Search Results (54)

Background/Objectives: ALK+ Anaplastic Large Cell Lymphoma (ALCL) is an aggressive T-cell lymphoma that is characterized by expression of the Anaplastic Lymphoma Kinase (ALK), which is induced by the t(2;5) chromosomal rearrangement, leading to the expression of the NPM-ALK fusion oncogene. Most previous preclinical models of ALK+ ALCL were based on overexpression of the NPM-ALK cDNA from heterologous promoters. Due to the enforced expression, this approach is prone to artifacts arising from synthetic overexpression, promoter competition and insertional variation. Methods: To improve the existing ALCL models and more closely recapitulate the oncogenic events in ALK+ ALCL, we employed CRISPR/Cas-based chromosomal engineering to selectively introduce translocations between the Npm1 and Alk gene loci in murine cells. Results: By inducing precise DNA cleavage at the syntenic loci on chromosome 11 and 17 in a murine IL-3-dependent Ba/F3 reporter cell line, we generated de novo Npm-Alk translocations in vivo, leading to IL-3-independent cell growth. To verify efficient recombination, we analyzed the expression of the NPM-ALK fusion protein in the recombined cells and could also show the t(11;17) in the IL-3 independent Ba/F3 cells. Subsequent functional testing of these cells using an Alk-inhibitor showed exquisite responsiveness towards Crizotinib, demonstrating strong dependence on the newly generated ALK fusion oncoprotein. Furthermore, a comparison of the gene expression pattern between Ba/F3 cells overexpressing the Npm-Alk cDNA with Ba/F3 cells transformed by CRISPR-mediated Npm-Alk translocation indicated that, while broadly overlapping, a set of pathways including the unfolded protein response pathway was increased in the Npm-Alk overexpression model, suggesting increased reactive changes induced by exogenous overexpression of Npm-Alk. Furthermore, we observed clustered expression changes in genes located in chromosomal regions close to the breakpoint in the new CRISPR-based model, indicating positional effects on gene expression mediated by the translocation event, which are not part of the older models. Conclusions: Thus, CRISPR-mediated recombination provides a novel and more faithful approach to model oncogenic translocations, which may lead to an improved understanding of the molecular pathogenesis of ALCL and enable more accurate therapeutic models of malignancies driven by oncogenic fusion proteins. Full article

Bovine enteroviruses (BEVs) are emerging pathogens with poorly understood evolutionary dynamics and zoonotic potential. Here, we report the discovery of a novel recombinant BEV strain, HeN-2022, isolated from cattle in China. Genomic analysis revealed that HeN-2022 is a primary hybrid of BEV-E1 (VG527, Ireland) and BEV-E4 (GX1901, China), with recombination breakpoints in the VP1 gene and 5′ UTR. Divergence dating traced its origin to 1991, predating closely related strains. Experimental infection in sheep demonstrated asymptomatic viral shedding (peak at 5 dpi) and robust neutralizing antibody responses, highlighting the potential cross-species adaptability. These findings underscore recombination as a potential key driver of BEV evolution and emphasize the need for global surveillance to address emerging livestock pathogens. Full article

The recent emergence of new HIV-1 recombinant strains presents a new challenge to the control of HIV-1/AIDS and the development of an effective vaccine. We employed a near full-length genomic sequence analysis of a newly identified CRF85_BC recombinant strain in Ningxia, China, to determine its recombination pattern. Blood samples were collected from HIV-infected or AIDS patients in Ningxia in 2023. CRF85_BC subtype strains were detected from three samples using an in-house method, and one sample’s near full-length genome sequence was also obtained. MEGA11, jpHMM, and Simplot software were used to identify subtypes and analyze recombination patterns. Neighbor-joining phylogenetic tree analysis showed that HIV-1 pol region sequences of three samples were CRF85_BC subtypes. One near full-length genome sequence of the recombinant strain was obtained, and jpHMM preliminarily judged that the recombinant strain was inserted with two subtype B fragments and two CRF01_AE fragments based on subtype C as the backbone. Further analysis using Simplot software revealed that the recombinant strain was the second-generation recombinant strain of CRF85_BC and CRF01_AE, and the recombination mode was based on the full-length genome of CRF85_BC, and CRF01_AE gene fragments that were inserted at positions 7365–8279 and 8431–9492, respectively. The results of the fragment phylogenetic tree verified its accuracy. One CRF01_AE and CRF85_BC second-generation recombinant strain was found in HIV-1 infected people in Ningxia, indicating that new HIV-1 recombinant strains continuously emerge and circulate in this region. Genomic surveillance of these recombinants should inform targeted interventions, such as prioritized contact tracing, to mitigate the formation of transmission clusters. Full article

