Centre de Recerca en Sanitat Animal

The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised the efficacy of many authorized monoclonal antibody products. This highlights the need for alternative strategies, especially for vulnerable populations such as immunocompromised individuals. Here, we optimized angiotensin-converting enzyme 2 (ACE2)-Fc fusion proteins by combining three engineering steps: in silico mutagenesis of the S protein binding interface to increase affinity, insertion of a flexible linker to improve protein stability and S protein accessibility, and generation of a tetrameric molecule to maximize avidity. Neutralizing activity was tested against a large panel of pre-Omicron and Omicron pseudoviruses and authentic viruses, including JN.1 and KP.2 variants. Optimized ACE2-Fc molecules demonstrated potent neutralizing activity, in the picomolar range, against all SARS-CoV-2 variants. Our molecules displayed similar potency but better resilience when compared to the monoclonal antibody Sipavibart. These findings support ACE2-Fc proteins as robust candidates for next-generation interventions against infection by an evolving SARS-CoV-2.

Abstract The ongoing panzootic of H5N1 clade 2.3.4.4b high pathogenicity avian influenza virus (HPAIV) has increasingly affected non-traditional hosts like seabirds and mammals, including recent outbreaks in fur farms, in which transmission from infected gulls were suspected. To assess bird-to-mammal spillover and mammal-to-bird spillback dynamics, we performed experimental infections in yellow-legged gulls (Larus michahellis) and American minks (Neogale vison) using HPAIV strains isolated from gulls (H5N1/gull) and a farm mink outbreak (H5N1/mink). Minks challenged with H5N1/gull developed subclinical infection with limited shedding, but viral neurotropism in two out of four animals led to spontaneous emergence of key mammalian adaptation mutations in the brain, which demonstrates that H5N1 clade 2.3.4.4b spillover events from gulls to minks can lead to fast mammalian adaptation. In contrast, mammal-to-bird spillback, modelled by challenging gulls with the H5N1/mink strain, yielded no evidence of infection, suggesting a lower risk of sustained bird-mammal-bird transmission chains of 2.3.4.4b H5N1 HPAIV.

IntroductionThe emergence of highly virulent strains of the porcine reproductive and respiratory syndrome virus has driven the need for new vaccines. This study evaluates the efficacy of an intranasal (IN) vaccine composed of a naturally attenuated PRRSV-2 isolate, compared to a commercially available intramuscularly administered (IM) PRRSV-1 vaccine, against a heterologous challenge with a highly virulent PRRSV-1 strain (R1).MethodsSixty-eight PRRSV-naïve pigs were divided into four groups: two non-vaccinated controls (NV/NCh, NV/Ch), one IM-vaccinated with a PRRSV-1 MLV (Por), and one intranasally (IN)-vaccinated with the PRRSV-2 vaccine (IL).ResultsClinical, pathological, and immunological outcomes were assessed post-challenge. Both vaccines significantly (p < 0.05) reduced fever duration (3–5 days less than controls, respectively), reduced the clinical scores after challenge, and mitigated weight loss (p < 0.05), though viral loads in serum and lungs remained comparable across groups. Macroscopic lung lesions at 10 days post-challenge (DPC) were reduced in vaccinated groups (13–14% of pneumonic lung on average in vaccinated groups vs. 35% in NV/Ch), yet microscopic lesions persisted, correlating with lung viral loads at 28 DPC (R2 = 0.54, p < 0.001). None of the tested vaccines achieved an efficient control of the viremia or nasal shedding compared to unvaccinated controls. Cross-reactive cell-mediated responses suggested shared epitopes between PRRSV-1 and PRRSV-2; however, the frequencies of interferon-gamma-secreting cells did not correlate with lesion severity.DiscussionThe IN vaccine demonstrated non-inferiority to IM vaccination in alleviating clinical signs and helped reduce weight losses, however, at later times control of viral replication was lower, underscoring limitations in heterologous protection. The dissociation between systemic immune markers and tissue-specific outcomes highlights the need for strategies targeting tissue-resident immunity. These findings advocate further exploration of mucosal vaccination as a complementary strategy for PRRSV control, particularly under heterologous challenge conditions, while emphasizing the persistent challenges posed by viral diversity and incomplete cross-protection.

Abstract Mycoplasma hyorhinis (homotypic synonym of Mesomycoplasma hyorhinis ) is a pathobiont from the upper respiratory tract of pigs. Under unclear circumstances, it can disseminate systemically and cause disease. Although some studies described different infectious capabilities, no factors have been directly linked to virulence. This study aimed to analyze the core and accessory genes of all available M. hyorhinis strains (pangenome) to identify potential virulence factors. We characterized the pangenome of 110 strains, including isolates from healthy (nasal cavity) and diseased (systemic organs, nasal cavity or lung) animals. Comparative analyses were performed according to the clinical background. Although most putative virulence genes were shared, we identified several genes absent in most health-associated strains related to DNA-processing mechanisms, including hsdM - hsdR restriction-modification system and various helicases. Furthermore, the particular analysis of variable lipoprotein ( vlp ) genes, revealed a similar presence in all strains but higher number of repeats in region III of vlpF and vlpC . Genome-scale metabolic models were used to infer the metabolic capabilities of the strains, showing highly conserved predicted reactomes, including growth capabilities and auxotrophies. In conclusion, although all strains may carry genes enabling disease, nasal strains from healthy animals lacked some DNA-processing genes and showed distinct vlp patterns. Additional genomes especially from strains isolated from healthy animals, would be needed to confirm these findings.

