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Introduction: The novel Porcine circovirus 3 (PCV-3) has been associated in the past years to different porcine diseases, including reproductive failure. The potential occurrence of PCV-3 in abortions from Swiss pig herds has not been investigated so far. Thus, we conducted a retrospective study on pig aborted cases submitted to our laboratory in the University of Bern during the last 10 years with the main aim of investigating the possible presence of PCV-3 in foetal and/or placental tissue. Twelve out of the 53 studied cases showed mild histopathological changes as previously described in PCV-3 positive cases. However, in none of the cases, PCV-3 genetic material could be detected in the examined formalin-fixed, paraffin-embedded tissues. In only one third of the cases, a cause for the abortion was found, which is similar to other studies. Our survey suggests that PCV-3 was not involved in the porcine abortion cases submitted over the last decade at our institution in Switzerland.

In China, a novel pathogen within the genus Circovirus has been identified as a causative agent of the 'novel acute hemorrhage syndrome' (NAHS) in aquacultured populations of turbot (Scophthalmus maximus L.). Histopathological examination using light microscopy revealed extensive necrosis within the cardiac, splenic, and renal tissues of the afflicted fish. Utilizing transmission electron microscopy (TEM), we detected the presence of circovirus particles within the cytoplasm of these cells, with the virions consistently exhibiting a spherical morphology of 20-40 nm in diameter. TEM inspections confirmed the predominance of these virions in the heart, spleen, and kidney. Subsequent molecular characterization through polymerase chain reaction (PCR) analysis corroborated the TEM findings, with positive signals in the aforementioned tissues, in stark contrast to the lack of detection in gill, fin, liver, and intestinal tissues. The TEM observations, supported by PCR electrophoresis data, strongly suggest that the spleen and kidney are the primary targets of the viral infection. Further characterization using biophysical, biochemical assays, and genomic sequencing confirmed the viral classification within the genus Circovirus, resulting in the nomenclature of turbot circovirus (TurCV). The current research endeavors to shed light on the pathogenesis of this pathogen, offering insights into the infection mechanisms of TurCV in this novel piscine host, thereby contributing to the broader understanding of its impact on turbot health and aquaculture., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen., Competing Interests: The authors declare no conflict of interest.

Porcine circovirus 3 (PCV3) was first reported in the United States in 2016; this virus is considered to be involved in diverse pathologies, such as multisystem inflammation, porcine dermatitis and nephropathy syndrome, and reproductive disorders. However, successful isolation of PCV3 using cultured cells has been rare. In this study, we aimed to isolate PCV3 using primary porcine bone marrow-derived cells. Mononuclear cells were isolated from the femur bones of clinically healthy pigs. These primary cells were cultured for 6-10 days post-seeding and infected with PCV3-containing tissue homogenates. The cells were cultured for up to 37 days, and the culture medium was changed every 3-4 days. The growth curve of PCV3 in porcine bone marrow cells revealed a decline in growth during the first 10 days post-infection, followed by an increase leading to > 10 10 genomic copies/mL of the cell culture supernatant; moreover, the virus was capable of passaging. The indirect fluorescent antibody assay for PCV3 infection revealed the presence of PCV3 capsid protein in the cytoplasm and nuclei of infected cells. Bone marrow cells were passaged for more than 20 generations (over 5 months), and PCV3 persistently infected the cells. PCV3-infected bone marrow cells expressed mesenchymal markers. These results reflect that primary porcine bone marrow-derived mesenchymal cells are permissive to PCV3 and continuously replicate a high copy number of the PCV3 genome. These findings regarding the high replication rate of PCV3 in bone marrow-derived mesenchymal cells could enhance our understanding of PCV3 pathogenicity., (© 2024. The Author(s).)

Since Porcine Circovirus 3 (PCV3) was first identified in 2016, our understanding of the humoral response is still relatively scarce. Current knowledge of the PCV3 humoral response is primarily based on field studies identifying the seroprevalence of PCV3 Cap-induced antibodies. Studies on the humoral response following experimental PCV3 infection have conflicting results where one study reports the development of the Cap IgG response 7 days postinfection with no concurrent Cap IgM response, while a second study shows a Cap IgM response at the same time point with no detection of Cap IgG. The dynamics of the PCV3 Cap and Rep IgG following maternal antibody transfer and experimental infection have not been well characterized. Additionally, the cross-reactivity of convalescent serum from PCV2 and PCV3 experimentally infected animals to serologic methods of the alternate PCV has limited evaluation. Here, we show that maternally derived antibodies were detectable in piglet serum 7-9 weeks postfarrowing for the Cap IgG and 5-weeks-post farrowing for the Rep IgG using Cap- and Rep-specific enzyme linked immunosorbent assays (ELISA) and immunofluorescent assays (IFA) methods. Following experimental inoculation, Cap IgG was detected at 2-weeks-post inoculation and Rep IgG detection was delayed until 4-weeks-post inoculation. Furthermore, convalescent serum from either PCV2 or PCV3 methods displayed no cross-reactivity by serological methods against the other PCV. The information gained in this study highlights the development of both the Cap- and Rep-specific antibodies following experimental infection and through the transfer of maternal antibodies. The increased understanding of the dynamics of maternal antibody transfer and development of the humoral response following infection gained in the present study may aid in the establishment of husbandry practices and potential application of prophylactics to control PCV3 clinical disease., Importance: Research on Porcine Circovirus 3 (PCV3) immunology is vital for understanding and controlling this virus. Previous studies primarily relied on field observations, but they have shown conflicting results about the immunological response against PCV3. This study helps fill those gaps by looking at how antibodies develop in pigs, especially those maternal-derived, and their impact in neonatal pigs preventing PCV3-associated disease in piglets. In addition, we look at the dynamics of antibodies in experimental infections mimicking infection in pigs in the grower-phase condition. Understanding this process can help to develop better strategies to prevent PCV3 infection. Also, this research found that PCV2 and PCV3 do not cross-react, which is crucial for serological test development and results interpretation. Overall, this work is essential for improving swine health and farming practices in the face of PCV3 infections., Competing Interests: The authors declare no conflict of interest.

