than in conventional farm pigs, which differ in their gut microbiome and their basal immune activation status. As shown previously using intramuscular infection, SPF pigs were more resilient to oronasal infection with the ASFV Estonia 2014 strain compared to farm pigs, which showed increased fever and clinical signs. All SPF and farm pigs nevertheless survived the infection and remained viremic for approximately 4 months. When all animals had no detectable viremia, both groups were rechallenged with the virulent ASFV Armenia 2008 strain. SPF pigs were fully protected against disease and showed little or no viremia upon re-challenge. In contrast, farm pigs developed high viremia, high proinflammatory cytokine responses, severe clinical signs, and 40% (2 of 5 pigs) reached humane endpoints. Our findings suggest that limited prior immune exposure to other pathogens and/or the microbiome composition of SPF pigs promotes resilience to infection with a moderately virulent strain such as Estonia 2014, and importantly promotes the development of a strong protective immune response against a second challenge with a virulent ASFV strain. In conclusion, testing safety and efficacy of live attenuated vaccine candidates should take into account the specific hygiene conditions and the associated changes of general immune status of pigs in clinical trials.

The Pfizer-BioNTech coronavirus vaccine (BNT162b2), one of the first nanoparticle-based vaccines approved by the World Health Organisation (WHO), demonstrated 95% efficacy in preventing against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the precise mechanism of action underlying its effectiveness remains poorly understood. This study investigated the early immune responses in the draining lymph node (dLN) and its role in mediating antiviral protection following vaccination. Here, we focused on the involvement of antigen-presenting cells (APCs) in adaptive immunity. In this study, we demonstrated that the Pfizer-BioNTech coronavirus vaccine is rapidly transported to the dLN and is primarily captured by leukocytes that initiate the expression of the viral antigenic spike protein. Notably, we demonstrated that plasmacytoid dendritic cells (pDCs) are key orchestrators of the inflammatory and humoral response, as their specific depletion led to impaired antibody production and diminished neutralization capacity. Furthermore, single-cell transcriptomic analysis revealed an interaction between pDCs and CD8+ T cells that facilitates T cell activation. In vivo experiments confirmed that pDCs expressing the viral spike protein directly engage with CD8+ T cells, promoting their differentiation and expansion. Moreover, the absence of pDCs affected the formation of antigen-specific memory T cells. Overall, these findings highlight that pDCs are essential players in mediating both adaptive and humoral responses to the Pfizer-BioNTech coronavirus vaccine, providing insights into the mechanistic functioning of mRNA vaccines and establishing a novel role for pDCs as professional APCs. ### Competing Interest Statement The authors have declared no competing interest.

Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/ or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients.

Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including ‘one-to-stop’ (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses.

SARS-CoV-2 3C-like protease (3CL(pro) or M-pro) cleaves the SARS-CoV-2 polyprotein and >300 intracellular host proteins to enhance viral replication. By lytic cell death following gasdermin (GSDM) pore formation in cell membranes, antiviral pyroptosis decreases 3CL(pro) expression and viral replication. Unexpectedly, 3CL(pro) and nucleocapsid proteins undergo unconventional secretion from infected cells via caspase-activated GSDMD/E pores in the absence of cell lysis. Bronchoalveolar lavage fluid of wild-type SARS-CoV-2-infected mice contains 3CL(pro), which decreases in Gsdmd(-/-)Gsdme(-/-) mice. We identify new 3CL(pro) cut-sites in GSDMD at LQ(29)down arrow(SS)-S-30, which blocks pore formation by 3CL(pro) cleavage at LH270 down arrow N lying adjacent to the caspase activation site (NFLTD275 down arrow G). Cleavage inactivation of GSDMD prevents excessive pore formation, thus countering antiviral pyroptosis and increasing 3CL(pro) secretion. Extracellular 3CL(pro) retains activity in serum, dampens platelet activation and aggregation, and inactivates antiviral interferon-lambda 1. Thus, in countering gasdermin pore formation and pyroptosis in SARS-CoV-2 infection, 3CL(pro) is secreted with extracellular pathological sequelae.

