Circular Rep-encoding Single-stranded DNA Research Articles

An endogenous DNA virus in an amphibian-killing fungus associated with pathogen genotype and virulence

The global panzootic lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single-stranded (CRESS)-like DNA virus, which we named Bd DNA virus 1 (BdDV-1). We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus-positive isolates were unsuccessful; however, phenotypic differences between naturally virus-positive and virus-negative Bd isolates suggested that BdDV-1 decreases the growth of its host invitro but increases the virulence of its host invivo. BdDV-1 is the first-described CRESS DNA mycovirus of zoosporic true fungi, with a distribution inversely associated with the emergence of the panzootic lineage.

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.

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.

Highly divergent CRESS DNA and picorna-like viruses associated with bleached thalli of the green seaweed Ulva.

Marine macroalgae (seaweeds) are important primary producers and foundation species in coastal ecosystems around the world. Seaweeds currently contribute to an estimated 51% of the global mariculture production, with a long-term growth rate of 6% per year, and an estimated market value of more than US$11.3 billion. Viral infections could have a substantial impact on the ecology and aquaculture of seaweeds, but surprisingly little is known about virus diversity in macroalgal hosts. Using metagenomic sequencing, we characterized viral communities associated with healthy and bleached specimens of the commercially important green seaweed Ulva. We identified 20 putative new and divergent viruses, of which the majority belonged to the Circular Rep-Encoding Single-Stranded (CRESS) DNA viruses [single-stranded (ss)DNA genomes], Durnavirales [double-stranded (ds)RNA], and Picornavirales (ssRNA). Other newly identified RNA viruses were related to the Ghabrivirales, the Mitoviridae, and the Tombusviridae. Bleached Ulva samples contained particularly high viral read numbers. While reads matching assembled CRESS DNA viruses and picorna-like viruses were nearly absent from the healthy Ulva samples (confirmed by qPCR), they were very abundant in the bleached specimens. Therefore, bleaching in Ulva could be caused by one or a combination of the identified viruses but may also be the result of another causative agent or abiotic stress, with the viruses simply proliferating in already unhealthy seaweed tissue. This study highlights how little we know about the diversity and ecology of seaweed viruses, especially in relation to the health and diseases of the algal host, and emphasizes the need to better characterize the algal virosphere. IMPORTANCE Green seaweeds of the genus Ulva are considered a model system to study microbial interactions with the algal host. Remarkably little is known, however, about viral communities associated with green seaweeds, especially in relation to the health of the host. In this study, we characterized the viral communities associated with healthy and bleached Ulva. Our findings revealed the presence of 20 putative novel viruses associated with Ulva, encompassing both DNA and RNA viruses. The majority of these viruses were found to be especially abundant in bleached Ulva specimens. This is the first step toward understanding the role of viruses in the ecology and aquaculture of this green seaweed.

A Diverse Virome Is Identified in Parasitic Flatworms of Domestic Animals in Xinjiang, China.

Parasitic flatworms infect diverse vertebrates and are major threats to animal and even human health; however, little is known about the virome of these lower life forms. Using viral metagenomic sequencing, we characterized the virome of the parasitic flatworms collected from major domestic animals, including Dicrocoelium lanceatum and Taenia hydatigena, Echinococcus granulosus sensu stricto and Echinococcus multilocularis. Seven and three different viruses were discovered from D. lanceatum and T. hydatigena, respectively, and no viral sequences were found in adult tapeworms and protoscoleces of E. granulosus sensu stricto and E. multilocularis. Two out of the five parasitic flatworm species carry viruses, showing a host specificity of these viruses. These viruses belong to the Parvoviridae, Circoviridae, unclassified circular, Rep-encoding single-stranded (CRESS) DNA virus, Rhabdoviridae, Endornaviridae, and unclassified RNA viruses. The presence of multiple highly divergent RNA viruses, especially those that cluster with viruses found in marine animals, implies a deep evolutionary history of parasite-associated viruses. In addition, we found viruses with high identity to common pathogens in dogs, including canine circovirus and canine parvovirus 2. The presence of these viruses in the parasites implies that they may infect parasitic flatworms but does not completely exclude the possibility of contamination from host intestinal contents. Furthermore, we demonstrated that certain viruses, such as CRESS DNA virus may integrate into the genome of their host. Our results expand the knowledge of viral diversity in parasites of important domestic animals, highlighting the need for further investigations of their prevalence among other parasites of key animals. IMPORTANCE Characterizing the virome of parasites is important for unveiling the viral diversity, evolution, and ecology and will help to understand the "Russian doll" pattern among viruses, parasites, and host animals. Our data indicate that diverse viruses are present in specific parasitic flatworms, including viruses that may have an ancient evolutionary history and viruses currently circulating in parasite-infected host animals. These data also raise the question of whether parasitic flatworms acquire and/or carry some viruses that may have transmission potential to animals. In addition, through the study of virus-parasite-host interactions, including the influence of viral infection on the life cycle of the parasite, as well as its fitness and pathogenicity to the host, we could find new strategies to prevent and control parasitic diseases.

