Evaluation of FTA cards as a laboratory and field sampling device for the detection of foot-and-mouth disease virus and serotyping by RT-PCR and real-time RT-PCR

Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for serodiagnosis pose a major problem in a tropical country like India, where there is maximum temperature fluctuation. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. The present study evaluated the usefulness of FTA Classic Cards for the collection, shipment, storage and identification of the FMDV genome by RT-PCR and real-time RT-PCR. The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards a useful option for transport of FMDV genome for identification and serotyping. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular document transport methods. Live virus cannot be isolated from samples collected in FTA cards, which is a limitation. This property can be viewed as an advantage as it limits the risk of transmission of live virus.     

A highly sensitive and specific multiplex RT-PCR to detect foot-and-mouth disease virus in tissue and food samples

Summary Three sets of primers to detect foot-and-mouth disease virus (FMDV) using multiplex RT-PCR were designed based on several reference nucleotide sequences, and their reaction conditions were determined. By testing ten-fold serial dilutions of FMDV, the sensitivity of multiplex RT-PCR is 100 times higher than conventional RT-PCR. Meanwhile, its specificity was confirmed compared with other related vesicular disease viruses. Furthermore, 30 field samples from different animals were tested, and the results supported the method’s potential applications in routine veterinary quarantine and epidemic surveillance of FMDV. Correspondence: Zai-Xin Liu, Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiologic Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China     

Inhibitors of foot-and-mouth disease virus

Summary In suspended secondary calf kidney cells infected with foot-and-mouth disease virus (FMDV) the temperature range for optimal virus growth is shifted down by 3 to 5° C in the presence of 1–2 mM guanidine. For some virus strains this shift is so effective that at infraoptimal temperatures virus yield in guanidine-treated cells exceeds that of the corresponding control by more than one log₁₀. On the contrary, at supraoptimal temperatures inhibition of virus growth by the drug is strongly enhanced.At a concentration of 1 to 2 mM guanidine virus yield reduction or enhancement is based on a decrease or increase, respectively, of the number of virus producing cells (infective centers; I.C.), while virus yield per I.C. is less affected.Besides this thermomimetic effect virus production is inhibited by guanidine depending on the concentration of this substance.A mutant of FMDV strain O₁L, resistant to 4.2 mM guanidine, did not differ from the original virus in its antigenic behaviour in the passive immunohemolysis test.With 9 Figures     

Inhibitors of foot-and-mouth disease virus

Summary The growth cycle of subtype C₀ of foot-and-mouth disease virus (FMDV) was studied comparing the wild type virus and a cold-adapted, temperature-sensitive (ts) mutant. Single cycle growth of these viruses in secondary calf kidney cells is obtained if cells are suspended in a modified Eagle's medium containing 1mm EDTA. By removal of divalent cations from cells, cellular RNA- and proteinsynthesis is reduced to 50 per cent of controls in 4 hours at 37° C. The exponential phase of the viral cycle is extended and the number of infective centers (I.C.) reduced to one third. Furthermore, the exponential phase of the cycle is affected by temperature and interferon, while the linear phase is strictly temperature-dependent, in the range from 23° to 41° C. Virus increase at the exponential phase is mainly determined by asynchronous formation of I.C. With strain C₀-ts, I.C. formation is reduced at supraoptimal temperature, 37° C, but not virus yield per cell. At infra-optimal temperature, 23° C, virus yield per I.C. is diminished, but no reduction in number of I. C. was found. The number of I.C. of wild type virus is, however, reduced at the relative infra-optimal temperature, 28° C. Pretreatment of cells with low concentrations of interferon led to a reduction of I.C. without affecting virus yield per I.C.Results show that formation of I.C. is a virusspecific function which is affected by different conditions and probably can be altered by mutation of the viral genome.     

