Search Results (176)

Background: Fowl typhoid (FT), a septicemic infection caused by Salmonella Gallinarum (SG), and H9N2 avian influenza are two economically important diseases that significantly affect the global poultry industry. Methods: We exploited the live attenuated Salmonella Gallinarum (SG) mutant JOL3062 (SG: ∆lon ∆pagL ∆asd) as a delivery system for H9N2 antigens to induce an immunoprotective response against both H9N2 and FT. To enhance immune protection against H9N2, a prokaryotic and eukaryotic dual expression plasmid, pJHL270, was employed. The hemagglutinin (HA) consensus sequence from South Korean avian influenza A virus (AIV) was cloned under the Ptrc promoter for prokaryotic expression, and the B cell epitope of neuraminidase (NA) linked with matrix protein 2 (M2e) was placed for eukaryotic expression. In vitro and in vivo expressions of the H9N2 antigens were validated by qRT-PCR and Western blot, respectively. Results: Oral immunization with JOL3121 induced a significant increase in SG and H9N2-specific serum IgY and cloacal swab IgA antibodies, confirming humoral and mucosal immune responses. Furthermore, FACS analysis showed increased CD4+ and CD8+ T cell populations. On day 28 post-immunization, there was a substantial rise in the hemagglutination inhibition titer in the immunized birds, demonstrating neutralization capabilities of immunization. Both IFN-γ and IL-4 demonstrated a significant increase, indicating a balance of Th1 and Th2 responses. Intranasal challenge with the H9N2 Y280 strain resulted in minimal to no clinical signs with significantly lower lung viral titer in the JOL3121 group. Upon SG wildtype challenge, the immunized birds in the JOL3121 group yielded 20% mortality, while 80% mortality was recorded in the PBS control group. Additionally, bacterial load in the spleen and liver was significantly lower in the immunized birds. Conclusions: The current vaccine model, designed with a host-specific pathogen, SG, delivers a robust immune boost that could enhance dual protection against FT and H9N2 infection, both being significant diseases in poultry, as well as ensure public health. Full article

Background: Annual influenza vaccine updates target viral drift, but immune responses may be biased by original antigenic sin (OAS). Few studies have explored this across closely related strains. This study examines how OAS shapes responses to sequential influenza variants in the context of seasonal vaccination. Methods: We conducted a prospective, longitudinal study to assess the humoral immune response to the 2023–2024 seasonal influenza vaccine containing the A/Victoria/4897/2022 (H1N1) strain. Bioinformatic analyses compared the hemagglutinin (HA) sequences of A/Victoria/4897/2022 and the antigenically related A/Victoria/2570/2019 strain. B-cell epitopes were mapped with BepiPred-3.0 and BepiBlast, and their physicochemical properties analyzed via accessibility, β-turns, flexibility, and hydrophilicity. Antibody responses were measured pre- and 28 days post-Vaxigrip Tetra vaccination in young (18–35) and older (>65) adults, stratified by cytomegalovirus (CMV) serostatus. HA sequences showed >97% identity, with variations mainly in the globular head. Predicted B-cell epitopes overlapped variable sites, suggesting possible immune escape. Despite having been vaccinated against the 2022 strain, serology showed higher antibody titers against the 2019 HA strain in all participants. This pattern suggests a potential antigen imprinting effect, though confirmation awaits further analysis. Age groups differed: older adults showed greater variability, while younger CMV+ individuals tended toward stronger 2019 HA responses. Conclusions: These findings suggest a complex interplay of factors shaping immune responses, though the imprinting effect and the potential role of CMV warrant further exploration in larger, more focused studies. Full article

