Continuous stable cell line for identification of infectious African swine fever virus in clinical samples
Inventors
Gladue, Douglas P. • RAI, AYUSHI • Borca, Manuel V.
Assignees
US Department of Agriculture USDA
Publication Number
US-11796548-B2
Publication Date
2023-10-24
Expiration Date
2041-04-21
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Abstract
Methods are provided herein utilizing a stable cell line capable of efficient infection by African swine fever virus (ASFV) and also provides for the detection of the presence of virus in samples applied to the cells. Detection of the virus by means such as red blood cell rosetting is a surprising result given that the cell line is derived from African green monkeys. This cell line provides a marked improvement over the currently available testing strategies.
Core Innovation
The invention provides a method and composition utilizing a stable cell line, specifically Ma-104 cells derived from African green monkeys, capable of supporting efficient infection by African swine fever virus (ASFV) and enabling the detection of infectious ASFV in samples applied to these cells. Detection methods include observing hemadsorption, where red blood cells adhere to infected Ma-104 cells, a surprising result given the non-swine origin of the cell line. This approach offers a marked improvement over the current reliance on primary swine macrophages for virus isolation.
The problem addressed is the limitation in current ASFV detection techniques, which depend primarily on real-time PCR that cannot distinguish between infectious and non-infectious virus. The only confirmatory test for infectious virus involves primary swine macrophages, which are labor-intensive to prepare, not readily available, and require fresh isolation from swine. Other established cell lines have shown low or no efficiency in detecting non-adapted field isolates of ASFV, thereby complicating virus identification and outbreak management.
The invention solves this problem by identifying the Ma-104 cell line as capable of virus isolation from field samples with sensitivity comparable to primary swine macrophages without requiring virus adaptation. The invention also provides methods for detecting ASFV infection using Ma-104 cells, including hemadsorption assays and immunohistochemical staining, enabling detection of various ASFV field isolates including the Georgia/2007 strain directly from clinical samples.
Claims Coverage
The patent includes one independent method claim and one independent composition claim. The inventive features focus on using Ma-104 cells for ASFV detection and providing a composition comprising these cells, ASFV, and red blood cells.
Method of detecting ASFV using Ma-104 cells
A method comprising: contacting a sample with Ma-104 cell culture under conditions supporting ASFV infection; incubating the mixture to allow ASFV detection; and analyzing the mixture for presence or absence of replicating ASFV.
Analyzing infected cells by hemadsorption
Analyzing step involves adding red blood cells to the Ma-104 and sample mixture and examining for hemadsorbed red blood cells on Ma-104 cells within 24 hours to detect infectious ASFV.
Use of Ma-104 cells for infection by unpassaged, non-attenuated ASFV strains
The method applies to ASFV strains that are not attenuated for growth in Ma-104 cells and have not been previously passaged in cell culture.
Composition comprising Ma-104 cells, ASFV, and red blood cells
A composition including Ma-104 cells, African Swine Fever Virus (ASFV), and red blood cells, wherein ASFV can be of the Georgia/2007 strain. The Ma-104 cells are genetically unmodified.
The claims cover a detection method employing Ma-104 cells for ASFV infection and analysis, particularly using hemadsorption, applicable to field isolates including Georgia/2007, alongside a relevant composition incorporating these elements.
Stated Advantages
Provides a stable cell line alternative to primary swine macrophages for efficient detection of infectious ASFV, reducing time and labor required.
Enables detection of infectious virus in field samples without the need for virus adaptation or multiple passages.
Offers sensitive detection comparable to primary swine macrophages, including for various ASFV genotypes.
Allows use of hemadsorption assays on a commercially available cell line for ASFV detection.
Documented Applications
Detection of African swine fever virus (ASFV) in clinical samples from swine using Ma-104 cells as a substrate for virus isolation and identification.
Use of Ma-104 cell cultures to detect infectious ASFV through hemadsorption assays involving red blood cells.
Immunohistochemical staining of ASFV-infected Ma-104 cells to detect virus proteins, notably for identification of isolates unable to produce hemadsorption.
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