Recombination, a process of genetic exchange between distinct organisms, has played a critical role in the emergence of SARS-CoV-2 variants such as the XEC recombinant. This study provides a detailed genomic and structural characterization of XEC, derived from the recombination of lineages KP.3.3 (donor) and KS.1.1 (acceptor). Phylogenomic analyses reveal that XEC and its descendant XEC.1 form a monophyletic clade with close evolutionary ties to KP.3.3. The genomic breakpoint, spanning nucleotide positions 22,363–22,463, marks the shift from KS.1.1 to KP.3.3 within the spike protein gene. Mutational analysis highlights shared traits with its parental lineages, including mutations associated with immune evasion, receptor affinity, and fusogenicity. Notable changes, such as Q493E and L455S, may confer unique immunogenic properties, though XEC’s overall immune escape potential is limited by the absence of new mutations in conserved epitopes. Despite these mutations, XEC demonstrates restricted geographical spread, low genetic variability, and an evolutionary trajectory indicative of an evolutionary dead-end. Bayesian Skyline Plot analysis corroborates this, showing stable but declining population size. These findings underscore the need for ongoing genomic surveillance to monitor recombinant variants’ characteristics and public health impact. This study contributes to understanding viral evolution and highlights the importance of distinguishing variants of concern from those with minimal epidemiological significance. Full article

Chloride channels (ClCs) have received global interest due to their significant role in the regulation of ion homeostasis, fluid transport, and electrical excitability of tissues and organs in different mammals and contributing to various functions, such as neuronal signaling, muscle contraction, and regulating the electrolytes’ balance in kidneys and other organs. In order to define the chloride voltage-gated channel (CLCN) gene family in buffalo, this study used in silico analyses to examine physicochemical properties, evolutionary patterns, and genome-wide identification. We identified eight CLCN genes in buffalo. The ProtParam tool analysis identified a number of important physicochemical properties of these proteins, including hydrophilicity, thermostability, in vitro instability, and basic nature. Based on their evolutionary relationships, a phylogenetic analysis divided the eight discovered genes into three subfamilies. Furthermore, a gene structure analysis, motif patterns, and conserved domains using TBtool demonstrated the significant conservation of this gene family among selected species over the course of evolution. A comparative amino acid analysis using ClustalW revealed similarities and differences between buffalo and cattle CLCN proteins. Three duplicated gene pairs were identified, all of which were segmental duplications except for CLCN4-CLCN5, which was a tandem duplication in buffalo. For each gene pair, the Ka/Ks test ratio findings showed that none of the ratios was more than one, indicating that these proteins were likely subject to positive selection. A synteny analysis confirmed a conserved pattern of genomic blocks between buffalo and cattle. Transcriptional control in cells relies on the binding of transcription factors to specific sites in the genome. The number of transcription factor binding sites (TFBSs) was higher in cattle compared to buffalo. Five main recombination breakpoints were identified at various places in the recombination analysis. The outcomes of our study provide new knowledge about the CLCN gene family in buffalo and open the door for further research on candidate genes in vertebrates through genome-wide studies. Full article

