Circular DNA Viruses Research Articles

First report of Cotton leaf curl Multan virus of Spinach in China.

Cotton leaf curl Multan virus(CLCuMuV; Begomovirus gossypimultanese, family Geminiviridea) is a single-stranded circular DNA virus, with a genome size of about 2.7 kb, CLCuMuV, which is commonly associated with its satellite DNA, Cotton leaf curl Multan betasatellite (CLCuMuB) (Mansoor et al., 2003), is a serious threat in cotton production causing cotton leaf curl disease (CLCuD) (Briddon et al., 2000). The spread of CLCuMuV is closely linked to its insect vector, whitefly (Bemisia tabaci), which is the exclusive vector species for CLCuMuV transmission (Pan et al., 2018). In May 2019, two spinach (Spinacia oleracea L) samples (XJBC01, XJBC02) showing upward curling of the leaf margins, vein thickening, and enation, symptoms were collected in Shihezi City, Xinjiang, China (Fig. 1B). A 570 bp fragment was amplified from the two symptomatic spinach samples using Begomovirus universal primer pair AV494 (5'-GCCYATRTAYAGRAAGCCMAG-3') and COPR (5'-GANGSATGHTRCADGCCAT ATA-3'), Sequences generated from these amplicons shared 99% nucleotide sequence identities with CLCuMuV DNA-A sequences, suggesting CLCuMuV infection in spinach. To our knowledge CLCuMuV has not been reported in spinach previously. The complete sequences of CLCuMuV and CLCuMuB were then sequenced using CLCuMuV-specific primers GD37-F (5'-GGATCCATTGTTAAACGAATTTCC-3') and GD37-R (5'-GGATCCCACATGTTTGAATTTGA-3') (Gu et al., 2015), as well as betasatellite universal primers β01 (5'-GGTACCACTACGCTACGCAGCAGCC-3') and β02 (5'-GGTACCTACCCTCCCAGGGGTACAC-3') (Zhou et al.,2003). The full length CLCuMuV DNA-A in spinach spans 2737 nt (GenBank accession number: MW561346), while CLCuMuB in spinach covers 1343 nt (GenBank accession number: MW561347). The 2737 nt full length CLCuMuV DNA-A and the associated 1343 nt CLCuMuB genome sequences generated from spinach samples were deposited in the GenBank with accession numbers MW561346 and MW561347. The MW561346 shared 99.5% sequence identity with CLCuMV GD37 from Hibiscus rosasinensis. Whereas the MW561347 shared 98.4% sequence identity with CLCuMuB GD37β. Therefore, we used infectious clones of CLCuMuV (GD37) and CLCuMuB (GD37β), provided by Xueping Zhou (Gu et al., 2015), to inoculate healthy spinach via Agrobacterium. Infected plants showed typical symptoms 14 days post-inoculation, including leaf edge curling, shrinkage, and vein enlargement, which is consistent with symptoms observed in infected spinach plants in the field (Fig. 1C). The expected 570 bp fragments were amplified in the uninoculated upper leaves of spinach showing symptoms, while not detected in the control spinach, indicating that the symptoms on spinach plants were caused by CLCuMuV associated with CLCuMuB. The transmission efficiency of CLCuMuV to spinach was assessed using two whitefly species, MEAM1 and MED, which were fed on h. rosasinensis infected with CLCuMuV. To compare the transmission efficiency between the two species, 14 spinach plants were inoculated with MEAM1, and 11 spinach plants were inoculated with MED. Each spinach plant was inoculated by releasing 10 whiteflies. After 30 days, MEAM1 transmitted CLCuMuV to spinach inducing typical symptoms (Fig. 1D), with a 78.57% (11/14) transmission efficiency. Similarly, MED also transmitted CLCuMuV to spinach but with a lower efficiency of 54.54% (6/11). These results suggested both MEAM1 and MED could transmit CLCuMuV to spinach, with MEAM1 demonstrating higher efficiency than MED. To the best of our knowledge, this study marks the first report of CLCuMuV infecting spinach, indicating an expanded host range for the virus.

