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Porcine circovirus type 4 (PCV4) is the newest member in the porcine circovirus family, first reported in 2020. To date, the presence of PCV4 has only been reported in China, South Korea and most recently in Thailand. Detection of PCV4 have been reported in various production stages of pigs from piglets, finishers to sows; associated with a myriad of clinical manifestations including porcine dermatitis and nephropathy syndrome (PDNS), postweaning multisystemic wasting syndrome (PMWS), respiratory, enteric and neurological diseases. While successful virus isolation and culture has yet to be reported, pathogenicity of PCV4 has been demonstrated through infectious clone studies. The objective of this study is to investigate the presence of PCV4 in Malaysian porcine population to update the epidemiology of porcine circoviruses in Malaysia. A total of 49 samples from commercial intensive pig farms, abattoir and wild boar population were subjected to conventional polymerase chain reaction assay to detect PCV4 capsid (cap) genome. Resulting cap nucleotide sequences were analyzed for maximum likelihood phylogeny relationship. Results revealed that PCV4 is present in Peninsular Malaysia at a molecular prevalence of 4.08% (2 / 49 samples). Both PCV4 positive samples originated from clinically healthy finishers. Malaysian PCV4 strains were classified as genotype PCV4b, and were found to be phylogenetically distinct from the China, South Korea and Thailand strains. With this latest update of the novel PCV4 in Malaysia, it is clear that more attention needs to be given to the investigation of novel porcine circoviruses (PCV) and management of PCV diseases.

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., (© 2023. The Author(s).)

Canine circovirus (CanineCV) is a single-stranded DNA virus that circulates in dogs and wild carnivores around the world. It has been suggested to be associated with diseases of respiratory and gastrointestinal systems, though its pathogenic potential remains unclear. Currently, CanineCV is divided into six genotypes (genotype 1-6), and genotypes 2, 3, and 4 have been described in China. In this study, 359 blood samples from pet dogs with or without clinical signs were collected in Harbin city. After PCR screening, a total of 34 samples were tested positive for CanineCV, and nine full-length genome sequences were recovered from positive samples. Pairwise sequence comparison showed that they shared 82.4-99.3% genome-wide identity with other CanineCVs available in GenBank. Additionally, recombination events were detected, all of which were determined to be associated with sequences obtained in China. The reconstructed phylogenetic tree based on the recombination-free complete genome sequences revealed that the complete genome sequences generated herein were clustered into genotypes 1 and 3. Furthermore, purifying selection was the dominant evolutionary pressure acting on the genomes of CanineCV. These results expand the knowledge about the genetic diversity of CanineCV circulating in China, and also promote us to better understand the evolution of CanineCV., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Emerging diseases are acknowledged as a growing threat to wildlife, with the continued identification of pathogenic and potentially pathogenic viruses in avian species resulting from ongoing advances in molecular diagnostic techniques. Parvoviruses under the genus Chaphamaparvovirus (subfamily Hamaparvovirinae) are highly divergent. The detection and characterisation of parvoviruses in psittacine birds is limited. This study reports a novel parvovirus, tentatively named psittaciform chaphamaparvovirus 3 (PsChV-3) under the genus Chaphamaparvovirus, identified in an Australian free-ranging little corella (Cacatua sanguinea). The PsChV-3 genome is 4277 bp in length and encompasses four predicted open-reading frames, including two major genes, a nonstructural replicase gene (NS1), and a structural capsid gene (VP1). The NS1 and VP1 genes showed the closest amino acid identities of 78.8% and 69.7%, respectively, with a recently sequenced psittaciform chaphamaparvovirus 2 from Australian Neophema species grass parrots. In addition, the presence of two complete novel beak and feather disease (BFDV) genomes, 1993 and 1868 nt in length, respectively, were detected from the same bird. Both these BFDV genomes contained two bidirectional ORFs encoding the putative Rep and Cap proteins. Phylogenetic analysis showed that the sequenced novel BFDV genomes clustered in a distinct subclade with other BFDVs isolated from Australian cockatoos. This study contributes to the characterisation chaphamaparvoviruses and BFDV in Australian parrots and supports the need for ongoing monitoring and molecular studies into the avian virome in native Australian psittacine bird species., (© 2023 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association.)

Background: Porcine circovirus type 2 (PCV2), a member of the genus Circovirus and family Circoviridae, is a closed, small, circular, and single-stranded DNA virus, and it is a crucial swine pathogen of porcine circovirus-associated diseases (PCVADs). PCV2 was first detected in PK-15(ATCC-CCL) cells in 1974, which has caused significant economic loss to the swine industry throughout the world. And the first case of PCV2 was reported in China in 2000. At present, PCV2d is the main genotype circulating widely in China., Methods: Lymph samples were obtained from piglets with emaciation and respiratory disease in Guangxi province, China. The main pathogens were detected via PCR from lymph samples, and then PCV2-single positive samples were used to inoculate with PK-15 cells. After successive generations, the isolate was subsequently identified by polymerase chain reaction (PCR), immunofluorescence assay (IFA), Western blot (WB), and transmission electron microscopic (TEM). The full-length genome and genetic characterization of isolates were analyzed by Sanger sequencing. The TCID 50 of the PCV2-GX-6 was determined by IFA, and the pathogenicity of PCV2 in BALB/c mice was analyzed via the mouse model., Results: The isolates were successfully isolated from clinical samples. The complete genome of PCV2-GX-4, PCV2-GX-6, PCV2-GX-7, PCV2-GX-11 and PCV2-GX-16 have been amplified, sequenced, and deposited in GenBank (accession no.: OR133747, OQ803314, OR133748, OR133749, OR133750). Homology and phylogenetic analysis with reference strains showed that the isolates belonged to the PCV2d genotype. The PCV2-GX-6 could be stably passaged more than 30 times in PK-15 cells. PCV2-GX-6 was identified by PCR, IFA, WB and TEM. The results of homology showed that PCV2-GX-6 was closely related to the reference strains PCV2-JS17-8 (GenBank accession no.: MH211363). Pathogenicity studies in mice have shown that PCV2-GX-6 can lead to growth inhibition of mice. Meanwhile PCV2-GX-6 caused the typical lesions of spleen, lung and kidney. The results of qPCR showed that PCV2 can effectively proliferate in the liver, spleen, lung, and kidney., Conclusion: PCV2-GX-6 can successfully infect BLAB/c mice, effectively proliferate in major organs, and possessed high pathogenicity. In conclusion, combined with the genotype and pathogenicity of PCV2d currently prevalent, PCV2-GX-6 can be used as a candidate vaccine strain., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Porcine circovirus 4 (PCV4), a newly emerging virus that was first discovered in 2019, may pose a potential threat to the pig industry. Droplet digital PCR (ddPCR) is an absolute quantitative method that has high sensitivity and accuracy. In this study, we developed a novel ddPCR assay to detect PCV4. Furthermore, we evaluated the detection limit, sensitivity, specificity and reproducibility of the ddPCR and TaqMan real-time quantitative PCR (qPCR) and tested 160 clinical samples to compare the detection rate of the two methods., Results: The detection limit for ddPCR was 0.54 copies/µL, 10.6 times greater sensitivity than qPCR. Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PCV4. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%)., Conclusions: This study successfully developed a sensitive, specific and repeatable ddPCR assay for PCV4 detection, which can be widely used in clinical diagnosis of PCV4 infections., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Porcine circovirus type 2 (PCV2) can cause porcine circovirus-associated disease (PCVAD), which causes significant economic losses to the global pig industry annually. There are no effective antiviral drugs used to control and treat PCV2, and prevention is mainly obtained through vaccination. PCV2 genome replicates through the rolling circle replication (RCR) mechanism involving Rep and Rep', so analyzing the holistic structure of Rep and Rep' will help us better understand the replication process of PCV2. However, there are no reports on the integral structure of Rep' and Rep, which seriously hinders the research of the viral replication. By using the x-ray diffraction method, the structure of the Rep' dimer was resolved by us in this study. Structural analysis revealed that Rep' is a dimer formed by the interaction of the C-terminal domain. The two Rep' form a positively charged groove, which may play an essential role in the viral binding of dsDNA. Together, this study help to understand the replication process of the virus and may also provide new insights into the development of antiviral drugs., (© 2023 Wiley Periodicals LLC.)