Introduction: Since their identification in 1974, circoviruses have caused clinicopathological diseases in various animal species, including humans. However, their origin, transmission, and genetic evolution remain poorly understood., Methods: In this study, the genome sequences of circovirus were obtained from GenBank, and the Bayesian stochastic search variable selection algorithm was employed to analyzed the evolution and origin of circovirus., Results: Here, the evolutionary origin, mode of transmission, and genetic recombination of the circovirus were determined based on the available circovirus genome sequences. The origin of circoviruses can be traced back to fish circovirus, which might derive from fish genome, and human contributes to transmission of fish circovirus to other species. Furthermore, mosquitos, ticks, bats, and/or rodents might play a role as intermediate hosts in circovirus intra- and inter-species transmission. Two major lineages (A and B) of circoviruses are identified, and frequent recombination events accelerate their variation and spread. The time to the most recent common ancestor of circoviruses can be traced back to around A.D. 600 and has been evolving at a rate of 10 -4 substitutions site -1 year -1 for a long time., Discussion: These comprehensive findings shed light on the evolutionary origin, population dynamics, transmission model, and genetic recombination of the circovirus providing valuable insights for the development of prevention and control strategies against circovirus infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Cui, Li, Xu, Xie, Wang, Hou, Zhou, Feng and Liu.)

Circoviruses cause severe disease in pigs and birds. Canine circovirus has thus far only been associated with respiratory and gastrointestinal disorders and systemic disease in dogs. The Iberian lynx (Lynx pardinus) is one of the most endangered carnivores in Europe and the most endangered felid worldwide. Exploring the virome of these animals may be important in terms of virus discovery and assessing the interspecies-circulation of viruses from related carnivores. In this study, 162 spleen samples from Iberian lynx were screened for CRESS DNA viruses. Overall, 11 (6.8%) of 162 samples tested positive using a consensus PCR. Partial rep sequences were tightly related to each other (96.6-100%). Specific molecular protocols were designed on the partial rep sequences of the novel virus, Iberian lynx-associated circovirus-1 (ILCV-1). By screening a subset of 45 spleen samples, the infection rate of ILCV-1 in Iberian lynxes was 57.8% (26/45). ILCV-1 strains formed a separate cluster intermingled with bat, rodent, mongoose, and felid circoviruses. The genome of the novel virus displayed the highest nucleotide identity (64.3-65.3%) to mongoose circoviruses, thus representing a novel candidate circovirus species. The detection of these viruses in the spleen tissues could suggest systemic infection in the animal host. Overall, these findings suggest that this novel circovirus is common in the Iberian lynx. Further studies are warranted to assess the possible health implications of ILCV-1 in this endangered species., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Goose circovirus (GoCV) is a common pathogen that causes immunosuppression and promotes secondary infections with other infectious agents in geese worldwide. In the present study, we identified GoCV in 2 out of 93 duck flocks from China and successfully sequenced the complete genomes of 2 strains (AH22du and HN20du). The whole genome of the two strains shared a high identity of 90.5 to 98.63% with China GoCV reference, and low identity of 58.98% with DuCV reference, respectively. Phylogenetic tree constructed on the two and other genome sequences of GoCV revealed three main branches. Both strains sequenced in this study were distributed on different sub-branches with most other Chinese GoCV strains, and AH22du clustered into an independent sub-branch within the cluster. Recombination analysis predicted that HN20du might potentially recombine from the major parent of yk4 (Zhejiang Province, China, 2007) and minor parent of GD/YJ/g2 (Guangdong Province, China, 2020). To the best of our knowledge, this is the first report of GoCV in ducks from China. This broadened host spectrum of GoCVs requires attention from the waterfowl industry and researchers., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Porcine circovirus type 2 (PCV2) infection leads to multi-system inflammation in pigs, and this effect can be achieved by upregulating host miR-21. The underlying mechanism of miR-21 regulates PCV2-induced inflammation is already known, however, how PCV2 regulates miR-21 levels and function using both autonomic and host factors remains to be further revealed. Here we present the first evidence that PCV2 ORF5 induces an inflammatory response by up-regulating miR-21 level through targeting nuclear miR-30d. In this study, we found that overexpression of ORF5 significantly increased miR-21 level and promoted the expression of inflammatory cytokines and activation of the NF-κB pathway, while ORF5 mutation had the opposite effect. Moreover, the differential expression of miR-21 could significantly change the pro-inflammatory effect of ORF5, indicating that ORF5 promotes inflammatory response by up-regulating miR-21. Bioinformatics analysis and clinical detection found that nuclear miR-30d was significantly down-regulated after ORF5 overexpression and PCV2 infection, and targeted pri-miR-21 and PCV2 ORF5. Functionally, we found that miR-30d inhibited the levels of miR-21 and inflammatory cytokines in cells. Mechanistically, we demonstrated that ORF5 inhibits miR-30d expression levels through direct binding but not via the circRNA pathway, and miR-30d inhibits miR-21 levels by targeting pri-miR-21. In summary, the present study revealed the molecular mechanism of ORF5 upregulation of miR-21, further refined the molecular chain of PCV2-induced inflammatory response and elucidated the role of miRNAs in it., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study was designed to investigate the different sequential order of infection for porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV). Thirty-six pigs were randomly assigned to six different treatment groups. The first (hereafter referred to as PRRSV-PCV2) group was inoculated with PRRSV first followed by PCV2d. The second (hereafter referred to as PCV2+PRRSV) group was co-infected with both viruses at the same timepoint (42 days of age). The third (hereafter referred to as PCV2-PRRSV) group was inoculated with PCV2d first followed by PRRSV. A fourth group was only inoculated with PCV2d at 42 days of age, while a fifth group was only inoculated with PRRSV at the same timepoint. The sixth group served as a negative control group. The most important observation discovered that PRRSV only had a potentiation effect on PCV2 in both PRRS-PCV2 and PCV2+PRRSV groups. Both PRRSV-PCV2 and PCV2+PRRSV groups experienced a significant reduction in growth performance compared with control pigs. In addition, PRRSV-PCV2 and PCV2+PRRSV groups exhibited a greater severity in their clinical signs, and/or had higher PCV2 blood and lymphoid viral loads that resulted in a stronger severity of lymphoid lesions compared with PCV2-PRRSV group. Serum TNF-α levels were significantly higher in both PRRS-PCV2 and PCV2+PRRSV groups compared with those in PCV2-PRRS, PCV2, and PRRSV groups. The results of this study demonstrated that divergent clinical outcomes are dependent on the sequential infection order of PCV2 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 © 2024 Elsevier B.V. All rights reserved.)

