A comprehensive analysis of Simple Sequence Repeats in Picornaviruses

doi:10.21203/rs.3.rs-1557265/v1

Genome-wide identification of simple sequence repeats (SSRs) of Picornaviruses was carried out to investigate type, distribution, and potential role in genome evolution. Investigation on 88 Picornavirus species revealed the presence of 2,488 SSRs and 100 compound SSRs. The relative abundance and relative density of SSR varied between 1.953 bp/kb-5.763 bp/kb and 13.39 bp/kb-45.02 bp/kb, while that of cSSR ranged from 0.108 bp/kb-0.636 bp/kb and 1.36 bp/kb-26.84 bp/kb. Regression analysis revealed a significant correlation of genome size and GC content with the incidence of SSRs. Motif duplication pattern, such as (C)-x-(C), (TG)-x-(TG), etc. and self-complementary motifs, such as (GC)-x-(CG), (TC)-x-(AG), etc. were observed in cSSR. Polymorphism analysis revealed that most of the cSSR were prone to instability, followed by consensus motifs. Finally, recombination analysis revealed that the breakpoints were rich in dinucleotide repeat, especially GT. However, further experimental validation is needed to elucidate the correlation between recombination hotspots and microsatellites.

Circos

Microsatellites

Picornaviridae

Polymorphism

Recombination

Non-enveloped virions with icosahedral capsids characterize the Picornaviridae family members. The capsid contains a small, single, and positive-stranded RNA as the genome. According to the International Committee on Taxonomy of Virus (ICTV) 2020, the family consists of 63 genera and 147 species. The family contains a range of human and animal pathogens such as foot and mouth disease virus (FMDV), human enterovirus 71 (EV-71), poliovirus, etc. The picornavirus genome range from 7-8.8 kb in size and share a typical pattern of gene arrangement (Alexandersen et al.,). The 5Ꞌ end of the genome is covalently linked to the small viral protein VPg (2KD size), and 3Ꞌ end is polyadenylated (Flanegan et al., 1977; Lee et al., 1977). The 5Ꞌ end of the genome is further anchored by untranslated region (5Ꞌ UTR), and a 5Ꞌ terminal domain; regulate viral replication and an internal ribosomal entry site (IRES) element that allows cap-independent translation activity (Toyoda et al., 2007). The 3Ꞌ UTR contains a pseudoknot structure involved in regulating replication and ends with a poly-A tail that is heterogeneous in length (Jacobson et al., 1993; Yogo and Wimmer 1972). Following virus entry to the permissible host cell, the cap-independent translation of the viral genome results in a precursor polyprotein synthesis. The precursor polyprotein is further proteolytically cleaved to form several precursor molecules and 10–12 mature proteins (Kitamura et al., 1981). These include structural proteins of the capsid (1A–1D or VP4–VP1) located at the amino-terminal portion of the polypeptide, followed by the nonstructural proteins (2A–2C and 3A–3D). In some genera, the polyprotein starts with a leader (L) peptide followed by VP0 structural protein. Upon synthesis of the viral proteins, the genome replicated in complexes associated with cytoplasmic membranes (Caliguiri and Tamm 1969). During viral genome replication, viral RNA dependent RNA polymerase (RdRp) frequently switches the template, which results in ''replicative recombination''. It not only helps to maintain not only stability but also the variation of picornavirus genomes. These recombination events, coupled with mutation, generate a large ensemble of genetically diverse genomes, contributing to the picornavirus evolution (Agol 2010; Simmonds 2010).

Simple sequence repeats (SSRs), also called microsatellites, are tandem repetitions of relatively short nucleotide motifs. They are distributed ubiquitously in the genome of eukaryotes and prokaryotes. SSRs are relatively rare in smaller viral genomes (Tóth et al., 2000; Mrázek et al., 2007; Chen et al., 2009). The microsatellites may be classified as either simple or compound, depending on the constituent of nucleotide sequences. Two or more microsatellites reside directly adjacent to each other form compound microsatellites, and often, they are interrupted by other repeats (Chambers and MacAvoy 2000). Compound microsatellites have been found in diverse taxa, including viruses, prokaryotes, and eukaryotes (Chen et al., 2012; Kofler et al., 2008). In the human genome, ~ 10% of microsatellites are categorized as compound microsatellites. These contain some highly polymorphic compound repeats, such as (dC-dA)n-(dG-dT)n (Weber 1990). Length variability in compound dinucleotide repeat was also reported in the human genome (Bull et al., 1999). Eukaryotic such as Homo sapiens, Macaca mulatta, Mus musculus, Rattus norvegicus, Ornithorhynchus anatinus, Gallus gallus, Daniorerio, and Drosophila melanogaster genomes show a higher frequency (4–25%) of compound microsatellites (Kofler et al., 2008). At the lower order taxa, Escherichia coli (E. coli) genomes show microsatellite frequency ranging from 1.75–2.85% (Chen et al., 2012). In a similar line, HIV-I isolates show a different pattern of compound microsatellites distribution, where the frequency varies from 0–24.24% for compound microsatellites (Chen et al., 2009; George et al., 2012; Zhao et al., 2011). Further, viral genome studies have unraveled many exciting facts about the distribution pattern, evolution, and regulatory role of microsatellites in the viral genome.

Length polymorphism of microsatellites may bring changes to DNA structure and influence protein functions (Mrázek et al., 2007). This phenomenon could trigger diseases like Huntington’s disease, dentatorubro-pallidoluysian atrophy, hereditary ataxias, and spinobulbar muscular atrophy (Li et al., 2004; Usdin 2008). Microsatellite instability through recombination processes or DNA polymerase slippage during DNA replication causes significant genetic variation (Ellegren 2004). Some reports suggest that recombination events between homologous microsatellites generate compound microsatellites (Jakupciak and Wells 1999). Microsatellites are regarded as recombination hot spots, where polymerase enzymes have a higher affinity for dinucleotide repeat sequences (Biet et al., 1999). Due to its unique characteristics, SSRs plays a significant role in meiotic recombination (Schultes and Szostak 1991; Kirkpatrick et al., 1999). The evolutionary role of microsatellites in various lower order species, including that of viruses, has recently come to the picture (Ellegren 2004; Bagshaw et al., 2008). Studies have identified the presence of microsatellite repeats in many viruses. These include Menovirus (Duke et al., 1990), vesicular stomatitis virus (Barr et al., 1997), hepatitis C virus (Yamada et al., 1996), and human respiratory syncytial virus (Garcia-Barreno et al., 1994). In the present study, we have systematically analyzed the occurrence, size, density, and motif types of different simple and compound microsatellites prevalent in picornaviruses. We also show the correlation between different parameters influencing the distribution of these repeats. We have identified the polymorphism of these microsatellites and hypothesized their functional significance in the viral genome. Further, the recombination breakpoints in the viral genome were identified, following the microsatellites' presence at these sites.

2.1 Genome sequences

The ICTV (2020) classification of the Picornaviridae family comprises 63 genera and 147 species (http://www.ictvonline.org/virusTaxonomy). In this study, we downloaded all 88 available complete genome sequences representing each species from GenBank (http://www.ncbi.nlm.nih.gov) (Table 1). To compare genomic sequences of different lengths, we calculated the relative density (RD) and relative abundance (RA) values. RD is defined as the total length (bp) contributed by each microsatellite per kb of sequence analyzed. In contrast, RA is the number of microsatellites present per kb of the genome (kb).

2.2 Microsatellite identification and investigation

The identification of perfect mono, di, tri, tetra, penta, hexa, and compound microsatellites, IMEx software was used (Mudunuri and Nagarajaram 2007). Microsatellite information was extracted by the ‘Advance-Mode’ of IMEx using the parameters previously utilized for RNA viruses (Chen et al., 2012; Alam et al., 2013). The parameters used are as follows: type of repeat: perfect; repeat size: all; minimum repeat number: 6, 3, 3, 3, 3, 3 for mono, di, tri, tetra, penta and hexanucleotide repeats, respectively. The maximum distance allowed between any two SSRs (dMAX) was ten nucleotides. Other parameters were set as default. Compound microsatellites (cSSR) were not standardized to determine original composition.

2.3 Estimation of the number of expected mononucleotide repeats

We randomly chose mono-nucleotide repeats to evaluate the over or under-represented motifs in the Picornavirus genome, compared the observed and expected number of microsatellites. The expected number of microsatellites composed of Mt (M is the motif of the microsatellite with repeat number of t, and its length is L) in a genome of length G was calculated using the formula given by de Wachter, 1981 (de Wachter 1981). The Z score indicates the statistical significance of the microsatellite representation (Mrázek et al., 2007).

Exp (Mt) = f (M)^t [1- f (M)] [GꞋ (1- f (M)+2L] ………………..... (1)

GꞋ = G-tL-2L+1 …………………………………………………. (2)

Z=O-E/√E ………………………………………………………. (3)

Where Exp (Mt) represents the expected number of microsatellites, and f (M) represents the probability of microsatellite.