Objectives: PCV2-associated disease (PCVAD), caused by porcine circovirus type 2 (PCV2) infection, is one of the major infectious diseases in the global swine industry. Nitric oxide (NO), as an important signalling molecule, has antiviral activities against a variety of viruses. To date, limited knowledge is available on the role of NO during PCV2 infection., Methods: This study was conducted to investigate the effects of exogenous NO on PCV2 replication in vitro. To exclude the possibility that the detected antiviral effects were due to cell toxicity, maximum non-cytotoxic concentrations of the drugs were determined. Kinetics of NO production were assessed after drug treatment. The antiviral activities of NO at different concentrations and at different time points were carefully assessed by measuring the virus titers, viral DNA copies and percentage of PCV2-infected cells. Regulation of NF-κB activity by exogenous NO was also investigated., Results: Kinetics of NO production indicated that S-nitroso-acetylpenicillamine (SNAP) produced NO in a dose-dependent manner, while NO was scavenged by its scavenger haemoglobin (Hb). An in vitro antiviral assay demonstrated that exogenous NO strongly inhibited PCV2 replication in a time-dependent and dose-dependent manner, whereas the inhibitory effects could be reversed by Hb. Furthermore, inhibition of NF-κB activity induced by NO contributed to a notable decrease in PCV2 replication., Conclusion: These findings provide a new potential antiviral therapy against PCV2 infection, and the antiviral effects of exogenous NO may be partly achieved by regulating NF-κB activity., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Canine circovirus (CanineCV) is a DNA virus affecting domestic dogs and other wild carnivore species. Despite the potential implications for dogs' health and wildlife conservation, data on CanineCV presence, epidemiology and genetic features from Africa is still poor. In the present study, biological specimens collected between 2020 and 2022 from a total of 32 jackals and 575 domestic dogs were tested for the presence of CanineCV DNA to evaluate its frequency. Furthermore, sequencing was conducted on positive samples to characterize the strains and compare them with publicly available sequences through phylogenetic analysis. A high CanineCV prevalence was observed both in jackals (43.75%; 95 CI: 28.17% - 60.67%) and domestic dogs (27.13%; 95 CI: 23.66% - 30.91%). All aside from one Namibian strain formed an independent clade, suggestive of extremely rare introduction events, followed by local persistence, circulation, and evolution. Remarkably, different recombination events were observed involving strains from both jackals and domestic dogs, which testify to the likely strain exchange between these populations. Distinctive amino acid residues were also observed in jackals. The limitations of the considered host populations however prevent a definitive conclusion on host adaptation, biological, and clinical features. Further studies should be performed to expand our current knowledge of the CanineCV disease scenario in Namibia, other African regions, and associated host species in Africa., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Porcine circovirus type-2 capsid protein contains a major immunodominant epitope used as a subunit vaccine. Transient expression in mammalian cells is an efficient process for producing recombinant proteins. However, there is still a lack of research on the efficient production of virus capsid proteins in mammalian cells. Here we present a comprehensive study to investigate and optimize the production process of a model "difficult-to-express" virus capsid protein, PCV2 capsid protein in HEK293F transient expression system. The study evaluated the transient expression of PCV2 capsid protein in the mammalian cell line HEK293F and investigated the subcellular distribution by confocal microscopy. In addition, the RNA sequencing (RNA-seq) was used to detect the differential expression of genes after cells transfected with pEGFP-N1-Capsid or empty vectors. The analysis revealed that the PCV2 capsid gene affected a panel of differential genes of HEK293F cells involved in protein folding, stress response, and translation process, such as SHP90β, GRP78, HSP47, and eIF4A. An integrated strategy of protein engineering combined with VPA addition was applied to promote the expression of PCV2 capsid protein in HEK293F. Moreover, this study significantly increased the production of the engineered PCV2 capsid protein in HEK293F cells, reaching a yield of 8.7 mg/L. Conclusively, this study may provide deep insight for other "difficult-to-express" virus capsid proteins in the mammalian cell system., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

There are three main genotypes of porcine circovirus type 2 (PCV2), namely PCV2a, PCV2b and PCV2d, of which PCV2b and PCV2d are currently the most common. There are antigenic differences between these different genotypes. To explore the effect of PCV2 antigen differences on the immune protection provided by vaccines, a cross-immune protection test was carried out in pigs. Three genotype strains, PCV2a-CL, PCV2b-MDJ and PCV2d-LNHC, were inactivated and emulsified to prepare inactivated vaccines to immunize pigs, who were then challenged with the circulating strains PCV2b-BY and PCV2d-LNHC. Immunoperoxidase monolayer assays (IPMAs) and micro-neutralization assays were used to detect antibodies against the three different genotypes of PCV2. The results showed that the three genotype vaccines induced pigs to produce antibodies against the same and different genotypes of PCV2, but the levels of IPMA and neutralizing antibodies against the same genotype were higher than those against different genotypes. Quantitative Polymerase Chain Reaction (qPCR), virus titration and immunohistochemistry were used to detect PCV2 genomic DNA, live virus and antigen, respectively, in inguinal lymph nodes of experimental pigs. Following challenge with the PCV2b-BY strain, the viral DNA load in the inguinal lymph nodes of pigs immunized with the three genotype vaccines was reduced by more than 99 % compared to the unimmunized group. Following challenge with the PCV2d-LNHC strain, the viral DNA loads in the inguinal lymph nodes of pigs immunized with PCV2a, PCV2b and PCV2d genotype vaccines were reduced by 93.8 %, 99.8 % and 98.3 %, respectively, compared to unimmunized controls. In addition, neither live PCV2 virus nor antigen were detected in the inguinal lymph nodes of pigs immunized with any of the genotype vaccines (0/18), but both were detected in the lymph nodes of experimental pigs in the unimmunized control group (6/6). These findings suggest that, although the antigenic differences of the three genotype strains induce significant differences in antibody levels, they seem to have little effect on cross-protection between different genotypes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The beak and feather disease virus (BFDV), causative agent of Psittacine beak and feather disease (PBFD), is a highly fatal and widespread virus that infects both the wild and captive Psittaciformes around the world. The BFDV genome is a ssDNA of approximately 2 kb in size, making it among the smallest known pathogenic viruses. Though, the virus is placed in Circoviridae family of the Circovirus genus, there is no classification system on clade and sub-clade level according to the International Committee on Taxonomy of Viruses and the strains are grouped on the bases of geographic locations. Thus, we provide the latest and robust phylogenetic classification of BFDVs in this study based on full-length genomic sequences, grouping all the available 454 strains detected during 1996-2022 into two distinct clades, e.g., GI and GII. The GI clade is further divided into six sub-clades (GI a-f), while GII into two sub-clades (GII a and b). In addition, the phylogeographic network identified high variability among the BFDV strains, showing several branches, where all the branches are connected to four strains, e.g., BFDV-ZA-PGM-70A(GenBank ID: HM748921.1, 2008-South Africa), BFDV-ZA-PGM-81A(GenBank ID: JX221009.1, 2008-South Africa), BFDV14(GenBank ID: GU015021.1, 2010-Thailand) and BFDV-isolate-9IT11(GenBank ID: KF723390.1, 2014-Italy). Furthermore, we identified 27 recombination events in the rep (replication-associated protein) and cap (capsid protein) coding regions using the complete genomes of BFDVs. Similarly, the amino acids variability analysis indicated that both the rep and cap regions are highly variable with values exceeding the variability coefficient estimation limit of 1.00, speculating the possible amino acids drift with the emergence of new strains. The findings provided in this study may offer the latest phylogenetic, phylogeographic and evolutionary landscape of the BFDVs., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Porcine circovirus-like virus P1 is the animal virus with the smallest genome discovered so far, and it has become widely distributed in the Chinese mainland in recent years., Results: In this study, a BALB/c mouse model was used to reveal P1 infection in female reproductive systems and the vertical transmission of the virus. The female reproductive system, including the ovary and uterus, was harvested on day 14 postinfection and examined for pathological lesions. One-day-old mice without colostrum born from infected or uninfected mothers were collected, and P1 virus distribution in the different organs was investigated. During the trials, all the mice showed no clinical symptoms or gross lesions. However, stillbirth did occur in groups infected with the P1 virus. P1 nucleic acid was detected in the heart, liver, spleen, lung, kidney, and brain tissues of 1-day-old mice born from infected mice. Microscopic lesions in P1-infected female mice were characterized by necrosis of the ovarian follicular granulosa cells and abscission, follicular atresia, necrosis of the endometrial epithelial and uterine glandular epithelial cells, and hyperplasia of the squamous endometrial epithelium. The spermatocytes in the seminiferous tubules of the infected male mice were disorderly arranged, and the germ and Sertoli cells were shed, necrotic, and decreased in number. Immunohistochemical results identified P1-positive particles in the nucleus and cytoplasm of cells from the ovary and uterus of female mice., Conclusions: This study shows that the P1 virus could cause pathological damage to the reproductive system of female mice and could be transmitted vertically., (© 2023. The Author(s).)

