Patent Grant
9121010
1. Field of the Invention
The invention relates to a transgenic cell line which has been engineered to constitutively express the αVβ6 integrin receptor, a principal bovine receptor for Foot and Mouth Disease Virus (FMDV). In particular, the invention relates to the transgenic fetal porcine kidney cell line, LFBK αvβ6, which is useful for the rapid and sensitive detection and identification of FMDV in diagnostic settings and also to identify serotypes and subtypes thereby facilitating vaccine selection procedures. LFBK αvβ6 is highly sensitive and permissive to infection by animal-derived FMDV from all seven serotypes in cell culture. The invention further relates to the transgenic LFBK αvβ6 cells for detection of FMDV from field samples.
2. Description of the Relevant Art
Foot and mouth disease virus (FMDV) is a severe economic concern for meat producing nations since the trade of animal products is prohibited from countries where the virus is confirmed. The rapid spread of the virus among susceptible animals results in severe morbidity and in some cases death, especially in young animals (Grubman and Baxt. 2004. Clin. Microbiol. Rev. 17: 465-493). Foot and mouth disease (FMD) is an extremely contagious viral disease of cloven-hoofed ungulates which include domestic animals (cattle, pigs, sheep, goats, and others) and a variety of wild animals. Infection or vaccination with one of the seven different serotypes does not confer cross-protection to other serotypes or even some subtypes of the same serotype. Vaccines for FMDV are widely used to prevent clinical disease, but since vaccines are serotype and subtype-specific, the virus(es) causing outbreaks must be isolated and serologically characterized for vaccine matching prior to selecting the appropriate vaccine antigen (reviewed in Rodriguez and Gay. 2011. Expert Rev. Vaccines 10:377-387).
Although molecular techniques such as PCR (polymerase chain reaction) coupled with genomic sequencing can be used in samples containing enough virus to rapidly identify the virus serotype and its relationship to other FMDV strains, appropriate vaccine prediction requires virus growth in cell culture to carry out neutralization tests using reference sera. Inefficient recovery of virus from animal samples can delay diagnosis and vaccine selection and thereby hamper rapid implementation of control measures; therefore, virus isolation protocols are designed for maximum sensitivity. Some primary cells, such as bovine thyroid (BTY) cells, are highly susceptible to a wide range of FMDV serotypes (Snowdon, W. A. 1966. Nature 210:1079-1080); however, they are difficult and costly to prepare and lose FMDV susceptibility after multiple passages (House and Yedloutschnig. 1982. Can. J. Comp. Med. 46:186-189). Primary lamb kidney (LK) cells are also very sensitive to FMDV. Unlike BTY cells, LK cells maintain their sensitivity to FMDV infection after cryopreservation; however, their sensitivity decreases after passage (House and House. 1989. Vet. Microbiol. 20:99-109). Immortalized cell lines (e.g. baby hamster kidney (BHK) fibroblasts and porcine kidney epithelial cells), while much easier to maintain, are in many cases less susceptible to specific animal-derived FMDV serotypes (Swaney, L. M. 1976. Amer. J. Vet. Res. 37:1319-1322; Ferris et al. 2006. Vet. Microbiol. 117:130-140; Ferris et al. 2002. Vet. Microbiol. 84:307-316; De Castro, M. P. 1964. Arch. Inst. Biol. San Paulo 31: 63-78).