2.4 Multiple sequence alignment and identification of polymorphic cSSR

The genomes having cSSR only were considered for identifying polymorphic microsatellites and consensus motifs. Species having more than five strains were considered for this investigation. Sequences were first transferred to BioEdit version 7.2.5 software and aligned by the CLUSTAL W module with some manual editing (Thompson et al., 2003). Finally, the consensus sequences were identified by the procedure followed by George et al., 2015 (George et al., 2015).

2.5 Recombination analysis

To elucidate the potential recombinant sequences and their parents (major and minor), the multiple sequence alignment files formed above were used as input for the RDP4 package (Martin et al., 2015). RDP4 consists of seven recombination detection methods such as RDP (Martin et al., 2015), GENCONV (Padidam et al., 1999), BOOTSCAN (Martin et al., 2005), MAXCHI (Smith 1992), CHIMAERA (Posada and Crandall 2001), SiScan (Gibbs et al., 2000) and 3SEQ (Boni et al. 2007), used to identify recombinant strain within the alignment. A sequence is considered a potential recombinant only if detected as significant (with the p-value cutoff of 0.00001) by at least three methods stated above.

2.6 Statistical analysis

All statistical analysis was performed using Graphpad Prism version 5 (La Jolla, CA, USA). Linear regression was used to reveal the correlation between incidence, RA, RD of simple as well as compound microsatellites with genome size and GC content.

3.1 Number, relative abundance and relative density of SSR in Picornaviruses

Genome-wide screening of 88 available picornavirus genomes revealed a total of 2,488 SSRs distributed across the species (Figure 1, Supplementary file 1). On average, 30 SSRs were observed per genome. The least incidence frequency of 14 was observed in Avisivirus C (V11), and a maximum incidence frequency of 46 was found in Salivirus A (V80) (Table 2). The RD of SSRs was observed, ranging from 13.39 bp/kb in Avisivirus C (V11) to 45.02 bp/kb in Cardiovirus A (V13). Similarly, relative abundance varied from a minimum of 1.953 in Avisivirus C (V11) to a maximum of 5.763 bp/kb in Parechovirus B (V69) (Table 2).

3.2 Number, relative abundance and relative density of cSSR in picornaviruses

The genome-wide scan revealed 1–5 compound microsatellites (cSSRs) in each analyzed sequence. Interestingly, 26 genomes lacked any cSSR. A total of 100 cSSRs were observed in 88 genomes (Table 2). The RD of cSSRs changed drastically in selected genomes, ranging from 1.36 bp/kb in Erbovirus A (V36) to 26.84 bp/kb in Cardiovirus A (V13). Similarly, the RA varied from 0.108 bp/kb in Ampivirus A (V3) to 0.636 bp/kb in the genome of Cardiovirus A (V13). The percentage of individual microsatellites being part of compound microsatellite (cSSR%) ranged from 2.439 in Gallivirus A (V37) to 15.15 in Cardiovirus A (V13) (Table 2).

3.3 Effect of dMAX on cSSR incidence

The dMAX is the maximum distance between any two adjacent microsatellites. If the distance separating two microsatellites is less than or equivalent to dMAX, these microsatellites are considered as cSSR (Kofler et al., 2008). To determine the impact of dMAX, five genome sequences such as bovine rhinitis B virus (V4), Cardiovirus A (V13), Enterovirus A (V21), Aichivirus A (V46), Salivirus A (V80) were chosen randomly to determine the variability of cSSR with increasing dMAX. It is noteworthy that the dMAX value in the IMEx software could only be set between 0 and 50 for analysis (Mudunuri and Nagarajaram 2007). Our study revealed an overall increase in the number of cSSRs with higher dMAX value in all selected Picornaviruses except for Enterovirus A (V21) (Figure 2).

3.4 Diversity of derived motifs

This study's derived microsatellites revealed the presence of mono to penta-nucleotide motif throughout the genome of the Picornaviridae family. Mono-nucleotide repeats were exhibited predominantly next to the dinucleotide motif. A 141 mono-nucleotide repeats stretch was observed in Cardiovirus A (V13) (Acc. no.X74312). Poly (G/C) repeats were found to be more prevalent than poly (A/T) repeats (Supplementary file 1). Within the dinucleotide repeats, CT/TC maintained the highest, whereas CG/GC repeats exhibited the lowest in terms of their distribution in the Picornaviridae. The CT/TC repeats were approximately 4.25 times more abundant than the least represented CG/GC repeats. The tri-nucleotide repeats were present abundantly next to mono-nucleotide repeats and contain 54 codon type repeats. Among them, GAA/AAG, and CAA/AAC represent the highest coding density, codes for glutamic acid/lysine and glutamine/asparagine, respectively.

In this study, seventeen tetra-nucleotide and six pentanucleotide motifs were observed among the picornaviruses. Among these, three tetra-nucleotide motifs such as CTCC, TCCT, and TTAG were localized in Aichivirus A (V46), and two pentanucleotide motifs such as GTTAA and TTAAG were localized in Aichivirus F (V51). Additionally, we have verified the distribution of SSRs throughout the non-genic and genic regions. The non-genic regions occupy 15.50% of SSR, and the rest 84.50% contribute toward the genic region. The 3D gene, which codes for the RdRp enzyme, contains maximum percentage (14.80%) followed by 2C (12.39%), then by VP3 (10.33%), which codes the protein for viral replication and capsid formation, respectively (Figure 3). We further examined the presence of mono-, di-, and tri-nucleotide repeats in the genic and non-genic regions depicted in Figure 4.

We have searched the biasness of the class of repeats to a genomic region. We found that the capsid protein VP4 is biased towards (A)₆ repeats, which code for lysine. While VP0, VP3, and 2A (codes for viral protease) were enriched with polyC, which codes for proline. We surveyed the conserved repeats at the 5' UTR and 3’ UTR, which were enriched with polyC and polyA, respectively. Except for VP0 (helping for RNA encapsidation), the gene enriched with dinucleotide repeats such as TC/CT, codes for serine/leucine, and rest of the regions were highly diverse with di- as well as tri-repeat types. The diversification of microsatellite indicates the rapid evolution of the species to adapt to a broad range of hosts.

3.5 Over and under-representation of mononucleotide repeat

We noticed a considerable variation in the numbers of mononucleotide repeats (≥6 nt), ranging from one (Enterovirus J, Enterovirus K, Hepatovirus D, Mosavirus A & Tremovirus A) to 22 (Parechovirus B). The ratio between the observed number of repeats and the expected number of repeats also varied considerably from -7.6407 (Rhinovirus A) to 14.8191 (Parechovirus B) (Table 3). Among the analyzed sequences, approximately 72.5% showed the under-represented distribution of MNRs. Broader host range and virus genome type might have influenced the ratio of the observed number of repeats to the expected number of repeats (O/E ratio) to some degree. The Z scores revealed the statistical significance of mononucleotide repeat representation. If the observed value is more than the expected value, the Z score is assigned Z>0 and vice versa. Thus, the greater the Z value, the higher is the statistical significance. Kunsagivirus A with the highest Z value of 4.85 denotes the most significant genome in terms of mononucleotide microsatellite evolution. Some of the viruses showed an over-representation of MNR loci compared to the expected value. This may arise due to the co-evolution of viral SSRs with that of the host genome.

3.6 Motif complexity and polymorphism of Picornavirus genome

Compound microsatellites (cSSRs) are termed by the presence of two or more adjacent individual microsatellites. The common patterns of cSSR are represented as m1-xn-m2, m1-xnm2-xn-m3 considered as '2-microsatellite', and '3-microsatellite', respectively (Kofler et al. 2008). The analysis of compound microsatellite complexity indicated that all surveyed genomes were rich in ‘2-microsatellite’ cSSR followed by a single ‘3-microsatellite’. Among the 88 species of Picornaviridae analyzed, the cSSR composition showed a random distribution pattern throughout the genome. The non-coding regions consist of 19% of the total microsatellite, while the rest 81% are present in the coding regions. Within the coding regions, VP3 occupied 14% of cSSRs, followed by 3D (13.23%) and VP1 (10.29%) region (Figure 5). The CTG-CAG compound microsatellite composed of self-complementary motifs has been proposed to be created by recombination (Jakupciak and Wells 1999). However, our study showed no such motifs, suggesting these compound microsatellites were not likely to be derived from recombination. The motif having the form [m1]n-xn-[m2]n can be termed as SSR-couples such as the compound microsatellite (A)6-x2-(TA)3 and (A)8-x2-(TA)5. We identified three types of SSR couples, such as (TG)-x-(CT), (TC)-x-(C), (CT)-x-(C) were presented twice in all analyzed genome. Some of the self-complementary motifs have been observed in Picornaviridae (GC)-x-(CG), (TC)-x-(AG), (GT)-x-(CA), and (TC)-x-(AG), which plays an important role in secondary structure formation. Motif duplication is one of the phenomena in which similar motifs are located on both ends of the spacer sequence, for example (CA)n-(X)y-(CA)z. About 13.23% of the total cSSR were made up of duplicated sequences having the motif pattern (C)-x-(C), (TG)-x-(TG), (AT)-x-(AT), (TC)-x-(TC) and (TG)-x-(TG) (Supplementary file 2).