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.

Background: Porcine circovirus 2 (PCV-2) is one of the pathogens that leads to a growing and persistent threat in pigs. Thus, the development of serological detection methods for PCV-2 is of great necessity for clinical diagnosis as well as epidemiological investigations. This study aimed to establish an indirect enzyme-linked immunosorbent assay (ELISA) to examine antibodies against PCV-2 based on virus-like particles (VLPs)., Results: Recombinant PCV-2 Cap protein was expressed in the baculovirus-insect cells system and PCV-2 VLPs were observed over transmission electron microscopy (TEM). The PCV-2 VLPs were shown to have good immunogenicity in mice and stimulated a high level of PCV-2 antibody titers. Using PCV-2 VLPs as coating antigen, the indirect ELISA can detect PCV-2 antibodies in animals with diagnostic sensitivity and specificity of 98.33% and 93.33% compared to immunofluorescence assay (IFA), respectively. The intra- and inter-assay coefficient variations (CVs) were < 10% in a batch, and < 15% in different batches, indicating good repeatability. There was no cross-reaction of this ELISA with antibodies against other porcine viruses. A total of 170 serum samples collected from different pig farms in China were tested for PCV-2 antibodies, and 151 (88.8%) samples were PCV-2 antibody positive., Conclusion: Our findings suggest that this ELISA was rapid, specific, and reproducible and can be used for large-scale serological investigations of PCV-2 antibodies in pigs., (© 2023. The Author(s).)

Wild boars can act as a reservoir of pathogenic viruses that affect the pig industry. Here, we assessed the presence of porcine circovirus 2, porcine parvovirus 1, and torque teno sus virus k2a in wild boars in northeastern Patagonia (Argentina). Total DNA was extracted from the tonsils of 27 animals (collected between early 2016 and mid-2019) and used to prepare sample pools, which were subjected to viral detection through two-round PCR assays. Sequencing of the amplification products and phylogenetic analysis confirmed the occurrence of all of the aforementioned infectious agents., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Porcine circovirus 4 (PCV4) is a recently discovered circovirus that was first reported in 2019 in several pigs with severe clinical disease in Hunan province of China, and also identified in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). To further investigate the epidemic profile and genetic characteristics of the two viruses, 150 clinical samples were collected from 9 swine farms in Shaanxi and Henan provinces of China, and a SYBR Green I-based duplex quantitative real-time polymerase chain reaction (qPCR) was developed for detecting PCV4 and PRRSV simultaneously. The results showed the limits of detection were 41.1 copies/μL and 81.5 copies/μL for PCV4 and PRRSV, respectively. The detection rates of PCV4 and PRRSV were 8.00% (12/150) and 12.00% (18/150) respectively, and a case of co-infection with PCV4 and PRRSV was found in the lung tissue of a suckling pig with respiratory symptom. Subsequently, the complete genomic sequences of five PCV4 strains were obtained, of which one PCV4 strain (SX-ZX) was from Shaanxi province, and these strains were 1770 nucleotides in length and had 97.7%-99.4% genomic identity with 59 PCV4 reference strains. The genome characteristic of the SX-ZX strain was evaluated from three aspects, a "stem-loop" structure, ORF1 and ORF2. As essential elements for the replication, the 17-bp iterative sequence was predicted as the stem structure, in which three non-tandem hexamers were found at downstream with H1/H2 (12-CGGCACACTTCGGCAC-27) as the minimal binding site. Three of the five PCV4 strains were clustered into PCV4b, which was composed of Suidae, fox, dairy cow, dog and raccoon dog. Phylogenetic analysis revealed that seven PRRSV strains from the present study were clustered into the PRRSV-2 genotype. Collectively, these data extend our understanding of the genome characteristic of PCV4 as well as the molecular epidemiology and the genetic profile of PCV4 and PRRSV., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Porcine circovirus type 2 (PCV-2) is among the most burdensome viruses of the swine industry globally. Several genotypes have been periodically emerging, but just three of them (PCV-2a, PCV-2b, and PCV-2d) seem to circulate worldwide and be associated with the disease. Conversely, the spatial-temporal distribution of minor genotypes appears limited and their clinical relevance is still unclear. Recently PCV-2e was incidentally detected for the first time in Europe in a breeding farm in Northeastern Italy, while no connection could be established with countries where this genotype had been previously detected. To investigate circulating genotypes in the neglected rural context and provide a comparison with the most explored industrial context, a molecular survey was performed on samples collected in rural (n = 72) and industrial farms (n = 110) located in the same geographic area. Phylogenetic analysis surprisingly evidenced PCV-2e circulation only in pigs reared in backyard farms (n = 5), while major genotypes (PCV-2a, -2b, -2d) circulate in both rearing contexts. However, the close genetic similarity between the herein detected PCV-2e strains and the previously reported one testify that, although unusual, such rural-to-industrial strains exchange affected also PCV-2e. The greater genetic and phenotypic diversity of PCV-2e genotype compared to other ones might threaten the protection granted by current vaccines. The present study suggests the rural context as an ecological niche for the circulation of PCV-2e, and even of other minor genotypes. PCV-2e detection in pigs with outdoor access further stresses the epidemiological role of backyard farms as interfaces for pathogen introduction, potentially ascribable to the different rearing approaches, lower managerial and biosecurity capabilities, and easier contacts with wildlife., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Porcine circovirus type 3 is a newly emerging pathogen of porcine circovirus associated disease (PCVAD). Currently, there is no commercially available vaccine, resulting in huge economic losses to the pig industry. Porcine circovirus type 3 capsid protein (Cap) can self-assemble into virus-like particles (VLPs). Therefore, the expression of the recombinant Cap protein is of great significance for the prevention, diagnosis and control of porcine circovirus type 3 associated diseases. In this study, the recombinant Cap protein was successfully expressed in Escherichia coli by deleting the nuclear localization sequence (NLS). The VLPs were observed by transmission electron microscopy. To evaluate the immunogenicity of the recombinant Cap protein, mice were immunized. As a result, the recombinant Cap protein can induce higher levels of humoral and cellular immune responses. A VLP-based ELISA method was developed for the detection of antibodies. The established ELISA method has good sensitivity, specificity, repeatability and clinical applicability. These results demonstrate the successful expression of the PCV3 recombinant Cap protein and the preparation of recombinant Cap protein VLPs, which can be used for the preparation of subunit vaccines. Meanwhile, the established I-ELISA method lays a foundation for the development of the commercial PCV3 serological antibody detection kit.

