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A continuous bovine kidney cell line constitutively expressing bovine ?v?6 integrin has increased susceptibility to foot-and-mouth disease virus.

ABSTRACT: Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. Since the ?V?6 integrin is a principal receptor for FMDV, we transduced a bovine kidney cell line to stably express both the ?V and ?6 bovine integrin subunits. This stable cell line (LFBK-?V?6) showed ?6 expression and enhanced susceptibility to FMDV infection for ? 100 cell passages. LFBK-?V?6 cells were highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophilic FMDV strain O/TAW/97. In comparison to other cell types that are currently used for virus isolation, LFBK-?V?6 cells were more effective at detecting FMDV in clinical samples, supporting their use as a more sensitive tool for virus isolation.

SUBMITTER: LaRocco M

PROVIDER: S-EPMC3716081 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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A continuous bovine kidney cell line constitutively expressing bovine αvβ6 integrin has increased susceptibility to foot-and-mouth disease virus.

LaRocco Michael M Krug Peter W PW Kramer Ed E Ahmed Zaheer Z Pacheco Juan M JM Duque Hernando H Baxt Barry B Rodriguez Luis L LL

Journal of clinical microbiology 20130320 6

Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. ...[more]

PMID: 23515553

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Project description:Three members of the alpha(V) integrin family of cellular receptors, alpha(V)beta(1), alpha(V)beta(3), and alpha(V)beta(6), have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid (RGD) amino acid sequence motif located within the betaG-betaH (G-H) loop of VP1. Other alpha(V) integrins, as well as several other integrins, recognize and bind to RGD motifs on their natural ligands and also may be candidate receptors for FMDV. To analyze the roles of the alpha(V) integrins from a susceptible species as viral receptors, we molecularly cloned the bovine beta(1), beta(5), and beta(6) integrin subunits. Using these subunits, along with previously cloned bovine alpha(V) and beta(3) subunits, in a transient expression assay system, we compared the efficiencies of infection mediated by alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), and alpha(V)beta(6) among three strains of FMDV serotype A and two strains of serotype O. While all the viruses could infect cells expressing these integrins, they exhibited different efficiencies of integrin utilization. All the type A viruses used alpha(V)beta(3) and alpha(V)beta(6) with relatively high efficiency, while only one virus utilized alpha(V)beta(1) with moderate efficiency. In contrast, both type O viruses utilized alpha(V)beta(6) and alpha(V)beta(1) with higher efficiency than alpha(V)beta(3). Only low levels of viral replication were detected in alpha(V)beta(5)-expressing cells infected with either serotype. Experiments in which the ligand-binding domains among the beta subunits were exchanged indicated that this region of the integrin subunit appears to contribute to the differences in integrin utilizations among strains. In contrast, the G-H loops of the different viruses do not appear to be involved in this phenomenon. Thus, the ability of the virus to utilize multiple integrins in vitro may be a reflection of the use of multiple receptors during the course of infection within the susceptible host.

Project description:BACKGROUND: Foot-and-mouth disease virus (FMDV) initiates infection via recognition of one of at least four cell-surface integrin molecules ?v?1, ?v?3, ?v?6, or ?v?8 by a highly conserved Arg-Gly-Asp (RGD) amino acid sequence motif located in the G-H loop of VP1. Within the animal host, the ?v?6 interaction is believed to be the most relevant. Sub-neutralizing levels of soluble secreted ?v?6 (ss?v?6) was used as a selective pressure during passages in vitro to explore the plasticity of that interaction. RESULTS: Genetically stable soluble integrin resistant (SIR) FMDV mutants derived from A24 Cruzeiro were selected after just 3 passages in cell culture in the presence of sub-neutralizing levels of ss?v?6. SIR mutants were characterized by: replication on selective cell lines, plaque morphology, relative sensitivity to ss?v?6 neutralization, relative ability to utilize ?v?6 for infection, as well as sequence and structural changes. All SIR mutants maintained an affinity for ?v?6. Some developed the ability to attach to cells expressing heparan sulfate (HS) proteoglycan, while others appear to have developed affinity for a still unknown third receptor. Two classes of SIR mutants were selected that were highly or moderately resistant to neutralization by ss?v?6. Highly resistant mutants displayed a G145D substitution (RGD to RDD), while moderately resistant viruses exhibited a L150P/R substitution at the conserved RGD + 4 position. VP1 G-H loop homology models for the A-type SIR mutants illustrated potential structural changes within the integrin-binding motif by these 2 groups of mutations. Treatment of O1 Campos with ss?v?6 resulted in 3 SIR mutants with a positively charged VP3 mutation allowing for HS binding. CONCLUSIONS: These findings illustrate how FMDV particles rapidly gain resistance to soluble receptor prophylactic measures in vitro. Two different serotypes developed distinct capsid mutations to circumvent the presence of sub-neutralizing levels of the soluble cognate receptor, all of which resulted in a modified receptor tropism that expanded the cell types susceptible to FMDV. The identification of some of these adaptive mutations in known FMDV isolates suggests these findings have implications beyond the cell culture system explored in these studies.

Project description:We have previously reported that Foot-and-mouth disease virus (FMDV), which is virulent for cattle and swine, can utilize the integrin alpha(v)beta(3) as a receptor on cultured cells. Since those studies were performed with the human integrin, we have molecularly cloned the bovine homolog of the integrin alpha(v)beta(3) and have compared the two receptors for utilization by FMDV. Both the alpha(v) and beta(3) subunits of the bovine integrin have high degrees of amino acid sequence similarity to their corresponding human subunits in the ectodomains (96%) and essentially identical transmembrane and cytoplasmic domains. Within the putative ligand-binding domains, the bovine and human alpha(v) subunits have a 98.8% amino acid sequence similarity while there is only a 93% similarity between the beta(3) subunits of these two species. COS cell cultures, which are not susceptible to FMDV infection, become susceptible if cotransfected with alpha(v) and beta(3) subunit cDNAs from a bovine or human source. Cultures cotransfected with the bovine alpha(v)beta(3) subunit cDNAs and infected with FMDV synthesize greater amounts of viral proteins than do infected cultures cotransfected with the human integrin subunits. Cells cotransfected with a bovine alpha(v) subunit and a human beta(3) subunit synthesize viral proteins at levels equivalent to those in cells expressing both human subunits. However, cells cotransfected with the human alpha(v) and the bovine beta(3) subunits synthesize amounts of viral proteins equivalent to those in cells expressing both bovine subunits, indicating that the bovine beta(3) subunit is responsible for the increased effectiveness of this receptor. By engineering chimeric bovine-human beta(3) subunits, we have shown that this increase in receptor efficiency is due to sequences encoding the C-terminal one-third of the subunit ectodomain, which contains a highly structured cysteine-rich repeat region. We postulate that amino acid sequence differences within this region may be responsible for structural differences between the human and bovine beta(3) subunit, leading to more efficient utilization of the bovine receptor by this bovine pathogen.

Project description:Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for the potential use of such mutants in improving the stability of SAT2 vaccines in countries where FMD is endemic, which rely heavily on the maintenance of the cold chain, with potential improvement to the duration of immune responses.

Dataset Information

A continuous bovine kidney cell line constitutively expressing bovine ?v?6 integrin has increased susceptibility to foot-and-mouth disease virus.

Publications

A continuous bovine kidney cell line constitutively expressing bovine αvβ6 integrin has increased susceptibility to foot-and-mouth disease virus.

Similar Datasets

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