Integrins of the αV subgroup have been demonstrated to be FMDV receptors by several laboratories including ours (Ruiz-Saenz et al. 2009. Intervirol. 52:201-212). Of the many αV integrins that have been shown to mediate FMDV attachment, the integrin αVβ6 has been shown to be one of the most efficient receptors for all FMDV serotypes (Jackson et al. 2000. J. Virol. 74:4949-4956; Ferris et al. 2005. J. Virological Methods 127:69-79) and high levels of αVβ6 expression are observed on epithelial cells at the sites of infection in cattle and swine (Monaghan et al. 2005. J. Gen. Virol. 86:2769-2780; O'Donnell et al. 2009. J. Comp. Path. 141:98-112). BTY cells, considered the most sensitive primary cells for FMDV isolation, have high levels of αVβ6 integrin surface expression (King et al. 2011. Vet. Immunol. Immunopath. 140:259-265). Moreover, transient expression of bovine αV and β6 integrin subunits in baby hamster kidney cells (BHK3-αVβ6) (Duque et al. 2004. J. Virol. 78:9773-9781) greatly increased the susceptibility of this cell line to a cow-passaged A24 Cruziero strain that contains an SGD motif in the VP1 (FMD Virus Protein 1) capsid protein (Rieder et al. 2005. J. Virol. 79:12989-12998). Although the BHK3-αVβ6 cells were initially more permissive to the A24-SGD virus than BHK-21 cells were, the BHK3-αVβ6 cells lost integrin expression and sensitivity to the A24-SGD virus after multiple passages (E. Rieder, personal communication).
Swaney derived an immortalized line of fetal porcine kidney (LFBK) cells that had high susceptibility to most FMDV serotypes and the susceptibility was maintained over many passages (Swaney, L. M. 1988. Vet. Microbiol. 18:1-14). Compared to BTY cells, LFBK cells had similar susceptibility to most FMDV serotypes and had equal or better susceptibility than the MVPK (Mengeling-Vaughn Porcine Kidney) cell line, the porcine kidney cell line, IB-RS-2, and fetal bovine kidney cells in the same experiments.
There is a need for a cell line that is easily maintained and is highly susceptible to all serotypes and subtypes of FMDV. The present invention, described below, combines the long-lived FMDV susceptibility of the LFBK cell line with a principal bovine receptor for FMDV, the αVβ6 integrin receptor, to provide a stable cell line which is highly susceptible to FMDV.
We have developed and characterized a stable transgenic cell line that is highly susceptible to animal-derived FMDV from all seven serotypes and discovered that this cell line greatly facilitates FMDV isolation and growth from field samples ensuring more accurate and more rapid diagnosis of the FMDV serotype involved in an outbreak, when compared to other cells used for diagnosis.
In accordance with this discovery, it is an object of the invention to provide the transgenic fetal porcine kidney cell line, LFBK αvβ6, engineered to express the αVβ6 integrin receptor, a principal bovine receptor for FMDV.
It is a further object of the invention to provide LFBK αvβ6 cells for the rapid isolation and growth of FMDV serotypes and subtypes from field samples thereby facilitating vaccine selection procedures. LFBK αvβ6 is sensitive and permissive to infection by animal-derived FMDV from all seven serotypes in cell culture.
It is another object of the invention to provide LFBK αvβ6 cells for the rapid and sensitive detection and identification of all seven serotypes of FMDV and multiple subtypes from field samples thereby facilitating vaccine selection procedures.
Other objects and advantages of this invention will become readily apparent from the ensuing description.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.
Here we report the engineering and comprehensive characterization of the LFBK-αVβ6 cell line, a fetal porcine kidney cell line stably transduced with the bovine αV and β6 integrin subunits. The expression of the β6 integrin subunit and the resulting enhanced infectivity of FMDV containing a SGD domain in VP1 were both maintained for at least 100 cell passages. We found that the LFBK-αVβ6 cells were more susceptible to all FMDV serotypes derived from experimentally-infected animals as compared to the LFBK cells from which they were derived and other cells commonly used for FMDV isolation. In a diagnostic sensitivity assay, LFBK-αVβ6 cells were more sensitive than primary lamb kidney cells, the porcine kidney cell line IB-RS-2, and the BHK cell line. LFBK-αVβ6 cells were also able to detect other vesicular disease viruses. Our results support the use of LFBK-αVβ6 cells for FMDV diagnostic purposes.