Porcine circovirus type 3 (PCV3) is a newly discovered pathogen that causes porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, multisystemic inflammation, and reproductive failure. Heme oxygenase-1 (HO-1), a stress-inducible enzyme, exerts protective functions by converting heme into carbon monoxide (CO), biliverdin (BV), and iron. However, the effects of HO-1 and its metabolites on PCV3 replication remain unknown. In this study, experiments involving specific inhibitors, lentivirus transduction, and small interfering RNA (siRNA) transfection revealed that active PCV3 infection reduced HO-1 expression and that the expression of HO-1 negatively regulated virus replication in cultured cells, depending on its enzymatic activity. Subsequently, the effects of the HO-1 metabolites (CO, BV, and iron) on PCV3 infection were investigated. The CO inducers (cobalt protoporphyrin IX [CoPP] or tricarbonyl dichloro ruthenium [II] dimer [CORM-2]) mediate PCV3 inhibition by generating CO, and this inhibition is reversed by hemoglobin (Hb; a CO scavenger). The inhibition of PCV3 replication by BV depended on BV-mediated reactive oxygen species (ROS) reduction, as N -acetyl-l-cysteine affected PCV3 replication while reducing ROS production. The reduction product of BV, bilirubin (BR), specifically promoted nitric oxide (NO) generation and further activated the cyclic GMP/protein kinase G (cGMP/PKG) pathway to attenuate PCV3 infection. Both the iron provided by FeCl 3 and the iron chelated by deferoxamine (DFO) with CoPP treatment failed to affect PCV3 replication. Our data demonstrate that the HO-1-CO-cGMP/PKG, HO-1-BV-ROS, and HO-1-BV-BR-NO-cGMP/PKG pathways contribute crucially to the inhibition of PCV3 replication. These results provide important insights regarding preventing and controlling PCV3 infection. IMPORTANCE The regulation of host protein expression by virus infection is the key to facilitating self-replication. As an important emerging pathogen of swine, clarification of the interaction between PCV3 infection and the host enables us to understand the viral life cycle and pathogenesis better. Heme oxygenase-1 (HO-1) and its metabolites carbon monoxide (CO), biliverdin (BV), and iron have been demonstrated to involve a wealth of viral replications. Here, we, for the first time, demonstrated that HO-1 expression decreases in PCV3-infected cells and negatively regulates PCV3 replication and that the HO-1 metabolic products CO and BV inhibit PCV3 replication by the CO- or BV/BR/NO-dependent cGMP/PKG pathway or BV-mediated ROS reduction, but the iron (the third metabolic product) does not. Specifically, PCV3 infection maintains normal proliferation by downregulating HO-1 expression. These findings clarify the mechanism by which HO-1 modulates PCV3 replication in cells and provide important targets for preventing and controlling PCV3 infection., Competing Interests: The authors declare no conflict of interest.

Like in many other African countries, pig production is increasing in Tanzania. To support farmers and stakeholders, it is important to increase our understanding of porcine pathogens present in the country. Currently, little is known about the circulation of porcine circovirus-2 (PCV-2) and porcine circovirus-3 (PCV-3). For this reason, samples from 124 pigs collected throughout the country between 2018 to 2022 were screened by PCR for the presence of PCV-2 and PCV-3. Sequencing and phylogenetic analysis of positive amplicons identified two PCV-2 genotypes (a and d). Limited genetic heterogenicity was observed among the PCV-3. This study provides important data on pathogens present in pigs in Tanzania and should be of use veterinary authorities involved in porcine disease management.

Porcine circovirus type 3 (PCV3) is a novel porcine circovirus that has been detected in pigs showing various clinical and pathological conditions, as well as in many asymptomatic pigs. The pathogenesis of PCV3 infection in pigs remains unclear. To evaluate the in vivo growth and pathogenicity of PCV3, we performed two experiments on PCV3 infection in laboratory-grade miniature pigs with strictly controlled genetic backgrounds and microbiological status. A PCV3 passage experiment confirmed PCV3 genome detection in the sera and multiple organs via in vivo serial passage generations. PCV3 was successively passaged in miniature pigs by inoculating tissue homogenates from infected pigs supporting Koch's principles. In the PCV3 infection experiment, viremia was observed in all the inoculated pigs, and transient neurological signs were observed in one of the three pigs. Histopathologically, all three pigs in the PCV3 inoculation group exhibited lung disorders such as interstitial pneumonia and lymphoplasmacytic perivasculitis. In addition, one pig with neurological signs in the PCV3 inoculation group showed focal thrombosis in the meninges of the cerebellum. Vascular lesions in both the lungs and brain suggest that PCV3 may cause injury to vascular tissues. In situ hybridization (ISH)-RNA analysis demonstrated that the PCV3 genome was localized in the lymph nodes of pigs inoculated with PCV3. The PCV3 in vivo passage system in NIBS miniature pigs will help investigate the pathogenicity of PCV3., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors are employees of Nippon Institute for Biological Science, whose affiliate, NISSEIKEN Co., Ltd., produces and markets NIBS miniature pigs., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Beak and feather disease virus (BFDV) is a 17-20 nm icosahedral virus belonging to the Circoviridae family. Psittacine beak and feather disease (PBFD) is caused by BFDV and its common symptoms include abnormal feather, beak, and claw development, as well as immunosuppression in various bird species. In this study, novel cell-penetrating peptides (CPPs) in the capsid protein (Cap) of BFDV were identified through bioinformatic analyses, after which they were experimentally characterized. The cell-penetrating activities of both CPP1 and CPP2 of BFDV were analyzed through flow cytometry and image analysis. The internalization of CPP1 and CPP2 was both dose- and time-dependent but their uptake efficiencies varied depending on the cell type. The cell-penetrating activities of BFDV CPP1 and CPP2 were both superior to that of a typical CPP-TAT originating from the viral protein of human immunodeficiency virus. The cellular uptake of 5 μM CPP1 was close to that of 25 μM TAT, albeit with less cytotoxicity. Using the identified CPPs, the pc-mCheery, pc-Rep, and pc-Cap plasmids were successfully delivered into the target cells for expression. Moreover, both the replication-associated protein with the tag and the Cap protein with the tag could also be successfully delivered into the cells by CPP1 and CPP2. Multiple endocytosis pathways and direct translocation were involved in the cell internalization of CPP1 and CPP2. Furthermore, the delivery of the apoptin gene using CPP1 and CPP2 effectively triggered apoptosis, thus confirming the potential of these CPPs as delivery vehicles. Similarly, green fluorescent protein (GFP) fused with CPP1 or CPP2 at their N-terminus successfully entered the cells. However, the cell internalization efficiency of CPP2-GFP was higher than that of CPP1-GFP. Taken together, our findings demonstrated that both CPP1 and CPP2 of BFDV have promising potential as novel CPPs., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Beak and feather disease virus (BFDV) belongs to the Circoviridae family, which has a relatively simple replication mechanism. As BFDV lacks a mature cell culture system, a novel mini-replicon system based on the reporter plasmid that contains the origin of replication, which can bind to the Rep protein expressed from another plasmid and thus trigger its replication and induce/increase luminescence was developed. The dual-luciferase assay was used in this system to measure replicative efficiency by comparing relative light units (RLU) of firefly luciferase. Linear relationships between the luciferase activity of the reporter plasmids with the BFDV origin of replication and the amounts of the Rep protein and vice versa were found, suggesting the mini-replicon system can be used to quantify viral replication. Moreover, the activities of reporter plasmids driven by mutated Rep proteins or the activities of reporter plasmids with mutations were significantly downregulated. The Rep and Cap promoter activities can be characterized using this luciferase reporter system. Notably, the RLU of the reporter plasmid was considerably inhibited in the presence of sodium orthovanadate (Na 3 VO 4 ). When BFDV-infected birds were treated with Na 3 VO 4 , the viral loads of BFDV rapidly decreased. In conclusion, this mini-replicon reporter gene-based system provides a practical means to screen for anti-viral drug candidates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The pathogenesis of porcine circovirus type 2b (PCV2b) and swine influenza A virus (SwIV) during co-infection in swine respiratory cells is poorly understood. To elucidate the impact of PCV2b/SwIV co-infection, newborn porcine tracheal epithelial cells (NPTr) and immortalized porcine alveolar macrophages (iPAM 3D4/21) were co-infected with PCV2b and SwIV (H1N1 or H3N2 genotype). Viral replication, cell viability and cytokine mRNA expression were determined and compared between single-infected and co-infected cells. Finally, 3'mRNA sequencing was performed to identify the modulation of gene expression and cellular pathways in co-infected cells. It was found that PCV2b significantly decreased or improved SwIV replication in co-infected NPTr and iPAM 3D4/21 cells, respectively, compared to single-infected cells. Interestingly, PCV2b/SwIV co-infection synergistically up-regulated IFN expression in NPTr cells, whereas in iPAM 3D4/21 cells, PCV2b impaired the SwIV IFN induced response, both correlating with SwIV replication modulation. RNA-sequencing analyses revealed that the modulation of gene expression and enriched cellular pathways during PCV2b/SwIV H1N1 co-infection is regulated in a cell-type-dependent manner. This study revealed different outcomes of PCV2b/SwIV co-infection in porcine epithelial cells and macrophages and provides new insights on porcine viral co-infections pathogenesis.

