Search Results (35)

Tomato leaf curl New Delhi virus (ToLCNDV) is among the most prevalent and widely distributed begomovirus infecting chili pepper (Capsicum annuum) and tomato in the Indian subcontinent. In this study, a guide RNA (gRNA) sequence-CRISPR-Cas9 approach was used to target and cleave two essential coding regions in the begomovirus genome. The gRNAs were designed to target conserved regions of the ToLCNDV replication-associated protein (rep) gene or ORF AC1, and/or the coat protein (cp) gene or AV1 ORF, respectively. Based on an alignment of 346 representative ToLCNDV genome sequences, all predicted single nucleotide polymorphisms off-target sites were identified and eliminated as potential gRNA targets. Based on the remaining genome regions, four candidate gRNAs were designed and used to build gRNA-Cas9 duplexed constructs, e.g., containing two gRNAs cloned in tandem, in different combinations (1–4). Two contained two gRNAs that targeted the coat protein gene (cp; AV1 ORF), while the other two constructs targeted both the cp and replication-associated protein gene (rep; AC1 ORF). These constructs were evaluated for the potential to suppress ToLCNDV infection in Nicotiana benthamiana plants in a transient expression-transfection assay. Among the plants inoculated with the duplexed gRNA construct designed to cleave ToLCNDV-AV1 or AC1-specific nucleotides, the construct designed to target both the cp (293–993 nt) and rep (1561–2324) showed the greatest reduction in virus accumulation, based on real-time quantitative PCR amplification, and attenuated disease symptoms, compared to plants inoculated with the DNA-A component alone or mock-inoculated, e.g., with buffer. The results demonstrate the potential for gRNA-mediated suppression of ToLCNDV infection in plants by targeting at least two viral coding regions, underscoring the great potential of CRISPR-Cas-mediated abatement of begomovirus infection in numerous crop species. Full article

Begomoviruses infect many crops and weeds globally, especially in the tropical and subtropical regions, where there are waves of epidemics. These begomovirus epidemics are frequently associated with three DNA satellites: betasatellites, alphasatellites, and deltasatellites. Except for the origin of replication, these satellites show no sequence identity with the helper begomovirus. Alphasatellites and betasatellites encode the $\mathsf{\alpha }$-Rep and βC1 proteins, respectively, while deltasatellites encode no proteins. $\mathsf{\alpha }$-Rep, which functions like the Rep of the helper begomoviruses, ensures alphasatellite replication autonomy, while betasatellites and deltasatellites depend wholly on the helper virus for replication. The betasatellite βC1 protein is a pathogenicity determinant and suppressor of RNA silencing. The associations between satellites and helper viruses vary, depending on the virus and the host, and the roles of these satellites in disease development are an active area of investigation. This review highlights current information on the role of DNA satellites in begomovirus diseases and examines commonalities and differences between and within these satellites under prevailing conditions. Furthermore, two episomes, SEGS-1 and SEGS-2, associated with cassava mosaic geminiviruses, and their possible status as DNA satellites are discussed. DNA satellites are a major factor in begomovirus infections, which are a major constraint to crop production, especially in tropical and subtropical regions. Thus, areas for future research efforts, as well as implications in the biotechnological management of these viruses, are discussed in this review. Full article

Red meat consumption is considered a risk factor for colorectal cancer (CRC) development and stimulated isolation of plasmid-like DNA molecules from bovine serum and milk, termed bovine meat and milk factors (BMMFs). BMMFs encode a conserved replication protein (Rep). Increased populations of Rep-expressing macrophages have been identified in the peritumor of CRC patients and pre-cancerous tissues when compared to the tissues of healthy individuals. This supports the concept that BMMFs increase cancer risk by indirect carcinogenesis, upon induction of chronic inflammation. However, the spread of Rep⁺ immune cells in tissues at greater distances from primary tumors has not yet been assessed. Here, we immunohistologically analyzed the presence of Rep⁺ immune cells in sets of tumor, peritumor and, additionally, distant tissues of CRC patients (n = 13). We identified consistently high numbers of BMMF-positive macrophages in mucosal tissues at distances of as much as 25 cm away from the primary tumors, at levels comparable to peritumors and associated with M2-like macrophage polarization. The broad distribution of BMMFs suggests that BMMF⁺ macrophages might already exist at stages of pre-cancerous dysplasia or before. Quantification of BMMF tissue expression during colonoscopy might help to preventively stratify individuals at risk of developing polyps/CRC and recommend them for enhanced surveillance and/or changes in dietary lifestyle. Full article