Understanding how sample type may influence the probability of influenza A virus (IAV) sequencing and isolation success can help improve the use of diagnostic tests and refine surveillance strategies in swine populations. The objective of this study was to evaluate the probability of success for IAV hemagglutinin (HA) and neuraminidase (NA) Sanger sequencing and virus isolation in Madin–Darby Canine Kidney (MDCK) cells across different porcine sample types submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2018 to 2024. Antemortem and postmortem sample types were selected and analyzed based on reverse transcription real-time polymerase chain reaction (RT-rtPCR) cycle threshold (Ct) values. The Ct values corresponding to 95%, 75%, and 50% probabilities of sequencing or virus isolation success were determined for each sample type. For antemortem samples, a 95% probability of success for HA Sanger sequencing on nasal swabs exhibited a Ct value of 27.8 from 1046 samples and 23.6 for NA sequencing based on 66 nasal swabs. Using oral fluids, HA and NA Sanger sequencing success was at Ct values of 27.3 from 3446 samples and 22.1 from 137 samples, respectively. For postmortem samples, lung tissue had the highest number of sequences for the HA and NA, with Ct values of 25.7 and 21.5, respectively. For a 95% probability of successful virus isolation, nasal swabs demonstrated a Ct value of 21.1 from 647 samples, while lungs had a Ct value of 18.7 from 5892 samples. This study determined that nasal swabs and lung tissue had the highest probability of IAV gene sequencing and virus isolation success, while oral fluids, a common swine diagnostic sample type that is easy to collect and welfare-friendly, can be effective for gene sequencing when using lower IAV RT-rtPCR Ct values, i.e., ≤27.3. These results provide practical expectations for successful IAV HA and NA gene sequencing and virus isolation at 95%, 75%, and 50% probabilities based on sample type and RT-rtPCR Ct values to improve diagnostic testing strategies in swine populations. Full article

The Highly Pathogenic Avian Influenza Virus (HPAIV) H5 clade 2.3.4.4b has caused severe outbreaks in domestic and wild birds worldwide since its emergence in 2014, and especially since 2020, with outbreaks in Europe and North America. The introduction of the virus into South America was reported for the first time in Colombia in October 2022, followed by outbreaks in other South American countries affecting poultry, wild birds, mammals, and humans. In this study, a phylogenetic and mutation analysis of the hemagglutinin (HA) gene of HPAIV H5N1 2.3.4.4b viruses isolated in South America was performed to analyze its evolution and its transmission and zoonotic potential. The analysis shows an increase in the viral effective population size between April and June 2022, which was followed by multiple outbreaks of HPAIV H5N1 clade 2.3.4.4b in South America. Moreover, the virus variants evolved from a recent common ancestor estimated to have existed in June 2017. The mean rate of evolution of the HA gene was 6.95 × 10⁻³ substitutions per site per year, and the sequence analysis of HA identified a mutation (D171N) located at antibody binding sites and viral oligomerization interfaces, with implications for immune response evasion and new host species infection. Additionally, viral strains from South America share the substitutions L104M, T156A, P181S, and V210A, compared to the vaccine strain A/chicken/Ghana/AVL763/2021. Understanding the dynamics of viral evolution and transmission is essential for effective prevention strategies to mitigate future outbreaks. Full article

Background/Objectives: Developing next-generation mRNA-based seasonal influenza vaccines remains challenging, primarily because of the relatively low immunogenicity of influenza B hemagglutinin (HA) antigens. We describe a systematic vaccine development strategy that combined vector and antigen design optimization. Methods: Novel untranslated region (UTR) sequences and a hybrid poly(A) tail were used to increase plasmid stability and mRNA expression. Fusion proteins containing HA antigens linked by T4 foldon domains were engineered to enhance the immune responses against influenza B HA antigens and to permit the expression of multiple HA ectodomains from a single mRNA species. The vaccine performance was verified in a traditional encapsulated lipid nanoparticle (LNP) formulation that requires long-term storage at temperatures below −15 °C as well as in a proprietary thermo-stable LNP formulation developed for the long-term storage of the mRNA vaccine at 2–8 °C. Results: In preclinical studies, our next-generation seasonal influenza vaccine tested alone or as a combination influenza/COVID-19 mRNA vaccine elicited hemagglutination inhibition (HAI) titers significantly higher than Fluzone HD, a commercial inactivated influenza vaccine, across all 2024/2025 seasonal influenza strains, including the B/Victoria lineage strain. At the same time, the combination mRNA vaccine demonstrated superior neutralizing antibody titers to 2023/2024 Spikevax, a commercial COVID-19 comparator mRNA vaccine. Conclusions: Our data demonstrate that the multimerization of antigens expressed as complex fusion proteins is a powerful antigen design approach that may be broadly applied toward mRNA vaccine development. Full article