Recombination is a pervasive phenomenon in RNA viruses and an important strategy for accelerating the evolution of RNA virus populations. Recombination in the porcine reproductive and respiratory syndrome virus (PRRSV) was first reported in 1999, and many case reports have been published in recent years. In this review, all the existing reports on PRRSV recombination events were collected, and the genotypes, parental strains, and locations of the recombination breakpoints have been summarized and analyzed. The results showed that the recombination pattern constantly changes; whether inter- or intra-lineage recombination, the recombination hotspots vary in different recombination patterns. The virulence of recombinant PRRSVs was higher than that of the parental strains, and the emergence of virulence reversion was caused by recombination after using MLV vaccines. This could be attributed to the enhanced adaptability of recombinant PRRSV for entry and replication, facilitating their rapid propagation. The aim of this paper was to identify common features of recombinant PRRSV strains, reduce the recombination risk, and provide a foundation for future research into the mechanism of PRRSV recombination. Full article

The most common form of hereditary spastic paraplegia (HSP), SPG4 is caused by single nucleotide variants and microrearrangements in the SPAST gene. The high percentage of multi-exonic deletions or duplications observed in SPG4 patients is predisposed by the presence of a high frequency of Alu sequences in the gene sequence. In the present study, we analyzed DNA and RNA samples collected from patients with different microrearrangements in SPAST to map gene breakpoints and evaluate the mutation mechanism. The study group consisted of 69 individuals, including 50 SPG4 patients and 19 healthy relatives from 18 families. Affected family members from 17 families carried varying ranges of microrearrangements in the SPAST gene, while one individual had a single nucleotide variant in the 5′UTR of SPAST. To detect the breakpoints of the SPAST gene, long-range PCR followed by sequencing was performed. The breakpoint sequence was detected for five different intragenic SPAST deletions and one duplication, revealing Alu-mediated microhomology at breakpoint junctions resulting from non-allelic homologous recombination in these patients. Furthermore, SPAST gene expression analysis was performed using patient RNA samples extracted from whole blood. Quantitative real-time PCR tests performed in 14 patients suggest no expression of transcripts with microrearrangements in 5 of them. The obtained data indicate that nonsense-mediated decay degradation is not the only mechanism of hereditary spastic paraplegia in patients with SPAST microrearrangements. Full article

Human coronaviruses (HCoVs) are seriously associated with respiratory diseases in humans and animals. The first human pathogenic SARS-CoV emerged in 2002–2003. The second was MERS-CoV, reported from Jeddah, the Kingdom of Saudi Arabia, in 2012, and the third one was SARS-CoV-2, identified from Wuhan City, China, in late December 2019. The HCoV-Spike (S) gene has the highest mutation/insertion/deletion rate and has been the most utilized target for vaccine/antiviral development. In this manuscript, we discuss the genetic diversity, phylogenetic relationships, and recombination patterns of selected HCoVs with emphasis on the S protein gene of MERS-CoV and SARS-CoV-2 to elucidate the possible emergence of new variants/strains of coronavirus in the near future. The findings showed that MERS-CoV and SARS-CoV-2 have significant sequence identity with the selected HCoVs. The phylogenetic tree analysis formed a separate cluster for each HCoV. The recombination pattern analysis showed that the HCoV-NL63-Japan was a probable recombinant. The HCoV-NL63-USA was identified as a major parent while the HCoV-NL63-Netherland was identified as a minor parent. The recombination breakpoints start in the viral genome at the 142 nucleotide position and end at the 1082 nucleotide position with a 99% CI and Bonferroni-corrected p-value of 0.05. The findings of this study provide insightful information about HCoV-S gene diversity, recombination, and evolutionary patterns. Based on these data, it can be concluded that the possible emergence of new strains/variants of HCoV is imminent. Full article