Unveiling the Virome of Wild Birds: Exploring CRESS-DNA Viral Dark Matter.

Amid global health concerns and the constant threat of zoonotic diseases, this study delves into the diversity of circular replicase-encoding single-stranded DNA (CRESS-DNA) viruses within Chinese wild bird populations. Employing viral metagenomics to tackle the challenge of "viral dark matter," the research collected and analyzed 3,404 cloacal swab specimens across 26 bird families. Metagenomic analysis uncovered a rich viral landscape, with 67.48% of reads classified as viral dark matter, spanning multiple taxonomic levels. Notably, certain viral families exhibited host-specific abundance patterns, with Galliformes displaying the highest diversity. Diversity analysis categorized samples into distinct groups, revealing significant differences in viral community structure, particularly noting higher diversity in terrestrial birds compared to songbirds and unique diversity in migratory birds versus perching birds. The identification of ten novel Circoviridae viruses, seven Smacoviridae viruses, and 167 Genomoviridae viruses, along with 100 unclassified CRESS-DNA viruses, underscores the expansion of knowledge on avian-associated circular DNA viruses. Phylogenetic and structural analyses of Rep proteins offered insights into evolutionary relationships and potential functional variations among CRESS-DNA viruses. In conclusion, this study significantly enhances our understanding of the avian virome, shedding light on the intricate relationships between viral communities and host characteristics in Chinese wild bird populations. The diverse array of CRESS-DNA viruses discovered opens avenues for future research into viral evolution, spread factors, and potential ecosystem impacts.

Clinical guidelines for hepatitis B virus infection in kidney transplant recipients (2023 edition)

The hepatitis B virus (HBV) is a liver-loving, double-stranded, circular DNA virus. The prevalence of hepatits B surface antigen(HBsAg)in the general population of China is approximately 6.1%, indicating a large base of HBV-infected individuals. The infection rate of HBV in the blood dialysis patient population is significantly higher than that in the general population. Kidney transplant recipients, being in an immunosuppressed state, are susceptible to HBV infection, and previous HBV infections may reactivate, affecting the long-term survival of the recipient and the transplanted kidney. To further standardize the diagnosis and treatment of HBV infection after kidney transplantation, the Transplantation Branch of the Chinese Medical Association has organized domestic experts to develop this guideline from aspects such as epidemiology, routes of HBV infection in kidney transplant recipients, indications for kidney transplantation in HBV-infected individuals, and the diagnosis, prevention, and treatment of HBV infection in kidney transplant recipients, in order to help kidney transplant professionals standardize and optimize the management of HBV infection.

Identification of new banana endogenous virus sequences highlights the hallmark gene encoded by retroviruses integrated in banana genomes

Endogenous pararetrovirus sequences (EPRVs) originated from DNA viruses of the family Caulimoviridae are widely present in plant genomes. Banana streak viruses (BSVs) are a group of circular double-stranded DNA viruses in the genus Badnavirus of the family Caulimoviridae. Banana endogenous virus sequences (BEVs) derived from the ancestral genes of badnaviruses and fixed in the genomes of various bananas. However, the genomic characteristics of BEVs remain unknown. In this study, we identified 2 new variants of BEVs GZ5 and GZ13 by sequences analyses, Southern blot, and fluorescent in situ hybridization (FISH). BEV GZ5 had one copy of integration in the BB genome of bananas, while BEV GZ13 was only present in the genome of the variety Dajiao. Importantly, BEV GZ5 contained a complete gene of reverse transcriptase (RT) and ribonuclease H (RNase H) (RT/RNase H). In addition, a 340-bp inverted repeat sequence partially overlapping with RNase H was found upstream and downstream of BEV GZ5. However, the amino acid sequences of BEV GZ5 had deletions and mutations compared with BSVs. The bioinformatics analyses showed that BEV GZ5 protein composed of 412 amino acids with a molecular weight of 47.37 kDa and an isoelectric point of 9.40. Leucine, isoleucine, and lysine (Lys) were the main amino acids of BEV GZ5 protein. The analyses revealed that BEV GZ5 protein contained 35 potential phosphorylation sites. Additionally, it was a hydrophilic protein without a signal peptide and transmembrane region. The secondary structure of BEV GZ5 protein consisted of 37.26% α-helix, followed by 36.25% random coil. To our knowledge, this is the first report that novel BEVs with the complete gene of RT/RNase H has been characterized, which provide a basis for further exploration the function and integration mechanism of BEVs in bananas.

