Search Results (709)

The classification systems currently in use for Streptococcus suis are primarily based on capsular polysaccharide (CPS) serotyping or multi-locus sequence typing (MLST). A genetic typing system based on the conserved Mac-1 domain of the Ide_Ssuis gene, which encodes an IgM-cleaving protease, could complement these approaches. Specifically, in this study, a genotyping system based on Mac-1 sequence homology was established, qualitative SYBR Green real-time PCR (qPCR) assays for standardized genotype classification were developed, and the global and European distribution of genotypes were analyzed. The Mac-1 classification system identified “two genotypes”, one of which has “two subgenotypes”, based on 427 unique sequences obtained from 4161 global sequences. GT1.1 was the most prevalent, having an amino acid similarity of 97.1%, followed by GT1.2 98.1%, and GT2 91.4%. The qPCR approach enabled reproducible, cross-laboratory-accessible typing. Among 5479 Streptococcus suis European isolates, 64% were GT1.1, 4.5% were GT1.2, and 29.6% were GT2, with a trend toward increasing GT1.1 detection rates between 2018 and 2023. Genotyping classification could complement current Streptococcus suis classification approaches to better support vaccine design and disease control strategies. Full article

Highly pathogenic avian influenza (HPAI) virus H5N1of clade 2.3.4.4b has emerged as the predominant lineage circulating in poultry flocks worldwide, raising concerns regarding the protective efficacy of currently available commercial vaccines, particularly in domestic ducks, which play an important role in virus maintenance and transmission. Thus, this study evaluated the immunogenicity along with the protective efficacy of four inactivated H5 vaccines against a recently isolated local HPAI-H5N1 (Newvalley-3-H5N1-2024, clade 2.3.4.4b) strain in Pekin ducks in Egypt. A total of 150 seronegative ducks were divided into vaccinated and control groups (10 groups) and vaccinated at 10 days of age. At 31 days of age, the vaccinated and positive control groups were challenged using 10^6.5 EID₅₀/0.5 mL/duck with the local isolate (Newvalley-3-H5N1-2024) via the oculo-nasal route. The vaccine efficacy was assessed through clinical signs, survival rate, hemagglutination inhibition (HI) antibody titer, tracheal and cloacal viral shedding quantified by real-time RT-PCR, and histopathological examination of trachea, lung, pancreas, and brain tissues. Generally, all ducks vaccinated with the ValleyVac Avian Flu H5 plus and MEFLUVAC^TM H5 PLUS 8 showed a significantly higher survival rate (100%) at 10 days post-vaccination (DPV) than those in the positive control (66.7% mortality rate). In contrast, ducks exhibited mortality rates ranging from 6.7% in the SERVAC Flu H5N1 group to 13.4% in the Sinder Fluvac group. The ValleyVac Avian Flu H5 plus and MEFLUVAC™ H5 PLUS 8 vaccines induced the highest HI antibody titers at 7, 14, 21, and 28 DPV in both homologous and heterologous AIV antigens, resulting in a significant reduction in viral load among all vaccinated duck groups (p-value < 0.05) comparable to the positive control group. Conversely, the SERVAC Flu H5N1 and Sinder Fluvac vaccines provided partial protection, suboptimal immunogenicity at different time points, and elevated viral shedding. Histopathological findings in ValleyVac Avian Flu H5 plus and MEFLUVAC™ H5 PLUS 8 vaccines exhibited mild tissue alterations following AIV challenge. Marked pathological lesions were observed in the SERVAC Flu H5N1 and Sinder Fluvac vaccinated groups. Among tested vaccines, both ValleyVac Avian Flu H5 plus and MEFLUVAC^TM H5 PLUS 8 showed the highest level of protective efficacy against the circulating AIV strain compared with other commercial vaccines. This study highlights the need for continuous molecular surveillance, antigenic matching, and regular updating of vaccine seed strains to ensure efficient HPAI control in Egypt. Full article