Cigarette smoke (CS) promotes the development of chronic pulmonary disease and has been associated with increased risk for influenza-related illness. Here, we directly addressed the impact of CS disordered microbiota on the severity of influenza A virus (IAV) infection. Specific and opportunistic pathogen-free (SOPF) C57BL/6J mice were exposed to CS or room air (RA) for 5.5 months. Each exposed mouse was then cohoused with a group of recipient germ-free (GF) mice for 1 month for microbial transfer. Colonized GF mice were then infected intranasally with IAV and disease development was monitored. Upper and lower airway and fecal microbiota were longitudinally investigated by 16S rRNA gene sequencing and bacterial cultures in donor and recipient mice. The bacterial family Streptococcaceae accounted for the largest difference between CS- and RA-exposed microbiota in the oropharynx. Analysis of the oropharynx and fecal microbiota indicated an efficient transfer to coprophagic recipient mice, which replicated the differences in microbiota composition observed in donor mice. Subsequent IAV infection revealed significantly higher weight loss for CS microbiota recipient mice at 8-10 days post infection (dpi) compared to control recipient mice. In addition, H1N1 infection inflicted substantial changes in the microbiota composition, especially at days 4 and 8 after infection. In conclusion, mice with a CS-associated microbiota suffer from higher disease severity upon IAV infection compared to mice colonized with a normal SOPF microbiota. Our data suggest that independently of CS exposure and concomitant structural lung damage, microbial distortion due to CS exposure may impact the severity of IAV disease course. IMPORTANCE It has been reported that chronic exposure to CS is associated with a disordered microbiota composition. In this study, we colonized germ-free (GF) mice with the microbiota from SOPF mice which were chronically exposed to CS or RA. This allowed disentangling the effect of the disordered microbiota from the immune-modulating effects of actual CS exposure. We observed a successful transfer of the microbiotas after cohousing including specific microbiota differences induced by CS exposure in formerly GF mice, which were never exposed to CS. We then investigated the effects of IAV infection on the disease course and microbiotas of formerly GF mice. We found that mice with CS-associated microbiota reveal worse disease course compared to the control group. We hypothesize that CS-induced disordering of the microbiota may, indeed, impact the severity of influenza A disease.

Wesselsbron virus (WSLV) is a zoonotic, mosquito-borne orthoflavivirus endemic to sub-Saharan Africa, causing abortions and stillbirths in small ruminants. The life cycle of WSLV involves Aedes mosquitoes and various wildlife and domestic animals. Seminal studies in the 1950s have shown the zoonotic potential of WSLV, notably in accidental infections of laboratory workers exposed to infected material. More recent epidemiological studies suggest the emergence of clade I WSLV strains in peri-domestic and rural areas of western and eastern Africa. The pathobiology of recent clade I WSLV strains is unknown and no virus isolate is available. To address these gaps, we generated a recombinant clade I WSLV SA999 infectious clone (rSA999) by reverse genetics. Subsequently, lactating ewes were inoculated intravenously with the WSLV rSA999 strain or the clade II SAH177 strain in insect-free biocontainment stables. Inoculated ewes developed fever, viremia, and showed high levels of viral RNA at mucosal surfaces, and elevated viral titers in milk. Milk production was reduced, which directly affected the growth of the lambs, particularly within the rSA999 group. The ewes with higher WSLV titers in their milk in each group transmitted the infection to their lambs, which developed fever, prolonged viremia, and virus secretion. All infected animals produced high antibody titers with cross-neutralizing activity against both WSLV strains. Histopathology and blood biochemistry analysis indicated liver damage associated with necrotizing hepatitis lesions and active viral replication in some cases, which was more pronounced in the rSA999 group. Notably, only the SAH177-infected animals exhibited lesions consistent with meningoencephalitis, suggesting that WSLV clade II strains are neurotropic and that clade I strain are more hepatotropic. These findings demonstrate a previously unrecognized mode of vector-free transmission of WSLV that raises significant concerns for public and animal health.