The virome of bubaline (Bubalus bubalis) tonsils reveals an unreported bubaline polyomavirus.

Water buffalo (Bubalus bubalis) farming is increasing in many regions of the world due to the species' ability to thrive in environments where bovine cattle would struggle. Despite water buffaloes being known for their resistance to diseases, there is a lack of data about the diversity of the microbiome of the species. In this study, we examined the virome diversity in palatine tonsils collected from animals from the island of Marajó, northern Pará state, Brazil, which harbors the largest bubaline flock in the country. Tonsil fragments from 60 clinically healthy bubalines were randomly selected from a sample of 293 animals. The samples were purified, extracted, and randomly amplified with phi29 DNA polymerase. After amplification, the products were purified and sequenced. Circular DNA viruses were predominant in the tonsils' virome. Sequences of genome segments representative of members of the genera Alphapolyomavirus (including a previously unreported bubaline polyomavirus genome) and Gemycircularvirus were identified, along with other not yet classified circular virus genomes. As the animals were clinically healthy at the time of sampling, such viruses likely constitute part of the normal tonsillar virome of water buffaloes inhabiting the Ilha do Marajó biome.

Molecular characterization of canine circovirus based on the Capsid gene in Thailand

BackgroundCanine circovirus (CanineCV) is a single-stranded circular DNA virus that infects domestic and wild canids in many countries. CanineCV is associated with gastroenteritis and diarrhea, respiratory disease, and generalized vasculitis leading to a fatal event. The Capsid protein (Cap) is a structural protein of the virus which has high genetic variability and plays a role in the canine immune response. In this study, we cloned the full-length CanineCV Capsid gene (Cap). In-silico analyses were used to explore the genomic and amino acid variability and natural selection acting on the Cap gene. The immune relevance for T-cell and B-cell epitopes was predicted by the immunoinformatic approach.ResultsAccording to the Cap gene, our results showed that CanineCV was separated into five phylogenetic groups. The obtained CanineCV strain from this study was grouped with the previously discovered Thai strain (MG737385), as supported by a haplotype network. Entropy analyses revealed high nucleotide and amino acid variability of the Capsid region. Selection pressure analysis revealed four codons at positions 24, 50, 103, and 111 in the Cap protein evolved under diversifying selection. Prediction of B-cell epitopes exhibited four consensus sequences based on physiochemical properties, and eleven peptide sequences were predicted as T-cell epitopes. In addition, the positive selection sites were located within T-cell and B-cell epitopes, suggesting the role of the host immune system as a driving force in virus evolution.ConclusionsOur study provides knowledge of CanineCV genetic diversity, virus evolution, and potential epitopes for host cell immune response.

A circular single-stranded DNA mycovirus infects plants and confers broad-spectrum fungal resistance

