**3. Results and discussion**

#### **3.1 Epidemiology**

Scientific publications on the infection of humans with the FMDV began to appear as early as 1869, 1872 [22, 23]. After some time, seven different serotypes of FMDV were identified, which have a different distribution. Some serotypes have a restricted geographical distribution, for example, Asia-1, whereas others, notably serotype O, occurred in many different regions [24].

FMDV is a very dangerous disease, and the data from the Pakistan showed that mortality due to the dominant FMDV serotype "O" was from 7.74 to 21.61% [25].

Studies in Nigeria showed a high prevalence of FMDV in the wild. Thus, the incidence of cattle ranges from 39.7 to 72.8% (in various animal breeds). Also, antibodies to FMDV were found in waterbucks, elephant, wildebeests, and other animals [26]. It is important to note that the protective properties of vaccines are effective only against infection with the same subtype [24].

#### **3.2 Genetic analysis**

The following is an analysis of the variability of the nucleotide sequences of various isolates for each of the virus serotypes (the list of isolates is shown in **Table 1**). For ease of orientation in the nucleotide sequence of the genomes of the various isolates of FMDV serotype A, the designations will be indicated relative to the isolate Zambia/90 (GenBank ID MH053307). For orientation in the nucleotide sequence of the genomes of various isolates of FMDV serotype Asia-1, designations will be indicated relative to isolate BAN/TA/Ma-167/2013 (GenBank ID MF782478). For orientation in the nucleotide sequence of the genomes of various isolates of FMDV serotype C, designations will be indicated relative to strain C-S8p200 (GenBank ID FJ824812). For orientation in the nucleotide sequence of the genomes of various isolates of FMDV serotype O, designations will be indicated relative to isolate UGA/3/2002 (GenBank ID MH053318). For orientation in the nucleotide sequence of the genomes of various isolates of FMDV SAT serotypes (1, 2, and 3), designations will be indicated with respect to SAT 3 serotype, isolate ZIM/P27/90 (DSA-31) (GenBank ID MH053352). Orientation in the nucleotide sequence of the genomes of various serotypes of FMDV, when analyzing the nucleotide sequence encoding the pathogenicity factor of the virus, will be labeled with respect to serotype A, isolate 4235 (GenBank ID JN099688). Analysis of the genomes of all serotypes is aimed at identifying the universal locus (marker nucleotide sequence), which is available in all FMDV serotypes. The marker locus should have minimal variability (minimum number of nucleotide substitutions in the annealing region of primers and probes). The size of the marker locus should not exceed 200 bp (base pair).

The identification of homologies among various types of virus, within the locus encoding the pathogenicity factor "VP1," is aimed at determining the locus, during amplification of which the maximum number of isolates (or all isolates) will be detected in all serotypes of FMDV. This nucleotide sequence is part of the nucleotide sequence encoding a viral capsid. The nucleotide sequence is located in the genome of the virus in the area from 2703 to 3353 bp. There were no significant homologies at this locus (40.5% homology), and the FMDV indication for this locus is not informative.

Further analysis was aimed at identifying homologies among the most abundant (largest number strains/isolates) serotype of the virus type, namely type O (179 strains/isolates). In the analysis, sequences were selected in which no more than two nucleotide substitutions were found in each region of the generation of oligonucleotide seeds (for different isolates). The first locus, in which it is possible to design oligonucleotide seeds, is localized in the region from 4132 to 4292 bp, while the maximum variability was observed in the strain with ID HQ412603 and isolate with ID AY593813. The second locus, in which it is possible to design oligonucleotide seeds, is localized in the region from 7810 to 7908 bp, while the maximum variability was observed in the isolate with ID KF985189; and the third locus, in which it is possible to design oligonucleotide seeds, is localized in the region from 7913 to 8043 bp; no significantly variable strains/isolates were detected in the nucleotide sequence of the locus.

Further homology was detected among the next most numerous virus strains/isolates, type A (117 strains/isolates). Homology was detected at three

**71**

*Genetic Polymorphisms of Foot-and-Mouth Disease Virus*

loci, which, based on the results of the analysis of type O virus, were able to design oligonucleotide seeds. By analyzing the first locus, which is localized in the region from 4151 to 4310 bp, one isolate with three nucleotide substitutions was identified in the region of the generation of the reverse primer (AY593793). Analysis of the second locus located in the region from 7829 to 7926 bp revealed three variable isolates (KC588943, KY322678, and KY404934). Analysis of the third locus located in the region from 7933 to 8061 bp revealed one isolate (MG725874) with three nucleotide substitutions in the region of generation of

The identification of homologies among virus isolates, type Asia-1 (59 strains/ isolates), was continued at the same three loci. By analyzing the first locus located in the region from 4162 to 4321 bp, one isolate (JN006720) with three nucleotide substitutions was detected in the region of generation of the oligonucleotide probe. The second locus, located in the region from 7840 to 7937 bp, was characterized by the variability of three nucleotides in the region of generation of the oligonucleotide probe in one strain (EF149009). Analysis of the third locus located in the region from 7944 to 8072 bp revealed polymorphism in the region of generation of the

