Whole-Genome Variants Discovery of FMD Virus Isolated from Cattle Population in Pakistan

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FMD virus may cause epidemics resulting in devastation of livestock industry so, it’s worthy to explore the genomic architecture of virus to harness the mortality and morbidity particularly in cattle from Pakistan. Epithelial scrapping samples of sick animals were taken from Punjab, Pakistan and cDNA of virus was sequenced through short-read NGS Illumina technology followed by variant calling analysis to reveal how novel variants give rise to new lineage in the region for a comprehensive insight of its genomic landscape. Haplotype-based variant discovery was performed by Genome Analysis Toolkit (GATK4) with Mutect2 using Pan Asia-II as reference genome. A total of 708 variants including 642 SNPs, 38 MNPs and 28 INDELs were observed. Furthermore, whole genome annotation revealed high, low, moderate and modifier impact variants count as 10(1.28%), 514(66.15%), 115(14.80%) and 138(17.76%) respectively which are distributed in VP3, 2C, 3B and 3D proteins of FMDV. Similarly, transitions-to-transversions ratio (3.75) and missense-to-silent ratio (0.1634) across the whole genome with 639 exonic, 3 downstream, 69 intergenic and 66 upstream effects were also identified. Whereas, high impact-frame shift mutations were concentrated in 5000-7000 nucleotide positions of the genome. A worth-mentioning deletion mutation of 75bp at 5276 position harbor 2C protein. The current whole genome variant discovery of FMDV will add new insight to understand the micro-evolution, speedy emergence of strains, mutation associated disease-severity and it’s lineage to prevent the prevalence of this catastrophe. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cloven hoofed animals are particularly affected by FMDV which was first ever known in 16th century along with the association of causative agent as a virus. Ongoing FMD episodes and their critical monetary impacts have opened up the worry of governments globally (Grubman and Baxt, 2004 ). In the months of religious event called Eid Al Azha, movement of animals is increased which results in outbreaks during months of January to March usually in Pakistan (Jamal et al., 2010 ). In South Asia, FMD remained endemic with outbreaks occurring regularly across the Indo-Pakistan subcontinents. Serotype O of FMDV is dispersed in all continents, particularly in the country of Middle east and south Asia (Rweyemamu et al., 2008 ). FMDV is a small positive sense ssRNA virus, approximately 8.3kb, belonging to Aphthovirus genus of Picornaviridae family. FMDV has seven antigenically distinct serotypes designated as A, O, C, Asia 1, and South African Types (SAT) types 1–3, and each serotype also contains different subtypes. OIE/FAO World Reference Laboratory for FMD (WRLFMD) reported 11 topo-types of FMDV serotype-O among which Pakistani strains are classified under Middle east-South Asia (ME-SA) topotype.(Samuel and Knowles, 2001 ) During 2002–2006 FMDV, type O virus has been isolated in Pakistan which belonged to pak-98, Pan-Asia-II circulated in 2006 or Iran-2001 Pan Asia lineages and now IND2001 (a, b, c, d, e) linages are evolving(Jamal et al., 2021 ) The virus genome codes for a single polyprotein which further proceeds to produce the set of intermediate and final mature proteins that are used for virus replication and assembly. The P1 region of polyprotein codes for four structural proteins (SP) which are VP1 (1D), VP2 (1B), VP3 (1C), and VP4 (1A). P2 and P3 regions codes for non-structural proteins (NSP) important for virus replication and cellular processes (Knipe et al., 1997 ). Conserved Arg-Gly-Asp (RGD) amino acid sequence play critical role in FMDV binding to host cells receptors called Integrins. According to the previous studies the use of nucleotide sequencing provides us the subsequent information for the identification of virus variants. These variant play very important role in the identification of trans- boundary virus transmission and generate form laboratory cell passages .These variants are also provide us strong evidence about the persistence of particle virus serotype. Nucleotide sequencing data is also play very important role to find origin of viral outbreak which is provide new approaches in inactivated vaccines (Han et al., 2021 ). Studies have shown that the higher throughput and lower cost of next generation sequencing (NGS) techniques is consider a most widely used approach for deep sequencing genomic investigation in future (Peng Li et al., 2016 ). Complete genome analysis of viruses circulating in the region provides platform for the recognition of new viral strain. Variant calling using Genome Analysis Tool Kit (GATK) pipeline provides comprehensive insight of variation between viruses from different time period (DePristo et al., 2011 ). Calling variants by GATK-4 is either done under category of UnifiedGenotyper (UG) or Haplotypecaller (HC) where later one is sophisticated and most used. If the sample size is more than 100, genome of diploid organism and pooled samples are used then UG is preferable. RNA genome viruses give rise to generation phylogenetically related but different variants called viral quasispecies due to RNA-dependent RNA polymerase (Biebricher and Eigen, 2006 ). Undoubtedly, use of whole genome sequencing (WGS) data and computational tools help in deeper understanding of viral outbreaks and reasons (Orton et al., 2020 ). In this study, query genome comparison against reference genome was held to analyze mutations between two lineages of FMDV using haplotype variant calling by GATK-4 pipeline. Materials And Methods 2.1 Sample collection, virus propagation and identification: Foot and Mouth Disease Virus (FMDV) samples were collected from the epithelium of cattle buccal cavity from different regions of Punjab-Pakistan. Further, collected samples were processed in Quality Operations Lab (QOL), University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan for virus isolation using liquid nitrogen pulverization. For extraction of virus from the cells, centrifugation was performed at 2000rpm for 10 minutes to remove cellular debris. Afterwards, samples were stored in cryovials at -80ºC. BHK21 cell line with 80–90% confluency was used for virus propagation at 37ºC with 5% CO 2 supply. Moreover, presence of virus was confirmed by cytopathic effects on BHK21 cell line and identification was done by IZSLER antigen detection ELISA- Pirbright assay. Propagated FMDV on BHK21 host cell line. 2.2 RNA extraction and reverse transcription for cDNA synthesis: RNA extraction was performed using QIAamp viral RNA kit (www.qiagen.com) as per manufacturer instructions. Similarly, cDNA synthesis was done using RevertAid First Strand cDNA synthesis kit (www.thermoscientific.com) as per manufacturer instructions. 2.3 Whole-Genome Sequencing using Illumina Platform: Library preparation Whole-genome RNA was extracted followed by cDNA synthesis as per sequencing platform requirements along with 5’ and 3’ adapter ligation. cDNA was sheared using Covaris ultrasonicator apparatus and library preparation was performed with TruSeq Nano DNA library kit. Adapter-ligated fragments are PCR amplified and further size selection was done for sequencing run using Illumina HiSeq 4000 NGS platform by Macrogen, Korea (www.macrogen.com). Selection of reference genome : Reference genome of FMDV with accession ID MH784404.1 was taken from https://www.ncbi.nlm.nih.gov/nuccore/MH784404.1, which belongs to PanAsia-II lineage. It’s FASTA and GFF3 annotated files were taken for further usage in GATK4 whole-genome variant calling pipeline. 