Japanese encephalitis virus (JEV), the prototype member of the JEV serogroup within the genus Orthoflavivirus (family Flaviviridae), is closely related to West Nile virus (WNV), Murray Valley encephalitis virus (MVEV), and St. Louis encephalitis virus (SLEV). To characterize antigenic cross-reactivity within this group, we evaluated 15 region-specific rabbit antisera, previously generated against nearly the entire JEV protein-coding region, by immunoblotting whole-cell lysates from BHK-21 cells infected with WNV, MVEV, or SLEV, with JEV included as a reference. Six antisera (α-E^N-term, α-NS2B, α-NS3^N-term, α-NS3^C-term, α-NS5^N-term, and α-NS5^C-term) robustly recognized homologous proteins across all three viruses. The remaining nine antisera displayed lineage-restricted or virus-specific reactivity: (a) α-C cross-reacted strongly with WNV and MVEV but weakly with SLEV; (b) α-M, α-NS1, α-NS4A, and α-NS4B^C-term cross-reacted with WNV and MVEV but not SLEV; (c) α-E^C-term and α-NS1′ cross-reacted only with WNV; (d) α-Pr cross-reacted exclusively with MVEV; and (e) α-NS4B^N-term showed no detectable cross-reactivity under the experimental conditions used. Notably, α-NS1 and α-NS1′ detected heat-labile multimers of NS1 and NS1′. These serological patterns mirror the established phylogeny of the JEV serogroup, with JEV clustering most closely with MVEV, followed by WNV and then SLEV. Together, these findings provide a comprehensive cross-reactivity map of JEV antigen-directed antisera and establish a practical framework for dissecting antigenic relationships among JEV serogroup members. These results enhance our understanding of orthoflavivirus antigenic evolution and support the future development of improved diagnostics, broad-acting vaccines, and experimental reagents for emerging and re-emerging encephalitic orthoflaviviruses. Full article

Introduction: The development of bivalent mucosal vaccines capable of providing protection against both influenza and SARS-CoV-2 is a major public health focus. While most COVID-19 vaccines target the spike (S) protein, the highly conserved nucleocapsid (N) protein represents a strategic target for cross-reactive, cell-mediated immunity. This study evaluates Corfluvec, an intranasal vaccine candidate based on an attenuated NS1-truncated influenza vector expressing a fragment of the SARS-CoV-2 N protein. Methods: Protective efficacy, including viral load and pathomorphological changes in the lungs and vessels, was evaluated in Syrian hamsters challenged with high and low doses of SARS-CoV-2 (lineage B.1.1). Cross-protective efficacy against homologous and heterologous influenza A strains (H1N1pdm09, H3N2, and A/PR/8/1934) was tested in a lethal murine model. Additionally, immunogenicity of Corfluvec applied via human-compatible delivery device was tested in cynomolgus macaques (Macaca fascicularis). Results: In Syrian hamsters, vaccination significantly reduced viral loads in the lungs and nasal turbinates. Histopathological analysis revealed a preservation of lung vascular integrity: vaccinated animals showed stable CD31 expression and controlled Ki-67 proliferative activity, accompanied by a marked reduction in vasculitis and perivascular edema compared to placebo controls. In mice, the vaccine provided 100% protection against homologous and heterologous influenza virus challenges. In macaques, the two-dose intranasal immunization was well-tolerated and induced significant systemic IgG and mucosal sIgA responses, alongside robust N-specific IFNγ+ T-cell activation. Conclusions: Corfluvec is a promising bivalent vaccine candidate that provides dual protection against influenza and COVID-19. Its ability to limit viral shedding from the upper respiratory tract and to mitigate SARS-CoV-2-induced pulmonary pathology, contributing to the preservation of lung vascular integrity, underscores the utility of mucosal immunization with Corfluvec as a valuable intranasal complement to current systemic vaccination strategies. Full article