Virus isolates, type C (23 strains/isolate), were further analyzed for homology. The first locus is localized in the region from 4100 to 4259 bp, with maximum variability observed in six virus isolates (AY593804, AY593810, KM268897, MH053308, MH 053309, and MH053310). The second locus, localized in the region from 7778 to 7875 bp, with maximum variability observed in three virus isolates (AY593810, MH053308, and MH053310). And the third locus, located in the region from 7882 to 8010 bp, in the nucleotide sequence of which no significantly variable strains/

The identification of homologies among virus isolates, type SAT, was carried out for a total of 74 strains/isolates for all three variants (SAT1, SAT2, and SAT3). The first locus is localized in the region from 4137 to 4296 bp, with maximum variability observed in more than 21 strain/isolates of the virus. The second locus is localized in the region from 7812 to 7909 bp, while the maximum variability was observed in 23 virus strains/isolates. And the third locus, localized in the region from 7916 to 8044 bp, with the maximum variability observed in six isolates of the virus (JX014255, JX014256, KC440884, MF678823, MF678824, and MF678825 were found to have identical nucleic substitutions, and the presented isolates had identical nucleotide sequences at the analyzed locus). An additional probe allows minimizing the effect of nucleotide substitutions on the FMDV indication (the nucleotide composition of the probe contains three substitutions characteristic of

Thus, the identification of homology among strains/isolates of all FMDV serotypes allowed us to determine the locus with minimal variability (in the text, this is the third locus). At this locus, oligonucleotides are complementary to the following positions in the GTA/3/2002 virus genome (GenBank ID MH053318): forward primer 7913–7934 bp, reverse primer 8026–8043 bp, and probe 7988–8024 bp.

All oligonucleotide seeds were analyzed for specificity in the nBLAST software utility. As a result of the analysis, the high specificity of the analyzed nucleotide sequences to the FMDV genome was established. In a more detailed analysis, excluding the nucleotide sequences of the FMDV genome in the search parameters, the complementarity of the forward and reverse primers was found for only one genome, *Triticum aestivum* (soft wheat), and more precisely, its second chromosome (GenBank ID LS480641.1); amplification is not possible in this case because of the large distance between the primers (527, 987, and 115 bp). The nucleotide sequences of the probes showed an identity only with the FMDV genome.

oligonucleotide probe in two virus isolates (DQ989310, DQ989311).

*DOI: http://dx.doi.org/10.5772/intechopen.93351*

the oligonucleotide probe.

isolates were detected.

the above isolates).

#### *Genetic Polymorphisms of Foot-and-Mouth Disease Virus DOI: http://dx.doi.org/10.5772/intechopen.93351*

*Some RNA Viruses*

**3.2 Genetic analysis**

Studies in Nigeria showed a high prevalence of FMDV in the wild. Thus, the incidence of cattle ranges from 39.7 to 72.8% (in various animal breeds). Also, antibodies to FMDV were found in waterbucks, elephant, wildebeests, and other animals [26]. It is important to note that the protective properties of vaccines are

The following is an analysis of the variability of the nucleotide sequences of various isolates for each of the virus serotypes (the list of isolates is shown in **Table 1**). For ease of orientation in the nucleotide sequence of the genomes of the various isolates of FMDV serotype A, the designations will be indicated relative to the isolate Zambia/90 (GenBank ID MH053307). For orientation in the nucleotide sequence of the genomes of various isolates of FMDV serotype Asia-1, designations will be indicated relative to isolate BAN/TA/Ma-167/2013 (GenBank ID MF782478). For orientation in the nucleotide sequence of the genomes of various isolates of FMDV serotype C, designations will be indicated relative to strain C-S8p200 (GenBank ID FJ824812). For orientation in the nucleotide sequence of the genomes of various isolates of FMDV serotype O, designations will be indicated relative to isolate UGA/3/2002 (GenBank ID MH053318). For orientation in the nucleotide sequence of the genomes of various isolates of FMDV SAT serotypes (1, 2, and 3), designations will be indicated with respect to SAT 3 serotype, isolate ZIM/P27/90 (DSA-31) (GenBank ID MH053352). Orientation in the nucleotide sequence of the genomes of various serotypes of FMDV, when analyzing the nucleotide sequence encoding the pathogenicity factor of the virus, will be labeled with respect to serotype A, isolate 4235 (GenBank ID JN099688). Analysis of the genomes of all serotypes is aimed at identifying the universal locus (marker nucleotide sequence), which is available in all FMDV serotypes. The marker locus should have minimal variability (minimum number of nucleotide substitutions in the annealing region of primers and probes).

effective only against infection with the same subtype [24].

The size of the marker locus should not exceed 200 bp (base pair).