To check polymorphism, the species with more than or equal to five strains were taken into consideration. Due to the limitation of sequence information, 12 out of 88 species got qualified for the detection of polymorphism. A sum of 141 sequences was analyzed to determine species-specific consensus microsatellite motifs (Supplementary file 3). We unraveled those five species contained the species-specific consensus sequences presented in the leader and VP3 regions, presumably being the most conserved microsatellite within the genome (Table 4). These conserved microsatellites could be used as potential biomarkers of virus identifications and population genetics study.

3.7 Recombination analysis

When the dataset of 141 complete genomes of 12 species was analyzed, three species were found lacking any recombination event. The rest of the species' recombination breakpoints were checked with major parent and minor parent information and their exact position in the genome. This analysis resulted in a total of 15 breakpoints, a majority of which associated with the structural proteins (VP4-VP1), followed by nonstructural capsid protein (2A-2C and 3A-3D) (Supplementary file 4). However, the least number of breakpoints were observed in the non-coding regions, followed by the leader sequence. Previous studies suggest that repetitive sequences are favored for the recombination and act as a hotspot, owing to the high affinity of recombinase enzymes towards dinucleotide repeats (Biet et al. 1999). This prompted us to check the repeats within the breakpoints present in nine species. Our results show that out of 15 breakpoints, three were devoid of any microsatellite.

In contrast, others are rich in dinucleotide repeats, which contribute 92%, followed by 4% tri-nucleotide repeats, and 4% mono-nucleotide repeats. Among the dinucleotide repeats, the GT/TG prevailed at the highest (25%) followed by AG/GA (18%), and CT/TC occurred at the least (2.2%) frequency. However, these data were not conclusive but laid the foundation for further research to know the relationship between microsatellite and recombination hotspot in the picornavirus family (Supplementary file 4).

3.8 Genomic parameters influencing SSR and cSSR distribution

Regression analysis shows significant correlation of genome size (R²= 0.258; P<0.05) and GC content (R²= 0.055; P<0.05) with incidence of SSR. There was no significant correlation observed between genome size, relative abundance RA (R²=0.027; P>0.05) and relative density RD (R²=0.014; P>0.05) (Figure 6A). Similarly, GC content, relative abundance (R²=0.020; P>0.05) and relative density (R²=0.022; P>0.05) were also found to be non-significantly correlated (Figure 6B). The regression analysis of cSSR revealed no significant correlation of incidence of cSSR with GC content (R²= 0.015; P>0.05) and genome size (R²= 0.031; P>0.05). Furthermore, genome size was also non-significantly correlated with cRA (R²= 0.007; P>0.05), cRD (R²= 0.007; P>0.05), percentage of cSSRs (R²= 0.003; P>0.05) (Figure 7A). Similarly, no significant correlation was observed in case of cRA (R²=0.009; P>0.05) and cRD (R²= 0.006; P>0.05) and percentage of cSSRs (R²= 0.000; P>0.05) with GC content of cSSRs (Figure 7B).

Picornaviridae, one of the largest RNA virus families and consisting of most diverse groups of species, is taken for study for the following reasons. Firstly, Picornaviruses have a broad host range, many of which cause serious health hazards to human and animals. Secondly, these viruses bear higher mutability properties. The higher mutation leads to create polymorphism in the microsatellite. Thus, it can be an excellent tool to study population genetics, evolution, and immune evasion strategies. However, virtually no comprehensive study of microsatellite analysis has been done in any of these viruses. Therefore, the microsatellites' analysis and their properties provide an excellent resource for further study of virus biology in general. For this reason, we have analyzed 88 complete genome sequences of Picornaviruses. As genome size is relatively small, fewer microsatellite repeats (2488 SSRs and 100 cSSR) with incidence frequency between 14–46 were observed. This incidence of microsatellites was proportional to the genome size, as seen in the case of Tobamovirus, having a lower number of SSRs than Potyviruses (Zhao et al., 2011). These observations come in line with the SSRs present in other viruses such as Flavivirus (27-67SSRs) (Alam et al. 2016) and HIV (22–48 SSRs) (Chen et al., 2009). The RD of SSR varied from 13.39 bp/kb-45.02 bp/kb on an average 26.29. Dinucleotide repeats were found to be more abundant than tri-nucleotide repeats in Picornaviruses. These indicate that dinucleotide repeats were more liable towards instability than the rest type of repeats, owing to a higher slippage rate (Katti et al., 2001). This type of diversification of motif might act as a molecular device for faster adaptation to environmental stresses in Picornavirus (Kashi et al., 1997; Li et al., 2004). In viruses, microsatellites were mostly accumulated in the coding regions, probably due to the high coding density of the viral genome (Chen et al., 2009; George et al., 2012). In the higher strata, the presence of SSRs in protein-coding regions are associated with functions such as social behavior in voles, sporulation efficiency and cell adhesion in yeast, skeletal morphology in domestic dogs, adaptive divergence in barley and wheat populations (Kashi and King 2006). The cSSR percentage of Picornaviruses ranged from 0-15.15%, which is lower in comparison to HIV-1 (0–24.24%) (Chen et al., 2012), Geminivirus (0-27.27%) (George et al., 2015), Herpesvirus (8.12–33.31%) (Ouyang et al., 2012) and other eukaryotic genomes (4–25%) (Kofler et al., 2008). However, the Picornaviruses cSSRs were in higher percentage compared to that of Flavivirus (0-12.82%) (Alam et al., 2016), Potexvirus (0-11.76%) (Alam et al., 2013) and prokaryotes such as E. coli (1.75–2.85%) (Chen et al., 2011). It was observed that the cSSR percentage increased with an increase in dMAX. However, this increase was not uniform in the five species. This indicates microsatellite distribution in Picornavirus genomes probably has diverse patterns. Similar observations were also made in the genome of eukaryotes and prokaryotes (Kofler et al., 2008; Kruglyak et al., 2000). Compound microsatellites present in Picornavirus have lower complexity as compared to the eukaryotes. The majority of cSSR consists of only two motifs. The largest cSSR was composed of three motifs. The prokaryotes show up to four cSSRs, while more than eight cSSRs are observed in eukaryotes. This indicates that the smaller genomes, the microsatellites are distributed far from one another. In general, the number of compound microsatellites decreases with an increase in complexity. Similar results have been demonstrated in HIV-1 genomes and E. coli, where smaller genome sizes and higher coding density probably were responsible for limiting the complexity of compound microsatellites. In Picornaviruses, 13.13% of cSSRs were composed of similar motifs, probably the result of genome duplication. Some study suggests that genome duplication may be helpful for the repeat tendency mechanism, which promotes the expansion of genome size such as yeast (Liti and Louis 2005; Fadda et al., 2003). In the evolutionary process, the occurrence of repeated sequences having a harmful or even lethal effect on the host might have been eliminated as a result of negative selection. This notion very well supports the fact that genome sizes do not increase abruptly. On the other hand, the SSRs showing neutral effect could be selected and fixed by genetic drift. Some longer repeat sequences could bring new functions to the organism, which may be beneficial for the organism's survival and could have been positively selected over time. The instability within the repeats creates polymorphism, which could act as a double-edged sword to drive adaptive variation in the genome through insertion, deletion, and substitution mutation and evades the host immune system. We could very well propose that the polymorphism directly or indirectly is related to important phenomena such as birth or death of microsatellite. Kelkar et al., 2008 (Kelkar et al. 2008) suggested that substitutions in short stretches of repeats be the predominant cause of births of microsatellites, whereas, insertions and deletions lead to death phase. Most of the microsatellites were found to be polymorphic owing to the higher mutability of Picornavirus. Each microsatellite type has its intrinsic properties, i.e., conserved microsatellite acting as a fingerprint in the viral genome to identify the viral strain. In contrast, polymorphic microsatellite can be of importance to study population genetic and complex evolution of the viruses.

This study shows a clear picture of type, relative abundance, relative density, and ubiquitous distribution pattern of microsatellite in Picornaviruses. The higher level of microsatellite polymorphism among Picornaviruses could be an excellent resource to address not only population genetics, and evolution related questions, but also solve, the complex mystery, such as birth or death of microsatellite. Breakpoints of these analyzed species are rich in dinucleotide repeats, which need further study to explore the relationship between recombination hotspot and microsatellite in this family. Finally, we can postulate that in-depth experimental analysis of SSR and cSSR in the diverse viral genome will pave the way to reveal the complex biological features such as host-pathogen interaction and the emergence of new epidemic strains.

Acknowledgements

BPS is thankful to the University Grant Commission (UGC, Govt. India) for providing Ph.D. fellowship as financial support.

Author Contributions Statement

BPS and BG did the bioinformatics analysis, and written the manuscript. PM, AM and RT constructed the figures. RS has generated the Circos plot. DN designed the study, executed, and written the manuscript.