Evolution of foot-and-mouth disease virus

Foot-and-mouth disease virus evolution is strongly influenced by high mutation rates and a quasispecies dynamics. Mutant swarms are subjected to positive selection, negative selection and random drift of genomes. Adaptation is the result of selective amplification of subpopulations of genomes. The extent of adaptation to a given environment is quantified by a relative fitness value. Fitness values depend on the virus and its physical and biological environment. Generally, infections involving large population passages result in fitness gain and population bottlenecks lead to fitness loss. Very different types of mutations tend to accumulate in the foot-and-mouth disease virus (FMDV) genome depending on the virus population size during replication. Quasispecies dynamics predict higher probability of success of antiviral strategies based on multivalent vaccines and combination therapy, and this has been supported by clinical and veterinary practice. Quasispecies suggest also new antiviral strategies based on virus entry into error catastrophe, and such procedures are under investigation. Studies with FMDV have contributed to the understanding of quasispecies dynamics and some of its biological implications.     

Cores in foot-and-mouth disease virus

Foot-and-mouth disease virions were dissociated with ammonium acetate and observed with the electron microscope. The major products of viral disassembly were 12 S viral subunits or skullcaps and cores. Cores appeared as spherical structures and were relatively unstable upon spreading, freezing and thawing, or treatment at low pH. Upon addition of dextran sulfate to the incubation mixture, it was possible to analyze cores by ultracentrifugation on glycerol gradients. Cores sedimented at 45 S but large amounts of filamentous structures, possibly unfolded cores, were also found at slower rates. The results of this work indicated that cores are predominantly formed by genomic RNA folded in a compact spherical configuration.     

Molecular epidemiology of foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is the most economically important veterinary pathogen due to its highly infectious nature, ability to cause persistent infections and long term effects on the condition and productivity of the many animal species it affects. Countries which have the disease have many trade restrictions placed upon them. In the last 15 years there have been significant advances in the understanding of FMD epidemiology. These have largely been due to the application of the molecular biological techniques of polymerase chain-reaction amplification and nucleotide sequencing. In the World Reference Laboratory for FMD (Pirbright, UK), a large sequence database has been built up. This database has been used to aid in the global tracing of virus movements. It has been possible to genetically group many FMDV's based on their geographic origin and this has led to their being referred to as topotypes. The implications of this are that inter-regional spread of viruses can often be easily recognised and any evolutionary changes which subsequently occur can be monitored. Using these techniques, for the first time, we have been able to unequivocally show the recent pandemic spread of a FMDV type O strain through the whole of Asia and into Africa and Europe. This type of surveillance will become increasingly important as further globalisation of markets occurs. An increased understanding of how FMDV strains move between geographic regions will play a pivotal role in the development of future disease control strategies.     

Temperature-dependent interferon-sensitivity of foot-and-mouth disease virus

Summary Several strains of foot-and-mouth disease virus (FMDV), types O, A, and C, grown in primary or secondary calf kidney cell cultures had an interferon-sensitivity which varied with incubation temperature. Some wild-type strains growing optimally at 37° C showed an increased interferon-sensitivity at 28° C. Strain C_L was more sensitive at both infra- and supraoptimal growth temperature. Temperature-sensitive mutants with optimal replication at 28° C were more sensitive at 37° C. Interferon-sensitivity moreover varied greatly among the virus strains tested even at optimal temperature. Results are interpreted by the hypothesis that the interferon-sensitivity depends on a virus specific product, presumably a protein, with a reduced optimal range of function (compared with virion formation) which acts as anti-interferon. Possible relationships with virulence of FMDV and temperature-sensitivity of viral proteins are discussed.     

Thermal stability of foot-and-mouth disease virus

The thermal stabilities of 146S component of seven strains of foot-and-mouth disease virus were found to differ considerably. Inactivation of infectivity with acetylethyleneimine (AEI) reduced the thermal stabilities of all but one of the viruses. Treatment of AEI inactivated and control virus preparations with glutaraldehyde stabilized 146S particles to a considerable extent, whereas treatment with dimethyl suberimidate was less effective. In similar experiments with 75S, natural empty particles, the thermal stabilities were lower than those of the corresponding 146 S particles. Treatment of 75S particles with AEI appeared to have no direct effect on the protein-protein interactions involved in 75S capsid integrity. As with 146S particles, glutaraldehyde stabilized 75S particles.     

