Evolutionary patterns and heterogeneity of Dengue Virus serotypes in Pakistan.

Comprehensive and systematic examination of Dengue virus (DENV) evolution is essential in the context of Pakistan as the virus presents a significant public health challenge with the ability to adapt and evolve. To shed light on intricate evolutionary patterns of all four DENV serotypes, we analyzed complete genome sequences (n=43) and envelope (E) gene sequences (n=44) of all four DENV serotypes collected in Pakistan from 1994 to 2023 providing a holistic view of their genetic evolution. Our findings revealed that all four serotypes of DENV co-circulate in Pakistan with a close evolutionary relationship between DENV-1 and DENV-3. Genetically distinct serotypes DENV-2 and DENV-4 indicate that DENV-4 stands out as the most genetically different, while DENV-2 exhibits greater complexity due to the presence of multiple genotypes and the possibility of temporal fluctuations in genotype prevalence. Selective pressure analysis in Envelope (E) gene revealed heterogeneity among sequences (n=44) highlighting 46 codons in the genome experiencing selective pressure, characterized by a bias towards balancing selection indicating genetic stability of the virus. Furthermore, our study suggested an intriguing evolutionary shift of DENV-4 towards the DENV-2 clade, potentially influenced by antibodies with cross-reactivity to multiple serotypes providing a critical insight into the complex factors shaping DENV evolution and contributing to the emergence of new serotype.

Deep learning guided prediction modeling of dengue virus evolving serotype.

Evolution remains an incessant process in viruses, allowing them to elude the host immune response and induce severe diseases, impacting the diagnostic and vaccine effectiveness. Emerging and re-emerging diseases are among the significant public health concerns globally. The revival of dengue is mainly due to the potential for naturally arising mutations to induce genotypic alterations in serotypes. These transformations could lead to future outbreaks, underscoring the significance of studying DENV evolution in endemic regions. Predicting the emerging Dengue Virus (DENV) genome is crucial as the virus disrupts host cells, leading to fatal outcomes. Deep learning has been applied to predict dengue fever cases; there has been relatively less emphasis on its significance in forecasting emerging DENV serotypes. While Recurrent Neural Networks (RNN) were initially designed for modeling temporal sequences, our proposed DL-DVE generative and classification model, trained on complete genome data of DENV, transcends traditional approaches by learning semantic relationships between nucleotides in a continuous vector space instead of representing the contextual meaning of nucleotide characters. Leveraging 2000 publicly available DENV complete genome sequences, our Long Short-Term Memory (LSTM) based generative and Feedforward Neural Network (FNN) based classification DL-DVE model showcases proficiency in learning intricate patterns and generating sequences for emerging serotype of DENV. The generated sequences were analyzed along with available DENV serotype sequences to find conserved motifs in the genome through MEME Suite (version 5.5.5). The generative model showed an accuracy of 93 %, and the classification model provided insight into the specific serotype label, corroborated by BLAST search verification. Evaluation metrics such as ROC-AUC value 0.818, accuracy, precision, recall and F1 score, all to be around 99.00 %, demonstrating the classification model's reliability. Our model classified the generated sequences as DENV-4, exhibiting 65.99 % similarity to DENV-4 and around 63-65 % similarity with other serotypes, indicating notable distinction from other serotypes. Moreover, the intra-serotype divergence of sequences with a minimum of 90 % similarity underscored their uniqueness.

Genomic selection pressure discovery using site-frequency spectrum and reduced local variability statistics in Pakistani Dera-Din-Panah goat.

BACKGROUND: Population geneticists have long sought to comprehend various selection traces accumulated in the goat genome due to natural or human driven artificial selection through breeding practices, which led the wild animals to domestication, so understanding evolutionary process may helpful to utilize the full genetic potential of goat genome. METHODS AND RESULTS: As a step forward to pinpoint the selection signals in Pakistani Dera-Din-Panah (DDP) goat, whole-genome pooled sequencing (n = 12) was performed, and 618,236,192 clean paired-end reads were mapped against ARS1 reference goat assembly. Five different selection signature statistics were applied using four site-frequency spectrum (SFS) methods (Tajima's D ([Formula: see text]), Fay and Wu's H ([Formula: see text]), Zeng's E ([Formula: see text]), [Formula: see text]) and one reduced local variability approach named pooled heterozygosity ([Formula: see text]). The under-selection regions were annotated with significant threshold values of [Formula: see text]≥4.7, [Formula: see text]≥6, [Formula: see text]≥2.5, Pool-HMM ≥ 12, and [Formula: see text]≥5 that resulted in accumulative 364 candidate gene hits. The highest genomic selection signals were observed on Chr. 4, 6, 10, 12, 15, 16, 18, 20, and 27 and harbor ADAMTS6, CWC27, RELN, MYCBP2, FGF14, STIM1, CFAP74, GNB1, CALML6, TMEM52, FAM149A, NADK, MMP23B, OPN3, FH, MFHAS1, KLKB1, RRM1, KMO, SPEF2, F11, KIT, KMO, ERI1, ATP8B4, and RHOG genes. Next, the validation of our captured genomic hits was also performed by more than one applied statistics which harbor meat production, immunity, and reproduction associated genes to strengthen our hypothesis of under-selection traits in this Pakistani goat breed. Furthermore, common candidate genes captured by more than one statistical method were subjected to gene ontology and KEGG pathway analysis to get insights of particular biological processes associated with this goat breed. CONCLUSION: Current perception of genomic architecture of DDP goat provides a better understanding to improve its genetic potential and other economically important traits of medium to large body size, milk, and fiber production by updating the genomic insight driven breeding strategies to boost the livestock and agriculture-based economy of the country.