Porcine circovirus type 4 (PCV4), a recently identified circovirus, is prevalent in numerous provinces in China, as well as in South Korea, Thailand, and Europe. PCV4 virus rescued from an infectious clone showed pathogenicity, suggesting the economic impact of PCV4. However, there remains a lack of understanding regarding the immunogenicity and epitopes of PCV4. This study generated a monoclonal antibody (MAb) 1D8 by immunizing mice with PCV4 virus-like particles (VLPs). Subsequently, the epitope recognized by the MAb 1D8 was identified by truncated protein expression and alanine scanning mutagenesis analysis. Results showed that the 225 PKQG 228 located at the C-terminus of the PCV4 Cap protein is the minimal motif binding to the MAb. Homology modeling analysis and immunoelectron microscopy revealed that the epitope extends beyond the outer surface of the PCV4 VLP. Moreover, the epitope is highly conserved among PCV4 strains and does not react with other PCVs. Together, the MAb 1D8 recognized epitope shows potential for detecting PCV4. These findings significantly contribute to the design of antigens for PCV4 detection and control strategies., Importance: Porcine circovirus type 4 (PCV4) is a novel circovirus. Although PCV4 has been identified in several countries, including China, Korea, Thailand, and Spain, no vaccine is available. Given the potential pathogenic effects of PCV4 on pigs, PCV4 could threaten the global pig farming industry, highlighting the urgency for further investigation. Thus, epitopes of PCV4 remain to be determined. Our finding of a conserved epitope significantly advances vaccine development and pathogen detection., Competing Interests: The authors declare no conflict of interest.

The aim of this study was to establish a rapid method for constructing infectious clones of porcine circovirus type 2 (PCV2). In this study, we constructed circular infectious clones of PCV2 by seamless cloning technology, using the clinically isolated strain PCV2-LX as a template. Meanwhile, this method was compared with the conventional restriction-ligation approach, focusing on the in vitro circularization (self-ligation) process of the genome and the growth characteristics of rescued viruses. The results showed that this method eliminates the need to analyze and introduce restriction endonuclease sites, thus avoiding the complexities associated with traditional restriction enzyme-based cloning steps. It offers a simple and rapid operation, enabling more efficient editing of the PCV2 genome. The infectious clones constructed using this method could be successfully rescued through liposome transfection, resulting in the production of recombinant viruses that could be stably passaged. Moreover, the recombinant viruses rescued by this method exhibited enhanced proliferative capacity in PK-15 cells and 3D4/31 cells (immortalized porcine alveolar macrophages). In conclusion, this study has established a novel reverse genetics system for PCV2, providing a new strategy for the development of PCV2 genetic engineering vaccines. Additionally, it serves as a reference for the construction of infectious clones for other emerging circoviruses such as PCV3 and PCV4.

Background: Canine circovirus (CanineCV), a non-enveloped virus with a circular DNA genome, has been identified in various avian and mammalian species, including domestic and wild canids. This study aimed to comprehensively analyze the prevalence of CanineCV across diverse animal species in 11 provinces of China., Results: A total of 1,666 serum samples were collected, revealing a 5.82% prevalence of CanineCV in dogs, with the highest rates being observed in southern and eastern China. Phylogenetic analysis of 266 global CanineCV genomes sourced from the NCBI identified six distinct genotypes, elucidating the complex dynamics of their evolution. Evidence suggested a potential bat origin for CanineCV, with positive selection and high rates of evolution being observed. Recombination analysis revealed dynamic genetic exchange, highlighting the intricate nature of CanineCV evolution. Mutational analysis identified key amino acid substitutions likely to influence the virus's adaptation. Additionally, glycosylation, palmitoylation, and SUMOylation sites were predicted, shedding light on crucial functional properties of the virus., Conclusions: This study provides a global perspective on the origin, genetic diversity, and evolutionary dynamics of CanineCV. Understanding these factors is crucial for elucidating its epidemiology and potential health risks., (© 2024. The Author(s).)

Background: Canine circovirus (CanineCV) is a single-stranded circular DNA virus that infects domestic and wild canids in many countries. CanineCV is associated with gastroenteritis and diarrhea, respiratory disease, and generalized vasculitis leading to a fatal event. The Capsid protein (Cap) is a structural protein of the virus which has high genetic variability and plays a role in the canine immune response. In this study, we cloned the full-length CanineCV Capsid gene (Cap). In-silico analyses were used to explore the genomic and amino acid variability and natural selection acting on the Cap gene. The immune relevance for T-cell and B-cell epitopes was predicted by the immunoinformatic approach., Results: According to the Cap gene, our results showed that CanineCV was separated into five phylogenetic groups. The obtained CanineCV strain from this study was grouped with the previously discovered Thai strain (MG737385), as supported by a haplotype network. Entropy analyses revealed high nucleotide and amino acid variability of the Capsid region. Selection pressure analysis revealed four codons at positions 24, 50, 103, and 111 in the Cap protein evolved under diversifying selection. Prediction of B-cell epitopes exhibited four consensus sequences based on physiochemical properties, and eleven peptide sequences were predicted as T-cell epitopes. In addition, the positive selection sites were located within T-cell and B-cell epitopes, suggesting the role of the host immune system as a driving force in virus evolution., Conclusions: Our study provides knowledge of CanineCV genetic diversity, virus evolution, and potential epitopes for host cell immune response., (© 2024. The Author(s).)

Since PCV4 was first described in 2019, the virus has been identified in several countries in Southeast Asia and Europe. Most studies have been limited to detecting PCV4 by PCR. Thus, PCV4 has an unclear association with clinical disease. This study utilized 512 porcine clinical lung, feces, spleen, serum, lymphoid tissue, and fetus samples submitted to the ISU-VDL from June-September 2023. PCV4 was detected in 8.6% of samples with an average Ct value of 33. While detection rates among sample types were variable, lymphoid tissue had the highest detection rate (18.7%). Two ORF2 sequences were obtained from lymphoid tissue samples and had 96.36-98.98% nucleotide identity with reference sequences. Direct detection of PCV4 by RNAscope revealed viral replication in B lymphocytes and macrophages in lymph node germinal centers and histiocytic and T lymphocyte infiltration in the lamina propria of the small intestine. PCV4 detection was most commonly observed in nursery to finishing aged pigs displaying respiratory and enteric disease. Coinfection with PCV2, PCV3, and other endemic pathogens was frequently observed, highlighting the complex interplay between different PCVs and their potential roles in disease pathogenesis. This study provides insights into the frequency of detection, tissue distribution, and genetic characteristics of PCV4 in the US., (© 2024. The Author(s).)

