Novel circular DNA virus identified in Opuntia discolor (Cactaceae) that codes for proteins with similarity to those of geminiviruses

Viral metagenomic studies have enabled the discovery of many unknown viruses and revealed that viral communities are much more diverse and ubiquitous than previously thought. Some viruses have multiple genome components that are encapsidated either in separate virions (multipartite viruses) or in the same virion (segmented viruses). In this study, we identify what is possibly a novel bipartite plant-associated circular single-stranded DNA virus in a wild prickly pear cactus, Opuntia discolor, that is endemic to the Chaco ecoregion in South America. Two ~1.8 kb virus-like circular DNA components were recovered, one encoding a replication-associated protein (Rep) and the other a capsid protein (CP). Both of the inferred protein sequences of the Rep and CP are homologous to those encoded by members of the family Geminiviridae. These two putatively cognate components each have a nonanucleotide sequence within a likely hairpin structure that is homologous to the origins of rolling-circle replication (RCR), found in diverse circular single-stranded DNA viruses. In addition, the two components share similar putative replication-associated iterative sequences (iterons), which in circular single-stranded DNA viruses are important for Rep binding during the initiation of RCR. Such molecular features provide support for the possible bipartite nature of this virus, which we named utkilio virus (common name of the Opuntia discolor in South America) components A and B. In the infectivity assays conducted in Nicotiana benthamiana plants, only the A component of utkilio virus, which encodes the Rep protein, was found to move and replicate systemically in N. benthamiana. This was not true for component B, for which we did not detect replication, which may have been due to this being a defective molecule or because of the model plants (N. benthamiana) used for the infection assays. Future experiments need to be conducted with other plants, including O. discolor, to understand more about the biology of these viral components.

Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions

Consumption of Eurasian bovine meat and milk has been associated with cancer development, in particular with colorectal cancer (CRC). In addition, zoonotic infectious agents from bovine products were proposed to cause colon cancer (zur Hausen et al., 2009). Bovine meat and milk factors (BMMF) are small episomal DNA molecules frequently isolated from bovine sera and milk products, and recently, also from colon cancer (de Villiers et al., 2019). BMMF are bioactive in human cells and were proposed to induce chronic inflammation in precancerous tissue leading to increased radical formation: for example, reactive oxygen and reactive nitrogen species and elevated levels of DNA mutations in replicating cells, such as cancer progenitor cells (zur Hausen et al., 2018). Mouse monoclonal antibodies against the replication (Rep) protein of H1MSB.1 (BMMF1) were used to analyze BMMF presence in different cohorts of CRC peritumor and tumor tissues and cancer-free individuals by immunohistochemistry and Western blot. BMMF DNA was isolated by laser microdissection from immunohistochemistry-positive tissue regions. We found BMMF Rep protein present specifically in close vicinity of CD68+ macrophages in the interstitial lamina propria adjacent to CRC tissues, suggesting the presence of local chronic inflammation. BMMF1 (modified H1MSB.1) DNA was isolated from the same tissue regions. Rep and CD68+ detection increased significantly in peritumor cancer tissues when compared to tissues of cancer-free individuals. This strengthens previous postulations that BMMF function as indirect carcinogens by inducing chronic inflammation and DNA damage in replicating cells, which represent progress to progenitor cells for adenoma (polyps) formation and cancer.

Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA

An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA–protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.

The 6th Amino Acid Mutation of Rep Protein Had No Effect on PCV2b but Enhanced PCV2d Virus Replication in Vitro

Porcine circovirus type 2 (PCV2) is the etiological agent that primary cause of post-weaning multisystemic wasting syndrome (PMWS). The major genotypes, PCV2a, PCV2b and PCV2d, are highly prevalent, but now replaced with 2b and 2d in swine population in worldwide. Rep protein is the key protein for viral replication. Compared a large number of Rep protein amino acid (aa) sequences, we found that there were three sites with regular changes between 2b and 2d. In order to analyze the effect of key sites on viral replication, we used site-directed mutagenesis to mutate the 6th aa of Rep (alternations with asparagine and serine) between PCV2b and PCV2d, Two wild-type and two mutant viruses infectious clones were rescued by non-contaminated porcine kidney-15 (PK-15) cells. Real-time quantitative PCR and a one-step growth curve were used to determine viral load to assess the replication of rescued viruses. The results showed that there was no significant difference between the PCV2b mutation and the wild-type PCV2b virus in vitro, while the mutation ofPCV2d enhanced viral replication.

Short Beak and Dwarfism Syndrome in Ducks in Poland Caused by Novel Goose Parvovirus

Short beak and dwarfism syndrome (SBDS), which was previously identified only in mule ducks, is now an emerging disease of Pekin ducks in China and Egypt. The disease is caused by the infection of ducks with a genetic variant of goose parvovirus—novel goose parvovirus (nGPV). In 2019, SBDS was observed for the first time in Poland in eight farms of Pekin ducks. Birds in the affected flock were found to show growth retardation and beak atrophy with tongue protrusions. Morbidity ranged between 15% and 40% (in one flock), while the mortality rate was 4–6%. Co-infection with duck circovirus, a known immunosuppressive agent, was observed in 85.7% of ducks. The complete coding regions of four isolates were sequenced and submitted to GenBank. The phylogenetic analysis revealed a close relationship of Polish viral sequences with the Chinese nGPV. Genomic sequence alignments showed 98.57–99.28% identity with the nGPV sequences obtained in China, and 96.42% identity with the classical GPV (cGPV; Derzsy’s disease). The rate of amino acid mutations in comparison to cGPV and Chinese nGPV was higher in the Rep protein than in the Vp1 protein. To our knowledge, this is the first report of nGPV infection in Pekin ducks in Poland and Europe. It should be emphasized that monitoring and sequencing of waterfowl parvoviruses is important for tracking the viral genetic changes that enable adaptation to new species of waterbirds.