2.4 Whole-Genome variant calling bioinformatics pipeline: GATK4 pipeline was used on Linux operating system with following protocols: Quality checks MultiQC version 1.11 was used for quality checks of forward and reverse fastq.gz files. Multiple parameters generated showing low quality, short and duplicate reads, insert size distribution, sequence quality, read GC & N-content and passed filters. Trimming Trimming was done by Fastp version 1.11. Jason file was produced for further processing. Mapping of filtered reads Indexing of reference genome and mapping was performed using Bowtie2 tool. Afterwards, SAM file was converted to BAM by Samtools while sorting and mark duplication steps were performed using Picard tool. Variant calling Variant calling was performed using Mutect2. Afterwards, vcf allelicpremitive step was performed before annotation. Functional annotation SnpEff software was used for annotating the vcf files. Moreover, SNPsift software was used to extract the required field from the resultant file. Results Whole-Genome Sequencing Data Summary: TruSeq Nano DNA library kit was used to sequence the whole-genome of FMDV by obeying manufacturer instructions using Illumina HiSeq 4000 NGS platform. Paired end (151x2) 37,223,254 reads were generated with 2.9 Gb data. Untrimmed raw FASTQ reads were obtained using bcl2fastq tool. Initial quality checks of reads revealed 42.35% of GC content, 95.95% of Q20 filter, and 90.1% of Q30 filter. Quality Checks by MultiQC: Sequencing Fastq files were analyzed, 98.40% of filtered reads were obtained with 1.60% low quality reads, none of the short reads and unknown nucleotides were observed. Similarly, less than 0.1% of duplicate reads with Phred score of more than 40. Average insert size was 151 with 78% coverage. Variant Identification and Annotation: After trimming and indexing, mapping of the clean reads of query FMDV against reference genome of PanAsia II was performed with Bowtie2 tool and variant calling was performed using Mutect2. As a result, 708 variants were obtained from the total genome size of 8194bp having variant rate of 1 variant every 11 bases. Four different type of markers were observed e.g. SNP , MNP, INS and DEL (shown in fig1.) which are equally distributed across the whole genome but major INDELS were observed at genome positions 5276, 5958, 6784, 6789, 7965, 7969, 7970 configuring 3.81%. Similarly, MNPs and SNPs were 5.38% and 90.67% respectively of total 708 variants. Detailed variants table is given in supplementary table S1. Categorization of variant by impact: Total 777 effects were calculated by impact and its functional class. High impact effects were measured as 10 (1.29%), low impact effects 514 (66.15%), moderate effects 115 (14.80%) and modifiers were 138 (17.76%). Moreover, functional class variants are classified as missense and silent effects with 83 (14.04%) and 508 (85.95%) respectively comprising missense/silent ratio of 0.1634. High impact variants are those who alter the splicing sites, loss or gain of start and stop codons. Low impact variants are synonymous changes in the coding region, start and stop codons whereas moderate impact variants are non-synonymous while modifiers are located in upstream, downstream, intergenic and 5’, 3’ UTR non coding regions of the genome. Details of variants impact and functional class are shown in Fig2 . Distribution of whole-genome variants by genomic region and type: Variants by genomic region: Maximum number of variants 639 (82.239%) were distributed in exonic region than intergenic, upstream and downstream region i.e. 69 (8.88%), 66 (8.494%) and 3 (0.386%) respectively. Variants by its type: Synonymous variants are in maximum number 514 (65.982%) followed by missense variant 108 (13.864%), intergenic region 69 (8.858%), upstream gene variant 66 (8.472%), frameshift variant 10 (1.284%), conservative in frame insertion 4 (0.513%), downstream gene variant 3 (0.385%), Stop gained 2 (0.275%), disruptive inframe deletion 2 (0.257%), conservative inframe deletion 1 (0.128%). As for as, insertion are concerned, a single 34 bp insertion was observed at position 7969. Similarly, one 75 bp deletion was observed at locus 5276.which might result in frameshifting of the harbor gene and further helpful for serotyping of this strain of FMDV. Transition to Transversion Ratio and Base change: Total 494 transitions (Ts) and 132 transversions (Tv) were observed with Ts/Tv ratio of 3.8. Base pair changes shown in (table 1) may affect codons and ultimately the protein structures and functions of the FMD virus. These changes might be responsible for altering sub-lineage, virus behavior, evolution and overall varying vaccine response in certain geographical region. Table 1. Display of Ts and Tv. Variable shaded cells indicate intensity of variation occurrence. Predicted genome wide codon and amino acid changes: Maximum of 22 times AAG→AAA codon changes were observed followed by 18 times GAC→GAG and 17 times GAG→GAA as shown in (Supplementary table S2). Moreover, maximum of 7 times Threonine and Valine (T & V) amino acids changes to Alanine (A) occurred as shown in (table 2). The aforementioned amino acid changes from Threonine to Alanine might affect the virus evolution because Threonine is hydroxyl as well as polar amino acid and Alanine is non polar. Although Valine and Alanine are both non-polar but Valine is aliphatic while Alanine is neither aliphatic nor aromatic. Table 2. Details of amino acid changes from reference genome MH784404.1 (rows) to PRJNA8560 query genome (columns) Variants distribution across genome: Distribution of 708 variants across the genome is even throughout genome. First variant was observed at locus180, last one at 8101 and all variants were equally distributed across the genome. High, moderate, low and modifier impact variants were observed where high impact variant are large chromosomal deletion, duplication and inversion, whole or partial deletion, duplication & inversion of exon, frameshift insertion, deletion, gene fusion, gene rearrangement, gene deletion, rare amino acid variant, splice acceptor variant, splice site donor variant, start & stop codon loss or gain. Whereas, moderate impact variants are inframe codon insertion, disruptive inframe insertion, codon disruptive deletion, missense variant, putative splice region 3’& 5’UTR deletion. Low impact variants are synonymous stop & start, synonymous coding, variation in splice site and non-synonymous start or stop of codon. Modifier impact variants hit often cds, downstream gene variation, exon variation, gene duplication or deletion, inter & intragenic variation, miRNA, intron variation, transcript and 3’ as well as 5’ UTR variation. High impact variants altered amino acids as p.Ser577fs, p.Leu578fs , p.Asp677fs, p.Leu1397fs, p.Val1622fs, p.Pro1898fs, p.Ala1900fs, p.Gly2291fs, p.Ala2293fs, p.Ala2293fs with genomic alteration at 2822, 2825, 3124 were in protein VP3, 5276 in protein 2C, 5958 in protein 3B, 67849, 6789, 7965,7969,7970 in protein 3D respectively. Details of high impact variants are given in table 3 whereas; low, moderate and modifier impact variants are given in supplementary table S3 . Table 3. Details of high impact variants Genomic Position Ref>Alt (Marker length) Marker Type Variant type & Impact Amino acid variation GenBank Ref. Protein ID & type 2822 GA>G (2) MNP Frameshift (High) p.Ser577fs, AYA84105 (Coding) 2825 T>TG (2) MNP Frameshift (High) p.Leu578fs, AYA84105 (Coding) 3124 ACACC>A (5) MNP Frameshift (High) p.Asp677fs, AYA84105 (Coding) 5276 ACCACCCCTGCAGAACGTGTACCAACTCGTA CAGGAGGTGAAAC GGGTCGAGCTCCATGAGAAAGTGTCGAG>A (75) MNP Frameshift (High) p.Leu1397fs, AYA84105 (Coding) 5958 G>GCCGTTAAGGAAGGCC (17) MNP Frameshift (High) p.Val1622fs, AYA84105 (Coding) 6784 GCCCT>G (5) MNP Frameshift (High) p.Pro1898fs, AYA84105 (Coding) 6789 GCTGCCTTGTCCAACAGGA>G (20) MNP Frameshift (High) p.Ala1900fs, AYA84105 (Coding) 7965 G>GAGCCATGACAGACAGT (17) MNP Frameshift (High) p.Gly2291fs, AYA84105 (Coding) 7969 T>TATAGAGTGTTTGAGTTTGAGATTAAAGTAAAGGA (34) MNP Frameshift (High) p.Ala2293fs, AYA84105 (Coding) 7970 C>CAG (3) MNP Frameshift (High) p.Ala2293fs AYA84105 (Coding) Phylogenetic analysis: The evolutionary history was inferred using the Neighbor-Joining method as shown in Fig 4. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis involved 15 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 8209 positions in the final dataset. Evolutionary analyses were conducted in MEGA11. Discussion Genomic variation in the FMD virus may lead to the evolution of new strain in the region. Since 2019, IND2001 sub lineage is reported first time from 10 districts of North-Eastern and North-Western Pakistan. FMDV samples were collected from different regions of Punjab-Pakistan and processed virus was cultured over BHK-21 cell line. WGS by Illumina MiSeq was performed from cDNA of confirmed FMDV serotype-O virus. Whole-genome variant calling analysis was performed using Pan-Asia II (MH784404.1) as reference genome. Current sequenced genome of FMDV from Pakistan is available on SRA project ID: PRJNA8560. Detailed insight of nucleotide base change and amino acid change analysis among both lineages illustrates that newly emerging lineage in Punjab have 708 variants including 777 major and minor effects as INDEL, SNP and MNP. Functional class effects were 14.044% missense and 85.956% silent. Frameshift variant 1.284%, conservative inframe deletion 0.128%, conservative inframe insertion 0.513%, disruptive inframe deletion 0.257%, downstream gene variant 0.385%, intergenic region variant 8.858%, missense variant 13.864%, stop gained 0.257%, synonymous variant 65.982% and upstream gene variant 8.472% were observed by effect and region type. In similar studies, it is indicated that major viral sequences which are called consensus sequence present in viral isolate but lack information about the variants. Difference between consensus sequence and individual sequences provide the strong evidence about the heterogeneity of virus population. High throughput sequencing plate form and more accurate calling analysis of variants is required to mark high divergent areas of viral genomes (Deng et al., 2021 ). Among short reads sequencing technologies, Illumina provide better results for variant calling analysis being more sensitive (Van den Hoecke et al., 2015 ). Variant calling initiative is identification and differentiation between true variant and noise of sequencing samples. These bioinformatics tools are key to check origin of the viral outbreaks. Production of more false positive variants (FPV) is more likely associated with sequencing protocols e.g. more amplification cycles may cause more FPV than caller type itself. Phylogenetic analysis along with mutation details in annotated files help us ruling out origin or divergence of new variants in viral outbreaks. In order to avoid sequencing errors and false positive occurrence on SNPs, different sample and library preparation methods could be used. (Wohl et al., 2016 ). To investigate any outbreak; quality and quantity of long genome reads of viruses for analysis, accurate library preparation methods, statistical phylogenetic analysis and reduction of missing data are key to success. In one of variant calling studies in Middle East on RNA-genome virus, 36 high impact variants were found out of 2200 distinct genome effects. Unlikely, 10 high impact variants were found in our study that inferred frameshift mutations in structural as well as non-structural proteins (Bindayna and Crinion, 2021 ). High rate of variants in Middle-East might be due to high temperature and multicultural region as compared to Pakistan. Previous studies from Pakistan have also shown genetic diversity of FMDV serotypes with high rate of evolution ≥ 6.65x10 3 and ~ 1.2x10 2 substitution per nucleotide per annum in circulating lineages, likewise current study provided variant rate of 1/11 bases with deeper understanding of evolution and emergence of new strain was deduced by analysis of SNPs, MNPs and INDELs. Moreover, it was assembly based variant calling which is demonstrated as beneficial and practical (Khan et al., 2018 ; Jamal et al., 2011 ; Heng Li, 2012 ). In another serotype of FMDV from Pakistan, P1 region of polyprotein is most divergent which is being used in identification and differentiation of FMDV serotypes, while in current study variants were more clustered in P1 region along with P2 and P3(Naqvi et al., 2022 ). Moreover, outbreak of new strain FMDV Serotype O/ME-SA/India2001 e have been reported in multiple regions of Punjab-Pakistan and our study provides plate form for novel emerging variant analysis through variant calling (Stenfeldt et al., 2022 ; Hicks et al., 2020 ). Taken together, our results show that deeper understanding of viral diversity through variant calling helps in finding evolution of novel viral strain in community. In this study, routinely recommended Haplotype caller i.e. Genome Analysis Tool Kit (GATK) was used using Mutect2 as variant analyzer to rule out lineage and sub-lineage of FMDV serotype-O in Pakistan. For future prospective, an optimum method for library preparation, sequencing and suitable variant caller should be declared by comparing error rate of methods to pick the best method for identification of evolution and viral genome variation. Statements And Declarations Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing interest: Authors have no financial or non-financial interest to disclose. Author Contribution: Anam Munir and Imran Altaf conceptualized the idea. Anam Munir, Imran Altaf and Aftab Ahmed Anjum designed methodology. First draft was written by Anam Munir. Aftab Ahmed Anjum, and Ali Raza Awan reviewed the manuscript. Acknowledgement: This work was supported by Higher Education Commission; Technology Development Fund 02-041. Special thanks to The Pirbright institute, UK. , Livestock and Dairy Development (L&DD), Lahore, Pakistan. Moreover, Dr. Rashid Saif from Decode Genomics provided his services for analysis of sequencing. Informed Consent: Informed consent was obtained from all authors. Ethics Approval: This is an observational study. The UVAS Research Ethics Committee has confirmed that no ethical approval is required. 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BMC genomics, 16 (1), 1-23. https://doi.org/10.1186/s12864-015-1284-z Wohl, S., Schaffner, S. F. & Sabeti, P. C. (2016). Genomic analysis of viral outbreaks. Annual review of virology, 3 (1), 173. https://doi.org/10.1146/annurev-virology-110615-035747 Supplementary Tables Supplementary Tables S1-S2 are not available with this version Additional Declarations No competing interests reported. Supplementary Files SupplementrytableS3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2396402","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":161772417,"identity":"c60c784d-d41e-4d4f-9027-b1c8323d5b03","order_by":0,"name":"Anam Munir","email":"","orcid":"","institution":"University of Veterinary and Animal Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anam","middleName":"","lastName":"Munir","suffix":""},{"id":161772418,"identity":"8f23e86a-8834-4208-be90-1d05b70225f0","order_by":1,"name":"Aftab Ahmed Anjum","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYDAC5gMMzCCasYGB8QFUzACIE3BrYUuGa2E2IE0LiClBlBb+Nv6DnwsY7PKYZ2SnVfO22dkzsDdvk2DckYZTi8QxZmbpGQzJxYwzcrfd5m1LTmzgOVYmwXgmB7fD7jczSPMwMCc2grTwnGFOYJDIMZNgbKvAqUMeaMtvHoZ6sJZinjP19gzyb/BrMTjGzAa05TBYCzNPxWHGBgkekBbcDjM8xmxmPcPgeGJjz9vNknMqjie28aQVWySewe19uWOMj28XVFQnbmzP3fjhjUG1PT/74Y03Pu5Ixu19iPOA1jVA2WwgIrEBp1oEkEfhMRKjZRSMglEwCkYKAAACZEz6wMwfowAAAABJRU5ErkJggg==","orcid":"","institution":"University