Equine monocyte-derived macrophage cultures and their applications for infectivity and neutralization studies of equine infectious anemia virus.
Abstract: Equine infectious anemia virus (EIAV) has been shown to infect cells of monocyte/macrophage lineage. These primary cells are intrinsically difficult to obtain, to purify and to culture in vitro for extended periods of time. As a result, most in vitro studies concerning this lentivirus make use of primary equine fibroblasts or transformed canine or feline cell lines. We describe methods that yield reproducibly pure cultures of equine blood monocytes from peripheral blood mononuclear cells. The in vitro differentiation of these cells into mature equine macrophage was verified using various cytochemical staining methods. The equine monocyte-derived macrophage (MDM) cultures were found to replicate cell-adapted and field strains of EIAV more efficiently than cultures of fully differentiated equine splenic macrophage. Having established reproducible and fully differentiated cultures of equine macrophage, in vitro assays of virus infectivity and serum neutralization were developed using the in vivo target cell of EIAV. These procedures, while developed for the EIAV system, should be equally useful for in vitro cultures of other macrophage-tropic pathogens of horses.
Publication Date: 1998-06-17 PubMed ID: 9628225DOI: 10.1016/s0166-0934(97)00204-8Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research article addresses the study of equine infectious anemia virus (EIAV) using pure cultures of equine blood monocytes derived from peripheral blood mononuclear cells. These cells, differentiated into mature equine macrophages, were found to replicate EIAV more effectively, offering insights into the virus’s behavior.
Objective of the Research
- The primary objective of this research article was to establish a more efficient in-vitro system for studying the equine infectious anemia virus (EIAV). The system involves the use of pure cultures of equine blood monocytes, differentiated into mature equine macrophages, which have been found to replicate the virus more efficiently.
Methods Used
- The authors developed methods to yield reproducibly pure cultures of equine blood monocytes from peripheral blood mononuclear cells. These cells were then transformed into mature equine macrophages through a process of in-vitro differentiation.
- The macrophages’ maturity was confirmed with cytochemical staining methods, ensuring that the cells developed in the cultures were not only genetically identical but also functionally equivalent to the macrophages that would typically interact with the EIAV in a living horse.
Results and Findings
- The resultant monocyte-derived macrophage (MDM) cultures were compared to cultures of fully differentiated equine splenic macrophages (which are currently used for most in-vitro EIAV studies). The results indicated that the MDM cultures replicated both cell-adapted and field strains of EIAV more efficiently.
- This provided a valuable insight into the virus’s behavior, exhibiting its preference for infection and replication in monocyte-derived macrophages.
Applications and Implications
- With the establishment of reproducible and fully differentiated cultures of equine macrophages, the researchers were able to develop in-vitro assays of virus infectivity and serum neutralization. This allowed them to study the interaction between the virus and its in-vivo target cells under controlled conditions, which would provide the ability to explore disease treatments or vaccines.
- While these procedures were developed specifically for the EIAV system, the authors state that they should also be useful for in-vitro cultures of other pathogens that infect macrophages in horses, thereby having wider potential applications in equine infectious disease research.
Cite This Article
APA
Raabe MR, Issel CJ, Montelaro RC.
(1998).
Equine monocyte-derived macrophage cultures and their applications for infectivity and neutralization studies of equine infectious anemia virus.
J Virol Methods, 71(1), 87-104.
https://doi.org/10.1016/s0166-0934(97)00204-8 Publication
Researcher Affiliations
- Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, Pennsylvania 15261, USA.
MeSH Terms
- Age Factors
- Animals
- Cell Line
- Female
- Horses
- Infectious Anemia Virus, Equine / pathogenicity
- Infectious Anemia Virus, Equine / physiology
- Macrophages / cytology
- Macrophages / physiology
- Macrophages / virology
- Monocytes / cytology
- Neutralization Tests / veterinary
- Reproducibility of Results
- Spleen / cytology
- Time Factors
- Virus Replication
Grant Funding
- 2R01 CA49296 / NCI NIH HHS
- 5RO1 AI25850 / NIAID NIH HHS
- 5T32 AIO7487 / NIAID NIH HHS
Citations
This article has been cited 19 times.- Du C, Duan Y, Wang XF, Lin Y, Na L, Wang X, Chen K, Wang X. Attenuation of Equine Lentivirus Alters Mitochondrial Protein Expression Profile from Inflammation to Apoptosis. J Virol 2019 Nov 1;93(21).