Pig production is increasing annually in Africa as it is recognized as a significant source of income, livelihood and food security, particularly in rural communities. Understanding the circulating swine pathogens is crucial for the success of this emerging industry. Although there is extensive data available on the African swine fever virus due to its devastating impact on pig production, knowledge about the presence of other viral swine pathogens on the continent is still extremely limited. This review discusses what is currently known about six swine pathogens in Africa: classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus-2, porcine circovirus-3, porcine parvovirus-1, and pseudorabies virus. Gaps in our knowledge are identified and topics of future focus discussed., 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 © 2024 Elsevier B.V. All rights reserved.)

Goose circovirus (GoCV), a potential immunosuppressive virus possessing a circular single-stranded DNA genome, is widely distributed in both domesticated and wild geese. This virus infection causes significant economic losses in the waterfowl industry. The codon usage patterns of viruses reflect the evolutionary history and genetic architecture, allowing them to adapt quickly to changes in the external environment, particularly to their hosts. In this study, we retrieved the coding sequences (Rep and Cap) and the genome of GoCV from GenBank, conducting comprehensive research to explore the codon usage patterns in 144 GoCV strains. The overall codon usage of the GoCV strains was relatively similar and exhibited a slight bias. The effective number of codons (ENC) indicated a low overall extent of codon usage bias (CUB) in GoCV. Combined with the base composition and relative synonymous codon usage (RSCU) analysis, the results revealed a bias toward A- and G-ending codons in the overall codon usage. Analysis of the ENC-GC 3s plot and neutrality plot suggested that natural selection plays an important role in shaping the codon usage pattern of GoCV, with mutation pressure having a minor influence. Furthermore, the correlations between ENC and relative indices, as well as correspondence analysis (COA), showed that hydrophobicity and geographical distribution also contribute to codon usage variation in GoCV, suggesting the possible involvement of natural selection. In conclusion, GoCV exhibits comparatively slight CUB, with natural selection being the major factor shaping the codon usage pattern of GoCV. Our research contributes to a deeper understanding of GoCV evolution and its host adaptation, providing valuable insights for future basic studies and vaccine design related to GoCV., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Porcine circovirus type 3 (PCV3) has become an important pathogen in the global swine industry and poses a threat to pig health, but its pathogenic mechanism remains unknown. In this study, we constructed an innovative, linear infectious clone of PCV3 for rescuing the virus, and explored the transcriptome of infected cells to gain insights into its pathogenic mechanisms. Subsequently, an in vivo experiment was conducted to evaluate the pathogenicity of the rescued virus in pig. PCV3 nucleic acid was distributed across various organs, indicating systemic circulation via the bloodstream and viremia. Immunohistochemical staining also revealed a significant presence of PCV3 antigens in the spleen, lungs, and lymph nodes, indicating that PCV3 had tropism for these organs. Transcriptome analysis of infected ST cells revealed differential expression of genes associated with apoptosis, immune responses, and cellular metabolism. Notably, upregulation of genes related to the hypoxia-inducible factor-1 pathway, glycolysis, and the AGE/RAGE pathway suggests activation of inflammatory responses, ultimately leading to onset of disease. These findings have expanded our understanding of PCV3 pathogenesis, and the interplay between PCV3 and host factors., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Chienjin Huang reports financial support was provided by National Science and Technology Council. If there are other authors, they 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 © 2024 Elsevier Ltd. All rights reserved.)

Avian leukemia virus subgroup J (ALV-J) and chicken infectious anemia virus (CIAV) can be vertically transmitted; however, the pathogenicity of vertically transmitted coinfection with these 2 pathogens has not been studied. In this study, we created a model of chick morbidity in which chicks carried either ALV-J, CIAV, or both viruses via embryo inoculation. Thereafter, we analyzed the effects of vertically transmitted coinfection with CIAV and ALV-J on the pathogenicity of ALV-J and performed a purification assay based on hatching, mortality viremia positivity, and detection of fecal ALV-p27 antigen rates, and body weight. The hatching rate of the ALV-J+CIAV group was 68.57%, lower than those of the single infection and control groups. The survival curve showed that the mortality rates of the CIAV and ALV-J coinfection groups were higher than those of the single infection and control groups. Body weight statistics showed that coinfection aggravated the 7-d growth inhibition effect. The results of ALV-p27 antigen detection in cell culture supernatants showed that the positivity rates of the ALV-J and ALV-J+CIAV groups were 100% at all ages and 0% in the control group. The results of ALV-p27 antigen detection by anal swabs showed that the positivity rates of the ALV-J group were 92.86, 90.90, 88.89, and 93.33% at all ages, and that the ALV-J p27 positivity detection rate of anal swabs was lower than that of plasma virus isolation. The immune organ index of the ALV-J+CIAV group was significantly or very significantly lower than those of the single infection and control groups. The immune organ viral load showed that coinfection with CIAV and ALV-J promoted the proliferation of ALV-J and CIAV in immune organs. Coinfection with ALV-J and CIAV reduced chicken embryo hatchability and increased chick mortality and growth inhibition relative to their respective single infections. Additionally, coinfection with ALV-J + CIAV was even more detrimental in inducing immune organ atrophy (e.g., the thymus, spleen, and bursa), and promoted individual virus replication during coinfection., (Copyright © 2024. Published by Elsevier Inc.)