Detection and genetic characterization of circoviruses in more than 80 bat species from eight countries on four continents

Several bat-associated circoviruses and circular rep-encoding single-stranded DNA (CRESS DNA) viruses have been described, but the exact diversity and host species of these viruses are often unknown. Our goal was to describe the diversity of bat-associated circoviruses and cirliviruses, thus, 424 bat samples from more than 80 species were collected on four continents. The samples were screened for circoviruses using PCR and the resulting amino acid sequences were subjected to phylogenetic analysis. The majority of bat strains were classified in the genus Circovirus and some strains in the genus Cyclovirus and the clades CRESS1 and CRESS3. Some strains, however, could only be classified at the taxonomic level of the order and were not classified in any of the accepted or proposed clades. In the family Circoviridae, 71 new species have been predicted. This screening of bat samples revealed a great diversity of circoviruses and cirliviruses. These studies underline the importance of the discovery and description of new cirliviruses and the need to establish new species and families in the order Cirlivirales.

A Novel Circular Rep-Encoding Single-Stranded DNA from Holstein Calves.

A novel, circular DNA molecule was identified in the serum of 4-week-old Holstein calves via Illumina sequencing. Comparisons with the NCBI nucleotide data bank indicate that the sequence is unique. The circle contains one predicted open reading frame (ORF), whose translated protein sequence shows high similarity to bacterial Rep proteins.

Watson-Crick Base-Pairing Requirements for ssDNA Recognition and Processing in Replication-Initiating HUH Endonucleases.

Replication-initiating HUH endonucleases (Reps) are sequence-specific nucleases that cleave and rejoin single-stranded DNA (ssDNA) during rolling-circle replication. These functions are mediated by covalent linkage of the Rep to its substrate post cleavage. Here, we describe the structures of the endonuclease domain from the Muscovy duck circovirus Rep in complex with its cognate ssDNA 10-mer with and without manganese in the active site. Structural and functional analyses demonstrate that divalent cations play both catalytic and structural roles in Reps by polarizing and positioning their substrate. Further structural comparisons highlight the importance of an intramolecular substrate Watson-Crick (WC) base pairing between the -4 and +1 positions. Subsequent kinetic and functional analyses demonstrate a functional dependency on WC base pairing between these positions regardless of the pair's identity (i.e., A·T, T·A, G·C, or C·G), highlighting a structural specificity for substrate interaction. Finally, considering how well WC swaps were tolerated in vitro, we sought to determine to what extent the canonical -4T·+1A pairing is conserved in circular Rep-encoding single-stranded DNA viruses and found evidence of noncanonical pairings in a minority of these genomes. Altogether, our data suggest that substrate intramolecular WC base pairing is a universal requirement for separation and reunion of ssDNA in Reps. IMPORTANCE Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses are a ubiquitous group of viruses that infect organisms across all domains of life. These viruses negatively impact both agriculture and human health. All members of this viral family employ a multifunctional nuclease (Rep) to initiate replication. Reps are structurally similar throughout this family, making them targets of interest for viral inhibition strategies. Here, we investigate the functional dependencies of the Rep protein from Muscovy duck circovirus for ssDNA interaction. We demonstrate that this Rep requires an intramolecular Watson-Crick base pairing for origin of replication (Ori) recognition and interaction. We show that noncognate base pair swaps are well tolerated, highlighting a local structural specificity over sequence specificity. Bioinformatic analysis found that the vast majority of CRESS-DNA Oris form base pairs in conserved positions, suggesting this pairing is a universal requirement for replication initiation in the CRESS-DNA virus family.