Circular single-stranded DNA (ssDNA) viruses have been rarely found in fungi, and the evolutionary and ecological relationships among ssDNA viruses infecting fungi and other organisms remain unclear. In this study, a novel circular ssDNA virus, tentatively named Diaporthe sojae circular DNA virus 1 (DsCDV1), was identified in the phytopathogenic fungus Diaporthe sojae isolated from pear trees. DsCDV1 has a monopartite genome (3185 nt in size) encapsidated in isometric virions (21–26 nm in diameter). The genome comprises seven putative open reading frames encoding a discrete replicase (Rep) split by an intergenic region, a putative capsid protein (CP), several proteins of unknown function (P1–P4), and a long intergenic region. Notably, the two split parts of DsCDV1 Rep share high identities with the Reps of Geminiviridae and Genomoviridae, respectively, indicating an evolutionary linkage with both families. Phylogenetic analysis based on Rep or CP sequences placed DsCDV1 in a unique cluster, supporting the establishment of a new family, tentatively named Gegemycoviridae, intermediate to both families. DsCDV1 significantly attenuates fungal growth and nearly erases fungal virulence when transfected into the host fungus. Remarkably, DsCDV1 can systematically infect tobacco and pear seedlings, providing broad-spectrum resistance to fungal diseases. Subcellular localization analysis revealed that DsCDV1 P3 is systematically localized in the plasmodesmata, while its expression in trans-complementation experiments could restore systematic infection of a movement-deficient plant virus, suggesting that P3 is a movement protein. DsCDV1 exhibits unique molecular and biological traits not observed in other ssDNA viruses, serving as a link between fungal and plant ssDNA viruses and presenting an evolutionary connection between ssDNA viruses and fungi. These findings contribute to expanding our understanding of ssDNA virus diversity and evolution, offering potential biocontrol applications for managing crucial plant diseases.

New duck papillomavirus type identified in a mallard in Missouri, USA.

Papillomaviruses are small circular DNA viruses that infect epithelial and mucosal cells and have co-evolved with their hosts. Some papillomaviruses in mammals are well studied (especially those associated with disease). However, there is limited information on papillomaviruses associated with avian hosts. From a cloacal swab sample of a mallard (Anas platyrhynchos) sampled in Missouri, USA (6 Jan 2023), we identified a papillomavirus (7839 nt) that shares ~68% genome-wide nucleotide sequence identity with Anas platyrhynchos papillomavirus 1 (AplaPV1) from a mallard sampled in Newfoundland (Canada) and ~40% with AplaPV2 from a mallard sampled in Minnesota (USA) with mesenchymal dermal tumors. The papillomavirus we identified shares 73.6% nucleotide sequence identity in the L1 gene with that of AplaPV1 and thus represents a new AplaPV type (AplaPV3). The genome sequence of AplaPV3 shares >97% identity with three partial PV genome sequences (1316, 1997, and 4241 nt) identified in a mallard in India, indicating that that virus was also AplaPV3.

Porcine circovirus type 2 infection promotes the SUMOylation of nucleophosmin-1 to facilitate the viral circular single-stranded DNA replication.

The mechanism of genome DNA replication in circular single-stranded DNA viruses is currently a mystery, except for the fact that it undergoes rolling-circle replication. Herein, we identified SUMOylated porcine nucleophosmin-1 (pNPM1), which is previously reported to be an interacting protein of the viral capsid protein, as a key regulator that promotes the genome DNA replication of porcine single-stranded DNA circovirus. Upon porcine circovirus type 2 (PCV2) infection, SUMO2/3 were recruited and conjugated with the K263 site of pNPM1's C-terminal domain to SUMOylate pNPM1, subsequently, the SUMOylated pNPM1 were translocated in nucleoli to promote the replication of PCV2 genome DNA. The mutation of the K263 site reduced the SUMOylation levels of pNPM1 and the nucleolar localization of pNPM1, resulting in a decrease in the level of PCV2 DNA replication. Meanwhile, the mutation of the K263 site prevented the interaction of pNPM1 with PCV2 DNA, but not the interaction of pNPM1 with PCV2 Cap. Mechanistically, PCV2 infection increased the expression levels of Ubc9, the only E2 enzyme involved in SUMOylation, through the Cap-mediated activation of ERK signaling. The upregulation of Ubc9 promoted the interaction between pNPM1 and TRIM24, a potential E3 ligase for SUMOylation, thereby facilitating the SUMOylation of pNPM1. The inhibition of ERK activation could significantly reduce the SUMOylation levels and the nucleolar localization of pNPM1, as well as the PCV2 DNA replication levels. These results provide new insights into the mechanism of circular single-stranded DNA virus replication and highlight NPM1 as a potential target for inhibiting PCV2 replication.

Characterization of the genomic sequence of a circo-like virus and of three chaphamaparvoviruses detected in mute swan (Cygnus olor).

