Ligilactobacillus salivarius UMNPBX2 Cell-Free Extract Has Antiviral Effects on H4N6 Low-Pathogenic Avian Influenza Virus Subtype in Madin–Darby Canine Kidney Cell Line and Embryonated Chicken Eggs
Source abstract
Abstract
Avian influenza (AI) is a highly infectious disease affecting birds. Some strains of AI virus (AIV) have zoonotic potential, posing a threat to humans. The H4N6 subtype is a low-pathogenic virus and causes mild infection in poultry. However, it has raised increasing concern due to its capability to infect pigs and its high potential for reassortment when co-infected with other strains. This study investigated the antiviral properties of turkey-derived Ligilactobacillus salivarius UMNPBX2 (L. salivarius UMNPBX2) cell-free extract (CFE) using both cell culture and in ovo methods. We assessed the growth kinetics of the H4N6 virus and the cytotoxicity of L. salivarius UMNPBX2 CFE in Madin–Darby Canine Kidney (MDCK) cells. The results revealed that the CFE from the 109 CFU/mL L. salivarius UMNPBX2 overnight culture had strong antiviral activities (p < 0.05). The CFE obtained from 107 to 105 CFU/mL of overnight culture also significantly reduced viral replication (p < 0.05), demonstrating dose-dependent inhibition of viral replication. Additionally, CFEs did not increase pro-inflammatory cytokine gene expression of IL-1β and IL-6 but rather tended to decrease it (IL-6). The embryo survivability experiments revealed a significant dose-dependent increase in survival rate (p < 0.05). The findings of this study highlight the antiviral properties of L. salivarius UMNPBX2 CFE, which contain potential postbiotics against the H4N6 virus, warranting in vivo studies.
Source: crossref · Quality: baseline
Research context
OpenAlex topics: Influenza Virus Research Studies · Animal Virus Infections Studies
SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.
Affiliations
Department of Animal Science, University of Minnesota, Saint Paul, MN 55108, USA
Department of Veterinary Population Medicine, University of Minnesota, Saint Paul, MN 55108, USA
Funding
Minnesota Agricultural Experiment Station · Minnesota Discovery, Research, and InnoVation Economy—Global Food Venture Graduate Professional Development (MnDRIVE-GFV) Fellowship
References (64)
Show source-provided references
1. (2024, September 13). CDC Bird Flu, Available online: https://www.cdc.gov/bird-flu/index.html.
2. World Health Organization (2025, September 01). Regional Office for the Western Pacific. 2024. Avian Influenza Weekly Update 2025. Available online: https://iris.who.int/handle/10665/380024.
3. Strategies for Enhancing Immunity against Avian Influenza Virus in Chickens: A Review10.1080/03079457.2022.2054309 ↗
4. Protection Afforded by Avian Influenza Vaccination Programmes Consisting of a Novel RNA Particle and an Inactivated Avian Influenza Vaccine against a Highly Pathogenic Avian Influenza Virus Challenge in Layer Chickens up to 18 Weeks Post-Vaccination10.1080/03079457.2019.1605148 ↗
5. Interactions between Lactobacilli and Chicken Macrophages Induce Antiviral Responses against Avian Influenza Virus10.1016/j.rvsc.2017.10.007 ↗
6. Majumder, R., Alam, M.B., Paudel, K.R., Ahmed, K.A., Devkota, H.P., Lee, S.-H., Hansbro, P.M., and Park, Y.-H. (2022). Anti-Influenza Virus Potential of Probiotic Strain Lactoplantibacillus plantarum YML015 Isolated from Korean Fermented Vegetable. Fermentation, 8.10.3390/fermentation8110572 ↗