of Veterinary and Animal Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Aftab","middleName":"Ahmed","lastName":"Anjum","suffix":""},{"id":161772421,"identity":"14828b48-f99f-4e16-a018-936decd6e4df","order_by":2,"name":"Imran Altaf","email":"","orcid":"","institution":"University of Veterinary and Animal Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Imran","middleName":"","lastName":"Altaf","suffix":""},{"id":161772422,"identity":"1d346a2b-1218-48d9-8300-f22aac698d28","order_by":3,"name":"Ali Raza Awan","email":"","orcid":"","institution":"University of Veterinary and Animal Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Raza","lastName":"Awan","suffix":""}],"badges":[],"createdAt":"2022-12-20 08:32:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2396402/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2396402/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30850255,"identity":"392f1dea-3ab4-472e-aacd-a379127b05b8","added_by":"auto","created_at":"2022-12-28 16:04:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18991,"visible":true,"origin":"","legend":"\u003cp\u003eObserved Variant and their types\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2396402/v1/85d13df5ebc996ba4db4fa4c.png"},{"id":30850251,"identity":"be2a7a7a-bb66-4d36-876a-49ebcb8642a8","added_by":"auto","created_at":"2022-12-28 16:04:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18285,"visible":true,"origin":"","legend":"\u003cp\u003eDetails of variants impact and their functional class\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2396402/v1/e647464d928ba1953c692a7c.png"},{"id":30850254,"identity":"9a8fb353-292c-499a-ad03-7a0152ccdd83","added_by":"auto","created_at":"2022-12-28 16:04:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32608,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of variants distribution by type and genomic region\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2396402/v1/5eb4c41e8ee829755b232314.png"},{"id":30850253,"identity":"095aae4d-fc93-491e-95db-2cfac905b17c","added_by":"auto","created_at":"2022-12-28 16:04:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":309203,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2396402/v1/3642d0aab8097aae2660804d.png"},{"id":30850258,"identity":"f3626667-745f-4ee6-be88-3c0887f2c8da","added_by":"auto","created_at":"2022-12-28 16:05:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":766679,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2396402/v1/8f3b6fab-9fe0-4c81-b4dc-49f563b99240.pdf"},{"id":30850257,"identity":"2a490767-49c7-4b3a-8ca3-bcfe6a61b028","added_by":"auto","created_at":"2022-12-28 16:05:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":766679,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2396402/v1/7ab80566-9e56-40a3-8117-07191dbcf2a9.pdf"},{"id":30850252,"identity":"8f5b6ca1-944f-4121-98ae-fec274fac8fa","added_by":"auto","created_at":"2022-12-28 16:04:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30926,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementrytableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-2396402/v1/978b0e1b8cab431deca53252.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole-Genome Variants Discovery of FMD Virus Isolated from Cattle Population in Pakistan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCloven hoofed animals are particularly affected by FMDV which was first ever known in 16th century along with the association of causative agent as a virus. Ongoing FMD episodes and their critical monetary impacts have opened up the worry of governments globally (Grubman and Baxt, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In the months of religious event called Eid Al Azha, movement of animals is increased which results in outbreaks during months of January to March usually in Pakistan (Jamal et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In South Asia, FMD remained endemic with outbreaks occurring regularly across the Indo-Pakistan subcontinents. Serotype O of FMDV is dispersed in all continents, particularly in the country of Middle east and south Asia (Rweyemamu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFMDV is a small positive sense ssRNA virus, approximately 8.3kb, belonging to \u003cem\u003eAphthovirus\u003c/em\u003e genus of \u003cem\u003ePicornaviridae\u003c/em\u003e family. FMDV has seven antigenically distinct serotypes designated as A, O, C, Asia 1, and South African Types (SAT) types 1\u0026ndash;3, and each serotype also contains different subtypes. OIE/FAO World Reference Laboratory for FMD (WRLFMD) reported 11 topo-types of FMDV serotype-O among which Pakistani strains are classified under Middle east-South Asia (ME-SA) topotype.(Samuel and Knowles, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) During 2002\u0026ndash;2006 FMDV, type O virus has been isolated in Pakistan which belonged to pak-98, Pan-Asia-II circulated in 2006 or Iran-2001 Pan Asia lineages and now IND2001 (a, b, c, d, e) linages are evolving(Jamal et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe virus genome codes for a single polyprotein which further proceeds to produce the set of intermediate and final mature proteins that are used for virus replication and assembly. The P1 region of polyprotein codes for four structural proteins (SP) which are VP1 (1D), VP2 (1B), VP3 (1C), and VP4 (1A). P2 and P3 regions codes for non-structural proteins (NSP) important for virus replication and cellular processes (Knipe et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Conserved Arg-Gly-Asp (RGD) amino acid sequence play critical role in FMDV binding to host cells receptors called Integrins.\u003c/p\u003e \u003cp\u003eAccording to the previous studies the use of nucleotide sequencing provides us the subsequent information for the identification of virus variants. These variant play very important role in the identification of trans- boundary virus transmission and generate form laboratory cell passages .These variants are also provide us strong evidence about the persistence of particle virus serotype. Nucleotide sequencing data is also play very important role to find origin of viral outbreak which is provide new approaches in inactivated vaccines (Han et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Studies have shown that the higher throughput and lower cost of next generation sequencing (NGS) techniques is consider a most widely used approach for deep sequencing genomic investigation in future (Peng Li et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Complete genome analysis of viruses circulating in the region provides platform for the recognition of new viral strain. Variant calling using Genome Analysis Tool Kit (GATK) pipeline provides comprehensive insight of variation between viruses from different time period (DePristo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Calling variants by GATK-4 is either done under category of UnifiedGenotyper (UG) or Haplotypecaller (HC) where later one is sophisticated and most used. If the sample size is more than 100, genome of diploid organism and pooled samples are used then UG is preferable. RNA genome viruses give rise to generation phylogenetically related but different variants called viral quasispecies due to RNA-dependent RNA polymerase (Biebricher and Eigen, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Undoubtedly, use of whole genome sequencing (WGS) data and computational tools help in deeper understanding of viral outbreaks and reasons (Orton et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, query genome comparison against reference genome was held to analyze mutations between two lineages of FMDV using haplotype variant calling by GATK-4 pipeline.