The identification of homologies among various types of virus, within the locus encoding the pathogenicity factor "VP1," is aimed at determining the locus, during amplification of which the maximum number of isolates (or all isolates) will be detected in all serotypes of FMDV. This nucleotide sequence is part of the nucleotide sequence encoding a viral capsid. The nucleotide sequence is located in the genome of the virus in the area from 2703 to 3353 bp. There were no significant homologies at this locus (40.5% homology), and the FMDV indication for this locus is not

Further analysis was aimed at identifying homologies among the most abundant (largest number strains/isolates) serotype of the virus type, namely type O (179 strains/isolates). In the analysis, sequences were selected in which no more than two nucleotide substitutions were found in each region of the generation of oligonucleotide seeds (for different isolates). The first locus, in which it is possible to design oligonucleotide seeds, is localized in the region from 4132 to 4292 bp, while the maximum variability was observed in the strain with ID HQ412603 and isolate with ID AY593813. The second locus, in which it is possible to design oligonucleotide seeds, is localized in the region from 7810 to 7908 bp, while the maximum variability was observed in the isolate with ID KF985189; and the third locus, in which it is possible to design oligonucleotide seeds, is localized in the region from 7913 to 8043 bp; no significantly variable strains/isolates were detected in the nucleotide

Further homology was detected among the next most numerous virus strains/isolates, type A (117 strains/isolates). Homology was detected at three

**70**

informative.

sequence of the locus.

loci, which, based on the results of the analysis of type O virus, were able to design oligonucleotide seeds. By analyzing the first locus, which is localized in the region from 4151 to 4310 bp, one isolate with three nucleotide substitutions was identified in the region of the generation of the reverse primer (AY593793). Analysis of the second locus located in the region from 7829 to 7926 bp revealed three variable isolates (KC588943, KY322678, and KY404934). Analysis of the third locus located in the region from 7933 to 8061 bp revealed one isolate (MG725874) with three nucleotide substitutions in the region of generation of the oligonucleotide probe.

The identification of homologies among virus isolates, type Asia-1 (59 strains/ isolates), was continued at the same three loci. By analyzing the first locus located in the region from 4162 to 4321 bp, one isolate (JN006720) with three nucleotide substitutions was detected in the region of generation of the oligonucleotide probe. The second locus, located in the region from 7840 to 7937 bp, was characterized by the variability of three nucleotides in the region of generation of the oligonucleotide probe in one strain (EF149009). Analysis of the third locus located in the region from 7944 to 8072 bp revealed polymorphism in the region of generation of the oligonucleotide probe in two virus isolates (DQ989310, DQ989311).

Virus isolates, type C (23 strains/isolate), were further analyzed for homology. The first locus is localized in the region from 4100 to 4259 bp, with maximum variability observed in six virus isolates (AY593804, AY593810, KM268897, MH053308, MH 053309, and MH053310). The second locus, localized in the region from 7778 to 7875 bp, with maximum variability observed in three virus isolates (AY593810, MH053308, and MH053310). And the third locus, located in the region from 7882 to 8010 bp, in the nucleotide sequence of which no significantly variable strains/ isolates were detected.

The identification of homologies among virus isolates, type SAT, was carried out for a total of 74 strains/isolates for all three variants (SAT1, SAT2, and SAT3). The first locus is localized in the region from 4137 to 4296 bp, with maximum variability observed in more than 21 strain/isolates of the virus. The second locus is localized in the region from 7812 to 7909 bp, while the maximum variability was observed in 23 virus strains/isolates. And the third locus, localized in the region from 7916 to 8044 bp, with the maximum variability observed in six isolates of the virus (JX014255, JX014256, KC440884, MF678823, MF678824, and MF678825 were found to have identical nucleic substitutions, and the presented isolates had identical nucleotide sequences at the analyzed locus). An additional probe allows minimizing the effect of nucleotide substitutions on the FMDV indication (the nucleotide composition of the probe contains three substitutions characteristic of the above isolates).

Thus, the identification of homology among strains/isolates of all FMDV serotypes allowed us to determine the locus with minimal variability (in the text, this is the third locus). At this locus, oligonucleotides are complementary to the following positions in the GTA/3/2002 virus genome (GenBank ID MH053318): forward primer 7913–7934 bp, reverse primer 8026–8043 bp, and probe 7988–8024 bp.

All oligonucleotide seeds were analyzed for specificity in the nBLAST software utility. As a result of the analysis, the high specificity of the analyzed nucleotide sequences to the FMDV genome was established. In a more detailed analysis, excluding the nucleotide sequences of the FMDV genome in the search parameters, the complementarity of the forward and reverse primers was found for only one genome, *Triticum aestivum* (soft wheat), and more precisely, its second chromosome (GenBank ID LS480641.1); amplification is not possible in this case because of the large distance between the primers (527, 987, and 115 bp). The nucleotide sequences of the probes showed an identity only with the FMDV genome.