Serotype-independent detection of foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) causes a highly contagious vesicular disease affecting cloven hoofed animals and is considered the most economically important disease worldwide. Recent FMD outbreaks in Europe and Taiwan and the associated need for rapid diagnostic turnaround have identified limitations that exist in current diagnostic capabilities. To aid improved diagnosis, a serotype-independent FMDV antigen capture assay was developed using antibodies directed against a highly conserved cross-reactive protein fragment (1AB') located within the structural protein 1AB. Cattle sera raised against all 7 serotypes of FMDV bound purified 1AB' demonstrating its immunogenicity in infected animals. Polyclonal anti-1AB' antiserum was produced in chickens and applied as a universal detector of FMDV antigen. Western blot analysis and ELISA both demonstrated that anti-1AB' serum could recognize FMDV antigens independent of serotype. Two recently characterized anti-FMDV monoclonal antibodies were also evaluated for their ability to capture FMDV antigen independently of serotype. When used in combination with chicken anti-1AB' antibodies in an antigen capture ELISA format, all serotypes of FMDV were detected. These data represent the first demonstration of the use of serotype-independent FMDV antigen capture reagents which may enable the development of rapid laboratory based assays or perhaps more significantly, rapid field-based pen-side or point of entry border control diagnostic tests.     

Development of a novel real-time RT-PCR assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues

Pigs are more difficult to immunise and more variable in their response to foot-and-mouth disease (FMD) than other livestock species. This has important consequences for FMD control during both prophylactic vaccination programmes in endemic situations and when emergency vaccination may be used as an adjunct to stamping out during outbreaks in countries normally free from the disease. The rapid and effective control of FMD in pigs is especially important in regions of high pig density since infected pigs have the potential to generate plumes of airborne virus and spread infection beyond the immediate control area. Increased knowledge of the kinetics of FMDV replication in pigs, especially in their respiratory tracts, could create opportunities for strategies to improve FMD vaccines for pigs. A fluorogenic TaqMan RT-PCR assay specific for the IRES (internal ribosome entry site) sequence of the O(1) Kaufbeuren/Lausanne strain of FMDV has been developed for this purpose. The assay had a sensitivity of 0.1 TCID(50)/ml, and a dynamic range of at least eight orders of magnitude. It was found that an established method of quantitation, relative to a housekeeping gene, exhibited two significant shortcomings when applied to a set of 16 anatomically highly diverse solid tissues: (i) tissue differences in housekeeping gene expression caused errors of up to 60-fold in estimated FMDV concentrations; and (ii) variability in total RNA yields caused unpredictable saturation of RT reactions, which in turn caused errors of up to 250-fold in the estimated FMDV concentration. A novel quantitation strategy, designated the C(t)(min) method, was developed to overcome these problems. The C(t)(min) method was based on the the RT-PCR examination of a dilution series of spectrophotometrically quantitated total RNA, spanning the optimum for the RT-PCR system used. The new method was influenced minimally by any tissue-specific RT-PCR inhibitors and was used to determine FMDV concentrations in tissues from four experimentally infected pigs. The results suggest that the lungs play a less important role in the replication of FMDV in pigs than was thought previously.     

Structure of foot-and-mouth disease virus capsid.

Electron microscope images of negative-stained particles of FMDV have been analyzed. The viral capsid appeared to be a pliant structure composed of monomers having an average width of about 2.5 nm and a length of 4 nm. Upon capsid disruption, a sequential and nonrandom detachment of skullcaps was observed. These skullcaps are presumably clusters of monomers, and they could be similar to those predicted for the 13–14 S subunits described for other picornaviruses. If this were the case, each FMDV capsid would be constituted by twelve skullcaps, making a total of 180 monomeric units for the outer part of the protein shell. However, the FMDV skullcap, compared with other picornavirus subunits, has a lower sedimentation coefficient (12 S) and shows high stability upon treatment with urea without production of the 4–5 S component characteristic of other related viruses.