Cell culture propagation of foot-and-mouth disease virus: adaptive amino acid substitutions in structural proteins and their functional implications.
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ABSTRACT:
SUBMITTER: Dill V
PROVIDER: S-EPMC6957568 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Infection by viruses depends on a balance between capsid stability and dynamics. This study investigated biologically and biotechnologically relevant aspects of the relationship in foot-and-mouth disease virus (FMDV) between capsid structure and thermostability and between thermostability and infectivity. In the FMDV capsid, a substantial number of amino acid side chains at the interfaces between pentameric subunits are charged at neutral pH. Here a mutational analysis revealed that the essential role for virus infection of most of the 8 tested charged groups is not related to substantial changes in capsid protein expression or processing or in capsid assembly or stability against a thermally induced dissociation into pentamers. However, the positively charged side chains of R2018 and H3141, located at the interpentamer interfaces close to the capsid 2-fold symmetry axes, were found to be critical both for virus infectivity and for keeping the capsid in a state of weak thermostability. A charge-restoring substitution (N2019H) that was repeatedly fixed during amplification of viral genomes carrying deleterious mutations reverted both the lethal and capsid-stabilizing effects of the substitution H3141A, leading to a double mutant virus with close to normal infectivity and thermolability. H3141A and other thermostabilizing substitutions had no detectable effect on capsid resistance to acid-induced dissociation into pentamers. The results suggest that FMDV infectivity requires limited local stability around the 2-fold axes at the interpentamer interfaces of the capsid. The implications for the mechanism of genome uncoating in FMDV and the development of thermostabilized vaccines against foot-and-mouth disease are discussed.IMPORTANCE This study provides novel insights into the little-known structural determinants of the balance between thermal stability and instability in the capsid of foot-and-mouth disease virus and into the relationship between capsid stability and virus infectivity. The results provide new guidelines for the development of thermostabilized empty capsid-based recombinant vaccines against foot-and-mouth disease, one of the economically most important animal diseases worldwide.
| S-EPMC6498042 | biostudies-literature
Project description:Foot-and-mouth disease virus (FMDV) is endemic in many parts of the world. Vaccination is an important control measure, limits viral spread, and can help to eradicate the disease. However, vaccination programs are cost-intensive because of the short shelf life of vaccines and the need for frequent re-vaccination. Animal-component-free (ACF) or chemically defined media (CDM) at high cell densities are a promising approach for the production of inexpensive high-quality vaccines, but the occurrence of cell density effects has been reported for various virus-cell systems in vaccine production. For FMDV, the use of CDM or ACF media for vaccine production has not been studied and no information about cell density effects is available. This work describes the propagation of FMDV in ACF or in CDM. Cells were grown at increasing cell densities and either 100% media exchange or addition of 30% fresh media was performed before infection with FMDV. Increasing cell densities reduced the viral titer and increased yield variability in all media except BHK300G. This effect can be mitigated by performing a 100% media exchange before infection or when using the controlled environment of a bioreactor. The media composition and also a fragile relationship between virus and cell metabolism seem to be causal for that phenomenon.
| S-EPMC6631978 | biostudies-literature
Project description:The integrins function as the primary receptor molecules for the pathogenic infection of foot-and-mouth disease virus (FMDV) in vivo, while the acquisition of a high affinity for heparan sulfate (HS) of some FMDV variants could be privileged to facilitate viral infection and expanded cell tropism in vitro. Here, we noted that a BHK-adapted Cathay topotype derivative (O/HN/CHA/93tc) but not its genetically engineered virus (rHN), was able to infect HS-positive CHO-K1 cells and mutant pgsD-677 cells. There were one or three residue changes in the capsid proteins of O/HN/CHA/93tc and rHN, as compared with that of their tissue-originated isolate (O/HN/CHA/93wt). The phenotypic properties of a set of site-directed mutants of rHN revealed that E83K of VP1 surrounding the fivefold symmetry axis was necessary for the integrin-independent infection of O/HN/CHA/93tc. L80 in VP2 was essential for the occurrence of E83K in VP1 during the adaptation of O/HN/CHA/93wt to BHK-21 cells. L80M in VP2 and D138G in VP1 of rHN was deleterious, which could be compensated by K83R of VP1 for restoring an efficient infection of integrin-negative CHO cell lines. These might have important implications for understanding the molecular and evolutionary mechanisms of the recognition and binding of FMDV with alternative cellular receptors.