Porcine circoviruses (PCVs) contain four types: PCV1, PCV2, PCV3, and PCV4, all of which can infect pigs. Among them, PCV1 is non-pathogenic, and PCV2 can cause porcine circovirus diseases (PCVD) or porcine circovirus-associated diseases (PCVAD). Although the pathogenicity of PCV3 and PCV4 is still controversial, increasing evidence shows that PCV3 and PCV4 can cause PCV-related disease. However, mixed infection of PCV2, PCV3, and PCV4 with other pathogens often occurs in large-scale pig breeding, bringing severe economic losses to the global pig industry. In this study, the soluble recombinant proteins of PCV2, PCV3, and PCV4 Cap were expressed by the prokaryotic expression system and biotinylated to combine with the Streptavidin magnetic beads, followed by immunogenicity evaluation of the recombinant proteins. Furthermore, we also assessed the efficacy and immunogenicity of trivalent recombinant proteins conjugated with different adjuvants in mice. The results showed that the highly effective anti-PCV serum was successfully prepared, and the recombinant proteins conjugated with different adjuvants produced various degrees of humoral and cellular immunity in mice. Three recombinant proteins are effective immunogens, and the trivalent proteins coupled with the aluminum adjuvant or GM-CSF-CpG for two-dose immunization can stimulate prominent humoral and cellular immunity against PCVs in vivo. The soluble recombinant proteins are the most promising candidate for developing a trivalent vaccine against PCVs (PCV2, PCV3, and PCV4) infection simultaneously., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. All authors have approved the final version of the manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome in piglets. Differences in the infectivity and horizontal transmissibility of different isolates of PCV2a, PCV2b, and PCV2d in pigs were evaluated by HE and IHC staining, PCR, virus titration, and IPMA to determine their clinical symptoms, pathological changes, levels of virus and antibody, and cohabitation infectivity. In the cohabitation infection experiment, weak viremia and low levels of antibodies were detected in the pigs challenged with PCV2a-CL, whereas no viremia or antibodies were detected in the corresponding cohabiting pigs. Furthermore, no PCV2 was isolated from any organ of pigs that were challenged with PCV2a-CL, as well as from those of their cohabiting pigs. In contrast, persistent viremia and pathological changes, including swollen inguinal lymph nodes, were detected in both the challenged and cohabiting pigs after PCV2b-BY or PCV2d-LNHC infection. Alive PCV2 was detected in the tonsils, inguinal lymph nodes, spleen, and kidneys of the experimental pigs by virus titration, and the highest viral titer was detected in the tonsils, followed by the inguinal lymph nodes. In a comparative analysis of the challenged and cohabiting pigs, a 1-week delay in viremia and specific antibodies was observed in the cohabiting pigs. Moreover, the number of viruses isolated from the tonsils and inguinal lymph nodes of the pigs cohabiting with PCV2d-LNHC-challenged pigs was significantly greater than those in the pigs that were directly challenged with PCV2d-LNHC in cohabitation infection experiment (P<0.05). Together, these results indicated that the infectivity and horizontal transmissibility of the strains PCV2b-BY and PCV2d-LNHC were much greater than those of the strain PCV2a-CL and provided some insights into PCV2 pathogenicity., 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 © 2024 Elsevier B.V. All rights reserved.)

This study was aimed to investigate the frequency of PiCV recombination, the kinetics of PiCV viremia and shedding and the correlation between viral replication and host immune response in young pigeons subclinically infected with various PiCV variants and kept under conditions mimicking the OLR system. Fifteen racing pigeons originating from five breeding facilities were housed together for six weeks. Blood and cloacal swab samples were collected from birds every seven days to recover complete PiCV genomes and determine PiCV genetic diversity and recombination dynamics, as well as to assess virus shedding rate, level of viremia, expression of selected genes and level of anti-PiCV antibodies. Three hundred and eighty-eight complete PiCV genomes were obtained and thirteen genotypes were distinguished. Twenty-five recombination events were detected. Recombinants emerged during the first three weeks of the experiment which was consistent with the peak level of viremia and viral shedding. A further decrease in viremia and shedding partially corresponded with IFN-γ and MX1 gene expression and antibody dynamics. Considering the role of OLR pigeon rearing system in spreading infectious agents and allowing their recombination, it would be reasonable to reflect on the relevance of pigeon racing from both an animal welfare and epidemiological perspective., (© 2024. The Author(s).)

Porcine parvoviruses (PPVs) are among the most important agents of reproductive failure in swine worldwide. PPVs comprise eight genetically different species ascribed to four genera: Protoparvovirus (PPV1, PPV8), Tetraparvovirus (PPV2-3), Copiparvovirus (PPV4-6), and Chaphamaparvovirus (PPV7). In 2016, PPV7 was firstly detected in the USA and afterwards in Europe, Asia, and South America. Recently, it was also identified in Italy in pig farms with reproductive failure. This study aimed to evaluate the circulation of PPV7 in domestic and wild pigs in Sardinia, Italy. In addition, its coinfection with Porcine Circovirus 2 (PCV2) and 3 (PCV3) was analysed, and PPV7 Italian strains were molecularly characterised. PPV7 was detected in domestic pigs and, for the first time, wild pigs in Italy. The PPV7 viral genome was detected in 20.59% of domestic and wild pig samples. PPV7 detection was significantly lower in domestic pigs, with higher PCV2/PCV3 co-infection rates observed in PPV7-positive than in PPV7-negative domestic pigs. Molecular characterisation of the NS1 gene showed a very high frequency of recombination that could presumably promote virus spreading.

Orf virus (ORFV), a member of the genus Parapoxvirus, possesses an excellent immune activation capability, which makes it a promising immunomodulation agent. In this study, we evaluated ORFV as a novel adjuvant to enhance the immune response of mice to a subunit vaccine using porcine circovirus type 2 (PCV2) capsid (Cap) protein as a model. Our results showed that both inactivated and live attenuated ORFV activated mouse bone marrow-derived dendritic cells and increased expression of immune-related cytokines interleukin (IL)-1β, IL-6, and TNF-α. Enhanced humoral and cellular immune responses were induced in mice immunized with PCV2 Cap protein combined with inactivated or live attenuated ORFV adjuvant compared with the aluminum adjuvant. Increased secretion of Th1 and Th2 cytokines by splenic lymphocytes in immunized mice further indicated that the ORFV adjuvant promoted a mixed Th1/Th2 immune response. Moreover, addition of the ORFV adjuvant to the PCV2 subunit vaccine significantly reduced the viral load in the spleen and lungs of PCV2-challenged mice and prevented pathological changes in lungs. This study demonstrates that ORFV enhances the immunogenicity of a PCV2 subunit vaccine by improving the adaptive immune response, suggesting the potential application of ORFV as a novel adjuvant., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gyroviruses are small single-stranded DNA (ssDNA) viruses that are largely associated with birds. Chicken anemia virus is the most extensively studied gyrovirus due to its disease impact on the poultry industry. However, we know much less about gyroviruses infecting other avian species. To investigate gyroviruses infecting waterfowl, we determined six complete genome sequences that fall into three gyrovirus groups, referred to as waterfowl gyrovirus 1 (n = 3), 2 (n = 2), and 3 (n = 1), in organs from hunter-harvested waterfowl from Arizona (USA). The waterfowl gyrovirus 1 variants were identified in multiple organs of a single American wigeon and represent a tentative new species. The waterfowl gyrovirus 2 variants were identified in the livers of two American wigeons and share >70% VP1 nucleotide sequence identity with gyrovirus 9, previously identified in the spleen of a Brazilian Pekin duck (MT318123) and a human fecal sample (KP742975). Waterfowl gyrovirus 3 was identified in a northern pintail spleen sample, and it shares >73% VP1 nucleotide sequence identity with two gyrovirus 13 sequences previously identified in Brazilian Pekin duck spleens (MT318125 and MT318127). These gyroviruses are the first to be identified in waterfowl in North America, as well as in American wigeons and northern pintails., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 10 1 and 1.27 × 10 1 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Human health is dependent on food safety and, therefore, on the health of farm animals. One of the most significant threats in regard to swine diseases is African swine fever (ASF). Infections caused by porcine circoviruses (PCVs) represent another important swine disease. Due to the ubiquitous nature of PCV2, it is not surprising that this virus has been detected in ASFV-affected pigs. However, recent data indicate that coinfection of PCV3 and ASFV also occurs. It is still unclear whether PCV infection plays a role in ASFV infection, and that subject requires further analysis. The aim of this study was to assess whether PCV3 and PCV4 are present in the wild boar population in Poland (real-time PCR). The analysis was performed on wild boar samples collected for routine ASF surveillance in Poland, between 2018 and 2021. By extension, the obtained data were compared in regard to ASFV presence in these samples, thus investigating the odds of ASFV infection on the grounds of the PCV carrier state in free-ranging Suidae in Poland. In addition, sequencing of PCV3 and phylogenetic analysis were performed, based on a full genome and a capsid gene. In the current study, we demonstrated the high prevalence of PCV3 in the wild boar population in Poland; meanwhile, PCV4 was not detected. The odds of ASFV infection on the grounds of the PCV3 carrier state in free-ranging Suidae in Poland was more than twice as high. Ten full genome sequences of PCV3 were obtained, all of them belonging to clade 3a. The similarity between them was in the range of 98.78-99.80%.