Porcine sapovirus (PoSaV) is one of the most significant pathogens causing piglet diarrhea, and one with limited genetic characterization. In this study, the prevalence, infection pattern, and genetic evolution of porcine sapovirus were elucidated in detail. The positive rate of PoSaV was 10.1% (20/198), with dual, triple, and quadruple infections of 45%, 40%, and 5%, respectively. To further explore the viral composition in the PoSaV-positive diarrhea feces, metagenomic sequencing was carried out. The results confirmed that RNA viruses accounted for a higher proportion (55.47%), including the two primary viruses of PoSaV (21.78%) and porcine astrovirus (PAstV) (24.54%) in the tested diarrhea feces samples. Afterward, a full-length sequence of the PoSaV isolate was amplified and named SHCM/Mega2023, and also given the identifier of GenBank No. PP388958. Phylogenetic analysis identified the prevalent PoSaV strain SHCM/Mega2023 in the GIII genogroup, involving a recombinant event with MK962338 and KT922089, with the breakpoint at 2969–5132 nucleotides (nt). The time tree revealed that the GIII genogroup exhibits the widest divergence time span, indicating a high likelihood of viral recombination. Moreover, SHCM/Mega2023 had three nucleotide “RPL” insertions at the 151–153 nt site in the VP2 gene, compared to the other GIII strains. Further selective pressure calculations demonstrate that the whole genome of the SHCM/Mega2023 strain was under purifying selection (dN/dS < 1), with seven positively selected sites in the VP1 protein, which might be related to antigenicity. In conclusion, this study presents a novel genomic evolution of PoSaV, offering valuable insights into antigenicity and for vaccine research. Full article

Birds (Aves) are the most speciose of terrestrial vertebrates, displaying Class-specific characteristics yet incredible external phenotypic diversity. Critical to agriculture and as model organisms, birds have adapted to many habitats. The only extant examples of dinosaurs, birds emerged ~150 mya and >10% are currently threatened with extinction. This review is a comprehensive overview of avian genome (“chromosomic”) organization research based mostly on chromosome painting and BAC-based studies. We discuss traditional and contemporary tools for reliably generating chromosome-level assemblies and analyzing multiple species at a higher resolution and wider phylogenetic distance than previously possible. These results permit more detailed investigations into inter- and intrachromosomal rearrangements, providing unique insights into evolution and speciation mechanisms. The ‘signature’ avian karyotype likely arose ~250 mya and remained largely unchanged in most groups including extinct dinosaurs. Exceptions include Psittaciformes, Falconiformes, Caprimulgiformes, Cuculiformes, Suliformes, occasional Passeriformes, Ciconiiformes, and Pelecaniformes. The reasons for this remarkable conservation may be the greater diploid chromosome number generating variation (the driver of natural selection) through a greater possible combination of gametes and/or an increase in recombination rate. A deeper understanding of avian genomic structure permits the exploration of fundamental biological questions pertaining to the role of evolutionary breakpoint regions and homologous synteny blocks. Full article

Molecular investigations of the HIV-1 pol region (2253–5250 in the HXB2 genome) were conducted on sequences obtained from 331 individuals infected with HIV-1 in Cyprus between 2017 and 2021. This study unveiled four distinct HIV-1 putative transmission clusters, encompassing 19 previously unidentified HIV-1 recombinants. These recombinants, each comprising eight, three, four, and four sequences, respectively, did not align with previously established Circulating Recombinant Forms (CRFs). To characterize these novel HIV-1 recombinants, near-full-length genome sequences were successfully obtained for 16 of the 19 recombinants (790–8795 in the HXB2 genome) using an in-house-developed RT-PCR assay. Phylogenetic analyses, employing MEGAX and Cluster-Picker, along with confirmatory neighbor-joining tree analyses of subregions, were conducted to identify distinct clusters and determine subtypes. The uniqueness of the HIV-1 recombinants was evident in their exclusive clustering within generated maximum likelihood trees. Recombination analyses highlighted the distinct chimeric nature of these recombinants, with consistent mosaic patterns observed across all sequences within each of the four putative transmission clusters. Conclusive genetic characterization identified four novel HIV-1 CRFs: CRF129_56G, CRF130_A1B, CRF131_A1B, and CRF138_cpx. CRF129_56G exhibited two recombination breakpoints and three fragments of subtypes CRF56_cpx and G. Both CRF130_A1B and CRF131_A1B featured seven recombination breakpoints and eight fragments of subtypes A1 and B. CRF138_cpx displayed five recombination breakpoints and six fragments of subtypes CRF22_01A1 and F2, along with an unclassified fragment. Additional BLAST analyses identified a Unique Recombinant Form (URF) of CRF138_cpx with three additional recombination sites, involving subtype F2, a fragment of unknown subtype origin, and CRF138_cpx. Post-identification, all putative transmission clusters remained active, with CRF130_A1B, CRF131_A1B, and CRF138_cpx clusters exhibiting further growth. Furthermore, international connections were identified through BLAST analyses, linking one sequence from the USA to the CRF130_A1B strain, and three sequences from Belgium and Cameroon to the CRF138_cpx strain. This study contributes valuable insights into the dynamic landscape of HIV-1 diversity and transmission patterns, emphasizing the need for ongoing molecular surveillance and global collaboration in tracking emerging viral variants. Full article