We report the complete genomes of four ssDNA viruses: a circular replication-associated protein-encoding single-stranded DNA virus belonging to a clade previously detected only in mammals, and three chaphamaparvoviruses, which were detected by viromic surveillance of mute swan (Cygnus olor) fecal samples from the United Kingdom.

Diverse Small Circular DNA Viruses Identified in an American Wigeon Fecal Sample.

American wigeons (Mareca americana) are waterfowls that are widely distributed throughout North America. Research of viruses associated with American wigeons has been limited to orthomyxoviruses, coronaviruses, and circoviruses. To address this poor knowledge of viruses associated with American wigeons, we undertook a pilot study to identify small circular DNA viruses in a fecal sample collected in January 2021 in the city of Tempe, Arizona (USA). We identified 64 diverse circular DNA viral genomes using a viral metagenomic workflow biased towards circular DNA viruses. Of these, 45 belong to the phylum Cressdnaviricota based on their replication-associated protein sequence, with 3 from the Genomoviridae family and the remaining 42 which currently cannot be assigned to any established virus group. It is most likely that these 45 viruses infect various organisms that are associated with their diet or environment. The remaining 19 virus genomes are part of the Microviridae family and likely associated with the gut enterobacteria of American wigeons.

Torque Teno Virus load is associated with CDC stage and CD4+ cell count in people living with HIV but unrelated to AIDS-defining events and Human Pegivirus load.

Torque Teno Virus (TTV) is a non-enveloped, circular single-strand DNA virus and part of the human virome. The replication of TTV was related to the immune status in patients treated with immunosuppressive drugs after organ transplantation. We hypothesize that TTV load could be an additional marker for immune function in people living with HIV (PLWH). In this analysis serum samples of PLWH from the RESINA multicenter cohort were reanalysed for TTV. Investigated clinical and epidemiological parameters included Pegivirus (HPgV) load, age, sex, HIV load, CD4+ cell count (CDC 1, 2, 3) and CDC clinical stages (1993 CDC classification system, A, B, C) before initiation of antiretroviral treatment. Regression analysis was used to detect possible associations among parameters. Our analysis confirmed TTV as a strong predictor of CD4+ cell count and CDC class 3. This relationship was used to propose a first classification of TTV load in regard to clinical stage. We found no association with clinical CDC stages A, B and C. HPgV load was inversely correlated with HIV load but not TTV load. TTV load was associated with immunodeficiency in PLWH. Neither TTV- nor HIV load were predictive for the clinical categories of HIV infection.

Metagenomic Identification of Novel Eukaryotic Viruses with Small DNA Genomes in Pheasants.

A panel of intestinal samples collected from common pheasants (Phasianus colchicus) between 2008 and 2017 was used for metagenomic investigation using an unbiased enrichment protocol and different bioinformatic pipelines. The number of sequence reads in the metagenomic analysis ranged from 1,419,265 to 17,507,704 with a viral sequence read rate ranging from 0.01% to 59%. When considering the sequence reads of eukaryotic viruses, RNA and DNA viruses were identified in the samples, including but not limited to coronaviruses, reoviruses, parvoviruses, and CRESS DNA viruses (i.e., circular Rep-encoding single-stranded DNA viruses). Partial or nearly complete genome sequences were reconstructed of at least three different parvoviruses (dependoparvovirus, aveparvovirus and chaphamaparvovirus), as well as gyroviruses and diverse CRESS DNA viruses. Generating information of virus diversity will serve as a basis for developing specific diagnostic tools and for structured epidemiological investigations, useful to assess the impact of these novel viruses on animal health.

Gyrovirus: current status and challenge.