Conflict of Interest Statement

The authors declare no conflict of interest.

Agol VI (2010) Picornaviruses as a model for studying the nature of RNA recombination. In: The Picornaviruses. American Society of Microbiology, pp 239-252
Alam C, Iqbal A, Thadari B, Ali S (2016) Imex based analysis of repeat sequences in flavivirus genomes, including dengue virus. J Data Mining Genomics Proteomics 7 (187):2153-0602.1000187
Alam CM, George B, Sharfuddin C, Jain S, Chakraborty S (2013) Occurrence and analysis of imperfect microsatellites in diverse potyvirus genomes. Gene 521 (2):238-244
Alexandersen S, Knowles NJ, Belsham GJ, Dekker A, Nfon C, Zhang Z, Koenen F Picornaviruses. In Diseases of Swine. doi:doi:10.1002/9781119350927.ch40
Bagshaw AT, Pitt JP, Gemmell NJ (2008) High frequency of microsatellites in S. cerevisiae meiotic recombination hotspots. BMC genomics 9 (1):49
Barr JN, Whelan S, Wertz GW (1997) cis-Acting signals involved in termination of vesicular stomatitis virus mRNA synthesis include the conserved AUAC and the U7 signal for polyadenylation. Journal of virology 71 (11):8718-8725
Biet E, Sun J-S, Dutreix M (1999) Conserved sequence preference in DNA binding among recombination proteins: an effect of ssDNA secondary structure. Nucleic Acids Research 27 (2):596-600
Boni MF, Posada D, Feldman MW (2007) An exact nonparametric method for inferring mosaic structure in sequence triplets. Genetics 176 (2):1035-1047
Caliguiri LA, Tamm I (1969) Membranous structures associated with translation and transcription of poliovirus RNA. Science 166 (3907):885-886
Chambers GK, MacAvoy ES (2000) Microsatellites: consensus and controversy. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 126 (4):455-476
Chen M, Tan Z, Jiang J, Li M, Chen H, Shen G, Yu R (2009) Similar distribution of simple sequence repeats in diverse completed Human Immunodeficiency Virus Type 1 genomes. FEBS letters 583 (17):2959-2963
Chen M, Tan Z, Zeng G, Zeng Z (2012) Differential distribution of compound microsatellites in various human immunodeficiency virus type 1 complete genomes. Infection, Genetics and Evolution 12 (7):1452-1457
Chen M, Zeng G, Tan Z, Jiang M, Zhang J, Zhang C, Lu L, Lin Y, Peng J (2011) Compound microsatellites in complete Escherichia coli genomes. FEBS letters 585 (7):1072-1076
de Wachter R (1981) The number of repeats expected in random nucleic acid sequences and found in genes. Journal of theoretical biology 91 (1):71-98
Duke GM, Osorio JE, Palmenberg AC (1990) Attenuation of Mengo virus through genetic engineering of the 5′ noncoding poly(C) tract. Nature 343 (6257):474-476. doi:10.1038/343474a0
Ellegren H (2004) Microsatellites: simple sequences with complex evolution. Nature reviews genetics 5 (6):435
Fadda Z, Daròs J, Flores R, Duran-Vila N (2003) Identification in eggplant of a variant of citrus exocortis viroid (CEVd) with a 96 nucleotide duplication in the right terminal region of the rod-like secondary structure. Virus research 97 (2):145-149
Flanegan JB, Petterson R, Ambros V, Hewlett N, Baltimore D (1977) Covalent linkage of a protein to a defined nucleotide sequence at the 5'-terminus of virion and replicative intermediate RNAs of poliovirus. Proceedings of the National Academy of Sciences 74 (3):961-965
Garcia-Barreno B, Delgado T, Melero JA (1994) Oligo (A) sequences of human respiratory syncytial virus G protein gene: assessment of their genetic stability in frameshift mutants. Journal of virology 68 (9):5460-5468
George B, Alam CM, Jain S, Sharfuddin C, Chakraborty S (2012) Differential distribution and occurrence of simple sequence repeats in diverse geminivirus genomes. Virus genes 45 (3):556-566
George B, Alam CM, Kumar RV, Gnanasekaran P, Chakraborty S (2015) Potential linkage between compound microsatellites and recombination in geminiviruses: Evidence from comparative analysis. Virology 482:41-50
Gibbs MJ, Armstrong JS, Gibbs AJ (2000) Sister-scanning: a Monte Carlo procedure for assessing signals in recombinant sequences. Bioinformatics 16 (7):573-582
Jacobson S, Konings D, Sarnow P (1993) Biochemical and genetic evidence for a pseudoknot structure at the 3'terminus of the poliovirus RNA genome and its role in viral RNA amplification. Journal of virology 67 (6):2961-2971
Jakupciak JP, Wells RD (1999) Genetic instabilities in (CTG· CAG) repeats occur by recombination. Journal of Biological Chemistry 274 (33):23468-23479
Kashi Y, King D, Soller M (1997) Simple sequence repeats as a source of quantitative genetic variation. Trends in genetics 13 (2):74-78
Kashi Y, King DG (2006) Simple sequence repeats as advantageous mutators in evolution. TRENDS in Genetics 22 (5):253-259
Katti MV, Ranjekar PK, Gupta VS (2001) Differential distribution of simple sequence repeats in eukaryotic genome sequences. Molecular biology and evolution 18 (7):1161-1167
Kelkar YD, Tyekucheva S, Chiaromonte F, Makova KD (2008) The genome-wide determinants of human and chimpanzee microsatellite evolution. Genome research 18 (1):30-38
Kirkpatrick DT, Wang Y-H, Dominska M, Griffith JD, Petes TD (1999) Control of meiotic recombination and gene expression in yeast by a simple repetitive DNA sequence that excludes nucleosomes. Molecular and cellular biology 19 (11):7661-7671
Kitamura N, Semler BL, Rothberg PG, Larsen GR, Adler CJ, Dorner AJ, Emini EA, Hanecak R, Lee JJ, van der Werf S, Anderson CW, Wimmer E (1981) Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature 291 (5816):547-553. doi:10.1038/291547a0
Kofler R, Schlötterer C, Luschützky E, Lelley T (2008) Survey of microsatellite clustering in eight fully sequenced species sheds light on the origin of compound microsatellites. BMC genomics 9 (1):612
Kruglyak S, Durrett R, Schug MD, Aquadro CF (2000) Distribution and abundance of microsatellites in the yeast genome can be explained by a balance between slippage events and point mutations. Molecular Biology and Evolution 17 (8):1210-1219
Lee YF, Nomoto A, Detjen BM, Wimmer E (1977) A protein covalently linked to poliovirus genome RNA. Proceedings of the National Academy of Sciences 74 (1):59-63
Li Y-C, Korol AB, Fahima T, Nevo E (2004) Microsatellites within genes: structure, function, and evolution. Molecular biology and evolution 21 (6):991-1007
Liti G, Louis EJ (2005) Yeast evolution and comparative genomics. Annu Rev Microbiol 59:135-153
Martin D, Murrell B, Golden M, Khoosal A, Muhire B (2015) RDP4: detection and analysis of recombination patterns in virus genomes. Virus Evol 1: vev003.
Martin D, Posada D, Crandall K, Williamson C (2005) A modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints. AIDS Research & Human Retroviruses 21 (1):98-102
Mrázek J, Guo X, Shah A (2007) Simple sequence repeats in prokaryotic genomes. Proceedings of the National Academy of Sciences 104 (20):8472-8477
Mudunuri SB, Nagarajaram HA (2007) IMEx: imperfect microsatellite extractor. Bioinformatics 23 (10):1181-1187
Ouyang Q, Zhao X, Feng H, Tian Y, Li D, Li M, Tan Z (2012) High GC content of simple sequence repeats in Herpes simplex virus type 1 genome. Gene 499 (1):37-40
Padidam M, Sawyer S, Fauquet CM (1999) Possible emergence of new geminiviruses by frequent recombination. Virology 265 (2):218-225
Posada D, Crandall KA (2001) Evaluation of methods for detecting recombination from DNA sequences: computer simulations. Proceedings of the National Academy of Sciences 98 (24):13757-13762
Schultes NP, Szostak JW (1991) A poly (dA. dT) tract is a component of the recombination initiation site at the ARG4 locus in Saccharomyces cerevisiae. Molecular and cellular biology 11 (1):322-328
Simmonds P (2010) Recombination in the evolution of picornaviruses. In: The Picornaviruses. American Society of Microbiology, pp 229-237
Smith JM (1992) Analyzing the mosaic structure of genes. Journal of molecular evolution 34 (2):126-129
Thompson JD, Gibson TJ, Higgins DG (2003) Multiple sequence alignment using ClustalW and ClustalX. Current protocols in bioinformatics (1):2.3. 1-2.3. 22
Tóth G, Gáspári Z, Jurka J (2000) Microsatellites in different eukaryotic genomes: survey and analysis. Genome research 10 (7):967-981
Toyoda H, Franco D, Fujita K, Paul AV, Wimmer E (2007) Replication of poliovirus requires binding of the poly (rC) binding protein to the cloverleaf as well as to the adjacent C-rich spacer sequence between the cloverleaf and the internal ribosomal entry site. Journal of virology 81 (18):10017-10028
Usdin K (2008) The biological effects of simple tandem repeats: lessons from the repeat expansion diseases. Genome research 18 (7):1011-1019
Weber JL (1990) Informativeness of human (dC-dA) n·(dG-dT) n polymorphisms. Genomics 7 (4):524-530
Yamada N, Tanihara K, Takada A, Yorihuzi T, Tsutsumi M, Shimomura H, Tsuji T, Date T (1996) Genetic organization and diversity of the 3′ noncoding region of the hepatitis C virus genome. Virology 223 (1):255-261
Yogo Y, Wimmer E (1972) Polyadenylic acid at the 3′-terminus of poliovirus RNA. Proceedings of the National Academy of Sciences 69 (7):1877-1882
Zhao X, Tan Z, Feng H, Yang R, Li M, Jiang J, Shen G, Yu R (2011) Microsatellites in different Potyvirus genomes: survey and analysis. Gene 488 (1-2):52-56

Table 1: Overview of selected 88 species of Picornaviridae family. The dataset includes specification of the viral strain, specific host, geo-location, isolation source, and disease caused.