In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1–13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing rep gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency. Full article

Porcine circovirus type 2 (PCV2) is a significant pathogen responsible for porcine circovirus-associated diseases (PCVAD), and it is widely prevalent in pig farms, leading to huge economic losses for the pig industry. Currently, the ability of PCV2 to enhance its own replication by using the antiviral inflammatory factors IFNα, IFNβ, and IL-2 and its complex immune escape mechanism remain unclear, which has attracted wide attention. Research has indicated that GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is involved in the innate immune response to a variety of viruses, primarily by regulating and composing stress granules (SGs) to inhibit viral replication. Our initial studies identified elevated G3BP1 expression during PCV2 infection, paradoxically promoting PCV2 replication. In light of this phenomenon, this study aims to elucidate how PCV2 regulates G3BP1 to enhance its replication. Our findings demonstrate that G3BP1 overexpression further activates PCV2-induced expression of RIG-I, MDA5, cGAS and STING, thereby promoting IFNβ production and affecting cell cycle arrest in the S phase, facilitating PCV2 replication. Moreover, interactions were observed between PCV2 Cap protein and G3BP1’s RGG domain, and between PCV2 Rep protein and G3BP1’s NTF2 and RRM domains, potentially promoting viral protein nuclear transfer. In summary, PCV2 enhances its replication by modulating G3BP1 to induce IFNβ production and directly binds viral proteins to promote viral protein nuclear transfer. This research provides a foundation for further investigation into the immune evasion mechanisms of PCV2. Full article

Adeno-Associated Viruses (AAVs, genus Dependoparvovirus) are the leading gene therapy vector. Until recently, efforts to enhance their capacity for gene delivery had focused on their capsids. However, efforts are increasingly shifting towards improving the viral replication protein, Rep78. We discovered that Rep78 and its shorter isoform Rep52 contain a strictly C-terminal sequence motif, DDx3EQ, conserved in most dependoparvoviruses. The motif is highly negatively charged and devoid of prolines. Its wide conservation suggests that it is required for the life cycle of dependoparvoviruses. Despite its short length, the motif’s strictly C-terminal position has the potential to endow it with a high recognition specificity. A candidate target of the DDx3EQ motif might be the DNA-binding interface of the origin-binding domain of Rep78, which is highly positively charged. Published studies suggest that this motif is not required for recombinant AAV production, but that substitutions within it might improve production. Full article

Adeno-associated viruses (AAVs) are small, non-enveloped viruses that package a single-stranded (ss)DNA genome of 4.7 kilobases (kb) within their T = 1 icosahedral capsid. AAVs are replication-deficient viruses that require a helper virus to complete their life cycle. Recombinant (r)AAVs have been utilized as gene delivery vectors for decades in gene therapy applications. So far, six rAAV-based gene medicines have been approved by the US FDA. The 4.7 kb ssDNA genome of AAV encodes nine proteins, including three viral structural/capsid proteins, VP1, VP2, and VP3; four large nonstructural proteins (replication-related proteins), Rep78/68 and Rep52/40; and two small nonstructural proteins. The two nonstructured proteins are viral accessory proteins, namely the assembly associated protein (AAP) and membrane-associated accessory protein (MAAP). Although the accessory proteins are conserved within AAV serotypes, their functions are largely obscure. In this review, we focus on the expression strategy and functional properties of the small nonstructural proteins of AAVs. Full article