Gyrovirus (GyV) is small, single-stranded circular DNA viruses that has recently been assigned to the family Anelloviridae. In the last decade, many GyVs that have an apparent pan-tropism at the host level were identified by high-throughput sequencing (HTS) technology. As of now, they have achieved global distribution. Several species of GyVs have been demonstrated to be pathogenic to poultry, particularly chicken anemia virus (CAV), causing significant economic losses to the global poultry industry. Although GyVs are highly prevalent in various birds worldwide, their direct involvement in the etiology of specific diseases and the reasons for their ubiquity and host diversity are not fully understood. This review summarizes current knowledge about GyVs, with a major emphasis on their morphofunctional properties, epidemiological characteristics, genetic evolution, pathogenicity, and immunopathogenesis. Additionally, the association between GyVs and various diseases, as well as its potential impact on the poultry industry, have been discussed. Future prevention and control strategies have also been explored. These insights underscore the importance of conducting research to establish a virus culture system, optimize surveillance, and develop vaccines for GyVs.

Identification of small circular DNA viruses in coyote fecal samples from Arizona (USA).

Coyotes (Canis latrans) have a broad geographic distribution across North and Central America. Despite their widespread presence in urban environments in the USA, there is limited information regarding viruses associated with coyotes in the USA and in particular the state of Arizona. To explore viruses associated with coyotes, particularly small DNA viruses, 44 scat samples were collected (April-June 2021 and November 2021-January 2022) along the Salt River near Phoenix, Arizona (USA), along 43 transects (500m). From these samples, we identified 11 viral genomes: two novel circoviruses, six unclassified cressdnaviruses, and two anelloviruses. One of the circoviruses is most closely related to a circovirus sequence identified from an aerosolized dust sample in Arizona, USA. The second circovirus is most closely related to a rodent-associated circovirus and canine circovirus. Of the unclassified cressdnaviruses, three encode replication-associated proteins that are similar to those found in protists (Histomonas meleagridis and Monocercomonoides exilis), implying an evolutionary relationship with or a connection to similar unidentified protist hosts. The two anelloviruses are most closely related to those found in rodents, and this suggests a diet-related identification.

Dynamics and Conformations of a Full-Length CRESS-DNA Replicase.

Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses encode for a Replicase (Rep) that is essential for viral replication. Rep is a helicase with three domains: an endonuclease, an oligomeric, and an ATPase domain (ED, OD, and AD). Our recent cryo-EM structure of the porcine circovirus 2 (PCV2) Rep provided the first structure of a CRESS-DNA Rep. The structure visualized the ED to be highly mobile, Rep to form a homo-hexamer, bound ssDNA and nucleotides, and the AD to adopt a staircase arrangement around the ssDNA. We proposed a hand-over-hand mechanism by the ADs for ssDNA translocation. The hand-over-hand mechanism requires extensive movement of the AD. Here, we scrutinize this mechanism using all-atom Molecular Dynamics (MD) simulation of Rep in three states: (1) Rep bound to ssDNA and ADP, (2) Rep bound to ssDNA, and (3) Rep by itself. Each of the 700 nsec simulations converges within 200 nsec and provides important insight into the dynamics of Rep, the dynamics of Rep in the presence of these biomolecules, and the importance of ssDNA and ADP in driving the AD to adopt the staircase arrangement around the ssDNA. To the best of our knowledge, this is the first example of an all-atom MD simulation of a CRESS-DNA Rep. This study sets the basis of further MD studies aimed at obtaining a chemical understanding of how Rep uses nucleotide binding and hydrolysis to translocate ssDNA.

Development and application of a self-assembling split-fluorescent protein toolkit to monitor geminiviral movement and infection in plant

Geminiviruses are a group of circular single-stranded DNA viruses that cause severe diseases in many crop plants. However, there is still no fluorescent protein tag suitable for labeling viral proteins endogenously due to the limited genomic space and structure of geminiviruses for foreign gene fragment insertion. Here, we established a split super-folder green fluorescent protein (sfGFP)-based imaging system that provides a platform to visualize the subcellular localization of geminiviral proteins in Nicotiana benthamiana. A short fragment of the GFP- coding sequence (GFP11) was inserted into a specific locus of the geminiviral genome, while the remainder of the GFP (GFP1–10) was transiently or constitutively expressed in N. benthamiana. Consequently, complementation fluorescence enables the examination of the subcellular localization of viral proteins in particular cells. Using this split sfGFP system, we examined the subcellular localization of the coat protein and BV1 protein of tomato golden mosaic virus (TGMV) and further monitored its intercellular and long-distance movement in N. benthamiana during viral infection. This approach allows us to study endogenously the subcellular localization and viral movement of geminiviruses in N. benthamiana. This new split sfGFP system may also hold the potential to provide orthogonal fluorescent proteins usable for geminiviral genome tagging in plants.