Sl. no.	Genus	Species	Organism	Acc. No.	Isolate/ Strain	Geo Location	Host	Isolation Source	Disease Caused
V1	Aalivirus	Aalivirus A	Duck aalivirus 1	NC_023985	GL/12	China	Anas platyrhynchos	Abdominal cavity	Unknown
V2	Ailurivirus	Ailurivirus A	Aimelvirus 1	MF327529	gpai001	China	Unknown	Faeces	Unknown
V3	Ampivirus	Ampivirus A	Ampivirus A1	NC_027214	NEWT/2013/HUN	Hungary	Newt	Faeces	Unknown
V4	Aphthovirus	Bovine rhinitis B virus	Bovine rhinitis B virus	NC_010354	EC11	United Kingdom	Bos taurus	Lungs	Respiratory disease in cattle
V5		Equine rhinitis A virus	Equine rhinitis A virus	KM269483	D1305-03	United Arab Emirates: Dubai	Camelus dromedarius	Unknown	Respiratory tract disease in horses
V6		Foot-and-mouth disease virus	Foot-and-mouth disease virus - type O	AF511039	Akesu/58	China	Cattle	Cattle epithelial blister	Foot and mouth disease
V7	Aquamavirus	Aquamavirus A	Seal picornavirus type 1	NC_009891	HO.02.21	Canada	Pusa hispida	Oronaso-pharynx, lymph, lung	Unknown
V8	Avihepatovirus	Avihepatovirus A	Avihepatovirus A	KC893553	12-01	China	Anatidae	Unknown	Duck hepatitis disease
V9	Avisivirus	Avisivirus A	Turkey avisivirus	KC465954	turkey/M176-TuASV/2011/HUN	Hungary	Meleagris gallopavo	Faeces	Turkey viral hepatitis
V10		Avisivirus B	Chicken picornavirus 2	NC_024766	44C	Hong Kong	Gallus gallus	Tracheal and cloacal swab	Diarrhoea in chicken
V11		Avisivirus C	Chicken picornavirus 3	NC_024767	45C	Hong Kong	Gallus gallus	Tracheal and cloacal swab	Unknown
V12	Bopivirus	Bopivirus A	Bopivirus A	KM589358	TCH6	USA	Bos taurus	Unknown	Unknown
V13	Cardiovirus	Cardiovirus A	Encephalomyocarditis virus	X74312	emcv-pv21	Germany	Unknown	By plague purification from "M-variant"	Myocarditis and encephalitis
V14		Cardiovirus B	Human TMEV-like cardiovirus	GU595289	HTCV-UC6	USA	Homo sapiens	Faeces	Encephalitis
V15		Cardiovirus C	Boone cardiovirus 1	JQ864242	BCV-1	USA	Rattus norvegicus	Unknown	Unknown
V16	Cosavirus	Cosavirus A	Cosavirus A	NC_012800	HCoSV-A1	N/A	Homo sapiens	Faeces	Unknown
V17		Cosavirus B	Human cosavirus B	NC_012801	HCoSV-B1	Pakistan	Homo sapiens	Faeces	Acute flaccid paralysis, acute diarrhoea and acute gastroenteritis
V18		Cosavirus D	Cosavirus D	NC_012802	HCoSV-D1	Pakistan	Homo sapiens	Faeces	Unknown
V19		Cosavirus E	Cosavirus E	NC_012798	HCoSV-E1	Australia	Homo sapiens	Stool specimen	Unknown
V20	Dicipivirus	Cadicivirus A	Canine picodicistrovirus	NC_021178	209	Hong Kong	Canis lupus familiaris	Faecal and urine specimen	Unknown
V21	Enterovirus	Enterovirus A	Coxsackievirus A8	KM609477	CVA8/SZ127/CHN/2012	China	Homo sapiens	Stool specimen	Aseptic meningitis
V22		Enterovirus B	Echovirus E14	KP289441	E14/P968/2013/China	China	Homo sapiens	Stool specimen	Hand, foot, mouth disease
V23		Enterovirus C	Enterovirus C	KP793687	Brunenders	N/A	Homo sapiens	Unknown	Acute flaccid paralysis
V24		Enterovirus D	Enterovirus D68	KT280503	2011-21186	China	Homo sapiens	Unknown	Acute haemorrhagic conjunctivitis
V25		Enterovirus E	Enterovirus E	KM667941	BEV/Egypt/2014	Egypt	Cattle	Faeces	Reproductive, respiratory, or enteric disease in cattle
V26		Enterovirus F	Bovine enterovirus type 2	HQ663846	BJ001	China	bovine	Faecal swabs	Respiratory disease
V27		Enterovirus G	Porcine enterovirus 9	HM131607	Ch-ah-f1	China	Swine	Faeces	Stillbirths, mummification of foetuses, embryonic death, and infertility in porcine
V28		Enterovirus H	Enterovirus H	NC_003988	N/A	N/A	Unknown	Unknown	Unknown
V29		Enterovirus I	Dromedary camel enterovirus 19CC	KP345887	19CC	United Arab Emirates: Dubai	Dromedary	Faeces	Unknown
V30		Enterovirus J	Enterovirus J	NC_010415	1631	N/A	Unknown	Unknown	Unknown
V31		Enterovirus K	Picornaviridae sp. rodent/Ee/PicoV/NX2015	NC_038989	rodent/Ee/ PicoV/NX2015	China	Rodent	Pharyngeal and anal swab specimens	Unknown
V32		Enterovirus L	Enterovirus SEV-gx	NC_029905	SEV-gx	China	Macaca mulatta	Faeces	Unknown
V33		Rhinovirus A	Human rhinovirus A1	FJ445111	ATCC VR-1559	USA	Homo sapiens	Naso-pharyngeal washings	Common cold
V34		Rhinovirus B	Human rhinovirus B27	FJ445186	ATCC VR-1137	USA	Homo sapiens	Presumed from human throat washings	Common cold
V35		Rhinovirus C	Rhinovirus C	EF582387	026	Hong Kong	Homo sapiens	Nasopharyngeal aspirates of patients	Respiratory tract disease
V36	Erbovirus	Erbovirus A	Equine rhinitis B virus 2	AF361253	P313/75	N/A	Equus caballus, rabbit (Laboratory host)	Unknown	Respiratory infection
V37	Gallivirus	Gallivirus A	Turkey gallivirus	NC_018400	turkey/M176/2011/HUN	Hungary	Meleagris gallopavo	Faeces	Gastrointestinal Disease
V38	Harkavirus	Harkavirus A	Falcovirus A1	NC_026921	kestrel/VOVE0622/2013/HUN	Hungary	Falco tinnunculus	cloacal sample	Unknown
V39	Hepatovirus	Hepatovirus A	Hepatovirus A	AB279735	HAJ85-1	Japan: Yamanashi	Homo sapiens	serum	Hepatitis
V40		Hepatovirus B	Phopivirus	NC_027818	NewEngland_USA/2011	USA	Phoca vitulina	lungs, livers, spleens, and oral mucosae	Unknown
V41		Hepatovirus C	Bat hepatovirus SMG18520Minmav2014	KT452742	SMG18520Minmav2014	Madagascar	Miniopterus cf. manavi	Tissue, Blood and faeces	Unknown
V42		Hepatovirus D	Rodent hepatovirus RMU101637Micarv2010	NC_028363	RMU101637Micarv2010	Germany	Microtus arvalis	Tissue, Blood and faeces	Unknown
V43		Hepatovirus F	Rodent hepatovirus KEF121Sigmas2012	NC_038315	KEF121Sigmas2012	Mexico	Sigmodon mascotensis	Tissue, Blood and faeces	Unknown