Meta-Transcriptomic Discovery of a Divergent Circovirus and a Chaphamaparvovirus in Captive Reptiles with Proliferative Respiratory Syndrome

Viral pathogens are being increasingly described in association with mass morbidity and mortality events in reptiles. However, our knowledge of reptile viruses remains limited. Herein, we describe the meta-transcriptomic investigation of a mass morbidity and mortality event in a colony of central bearded dragons (Pogona vitticeps) in 2014. Severe, extensive proliferation of the respiratory epithelium was consistently found in affected dragons. Similar proliferative lung lesions were identified in bearded dragons from the same colony in 2020 in association with increased intermittent mortality. Total RNA sequencing identified two divergent DNA viruses: a reptile-infecting circovirus, denoted bearded dragon circovirus (BDCV), and the first exogeneous reptilian chaphamaparvovirus—bearded dragon chaphamaparvovirus (BDchPV). Phylogenetic analysis revealed that BDCV was most closely related to bat-associated circoviruses, exhibiting 70% amino acid sequence identity in the Replicase (Rep) protein. In contrast, in the nonstructural (NS) protein, the newly discovered BDchPV showed approximately 31%–35% identity to parvoviruses obtained from tilapia fish and crocodiles in China. Subsequent specific PCR assays revealed BDCV and BDchPV in both diseased and apparently normal captive reptiles, although only BDCV was found in those animals with proliferative pulmonary lesions and respiratory disease. This study expands our understanding of viral diversity in captive reptiles.

Unveiling Crucivirus Diversity by Mining Metagenomic Data

ABSTRACT The discovery of cruciviruses revealed the most explicit example of a common protein homologue between DNA and RNA viruses to date. Cruciviruses are a novel group of circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) viruses that encode capsid proteins that are most closely related to those encoded by RNA viruses in the family Tombusviridae. The apparent chimeric nature of the two core proteins encoded by crucivirus genomes suggests horizontal gene transfer of capsid genes between DNA and RNA viruses. Here, we identified and characterized 451 new crucivirus genomes and 10 capsid-encoding circular genetic elements through de novo assembly and mining of metagenomic data. These genomes are highly diverse, as demonstrated by sequence comparisons and phylogenetic analysis of subsets of the protein sequences they encode. Most of the variation is reflected in the replication-associated protein (Rep) sequences, and much of the sequence diversity appears to be due to recombination. Our results suggest that recombination tends to occur more frequently among groups of cruciviruses with relatively similar capsid proteins and that the exchange of Rep protein domains between cruciviruses is rarer than intergenic recombination. Additionally, we suggest members of the stramenopiles/alveolates/Rhizaria supergroup as possible crucivirus hosts. Altogether, we provide a comprehensive and descriptive characterization of cruciviruses. IMPORTANCE Viruses are the most abundant biological entities on Earth. In addition to their impact on animal and plant health, viruses have important roles in ecosystem dynamics as well as in the evolution of the biosphere. Circular Rep-encoding single-stranded (CRESS) DNA viruses are ubiquitous in nature, many are agriculturally important, and they appear to have multiple origins from prokaryotic plasmids. A subset of CRESS-DNA viruses, the cruciviruses, have homologues of capsid proteins encoded by RNA viruses. The genetic structure of cruciviruses attests to the transfer of capsid genes between disparate groups of viruses. However, the evolutionary history of cruciviruses is still unclear. By collecting and analyzing cruciviral sequence data, we provide a deeper insight into the evolutionary intricacies of cruciviruses. Our results reveal an unexpected diversity of this virus group, with frequent recombination as an important determinant of variability.

Establishment of a Rep′ protein antibody detection method to distinguish natural infection with PCV2 from subunit vaccine immunization

Introduction. PCV2 is a DNA virus that exists widely in pigs and has caused great economic losses to the pig industry worldwide. In the existing commercial PCV2 enzyme-linked immunosorbent assay (ELISA) kits both natural infection with PCV2 and vaccine immunization produce results that are positive for PCV2 Cap antibodies and therefore they cannot diagnose PCV2 infection in immunized pig farms. Aim. To establish a PCV2 non-structural protein antibody detection method that distinguishes between antibodies resulting from natural prior exposure (infection) and those induced by subunit vaccine immunization. Methodology. Based on the non-structural Rep′ protein, we established an indirect ELISA (iELISA) using sera from guinea pigs and piglets. Results. The results for iELISA for guinea pig serum showed that animals vaccinated with a whole-virus inactivated PCV2 vaccine had 100 % (10/10) Cap antibody positivity and 100 % (10/10) Rep′ antibody positivity. Guinea pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (10/10) Cap antibody positivity, while no (0/10) guinea pigs were Rep′ antibody-positive. The combined detection results for the Rep′ iELISA and a PCV2 Antibody Test kit (Commercial) showed that pigs vaccinated with a whole-virus inactivated PCV2 vaccine or PCV2 SD/2017 had 100 % (5/5) Cap antibody positivity and 100 % (5/5) Rep′ antibody positivity. Pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (5/5) Cap antibody positivity, while no (0/10) pigs were Rep′ antibody-positive. Conclusion. This paper describes an effective iELISA method that can distinguish natural infection with PCV2 (Cap and Rep positive) or inoculation with a whole-virus inactivated vaccine (Cap and Rep positive) from subunit vaccine immunization (Cap-positive, Rep-negative). These comparative assays could be very useful in the control of PCV2 in pig herds.