Quantification of Virion-Sense and Complementary-Sense DNA Strands of Circular Single-Stranded DNA Viruses.

Circular ssDNA viruses are ubiquitous and can be found in both prokaryotes and eukaryotes. To understand the interaction of ssDNA viruses with their hosts, it is important to characterize the dynamics of viral sense (VS) and complementary-sense (CS) viral strands during the infection process. Here, we present a simple and rapid protocol that allows sensitive and accurate determination of the VS and CS strands generated during viral infection.The method consists of a two-step qPCR in which the first step uses a strand-specific (CS or VS) labeled primer and T4 DNA polymerase that lacks strand displacement activity and makes a single copy per VS or CS strand. Next, the T4 DNA polymerase and unincorporated oligonucleotides are removed by a silica membrane spin column. Finally, the purified VS or CS strands are quantified by qPCR in a second step in which amplification uses a tag primer and a specific primer. Absolute quantification of VS and CS strands is obtained by extrapolating the Cq data to a standard curve of ssDNA, which can be generated by phagemid expression. Quantification of VS and CS strands of two geminiviruses in infections of Solanum lycopersicum (tomato) and Nicotiana benthamiana plants using this method is shown.

Digital CRISPR/Cas13a-RPA based assay for rapid and sensitive detection of JC virus in renal transplant patients

JC viruses (JCVs), members of the Polyomaviruses family, are a type of circular double-stranded DNA (dsDNA) viruses and can lead to opportunistic infections. Infection of JCVs in kidney transplant patients can lead to polyomavirus-associated nephropathy. Thus, early detection and quantification of JC virus in urine or blood will effectively improve the survival rate of kidney transplant patients and reduce the mortality of patients. At present, PCR has been used as a routine method for JC virus detection in clinical practice. However, PCR cannot achieve absolute quantitative detection of viral nucleic acid. rendering it unsuitable for certain clinical applications with specific requirements. Herein, we proposed a JC virus nucleic acid testing platform, named MdCaR, which couples a microfluidic droplet device with CRISPR-Cas13a and recombinase polymerase amplification. MdCaR can quantitatively detect JC virus through digital droplet. The detection limit achieves 1 copy/mL, and the assay takes only 40 min. Twelve renal transplant patients and 20 control subjects’ specimens were analyzed. MdCaR conveyed a good performance in testing these samples, especially for detecting the low viral load samples identified as negative by qPCR. Taken altogether, MdCaR could be regarded as a promising, rapid JCV detection strategy and widely applied for diagnosis for JCV infection.

Circular replication-associated protein-encoding single-stranded DNA virus with risk of spillover is widely prevalent in domestic animals in China

Circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses are highly diverse and have a broad range of hosts. In this study, we report the detection of Bo-Circo-like virus AH20-1 in the feces of diarrheal cattle. The virus has a circular genome of 3,912 nucleotides, three major putative open reading frames, and encodes a Rep gene of 310 amino acids. We found that the virus is closely related to the Bo-Circo-like virus CH strain, which belongs to the novel Kirkoviridae family. Furthermore, we conducted a nationwide surveillance program and found that the virus is prevalent in China (23.6%, 205/868), with the BCLa subtype being the predominant strain. Our findings suggest that the virus can infect sheep, highlighting the potential for cross-species transmission. Our pressure analysis indicates that the CRESS-DNA Kirkoviridae family has broad host adaptation, and that selection pressure played an important role in the evolution of its Rep genes. Our study underscores the need for continued epidemiological surveillance of this virus due to its widespread prevalence in our ruminant population and potential for cross-species transmission.