Porcine circovirus 4 (PCV4) is a recently discovered circovirus that was first reported in 2019 in several pigs in Hunan province of China and has also been identified in pigs infected with porcine epidemic diarrhea virus (PEDV). To further investigate the coinfection and genetic diversity of these two viruses, 65 clinical samples (including feces and intestinal tissues) were collected from diseased piglets on 19 large-scale pig farms in Henan province of China, and a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (qPCR) assay was developed for detecting PEDV and PCV4 simultaneously. The results showed that the limit of detection was 55.2 copies/μL and 44.1 copies/μL for PEDV and PCV4, respectively. The detection rate for PEDV and PCV4 was 40% (26/65) and 38% (25/65), respectively, and the coinfection rate for the two viruses was 34% (22/65). Subsequently, the full-length spike (S) gene of eight PEDV strains and a portion of the genome containing the capsid (Cap) gene of three PCV4 strains were sequenced and analyzed. Phylogenetic analysis showed that all of the PEDV strains from the present study clustered in the G2a subgroup and were closely related to most of the PEDV reference strains from China from 2011 to 2021, but they differed genetically from a vaccine strain (CV777), a Korean strain (virulent DR1), and two Chinese strains (SD-M and LZC). It is noteworthy that two PEDV strains (HEXX-24 and HNXX-24XIA) were identified in one sample, and the HNXX-24XIA strain had a large deletion at amino acids 31-229 of the S protein. Moreover, a recombination event was observed in strain HEXX-24. Phylogenetic analysis based on the amino acid sequence of the PCV4 Cap protein revealed that PCV4 strains were divided into three genotypes: PCV4a1, PCV4a2, and PCV4b. Three strains in the present study belonged to PCV4a1, and they had a high degree of sequence similarity (>98% identity) to other PCV4 reference strains. This study not only provides technical support for field investigation of PEDV and PCV4 coinfection but also provides data for their prevention and control., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Hepatitis of undetermined origin can be caused by a wide variety of pathogens, sometimes emerging pathogens. We report the discovery, by means of routine shotgun metagenomics, of a new virus belonging to the family Circoviridae, genus Circovirus, in a patient in France who had acute hepatitis of unknown origin.

We identified a novel circovirus (human-associated circovirus 2 [HuCV2]) from the blood of 2 intravenous drug users in China who were infected with HIV-1, hepatitis C virus, or both. HuCV2 is most closely related to porcine circovirus 3. Our findings underscore the risk for HuCV2 and other emerging viruses among this population.

Duck circovirus genotype 2 (DuCV2) belongs to the genus Circovirus, family Circoviridae. It can generally cause lymphocyte atrophy and necrosis in ducks, which leads to immunosuppression. The function of the DuCV2 open reading frame 3 (ORF3) protein in viral pathogenesis in host cells remains unclear. Therefore, a series of studies based on ORF3 of the isolate DuCV GH01 strain (belonging to DuCV2) were carried out in duck embryo fibroblasts (DEFs) in this study. The results showed that the ORF3 protein could induce nuclear shrinkage and fragmentation in DEFs. Chromosomal DNA breakage was observed by TUNEL assay. The expression levels of caspase-related genes showed that ORF3 primarily promoted caspase 3 and caspase 9 expression. Furthermore, the protein expression levels of cleaved caspase 3 and cleaved caspase 9 in DEFs were enhanced by ORF3. Thus, ORF3 may activate the mitochondrial apoptosis pathway. When the 20 amino acid residues at the C-terminus of ORF3 (ORF3ΔC 20 ) were deleted, the apoptosis rates were decreased. Moreover, compared to ORF3, ORF3ΔC 20 downregulated the mRNA levels of cytochrome c (Cyt c), poly ADP-ribose polymerase (PARP) and apoptosis protease activating factor 1 (Apaf-1), which are the key molecules in the mitochondrial apoptotic pathway. Further study showed that ORF3ΔC 20 could reduce the mitochondrial membrane potential (MMP). This study suggested that the DuCV2 ORF3 protein may primarily activate apoptosis through the mitochondrial pathway in DEFs, and this function is ORF3 C 20 dependent., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVADs) which affect pigs worldwide. Currently, there is a worldwide genotype prevalence switch from PCV2b to PCV2d, which has led to increased virulence of the circulating virus strains leading to vaccine failures and selection pressure. In the present study, the PCV2 genotypes circulating in north eastern region (NER) of India particularly the states of Assam and Arunachal Pradesh was characterized by isolation, sequencing and phylogenetic analysis of cap gene. The phylogenetic analysis revealed that the PCV2 isolates circulating in pigs of Assam and Arunachal Pradesh were mostly of PCV2d genotype. Hence, it can be concluded that PCV2d genotype is the most dominating genotype in NER and priority should be given to this genotype for development of future vaccine candidate against PCV2 in India.