Canine distemper virus (CDV) is a highly contagious pathogen and causes a fatal systemic disease in domestic dogs and wild carnivores worldwide. Despite CDV infections being monitored globally, studies on CDV in Vietnam seem to be limited. This study, therefore, investigated the epidemiological, clinical, and molecular characteristics of CDV in the Mekong Delta (MD) region of Vietnam. A total of 6687 ocular/nasal swabs were collected from CDV-suspected dogs across seven cities/provinces. CDV infection was detected in 6.19% (414 dogs) of suspected dogs using a commercially available rapid kit, with infection associated with age, roaming status, and vaccination status. Hematological and blood biochemical analysis of CDV-infected dogs revealed anemia, leukopenia, neutrophilia, thrombocytopenia, a slight increase in aspartate aminotransferase (AST) levels, and a significant increase in blood urea nitrogen (BUN) levels. Molecular characterization of partial hemagglutinin (H) and fusion (F) genes exhibited high nucleotide and amino acid homology with the Asia-1 genotype. Phylogenetic analysis confirmed that the field sequences were clustered into the Asia-1 genotype together with the neighboring countries. These findings provide important insights into the current epidemiological, clinical, and molecular features of CDV circulating in Vietnam. Full article

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses threaten animal and human health worldwide. The first documented cases in the Middle East were reported in 2005; however, despite extensive phylogenetic studies, there is limited information on the transmission dynamics of the virus within this region. We analyzed HA and NA gene sequences from various hosts to address this gap and to understand the virus’s spread and evolution in the Middle East. We hypothesized that H5N1 transmission exhibits host-specific or geographically influenced clade structures in this region. This study traced transmission pathways of HPAI A/H5N1 through a phylogenetic and amino acid sequence analysis of HA and NA gene segments from isolates across different hosts in Middle Eastern countries, using the MUSCLE algorithm for alignments and MEGA11 software for phylogenetic analysis. Sequences were selected from NCBI’s virus database based on geographic and host diversity, including those from birds, humans, and other mammals, and were collected at different time points, predominantly after the early 2000s. An amino acid phylogenetic tree was also constructed to examine the conservation of key HA and NA protein residues, identifying distinct clades linked to specific countries and host species, suggesting a possible interspecies transmission and cross-border spread distinct between Egypt and neighboring countries. These findings underscore the role of migratory birds in regional transmission and point to the need for more targeted surveillance and biosecurity efforts, offering more genomic insights into the spread of HPAI A/H5N1 and contributing valuable information for future prevention strategies. Full article

Background/Objetives: Nanobodies (VHH) have become an excellent tool for diagnosis, therapy, and research since VHH shows a high capability of recognizing and neutralizing antigens. VHHs are highly soluble and stable at high temperatures, and in the presence of chaotropic agents, they offer significant advantages over other biological therapeutic agents. This study aimed to identify and humanize VHH fragments with neutralizing potential against the influenza A/H1N1 virus. Methods: A library of VHH antibody fragments was produced by phage display technique against an inactivated influenza A/H1N1 vaccine. Three VHH sequences were selected and humanized. Specifically, the recognition capacity of the antibodies denominated 2-C10 and 2-C10H was confirmed by ELISA and western blot (WB), as well as their microneutralization capacity in a cellular model, suggesting their potential therapeutic use in patients infected with the influenza A/H1N1 virus. Molecular docking assays were used to support the mechanism of viral inhibition. Results: The VHHs 2-C10 and 2-C10H showed specific recognition of influenza A/H1N1 antigens by ELISA and Western Blot and demonstrated neutralizing activity in vitro. The optimal VHH, 2-C10H, showed 75% neutralization capacity at a concentration of 1.56 μg/mL against the A/H1N1 viral strain, potentially through the inactivation of hemagglutinin protein, a phenomenon supported by molecular docking assays. Conclusions: This study presents a strategic approach to identify VHH candidates that may be useful for diagnosing and potentially treating patients already infected by the A/H1N1 virus, as it may reduce the severity of their symptoms. Full article