V44		Hepatovirus I	Shrew hepatovirus KS121232Sorara2012	NC_028364	KS121232Sorara2012	Germany	Sorex araneus	Tissue, Blood and faeces	Unknown
V45	Hunnivirus	Hunnivirus A	Bovine hungarovirus 1	NC_018668	BHUV1/2008/HUN	Hungary	Bos taurus	Faeces	Unknown
V46	Kobuvirus	Aichivirus A	Aichi virus 1	NC_001918	A846/88	N/A	Homo sapiens	Unknown	Gastroenteritis (the stomach flu)
V47		Aichivirus B	Aichivirus B	NC_004421	U-1	N/A	bovine	Unknown	Gastroenteritis (the stomach flu)
V48		Aichivirus C	Porcine kobuvirus	KJ452348	CH/DX/2012	China	pig	Faeces	Gastroenteritis (the stomach flu)
V49		Aichivirus D	Kobuvirus cattle/Kagoshima-1-22-KoV/2014/JPN	NC_027919	Kagoshima-1-22-KoV/2014/JPN	Japan: Kagoshima	Bos taurus	Faeces	Bovine diarrhoea
V50		Aichivirus E	Rabbit picornavirus	KT325852	Rabbit01/2013/HUN	Hungary	Oryctolagus cuniculus var. domestica	Faeces	Unknown
V51		Aichivirus F	Bat picornavirus	KJ641687	BtMf-PicoV/FJ2012	China	Miniopterus fuliginosus	Pharyngeal and anal swab specimens	Unknown
V52	Kunsagivirus	Kunsagivirus A	Kunsagivirus A	KC935379	roller/SZAL6-KuV/2011/HUN	Hungary	Coracias garrulus	Faeces	Unknown
V53		Kunsagivirus C	Bakunsa virus	NC_034206	baboon/M27-KuV/1986/TAN	Tanzania: Mikumi National Park	Papio cynocephalus	Unknown	Unknown
V54	Limnipivirus	Limnipivirus A	Bluegill picornavirus	JX134222	04-032	USA	Lepomis macrochirus	Unknown	Unknown
V55		Limnipivirus B	Carp picornavirus 1	NC_023162	F37/06	Germany	Cyprinus carpio	heart, brain and liver	Unknown
V56		Limnipivirus C	Fathead minnow picornavirus	NC_039212	FHMPV-1	USA	Pimephales promelas	kidney/spleen or entire viscera	Unknown
V57	Livupivirus	Livupivirus A	Livupivirus A	NC_032126	newt/II-5-Pilis/2014/HUN	Hungary	Lissotriton vulgaris	Faeces	Unknown
V58	Megrivirus	Megrivirus A	Megrivirus A	KC663628	LY	N/A	Anas platyrhynchos	Unknown	Unknown
V59		Megrivirus B	Picornavirus HK21	NC_038957	HK21	Hong Kong	pigeon	Faeces	Unknown
V60		Megrivirus D	Harrier picornavirus 1	NC_034617	harrier/MR-01/HUN/2014	Hungary	Circus aeruginosus	cloacal sample	Unknown
V61		Megrivirus E	Penguin megrivirus	NC_039004	KGI-Bel-P5/2015	N/A	Pygoscelis adeliae	Unknown	Unknown
V62		Melegrivirus A	Turkey hepatitis virus 2993D	NC_021201	124	USA	Turkey poultry	liver	Hepatitis
V63	Mischivirus	Mischivirus A	Miniopterus schreibersii picornavirus 1	JQ814851	Unknown	China	Miniopterus schreibersii	Unknown	Unknown
V64		Mischivirus C	African bat icavirus PREDICT-06105	NC_026470	PREDICT-06105	Democratic Republic of the Congo	Hipposideros gigas	oral swab	Unknown
V65	Mosavirus	Mosavirus A	Mosavirus A2	NC_023987	SZAL6-MoV/2011/HUN	Hungary	Coracias garrulus	Faeces	Unknown
V66	Orivirus	Orivirus A	Orivirus A	KM203656	chicken/Pf-CHK1/2013/HUN	Hungary	Gallus gallus domesticus	cloacal sample	Common cold
V67	Oscivirus	Oscivirus A	Oscivirus A1	GU182409	007167	Hong Kong	Oriental Magpie Robin	Unknown	Unknown
V68	Parechovirus	Parechovirus A	Human parechovirus 1	JX575746	CAU10-NN	South Korea	Homo sapiens	Unknown	Respiratory or gastrointestinal illness
V69		Parechovirus B	Ljungan virus	EF202833	87-012G	Sweden	Clethrionomys glareolus	Unknown	Malformations, intrauterine foetal death
V70		Parechovirus C	Sebokele virus 1	NC_021482	1	Central African Republic	Hylomyscus, suckling newborn mice (Laboratory host)	Unknown	Unknown
V71	Pasivirus	Pasivirus A	Pasivirus A	LT898442	SPaV-A/GER/L00721/2014	N/A	Unknown	Unknown	Diarrhoea
V72	Passerivirus	Passerivirus A	Passerivirus A1	NC_014411	00356	Hong Kong	Pale Thrush	Unknown	Unknown
V73	Poecivirus	Poecivirus A	Poecivirus BCCH-449	KU977108	BCCH-449	USA: Alaska	Black-capped chickadee	beak	Avian keratin disorder (AKD)
V74	Potamipivirus	Potamipivirus A	Potamipivirus A	MK189163	TSPV	USA: Alaska	Gasterosteus- aculeatus	intestine	Unknown
V75	Rabovirus	Rabovirus A	Rabovirus A	NC_026314	Berlin/Jan2011/0572	Germany	Rattus norvegicus	Faeces	Unknown
V76	Rosavirus	Rosavirus A	Rosavirus A2	NC_024070	GA7403	Gambia	Homo sapiens	Faeces	Unknown
V77		Rosavirus B	Rosavirus B	NC_031105	RNCW0602091R	Hong Kong	Rattus norvegicus	Unknown	Multisystemic dissemination
V78		Rosavirus C	Rosavirus C	NC_031106	RATLC11A	Hong Kong	Rattus andamanensis	Unknown	Multisystemic dissemination
V79	Sakobuvirus	Sakobuvirus A	Feline sakobuvirus A	NC_022802	FFUP1	Portugal	Felis catus	Unknown	Unknown
V80	Salivirus	Salivirus A	Salivirus A	NC_012986	02394-01	USA	Homo sapiens	stool specimen	Gastroenteritis
V81	Sapelovirus	Avian sapelovirus	Avian sapelovirus	NC_006553	TW90A	N/A	Unknown	Unknown	Unknown
V82		Sapelovirus A	Porcine sapelovirus 1	NC_003987	V13	N/A	Unknown	Unknown	Diarrhoea, polio encephalomyelitis, pneumonia
V83		Sapelovirus B	Simian sapelovirus 1	NC_004451	2383	N/A	Unknown	Unknown	Unknown
V84	Senecavirus	Senecavirus A	Senecavirus A	KC667560	11-55910-3	Canada	swine	brain	Vesicular lesions in pigs
V85	Sicinivirus	Sicinivirus A	Sicinivirus Pf-CHK1/SiV	KT880665	Pf-CHK1/SiV	Hungary	Gallus gallus domesticus	cloacal sample	Unknown
V86	Teschovirus	Teschovirus A	Teschovirus A	KC667562	13-3064-3	Dominican Republic	swine	spinal cord	Encephalomyelitis in pigs
V87	Torchivirus	Torchivirus A	Tortoise picornavirus	KM873611	14-04	Germany	Testudo hermanni	Unknown	Unknown
V88	Tremovirus	Tremovirus A	Tremovirus A	KF979338	204C	Hong Kong	Gallus gallus	Unknown	Encephalomyelitis