Our data indicate that the LFBK-αVβ6 cells are excellent for detecting FMDV serotype O isolates. A previous study at PIADC (Plum Island Animal Disease Center) showed that LFBK cell sensitivity to O1 Manisa isolated from cattle was close to the ID50 observed in primary bovine tongue cells (Pacheco et al. 2010. Vet. J. 183:46-53); however, we have observed that the transgenic LFBK-αVβ6 cells detect O1 Manisa over one log10 more efficiently than LFBK cells. Further, Burman and coworkers showed that the integrin-binding domain on the VP1 capsid protein from O1 FMDV binds αVβ6 with the highest affinity among the αVβ3, αVβ6 and αVβ8 integrins (Burman et al. 2006. J. Virol. 80:9798-9810). In addition, in our experiments (
BHK-21 cells are widely used for FMDV virus isolation, due to their rapid growth properties and sufficient susceptibility to most serotypes of FMDV. BHK cells generally performed very well in our sensitivity experiments (
FMDV strain O/TAW/97 does not grow well in primary bovine thyroid cells (BTY) or in cattle; yet, O/TAW/97 grows in the IB-RS-2 porcine kidney cell line and is extremely virulent in swine (Dunn and Donaldson. 1997. Vet. Rec. 141:174-175). As such, O/TAW/97 has been referred to as a “porcinophillic” virus (Dunn and Donaldson, supra; Knowles et al. 2001. J. Virol. 75:1551-1556; Pacheco and Mason. 2010. J. Vet. Sci. 11:133-142). In our experiments this virus also grew poorly in primary lamb kidney cells, forming extremely small plaques. However, O/TAW/97 grew better in the porcine-derived LFBK cells than in the cell lines of swine origin, namely, MVPK and IB-RS-2. The expression of αVβ6 in the LFBK-αVβ6 cells enhanced infectivity of the O/TAW/97 virus over the infectivity levels observed in LFBK cells to a level about the same as seen with the O1 Manisa and O/UKG/2001 viruses.
LFBK-αVβ6 cells have been in use at PIADC for all aspects of FMDV virology with tremendous success. They support the replication of animal-derived virus strains that do not grow well in other cell types (e.g. O1 Manisa, O/TAW/97) and maintain high sensitivity to FMDV for at least 100 subculture passages. They do not require the extraction of animal organs to make primary cells, they grow as efficiently as standard LFBK cells, and they have no special media requirements. We have characterized the FMDV susceptibility of this transduced cell line by infection with animal-derived FMDV from all 7 serotypes as well as recent diagnostic field samples and compared its susceptibility to that of other cell types used for diagnostic FMDV virus isolation. Our results indicate that LFBK-αvβ6 cells are highly permissive for all FMDV serotypes and have excellent performance in a diagnostic setting. Based on the data presented here, LFBK-αVβ6 cells are a valuable tool for the rapid detection and/or isolation of FMDV serotypes in clinical laboratories and are exceptionally suited for all routine FMDV diagnostic and research-based cell applications.
The terms “sample” and “specimen” in the present specification and claims are used in their broadest sense to include any composition that is obtained and/or derived from biological or environmental source, as well as sampling devices (e.g., swabs) which are brought into contact with biological or environmental samples. “Biological samples” include those obtained from an animal, including cloven-hoofed ungulates which include domestic animals (cattle, pigs, sheep, goats, and others) and a variety of wild animals, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, sputum, and saliva, as well as solid tissue. Also included are samples obtained from food products and food ingredients such as dairy items, meat, meat by-products, and waste. “Environmental samples” include environmental material such as surface matter, soil, water, and industrial materials, as well as material obtained from food and dairy processing instruments, apparatus, equipment, disposable, and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
As used herein, the term “cell type,” refers to any cell, regardless of its source or characteristics.
As used herein, the term “microorganism” refers to any organism of microscopic or ultramicroscopic size including, but not limited to, viruses, bacteria, and protozoa.