Whole-genome sequencing and variant discovery of Citrus reticulata "Kinnow" from Pakistan.

Citrus is a source of nutritional and medicinal advantages, cultivated worldwide with major groups of sweet oranges, mandarins, grapefruits, kumquats, lemons and limes. Pakistan produces all major citrus groups with mandarin (Citrus reticulata) being the prominent group that includes local commercial cultivars Feutral's Early, Dancy, Honey, and Kinnow. The present study designed to understand the genetic architecture of this unique variety of Citrus reticulata 'Kinnow.' The whole-genome resequencing and variant calling was performed to map the genomic variability that might be responsible for its particular characteristics like taste, seedlessness, juice content, thickness of peel, and shelf-life. A total of 139,436,350 raw sequence reads were generated with 20.9 Gb data in Fastq format having 98% effectiveness and 0.2% base call error rate. Overall, 3,503,033 SNPs, 176,949 MNPs, 323,287 INS, and 333,083 DEL were identified using the GATK4 variant calling pipeline against Citrus clementina. Furthermore, g:Profiler was applied for annotating the newly found variants, harbor genes/transcripts and their involved pathways. A total of 73,864 transcripts harbors 4,336,352 variants, most of the observed variants were predicted in non-coding regions and 1009 transcripts were found well annotated by different databases. Out of total aforementioned transcripts, 588 involved in biological processes, 234 in molecular functions and 167 transcripts in cellular components. In a nutshell, 18,153 high impact variants and 216 genic variants found in the current study, which may be used after its functional validation for marker-assisted breeding programs of "Kinnow" to propagate its valued traits for the improvement of contemporary citrus varieties in the region.

Detection of whole genome selection signatures of Pakistani Teddy goat.

BACKGROUND: Natural and artificial selection tend to cause variability that contributes to shape the genome of livestock in a way that differentiates them among the animal kingdom. The particular aim here is to identify positive selection signatures with whole genome pooled-sequence data of Pakistani Teddy goat. METHODS AND RESULTS: Paired-end alignment of 635,357,043 reads of Teddy goat with (ARS1) reference genome assembly was carried out. Pooled-Heterozygosity (Hp) and Tajima's D (TD) are applied for validation and getting better hits of selection signals, while pairwise FST statistics is conducted on Teddy vs. Bezoar (wild goat ancestor) for genomic differentiation, moreover annotation of regions under positive selection was also performed. Hp score with - ZHp > 5 detected six windows having highest hits on Chr. 29, 9, 25, 15 and 14 that harbor HRASLS5, LACE1 and AXIN1 genes which are candidate for embryonic development, lactation and body height. Secondly, - ZTD value of > 3.3 showed 4 windows with very strong hits on Chr.5 & 9 which harbor STIM1 and ADM genes related to body mass and weight. Lastly, - ZFST < - 5 generated four strong signals on Chr.5 & 12 harbor LOC102183233 gene. Other significant selection signatures encompass genes associated with wool production, prolificacy and coat colors traits in this breed. CONCLUSIONS: In brief, this study identified the genes under selection in Pakistani Teddy goat that will be helpful to refining the marker-assisted breeding policies and converging required production traits within and across other goat breeds and to explore full genetic potential of this valued species of livestock.

Whole genome comparison of Pakistani Corona virus with Chinese and US Strains along with its predictive severity of COVID-19.