To the Editor, Early in the coronavirus disease 2019 (COVID19) pandemic, age has been recognized as one of the major risk factors for poor clinical outcome.1 Based on hospitalization rates, it has also rapidly become evident that fewer women than men were affected by severe disease manifestation.2 With the primary goal to protect the most vulnerable populations, those older than 65, scientists around the world have successfully developed different vaccines with unprecedented speed.3 Although it is well established that immune responses against infections decline with age,4 it is less clear how vaccineelicited immunity varies between different sex and age groups.5 Given the importance of understanding these biological parameters, which may directly affect translatability of research findings into the clinic, we sought to investigate the immune response against severe acute respiratory syndrome coronavirus 2 (SARSCoV2) in a proteinbased and vesicular stomatitis virus (VSV)vectored vaccination approach in young and aged mice of both sexes. First, we used the recombinant receptorbinding domain (RBD) of the SARSCoV2 spike protein from the original reference strain emulsified in an aluminum hydroxide containing wet gel suspension (i.e. Alum) to subcutaneously immunize C57BL/6 mice (Figure S1). Seven days later, they received a booster injection and the vaccine response was assessed on day 28 (Figure 1A,B). To test the induction of humoral immunity as a function of age, we measured antigenspecific IgG in young (2 months old) and aged (18– 19 months old) mice by ELISA. Consistent with other studies, the systemic RBDspecific IgG response was significantly diminished in aged mice (Figure 1C,D). This agerelated decline in total RBDspecific IgG is primarily due to a loss of IgG1 production since the other subclasses remained barely detectable (Figure S2). To further characterize humoral immunity, we measured the total number of plasma Bcells in spleen by flow cytometry and quantified RBDspecific plasma Bcells in the spleen of immunized mice by ELISpot. While the total number of splenic plasma Bcells was increased in aged mice the RBDspecific IgG positive Bcells were significantly diminished (Figure 1E,F) and correlated with serum IgG levels (Figure 1G), suggesting that the agerelated reduction of RBDspecific plasma cell formation might contribute to the concomitant decrease in antibody titers. Previous studies have reported significant alterations in T follicular helper (Tfh) and regulatory (Tfr) cell numbers in lymphoid organs in aged mice contributing to impaired plasma Bcell generation and defective antibody production.6 Indeed, we measured an agerelated increase in both Tfr and Tfh populations in the spleen as quantified by flow cytometry, while the number of classical T regulatory cells (Tregs) remained unchanged (Figure S3A). Most importantly, the live SARSCoV2 neutralization potency of serum from aged mice was significantly reduced for the original reference strain and different other variants of concern (i.e. alpha, gamma and delta), which is in line with the agerelated decrease in RBDspecific serum antibody titers and plasma Bcells (Figure 1H). Additionally, we evaluated sexspecific differences in vaccination response across age in the same cohorts of immunized C57BL/6 mice (Figure 2A). While RBDspecific IgG and IgG1 responses in serum were higher in young females as compared to young male controls (Figure 2B,C), these differences were no longer apparent in the aged mice, and there were no detectable sexspecific differences in the number of splenic plasma Bcells (Figure 2D). In line with higher RBDspecific antibody titers, young female C57BL/6 mice also showed more potent virus neutralization of the SARSCoV2 reference strain when subcutaneously immunized with a proteinbased vaccine or intramuscularly injected with two VSVvectored COVID19 vaccine candidates (i.e., VSVSD21 and VSVMqSD21) as compared to male controls (Figure 2E and Figure S4A,B). However, neutralization of the other tested variants of concern was diminished and equally weak in both sexes (Figure 2E), indicating that mutations in the RBD domain of these variants were sufficient to escape the vaccineinduced antibody response. To test whether these findings were conserved across different mouse strains, we repeated the same immunization regimen in young BALB/c mice. The observed outcome was essentially the same with females showing a better vaccination response than male mice (Figure S5A– E). The sexspecific differences in humoral immune response of young C57BL/6 mice persisted even after an additional injection with a proteinbased vaccine 21 days after the first boost as assessed on day 42 (Figure 2F– J). Strikingly, we found increased numbers of RBDspecific plasma Bcells in the bone marrow of young female mice in this context. In summary, our data demonstrate significant ageand sexrelated differences in the humoral immune response to different

The 2023 International African Swine Fever Workshop (IASFW) took place in Beijing, China, on 18–20 September 2023. It was jointly organized by the U.S.-China Center for Animal Health (USCCAH) at Kansas State University (KSU) and the Chinese Veterinary Drug Association (CVDA) and sponsored by the United States Department of Agriculture Foreign Agricultural Service (USDA-FAS), Harbin Veterinary Research Institute, and Zoetis Inc. The objective of this workshop was to provide a platform for ASF researchers around the world to unite and share their knowledge and expertise on ASF control and prevention. A total of 24 outstanding ASF research scientists and experts from 10 countries attended this meeting. The workshop included presentations on current ASF research, opportunities for scientific collaboration, and discussions of lessons and experiences learned from China/Asia, Africa, and Europe. This article summarizes the meeting highlights and presents some critical issues that need to be addressed for ASF control and prevention in the future.