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv\u003e \u003ch2\u003e2.1 Sample collection, virus propagation and identification:\u003c/h2\u003e \u003cp\u003eFoot and Mouth Disease Virus (FMDV) samples were collected from the epithelium of cattle buccal cavity from different regions of Punjab-Pakistan. Further, collected samples were processed in Quality Operations Lab (QOL), University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan for virus isolation using liquid nitrogen pulverization. For extraction of virus from the cells, centrifugation was performed at 2000rpm for 10 minutes to remove cellular debris. Afterwards, samples were stored in cryovials at -80ºC. BHK21 cell line with 80–90% confluency was used for virus propagation at 37ºC with 5% CO\u003csub\u003e2\u003c/sub\u003e supply. Moreover, presence of virus was confirmed by cytopathic effects on BHK21 cell line and identification was done by IZSLER antigen detection ELISA- Pirbright assay. Propagated FMDV on BHK21 host cell line.\u003c/p\u003e \u003cdiv\u003e \u003ch2\u003e2.2 RNA extraction and reverse transcription for cDNA synthesis:\u003c/h2\u003e \u003cp\u003eRNA extraction was performed using QIAamp viral RNA kit (www.qiagen.com) as per manufacturer instructions. Similarly, cDNA synthesis was done using RevertAid First Strand cDNA synthesis kit (www.thermoscientific.com) as per manufacturer instructions.\u003c/p\u003e \u003cdiv\u003e \u003ch2\u003e2.3 Whole-Genome Sequencing using Illumina Platform:\u003c/h2\u003e \u003cp\u003e\u003cstrong\u003eLibrary preparation\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eWhole-genome RNA was extracted followed by cDNA synthesis as per sequencing platform requirements along with 5’ and 3’ adapter ligation. cDNA was sheared using Covaris ultrasonicator apparatus and library preparation was performed with TruSeq Nano DNA library kit. Adapter-ligated fragments are PCR amplified and further size selection was done for sequencing run using Illumina HiSeq 4000 NGS platform by Macrogen, Korea (www.macrogen.com).\u003c/p\u003e \u003cp\u003e\u003cem\u003eSelection of reference genome\u003c/em\u003e: Reference genome of FMDV with accession ID MH784404.1 was taken from https://www.ncbi.nlm.nih.gov/nuccore/MH784404.1, which belongs to PanAsia-II lineage. It’s FASTA and GFF3 annotated files were taken for further usage in GATK4 whole-genome variant calling pipeline.\u003c/p\u003e \u003cdiv\u003e \u003ch2\u003e2.4 Whole-Genome variant calling bioinformatics pipeline:\u003c/h2\u003e \u003cp\u003eGATK4 pipeline was used on Linux operating system with following protocols:\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eQuality checks\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eMultiQC version 1.11 was used for quality checks of forward and reverse fastq.gz files. Multiple parameters generated showing low quality, short and duplicate reads, insert size distribution, sequence quality, read GC \u0026amp; N-content and passed filters.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eTrimming\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eTrimming was done by Fastp version 1.11. Jason file was produced for further processing.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eMapping of filtered reads\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eIndexing of reference genome and mapping was performed using Bowtie2 tool. Afterwards, SAM file was converted to BAM by Samtools while sorting and mark duplication steps were performed using Picard tool.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eVariant calling\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eVariant calling was performed using Mutect2. Afterwards, vcf allelicpremitive step was performed before annotation.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eFunctional annotation\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eSnpEff software was used for annotating the vcf files. Moreover, SNPsift software was used to extract the required field from the resultant file.\u003c/p\u003e \u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eWhole-Genome Sequencing Data Summary:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTruSeq Nano DNA library kit was used to sequence the whole-genome of FMDV by obeying manufacturer instructions using Illumina HiSeq 4000\u0026nbsp;NGS platform. Paired end (151x2) 37,223,254 reads were generated with 2.9 Gb data. Untrimmed raw FASTQ reads were obtained using bcl2fastq tool. Initial quality checks of reads revealed 42.35% of GC content, 95.95% of Q20 filter, and 90.1% of Q30 filter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuality Checks by MultiQC:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequencing Fastq files were analyzed, 98.40% of filtered reads were obtained with 1.60% low quality reads, none of the short reads and unknown nucleotides were observed. Similarly, less than 0.1% of duplicate reads with Phred score of more than 40. Average insert size was 151 with 78% coverage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVariant Identification and Annotation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter trimming and indexing, mapping of the clean reads of query FMDV against reference genome of PanAsia II was performed with Bowtie2 tool and variant calling was performed using Mutect2. As a result, 708 variants were obtained from the total genome size of 8194bp having variant rate of 1 variant every 11 bases. Four different type of markers were observed e.g. SNP , MNP, INS and DEL (shown in fig1.) which are equally distributed across the whole genome but major INDELS \u0026nbsp;were observed at genome positions 5276, 5958, 6784, 6789, 7965, 7969, 7970 configuring 3.81%. Similarly, MNPs and SNPs were 5.38% and 90.67% respectively of total 708 variants. Detailed variants table is given in supplementary table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCategorization of variant by impact:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal 777 effects were calculated by impact and its functional class. High impact effects were measured as 10 (1.29%), low impact effects 514 (66.15%), moderate effects 115 (14.80%) and modifiers were 138 (17.76%). Moreover, functional class variants are classified as missense and silent effects with 83 (14.04%) and 508 (85.95%) respectively comprising missense/silent ratio of 0.1634. High impact variants are those who alter the splicing sites, loss or gain of start and stop codons. Low impact variants are synonymous changes in the coding region, start and stop codons whereas moderate impact variants are non-synonymous while modifiers are located in upstream, downstream, intergenic and 5\u0026rsquo;, 3\u0026rsquo; UTR non coding regions of the genome. Details of variants impact and functional class are shown in\u003cstrong\u003e\u0026nbsp;Fig2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of whole-genome variants by genomic region and type:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVariants by genomic region:\u0026nbsp;\u003c/em\u003eMaximum number of variants 639 (82.239%) were distributed in exonic region than intergenic, upstream and downstream region i.e. 69 (8.88%), 66 (8.494%) and 3 (0.386%) respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVariants by its type:\u003c/em\u003e Synonymous variants are in maximum number 514 (65.982%) followed by missense variant 108 (13.864%), intergenic region 69 (8.858%), upstream gene variant 66 (8.472%), frameshift variant 10 (1.284%), conservative in frame insertion 4 (0.513%), downstream gene variant 3 (0.385%), Stop gained 2 (0.275%), disruptive inframe deletion 2 (0.257%), conservative inframe deletion 1 (0.128%). As for as, insertion are concerned, a single 34 bp insertion was observed at position 7969. Similarly, one 75 bp deletion was observed at locus 5276.which might result in frameshifting of the harbor gene and further helpful for serotyping of this strain of FMDV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransition to Transversion Ratio and Base change:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal 494 transitions (Ts) and 132 transversions (Tv) were observed with Ts/Tv ratio of 3.8. Base pair changes shown in (table 1) may affect codons and ultimately the protein structures and functions of the FMD virus. These changes might be responsible for altering sub-lineage, virus behavior, evolution and overall varying vaccine response in certain geographical region.