CRESS-DNA encompasses a broad spectrum of viruses documented across diverse organisms such as animals, plants, diatoms, fungi, and marine invertebrates. Despite this prevalence, the full extent of these viruses’ impact on the environment and their respective hosts remains incompletely understood. Furthermore, an increasing number of viruses within this category lack detailed characterization. This investigation focuses on unveiling and characterizing viruses affiliated with the Genomoviridae family identified in liver samples from the bat Molossus molossus. Leveraging viral metagenomics, we identified seven sequences (MmGmV-PA) featuring a circular DNA genome housing two ORFs encoding replication-associated protein (Rep) and capsid protein (Cap). Predictions based on conserved domains typical of the Genomoviridae family were established. Phylogenetic analysis revealed the segregation of these sequences into two clades aligning with the genera Gemycirculavirus (MmGmV-06-PA and MmGmV-07-PA) and Gemykibivirus (MmGmV-01-PA, MmGmV-02-PA, MmGmV-03-PA, MmGmV-05-PA, and MmGmV-09-PA). At the species level, pairwise comparisons based on complete nucleotide sequences indicated the potential existence of three novel species. In summary, our study significantly contributes to an enhanced understanding of the diversity of Genomoviridae within bat samples, shedding light on previously undiscovered viral entities and their potential ecological implications. Full article

Apple chlorotic leaf spot virus (ACLSV) (genus, Trichovirus; family, Betaflexiviridae) is a widespread, deleterious, and the most damaging pathogen of pome and fruit trees including domesticated apple (Malus × domestica Borkh.), to which it is transmitted by grafting and pruning. The positive-sense, single-stranded RNA virus is 600–700 nm long and has a genome of 74.7–7.56 kbp in size, minus the poly-A tail and 3′- and 5′-untranslated regions. The genome has three overlapping open reading frames (ORFs) that encode a replication-associated protein (Rep), movement protein (MP), and coat protein (CP). RNA interference (RNAi)-mediated antiviral defense in eukaryotes has evolved to control infections in plant viruses. The objective of this study was to analyze locus-derived microRNAs (mdm-miRNAs) in the apple genome with potential for targeting ACLSV +ssRNA-encoded mRNAs, using a predictive approach that involves four algorithms. The goal is to mobilize the in silico-predicted endogenous mdm-miRNAs and trigger the RNAi pathway experimentally in apple trees to evaluate antiviral resistance to ACLSV. Experimentally validated apple (2n = 2X = 34) mdm-miRNAs (n = 322) were obtained from the miRBase database and aligned to the ACLSV genome (KU870525). Of the 322 targeting mature locus-derived mdm-miRNAs analyzed, nine apple mdm-miRNA homologs (mdm-miR395k, mdm-miR5225c, and mdm-miR7121 (a, b, c, d, e, f, g, h) were predicted by all “four algorithms”, whereas fifty-eight mdm-miRNAs were identified as consensus binding sites by the combined results of two algorithms. The miRanda, RNA22, and TAPIR algorithms predicted binding of mdm-miR395k at nucleotide position 4691 and identified it as the most effective interacting mdm-miRNA targeting the virus ORF1 sequence. An integrated Circos plot was generated to validate the accuracy of target prediction and determine if apple mdm-miRNAs could bind to the predicted ACLSV mRNA target(s). A genome-wide in silico-predicted miRNA-mediated target gene regulatory network was implicated to validate interactions necessary to warrant in vivo analysis. The availability of validated locus-derived microRNAs (mdm-miRNAs) with predicted potential to target ACLSV in infected apple trees represents the first step toward development of ACLSV-resistant apple trees. Full article