As used herein, the term “culture” refers to a composition, whether liquid, gel, or solid, which contains one or more microorganisms and/or one or more cells. A culture of organisms and/or cells can be pure or mixed. For example, a “pure culture” of an organism as used herein refers to a culture in which the organisms present are of only one strain of a single species of a particular genus. This is in contrast to a “mixed culture” of organisms which refers to a culture in which more than one strain of a single genus and/or species of microorganism is present.
As used herein, the terms “culture media,” and “cell culture media,” refer to media that are suitable to support maintenance and/or growth of cells in vitro (i.e., cell cultures).
A “primary cell” is a cell which is directly obtained from a tissue or organ of an animal whether or not the cell is in culture.
A “cultured cell” is a cell which has been maintained and/or propagated in vitro. Cultured cells include primary cultured cells and cell lines.
“Primary cultured cells” are primary cells which are in in vitro culture and which preferably, though not necessarily, are capable of undergoing ten or fewer passages in in vitro culture before senescence and/or cessation of proliferation.
The terms “cell line” and “immortalized cell” refer to a cell which is capable of a greater number of cell divisions in vitro before cessation of proliferation and/or senescence as compared to a primary cell from the same source. A cell line includes, but does not require, that the cells be capable of an infinite number of cell divisions in culture. The number of cell divisions may be determined by the number of times a cell population may be passaged (i.e., subcultured) in in vitro culture. Passaging of cells is accomplished by methods known in the art. Briefly, a confluent or subconfluent population of cells which is adhered to a solid substrate (e.g., plastic Petri dish) is released from the substrate (e.g., by enzymatic digestion), and a proportion (e.g., 10%) of the released cells is seeded onto a fresh substrate. The cells are allowed to adhere to the substrate, and to proliferate in the presence of appropriate culture medium. The ability of adhered cells to proliferate may be determined visually by observing increased coverage of the solid substrate over a period of time by the adhered cells. Alternatively, proliferation of adhered cells may be determined by maintaining the initially adhered cells on the solid support over a period of time, removing and counting the adhered cells and observing an increase in the number of maintained adhered cells as compared to the number of initially adhered cells.
Cell lines may be generated spontaneously or by transformation. A “spontaneous cell line” is a cell line which arises during routine culture of cells. A “transformed cell line” refers to a cell line which is generated by the introduction of a “transgene” comprising nucleic acid (usually DNA) into a primary cell or into a finite cell line by way of human intervention
Cell lines include, but are not limited to, finite cell lines and continuous cell lines. As used herein, the term “finite cell line” refers to a cell line which is capable of a limited number of cell divisions prior to senescence.
The term “continuous cell line” refer to a cell line which is capable of more than about 50 (and more preferably, an infinite number of) cell divisions.
The term “transgene” is understood to describe genetic material which has been or is about to be artificially inserted into the genome of a non-human animal, and particularly into a cell of a living non-human mammal. It is to be understood that as used herein the term “transgenic” includes any cell, cell line, or tissue, the genotype of which has been altered by the presence of a heterologous nucleic acid. A transgene may be an “endogenous DNA sequence” or a “heterologous DNA sequence” (i.e., “foreign DNA”). The term “endogenous DNA sequence” refers to a nucleotide sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence. The term “heterologous DNA sequence” refers to a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Heterologous DNA also includes an endogenous DNA sequence which contains some modification. Generally, although not necessarily, heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed.
The term “transduction” is used to refer to the introduction of genetic material into a cell by using a viral vector. As used herein a transduced cell results from a transduction process and contains genetic material it did not contain before the transduction process, whether stably integrated or not.