Porcine circoviruses (PCVs) are a significant cause of concern for swine health, with four genotypes currently recognized. Two of these, PCV3 and PCV4, have been detected in pigs across all age groups, in both healthy and diseased animals. These viruses have been associated with various clinical manifestations, including porcine dermatitis and nephropathy syndrome (PDNS) and respiratory and enteric signs. In this study, we detected PCV3 and PCV4 in central China between January 2022 and February 2023. We tested fecal swabs and tissue samples from growing-finishing and suckling pigs with or without respiratory and systemic manifestations and found the prevalence of PCV3 to be 15.15% (15/99) and that of PCV3/PCV4 coinfection to be 4.04% (4/99). This relatively low prevalence might be attributed to the fact that most of the clinical samples were collected from pigs exhibiting respiratory signs, with only a few samples having been obtained from pigs with diarrhea. In some cases, PCV2 was also detected, and the coinfection rates of PCV2/3, PCV2/4, and PCV2/3/4 were 6.06% (6/99), 5.05% (5/99), and 3.03% (3/99), respectively. The complete genomic sequences of four PCV3 and two PCV4 isolates were determined. All four of the PCV3 isolates were of subtype PCV3b, and the two PCV4 isolates were of subtype PCV4b. Two mutations (A24V and R27K) were found in antibody recognition domains of PCV3, suggesting that they might be associated with immune escape. This study provides valuable insights into the molecular epidemiology and evolution of PCV3 and PCV4 that will be useful in future investigations of genotyping, immunogenicity, and immune evasion strategies., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

We report the first detection and prevalence of Beak and feather disease virus (BFDV) in Australia's Red Goshawk (Erythrotriorchis radiatus). This is a new host for this pervasive pathogen amongst a growing list of non-psittacine species including birds of prey from the orders Accipitriformes (hawks, eagles, kites), Falconiformes (falcons and caracas), and Strigiformes (owls). The Red Goshawk is the first non-psittacine species listed as Endangered to be diagnosed with BFDV. We report an initial case of infection discovered post-mortem in a dead nestling and subsequent surveillance of birds from across northern Australia. We reveal BFDV prevalence rates in a wild raptor population for the first time, with detections in 25% (n = 7/28) of Red Goshawks sampled. Prevalence appears higher in juveniles compared to adults, although not statistically significant, but is consistent with studies of wild psittacines. BFDV genotypes were associated with the Loriinae (lorikeets, budgerigar, and fig parrots), Cacatuini (Cockatoos), and Polytelini (long-tailed parrots) tribes; species which are preyed upon by Red Goshawks. A positive BFDV status may be associated with lower body mass but small sample sizes precluded robust statistical analysis. We postulate the possible impacts of the virus on Red Goshawks and discuss future research priorities given these preliminary observations., (© 2024. The Author(s).)

Porcine circovirus type 2 (PCV2) infection can cause immunosuppressive diseases in pigs. Vascular endothelial cells (VECs), as the target cells for PCV2, play an important role in the immune response and inflammatory regulation. Endothelial IL-8, which is produced by porcine hip artery endothelial cells (PIECs) infected with PCV2, can inhibit the maturation of monocyte-derived dendritic cells (MoDCs). Here, we established a co-culture system of MoDCs and different groups of PIECs to further investigate the PCV2-induced endothelial IL-8 signaling pathway that drives the inhibition of MoDC maturation. The differentially expressed genes related to MoDC maturation were mainly enriched in the NF-κB and JAK2-STAT3 signaling pathways. Both the NF-κB related factor RELA and JAK2-STAT3 signaling pathway related factors (IL2RA, JAK, STAT2, STAT5, IL23A, IL7, etc.) decreased significantly in the IL-8 up-regulated group, and increased significantly in the down-regulated group. The expression of NF-κB p65 in the IL-8 up-regulated group was reduced significantly, and the expression of IκBα was increased significantly. Nuclear translocation of NF-κB p65 was inhibited, while the nuclear translocation of p-STAT3 was increased in MoDCs in the PCV2-induced endothelial IL-8 group. The results of treatment with NF-κB signaling pathway inhibitors showed that the maturation of MoDCs was inhibited and the expression of IL-12 and GM-CSF at mRNA level were lower. Inhibition of the JAK2-STAT3 signaling pathway had no significant effect on maturation, and the expression of IL-12 and GM-CSF at mRNA level produced no significant change. In summary, the NF-κB signaling pathway is the main signaling pathway of MoDC maturation, and is inhibited by the PCV2-induced up-regulation of endothelial-derived IL-8.