Zucchini yellow mosaic virus (ZYMV) is a severe threat to cucurbit crops worldwide, including Pakistan. This study was pursued to evaluate the prevalence, geographic distribution, and molecular diversity of ZYMV isolates infecting cucurbits in Pakistan’s Pothwar region. Almost all the plant viruses act as a biotic stress on the host plants, which results in a yield loss. These viruses cause losses in single-infection or in mixed-infection cucurbit crops, and we have found a number of mixed-infected samples belonging to the Curubitaceae family. Serological detection of the tested potyviruses in the collected cucurbit samples revealed that ZYMV was the most prevalent virus, with a disease incidence (DI) at 35.2%, followed by Papaya ringspot virus (PRSV) with an incidence of 2.2%, and Watermelon mosaic virus (WMV) having an incidence as little as 0.5% in 2016. In the year 2017, a relatively higher disease incidence of 39.7%, 2.4%, and 0.3% for ZYMV, WMV, and PRSV, respectively, was recorded. ZYMV was the most prevalent virus with the highest incidence in Attock, Rawalpindi, and Islamabad, while PRSV was observed to be the highest in Islamabad and Jhelum. WMV infection was observed only in Rawalpindi and Chakwal. Newly detected Pakistani ZYMV isolates shared 95.8–97.0% nucleotide identities among themselves and 77.1–97.8% with other isolates retrieved from GenBank. Phylogenetic relationships obtained using different ZYMV isolates retrieved from GenBank and validated by in silico restriction analysis revealed that four Pakistani isolates clustered with other ZYMV isolates in group IIb with Chinese, Italian, Polish, and French isolates, while another isolate (MK848239) formed a separate minor clade within IIb. The isolate MK8482490, reported to infect bitter gourd in Pakistan, shared a minor clade with a Chinese isolate (KX884570). Recombination analysis revealed that the recently found ZYMV isolate (MK848239) is most likely a recombinant of Pakistani (MK848237) and Italian (MK956829) isolates, with a recombinant breakpoint between 266 and 814 nucleotide positions. Local isolate comparison and recombination detection may aid in the development of a breeding program that identifies resistant sources against recombinant isolates because the ZYMV is prevalent in a few cucurbit species grown in the surveyed areas and causes heavy losses and economic damage to the agricultural community. Full article

A complete genome sequence of an avian coronavirus (AvCoV; 27,663 bp excluding 3′ poly(A) tail) was determined using nontargeted next-generation sequencing (NGS) of an oropharyngeal swab from a backyard chicken in a live bird market in Arusha, Tanzania. The open reading frames (ORFs) of the Tanzanian strain TZ/CA127/19 are organized as typical of gammaCoVs (Coronaviridae family): 5′UTR-[ORFs 1a/1b encoding replicase complex (Rep1ab) non-structural peptides nsp2-16]-[spike (S) protein]-[ORFs 3a/3b]-[small envelop (E) protein]-[membrane (M) protein]-[ORFs 4a/4c]-[ORFs 5a/5b]-[nucleocapsid (N) protein]-[ORF6b]-3′UTR. The structural (S, E, M and N) and Rep1ab proteins of TZ/CA127/19 contain features typically conserved in AvCoVs, including the cleavage sites and functional motifs in Rep1ab and S. Its genome backbone (non-spike region) is closest to Asian GI-7 and GI-19 infectious bronchitis viruses (IBVs) with 87.2–89.7% nucleotide (nt) identities, but it has a S gene closest (98.9% nt identity) to the recombinant strain ck/CN/ahysx-1/16. Its 3a, 3b E and 4c sequences are closest to the duck CoV strain DK/GD/27/14 at 99.43%, 100%, 99.65% and 99.38% nt identities, respectively. Whereas its S gene phylogenetically cluster with North American TCoVs and French guineafowl COVs, all other viral genes group monophyletically with Eurasian GI-7/GI-19 IBVs and Chinese recombinant AvCoVs. Detection of a 4445 nt-long recombinant fragment with breakpoints at positions 19,961 and 24,405 (C- and N-terminus of nsp16 and E, respectively) strongly suggested that TZ/CA127/19 acquired its genome backbone from an LX4-type (GI-19) field strain via recombination with an unknown AvCoV. This is the first report of AvCoV in Tanzania and leaves unanswered the questions of its emergence and the biological significance. Full article