7. Alqazlan, N., Alizadeh, M., Boodhoo, N., Taha-Abdelaziz, K., Nagy, E., Bridle, B., and Sharif, S. (2020). Probiotic Lactobacilli Limit Avian Influenza Virus Subtype H9N2 Replication in Chicken Cecal Tonsil Mononuclear Cells. Vaccines, 8.10.3390/vaccines8040605 ↗
8. Synbiotic Enhances Immune Responses against Infectious Bronchitis, Infectious Bursal Disease, Newcastle Disease and Avian Influenza in Broiler Chickens
9. Effects of Dietary Supplementation of Mannan-Oligosaccharide on Virus Shedding in Avian Influenza (H9N2) Challenged Broilers
10. Safety and Antiviral Activity of Essential Oil Against Avian Influenza and NewCastle Disease Viruses
11. Antiviral Activity of Lavandula angustifolia L. and Salvia officinalis L. Essential Oils against Avian Influenza H5N1 Virus
12. Raj, S., Matsuyama-Kato, A., Alizadeh, M., Boodhoo, N., Nagy, E., Mubareka, S., Karimi, K., Behboudi, S., and Sharif, S. (2023). Treatment with Toll-like Receptor (TLR) Ligands 3 and 21 Prevents Fecal Contact Transmission of Low Pathogenic H9N2 Avian Influenza Virus (AIV) in Chickens. Viruses, 15.10.3390/v15040977 ↗
13. Antiviral Drugs for the Treatment and Prevention of Influenza10.1007/s40506-017-0129-5 ↗
14. Drug Resistance in Influenza A Virus: The Epidemiology and Management10.2147/IDR.S105473 ↗
15. Evaluation of Leuconostoc mesenteroides YML003 as a Probiotic against Low-Pathogenic Avian Influenza (H9N2) Virus in Chickens: Probiotic against H9N2 Virus in Chickens10.1111/j.1365-2672.2012.05326.x ↗
16. Selective Enumeration of Lactobacillus Delbrueckii Ssp. Bulgaricus, Streptococcus Thermophilus, Lactobacillus Acidophilus, Bifidobacteria, Lactobacillus Casei, Lactobacillus Rhamnosus, and Propionibacteria10.3168/jds.S0022-0302(03)73821-1 ↗
17. Effect of Propionibacterium Freudenreichii on Salmonella Multiplication, Motility, and Association with Avian Epithelial Cells110.3382/ps/pew367 ↗
18. Comparative Study of Influenza Virus Replication in Vero and MDCK Cell Lines10.1016/j.jviromet.2004.03.011 ↗
19. Influenza: Propagation, Quantification, and Storage10.1002/9780471729259.mc15g01s29 ↗
20. Lugovtsev, V.Y., Melnyk, D., and Weir, J.P. (2013). Heterogeneity of the MDCK Cell Line and Its Applicability for Influenza Virus Research. PLoS ONE, 8.10.1371/journal.pone.0075014 ↗
21. Xue, J., Chambers, B.S., Hensley, S.E., and López, C.B. (2016). Propagation and Characterization of Influenza Virus Stocks That Lack High Levels of Defective Viral Genomes and Hemagglutinin Mutations. Front. Microbiol., 7.10.3389/fmicb.2016.00326 ↗
22. Rather, I.A., Kamli, M.R., Sabir, J.S.M., and Ali, S. (2022). Evaluation of Lactiplantibacillus plantarum KAU007 against Low-Pathogenic Avian Influenza Virus (H9N2). Pathogens, 11.10.3390/pathogens11111246 ↗
23. Spackman, E. (2020). Animal Influenza Virus: Methods and Protocols, Springer. Methods in Molecular Biology.10.1007/978-1-0716-0346-8 ↗
24. Use of Crystal Violet to Improve Visual Cytopathic Effect-Based Reading for Viral Titration Using TCID50 Assays
25. Krishna, V.D., Roach, E., Zaidman, N.A., Panoskaltsis-Mortari, A., Rotschafer, J.H., O’Grady, S.M., and Cheeran, M.C.-J. (2015). Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells. PLoS ONE, 10.10.1371/journal.pone.0138704 ↗