Porcine epidemic diarrhea virus (PEDV) G2c variants have recently emerged, posing significant challenges to swine health management. As a major coronavirus affecting the swine industry, PEDV exhibits extensive genetic variability, which has greatly complicated disease control. Current vaccines provide suboptimal protection under field conditions. Therefore, the isolation of recently circulating strains and the establishment of a robust reverse genetics system are critical for advancing the study of emerging variants and facilitating rational vaccine development. In this study, a PEDV field strain designated PEDV-BJ-2023 was isolated from diarrheic piglets in Guizhou, China. Phylogenetic analysis based on the complete genome and spike gene classified PEDV-BJ-2023 within the emerging G2c lineage. To facilitate functional studies, a full-length infectious cDNA clone was constructed using transformation-associated recombination cloning in yeast. Furthermore, an enhanced green fluorescent protein reporter virus was generated via CRISPR/Cas9-assisted homologous recombination by inserting an EGFP-2A cassette upstream of the nucleocapsid gene. The recombinant viruses displayed virion morphology and plaque characteristics similar to those of the parental wild-type PEDV-BJ-2023 strain, although the parental virus exhibited faster replication during the early stage of infection in vitro. In 5-day-old piglets, all three viruses caused severe diarrhea, weight loss, and intestinal lesions; however, recombinant viruses exhibited slightly reduced viral shedding and pathogenicity, with rPEDV-EGFP being the most attenuated. Notably, rPEDV-EGFP maintained stable EGFP expression over eight serial passages. This study establishes a reverse genetics platform for an emerging G2c PEDV strain and provides a stable fluorescent reporter virus, offering valuable tools for visualizing viral infection and investigating virus–host interactions. Full article

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that poses serious risks to immunocompromised individuals and the global livestock industry. Our previous study showed that AP2X-1 regulates stage conversion and virulence of T. gondii; however, whether AP2X-1-deficient strains can serve as live-attenuated vaccine candidates is unknown. Here, we evaluated the in vivo virulence, protective efficacy, and induced immunity of the ap2X-1 knockout strain PruΔap2X-1. In Kunming mice, infection with even the highest tested dose (5 × 10⁶ tachyzoites) of PruΔap2X-1 resulted in 100% survival without detectable brain cysts. Vaccination with 10⁶ PruΔap2X-1 tachyzoites conferred complete protection (100% survival) against acute challenge with homologous type II Pru or heterologous ToxoDB#9 PYS strains, whereas control mice exhibited only 0% to 16.7% survival. Moreover, immunized mice orally challenged with Pru cysts showed 100% survival, and no brain cysts were detected under the conditions tested, compared with only 30% survival in control mice challenged with 10 cysts and no survival with 40 cysts. The vaccination induced a Th1-biased response as evidenced by significantly elevated T. gondii-specific total IgG, IgG2a, and the Th1 cytokines IFN-γ and IL-2, along with a significantly reduced IgG1/IgG2a ratio. Collectively, the attenuated PruΔap2X-1 strain generates strong protective immunity against acute and chronic infection with low-virulence strains (the type II Pru and ToxoDB#9 PYS), as well as partial protection against the hypervirulent type I RH strain, positioning it as a potential candidate for a live-attenuated vaccine, with a favorable safety profile observed under the experimental conditions. Full article

H7N9 avian influenza virus (AIV) poses a persistent threat to poultry and public health. Despite widespread vaccination in China, rapid antigenic drift and reassortment necessitate frequent updates of vaccine strains. Phylogenetic analysis of isolates from Chinese provinces (from 2019 to 2023) showed that while surface genes diversified, the internal gene cassette remained conserved yet actively reassorted with other subtypes, suggesting internal gene compatibility may influence vaccine performance. We selected the H7N9 strain A/chicken/Northeast China/19854-6/2019 (E2), which harbors a polybasic Hemagglutinin (HA) cleavage site and predicted dual receptor-binding affinity. To enable safe vaccine development, we modified the HA cleavage site to generate a low-pathogenicity strain (E2-Δ). Using reverse genetics, we constructed three recombinant viruses: E2-Δ (retaining contemporary internal genes), CVI-E2, and CVII-E2 (containing internal genes from commercially used donor strains CVI and CVII, respectively). Inactivated vaccines were evaluated in specific-pathogen-free (SPF) chickens. E2-Δ induced HI antibody titers comparable to those of CVI-E2 and significantly higher than those of CVII-E2, and showed modestly higher cross-reactive HI titers against some recent H7N9 variants in exploratory analyses. All vaccines provided complete homologous protection with reduced viral shedding and no clinical signs in challenge trials. Our findings suggest that internal gene backbone compatibility may influence vaccine immunogenicity. While E2-Δ outperformed CVII-E2, it was comparable to CVI-E2, indicating that certain traditional backbones may still be suitable for H7N9 vaccine development. This approach warrants further validation to support a refined vaccine design strategy for H7N9 and potentially other avian influenza subtypes. Full article