- Sponseller BA, Clark SK, Gilbertie J, Wong DM, Hepworth K, Wiechert S, Chandramani P, Sponseller BT, Alcott CJ, Bellaire B, Petersen AC, Jones DE. Macrophage effector responses of horses are influenced by expression of CD154. Vet Immunol Immunopathol 2016 Nov 1;180:40-44.
- Craigo JK, Ezzelarab C, Cook SJ, Chong L, Horohov D, Issel CJ, Montelaro RC. Envelope determinants of equine lentiviral vaccine protection. PLoS One 2013;8(6):e66093.
- Flaminio MJ, Nydam DV, Marquis H, Matychak MB, Giguère S. Foal monocyte-derived dendritic cells become activated upon Rhodococcus equi infection. Clin Vaccine Immunol 2009 Feb;16(2):176-83.
- Fidalgo-Carvalho I, Craigo JK, Barnes S, Costa-Ramos C, Montelaro RC. Characterization of an equine macrophage cell line: application to studies of EIAV infection. Vet Microbiol 2009 Apr 14;136(1-2):8-19.
- Bogerd HP, Tallmadge RL, Oaks JL, Carpenter S, Cullen BR. Equine infectious anemia virus resists the antiretroviral activity of equine APOBEC3 proteins through a packaging-independent mechanism. J Virol 2008 Dec;82(23):11889-901.
- Allen CA, Payne SL, Harville M, Cohen N, Russell KE. Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages. J Immunol Methods 2007 Dec 1;328(1-2):59-69.
- Craigo JK, Durkin S, Sturgeon TJ, Tagmyer T, Cook SJ, Issel CJ, Montelaro RC. Immune suppression of challenged vaccinates as a rigorous assessment of sterile protection by lentiviral vaccines. Vaccine 2007 Jan 15;25(5):834-45.
- Jin S, Zhang B, Weisz OA, Montelaro RC. Receptor-mediated entry by equine infectious anemia virus utilizes a pH-dependent endocytic pathway. J Virol 2005 Dec;79(23):14489-97.
- Zhang B, Jin S, Jin J, Li F, Montelaro RC. A tumor necrosis factor receptor family protein serves as a cellular receptor for the macrophage-tropic equine lentivirus. Proc Natl Acad Sci U S A 2005 Jul 12;102(28):9918-23.
- Patton KM, McGuire TC, Hines MT, Mealey RH, Hines SA. Rhodococcus equi-specific cytotoxic T lymphocytes in immune horses and development in asymptomatic foals. Infect Immun 2005 Apr;73(4):2083-93.
- Patton KM, McGuire TC, Fraser DG, Hines SA. Rhodococcus equi-infected macrophages are recognized and killed by CD8+ T lymphocytes in a major histocompatibility complex class I-unrestricted fashion. Infect Immun 2004 Dec;72(12):7073-83.
- Chen C, Weisz OA, Stolz DB, Watkins SC, Montelaro RC. Differential effects of actin cytoskeleton dynamics on equine infectious anemia virus particle production. J Virol 2004 Jan;78(2):882-91.
- Li F, Chen C, Puffer BA, Montelaro RC. Functional replacement and positional dependence of homologous and heterologous L domains in equine infectious anemia virus replication. J Virol 2002 Feb;76(4):1569-77.
- Leroux C, Craigo JK, Issel CJ, Montelaro RC. Equine infectious anemia virus genomic evolution in progressor and nonprogressor ponies. J Virol 2001 May;75(10):4570-83.
- Harrold SM, Cook SJ, Cook RF, Rushlow KE, Issel CJ, Montelaro RC. Tissue sites of persistent infection and active replication of equine infectious anemia virus during acute disease and asymptomatic infection in experimentally infected equids. J Virol 2000 Apr;74(7):3112-21.
- Li F, Leroux C, Craigo JK, Cook SJ, Issel CJ, Montelaro RC. The S2 gene of equine infectious anemia virus is a highly conserved determinant of viral replication and virulence properties in experimentally infected ponies. J Virol 2000 Jan;74(1):573-9.
- Li F, Puffer BA, Montelaro RC. The S2 gene of equine infectious anemia virus is dispensable for viral replication in vitro. J Virol 1998 Oct;72(10):8344-8.
- Cardeti G, Manna G, Cersini A, Nardini R, Rosati S, Reina R, Cittadini M, Sittinieri S, Altigeri A, Marcario GA, Scicluna MT. Horse Innate Immunity in the Control of Equine Infectious Anemia Virus Infection: A Preliminary Study. Viruses 2024 Nov 21;16(12).
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