Table 2: Survey of various microsatellites (SSRs and cSSRs) with their relative abundance, relative density and cSSR % in selected Picornaviridae family.

Sl. No.	Acc. No.	Genome size (bp)	GC (%)	Total no of SSR	Total no of cSSR	RA (SSR)	RD (SSR)	RA (cSSR)	RD (cSSR)	% of cSSR
V1	NC_023985	8976	43.17	29	1	3.231	24.06	0.111	2.78	3.448
V2	MF327529	8029	45.19	29	1	3.612	24.909	0.125	2.49	3.448
V3	NC_027214	9246	45.14	31	1	3.353	21.84	0.108	2.16	3.226
V4	NC_010354	7556	45.55	32	0	4.235	33.24	0.000	0	0.000
V5	KM269483	7681	47.57	38	3	4.947	31.62	0.391	7.16	7.895
V6	AF511039	8147	53.38	24	0	2.946	20.6	0.000	0	0.000
V7	NC_009891	6718	43.68	35	1	5.210	39.27	0.149	2.23	2.857
V8	KC893553	7800	43.01	38	2	4.872	34.47	0.256	3.46	5.263
V9	KC465954	7532	44.96	23	0	3.054	21.1	0.000	0	0.000
V10	NC_024766	7310	48.52	23	0	3.146	19.69	0.000	0	0.000
V11	NC_024767	7167	45.21	14	1	1.953	13.39	0.140	1.95	7.143
V12	KM589358	7018	50.37	21	0	2.992	22.08	0.000	0	0.000
V13	X74312	7861	49.73	33	5	4.198	45.02	0.636	26.84	15.152
V14	GU595289	8047	43.85	20	0	2.485	16.89	0.000	0	0.000
V15	JQ864242	8530	47.74	29	1	3.400	26.49	0.117	1.87	3.448
V16	NC_012800	7632	43.75	28	0	3.669	27.64	0.000	0	0.000
V17	NC_012801	7205	43.78	27	1	3.747	27.48	0.139	4.99	3.704
V18	NC_012802	7215	42.47	27	1	3.742	25.502	0.139	2.91	3.703
V19	NC_012798	6580	41.93	26	1	3.951	29.93	0.152	1.82	3.846
V20	NC_021178	8785	41.58	33	2	3.756	27.66	0.228	5.69	6.061
V21	KM609477	7396	47.64	21	1	2.839	19.06	0.135	2.02	4.762
V22	KP289441	7451	47.6	27	1	3.624	24.15	0.134	1.87	3.704
V23	KP793687	7507	46.09	21	0	2.797	28.22	0.000	0	0.000
V24	KT280503	7332	41.8	24	1	3.273	18.67	0.136	2.04	4.167
V25	KM667941	7417	50.41	20	1	2.697	20.89	0.135	1.69	5.000
V26	HQ663846	7430	50.76	18	0	2.423	19.77	0.000	0	0.000
V27	HM131607	7390	45.27	19	1	2.571	17.18	0.135	2.57	5.263
V28	NC_003988	7374	42.93	26	2	3.526	29.96	0.271	3.25	7.692
V29	KP345887	7441	45.39	26	0	3.494	26.86	0.000	0	0.000
V30	NC_010415	7351	45.28	20	0	2.721	16.99	0.000	0	0.000
V31	NC_038989	7805	46.09	17	0	2.178	16.14	0.000	0	0.000
V32	NC_029905	7367	43.14	21	1	2.851	19.95	0.136	2.03	4.762
V33	FJ445111	7137	37.43	31	2	4.344	29.704	0.280	4.2	6.452
V34	FJ445186	7217	39.46	26	1	3.603	21.05	0.139	1.66	3.846
V35	EF582387	7086	42.98	27	0	3.810	22.45	0.000	0	0.000
V36	AF361253	8821	50.4	35	1	3.968	27.2	0.113	1.36	2.857
V37	NC_018400	8496	48.28	41	1	4.826	32.71	0.118	1.41	2.439
V38	NC_026921	8003	41.80	29	3	3.624	25.61	0.375	7.74	10.345
V39	AB279735	7478	38.2	22	1	2.942	22.45	0.134	2	4.545
V40	NC_027818	7475	36.80	16	1	2.140	17.25	0.134	1.605	6.250
V41	KT452742	7570	38.08	34	0	4.491	27.73	0.000	0	0.000
V42	NC_028363	7656	36.10	26	2	3.396	26.907	0.261	5.09	7.692
V43	NC_038315	7563	35.11	35	2	4.628	31.46	0.264	4.23	5.714
V44	NC_028364	7810	36.71	36	1	4.609	29.705	0.128	1.92	2.778
V45	NC_018668	7583	45.57	24	0	3.165	22.41	0.000	0	0.000
V46	NC_001918	8251	58.9	45	4	5.454	36.59	0.485	6.54	8.889
V47	NC_004421	8374	54.6	38	2	4.538	29.83	0.239	3.22	5.263
V48	KJ452348	8124	51.82	30	0	3.693	25.72	0.000	0	0.000
V49	NC_027919	8087	55.19	40	1	4.946	34.87	0.124	2.59	2.500
V50	KT325852	8364	55.17	35	1	4.185	29.41	0.120	2.51	2.857
V51	KJ641687	7435	49.37	18	0	2.421	24.209	0.000	0	0.000
V52	KC935379	7272	53.01	35	1	4.813	33.69	0.138	2.47	2.857
V53	NC_034206	7429	49.02	25	1	3.365	22.47	0.135	2.42	4.000
V54	JX134222	8050	44.01	36	0	4.472	32.79	0.000	0	0.000
V55	NC_023162	7697	45.01	20	1	2.598	25.98	0.130	2.2	5.000
V56	NC_039212	7746	45.82	18	1	2.324	15.87	0.129	1.93	5.556
V57	NC_032126	7768	46.77	24	0	3.090	20.85	0.000	0	0.000
V58	KC663628	9700	45.26	44	0	4.536	30.309	0.000	0	0.000
V59	NC_038957	9100	47.37	34	0	3.736	27.36	0.000	0	0.000
V60	NC_034617	8541	45.51	28	1	3.278	20.25	0.117	1.404	3.571
V61	NC_039004	9702	43.22	41	3	4.226	29.16	0.309	5.66	7.317
V62	NC_021201	9075	46.07	39	1	4.297	28.98	0.110	2.09	2.564
V63	JQ814851	8468	47.43	38	2	4.487	31.52	0.236	3.18	5.263
V64	NC_026470	8096	47.31	33	1	4.076	26.92	0.124	2.09	3.030
V65	NC_023987	8398	45.47	24	0	2.858	21.18	0.000	0	0.000
V66	KM203656	7037	49.8	29	3	4.121	26.57	0.426	7.1	10.345
V67	GU182409	7640	46.58	34	2	4.450	32.32	0.262	3.14	5.882
V68	JX575746	7348	39.75	24	0	3.266	23.38	0.000	0	0.000
V69	EF202833	7635	42.58	44	4	5.763	37.19	0.524	6.67	9.091
V70	NC_021482	7537	45.75	27	4	3.582	23.61	0.530	8.35	14.815
V71	LT898442	6917	43.02	27	0	3.903	25.01	0.000	0	0.000
V72	NC_014411	8035	57.92	26	2	3.236	23.26	0.249	3.23	7.692
V73	KU977108	7653	43.45	34	1	4.443	29.26	0.131	1.56	2.941
V74	MK189163	8532	43.33	30	1	3.516	27.89	0.117	2.1	3.333
V75	NC_026314	7853	43.23	32	1	4.074	28.77	0.127	2.54	3.125
V76	NC_024070	8930	51.41	40	2	4.479	28.55	0.224	4.25	5.000
V77	NC_031105	8938	50.92	34	3	3.803	24.83	0.335	7.04	8.824
V78	NC_031106	9112	50.98	33	1	3.622	26.44	0.110	1.86	3.030
V79	NC_022802	7807	55.51	40	1	5.124	35.34	0.128	1.53	2.500
V80	NC_012986	7989	56.67	46	2	5.758	39.04	0.250	3.37	4.348
V81	NC_006553	8289	42.69	35	2	4.222	34.97	0.241	2.89	5.714
V82	NC_003987	7491	41.03	26	2	3.471	22.01	0.267	3.6	7.692
V83	NC_004451	8126	40.38	29	1	3.569	23.12	0.123	1.84	3.448
V84	KC667560	7356	51.54	25	1	3.399	25.55	0.136	1.63	4.000
V85	KT880665	9883	53.81	35	2	3.541	22.35	0.202	2.42	5.714
V86	KC667562	7155	44.55	21	0	2.935	25.7	0.000	0	0.000
V87	KM873611	7093	36.04	30	0	4.230	30.17	0.000	0	0.000
V88	KF979338	6955	44.18	25	1	3.595	23.28	0.144	1.72	4.000

Table 3: Mono-nucleotide microsatellite representation in Picornaviridae family.