The term “transformation” refers to a permanent or transient genetic change induced in a cell following the incorporation of new DNA (i.e. DNA exogenous to the cell). Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Thus, isolated polynucleotides can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
As used herein, the terms “nucleic acid molecule”, “nucleic acid sequence”, “polynucleotide”, “polynucleotide sequence”, “nucleic acid fragment”, “isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded and that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
The term “isolated” polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as other chromosomal and extrachromosomal DNA and RNA, that normally accompany or interact with it as found in its naturally occurring environment. However, isolated polynucleotides may contain polynucleotide sequences which may have originally existed as extrachromosomal DNA but exist as a nucleotide insertion within the isolated polynucleotide. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
As used herein, “recombinant” refers to a nucleic acid molecule which has been obtained by manipulation of genetic material using restriction enzymes, ligases, and similar genetic engineering techniques as described by, for example, Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. or DNA Cloning: A Practical Approach, Vol. I and II (Ed. D. N. Glover), IRL Press, Oxford, 1985. “Recombinant,” as used herein, does not refer to naturally occurring genetic recombinations.
As used herein, the term “chimeric” refers to two or more DNA molecules which are derived from different sources, strains, or species, which do not recombine under natural conditions, or to two or more DNA molecules from the same species, which are linked in a manner that does not occur in the native genome. A “construct” refers to a recombinant nucleic acid, generally recombinant DNA, that has been generated for the purpose of the expression of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences. A “chimeric gene construct” refers to a nucleic acid sequence encoding a protein, operably linked to a promoter and/or other regulatory sequences.
As used herein, the term “express” or “expression” is defined to mean transcription alone. “Altered levels” or “altered expression” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
As used herein, the terms “encoding”, “coding”, or “encoded” when used in the context of a specified nucleic acid mean that the nucleic acid comprises the requisite information to guide translation of the nucleotide sequence into a specified protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA).
The term “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
“Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
“Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.
The “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
The “3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor.
As used herein, the term “FMD” encompasses disease symptoms in cattle and swine caused by a FMDV infection. Examples of such symptoms include, but are not limited to, vesicles in the mouth, and on the feet. As used herein, a FMDV that is “unable to produce FMD” refers to a virus that can infect a pig, but which does not produce any disease symptoms normally associated with a FMD infection in the pig, or produces such symptoms, but to a lesser degree, or produces a fewer number of such symptoms, or both.
The terms “porcine” and “swine” are used interchangeably herein and refer to any animal that is a member of the family Suidae such as, for example, a pig. “Mammals” include any warm-blooded vertebrates of the Mammalia class, including humans.
The terms “foot and mouth disease virus” and “FMDV”, as used herein, unless otherwise indicated, mean any strain of FMD viruses.
Terms such as “suitable host cell” and “appropriate host cell”, unless otherwise indicated, refer to cells into which RNA molecules (or isolated polynucleotide molecules or viral vectors comprising DNA sequences encoding such RNA molecules) of the present invention can be transformed or transfected. “Suitable host cells” for transfection with such RNA molecules, isolated polynucleotide molecules, or viral vectors, include mammalian, particularly bovine and porcine cells, and are described in further detail below.
A “functional virion” is a virus particle that is able to enter a cell capable of hosting a FMDV, and express genes of its particular RNA genome (either an unmodified genome or a genetically modified genome) within the cell.
In summary, we provide a stable transgenic fetal porcine kidney cell line, LFBK αvβ6, useful for the rapid isolation and sensitive detection and identification of all seven FMDV serotypes and multiple subtypes in cell culture. The invention provides for highly sensitive detection of FMDV from field samples.
Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.
LFBK cells were propagated in DMEM supplemented with 10% fetal bovine serum and antibiotics as previously described (Piccone et al. 2009. J. Virol. 83:6681-6688) and used for these experiments between passages 64 and 71. LFBK-αVβ6 cells were propagated in the same manner as LFBK cells and used at the passage indicated in each experiment. BHK cells, used between passages 62 and 67, were propagated in MEM supplemented with 10% calf serum, 10% tryptose phosphate broth and antibiotics. Primary lamb kidney (LK) cells, generously supplied by the APHIS Diagnostic Service Section at PIADC, were propagated in DMEM supplemented with 10% fetal bovine serum, antibiotics and sodium pyruvate and used directly from cryovials or at passage 1 as indicated. IB-RS-2 cells were used between passage 129 and 137 and MVPK cells were used between passages 38 and 41; both of these cell lines were propagated in MEM supplemented with 10% fetal calf serum, nonessential amino acids and antibiotics.