Initially submitted 784 SARS-nCoV2 whole genome sequences on NCBI Virus database were selected for phylogenetic analysis to look into their similarities with two of Pakistani sequenced coronavirus strains having accessions of MT240479 and MT262993. The MT240479 named (Gilgit1-Pak) was found in close proximity to MT184913 named (CruiseA-USA), while MT262993 named (Manga-Pak) was in neighboring to MT039887 named (WI-USA) strain, which were further chosen for variant calling analysis along with reference genome NC_045512 as out-group to construct concluding cladogram and looked for evolutionary distance with PAUP software in this article. Aforementioned Pakistani strains each of having 29,836 bases were compared with MT263429 (WI-USA) of 29,889 bases and MT259229 (Wuhan-P.R. China) of 29,864 bases. Whole genome variant calling pipeline revealed 31 variants in both Pakistani strains collectively (Manga-Pak vs USA having 2del & 7SNPs, while different from Chinese strain with 2del & 2SNPs, similarly Gilgit1-Pak vs USA having 10SNPs, while different from Chinese strains having 8SNPs). These variants harbour ORF1ab, ORF1a and N genes having their role is viral replication/translation, host innate immunity and viral capsid formation respectively. These novel variants may be one of the reasons for low mortality rate in Pakistan with 385 deaths as compared to USA with 63,871 and P.R. China with 4633 by May 01, 2020. However functional characterization of these variants and their integrations with other viral proteins including variability of human receptors (ACE2 & NRP1) may be the other reasons for unlikely COVID-19 statistics in Pakistan which need further confirmatory studies. Moreover, mutated N and ORF1a proteins in Pakistani strains were also analyzed by 3D structure modeling, which give another dimension of comparing these alterations at amino acid level. In a nutshell, these novel variants are correlated with reduced mortality of COVID-19 severity in Pakistan while more robust results can be obtained by wet lab experimentation. This also gives insight of genomic landscape of these indigenous strains to develop diagnostics kits, vaccines and therapeutic interventions.

The LCORL Locus is under Selection in Large-Sized Pakistani Goat Breeds.

Goat domestication and human selection for valued traits have formed diverse breeds with characteristic phenotypes. This process led to the fixation of causative genetic variants controlling breed-specific traits within regions of reduced genetic diversity-so-called "selection signatures". We previously reported an analysis of selection signatures based on pooled whole-genome sequencing data of 20 goat breeds and bezoar goats. In the present study, we reanalyzed the data and focused on a subset of eight Pakistani goat breeds (Angora, Barbari, Beetal, Dera Din Panah, Kamori, Nachi, Pahari, Teddy). We identified 749 selection signatures based on reduced heterozygosity in these breeds. A search for signatures that are shared across large-sized goat breeds revealed that five medium-to-large-sized Pakistani goat breeds had a common selection signature on chromosome 6 in a region harboring the LCORL gene, which has been shown to modulate height or body size in several mammalian species. Fine-mapping of the region confirmed that all five goat breeds with the selection signature were nearly fixed for the same haplotype in a ~191 kb region spanning positions 37,747,447-37,938,449. From the pool sequencing data, we identified a frame-shifting single base insertion into an isoform-specific exon of LCORL as a potential candidate causal variant mediating the size-increasing effect. If this preliminary result can be confirmed in independent replication studies, genotyping of this variant might be used to improve breeding programs and the selection for stature in goats.

Selection signatures in goats reveal copy number variants underlying breed-defining coat color phenotypes.

Domestication and human selection have formed diverse goat breeds with characteristic phenotypes. This process correlated with the fixation of causative genetic variants controlling breed-specific traits within regions of reduced genetic diversity, so called selection signatures or selective sweeps. Using whole genome sequencing of DNA pools (pool-seq) from 20 genetically diverse modern goat breeds and bezoars, we identified 2,239 putative selection signatures. In two Pakistani goat breeds, Pak Angora and Barbari, we found selection signatures in a region harboring KIT, a gene involved in melanoblast development, migration, and survival. The search for candidate causative variants responsible for these selective sweeps revealed two different copy number variants (CNVs) downstream of KIT that were exclusively present in white Pak Angora and white-spotted Barbari goats. Several Swiss goat breeds selected for specific coat colors showed selection signatures at the ASIP locus encoding the agouti signaling protein. Analysis of these selective sweeps revealed four different CNVs associated with the white or tan (AWt), Swiss markings (Asm), badgerface (Ab), and the newly proposed peacock (Apc) allele. RNA-seq analyses on skin samples from goats with the different CNV alleles suggest that the identified structural variants lead to an altered expression of ASIP between eumelanistic and pheomelanistic body areas. Our study yields novel insights into the genetic control of pigmentation by identifying six functionally relevant CNVs. It illustrates how structural changes of the genome have contributed to phenotypic evolution in domestic goats.