Importance: Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes multisystem disease in pigs and poses a severe threat to the swine industry. However, the mechanisms of how PCV3 uses host proteins to regulate its own life cycle are not well understood. In this study, we found that PCV3 capsid protein interacts with nucleolin and degrades it. Degradation of nucleolin by the PCV3 capsid protein requires recruitment of the enzyme RNF34, which is transported to the nucleolus from the cytoplasm in the presence of the PCV3 capsid protein. Nucleolin also decreases PCV3 replication by promoting the release of interferon β. These findings clarify the mechanism by which nucleolin modulates PCV3 replication in cells, thereby facilitating to provide an important strategy for preventing and controlling PCV3 infection., Competing Interests: The authors declare no conflict of interest.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), is known to induce oxidative stress, activate p53 with induction of cell cycle arrest, and trigger the PERK (protein kinase R-like endoplasmic reticulum kinase) branch of the endoplasmic reticulum (ER) stress pathway. All these cellular responses could enhance PCV2 replication. However, it remains unknown whether PERK activation by PCV2 is involved in p53 signaling with subsequent changes of cell cycle. Here, we demonstrate that PCV2 infection induced cell cycle arrest at S phase to favor its replication via the PERK-reactive oxygen species (ROS)-p53 nexus. PCV2 infection promoted phosphorylation of p53 (p-p53) at Ser15 in porcine alveolar macrophages. Inhibition of PERK by RNA silencing downregulated total p53 (t-p53) and p-p53. Treatment with the MDM2 inhibitor nutlin-3 led to partial recovery of t-p53 in perk -silenced and PCV2-infected cells. perk silencing markedly downregulated ROS production. Scavenging of ROS with N -acetylcysteine (NAC) of PCV2-infected cells downregulated t-p53 and p-p53. Increased accumulation of p-p53 in the nuclei during PCV2 infection could be downregulated by silencing of perk or NAC treatment. Further studies showed that perk silencing or NAC treatment alleviated S phase accumulation and downregulated cyclins E1 and A2 in PCV2-infected cells. These findings indicate that the PCV2-activated PERK-ROS axis promotes p-p53 and contributes to cell cycle accumulation at S phase when more cellular enzymes are available to favor viral DNA synthesis. Overall, our study provides a novel insight into the mechanism how PCV2 manipulates the host PERK-ROS-p53 signaling nexus to benefit its own replication via cell cycle arrest. IMPORTANCE Coinfections or noninfectious triggers have long been considered to potentiate PCV2 infection, leading to manifestation of PCVAD. The triggering mechanisms remain largely unknown. Recent studies have revealed that PERK-mediated ER stress, oxidative stress, and cell cycle arrest during PCV2 infection are conducive to viral replication. However, how PCV2 employs such host cell responses requires further research. Here, we provide a novel mechanism of PCV2-induced ER stress and enhanced viral replication: the PCV2-activated PERK-ROS-p53 nexus increases S phase cell population, a cell cycle period of DNA synthesis favorable for PCV2 replication. The fact that PCV2 deploys the simple ROS molecules to activate p53 to benefit its replication provides novel insights into the triggering factors, that is, certain stimuli or management measures that induce ER stress with subsequent generation of ROS would exacerbate PCVAD. Use of antioxidants is justified on farms where PCVAD is severe.

Porcine Circovirus 2 (PCV2) is a crucial swine pathogen and considered a primary causative agent of porcine circovirus-associated diseases (PCVADs), posing a serious economic threat to the swine industry across globe. The world's biggest agricultural conglomerates have teamed up to create giant commercial pig farms across Shanghai due to the proximity of this region to more affluent lean-pork markets. Since its discovery, PCV2 has displayed extraordinary genetic diversity, and its genome is swiftly evolving through a series of mutations and recombinations. However, limited information on epidemiology, molecular characteristics, vaccine cross-protection, and the co-infection rate of PCV2 with other lethal swine diseases can adversely impact the pig production in the region. To investigate the molecular epidemic characteristics and genetic evolution of PCV2, pigs with doubtful symptoms of PCVADs were sampled from various commercial pig farms with a history of PWMS and/or PDNS across Shanghai from 2014 to 2018. Our results revealed the coexistence of multiple PCV2 genotypes (PCV2b, PCV2e, and PCV2d) among Shanghai pig herds and dominance of PCV2d among them. We also found critical amino acid substitutions in epitope regions of important capsid proteins in PCV2 isolates involved in viral replication and host immune escape. Spotted mutations may favor the prevalence and survival of various PCV2 genotypes despite availability of commercial vaccines. This study also provides insight into the co-infection status of PCV2 with major lethal swine viral diseases such as PPV and PPRSV. Collectively, these investigations will contribute to understanding the molecular epidemiology and evolution of PCV2 across the region.

Porcine circovirus type 2 (PCV2) plays a key role in PCV2-associated disease (PCVAD) etiology and has yielded significant losses in the pig husbandry in the last 20 years. However, the impact of two recently described species of porcine circoviruses, PCV3 and PCV4, on the pork industry remains unknown. The presence of PCV3 has been associated with several clinical presentations in pigs. Reproductive failure and multisystemic inflammation have been reported most consistently. The clinical symptoms, anatomopathological changes and interaction with other pathogens during PCV3 infection in pigs indicate that PCV3 might be pathogenic for these animals and can cause economic losses in the swine industry similar to PCV2, which makes PCV3 worth including in the differential list as a cause of clinical disorders in reproductive swine herds. Moreover, subsequent studies indicate interspecies transmission and worldwide spreading of PCV3. To date, research related to PCV3 and PCV4 vaccine design is at early stage, and numerous aspects regarding immune response and virus characteristics remain unknown.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCVs) are two major viruses that affect pigs. Coinfections between PRRSV and PCV2 are frequently reported in most outbreaks, with clinical presentations involving dyspnea, fever, reduced feed intake, weight loss, and death in fattening pigs. The NADC30-like PRRSV and PCV2d are the main circulating virus strains found in China. This study determines the impact of NADC30-like PRRSV and PCV2d mono-infection and coinfection on the immune system, organ pathology, and viral shedding in five-week-old post-weaned pigs. Pigs were randomly divided into six groups: PBS, PRRSV, PCV2, PRRSV-PCV2 coinfection (co), and PRRSV-PCV2 or PCV2-PRRSV sequential infections. Fever, dyspnea, decreased feed intake, weight loss, and pig deaths occurred in groups infected with PRRSV, Co-PRRSV-PCV2, and PRRSV-PCV2. The viral load was higher in Co-PRRSV-PCV2, PRRSV-PCV2, and PCV2-PRRSV than those mono-infected with PRRSV or PCV2. Additionally, cytokines (IFN-γ, TNF-α, IL-4, and IL-10) produced by pigs under Co-PRRSV-PCV2 and PRRSV-PCV2 groups were more intense than the other groups. Necropsy findings showed hemorrhage, emphysema, and pulmonary adhesions in the lungs of pigs infected with PRRSV. Smaller alveoli and widened lung interstitium were found in the Co-PRRSV-PCV2 and PRRSV-PCV2 groups. In conclusion, PRRSV and PCV2 coinfection and sequential infection significantly increased viral pathogenicity and cytokine responses, resulting in severe clinical signs, lung pathology, and death.