Bycatch is the most common cause of death of small delphinids worldwide, including the Mediterranean Sea. The diagnosis of bycatch as cause of death in stranded cetaceans depends on the cumulative presence of multiple findings, termed bycatch criteria. In this study, we retrospectively evaluated the presence of bycatch criteria in 138 necropsied cetaceans, 136 stranded and 2 confirmed bycaught, in the Catalan Mediterranean Sea across a 13-year period. With the aim of identifying the most specific and reliable bycatch criteria, the animals' cause of death was classified as either bycaught or other causes. Animals were necropsied according to standard procedures with complete histopathological examination and ancillary diagnostic techniques. We reviewed the necropsy reports and photographs of 138 cetaceans of seven species. Bycatch had been determined as the cause of death/stranding in 40 (29%) necropsied cetaceans. Both sexes were equally represented in the bycatch group. Bycatch was diagnosed in the Mediterranean common bottlenose dolphin (10/14; 71.4%), striped dolphin (29/108; 26.9%), and Risso's dolphin (1/11; 9.1%). Sixty-seven out of 98 (68.3%) cetaceans that had been classified as non-bycatch had one or two bycatch criteria. Cetaceans with two and three major criteria had an overlap of causes of death, as some animals were diagnosed with bycatch and others with other causes of mortality. Animals with four criteria were invariably diagnosed as being bycaught. Recent feeding, absence of disease, good nutritional status, marks of fishing gear, multiorgan intravascular gas bubbles, hyphema and amputations or sharp incisions presumably inflicted by humans were significantly more likely to result in a diagnosis of bycatch, while loss of teeth and cranial fractures were not. None of the dolphins diagnosed as bycatch had ingested fishing gear. Our results highlight the relevance of bycatch as the cause of death of dolphins in the Mediterranean and suggest that some criteria traditionally linked to bycatch are not specific for bycatch in our region.

Influenza is still a chronic global health threat, inducing a sustained search for effective antiviral therapeutics. Computational methods have played a pivotal role in developing small molecule therapeutics. In this study, we applied a combined in silico and in vitro approach to explore the potential anti-influenza activity of cyproheptadine, a clinically used histamine H1 receptor antagonist. Virtual screening based on the average quasivalence number (AQVN) and electron–ion interaction potential (EIIP) descriptors suggests similarities between cyproheptadine and several established anti-influenza agents. The subsequent ligand-based pharmacophore screening of a focused H1 antagonist library was aligned with the bioinformatics prediction, and further experimental in vitro evaluation of cyproheptadine demonstrated its anti-influenza activity. These findings provide proof of concept for cyproheptadine’s in silico-predicted antiviral potential and underscore the value of integrating computational predictions with experimental validation. The results of the current study provide a preliminary proof of concept for the predicted anti-influenza potential based on computational analysis and emphasize the utility of integrating in silico screening with experimental validation in the early stages of drug repurposing efforts.

Globalization has increased the incidence of infectious diseases in livestock, further aggravated by the reduction of antibiotic usage. To minimize the resulting economic consequences to the meat production industry, as well as the risk of zoonotic events, the use of immunostimulants has emerged as a potential strategy to enhance animal resilience to diseases. In particular, the capability of bacterial-based immunostimulants to modulate innate immune cells functionality makes them cost-effective candidates as vaccine adjuvants, antimicrobials, or preventive immunostimulators inducing long-term innate immune memory in livestock. However, further research is required to identify novel bacterial strains with immunostimulatory properties. Here we characterized in vitro the immunostimulatory properties of Rothia nasimurium isolated from warthog fecal microbiota. Stimulation with heat-inactivated Rothia induced cytokine production by porcine immune cells, and a robust innate immune transcriptomic signature in porcine alveolar macrophages. Interestingly, the bacteria induced inflammasome activation and IL-1β production, thus confirming its pro-inflammatory properties, and suggesting its potential as vaccine adjuvant. Importantly, this immunostimulatory status functionally resulted in an antimicrobial state, enhancing the phagocytic capability of alveolar macrophages, and hampering the replication levels of two major porcine viral pathogens: the porcine reproductive and respiratory syndrome virus (PRRSV) and the African swine fever virus (ASFV). Moreover, macrophages showed an enhanced cytokine response upon ASFV infection several days after heat-inactivated Rothia stimulation, suggesting the induction of an innate immune memory phenotype. This nonspecific response resulted in a significant reduction of ASFV replication kinetics, demonstrating the capacity of the bacteria to induce a more resistant state in macrophages against a virus infection. Altogether, these results demonstrate the immunostimulatory capability of heat-inactivated R. nasimurium in porcine macrophages, showing potential to enhance animal resilience to diseases through the modulation of innate immune cells responsiveness to infections.

African swine fever (ASF) is a disease that affects both domestic and wild swine. It was recently reported in the Dominican Republic and Haiti (2021), representing a substantial risk to America. The goal of this study was to produce a truncated form of the ASF-p72 recombinant protein based on the ASF strain genotype II (Georgia 2017) as well as to develop and validate a sensitive and specific ASF indirect-ELISA (iELISA) for early detection of ASF. The truncated ASF-p72 recombinant protein was successfully expressed in E. coli BL21/DE3 cells using the pET-SUMO plasmid. Bioinformatics analysis showed 100% homology among the new isolates of ASFV from genotype II. The ASF-p72-truncated protein was used to develop an iELISA, which had a high sensitivity (88%) and strong specificity (97%); the concordance index kappa was K = 0.872, indicating nearly perfect agreement compared to the WOAH confirmatory immunoperoxidase test. The validation results utilizing the reference sera panel from the OIE-ASF Reference Laboratory show the excellent detection capabilities of ASF antibodies up to a 1:1000 serum dilution. The inter-assay coefficient of variation (CV 10.4%) and intra-assay CV (2.8%) data show that the assay is precise and reproducible. This biotechnology advancement can be used to conduct future epidemiological research for ASF surveillance in ASF-free American countries.