Porcine circovirus 4 (PCV4) is considered a novel PCV, firstly found in China in 2019 and later discovered in Korea. This present study investigated the prevalence and genetic characteristics of PCV4 from high pig-density areas in Thailand during 2019-2020. From 734 samples, three samples (0.4%) from aborted fetuses and porcine respiratory disease complex (PRDC) cases were found positive for PCV4, two of the PCV4-positive samples were coinfected with both PCV2 and PRRSV, and the other PCV4-positive sample was found coinfected with PCV2. In situ hybridization (ISH) revealed the presence of PCV4 in the bronchial epithelial cells and in lymphocytes and histiocyte-like cells in the lymphoid follicles of the PRDC-affected pig. The complete Thai PCV4 genome had over 98% nucleotide identity with other PCV4 strains and was closely related to the Korean and Chinese PCV4b strains. Importantly, the amino acid residue at position 212 of the Cap gene is recommended for differentiating PCV4a ( 212 L) from PCV4b ( 212 M) based on currently available PCV4 genome sequences. These findings provide important clues for the pathogenesis, epidemiology, and genetic characteristics of PCV4 in Thailand., (© 2023. The Author(s).)

We investigated the prevalence of porcine circovirus 2 (PCV2) and 3 (PCV3) in wild boars captured or found dead in Nagano Prefecture in 2020. Based on PCR testing, 21 of 254 (8.3%) wild boars were PCV2-positive and 43 of 256 (16.8%) wild boars were PCV3-positive, 5 of 253 (2.0%) wild boars were both PCV2-positive and PCV3-positive. The frequency of detecting PCV3 in wild boars was significantly higher in adults than in juveniles (P=0.014). The PCV2-positive wild boars were found in all districts except for the North Alps and Hokushin, while PCV3-positive wild boars were found in all districts except for Suwa. This is the first report of PCV2 and PCV3 detected in wild boars in Japan.

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.

The continuous discovery of new viruses during the last decades has increased the need for new classification approaches and rules. Currently, the International Committee on Taxonomy of Viruses classifies viruses up to the species level. However, because of the higher variability of most of these infectious agents, a below-species categorization is often required for proper epidemiological investigations. Unfortunately, variable criteria are typically proposed by different research groups, leading to misleading and poorly reproducible results. This scenario occurred for the recently identified Porcine circovirus 3. Although genotype definition standards had been defined by a group of experts in the field, recent articles have been published introducing new genotypes, whose classification rules are not reported. We therefore would like to stress the usefulness of defining and maintaining a common language to allow proper results comparison among groups. We consider the consensus opinion of a heterogeneous expert team as the most valuable approach. Nevertheless, if other approaches are proposed, the disclosure of the criteria and the comparison with previous literature should be deemed mandatory to allow effective results reproducibility, interpretation and sharing., (© 2023. The Author(s).)

Like other alpha herpesviruses, pseudorabies virus (PRV) establishes lifelong latency in trigeminal ganglionic (TG) neurons. Upon stress, the latent viruses in the TG neurons reactivate and are transported anterograde from the neuron cell bodies to the nerve endings in the nasal mucosa, where they replicate and are discharged in the nasal and oral secretions. Consequently, the virus is transmitted to other naïve animals. This cycle of latency and reactivation continues until the animal dies or is slaughtered. We have constructed a PRV triple mutant virus (PRVtmv) and used it as a live subunit vaccine vector against porcine circovirus 2b (PCV2b) and classical swine fever virus (CSFV) (PRVtmv+). We compared the latency reactivation properties of PRVtmv+ with its parent wild-type (wt) Becker strain following intranasal infection. The results showed that PRV wt and PRVtmv+ established latency in the TG neurons. Based on nasal virus shedding, immediate early (infected cell protein 0; ICP0) and late genes, MCP (major capsid protein) and gC (glycoprotein C) transcriptions, and viral DNA copy numbers in the TGs of latently infected and dexamethasone (Dex)-treated pigs, both PRV wt and PRVtmv+ reactivated from latency. We noticed that PRV wt virus replicated productively in the terminally differentiated, postmitotic TG neurons, but PRVtmv+ failed to replicate and, therefore, there was no virus production in the TG. In addition, we found that only the PRV wt virus was shed in the nasal secretions following the Dex-induced reactivation. Our results demonstrated that the PRVtmv+ is safe as a live viral subunit vaccine vector without the possibility of productive replication in the TG upon reactivation from latency and without subsequent nasal virus shedding. This property of PRVtmv+ precludes the possibility of vaccine virus circulation in pigs and the risk of reversion to virulence.

The commercial farming and trading of parrots and ornamental birds as companion animals are important economic activities in many countries. Some of the bird species farmed/traded are captured from the wild or are closely related to wild birds and therefore represent a risk of pathogen exchange/introduction. Beak and feather disease virus (BFDV) and avian poliomavirus (APV) are among the viruses with the biggest impact on companion bird populations and have been detected in different hosts worldwide. Despite their relevance for both domesticated and wild birds, our knowledge of BFDV and APV epidemiology remains limited in several African countries. In the present study, 143 cloacal swabs were collected from companion birds in Windhoek, Namibia, and tested by polymerase chain reaction for BFDV and APV. Of the samples tested, 35/143 (24.48%) tested positive for BFDV; 11/143 (7.69%) were positive for APV; and 6/143 (4.2%) tested positive for both pathogens. Positive amplicons, consisting of segments of the ORF1 and VP1 genes, were sequenced and compared with sequences from viruses identified in other countries. Four Namibian-only clades of BFDV were identified, loosely related to foreign strains, which suggest the occurrence of multiple introduction events in the past, potentially from South Africa, followed by local, independent evolution. In contrast, the Namibian APV sequences were identical to each other and form a single clade. In both instances, no correlation was observed between the sampling host and the viral phylogeny, suggesting the absence of host-specific adaptation and a remarkable, unconstrained viral circulation within Namibian borders. Therefore, while regulations and control measures developed against foreign strain introduction have proven to be effective over time, the spread of BFDV and APV within Namibia's borders appears undeterred. Additional resources should be dedicated to limit strain circulation in commercial farming facilities, markets and small-scale traders., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In March 2022, a 61-year-old woman in France who had received a heart-lung transplant sought treatment with chronic hepatitis mainly characterized by increased liver enzymes. After ruling out common etiologies, we used metagenomic next-generation sequencing to analyze a liver biopsy sample and identified an unknown species of circovirus, tentatively named human circovirus 1 (HCirV-1). We found no other viral or bacterial sequences. HCirV-1 shared 70% amino acid identity with the closest known viral sequences. The viral genome was undetectable in blood samples from 2017-2019, then became detectable at low levels in September 2020 and peaked at very high titers (10 10 genome copies/mL) in January 2022. In March 2022, we found >10 8 genome copies/g or mL in the liver and blood, concomitant with hepatic cytolysis. We detected HCirV-1 transcripts in 2% of hepatocytes, demonstrating viral replication and supporting the role of HCirV-1 in liver damage.

