Aug 2026· Frontiers in Veterinary Science· Vol 13· 0 citations· 32 references
Medicine
TL;DR
It is demonstrated that the developed subunit vaccine induces robust and durable humoral and cellular immune responses and represents a promising candidate for the prevention and control of aMPV subtype B infection in poultry.
Abstract
Objectives aMPV subtype B remains one of the major respiratory pathogens affecting commercial poultry worldwide despite the widespread implementation of vaccination programs. In the present study, epidemiological surveillance, virus isolation, and immunogenic evaluation of a newly developed subunit vaccine against aMPV subtype B were conducted. Methods Clinical samples, representing 115 chicken flocks from 16 commercial poultry farms located in three regions of Kazakhstan, were screened for aMPV subtype B using multiplex real-time RT-PCR. Approximately 20% of the samples collected during the spring season tested positive for aMPV, with subtype B predominating among the detected strains. Phylogenetic analysis revealed that the identified viruses clustered within subtype B and were closely related to vaccine-like strains while forming a distinct monophyletic group. For virus isolation, field samples that tested negative for IBV, NDV, ILTV, and Mycoplasma gallisepticum were propagated in Vero cells. Cytopathic effects were observed after 3–4 passages, and one isolate, designated Ck/B/Kaz/256/25, was successfully recovered from a laying hen. The isolate was subsequently confirmed by electron microscopy, gel electrophoresis, and was used for the development of a subunit vaccine. Results The immunogenicity of the vaccine was evaluated in chickens following a prime–boost immunization regimen and compared with that of a commercial live vaccine. The subunit vaccine induced 100% seroconversion by day 14 post-vaccination and elicited significantly higher IgG and virus-neutralizing antibody titers than the live vaccine. Neutralizing antibody titers were 8-fold higher after primary immunization and 16-fold higher after booster immunization compared with those in the live vaccine group. Furthermore, the subunit vaccine significantly enhanced the expression of IFN-γ and IL-4, indicating activation of both Th1- and Th2-mediated immune responses. Conclusion Overall, these findings demonstrate that the developed subunit vaccine induces robust and durable humoral and cellular immune responses and represents a promising candidate for the prevention and control of aMPV subtype B infection in poultry.
Canine Parvovirus type 2 (CPV-2) is a significant pathogen in dogs, inducing fatal hemorrhagic enteritis. Earlier studies confirmed the circulation of all three CPV variants in Iran. Vaccination is the primary preventive strategy against CPV infection; however, emerging substitutions challenge the effectiveness of conventional vaccines. This study aimed to detect and genetically characterize circulating variants of CPV-2 from clinically infected dogs in Iran and subsequently in silico design a multi-epitope vaccine against detected variants. Forty rectal samples were collected from clinically suspected to CPV infection and confirmed utilizing PCR assay. CPV variants were distinguished using established primer sets, with CPV-2c further confirmed by restriction analysis. The VP2 gene of positive CPV samples was used to construct a Maximum-Likelihood phylogenetic tree. Subsequently, using several bioinformatics tools, specific, non-toxic, non-allergenic, and highly antigenic B-cell and T-cell epitopes from amino acid sequences of detected variants were identified and chosen for vaccine construction. AAY, GPGPG, KK and EAAK linkers were employed to join the selected epitopes, the TLR 4-agonist and the PADRE adjuvant to achieve the final vaccine construct. The designed vaccine construct was in silico evaluated in terms of physicochemical properties, 3D structures and ability to interact with MHC II and I immune receptors.
Among the 15 positive samples, 10 were sequenced and submitted to NCBI (MW924816.1 and OL330974.1–OL330982.1), and a phylogenetic tree was constructed. The multi-epitope vaccine construct was successfully designed and predicted to exhibit stability, solubility, and a hydrophilic profile. Also, modeled 3D structure of designed vaccine and Ramachandran analysis showed 90.99% of residues in the favored regions, indicating high quality and reliable structure modeling. Molecular docking analysis demonstrated a strong interaction potential between the vaccine construct and the MHC class I and II canine immune cell receptors, exhibiting a docking score of -273.99 and − 318.5 with confidence score of 0.92 and 0.96 respectively.