To date, only a handful of viruses have been identified in sea turtles. Although eukaryotic circular Rep (replication initiation protein)-encoding single-stranded DNA (CRESS DNA) viruses have been reported from a wide variety of terrestrial species, and some of these viruses have been associated with clinical conditions in certain animals, limited information is available on CRESS DNA viruses from marine life. The present study aimed to investigate the presence of CRESS DNA viruses in sea turtles. In the present study, two (samples T3 and T33) of the 34 cloacal samples from 31 sea turtles (found in ocean waters around the Caribbean Islands of St. Kitts and Nevis) tested positive for CRESS DNA viruses by a pan-rep nested PCR assay. The partial Rep sequence of T3 shared 75.78% of a deduced amino acid (aa) identity with that of a CRESS DNA virus (classified under family Circoviridae) from a mollusk. On the other hand, the complete genome (2428 bp) of T33 was determined by an inverse nested PCR assay. The genomic organization of T33 mirrored those of type II CRESS DNA viral genomes of cycloviruses, characterized by the putative “origin of replication” in the 5’-intergenic region, and the putative Capsid (Cap)- and Rep-encoding open reading frame on the virion-sense- and antisense-strand, respectively. The putative Rep (322 aa) of T33 retained the conserved “HUH endonuclease” and the “super 3 family helicase” domains and shared pairwise aa identities of ~57% with unclassified CRESS DNA viruses from benthic sediment and mollusks. Phylogenetically, the T33 Rep formed a distinct branch within an isolated cluster of unclassified CRESS DNA viruses. The putative Cap (370 aa) of T33 shared maximum pairwise aa identity of 30.51% with an unclassified CRESS DNA virus from a capybara. Except for a blood sample from T33 that tested negative for CRESS DNA viruses, other tissue samples were not available from the sea turtles. Therefore, we could not establish whether the T3 and T33 viral strains infected the sea turtles or were of dietary origin. To our knowledge, this is the first report on the detection of CRESS DNA viruses from sea turtles, adding yet another animal species to the rapidly expanding host range of these viruses. Complete genome analysis of T33 identified a novel, unclassified CRESS DNA virus, providing insights into the high genetic diversity between viruses within the phylum Cressdnaviricota. Considering that sea turtles are an at-risk species, extensive studies on virus discovery, surveillance, and pathogenesis in these marine animals are of the utmost importance. Full article

Klebsiella pneumoniae is an emergent pathogen causing respiratory tract, bloodstream, and urinary tract infections in humans. This study defines the genomic sequence data, genotypic and phenotypic characterization of K. pneumoniae clinically isolated from Al-Kharj, KSA. Whole-genome analysis of four K. pneumoniae strains was performed, including de novo assembly, functional annotation, whole-genome-phylogenetic analysis, antibiotic-resistant gene identification, prophage regions, virulent factor, and pan-genome analysis. The results showed that K6 and K7 strains were MDR and ESBL producers, K16 was an ESBL producer, and K8 was sensitive to all tested drugs except ampicillin. K6 and K7 were identified with sequence type (ST) 23, while K16 and K8 were identified with STs 353 and 592, respectively. K6 and K7 were identified with the K1 (wzi1 genotype) capsule and O1 serotype, while K8 was identified with the K57 (wzi206 genotype) capsule and O3b. K6 isolates harbored 10 antimicrobial resistance genes (ARGs) associated with four different plasmids; the chloramphenicol acetyltransferase (catB3), bla_OXA-1 and aac(6′)-Ib-cr genes were detected in plasmid pB-8922_OXA-48. K6 and K7 also carried a similar gene cassette in plasmid pC1K6P0122-2; the gene cassettes were the trimethoprim-resistant gene (dfrA14), integron integrase (IntI1), insertion sequence (IS1), transposase protein, and replication initiation protein (RepE). Two hypervirulent plasmids were reported in isolates K6 and K7 that carried synthesis genes (iucA, iucB, iucC, iucD, and iutA) and iron siderophore genes (iroB, iroC, iroD, and iroN). The presence of these plasmids in high-risk clones suggests their dissemination in our region, which represents a serious health problem. Full article

We report here high rates (47.5%, 48/101) of detection of porcine circovirus 2 (PCV2) in diarrheic pigs from three pig farms in the Dominican Republic. Seventeen of the PCV2 positive samples, representing the three pig farms, different age groups and sampling periods (2020–2021), were amplified for the complete PCV2 genome. Based on analysis of open reading frame 2 and complete genome sequences, the 17 PCV2 strains were assigned to the PCV2d genotype. Significant differences were observed in PCV2 detection rates between the vaccinated (20% (10/50)) and unvaccinated (62.5% (10/16) and 80% (28/35)) farms, corroborating previous observations that PCV2a-based vaccines confer protection against heterologous PCV2 genotypes. The present study is the first to report detection and molecular characterization of PCV2 from the Dominican Republic, warranting large-scale molecular epidemiological studies on PCV2 in pig farms and backyard systems across the country. For the first time, PCV2d was identified as the predominant PCV2 genotype in a study from the Caribbean region, suggesting that a genotype shift from PCV2b to PCV2d might be happening in the Caribbean region, which mirrored the current PCV2 genotype scenario in many other parts of the world. Besides PCV2, we also identified a pigeon circovirus-like virus, and a circular Replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus, which was characterized for the complete genome. The CRESS DNA virus shared a similar genomic organization and was related to unclassified CRESSV2 DNA viruses (belonging to the Order Cirlivirales) from porcine feces in Hungary, indicating that related unclassified CRESS DNA viruses are circulating among pigs in different geographical regions, warranting further studies on the epidemiology and biology of these novel viruses. Full article