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Display of Ts and Tv. Variable shaded cells indicate intensity of variation occurrence.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePredicted genome wide codon and amino acid changes:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaximum of 22 times AAG\u0026rarr;AAA codon changes were observed followed by 18 times GAC\u0026rarr;GAG and 17 times GAG\u0026rarr;GAA as shown in (Supplementary table S2). Moreover, maximum of 7 times Threonine and Valine (T \u0026amp; V) amino acids changes to Alanine (A) occurred as shown in (table 2). The aforementioned amino acid changes from Threonine to Alanine might affect the virus evolution because Threonine is hydroxyl as well as polar amino acid and Alanine is non polar. Although Valine and Alanine are both non-polar but Valine is aliphatic while Alanine is neither aliphatic nor aromatic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Details of amino acid changes from reference genome MH784404.1 (rows) to PRJNA8560 query genome (columns)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVariants distribution across genome:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDistribution of 708 variants across the genome is even throughout genome. First variant was observed at locus180, last one at 8101 and all variants were equally distributed across the genome. High, moderate, low and modifier impact variants were observed where high impact variant are large chromosomal deletion, duplication and inversion, whole or partial deletion, duplication \u0026amp; inversion of exon, frameshift insertion, deletion, gene fusion, gene rearrangement, gene deletion, rare amino acid variant, splice acceptor variant, splice site donor variant, start \u0026amp; stop codon loss or gain. Whereas, moderate impact variants are inframe codon insertion, disruptive inframe insertion, codon disruptive deletion, missense variant, putative splice region 3\u0026rsquo;\u0026amp; 5\u0026rsquo;UTR deletion. Low impact variants are synonymous stop \u0026amp; start, synonymous coding, variation in splice site and non-synonymous start or stop of codon. Modifier impact variants hit often cds, downstream gene variation, exon variation, gene duplication or deletion, inter \u0026amp; intragenic variation, miRNA, intron variation, transcript and 3\u0026rsquo; as well as 5\u0026rsquo; UTR variation. High impact variants altered amino acids as p.Ser577fs, p.Leu578fs , p.Asp677fs, p.Leu1397fs, p.Val1622fs, p.Pro1898fs, p.Ala1900fs, p.Gly2291fs, p.Ala2293fs, p.Ala2293fs with genomic alteration at 2822, 2825, 3124 were in protein VP3, 5276 in protein 2C, 5958 in protein 3B, 67849, 6789, 7965,7969,7970 in protein 3D respectively. Details of high impact variants are given in table 3 whereas; low, moderate and modifier impact variants are given in supplementary table \u003cstrong\u003eS3\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Details of high impact variants\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" style=\"margin-right: calc(9%); width: 91%;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenomic Position\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRef\u0026gt;Alt\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Marker length)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMarker Type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariant type \u0026amp; Impact\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmino acid variation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenBank Ref. Protein ID \u0026amp; type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e2822\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eGA\u0026gt;G\u003c/p\u003e\n \u003cp\u003e(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Ser577fs,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e2825\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eT\u0026gt;TG\u003c/p\u003e\n \u003cp\u003e(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Leu578fs,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e3124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eACACC\u0026gt;A\u003c/p\u003e\n \u003cp\u003e(5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Asp677fs,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e5276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eACCACCCCTGCAGAACGTGTACCAACTCGTA\u003cbr\u003eCAGGAGGTGAAAC\u003cbr\u003eGGGTCGAGCTCCATGAGAAAGTGTCGAG\u0026gt;A\u003c/p\u003e\n \u003cp\u003e(75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Leu1397fs,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e5958\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eG\u0026gt;GCCGTTAAGGAAGGCC\u003c/p\u003e\n \u003cp\u003e(17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Val1622fs,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e6784\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eGCCCT\u0026gt;G\u003c/p\u003e\n \u003cp\u003e(5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Pro1898fs,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e6789\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eGCTGCCTTGTCCAACAGGA\u0026gt;G\u003c/p\u003e\n \u003cp\u003e(20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Ala1900fs,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e7965\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eG\u0026gt;GAGCCATGACAGACAGT\u003c/p\u003e\n \u003cp\u003e(17)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Gly2291fs,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e7969\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eT\u0026gt;TATAGAGTGTTTGAGTTTGAGATTAAAGTAAAGGA\u003c/p\u003e\n \u003cp\u003e(34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Ala2293fs,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.66294227188082%\"\u003e\n \u003cp\u003e7970\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36.0151%;\" valign=\"top\" width=\"20.85661080074488%\"\u003e\n \u003cp\u003eC\u0026gt;CAG\u003c/p\u003e\n \u003cp\u003e(3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3312%;\" valign=\"top\" width=\"12.476722532588454%\"\u003e\n \u003cp\u003eMNP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.8271%;\" valign=\"top\" width=\"13.780260707635009%\"\u003e\n \u003cp\u003eFrameshift\u003c/p\u003e\n \u003cp\u003e(High)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8254%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003ep.Ala2293fs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.128%;\" valign=\"top\" width=\"20.11173184357542%\"\u003e\n \u003cp\u003eAYA84105\u003c/p\u003e\n \u003cp\u003e(Coding)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe evolutionary history was inferred using the Neighbor-Joining method as shown in Fig 4. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis involved 15 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 8209 positions in the final dataset. Evolutionary analyses were conducted in MEGA11.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGenomic variation in the FMD virus may lead to the evolution of new strain in the region. Since 2019, IND2001 sub lineage is reported first time from 10 districts of North-Eastern and North-Western Pakistan. FMDV samples were collected from different regions of Punjab-Pakistan and processed virus was cultured over BHK-21 cell line. WGS by Illumina MiSeq was performed from cDNA of confirmed FMDV serotype-O virus. Whole-genome variant calling analysis was performed using Pan-Asia II (MH784404.1) as reference genome. Current sequenced genome of FMDV from Pakistan is available on SRA project ID: PRJNA8560. Detailed insight of nucleotide base change and amino acid change analysis among both lineages illustrates that newly emerging lineage in Punjab have 708 variants including 777 major and minor effects as INDEL, SNP and MNP. Functional class effects were 14.044% missense and 85.956% silent. Frameshift variant 1.284%, conservative inframe deletion 0.128%, conservative inframe insertion 0.513%, disruptive inframe deletion 0.257%, downstream gene variant 0.385%, intergenic region variant 8.858%, missense variant 13.864%, stop gained 0.257%, synonymous variant 65.982% and upstream gene variant 8.472% were observed by effect and region type.\u003c/p\u003e \u003cp\u003eIn similar studies, it is indicated that major viral sequences which are called consensus sequence present in viral isolate but lack information about the variants. Difference between consensus sequence and individual sequences provide the strong evidence about the heterogeneity of virus population. High throughput sequencing plate form and more accurate calling analysis of variants is required to mark high divergent areas of viral genomes (Deng et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Among short reads sequencing technologies, Illumina provide better results for variant calling analysis being more sensitive (Van den Hoecke et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Variant calling initiative is identification and differentiation between true variant and noise of sequencing samples. These bioinformatics tools are key to check origin of the viral outbreaks. Production of more false positive variants (FPV) is more likely associated with sequencing protocols e.g. more amplification cycles may cause more FPV than caller type itself. Phylogenetic analysis along with mutation details in annotated files help us ruling out origin or divergence of new variants in viral outbreaks. In order to avoid sequencing errors and false positive occurrence on SNPs, different sample and library preparation methods could be used. (Wohl et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo investigate any outbreak; quality and quantity of long genome reads of viruses for analysis, accurate library preparation methods, statistical phylogenetic analysis and reduction of missing data are key to success. In one of variant calling studies in Middle East on RNA-genome virus, 36 high impact variants were found out of 2200 distinct genome effects. Unlikely, 10 high impact variants were found in our study that inferred frameshift mutations in structural as well as non-structural proteins (Bindayna and Crinion, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). High rate of variants in Middle-East might be due to high temperature and multicultural region as compared to Pakistan. Previous studies from Pakistan have also shown genetic diversity of FMDV serotypes with high rate of evolution\u0026thinsp;\u0026ge;\u0026thinsp;6.65x10\u003csup\u003e3\u003c/sup\u003e and ~\u0026thinsp;1.2x10\u003csup\u003e2\u003c/sup\u003e substitution per nucleotide per annum in circulating lineages, likewise current study provided variant rate of 1/11 bases with deeper understanding of evolution and emergence of new strain was deduced by analysis of SNPs, MNPs and INDELs. Moreover, it was assembly based variant calling which is demonstrated as beneficial and practical (Khan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jamal et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Heng Li, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In another serotype of FMDV from Pakistan, P1 region of polyprotein is most divergent which is being used in identification and differentiation of FMDV serotypes, while in current study variants were more clustered in P1 region along with P2 and P3(Naqvi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, outbreak of new strain FMDV Serotype O/ME-SA/India2001\u003csup\u003ee\u003c/sup\u003e have been reported in multiple regions of Punjab-Pakistan and our study provides plate form for novel emerging variant analysis through variant calling (Stenfeldt et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hicks et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, our results show that deeper understanding of viral diversity through variant calling helps in finding evolution of novel viral strain in community. In this study, routinely recommended Haplotype caller i.e. Genome Analysis Tool Kit (GATK) was used using Mutect2 as variant analyzer to rule out lineage and sub-lineage of FMDV serotype-O in Pakistan. For future prospective, an optimum method for library preparation, sequencing and suitable variant caller should be declared by comparing error rate of methods to pick the best method for identification of evolution and viral genome variation.\u003c/p\u003e"},{"header":"Statements And Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompeting interest:\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;Authors have no financial or non-financial interest to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAuthor Contribution:\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnam Munir and Imran Altaf conceptualized the idea. Anam Munir, Imran Altaf and Aftab Ahmed Anjum designed methodology. First draft was written by Anam Munir. \u0026nbsp;Aftab Ahmed Anjum, and Ali Raza Awan reviewed the manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAcknowledgement:\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Higher Education Commission; Technology Development Fund 02-041. Special thanks to The Pirbright institute, UK. , Livestock and Dairy Development (L\u0026amp;DD), Lahore, Pakistan. Moreover, Dr. Rashid Saif from Decode Genomics provided his services for analysis of sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eInformed Consent:\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInformed consent was obtained from all authors.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthics Approval:\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis is an observational study. The UVAS Research Ethics Committee has confirmed that no ethical approval is required.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eInternal Review Board approval:\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe study was approved by Advance Studies and Research Board (ASRB), UVAS (DAS/7091).\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBiebricher, C. K. \u0026amp; Eigen, M. (2006). What is a quasispecies? Quasispecies: concept and implications for virology, 1-31. https://doi.org/10.1007/3-540-26397-7_1\u003c/li\u003e\n \u003cli\u003eBindayna, K. M. \u0026amp; Crinion, S. (2021). Variant analysis of SARS-CoV-2 genomes in the Middle East. Microbial Pathogenesis, 153, 104741. https://doi.org/10.1016/j.micpath.2021.104741\u003c/li\u003e\n \u003cli\u003eDeng, Z.