Widespread, human-associated redondoviruses infect the commensal protozoan Entamoeba gingivalis

Redondoviruses are circular Rep-encoding single-stranded DNA (CRESS) viruses of high prevalence in healthy humans. Redondovirus abundance is increased in oro-respiratory samples from individuals with periodontitis, acute illness, and severe COVID-19. We investigated potential host cells supporting redondovirus replication in oro-respiratory samples and uncovered the oral amoeba Entamoeba gingivalis as a likely host. Redondoviruses are closely related to viruses of Entamoeba and contain reduced GC nucleotide content, consistent with Entamoeba hosts. Redondovirus and E.gingivalis co-occur in metagenomic data from oral disease and healthy human cohorts. When grown in xenic cultures with feeder bacteria, E.gingivalis was robustly positive for redondovirus RNA and DNA. A DNA proximity-ligation assay (Hi-C) on xenic culture cells showed enriched cross-linking of redondovirus and Entamoeba DNA, supporting E.gingivalis as the redondovirus host. While bacteria are established hosts for bacteriophages within the human virome, this work shows that eukaryotic commensals also contribute an abundant human-associated virus.

CRUISE, a Tool for the Detection of Iterons in Circular Rep-Encoding Single-Stranded DNA Viruses.

Iterons are short, repeated DNA sequences that are important for the replication of circular single-stranded DNA viruses. No tools that can reliably predict iterons are currently available. The CRUcivirus Iteron SEarch (CRUISE) tool is a computational tool that identifies iteron candidates near stem-loop structures in viral genomes.

A variety of highly divergent eukaryotic ssDNA viruses in sera of pigs

Over the last decade, viral metagenomics has been established as a non-targeted approach for identifying viruses in stock animals, including pigs. This has led to the identification of a vast diversity of small circular ssDNA viruses. The present study focuses on the investigation of eukaryotic circular Rep-encoding single-stranded (CRESS) DNA viral genomes present in serum of commercially reared pigs from southern Brazil. Several CRESS DNA viral genomes were detected, including representatives of the families Smacoviridae (n=5), Genomoviridae (n=3), Redondoviridae (n=1), Nenyaviridae (n=1) and other yet unclassified genomes (n=9), plus a circular DNA molecule, which probably belongs to the phylum Cressdnaviricota. A novel genus within the family Smacoviridae, tentatively named 'Suismacovirus', comprising 21 potential new species, is proposed. Although the reported genomes were recovered from pigs with clinical signs of respiratory disease, further studies should examine their potential role as pathogens. Nonetheless, these findings highlight the diversity of circular ssDNA viruses in serum of domestic pigs, expand the knowledge on CRESS DNA viruses' genetic diversity and distribution and contribute to the global picture of the virome of commercially reared pigs.

Epidemiology and Evolution of Emerging Porcine Circovirus-like Viruses in Pigs with Hemorrhagic Dysentery and Diarrhea Symptoms in Central China from 2018 to 2021.

Porcine circovirus-like virus (PCLV) is a type of circular Rep-encoding single-stranded DNA virus and may be associated with the development of diarrheal symptoms in pigs. In this study, we retrospectively analyzed three years of past cases in Anhui, China, and reported a case of hemorrhagic enteritis and death in a pregnant sow possibly caused by PCLV. In addition, we analyzed the evolutionary characteristics of PCLV and found that mutation, recombination and selective pressure all played an important role in the evolution of PCLV. We identified N15D and T17S as well as L56T, T58R, K59Q, M62R, L75I and R190K mutations in two different branches, and we noted recombination events in the Rep of a group of Chinese strains. Analysis of selection pressure revealed that PCLV gained more positive selection, indicating that the virus is in a continuous evolutionary state. The PR2 plot, ENC-plot and neutrality analysis showed a greater role of natural selection than that of mutational pressure in the formation of codon usage patterns. This study is the first to identify PCLV in sows with hemorrhagic dysentery and death, and it provides new epidemiological information on PCLV infection in pigs in China.

Novel Cyclovirus Species in Dogs with Hemorrhagic Gastroenteritis.

Nested PCRs with circovirus/cyclovirus pan-rep (replicase gene) primers detected eukaryotic circular Rep-encoding single-stranded DNA (CRESS DNA) viruses in three (samples CN9E, CN16E and CN34) of 18 canine parvovirus-2-positive fecal samples from household dogs with hemorrhagic gastroenteritis on the Caribbean island of Nevis. The complete genomes of CRESS DNA virus CN9E, CN16E and CN34 were determined by inverse nested PCRs. Based on (i) genome organization, (ii) location of the putative origin of replication, (iii) pairwise genome-wide sequence identities, (iv) the presence of conserved motifs in the putative replication-associated protein (Rep) and the arginine-rich region in the amino terminus of the putative capsid protein (Cp) and (v) a phylogenetic analysis, CN9E, CN16E and CN34 were classified as cycloviruses. Canine-associated cycloviruses CN16E and CN34 were closely related to each other and shared low genome-wide nucleotide (59.642–59.704%), deduced Rep (35.018–35.379%) and Cp (26.601%) amino acid sequence identities with CN9E. All the three canine-associated cycloviruses shared < 80% genome-wide pairwise nucleotide sequence identities with cycloviruses from other animals/environmental samples, constituting two novel species (CN9E and CN16E/34) within the genus Cyclovirus. Considering the feeding habits of dogs, we could not determine whether the cycloviruses were of dietary origin or infected the host. Interestingly, the CN9E putative Rep-encoding open reading frame was found to use the invertebrate mitochondrial genetic code with an alternative initiation codon (ATA) for translation, corroborating the hypothesis that cycloviruses are actually arthropod-infecting viruses. To our knowledge, this is the first report on the detection and complete genome analysis of cycloviruses from domestic dogs.