Putative replication-associated protein (REP) and capsid-like (CAP) proteins are encoded by circular single-stranded DNA viruses (CRESS DNA), which have been found in samples from most eukaryotic groups. However, the details of these viruses’ life cycles and their significance in diseases have yet to be established. We presented and analyzed two full-length CRESS DNA genomes acquired from two children diagnosed with acute gastroenteritis (GI) in the northeast state of Tocantins, Brazil, using next-generation sequencing and a virus-like filtration approach. Both sequences (named SmaCV3BR08 and SmaCV3BR291) are closely similar to a prior CRESS DNA sequence discovered in the feces of a new world monkey (Alouatta caraya) from the United States in 2009 and termed Howler monkey-associated porprismacovirus 1 (Genbank ID: NC 026317). According to our comparative study, these porprismacovirus genomes deviate by 10% at the nucleotide level. For comparative reasons, the divergence between our sequences (SmaCV3BR08 and SmaCV3BR291) and a porprismacovirus recently identified in a human fecal sample from Peru is 37%. These data suggest that there is a great diversity of porprismacoviruses in South America, perhaps more than two species. In addition, the finding of closely related sequences of porprismacoviruses in humans and native monkeys highlights the zoonotic potential of these viruses. Full article

Alphasatellites are small single-stranded circular DNA molecules associated with geminiviruses and nanoviruses. In this study, a meta-analysis of known alphasatellites isolated from the genus Gossypium (cotton) over the last two decades was performed. The phylogenetic and pairwise sequence identity analysis suggested that cotton-infecting begomoviruses were associated with at least 12 different alphasatellites globally. Three out of twelve alphasatellite were associated with cotton leaf curl geminiviruses but were not isolated from cotton plants. The cotton leaf curl Multan alphasatellite, which was initially isolated from cotton, has now been reported in several plant species, including monocot plants such as sugarcane. Our recombination analysis suggested that four alphasatellites, namely cotton leaf curl Lucknow alphasatellites, cotton leaf curl Multan alphasatellites, Ageratum yellow vein Indian alphasatellites and Ageratum enation alphasatellites, evolved through recombination. Additionally, high genetic variability was detected among the cotton-infecting alphasatellites at the genome level. The nucleotide substitution rate for the replication protein of alphasatellites (alpha-Rep) was estimated to be relatively high (~1.56 × 10⁻³). However, unlike other begomoviruses and satellites, the first codon position of alpha-Rep rapidly changed compared to the second and third codon positions. This study highlights the biodiversity and recombination of alphasatellites associated with the leaf curl diseases of cotton crops. Full article

Canine circovirus (CanineCV) is an emerging virus in canines. Since the first strain of CanineCV was reported in 2012, CanineCV infection has shown a trend toward becoming a global epidemic. CanineCV infection often occurs with coinfection with other pathogens that may aggravate the symptoms of disease in affected dogs. Currently, CanineCV has not been successfully isolated by laboratories, resulting in a lack of clarity regarding its physicochemical properties, replication process, and pathogenic characteristics. To address this knowledge gap, the following results were obtained in this study. First, a CanineCV strain was rescued in F81 cells using infectious clone plasmids. Second, the Rep protein produced by the viral packaging rescue process was found to be associated with cytopathic effects. Additionally, the Rep protein and CanineCV inhibited the activation of the type I interferon (IFN-I) promoter, blocking subsequent expression of interferon-stimulated genes (ISGs). Furthermore, Rep was found to broadly inhibit host protein expression. We speculate that in CanineCV and canine parvovirus type 2 (CPV-2) coinfection cases, CanineCV promotes CPV-2 replication by inducing immunosuppression, which may increase the severity of clinical symptoms. Full article