| S-EPMC7650640 | biostudies-literature
Project description:BackgroundSuspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease.MethodsChanges to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation.ResultsCapsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A24-2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media.ConclusionThe selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization profile were unaffected, but the 146S particle yield differed for one of the tested serotypes.
| S-EPMC5857075 | biostudies-literature
Project description:Analysis of full-genome sequences was previously used to trace the origin and transmission pathways of foot-and-mouth disease virus (FMDV) outbreaks in the UK in 2001 and 2007. Interpretation of these data was sometimes at variance with conventional epidemiological tracing, and was also used to predict the presence of undisclosed infected premises that were later discovered during serological surveillance. Here we report the genome changes associated with sequential passage of a highly BHK-21-cell-adapted (heparan sulphate-binding) strain of FMDV arising from two independent transmission chains in cattle. In vivo virus replication rapidly selected for a wild-type variant with an amino acid substitution at VP3(56). Full-genome sequence analysis clearly demonstrated sequence divergence during parallel passage. The genetic diversity generated over the course of infection and the rate at which these changes became fixed and were transmitted between cattle occurred at a rate sufficient to enable reliable tracing of transmission pathways at the level of the individual animal. However, tracing of transmission pathways was only clear when sequences from epithelial lesions were compared. Sequences derived from oesophageal-pharyngeal scrapings were problematic to interpret, with a varying number of ambiguities suggestive of a more diverse virus population. These findings will help to correctly interpret full-genome sequence analyses to resolve transmission pathways within future FMDV epidemics.
| S-EPMC3542721 | biostudies-literature
Project description:BackgroundFoot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV.MethodsTwo serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice.ResultsThree amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain.ConclusionsThese results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine.
| S-EPMC3489780 | biostudies-literature
Project description:The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D(pol)) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D(pol) coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp(26)?Glu substitution in a beta sheet located within a small groove of the 3D(pol) protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.
| S-EPMC3232396 | biostudies-literature
Project description:The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time.
| S-EPMC241137 | biostudies-other
Project description:The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3, of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed using Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008 and 3060 and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1, VP2, and VP3 (VP1-3), as well as between the internal VP4 and the three external capsid proteins of FMDV, might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance compared to the Y2079-encoding viruses. Interestingly, none of all the 16 rescued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s) and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N terminus of VP4 and several potential domains of VP1-3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity, as well as acid sensitivity correlated with NH4Cl resistance, whereas there was no inevitable correlation in viral plaque and acid-sensitive phenotypes. The high affinity of non-integrin-dependent FMDVs for heparin might be explained by the differences in structures of heparan sulfate proteoglycans on the surfaces of different cell lines. These results may contribute to our understanding of the distinct phenotypic properties of FMDV in vitro and in vivo.
| S-EPMC6430551 | biostudies-literature
Project description:BackgroundFoot-and-mouth disease is a highly contagious and economically devastating disease with endemic occurrence in many parts of the world. Vaccination is the method of choice to eradicate the disease and to limit the viral spread. The vaccine production process is based on mammalian cell culture, in which the viral yield varies in dependence of the composition of the culture media. For foot-and-mouth disease virus (FMDV), very little is known about the culture media components that are necessary to grow the virus to high titers in cell culture.ResultsThis study examined the influence of increasing concentrations of glucose, glutamine, ammonium chloride and different cell densities on the yield of FMDV. While an excess of glucose or glutamine does not affect the viral yield, increasing cell density reduces the viral titer by a log10 step at a cell density of 3 × 106 cells/mL. This can be mitigated by performing a 100% media exchange before infection of the cells.ConclusionsThe reasons for the diminished viral growth, if no complete media exchange has been performed prior to infection, remain unclear and further studies are necessary to investigate the causes more deeply. For now, the results argue for a vaccine production process with 100% media exchange to reliably obtain high viral titers.
| S-EPMC6555971 | biostudies-literature