The integrin αVβ6 is an important receptor for FMDV in relevant tissues in vivo. LFBK cells are a transformed cell line that has high sensitivity to most FMDV serotypes but does not express high levels of β6 integrin protein. In order to merge the enhanced susceptibility of αVβ6 expression with the transformed phenotype of LFBK cells, the bovine αV and β6 integrin subunits were transduced into LFBK cells. Individual retroviruses expressing the bovine αV (Gen Bank AF239958) or bovine β6 (GenBank AF468060) integrin subunits (Duque et al., supra) were created with the Pantropic Retroviral Expression System (Clontech PT3346-5) following the manufacturer's protocol. LFBK cells at passage 145 were infected with the αV-expressing retrovirus and neomycin selection was used to select against non-transduced cells. A pool of these cells was infected with the β6-expressing retrovirus, then cloned to single cells by terminal dilution. Clones were chosen that showed consistent expression of the β6 subunit by immunohistochemistry.
Immunohistochemical staining was used to demonstrate the long-term maintenance of β6 expression in LFBK-αVβ6 cells. The Vectastain ABC kit (Vector Labs, PK-6102) and Vector VIP peroxidase substrate kit (Vector Labs, SK-4600) were used according to the manufacturer's protocols. Mouse anti-human β6 (Chemicon, CSb6) was used to detect the bovine integrin subunit β6 at a 1:300 dilution. The mouse monoclonal antibody F19-51 (Yang et al. 2007. J. Immunol. Methods 321:174-181) was used to detect the FMDV 3D protein at a 1:200 dilution.
Immunohistochemical staining demonstrated the long-term maintenance of β6 expression in the LFBK-αVβ6 cells for at least 102 cell passages as compared to the non-transduced LFBK cells (
FMDV A24-SGD, an FMDV A24 Cruziero strain serially passed in cattle, has an SGD motif in the G-H loop of VP1 and enhanced infectivity in cells expressing the αVβ6 integrin (Rieder et al., supra). LFBK-αVβ6 cells at various subculture passages and non-transduced LFBK cells were infected with A24-SGD or a cell culture grown control virus (A24-BHK) to confirm the long-term functional maintenance of the αVβ6 integrin.
Cells were seeded 72 hours before infection in 6-well cell culture plates unless otherwise noted. Inoculum volume was 200 μl. 1 hour post adsorption, the inoculum was removed and the cells were overlaid with 50/50 1.2% Gum Tragacanth/2×MEM supplemented with antibiotic/antimycotic. Plates were incubated for 24 hours unless otherwise indicated then simultaneously fixed and stained with Histo-Choice tissue fixative (AMRESCO) containing crystal violet and plaques were counted.
The results of these experiments showed that the LFBK-αVβ6 cells have a 2.5 log increased susceptibility to A24-SGD compared to LFBK cells and that the increased susceptibility is maintained for over 100 passages (
In order to show that the LFBK-αVβ6 cells support the normal growth progression of FMDV, a multi-step growth curve was performed (
In this experiment, the replication of A24-BHK was similar in both LFBK and LFBK-αVβ6 cells. While A24-SGD replicated slowly in the non-transduced LFBK cells, this virus grew normally in LFBK-αVβ6 cells, demonstrating that the expression of αVβ6 in LFBK cells complements the defect of non-transduced LFBK cells to support efficient A24-SGD replication.
In order to determine the susceptibility of LFBK-αVβ6 cells to animal-derived FMDV strains and compare with that of LFBK, primary lamb kidney (LK), BHK, and two swine kidney cells lines (IB-RS-2 and MVPK), each cell type was infected with serial dilutions of infected tissue macerates or vesicular fluid obtained from animals experimentally infected with each of the FMDV serotypes. The viruses used in this experiment are listed in Table 1.