Seroepidemiology of foot and mouth disease (FMD) virus non-structural protein (NSP) antibodies in the livestock of Oman.

Foot and mouth disease (FMD) is a highly contagious acute viral disease that causes severe economic losses to the affected countries. To estimate the serological prevalence of the local livestock to the natural infection with FMD virus, a cross-sectional study was conducted from 2015 to 2017 in Oman. Sera from 5807 randomly selected animals (1792 cattle, 2119 goats and 1896 sheep) belonging to 884 herds were tested for the presence of antibodies against 3ABC non-structural protein (NSP) of the FMD virus by a Competition ELISA. Prevalence along with confidence intervals (CI) and odds ratio (OR) was calculated, and the data were further analysed through univariable and multivariable techniques. The herd-level seroprevalence of NSP (41.4%) varied significantly among 11 governorates of Oman. At herd-level seroprevalence varied significantly among cattle (55.2%), goats (38.2%) and sheep (37.7%). At animal-level, the significantly higher prevalence was recorded in cattle (26.8%) followed by sheep (17.9%) and goats (17.3%). Binary logistic regression analysis at the individual level indicated that cattle (OR: 1.88), livestock above one year of age (OR: 3.47), imported breeds (OR: 1.82) and females (OR: 1.27) were the risk factors associated with higher prevalence. Whereas, sedentary (OR: 2.63) farming system, vaccination against FMD (OR: 5.98) and previous history of FMD (OR: 5.55) were the variables found associated with higher seroprevalence at the herd-level. We conclude that natural infection of FMDV is widespread in Oman and in future molecular typing of FMD virus outbreaks strains and vaccine matching studies should be employed to develop an effective control program in Oman.

Author Correction: Applications and efficiencies of the first cat 63 K DNA array.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Author Correction: Applications and efficiencies of the first cat 63K DNA array.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Applications and efficiencies of the first cat 63K DNA array.

The development of high throughput SNP genotyping technologies has improved the genetic dissection of simple and complex traits in many species including cats. The properties of feline 62,897 SNPs Illumina Infinium iSelect DNA array are described using a dataset of over 2,000 feline samples, the most extensive to date, representing 41 cat breeds, a random bred population, and four wild felid species. Accuracy and efficiency of the array's genotypes and its utility in performing population-based analyses were evaluated. Average marker distance across the array was 37,741 Kb, and across the dataset, only 1% (625) of the markers exhibited poor genotyping and only 0.35% (221) showed Mendelian errors. Marker polymorphism varied across cat breeds and the average minor allele frequency (MAF) of all markers across domestic cats was 0.21. Population structure analysis confirmed a Western to Eastern structural continuum of cat breeds. Genome-wide linkage disequilibrium ranged from 50-1,500 Kb for domestic cats and 750 Kb for European wildcats (Felis silvestris silvestris). Array use in trait association mapping was investigated under different modes of inheritance, selection and population sizes. The efficient array design and cat genotype dataset continues to advance the understanding of cat breeds and will support monogenic health studies across feline breeds and populations.

Expression Profiling of Hspb1 and Tp53 Genes through RT-qPCR in Different Cancer Types of Canis familiaris.

Background: Diagnostic molecular marker studies are in vogue to have insight of most prevalent animal diseases including cancer. Objectives: Gene expression profiling of pro and anti-apoptotic genes was conducted in dog Lymphoma, CTVT, SCC, granuloma, perianal adenocarcinoma and mammary tumors. Materials and Methods: Cancerous tissues of 21 affected animals were obtained. Total RNA was extracted followed by cDNA synthesis. Comparative Ct method via Taqman assay (RT-qPCR) was used to quantify corresponding mRNA molecules, Tp53 and Hspb1, as normalized by GAPDH as the reference gene . Results:Hspb1 showed ectopic expression in lymphoma, CTVT and mammary tumors; its down-regulation was observed in granuloma and oral SCC with fold difference (FD) of ±35. Similarly, Tp53 as the tumor suppressor gene with pro-apoptotic properties, showed up-regulation in all tumor types, notably 80% of mammary tumors and 60% of CTVT. The FD values were 33.31 and 2.27, respectively. Conclusion: Altered transcriptomic response of Hspb1 and Tp53 was observed in all cancer types of Canis familiaris. The resulting profile depicts the involvement of the genes in cancer pathways. Thus, the data might be helpful for diagnosis, prognosis, identification and classification of these widespread neoplasms in this species.

Hspb1 and Tp53 Mutation and Expression Analysis in Cat Mammary Tumors.