Background: DNA vaccines are widely used due to their low production cost, ease of large-scale manufacturing, and rapid responsiveness to emerging epidemics. However, their relatively modest immunogenicity in larger species often requires potent adjuvants to elicit robust protective responses. GM-CSF and Flt3L are two cytokine adjuvants targeting dendritic cells (DCs). Although their combination has been reported to augment immune responses, the mechanism underlying the complementary effects of a genetically fused GM-CSF-Flt3L adjuvant plasmid remains incompletely understood. Methods: We constructed a GM-CSF-Flt3L fusion adjuvant plasmid and compared it with single-adjuvant plasmids for their effects on innate, humoral, and cellular immunity, as well as protective efficacy against lethal homologous virus challenge. Results: Compared with single-adjuvant plasmids, GM-CSF-Flt3L synergistically promoted the maturation of bone marrow-derived dendritic cells (BMDCs) and activated draining lymph node DCs, differentially expanded DC subsets, and enhanced the recruitment of migratory DCs. This adjuvant significantly elevated hemagglutinin (HA)-specific serum IgG titers, hemagglutination inhibition (HI) titers, and the responses of T follicular helper (TFH) cells and germinal center B (GCB) cells, while also enhancing the capacity of HA-specific CD4⁺ and CD8⁺ T cells to secrete IFN-γ and TNF-α. Following lethal homologous virus challenge, the GM-CSF-Flt3L group exhibited markedly reduced lung viral loads and no significant pathological damage in lung tissues. Conclusions: These findings demonstrate that the GM-CSF-Flt3L fusion adjuvant complementarily enhances humoral and cellular immune responses induced by the influenza DNA vaccine and improves protective efficacy, highlighting its potential as an effective DNA vaccine adjuvant. Full article

Influenza A(H3N2) remains a significant public health threat due to its rapid antigenic drift, which often compromises vaccine effectiveness. This study characterized the molecular and epidemiological profile of hemagglutinin (HA) variants circulating in Western Mexico throughout 2022. Among 476 positive cases, A(H3N2) was the predominant subtype (88%), with infection peaks during epidemiological weeks (EW) 1, 45, and 46. Sanger sequencing of the HA gene identified 64 amino acid substitutions, with 85.9% of the substitutions located in the HA1 subunit, primarily within the receptor-binding domain (RBD). Homology modeling and molecular docking were performed on five representative variants: C156S, D158N, Y159N, C136S, and L227P. All variants exhibited a slight decrease in binding affinity for sialic acid compared to the 1HGE reference. Notably, while mutations such as D158N and Y159N remodeled the interaction network, Glu190 and His183 remained critical for stabilizing the HA-ligand complex through hydrogen bonds and non-covalent interactions in the structural models. These structural findings suggest that contemporary mutations in the RBD may contribute to changes in receptor-binding interactions while preserving key structural features associated with host cell attachment. Full article