The cross-species transmission of influenza viruses represents a critical link in the pandemic of zoonotic diseases. This mechanism involves multi-level interactions, including viral genetic adaptability, host–receptor compatibility, and ecological drivers. Recent studies have highlighted the essential role of mutations in hemagglutinin and neuraminidase in overcoming host barriers, while elucidating the differences in the distribution of host sialic acid receptors. Furthermore, the “mixer” function of intermediate hosts, such as pigs, plays a significant role in viral redistribution. Advances in high-throughput sequencing and structural biology technologies have gradually resolved key molecular markers and host restriction factors associated with these viruses. However, challenges remain in understanding the dynamic evolutionary patterns of virus–host interaction networks, developing real-time early warning capabilities for cross-species transmission, and formulating broad-spectrum prevention and control strategies. Moving forward, it is essential to integrate multidisciplinary approaches to establish a multi-level defense system, leveraging the ‘One Health’ monitoring network, artificial intelligence prediction models, and new vaccine research and development to address the ongoing threat of cross-species transmission of influenza viruses. This paper systematically reviews the research progress and discusses bottlenecks in this field, providing a theoretical foundation for optimizing future prevention and control strategies. Full article

Influenza is a communicable disease caused by RNA viruses. Strains A (affecting animals, humans), B (affecting humans), C (affecting rarely humans and pigs), and D (affecting cattle) comprise a variety of substrains each. Influenza A strain, affecting both humans and animals, is considered the most infectious, causing pandemics. There is an emerging need for the accurate classification of the different influenza A virus (IAV) subtypes, elucidating their mode of infection, as well as their fast and accurate diagnosis. Notably, in recent years, oligomeric sequences (words) that are present in the pathogen genomes and entirely absent from the host human genome were suggested to provide robust biomarkers for virus classification and rapid detection. To this end, we performed updated phylogenetic analyses of the IAV hemagglutinin genes, focusing on the sub H1N1 and H5N1. More importantly, we applied in silico methods to identify minimum length “words” that exist consistently in the IAV genomes and are entirely absent from the human genome; these sequences identified in our current analysis may represent minimal signatures that can be utilized to distinguish IAV from other influenza viruses, as well as to perform rapid diagnostic tests. Full article

The genetic diversity of influenza A virus is a major obstacle that makes vaccine effectiveness variable and unpredictable. Objectives: Current vaccines induce strain-specific immunity that oftentimes fail to protect against divergent strains. Our previous research explored synthetic centralized consensus (CC) vaccines to minimize immunogen-strain divergence and focused on the viral glycoprotein hemagglutinin. Methods: Recently, emerging evidence of neuraminidase (NA)-mediated immunity has shifted vaccine strategies, prompting our development of a CC NA type 1 (N1CC) vaccine based on ancestral N1 sequences and delivered using a human adenovirus type 5 vector Results: The N1CC vaccine elicited antibody responses with NA inhibition activity and induced NA-specific T-cell responses. In lethal influenza challenge models, N1CC fully protected mice from death against human, swine, and avian influenza H1N1 and H5N1 strains. Conclusions: These findings support NA as a protective immunogen and demonstrate the power and efficacy of a centralized consensus NA design. Full article

Highly pathogenic avian influenza (HPAI) H5 viruses have been found to have a substantial geographic distribution since they were first reported in Guangdong Province, China. The emergence of new genotypes threatens the poultry industry and human health worldwide. Here, we report five HPAI H5N1 variants isolated from Anser indicus in Yunnan Province, China. A phylogenetic analysis of the hemagglutinin (HA) gene showed that all isolates belong to the highly pathogenic H5 clade 2.3.4.4b and formed two distinct genetic clusters. Bayesian phylogenetic analysis also revealed that the viruses were initially disseminated from wild birds to Anser indicus, implying that infected birds most likely contributed to viral transmission in the region. Genomic sequence analysis revealed several amino acid substitutions, also implying that the infected birds contributed to the spread of the virus throughout the region. Substitutions in the HA glycoprotein increased the virus’s binding affinity to human α-2,6 sialic acid residues. Substitutions in the PB1, PA, and PB2 motifs increased viral polymerase activity and replication in hosts, whereas substitutions in the NP, M1, and NS motifs increased viral pathogenicity in chickens and mice. Full article