BACKGROUND: Molecular marker based cancer diagnosis gaining more attention in the current genomics era. So, Hspb1 and Tp53 gene characterization and their mRNA expression might be helpful in diagnosis and prognosis of cat mammary adenocarcinoma. It will also add information in comparative cancer genetics and genomics. OBJECTIVES: Eight tumors of Siamese cats were analyzed to ascertain germ-line and tissue-specific somatic DNA variations of Hspb1 and Tp53 genes along with the ectopic differential expression in tumorous and normal tissues were also analyzed. MATERIALS AND METHODS: Tumorous tissues and peripheral blood from mammary adenocarcinoma affected Siamese cats were collected from the Pet center-UVAS. DNA and RNA were extracted from these tissues to analyze the Hspb1 and Tp53 DNA variants and ectopic expression of their mRNA within cancerous and normal tissues. RESULTS: Exon 1 and 3 revealed as hotspots in Hspb1 gene. The 5´UTR region of the exon1 bear six mutation including 3 transitions, 2 transversion and one heterozygous synonymous transversion in two samples at locus c.34C>C/A. Exon 3 has 1 transversion at c.773A>A/T, 3´UTR of this exon harbor two point mutations at 1868A>T and 2193C>T loci. Intron 2 has two alterations at 1490C>C/T and GTCT4del at 1514. Overall up-regulation of Hspb1 gene was observed. While exons 3, 4 and 7 of Tp53 harbor a single variationat c.105A>A/G, c.465T>T/C and c.859G>T respectively. The locus c.1050G>G/A in exon 9 is a heterozygous (G/A) in 3 samples and homozygous (G) in 2 other tumours. Introns 3, 5, 7 and 9 harbor 3, 4, 2 and 7 altered loci respectively. Sixty percent of cancers showed up-regulated trend of Tp53 gene. CONCLUSIONS: Tumor specific mutations and ectopic expression of Hspb1 and Tp53 genes might be helpful in the diagnosis of the mammary lesions and endorse their involvement in cat mammary neoplasm.

Molecular phylogeny of Pakistani riverine buffalo based on genetic variability of mitochondrial cytochrome b gene.

Mitochondrial cytochrome b gene is considered to be one of the best markers for breed characterization as well as studying the ancestry in the vertebrates due to its exclusive maternal inheritance. DNA fingerprinting by single nucleotide polymorphism is most reliable and widely used molecular technique in modern forensics and is being considered in this study. Partial sequencing of 1,061 bp of aforementioned gene from 14580 to 15643 was conducted in two famous Pakistani buffalo breeds named Nili-Ravi and Kundi. In which we explore seven haplotypes within earlier and none in the latter breed. Nili-Ravi is polymorphic at four codons of this gene, and the protein translation is also different from the reference sample while monomorphic at three codons with no amino acid replacement. Haplotypes frequency distribution of these four haplotypes named NR3, NR4, NR5, NR7 revealed that the prevalence of each haplotype is 0.04 % in the Pakistani buffalo population of this Nili-Ravi breed while complete homoplasmy was observed in the Kundi breed population. Nili-Ravi breed of buffalo is genetically more variable than the Kundi breed as far as the gene in subject is concerned. It means later breed has spent more time to propagate its wild type haplotype which make this breed more ancestral as compare to Nili-Ravi. Secondly both breeds share their common ancestors with regional water buffalo rather than the swamp one.

DNA fingerprinting of Pakistani buffalo breeds (Nili-Ravi, Kundi) using microsatellite and cytochrome b gene markers.

Cytochrome b gene markers have been proved as an efficient and powerful tool for breed characterization and species identification of buffaloes. This study represents the substantial analysis of mitochondrial DNA variation in Pakistani buffalo breeds and provides information about their genetic diversity. In this study partial amplification of cytochrome b gene of 1,061 bp was done and sequencing results showed ten haplotypes. Comparing all fifty samples from two buffalo breeds of Pakistan, fifteen polymorphic sites were observed out of which, twelve codons 42, 71, 118, 120, 199, 235, 269, 297, 318, 327, 350, 355 of mitochondrial cytochrome b gene are monomorphic which translate same amino acids as in the reference protein sequence due to silent mutation while different in DNA sequence. Similarly three codons 163, 246, 337 of mitochondrial cytochrome b are polymorphic and different from the reference sequence with respect to DNA as well as protein sequence. For the further confirmation a panel of nine microsatellite markers was used with high polymorphism information content (PIC). The frequency distribution of these alleles varies from three to eight allele at locus CSSM66 and ILST029 respectively. The results obtained from this study may contribute to the establishment of routine genotyping service of buffalo breeds for buffalo farmers for animal forensic application in case of any dispute. Additionally this study may help for breed characterization and phylogeny of aforementioned breeds of buffalo.