Vaccination remains the primary strategy for poliomyelitis prevention. The D-antigen of poliovirus is a critical component of inactivated polio vaccines, as it induces the production of neutralizing antibodies and provides protective immunity. Therefore, the development of quantitative immunoassays for monitoring D-antigen content during vaccine production is an important task. In this study, we generated recombinant camelid single-domain antibodies (VHHs) specific for the D-antigen of Sabin poliovirus type 1 and evaluated their antigen specificity. The obtained VHH antibodies demonstrated strong binding to the native D-antigen of Sabin type 1 poliovirus. Furthermore, the use of clone S1-C7 as a capture antibody in combination with Sabin type 1-specific polyclonal antibodies for detection revealed differential recognition of the vaccine-derived Sabin type 1 and homologous wild-type Mahoney strains. Notably, such discrimination was not observed when polyclonal antibodies were used alone, indicating that incorporation of VHH S1-C7 into the assay enhances strain-specific antigen recognition. In a neutralization assay, clone S1-C7 exhibited in vitro neutralizing activity against Sabin type 1 poliovirus. One clone, S1-D9, demonstrated cross-reactivity with all three poliovirus serotypes, suggesting recognition of a conserved epitope in the capsid and potential utility as a universal detection antibody. The generated VHH antibodies represent promising analytical tools for poliovirus antigen characterization. Together with S1-C7, they enable the discrimination of both D- and C-antigen forms as well as vaccine-derived and wild-type strains. Full article

Background/Objectives: Pandemic influenza remains a persistent global threat, and while vaccination is the primary preventive measure, conventional vaccines often induce narrow, strain-specific immunity. This study evaluated the immunogenicity, protective efficacy, and cross-protective potential of a CpG-adjuvanted trivalent inactivated influenza vaccine (CpG-TIV) administered intramuscularly at high and low doses in ferrets. Methods: Groups of influenza-seronegative ferrets received two intramuscular injections, 3 weeks apart, of high- or low-dose CpG-TIV or a commercial non-adjuvanted trivalent vaccine. Three weeks after the second immunization (Day 42), serum was obtained, and the ferrets were subsequently challenged intranasally with homologous H1N1 and influenza B viruses, as well as a heterologous drifted H3N2 strain. Clinical signs, body weight, nasal viral load, and lung histopathology were monitored following the viral challenge. Results: CpG-TIV induced significantly higher dose-dependent HI and IgG antibodies than the commercial unadjuvanted vaccine. High-dose CpG-TIV markedly reduced weight loss, clinical symptoms, nasal viral load (by up to 99%), and lung pathological damage. Notably, high-dose CpG-TIV provided significant cross-protection against heterologous H3N2, whereas the commercial vaccine showed no protective effect. At Day 42, HI GMTs in the high-dose group reached 500, 254, and 594 against H1N1, H3N2, and B strains, respectively, with a maximal 2.58 log10 reduction in H1N1 viral load. Conclusions: High-dose CpG-TIV demonstrates strong immunogenicity and robust dose-dependent homologous and heterologous cross-protection in ferrets. The combination of a CpG adjuvant and high-dose antigen broadens protection against drifted influenza viruses, overcoming the narrow coverage of conventional vaccines. These data support further clinical development of this broad-spectrum influenza vaccine candidate. Full article

Outbreaks of avian infectious bronchitis virus (IBV) often occur in vaccinated flocks. The antibody-dependent enhancement (ADE) has been proposed as a potential mechanism underlying coronavirus vaccine failure. However, this hypothesis has yet to be substantiated in flocks. This study demonstrates ADE occurrence in IBV (gamacoronavirus) in vitro and in vivo. Using the SPF chicken host model, primary infection with an O-glycosylation-modified attenuated strain enhanced pathogenesis upon secondary homologous/heterologous virulent challenge, increasing morbidity/mortality (≥30%), pathological lesions, and viral loads. Notably, sequentially attenuated infections also induced ADE, suggesting live attenuated vaccine risks. The immune serum raised against the O-glycosylation-modified attenuated strain was also pre-mixed with the challenge strain, and the mixtures were then inoculated into target cells, non-susceptible macrophage cells, or a co-culture of both cell types. The serum-virus complexes replicated poorly in macrophages, yet immune cells amplified the expression of inflammatory factors and ADE-mediated viral replication in target cells, indicating a significant promoting role of immune cells in this process. The concentrations of complement component C3 and neutralizing antibodies in the immune serum were also measured, and results showed that the induction of this ADE is associated with high complement component C3 and low neutralizing antibody titers. These findings highlight risks for vaccines and antibody-based therapeutic strategies of coronavirus infection. Full article

Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis, a widespread zoonotic disease leading to serious public health concerns and economic losses to animal husbandry. Currently, highly effective vaccines against toxoplasmosis remain unavailable. This study aimed to investigate the safety, immunogenicity, and protective efficacy of the gra47-deficient mutant strain PruΔgra47 as a live-attenuated vaccine candidate. We evaluated the virulence of PruΔgra47 in a mouse model, determined the optimal immunization dose, and measured serum antibody levels and cytokine profiles. Then, mice immunized with PruΔgra47 were challenged with different T. gondii strains to assess protection against acute and chronic infection. PruΔgra47 displayed significantly attenuated virulence and its ability to form cysts was weakened. Vaccination with 5 × 10⁶ tachyzoites elicited predominantly Th1-skewed immune responses. Immunization with PruΔgra47 provided complete protection against challenge infection with relatively low-virulent PYS strain and homologous Pru strain, prolonged survival against the highly virulent RH strain, and achieved a 90% survival rate with reduced brain cyst burden under chronic challenge. In conclusion, PruΔgra47 is relatively safe and immunogenic in the murine model, and is worth being evaluated in food-producing animals and cats. Full article

Background/Objectives: African swine fever (ASF) is a highly lethal disease of domestic pigs and wild suids that continues to cause substantial economic losses worldwide. Despite recent progress in live attenuated ASF vaccine development, evidence supporting durable protection under repeated exposure conditions representative of endemic settings remains limited. Here, we assessed the long-term safety and protective efficacy of a live attenuated ASFV-G-ΔI177L/ΔLVR vaccine using a repeated-challenge experimental design intended to model re-exposure in ASF-endemic regions. Methods: Vaccinated pigs were subjected to homologous virulent ASF virus challenges at multiple intervals, including repeated challenges (three sequential inoculations) and single challenges administered at 8 and 12 weeks post-vaccination. Results: Across all challenge regimens, vaccinated animals survived and remained clinically healthy, including those receiving three challenges, supporting sustained protection under repeated exposure pressure. Animals challenged at 8 or 12 weeks post-vaccination likewise exhibited complete survival, indicating maintained efficacy through at least 12 weeks. No vaccine-associated adverse clinical outcomes were detected over the study period, and post-challenge viral shedding was minimal. Conclusions: Overall, these data demonstrate that the candidate live attenuated ASF vaccine provides excellent protective efficacy and confers sustained protection against homologous ASF virus infection. This result is expected to be equally applicable under repeated exposure conditions in regions with unstable ASF biosecurity, making it a sufficiently promising model experiment for field application in ASF epidemic areas. However, this is still a vaccine variant, and further studies are planned to evaluate its genomic stability and transmissibility. Full article

Senecavirus A (SVA) is a newly emerging picornavirus associated with porcine idiopathic vesicular disease and sudden death in newborn piglets. Currently, no specific vaccines or drugs are available against SVA, highlighting the importance of investigating the immunological characteristics of its key proteins. The VP1 protein of SVA exhibits strong immunogenicity and high sequence conservation, and it is indispensable to the viral life cycle. In the present study, a monoclonal antibody (mAb) against VP1 was generated. A series of truncated VP1 proteins was then expressed to precisely map the epitope recognized by this mAb. The minimal reactive unit was identified as ¹⁶DTDFSGELA²⁴. Homology analysis further revealed that this epitope is conserved among different SVA isolates deposited in GenBank. Moreover, AlphaFold prediction, along with PyMOL (Version 3.0.3) and GETAREA analyses, reveals that this epitope resides in the α-helix and loop regions of the three-dimensional structure of the VP1 protein and is surface-exposed. Collectively, these findings indicate that the mAb and its recognized epitope represent valuable tools for investigating SVA etiology and VP1 protein function. Full article