26. Characterization of MDCK Cells and Evaluation of Their Ability to Respond to Infectious and Non-Infectious Stressors10.1007/s10616-019-00360-z ↗
27. Cytokine Expression in an Ex Vivo Culture System of Duodenal Samples from Dogs with Chronic Enteropathies: Modulation by Probiotic Bacteria10.1016/j.domaniend.2005.04.006 ↗
28. Cytokine and Transcription Factor Profiles in the Skin of Dogs Naturally Infected by Leishmania (Leishmania) Chagasi Presenting Distinct Cutaneous Parasite Density and Clinical Status10.1016/j.vetpar.2010.11.025 ↗
29. Activation of the Damage-Associated Molecular Pattern Receptor P2X7 Induces Interleukin-1β Release from Canine Monocytes10.1016/j.vetimm.2012.05.004 ↗
30. Cavalcanti, A.S., Ribeiro-Alves, M., de Pereira, L.O.R., Mestre, G.L., Ferreira, A.B.R., Morgado, F.N., Boité, M.C., Cupolillo, E., Moraes, M.O., and Porrozzi, R. (2015). Parasite Load Induces Progressive Spleen Architecture Breakage and Impairs Cytokine mRNA Expression in Leishmania infantum-Naturally Infected Dogs. PLoS ONE, 10.10.1371/journal.pone.0123009 ↗
31. Ggplot210.1002/wics.147 ↗
32. Differential Replication Properties among H9N2 Avian Influenza Viruses of Eurasian Origin10.1186/s13567-015-0198-8 ↗
33. Differences in Sialic Acid-Galactose Linkages in the Chicken Egg Amnion and Allantois Influence Human Influenza Virus Receptor Specificity and Variant Selection10.1128/jvi.71.4.3357-3362.1997 ↗
34. Avian-Virus-like Receptor Specificity of the Hemagglutinin Impedes Influenza Virus Replication in Cultures of Human Airway Epithelium10.1016/j.virol.2006.11.030 ↗
35. Influenza A Virus Isolation, Culture and Identification10.1038/nprot.2014.180 ↗
36. Li, T., Zhao, C., Guo, Y., Dong, J., Du, F., Zhou, Y., Shu, S., Liu, Y., Cheng, Y., and Cao, Z. (2024). Genetic and Biological Characteristics of Duck-Origin H4N6 Avian Influenza Virus Isolated in China in 2022. Viruses, 16.10.3390/v16020207 ↗
37. Genetics and Pathogenicity of Influenza A (H4N6) Virus Isolated from Wild Birds in Jiangsu Province, China, 202310.1155/2024/7421277 ↗
38. Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium10.1128/JVI.01477-12 ↗
39. Monitoring Virus Harvesting Time in Embryonated Chicken Eggs Inoculated with Avian Influenza H9N2 Vaccine Strain
40. Growth Characteristics of the H5N1 Avian Influenza Virus in Chicken Embryonic Eggs and MDCK Cells10.56808/2985-1130.2184 ↗
41. Growth Curves of Influenza Virus Based on Hemagglutination Titers in Individual Embryonated Eggs
42. Different Incubation Temperatures Affect Viral Polymerase Activity and Yields of Low-Pathogenic Avian Influenza Viruses in Embryonated Chicken Eggs10.1007/s00705-011-0933-z ↗
43. Proteolytic Enzymes in Embryonated Chicken Eggs Sustain the Replication of Egg-Grown Low-Pathogenicity Avian Influenza Viruses in Cells in the Absence of Exogenous Proteases10.1016/j.jviromet.2014.02.023 ↗
44. Evaluation of Different Embryonating Bird Eggs and Cell Cultures for Isolation Efficiency of Avian Influenza A Virus and Avian Paramyxovirus Serotype 1 from Real-Time Reverse Transcription Polymerase Chain Reaction–Positive Wild Bird Surveillance Samples10.1177/1040638712440991 ↗
45. Tsai, H.-C., Lehman, C.W., Lin, C.-C., Tsai, S.-W., and Chen, C.-M. (2019). Functional Evaluation for Adequacy of MDCK-Lineage Cells in Influenza Research. BMC Res. Notes, 12.10.1186/s13104-019-4134-2 ↗