-L., Dhingra, A., Fritz, A., G\u0026ouml;tting, J., M\u0026uuml;nch, P. C., et al. (2021). Evaluating assembly and variant calling software for strain-resolved analysis of large DNA viruses. Briefings in bioinformatics, 22 (3), bbaa123. https://doi.org/10.1093/bib/bbaa123\u003c/li\u003e\n \u003cli\u003eDePristo, M. A., Banks, E., Poplin, R., Garimella, K. V., Maguire, J. R., et al. (2011). A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature genetics, 43 (5), 491-498. https://doi.org/10.1038/ng.806\u003c/li\u003e\n \u003cli\u003eGrubman, M. J. \u0026amp; Baxt, B. (2004). Foot-and-mouth disease. Clinical microbiology reviews, 17 (2), 465-493. https://doi.org/10.1128/cmr.17.2.465-493.2004\u003c/li\u003e\n \u003cli\u003eHan, L., Yuan, Y., Hu, J., Li, J., Zhu, S., et al. (2021). Next-generation sequencing sheds light on the interaction between virus and cell during foot-and-mouth disease virus persistent infection. Veterinary Microbiology, 263, 109247. https://doi.org/10.1016/j.vetmic.2021.109247\u003c/li\u003e\n \u003cli\u003eHicks, H. M., Wadsworth, J., Azhar, M., Afzal, M., Manzoor, S., et al. (2020). Genome sequences of foot-and-mouth disease virus O/ME-SA/Ind-2001e strains isolated in Pakistan. Microbiology Resource Announcements, 9 (18), e00165-20. https://doi.org/10.1128/mra.00165-20\u003c/li\u003e\n \u003cli\u003eJamal, S. M., Ahmed, S., Hussain, M. \u0026amp; Ali, Q. (2010). Status of foot-and-mouth disease in Pakistan. Archives of virology, 155 (9), 1487-1491. https://doi.org/10.1007/s00705-010-0732-y\u003c/li\u003e\n \u003cli\u003eJamal, S. M., Ferrari, G., Ahmed, S., Normann, P., Curry, S., et al. (2011). Evolutionary analysis of serotype A foot-and-mouth disease viruses circulating in Pakistan and Afghanistan during 2002\u0026ndash;2009. Journal of General Virology, 92 (12), 2849-2864. https://doi.org/10.1099/vir.0.035626-0\u003c/li\u003e\n \u003cli\u003eJamal, S. M., Khan, S., Knowles, N. J., Wadsworth, J., Hicks, H. M., et al. (2021). Foot‐and‐mouth disease viruses of the O/ME‐SA/Ind‐2001e sublineage in Pakistan. Transboundary and Emerging Diseases, 68 (6), 3126-3135. https://doi.org/10.1111/tbed.14134\u003c/li\u003e\n \u003cli\u003eKhan, M., Shahid, K., Faiza, A., Sibghat, U., Bait, U., et al. (2018). Emergence of new variants in foot and mouth disease virus serotype\u0026apos;O\u0026apos;in Khyber Pakhtunkhwa-Pakistan, 2012 to 2015. Asian Journal of Agriculture and Biology, 6 (2), 181-188. https://doi.org/10.12980/apjtd.7.2017d7-2\u003c/li\u003e\n \u003cli\u003eKnipe, T., Rieder, E., Baxt, B., Ward, G. \u0026amp; Mason, P. W. (1997). Characterization of synthetic foot-and-mouth disease virus provirions separates acid-mediated disassembly from infectivity. Journal of virology, 71 (4), 2851-2856. https://doi.org/10.1128/jvi.71.4.2851-2856.1997\u003c/li\u003e\n \u003cli\u003eLi, H. (2012). Exploring single-sample SNP and INDEL calling with whole-genome de novo assembly. Bioinformatics, 28 (14), 1838-1844. https://doi.org/10.1093/bioinformatics/bts280\u003c/li\u003e\n \u003cli\u003eLi, P., Yue, Y., Song, N., Li, B., Meng, H., et al. (2016). Genome analysis of enterovirus 71 strains differing in mouse pathogenicity. Virus Genes, 52 (2), 161-171. https://doi.org/10.1007/s11262-015-1271-0\u003c/li\u003e\n \u003cli\u003eNaqvi, S. S., Bostan, N., Fukai, K., Ali, Q., Morioka, K., et al. (2022). Evolutionary Dynamics of Foot and Mouth Disease Virus Serotype A and Its Endemic Sub-Lineage A/ASIA/Iran-05/SIS-13 in Pakistan. Viruses, 14 (8), 1634. https://doi.org/10.3390/v14081634\u003c/li\u003e\n \u003cli\u003eOrton, R. J., Wright, C. F., King, D. P. \u0026amp; Haydon, D. T. (2020). Estimating viral bottleneck sizes for FMDV transmission within and between hosts and implications for the rate of viral evolution. Interface Focus, 10 (1), 20190066. https://doi.org/10.1098/rsfs.2019.0066\u003c/li\u003e\n \u003cli\u003eRweyemamu, M., Roeder, P., Mackay, D., Sumption, K., Brownlie, J., et al. (2008). Epidemiological patterns of foot‐and‐mouth disease worldwide. Transboundary and emerging diseases, 55 (1), 57-72. https://doi.org/10.1111/j.1865-1682.2007.01013.x\u003c/li\u003e\n \u003cli\u003eSamuel, A. \u0026amp; Knowles, N. (2001). Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes). Journal of General Virology, 82 (3), 609-621. https://doi.org/10.1099/0022-1317-82-3-609\u003c/li\u003e\n \u003cli\u003eStenfeldt, C., Bertram, M., Holinka-Patterson, L., Fish, I., Farooq, U., et al. (2022). Foot-and-Mouth Disease Virus Serotypes O and A from Outbreaks in Pakistan 2011\u0026ndash;2012. Microbiology Resource Announcements, 11 (8), e00574-22. https://doi.org/10.1128/mra.00574-22\u003c/li\u003e\n \u003cli\u003eVan den Hoecke, S., Verhelst, J., Vuylsteke, M. \u0026amp; Saelens, X. (2015). Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing. BMC genomics, 16 (1), 1-23. https://doi.org/10.1186/s12864-015-1284-z\u003c/li\u003e\n \u003cli\u003eWohl, S., Schaffner, S. F. \u0026amp; Sabeti, P. C. (2016). Genomic analysis of viral outbreaks. Annual review of virology, 3 (1), 173. https://doi.org/10.1146/annurev-virology-110615-035747\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Tables","content":"\u003cp\u003eSupplementary Tables S1-S2 are not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2396402/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2396402/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFoot-and-mouth disease (FMD) is extremely contagious and multispecies that has a severe impact on animal trade across the borders. FMD virus may cause epidemics resulting in devastation of livestock industry so, it’s worthy to explore the genomic architecture of virus to harness the mortality and morbidity particularly in cattle from Pakistan. Epithelial scrapping samples of sick animals were taken from Punjab, Pakistan and cDNA of virus was sequenced through short-read NGS Illumina technology followed by variant calling analysis to reveal how novel variants give rise to new lineage in the region for a comprehensive insight of its genomic landscape. Haplotype-based variant discovery was performed by Genome Analysis Toolkit (GATK4) with Mutect2 using Pan Asia-II as reference genome. A total of 708 variants including 642 SNPs, 38 MNPs and 28 INDELs were observed. Furthermore, whole genome annotation revealed high, low, moderate and modifier impact variants count as 10(1.28%), 514(66.15%), 115(14.80%) and 138(17.76%) respectively which are distributed in VP3, 2C, 3B and 3D proteins of FMDV. Similarly, transitions-to-transversions ratio (3.75) and missense-to-silent ratio (0.1634) across the whole genome with 639 exonic, 3 downstream, 69 intergenic and 66 upstream effects were also identified. Whereas, high impact-frame shift mutations were concentrated in 5000-7000 nucleotide positions of the genome. A worth-mentioning deletion mutation of 75bp at 5276 position harbor 2C protein. The current whole genome variant discovery of FMDV will add new insight to understand the micro-evolution, speedy emergence of strains, mutation associated disease-severity and it’s lineage to prevent the prevalence of this catastrophe.\u003c/p\u003e","manuscriptTitle":"Whole-Genome Variants Discovery of FMD Virus Isolated from Cattle Population in Pakistan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-28 16:04:52","doi":"10.21203/rs.3.rs-2396402/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"df6ab665-50d5-4050-a7fb-7bcc67cb11cb","owner":[],"postedDate":"December 28th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-28T16:04:55+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-28 16:04:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2396402","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2396402","identity":"rs-2396402","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}