Influenza poses a serious threat to both individual and public health. This study aimed to investigate the virological and epidemiological characteristics of influenza infections and to explore the genetic diversity of the circulating influenza viruses. In total, 1886 nasopharyngeal specimens from patients with acute respiratory illnesses were tested against 13 respiratory viruses using a multiplex real-time PCR. Whole-genome sequencing, phylogenetic, and amino acid analyses of representative influenza strains were performed. At least one respiratory virus was detected in 869 (46.1%) patients; 87 (4.6%) were co-infected with two or three viruses. Influenza A(H1N1)pdm09 was the most prevalent virus (16.1%), followed by rhinoviruses (8.1%) and RSV (6.7%). Hemagglutinin (HA) genes of the 74 influenza A(H1N1)pdm09 viruses were categorized in subclades C.1.8, C.1.9, and C.1 within clade 5a.2a and D1, D.2, and D.3 within clade 5a.2a.1. The A(H3N2) viruses analyzed belonged to clade 2a.3a.1, subclades J.2 and J.1. The sequenced B/Victoria lineage viruses fell into clade V1A.3a.2, subclades C.5.6 and C.5.7. Amino acid substitutions in most viral proteins were identified compared with the vaccine strains, including in the HA antigenic sites. This study demonstrated the dominant distribution of the influenza A(H1N1)pdm09 virus among the respiratory viruses studied and the genetic diversity of the circulating influenza viruses. Full article

Despite annual vaccines, Influenza B viruses (IBVs) continue to cause severe infections and have a significant impact and burden on the healthcare system. Improving IBV vaccine effectiveness is a key focus, with various strategies under investigation. In this research, we used a computational design to select wildtype sequences for a multivalent viral-vectored vaccine (rAd-Tri-Vic) targeting the Victoria-like (Vic) hemagglutinin (HA) protein. In mouse models, the vaccine induced strong antibody and T cell responses, providing complete protection against both lineage-specific and cross-lineage (Yamagata-like) lethal challenges. The immune responses remained robust for up to six months, which demonstrated sustained protection. These results highlight the potential of HA-targeted multivalent vaccines to enhance the IBV efficacy and address protection against antigenically diverse IBV strains. Full article

Vaccination is a common influenza A virus (IAV) control strategy for pigs. Vaccine efficacy depends on strain cross-protection and effective vaccination program implementation. We evaluated a multi-faceted IAV vaccination strategy which included (a) monthly surveillance of pigs at weaning, (b) selection of epidemiologically relevant strains from farms under surveillance, (c) updating IAV strains in custom-made vaccines, and (d) seasonal mass vaccination with custom-made vaccines given to sows in 35 farrow-to-wean farms within an integrated swine farm system. Reduction of IAV in pigs from vaccinated sows was determined by monthly monitoring of farms for 30 months by IAV rRT-PCR (PCR) testing of nasal wipes collected from litters of piglets at weaning. Hemagglutinin (HA) nucleotide and amino acid (AA) sequence homology of the circulating and vaccine strains was determined by pairwise alignment and AA comparison at antigenic sites. Of the 35 farms monitored, 28 (80%) tested positive at least once, and 481 (5.75%) of 8352 PCR tests were IAV positive. Complete HA sequences were obtained from 54 H1 (22 H1-δ_1B.2.1, 28 H1-γ_1A.3.3.3, and 4 H1-pdm_1A.3.3.2 clades) and 14 H3 (12 IV-A 3.1990.4.1 and 2 IV-B 3.1990.4.2 clades) circulating IAV strains. During the study, custom-made vaccines were updated three times (eight strains total) and administered to sows at five distinct time periods. The HA AA similarity between vaccine and circulating strains ranged from 95% to 99%; however, the 0 to 71% similarity at HA antigenic sites prompted the vaccine updates. Herd IAV prevalence decreased from 40% (14/35) to 2.9% (1/35), accompanied by a numerical reduction in IAV-positive samples post-vaccination. Our results support having a comprehensive approach to controlling influenza in swine herds that includes surveillance, vaccination, and careful program implementation to reduce IAV in pigs. Full article