Overall, we have identified circulating CPV variants in Iran and computationally designed a specific multi-epitope vaccine construct against these variants potentially applicable for targeting the canine immune receptors. Further experimental investigations are required to confirm the potential application of this newly designed multi-epitope protein for development of effective vaccines against the CPV infection.
Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive condition in chickens, caused by the infectious bursal disease virus (IBDV). The recent emergence of novel variant IBDV (nVarIBDV) poses a significant threat to the global poultry industry. However, currently available vaccines provide only limited protection against nVarIBDV strains. In this study, a recombinant avian metapneumovirus subtype B (aMPV/B) expressing the nVarIBDV VP2 gene (rLN16A-nVarVP2) was successfully rescued by inserting the gene between the G and L genes of the attenuated aMPV/B strain LN16-A. Immunofluorescence and Western blotting analyses confirmed stable VP2 expression in vitro. Further evaluation showed that VP2 insertion did not alter the growth kinetics of the parental virus, and the expression remained stable after 20 serial passages. A single immunization with rLN16A-nVarVP2 elicited robust humoral and cellular immune responses in specific pathogen-free chickens, inducing high levels of neutralizing antibodies against both nVarIBDV and aMPV/B, as well as Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-6) cytokines. Moreover, rLN16A-nVarVP2 conferred complete (100%) protective efficacy against both nVarIBDV and aMPV/B, fully prevented bursal atrophy and clinical signs associated with aMPV/B infection, and markedly decreased bursa viral load (nVarIBDV) and turbinate viral shedding (aMPV/B). Our findings suggest that rLN16A-nVarVP2 represents a promising live attenuated bivalent vaccine candidate for preventing infections caused by nVarIBDV and aMPV/B.
Newcastle disease remains a major constraint on poultry production worldwide, with recurrent outbreaks reported even in vaccinated flocks. In Pakistan, the persistence of Newcastle disease raises concerns about the ongoing evolution of Newcastle disease virus and the effectiveness of existing vaccines. This study aimed to investigate the molecular epidemiology and genetic characteristics of Newcastle disease virus circulating in commercial poultry farms in Punjab Province, Pakistan. A cross-sectional molecular surveillance study was conducted from May to November 2025 across five major poultry-producing districts. Clinical samples consisting of pooled oropharyngeal swabs and tissue homogenates were collected from affected flocks and organized into 100 pooled samples obtained from 25 commercial farms experiencing severe disease characterized by respiratory and neurological signs with high mortality (>40%), despite routine vaccination, and clinically suspected of Newcastle disease. Reverse transcription–polymerase chain reaction targeting the fusion (F) gene detected Newcastle disease virus in 91.1% of the prioritized tissue homogenates and oropharyngeal swabs from the sampled flocks. Based on RNA quality, successful amplification of the target region, geographic distribution, and epidemiological relevance, four representative reverse transcription–polymerase chain reaction-positive clinical samples were selected for complete F-gene sequencing. All sequenced samples possessed the polybasic cleavage site motif 112RRQKR↓F117, indicating a molecular signature consistent with a velogenic pathotype. Phylogenetic analysis consistently clustered the Newcastle disease virus sequences within Class II, further divided into sub-genotypes VII.2 and VII.1.1, indicating the co-circulation of at least two endemic lineages. Comparative sequence analysis revealed substantial genetic divergence between field samples and commonly used vaccine strains, with an average nucleotide difference of 10.7% relative to the LaSota vaccine strain. Additionally, several amino acid substitutions were identified in known neutralizing epitopes, along with evidence of positive selection at antigenically significant sites. These findings demonstrate the endemic circulation of genetically diverse genotype VII Newcastle disease virus strains in Punjab and provide molecular insights into potential antigenic divergence between vaccine and field strains, highlighting the need for further functional efficacy studies. Continuous molecular surveillance and the reassessment of vaccination strategies are essential for achieving effective and sustainable control of Newcastle disease in Pakistan.
Cite this article as: Naveed, M. T., Gulzar, M. W., Ahmad, M. M., Azeem, M. W., Ali, M. A., Anwar, M. N., Riaz, S., Shah, M. S., & Habib, M. (2026). Molecular and phylogenetic evidence of endemic genotype VII Newcastle disease virus circulation and vaccine mismatch in Punjab, Pakistan. Acta Veterinaria Eurasia, 52, 0072, doi: 10.5152/actavet.2026.26072.