To better understand the evolution of the SARS-CoV-2 Omicron subvariants, we performed molecular evolutionary analyses of the spike (S) protein gene/S protein using advanced bioinformatics technologies. First, time-scaled phylogenetic analysis estimated that a common ancestor of the Wuhan, Alpha, Beta, Delta variants, and Omicron variants/subvariants diverged in May 2020. After that, a common ancestor of the Omicron variant generated various Omicron subvariants over one year. Furthermore, a chimeric virus between the BM.1.1.1 and BJ.1 subvariants, known as XBB, diverged in July 2021, leading to the emergence of the prevalent subvariants XBB.1.5 and XBB.1.16. Next, similarity plot (SimPlot) data estimated that the recombination point (breakpoint) corresponded to nucleotide position 1373. As a result, XBB.1.5 subvariants had the 5′ nucleotide side from the breakpoint as a strain with a BJ.1 sequence and the 3′ nucleotide side as a strain with a BM.1.1.1 sequence. Genome network data showed that Omicron subvariants were genetically linked with the common ancestors of the Wuhan and Delta variants, resulting in many amino acid mutations. Selective pressure analysis estimated that the prevalent subvariants, XBB.1.5 and XBB.1.16, had specific amino acid mutations, such as V445P, G446S, N460K, and F486P, located in the RBD when compared with the BA.4 and BA.5 subvariants. Moreover, some representative immunogenicity-associated amino acid mutations, including L452R, F486V, R493Q, and V490S, were also found in these subvariants. These substitutions were involved in the conformational epitopes, implying that these mutations affect immunogenicity and vaccine evasion. Furthermore, these mutations were identified as positive selection sites. These results suggest that the S gene/S protein Omicron subvariants rapidly evolved, and mutations observed in the conformational epitopes may reduce the effectiveness of the current vaccine, including bivalent vaccines such as mRNA vaccines containing the BA.4/BA.5 subvariants. Full article

The Sox gene family constitutes transcription factors with a conserved high mobility group box (HMG) that regulate a variety of developmental processes, including sex differentiation, neural, cartilage, and early embryonic development. In this study, we systematically analyzed and characterized the 20 Sox genes from the whole buffalo genome, using comparative genomic and evolutionary analyses. All the buffalo Sox genes were divided into nine sub-groups, and each gene had a specific number of exons and introns, which contributed to different gene structures. Molecular phylogeny revealed more sequence similarity of buffalo Sox genes with those of cattle. Furthermore, evolutionary analysis revealed that the HMG domain remained conserved in the all members of the Sox gene family. Similarly, all the genes are under strong purifying selection pressure; seven segmental duplications occurred from 9.65 to 21.41 million years ago (MYA), and four potential recombination breakpoints were also predicted. Mutational analysis revealed twenty non-synonymous mutations with potential effects on physiological functions, including embryonic development and cell differentiation in the buffalo. The present study provides insights into the genetic architecture of the Sox gene family in buffalo, highlights the significance of mutations, and provides their potential utility for marker-assisted selection for targeted genetic improvement in buffalo. Full article