Porcine circovirus-like virus (PCLV) is a recently discovered virus that may be associated with diarrhea in pigs. To investigate the epidemic profile and genetic characteristics of this virus, 175 clinical samples (141 intestinal samples, 17 blood samples, and 17 fecal samples) were collected from diseased piglets during outbreaks of diarrhea from 33 pig farms in 19 cities of Henan and Shanxi provinces of China between 2016 and 2021 and were screened by PCR for the presence of PCLV. The results showed that the positive rate for PCLV was 32% (56/175) at the sample level, 60.6% (20/33) at the farm level, and 57.9% (11/19) at the city level, which varied from 5.88% to 44.12% between 2016 and 2021. It was also found that PCLV occurred in coinfections with porcine circovirus type 2 (PCV2), PCV3, PCV4, porcine epidemic diarrhea virus, and porcine reproductive and respiratory syndrome virus, but no nucleic acids were detected for transmissible gastroenteritis virus, porcine deltacoronavirus, or porcine rotavirus in piglets with diarrhea. Notably, PCLV was detected in 13 diarrheal piglets from four different farms that were negative for the other porcine viruses. These findings suggest that PCLV may be associated with porcine diarrhea and that it has been circulating in piglets in Henan and Shanxi provinces of China. In addition, the complete genomes of 13 PCLV strains were sequenced and found to share 35.4%-91.0% nucleotide sequence identity with sequences available in the GenBank database. Phylogenetic analysis based on Rep amino acid sequences revealed that the 13 PCLV strains from this study clustered in group 1 and were closely related to eight Chinese PCLV strains, Bo-Circo-like virus CH, American strains 21 and 22, and Hungarian strains 288&#95;4 and 302&#95;4, but they differed genetically from seven other foreign PCLV strains. The whole genome and rep gene of 13 PCLV strains in this study were 72.2%-82% and 83.8%-89.7% identical, respectively, to those of Bo-Circo-like virus strain CH, indicating that PCLV is a novel virus in pigs that may be involved in cross-species transmission. Evidence of a recombination event was found in the rep region of the 13 PCLV strains sequenced. This study enriches the epidemiological data on PCLV infection in pigs in China and lays a foundation for further study on the pathogenesis of PCLV., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The objective of the current study was to compare the virulence of four porcine circovirus type 2 (PCV2) genotypes (PCV2a, 2b, 2d, and 2e) in pigs. Pigs were inoculated at 42 days of age with one of four PCV2 genotypes, then necropsied at 63 days of age. PCV2 genotype groups were evaluated through a comparison of clinical outcomes, antibody titers, level of PCV2 loads in blood and lymph nodes, and lymphoid lesion severity. Statistical differences did not occur between the evaluated genotype groups. Pigs inoculated with PCV2a, PCV2b, or PCV2d had a significantly (P<0.05) higher levels of PCV2 loads in blood and lymph node compared to pigs inoculated with PCV2e. The results of this study indicated that the PCV2a, PCV2b, and PCV2d are more virulent than PCV2e based on blood and lymphoid viral load of PCV2.

Porcine circovirus type 3 (PCV3) was first detected in 2016 and has been reported in many pig-producing countries around the world, including Vietnam. PCV3 has been found in complex cases with multiple clinical syndromes in swine. In this study, we investigated the genetic diversity of PCV3 strains circulating in Vietnam. A total of 249 samples were collected from swine farms located in eight provinces of Vietnam, and 11.65% (29/249) of these samples were found to contain PCV3. The ORF2 genes from the 29 PCV3-positive samples were amplified, purified, and sequenced. Phylogenetic analysis showed that 23 of these strains belonged to the PCV3b subtype, while the remaining six strains belonged to subtype c and subtype a (a-1 and a-2). Analysis of the ORF2 genes indicated that the 29 PCV3 strains had high sequence identity (96.90-100% at the genomic level and 96.19-100% at the amino acid level). Fifteen amino acid substitutions were found in predicted B-cell epitopes in the capsid proteins of the Vietnamese PCV3 strains., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Replication of porcine circovirus type 2 (PCV2), an important worldwide swine pathogen, has been demonstrated to be influenced by host genotype. Specifically, a missense DNA polymorphism (SYNGR2 p.Arg63Cys) within the SYNGR2 gene was demonstrated to contribute to variation in PCV2b viral load and subsequent immune response following infection. PCV2 is known to induce immunosuppression leading to an increase in susceptibility to subsequent infections with other viral pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV). In order to assess the role of SYNGR2 p.Arg63Cys in co-infections, pigs homozygous for the favorable SYNGR2 p.63Cys (N = 30) and unfavorable SYNGR2 p.63Arg (N = 29) alleles were infected with PCV2b followed a week later by a challenge with PRRSV. A lower PCV2b viremia (P < 0.001) and PCV2-specific IgM antibodies (P < 0.005) were observed in SYNGR2 p.63Cys compared to SYNGR2 p.63Arg genotypes. No significant differences in PRRSV viremia and specific IgG antibodies were observed between SYNGR2 genotypes. Lung histology score, an indicator of disease severity, was lower in the pigs with SYNGR2 p.63Cys genotypes (P < 0.05). Variation in the lung histology scores within SYNGR2 genotypes suggests that additional factors, environmental and/or genetic, could be involved in disease severity., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Viruses in the family Circoviridae have small circular single-stranded DNA (ssDNA) genomes. Circoviruses are known to infect a wide variety of animals, with notable disease pathology in psittacine (psittacine beak and feather disease) and porcine (postweaning multisystemic wasting syndrome) species. There is still a dearth of research investigating circoviruses associated with felid species. In six fecal samples collected from bobcats (Lynx rufus) in California from 2010 to 2011, we identified six viruses belonging to the genera Circovirus (n = 1) and Cyclovirus (n = 5), using a high-throughput-sequencing-based approach. Of these, the virus in the genus Circovirus represents a new species, as it shares only 54-60% genome-wide sequence identity with the other members of this genus. The five viruses in the genus Cyclovirus represent three new species, sharing <73% genome-wide sequence identity with all other cycloviruses. Three of the cycloviruses belong to a single putative species and were obtained from the feces of three individual bobcats, sharing 95.7-99.9% sequence identity, whereas the other two unique cycloviruses were identified in a single fecal sample. At present, it is unknown whether the identified viruses infect bobcats, their prey, or their gut parasites., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The objective of this study was to compare the virulence of 4 porcine circovirus type 2 (PCV-2) genotypes (2a, 2b, 2d, and 2e) in pigs singly infected with 1 of these 4 PCV-2 genotypes and pigs dually infected with a combination of 1 of the 4 PCV-2 genotypes and porcine reproductive and respiratory syndrome virus (PRRSV). Virulence was determined based on levels of PCV-2 loads in the blood and lymph nodes and the severity of lymphoid lesion. Within the singly infected groups, PCV-2a, PCV-2b, and PCV-2d resulted in a similar virulence to each other and all were more virulent than the PCV-2e groups. Within the dually infected groups, the combination of PCV-2d and PRRSV was more virulent than the other 3 PCV-2 genotypes (2a, 2b, and 2e), each in combination with PRRSV. Both PCV-2a+PRRSV and PCV-2b+PRRSV were more virulent than PCV-2e+PRRSV in dually infected pigs. This increased virulence of PCV-2d compared to the other 3 PCV-2 genotypes (2a, 2b, and 2e) may be attributed to an extra amino acid (lysine residue) found within open reading frame 2 (ORF2) of PCV-2d. In contrast, extra amino acids in ORF2 may decrease the virulence of PCV-2e when compared to the other 3 PCV-2 genotypes (2a, 2b, and 2d). The results of this study demonstrated that PCV-2d was the most virulent PCV-2 genotype in pigs co-infected with PRRSV. The results also suggest that genetic differences in the ORF2 of PCV-2 may affect the virulence of PCV-2 genotypes., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Duck circovirus (DuCV) is an immunosuppressive virus and can induce immunosuppression increasing rates of infection caused by other pathogens, which has resulted in gross economic losses in poultry industries. The genome sizes of DuCVs varied from 1987 to 1996 nucleotides (nt). Up to date two DuCV genotypes/lineages, DuCV1 and DuCV2, have been defined, with genome identities of around 83% between each other. In this study, a novel duck circovirus having a genome size of 1755 nt was identified from laying ducks with a disease of egg production declining or abrogation from Hunan province, China. Two major open reading frames (ORFs) were identified, with ORF1 (882 nt) and ORF2 (738 nt) encoding replicase (Rep) and capsid protein (Cap), respectively. Its genome showed the highest identities of 62.3-63.7% and 66.3%- 67.8% to the known genomes of DuCVs and goose circoviruses (GoCVs) available in GenBank, respectively, while it showed less than 50% identities to the genomes of other circoviruses. The amino acid sequence of capsid of this virus showed the highest identities of 45.4%- 47.3% and 42.9%- 44.5% to capsids of the known DuCVs and GoCVs, respectively, while it showed less than 27% identity to the capsid proteins of other circoviruses available in GenBank. Further phylogenetic analyses based on genomes, amino acid sequences of Rep and Cap proteins demonstrated that the present duck circovirus was clustered in a separate clade, distinct from other DuCVs and GoCVs, corroborating it is a distinct novel circovirus species in ducks, tentatively designated as duck cirovirus 3 (DuCV3). The clinical significance and pathogenesis of this virus needs further investigation., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Canine distemper virus (CDV) and canine circovirus (CanineCV) have been described worldwide in multi-systemic disease in dogs. Both agents may be occasionally associated with other viral pathogens, but reports of coinfection by CDV and CanineCV associated with disease are rare. In this article, we report a coinfection between CDV and CanineCV detected during an investigation of viral agents involved in multisystemic disease in dogs in Southern Brazil. Molecular testing by PCR in lungs and intestines of 77 dogs necropsied in pathology services (2015-2020) revealed several single and mixed viral infections, including a CDV/CanineCV coinfection. In the case reported here, gross and histological findings were compatible with CDV pathology (bronchointerstitial pneumonia and viral intracytoplasmatic inclusions in pneumocytes and transitional epithelial cells of urinary bladder). CanineCV DNA and CDV antigens were detected in lung and intestine fragments by PCR and immunohistochemistry, respectively. CanineCV PCR amplicons subjected to nucleotide sequencing showed > 98.6% nucleotide identity with CanineCV sequences from GenBank. Although the role of CanineCV in the pathogenesis of the reported case could not be determined, our results show that CanineCV may be associated with other viral infections in cases of multisystemic disease in dogs. These results reinforce the circulation of CanineCV in dogs in Brazil and highlight the importance of including this virus in the list of differential diagnoses of respiratory and gastroenteric infectious diseases in dogs., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Background: Porcine circovirus 2 is globally noted swine pathogen with multiple genotypes associated with vast clinical and subclinical outcomes. This study aimed to isolate and characterize PCV2 genotypes circulating in southern states of India., Methods and Results: A total of 434 field samples comprising of serum (n = 273), tissues (n = 109) and swabs (n = 52) collected from swine during 2019 to 2021 from southern states of India were screened for PCV2 by specific polymerase chain reaction (PCR) assay. Molecular prevalence of PCV2 in southern India was found to be 12.21% (n = 53). All the 53 PCV2 positive samples were further subjected to the PCR assay with designed primers targeting full length amplification of ORF2 gene of PCV2 for molecular characterization. Randomly 32 positive samples by full length PCV2-ORF2 gene PCR were sequenced for genotyping. Signature motif and phylogenetic analysis of 32 PCV2 sequences revealed 62.5% (n = 20) prevalence of PCV2d genotype followed by 21.8% (n = 7) of PCV2h or PCV2-IM1 and 15.6% (n = 5) of PCV2b genotypes. Twenty five PCR positive field samples were subjected for virus isolation in PK15 cells and characterized. Out of 25 samples processed 5 (20%) PCV2 isolates obtained in this study were confirmed by PCR and immune fluorescence assay. Molecular characterization of PK15 adapted five PCV2 isolates confirmed circulation of PCV2d, PCV2h and PCV2b genotypes in pigs under field conditions in southern India., Conclusions: Isolation and molecular epidemiological study of PCV2 in southern states of India evidences high circulation of PCV2d genotypes in field conditions in comparison to other genotypes., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