Sl. No.	Observed Total	Real Expected	O/E	O-E	Square Root of E	Z score
V1	6	8.792490113	0.682401	-2.79249	2.965213333	-0.941750154
V2	2	5.853882275	0.341654	-3.85388	2.419479753	-1.592855766
V3	5	6.950827321	0.719339	-1.95083	2.636442171	-0.739946942
V4	6	6.079360927	0.986946	-0.07936	2.465636009	-0.032186798
V5	10	5.774709596	1.731689	4.22529	2.403062545	1.758293979
V6	3	6.305285261	0.475791	-3.30529	2.511032708	-1.31630514
V7	7	5.921120397	1.182209	1.07888	2.433335241	0.443374832
V8	7	7.148990812	0.979159	-0.14899	2.673759677	-0.055723337
V9	8	6.221224962	1.28592	1.778775	2.494238353	0.713153591
V10	5	6.34132674	0.788479	-1.34133	2.518199107	-0.532653171
V11	4	5.67221231	0.705192	-1.67221	2.381640676	-0.702126197
V12	3	6.75032156	0.444423	-3.75032	2.598138095	-1.44346506
V13	9	5.738697585	1.5683	3.261302	2.395557886	1.361395788
V14	4	7.032841022	0.56876	-3.03284	2.651950418	-1.143626593
V15	6	6.67352211	0.899075	-0.67352	2.583316107	-0.260719975
V16	4	5.34522324	0.748332	-1.34522	2.311973884	-0.581850534
V17	4	5.87240551	0.681152	-1.87241	2.423304667	-0.772666159
V18	4	5.95257341	0.671978	-1.95257	2.439789624	-0.800304006
V19	5	6.45245031	0.774899	-1.45245	2.540167378	-0.571793151
V20	3	6.026337571	0.497815	-3.02634	2.45485999	-1.232794368
V21	2	5.550081918	0.360355	-3.55008	2.355861184	-1.506914729
V22	3	5.596912349	0.53601	-2.59691	2.365779438	-1.097698419
V23	3	5.910549533	0.507567	-2.91055	2.431162177	-1.197184441
V24	2	7.243071394	0.276126	-5.24307	2.691295486	-1.948158952
V25	4	5.416504044	0.738484	-1.4165	2.327338403	-0.608636906
V26	2	5.437974326	0.367784	-3.43797	2.331946467	-1.474293846
V27	2	5.76785342	0.346749	-3.76785	2.401635572	-1.568869759
V28	7	6.788938974	1.031089	0.211061	2.605559244	0.081004117
V29	5	6.027594892	0.829518	-1.02759	2.455116065	-0.41855247
V30	1	5.98317411	0.167135	-4.98317	2.446052761	-2.037230835
V31	1	5.23123021	0.19116	-4.23123	2.287188276	-1.849970225
V32	6	6.2352211	0.962275	-0.23522	2.497042471	-0.09419988
V33	2	9.640702298	0.207454	-7.6407	3.104948035	-2.460814871
V34	4	8.380757236	0.477284	-4.38076	2.894953754	-1.513239108
V35	2	6.503150558	0.307543	-4.50315	2.550127557	-1.765853063
V36	7	6.446453953	1.085868	0.553546	2.538986797	0.218018482
V37	13	6.295819278	2.064862	6.704181	2.509147122	2.671896225
V38	5	9.532580223	0.524517	-4.53258	3.087487688	-1.468048032
V39	5	6.858401211	0.729033	-1.8584	2.618854943	-0.709623577
V40	2	6.87223112	0.291026	-4.87223	2.621494063	-1.858570343
V41	6	9.73778124	0.616157	-3.73778	3.120541818	-1.197798798
V42	1	5.73224502	0.174452	-4.73225	2.39421073	-1.976536552
V43	11	5.894062032	1.866285	5.105938	2.427768941	2.103139999
V44	11	6.23342312	1.76468	4.766577	2.496682423	1.909164272
V45	2	6.096045186	0.328082	-4.09605	2.469017049	-1.658978089
V46	14	8.570159884	1.633575	5.42984	2.927483541	1.854780749
V47	5	6.795861417	0.735742	-1.79586	2.606887304	-0.688891083
V48	4	6.034100908	0.662899	-2.0341	2.456440699	-0.828068395
V49	11	4.32408912	2.543888	6.675911	2.079444426	3.210430054
V50	10	5.98	1.672241	4.02	2.445403852	1.643900248
V51	2	6.34	0.315457	-4.34	2.517935662	-1.723634192
V52	17	5.559061712	3.05807	11.44094	2.357766255	4.85244806
V53	11	5.234853212	2.101301	5.765147	2.28798016	2.519753838
V54	6	5.711408548	1.050529	0.288591	2.38985534	0.120756871
V55	5	6.345861415	0.787915	-1.34586	2.519099326	-0.534262941
V56	2	6.36221342	0.314356	-4.36221	2.522342843	-1.729429221
V57	5	6.24513412	0.800623	-1.24513	2.499026635	-0.498247639
V58	9	5.2365712	1.718682	3.763429	2.288355567	1.644599666
V59	4	6.74127623	0.593359	-2.74128	2.596396778	-1.055800197
V60	7	6.2412311	1.121574	0.758769	2.498245604	0.303720699
V61	6	6.8751218	0.872712	-0.87512	2.622045347	-0.333755402
V62	2	6.5869412	0.303631	-4.58694	2.566503692	-1.787233431
V63	6	6.391546817	0.93874	-0.39155	2.528150869	-0.154874783
V64	5	6.57221347	0.760779	-1.57221	2.563632866	-0.613275595
V65	1	6.783046221	0.147426	-5.78305	2.604428195	-2.220466755
V66	7	6.546553953	1.069265	0.453446	2.558623449	0.177222657
V67	6	5.911408748	1.014986	0.088591	2.43133888	0.036437229
V68	3	8.356769708	0.35899	-5.35677	2.890807795	-1.853035583
V69	22	7.180837653	3.063709	14.81916	2.679708501	5.530139692
V70	4	6.3652329	0.628414	-2.36523	2.522941319	-0.937490255
V71	8	6.321524062	1.265518	1.678476	2.514264119	0.667581391
V72	9	7.821327733	1.1507	1.178672	2.796663679	0.421456565
V73	14	8.79219342	1.592322	5.207807	2.965163304	1.756330443
V74	6	5.920408748	1.013444	0.079591	2.433189008	0.032710674
V75	4	6.76219785	0.591524	-2.7622	2.600422629	-1.062211126
V76	14	8.2323145	1.700615	5.767686	2.869201021	2.010206137
V77	13	6.5312134	1.990442	6.468787	2.555623877	2.531196652
V78	10	5.3423145	1.871848	4.657686	2.311344738	2.015140979
V79	5	6.611529168	0.756255	-1.61153	2.571289398	-0.626739709
V80	9	7.205316295	1.249078	1.794684	2.684272023	0.668592337
V81	2	7.746547446	0.25818	-5.74655	2.783262015	-2.064680729
V82	3	7.789803711	0.385119	-4.7898	2.791021983	-1.716146895
V83	6	8.841380113	0.678627	-2.84138	2.973445832	-0.955584959
V84	6	5.434544771	1.104048	0.565455	2.331211009	0.242558579
V85	16	7.772379513	2.058572	8.22762	2.787898763	2.951190551
V86	3	6.026337571	0.497815	-3.02634	2.45485999	-1.232794368
V87	8	6.291234962	1.27161	1.708765	2.508233434	0.681262364
V88	1	5.968473567	0.167547	-4.96847	2.443045961	-2.033720874

Table 4: The existence of the consensus motif in diverse species of Picornaviridae family.

Species	Compound microsatellite	Start	End	Location	(a/b)	Consensus motif
Avihepatovirus A	(CT)3-x-1-(T)6	1740	1750	VP3	(17/9)	(CT)3-x-1-(T)6
	(TGA)3-x4-(GT)3	2152	2170	VP1	(17/5)	absent
Enterovirus A	(GC)3-x3-(CAA)3	2128	2145	VP3	(10/5)	(GC)3-x3-(CAA)3
Enterovirus B	(TG)3-x1-(TG)4	2229	2243	VP3	(15/2)	absent
Enterovirus D	(GAA)3-x5-(AT)3	6251	6270	3D	(17/16)	(GAA)3-x5-(AT)3
Rhinovirus B	(AT)3-x3-(AT)3	604	618	Non-coding	(12/5)	absent
Hepatovirus A	(TG)3-x-3-(GTG)3	5784	5795	3C	(21/1)	absent
Aichivirus A	(C)6-x1-(C)6	95	107	Non-coding	(6/2)	absent
	(C)6-x2-(C)6	2706	2719	VP-3	(6/2)	absent
	(GC)3-x0-(CG)3	5371	5382	2C	(6/2)	absent
	(C)6-x5-(TCCT)3	7619	7641	3D	(6/2)	absent
Parechovirus B	(GT)3-x6-(GA)3	1760	1777	VP3	(6/2)	absent
	(TC)3-x2-(CA)3	2881	2894	VP1	(6/2)	absent
	(T)8-x9-(TG)3	3681	3703	2B	(6/5)	(T)8-x9-(TG)3
	(A)7-x4-(TG)3	5964	5980	3C	(6/2)	absent
Salivirus A	(CT)3-x5-(TC)4	2449	2467	VP3	(9/2)	absent
	(CT)3-x3-(ACT)3	7201	7218	3D	(9/5)	(CT)3-x3-(ACT)3
Sapelovirus A	(C)6-x7-(CTA)3	438	459	Leader	(9/9)	(C)n-x7-(CTA)3 (n=6-7)
	(AG)3-x3-(GT)3	493	507	Leader	(9/8)	(AG)3-x3-(GT)3
Senecavirus A	(GT)3-x0-(TG)3	1205	1216	Leader	(19/19)	(GT)3-x0-(TG)3
Sicinivirus A	(GT)3-x7-(CA)3	3231	3249	VP3	(5/1)	absent
	(T)6-x6-(GT)3	3319	3336	VP3	(5/1)	absent
Tremovirus A	(TA)3-x5-(GT)3	4549	4565	3A	(5/1)	absent
a/b indicates the number of strains per species used/number of strains having cSSR

SupplementaryFile.xlsx

A comprehensive analysis of Simple Sequence Repeats in Picornaviruses

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusion

Declarations

References

Tables

Supplementary Files

Status:

Version 1