Mechanism of DNA Interaction and Translocation by the Replicase of a Circular Rep-Encoding Single-Stranded DNA Virus

ABSTRACTCircular Rep-encoding single-stranded DNA (CRESS-DNA) viruses infect members from all three domains of life (Archaea, Prokarya, and Eukarya). The replicase (Rep) from these viruses is responsible for initiating rolling circle replication (RCR) of their genomes. Rep is a multifunctional enzyme responsible for nicking and ligating ssDNA and unwinding double-stranded DNA (dsDNA). We report the structure of porcine circovirus 2 (PCV2) Rep bound to ADP and single-stranded DNA (ssDNA), and Rep bound to ADP and double-stranded DNA (dsDNA). The structures demonstrate Rep to be a member of the superfamily 3 (SF3) of ATPases Associated with diverse cellular Activities (AAA+) superfamily clade 4. At the Rep N terminus is an endonuclease domain (ED) that is responsible for ssDNA nicking and ligation, in the center of Rep is an oligomerization domain (OD) responsible for hexamerization, and at the C terminus is an ATPase domain (AD) responsible for ssDNA/dsDNA interaction and translocation. The Rep AD binds to DNA such that the ED faces the replication fork. The six AD spiral around the DNA to interact with the backbone phosphates from four consecutive nucleotides. Three of the six AD are able to sense the backbone phosphates from the second strand of dsDNA. Heterogeneous classification of the data demonstrates the ED and AD to be mobile. Furthermore, we demonstrate that Rep exhibits basal nucleoside triphosphatase (NTPase) activity.

Redondovirus DNA in human respiratory samples

Redondovirus (ReDoV) is a recently discovered circular, Rep-encoding single-stranded DNA (CRESS-DNA) virus in humans. Its pathogenesis and clinical associations are still completely unknown. The presence of ReDoV DNA was investigated in biological specimens of 543 Italian subjects by in-house developed PCR assays. The overall ReDoV prevalence was about 4% (23 of 543 samples). The virus was detected in 22 of 209 (11 %) respiratory samples. One stool sample was also ReDoV positive. Viral DNA was not found in blood samples from immunocompetent and immunosuppressed subjects and cerebrospinal fluids from patients with neurological diseases. Genomic nucleotide differences were detected among the ReDoV isolates by sequencing a 582-nucleotide fragment of the capsid gene of the viral genome. The results demonstrate that ReDoV is mainly present in the respiratory tract of infected people. Further investigations are needed to reveal possible clinical implications of this new CRESS-DNA virus in humans.

Virome of crab-eating (Cerdocyon thous) and pampas foxes (Lycalopex gymnocercus) from southern Brazil and Uruguay

Crab-eating (Cerdocyon thous) and Pampas foxes (Lycalopex gymnocercus) are wild canids distributed in South America. Domestic dogs (Canis lupus familiaris) and wild canids may share viral pathogens, including rabies virus (RABV), canine distemper virus (CDV), and canine parvovirus 2 (CPV-2). To characterize the virome of these wild canid species, the present work evaluated the spleen and mesenteric lymph node virome of 17 crab-eating and five Pampas foxes using high-throughput sequencing (HTS). Organ samples were pooled and sequenced using an Illumina MiSeq platform. Additional PCR analyses were performed to identify the frequencies and host origin for each virus detected by HTS. Sequences more closely related to the Paramyxoviridae, Parvoviridae and Anelloviridae families were detected, as well as circular Rep-encoding single-stranded (CRESS) DNA viruses. CDV was found only in crab-eating foxes, whereas CPV-2 was found in both canid species; both viruses were closely related to sequences reported in domestic dogs from southern Brazil. Moreover, the present work reported the detection of canine bocavirus (CBoV) strains that were genetically divergent from CBoV-1 and 2 lineages. Finally, we also characterized CRESS DNA viruses and anelloviruses with marked diversity. The results of this study contribute to the body of knowledge regarding wild canid viruses that can potentially be shared with domestic canids or other species.