As pig production increases in Africa, it is essential to identify the pathogens that are circulating in the swine population to assess pig welfare and implement targeted control measures. For this reason, DNA samples collected from pigs in Nigeria in the context of African swine fever monitoring were further screened by PCR for porcine circovirus 2 (PCV-2), porcine circovirus 3 (PCV-3), and porcine parvovirus 1 (PPV1). Forty-seven (45%) pigs were positive for two or more pathogens. Sequence analysis identified PCV-2 genotypes a, b, and d, while limited genetic heterogenicity was observed among PCV-3 strains. All except one of the PPV1 sequences were genetically distinct from those previously identified in other countries., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Porcine circovirus type 2 (PCV2) is capable of causing porcine circovirus-associated disease (PCVAD) and is one of the major threats to the global pig industry. The nucleocapsid protein Cap encoded by the PCV2 ORF2 gene is an ideal antigen for the development of PCV2 subunit vaccines, and its N-terminal nuclear localization sequence (NLS) structural domain is essential for the formation of self-assembling VLPs. In the present study, we systematically expressed and characterized full-length PCV2 Cap proteins fused to dominant T and B cell antigenic epitopes and porcine-derived CD154 molecules using baculovirus and found that the Cap proteins fusing epitopes were still capable of forming virus-like particles (VLPs). Both piglet and mice experiments showed that the Cap proteins fusing epitopes or paired with the molecular adjuvant CD154 were able to induce higher levels of humoral and cellular responses, particularly the secretion of PCV2-specific IFN-γ and IL-4. In addition, vaccination significantly reduced clinical signs and the viral load of PCV2 in the blood and tissues of challenged piglets. The results of the study provide new ideas for the development of a more efficient, safe and broad-spectrum next-generation PCV2 subunit vaccine.

Porcine circovirus 3 (PCV3) is a newly emerging virus and has been found associated with porcine dermatitis and nephropathy syndrome in pigs. Compared with PCV2, research into PCV3 cap gene sequencing is deficient. To investigate the prevalence and genotype distribution of PCV3, we collected 1291 samples from 211 pig farms throughout 15 provinces and municipalities. 312 out of 1291 samples were tested positive by PCR. We further sequenced and analyzed 164 PCR-positive samples. The majority (61.8%) of isolates we sequenced belong to genotype PCV3c. PCV3c is also the dominant genotype in Hubei, Hunan, Hebei province and Chongqing city. We found 3 sites under positive selection and located in predicted epitope peptide, revealing that the pig's immunity may be a reason those sites are undergoing highly positive selection., (© 2022. The Author(s).)

This study evaluates the efficacy of intradermal all-in-one vaccine (MHYOSPHERE® PCV ID, Laboratorios Hipra S.A. Amer, Spain) based on the strain Nexhyon, an inactivated recombinant M. hyopneumoniae strain with an embedded/integrated PCV2a capsid protein thereof, as the single active substance. Pigs were administered the vaccine intradermally at 21 days of age with 0.2 mL, then challenged at 49 days of age with either M. hyopneumoniae (intratracheal route), PCV2d (intranasal route), or both. Upon dual challenge, growth performance was improved when the intradermal all in one vaccine was administered compared to the unvaccinated group. In pigs receiving single or dual challenge, vaccination increased neutralizing antibodies against PCV2d and specific interferon-γ secreting cells for each pathogen. In contrast, viral load of PCV2d in the blood, M. hyopneumoniae load in the larynx, and the severity of pulmonary and lymphoid lesions were decreased. Vaccination provided good protection against challenge with M. hyopneumoniae and PCV2d., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)