M. Naveed, Muhammad Wasif Gulzar, Muhammad Mubeen Ahmad et al.· Acta Veterinaria Eurasia· 0 citations
The findings suggest that internal gene backbone compatibility may influence vaccine immunogenicity and warrant further validation to support a refined vaccine design strategy for H7N9 and potentially other avian influenza subtypes.
Yi Liu, Meng-Yuan Bai, Tao Zhang et al.· Microorganisms· 0 citations
Infectious bursal disease virus (IBDV) causes severe immunosuppression diseases in poultry, resulting in substantial economic losses for the global poultry industry. This study aimed to characterize circulating IBDV strains in Egyptian chicken flocks and assess their potential impact on poultry health and production. We conducted a passive surveillance study by collecting bursal samples from a total of 30 flocks, including commercial broilers, layers, and Baladi chickens, across nine Egyptian governorates in 2025. These flocks exhibited clinical signs of depression, along with kidney and bursal lesions indicative of IBDV infection. Pooled bursal homogenates were tested using RT-PCR with VP2-specific primers, revealing that 20 flocks (66.6%) tested positive for IBDV. Field and vaccine strains were distinguished by phylogenetic clustering of the VP2 hypervariable region (HVR) sequences against reference vaccine strains (D78, Winterfield 228, VAXXITEK), strains that cluster closely with reference vaccine strains are classified as vaccine-origin, whereas field strains are distinct branches indicate circulating wild-type or vvIBDV. Ten representative positive samples were inoculated in specific pathogen-free embryonated chicken eggs (SPF-ECE). The inoculated embryos exhibited haemorrhage, skull swelling, and liver necrosis with a pale-yellow appearance, along with congestion and thickening of the chorioallantoic membrane (CAM). Phylogenetic analysis of the partial VP2 gene classified the isolates according to the unified genogroup nomenclature: the majority of isolates belonged to the A3 (vvIBDV) genogroup, while one isolate clustered within the A2d (nVarIBDV) genogroup. These findings highlight the co-circulation of both virulent and variant IBDV strains in Egyptian chicken flocks, complicating disease control and demanding a continued surveillance and periodic re-evaluation of vaccination programs.
Amr H. Abd El-Fatah, Bouran Salama, Mariam O. Elnahhas et al.· Scientific Reports· 0 citations
Brucellosis is one of the most severe Class B infectious diseases prevalent in the agricultural and pastoral areas of northern China. Current attenuated live vaccines (e.g., M5, S19) have defects such as residual virulence, causing abortion in pregnant animals, and the inability to differentiate between natural infection and vaccination (DIVA). Relying on the ABSL-3 laboratory of the Inner Mongolia Center for Disease Control and Prevention, this study established a chronic infection model in C57BL/6J mice using the virulent strain Brucella melitensis M16. Single-cell transcriptome sequencing (10x Genomics) was employed to map the heterogeneity of splenic immune cells. Whole-genome scanning and pangenomic analysis were performed on four strains with different virulence levels (M16, 544 A, M5, 104 M) using second-generation sequencing. Membrane/secreted proteins unique and conserved in virulent strains were screened as candidate antigens, prepared via prokaryotic expression systems, and their humoral and cellular immune levels were detected by indirect ELISA and flow cytometry. A stable chronic infection model was successfully constructed (bacterial load Log10 CFU > 4.5). Single-cell sequencing yielded 45,231 cells, annotated into 12 immune cell subpopulations, revealing significant expansion of Effector CD4 + T cells (P < 0.01) and high expression of the Ifng gene. Pangenomic analysis identified three candidate antigens (BMEI0021, BMEI1943, BMEI0367) that are 100% conserved in virulent strains but absent in the vaccine strain M5. Immunogenicity assays showed that BMEI1943 induced high levels of IgG2a subtype antibodies (titer Log2 13.45 ± 0.52) and IFN-γ + CD4 + T cell responses (frequency 15.80% ± 2.10%). Through multi-omics integration analysis, this study successfully identified a novel candidate antigen, BMEI1943, with strong Th1-type immunogenicity, providing an experimental basis for the development of safe and efficient subunit vaccines against brucellosis.
Zhiheng Dong, Sha Li, Jiarong Guo et al.· Brazilian Journal of Microbi...· 0 citations
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