For each strain of virus, the LFBK-αVβ6 cells had equal or higher sensitivity to animal-derived FMDV compared to the other cells tested. In some cases, especially with the O serotype FMDV strains, the LFBK-αVβ6 cells supported FMDV replication greater than tenfold higher than most cell types tested (
#log10 PFU/ml ± max-min
These data confirm that LFBK-αVβ6 cells can readily detect all FMDV serotypes in tissues from experimentally-infected animals.
LK cells, BHK, IB-RS-2 and LFBK-αVβ6 were seeded onto 48-well cell culture plates 48 hours prior to infection. LK cells were seeded directly from storage cryovials. LFBK-αVβ6 cells were seeded at passage 32, IB-RS-2 at passage 136 and BHK-21 at passage 66. Diagnostic lesion tissues were disrupted and virus isolated after centrifugation through a Spin-X purification column (Costar) as described in (Pacheco et al. 2010, supra). 100 μl of a 1:10 dilution of these tissue macerates were used to infect each cell type for 1 hour at 37° C. After adsorption, 400 μl of growth media was added to each well and the plates were incubated at 37° C. Starting at 4 HPI, visual evidence of cytopathic effects was recorded every 2 hours until 20 HPI, then at 24, 28 and 48 HPI. At 48 HPI, all wells were fixed with 50% acetone:50% methanol and wells negative for cytopathic effects were immunohistochemically stained with a monoclonal antibody to FMDV 3D protein to confirm negative results.
Samples from experimentally-infected animals tend to be very high titer and have better integrity than field samples. To more closely mimic diagnostic conditions, we obtained field diagnostic samples from Afghanistan, Bolivia and Pakistan (Table 3).
These samples were processed from tissue according to standard virus isolation procedures and used to infect LFBK-αVβ6 cells and also other cells commonly used for FMDV diagnostics, including LK, IB-RS-2 and BHK cells.
Animals exhibiting vesicular lesions may be infected with other agents besides FMDV. In order to determine if LFBK-αVβ6 cells could detect other viruses causing vesicular disease, we inoculated BHK, LK, IB-RS-2, LFBK or LFBK-αVβ6 cells with each of 5 non-FMD viruses causing vesicular disease.
We found that vesicular exanthema of swine virus (VESV) and swine vesicular disease virus (SVDV) replicated as well in LFBK-αVβ6 cells as they did in IB-RS-2 cells (Table 4). Vesicular stomatitis viruses serotype New Jersey (VSV-NJ) grew to similar titers in all the cell lines. LK, LFBK and LFBK-αVβ6 cells supported the growth of bovine papular stomatitis virus (BPSV) as evidenced by the formation of plaques; BPSV grew to a slightly higher titer in LK cells than in LFBK-αVβ6 cells. Infection with bluetongue virus (BTV) only induced cytopathic effects in IB-RS-2 and LK cells by 96 HPI.
6.15a
NDb
aVirus titers in log10TCID50/ml.
bND, not detected. Limit of detection in this assay is 1.8 log10TCID50/ml.
cThis virus obtained from a pool of experimentally infected animal tissues.
Thus, LFBKαvβ6 cells can support the growth of VESV, SVDV, VSV-NJ and BPSV) as well or better than LFBK, IB-RS-2, BHK and LK.
The LFBKαvβ6 cell line has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 on Jul. 11, 2012, under accession number PTA-13047, as a patent deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The subject cell line has been deposited under conditions that assure that access to the cell line will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposit(s). All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
The foregoing description and certain representative embodiments and details of the invention have been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be apparent to practitioners skilled in this art that modifications and variations may be made therein without departing from the scope of the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20040071659 | Chang et al. | Apr 2004 | A1 |
| 20080176962 | Cohen et al. | Jul 2008 | A1 |
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| Rhodes et al. Mammalian expression of transmembrane receptors for pharmaceutical applications. Biochem Soc Trans. Nov. 1998;26(4):699-704. |