46. Cree, I.A. (2011). Cell Sensitivity Assays: The MTT Assay. Cancer Cell Culture: Methods and Protocols, Humana Press.10.1007/978-1-61779-080-5 ↗
47. Franco-Robles, E. (2022). Prebiotics and Probiotics: From Food to Health, IntechOpen.10.5772/intechopen.94725 ↗
48. Tindjau, R., Chua, J.-Y., and Liu, S.-Q. (2024). Co-Culturing Propionibacterium Freudenreichii and Bifidobacterium Animalis Subsp. Lactis Improves Short-Chain Fatty Acids and Vitamin B12 Contents in Soy Whey. Food Microbiol., 121.10.1016/j.fm.2024.104525 ↗
49. Emerging Lactic Acid Bacteria Bacteriocins as Anti-Cancer and Anti-Tumor Agents for Human Health10.1016/j.heliyon.2024.e37054 ↗
50. Anti-Influenza Activity of a Bacillus subtilis Probiotic Strain10.1128/AAC.00539-17 ↗
51. Rather, I.A., Kamli, M.R., Sabir, J.S.M., and Paray, B.A. (2022). Potential Antiviral Activity of Lactiplantibacillus plantarum KAU007 against Influenza Virus H1N1. Vaccines, 10.10.3390/vaccines10030456 ↗
52. Anti–Influenza Activity of Enterocin B In Vitro and Protective Effect of Bacteriocinogenic Enterococcal Probiotic Strain on Influenza Infection in Mouse Model10.1007/s12602-018-9457-0 ↗
53. Antiviral Activities of Cell-Free Supernatants of Yogurts Metabolites against Some RNA Viruses10.1007/s00217-009-1009-0 ↗
54. The Host Immune Response in Respiratory Virus Infection: Balancing Virus Clearance and Immunopathology10.1007/s00281-016-0558-0 ↗
55. The Relationship among Avian Influenza, Gut Microbiota and Chicken Immunity: An Updated Overview10.1016/j.psj.2022.102021 ↗
56. Dairy-origin Propionibacterium freudenreichii, Turkey-Origin Lactobacillus salivarius, and a Salmonella Typhimurium Vaccine Elicit Comparable Colonization Resistance on Drug-Resistant Salmonella serotypes (S. Reading, S. Agona, and S. Saintpaul) in Growing Turkeys After Oral Challenge10.1016/j.japr.2024.100428 ↗
57. Muringattu Prabhakaran, D., Kollanoor Johny, A., Nair, D.V.T., Manjankattil, S., Johnson, T.J., Noll, S., and Reed, K.M. (2025). Beneficial Cecal Microbiome Modulation in Turkeys Exposed to Probiotics and Vaccine After Multidrug-Resistant Salmonella Heidelberg Challenge. Microbiol. Res., 16.10.3390/microbiolres16070136 ↗
58. Dewi, G., Ramanathan, R., and Kollanoor Johny, A. (2025). Cecal Metabolome Profiles of Turkey Poults in Response to Salmonella Heidelberg Challenge with or Without Turkey-Derived Lactobacillus Probiotic and Trans-Cinnamaldehyde. Animals, 15.10.3390/ani15142016 ↗
59. Lactobacillus-Deficient Cervicovaginal Bacterial Communities Are Associated with Increased HIV Acquisition in Young South African Women10.1016/j.immuni.2016.12.013 ↗
60. Wang, Y., Moon, A., Huang, J., Sun, Y., and Qiu, H.-J. (2022). Antiviral Effects and Underlying Mechanisms of Probiotics as Promising Antivirals. Front. Cell Infect. Microbiol., 12.10.3389/fcimb.2022.928050 ↗
61. Lu, W., Fang, Z., Liu, X., Li, L., Zhang, P., Zhao, J., Zhang, H., and Chen, W. (2021). The Potential Role of Probiotics in Protection against Influenza a Virus Infection in Mice. Foods, 10.10.3390/foods10040902 ↗
62. The Role of Lactic Acid Production by Probiotic Lactobacillus Species in Vaginal Health10.1016/j.resmic.2017.04.001 ↗
63. Immune System Stimulation by Probiotic Microorganisms10.1080/10408398.2011.619671 ↗
64. The Influence of Commensal Bacteria on Infection with Enteric Viruses10.1038/nrmicro.2015.25 ↗