A novel circular Rep-encoding single-stranded DNA virus detected in Agrotis ipsilon (Lepidoptera: Noctuidae) in China.

Long-distance migratory insects carry microorganisms that can potentially play a crucial role in the life cycles of their hosts. Here, we used Illumina and Sanger sequencing to determine the complete genome sequence of a novel circular Rep-encoding single-stranded (ss) DNA virus from an important migratory pest, Agrotis ipsilon (Hufnagel). The full genome of this new virus is about 2, 242 nt in length and shares 55-75% genome-wide pairwise sequence identity with members of the family Genomoviridae but 91% nucleotide sequence identity with finch-associated genomovirus 3 isolate S30P_D, which is tentatively abbreviated "FaGmV-3". Viral infection rates in A. ipsilon from Yantai, Langfang and Xinxiang were 4.5% (n = 88), 11.8% (n = 85) and 0% (n = 35), respectively. Phylogenetic analysis based on the deduced amino acid sequence of Rep indicated that the Agrotis ipsilon-associated virus is closely related to members of the genus Gemykibivirus, and we propose it to be a new member of this genus. Hence, it is tentatively named "Agrotis ipsilon-associated genomovirus" (AiGmV).

Novel Circular Rep-Encoding Single-Stranded DNA Viruses Detected in Treated Wastewater.

Here, we present the complete genome sequences of three circular replication-associated protein (Rep)-encoding single-stranded DNA (CRESS DNA) viruses detected in secondary treated and disinfected wastewater effluent. The discovered viruses, named wastewater CRESS DNA virus (WCDV)-1 to -3, represent novel viral species that seem to persist in wastewater effluent.

Viral metagenomics reveals significant viruses in the genital tract of apparently healthy dairy cows.

The virome in genital tract secretion samples collected from 80 dairycattle in Shanghai, China, was characterized. Viruses detected included members of the families Papillomaviridae, Polyomaviridae, Hepeviridae, Parvoviridae, Astroviridae, Picornaviridae, and Picobirnaviridae. A member of a new species within the genus Dyoxipapillomavirus and six circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) viral genomes were fully sequencedand phylogenetically analyzed. The prevalence of bovine polyomaviruses 1 and 2 was measured by PCR to be 10% (8/80) and 6.25% (5/80), respectively.PCR screening also indicated that the novel papillomavirus ujs-21015 and bovine herpesvirus 6 were present in three and two out of the 80 samples, respectively.

Evaluation of the serum virome in calves persistently infected with Pestivirus A, presenting or not presenting mucosal disease

Bovine viral diarrhea virus 1, reclassified as Pestivirus A, causes an economically important cattle disease that is distributed worldwide. Pestivirus A may cause persistent infection in that calves excrete the virus throughout their lives, spreading the infection in the herd. Many persistently infected (PI) calves die in the first 2years of life from mucosal disease (MD) or secondary infections, probably as a consequence of virus-induced immune depression. Here, high-throughput sequencing (HTS) was applied for evaluation of the total virome in sera of (i) PI calves displaying clinically apparent MD (n = 8); (ii) PI calves with no signs of MD (n = 8); and (iii) control, Pestivirus A-free calves (n = 8). All the groups were collected at the same time and from the same herd. Serum samples from calves in each of the groups were pooled, submitted to viral RNA/DNA enrichment, and sequenced by HTS. Viral genomes of Pestivirus A, Ungulate erythroparvovirus 1, bosavirus (BosV), and hypothetical circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses were identified. Specific real-time PCR assays were developed to determine the frequency of occurrence of such viruses in each of the groups. The absolute number of distinct viral genomes detected in both PI calf groups was higher than in the control group, as revealed by higher number of reads, contigs, and genomes, representing a wider range of taxons. Genomes representing members of the family Parvoviridae, such as U. erythroparvovirus 1 and BosV, were most frequently detected in all the three groups of calves. Only in MD-affected PI calves, we found two previously unreported Hypothetical single-stranded DNA genomes clustered along with CRESS-DNA viruses. These findings reveal that parvoviruses were the most frequently detected viral genomes in cattle serum; its frequency of detection bears no statistical correlation with the status of calves in relation to Pestivirus A infection, since clinically normal or MD-affected/non-affected PI calves were infected with similar U. erythroparvovirus 1 genome loads. Moreover, MD-affected PI calves were shown to support viremia of CRESS-DNA viral genomes; however, the meaning of such correlation remains to be established.