Porcine circovirus 4 (PCV4) was identified as a novel porcine circovirus in China in 2019. To investigate the prevalence and genetic characteristics of PCV2 and PCV4, 133 clinical samples (103 tissue samples and 30 serum samples) were collected from 30 different pig farms in Henan province of China, and a SYBR Green I-based duplex quantitative real-time polymerase chain reaction assay was established to detect PCV2 and PCV4 genomes simultaneously. The complete genome sequences of 20 PCV2 and 6 PCV4 strains from 19 and 6 clinical samples respectively were sequenced and analyzed. The results showed the detection limits of this assay were 80.2 copies/μL for PCV2 and 58.6 copies/μL for PCV4. The detection results of clinical samples revealed the PCV2 positive rate was 63.16% (84/133), the PCV4 positive rate was 33.33% (45/133), and the PCV2 and PCV4 co-infection positive rate was 21.05% (28/133). Among 20 PCV2 strains, 6 belonged to PCV2a, 6 belonged to PCV2b and 8 belonged to PCV2d. Co-infection with JZ1 (PCV2b) and JZ2 (PCV2d) strains was identified in one sample (JZ-1). Eleven putative recombination events were found through the recombination analysis, suggesting that the new PCV2 variant strains had circulated in Henan province, which contributes to our understanding of evolutionary characteristics of PCV2 in China. The possible genotypes of PCV4 strains were determined based on genomic sequences of 6 PCV4 strains in this study and 29 PCV4 reference strains available at GenBank. According to three different phylogenetic trees (ORF1, ORF2 and complete genome), all 35 PCV4 strains were clustered into two major genotypes (PCV4a and PCV4b), and 6 PCV4 strains in this study belonged to PCV4a. Additionally, the functional regions of PCV4 strains were predicted by comparison with other circoviruses, which are conducive to the further study of the biological functions of PCV4 genome., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Canine circovirus is reported in dogs in many countries, including the USA, China and Thailand. It has been detected in healthy dogs and dogs with diarrhea, hemorrhagic gastroenteritis, and vasculitis. It comprises five genotypes and is frequently found as a coinfection with canine parvovirus-2 (CPV-2)., Aim: To characterize canine circovirus genotypes co-circulating with CPV-2 in Vietnam., Method: PCR assessment of 81 CPV-2-positive fecal samples from Vietnamese diarrheic dogs up to seven months of age for other viral enteric pathogens, including canine bocavirus, canine adenovirus, paramyxovirus, canine coronavirus, porcine circovirus-3 and canine circovirus. In addition, eight selected full genome sequences of Vietnamese canine circovirus were analyzed and used for phylogeny., Results: In total 19.8% of samples were found to be positive for canine circovirus. Phylogeny revealed that the Vietnamese canine circovirus strains were clustered in two different genotypes (genotype-1 and -3). The genetic diversity among Vietnamese canine circovirus was 86.0-87.2%. The nucleotide discrepancy among both genotypes altered the deduced amino acid sequence in 14 and ten residues of the replicase and capsid proteins, respectively. Genetic recombination analysis revealed that the Vietnamese canine circovirus-6 strain has the American and Chinese canine circovirus as its major and minor parents, respectively. Only a single dog revealed triple detections of CPV-2c, Canine circovirus and canine adenovirus (1.2%)., Conclusion: The co-circulation of two different genotypes of canine circovirus and CPV-2c in dogs in Vietnam has been illustrated., Clinical Relevance: The mortality rate with CPV-2 only (22%) doubled in dogs with canine circovirus and CPV-2 co-infection.

Porcine circovirus-2 (PCV2) is a widespread virus and presents sub-clinically in most of the swineherd. Globally, eight genotypes of PCV2 have been identified that is PCV2a to 2h. To determine the region-wide genotype distribution of PCV2 infection, with additional reference to indigenous breeds, a total of 1314 pig's clinical samples from the eight states of North-eastern India between 2011 and 2014; were analyzed. The overall prevalence rate of PCV2 in this region was 28.2% (370/1314) by PCR. The state-wise PCR based PCV2 prevalence rate was: Tripura (20.8%), Nagaland (25.0%), Meghalaya (25.8%), Assam (32.1%), Sikkim (32.6%), Manipur (33.3%), Mizoram (36.7%) and Arunachal Pradesh (42.3%). Subsequently, a total of 29 complete genomes of PCV2 were amplified and sequenced from these PCV2 positive samples. The phylogenetic tree represents that the 29 PCV2 isolates of this study were divided into four distinct genetic groups; PCV2a, PCV2b, PCV2d, and PCV2f. Among these, 14 PCV2 strains were classified as PCV2f, 13 classified as PCV2d, and one isolate of each classified as PCV2a and PCV2b. All the 14 PCV2f strains appeared from indigenous pigs of this region. Based on the date of collection, the present study further describes that the PCV2f genotypes circulate in the indigenous pigs' population back in 2011. The amino acid residues and the atomic coordinate structure (3D model) of PCV2f capsid protein represents similarity to PCV2d capsid protein support the efficacy of the existing PCV2 vaccine against the PCV2f. The observation of this study helps to understand the genotype distribution of PCV2 and stands as a reference for future molecular epidemiological studies in North-eastern India., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In recent years, several novel circular single-stranded DNA viruses have been detected in various mammals, birds, insects, and environmental samples using metagenomic and high-throughput sequencing approaches. In this study, we tested for the presence of circoviruses in 243 bat fecal samples collected between 2018 and 2019 from 48 sampling sites across Korea. To detect circoviruses, nested PCR was performed with degenerate primers targeting a conserved replication-associated protein (rep) gene of circovirus/cyclovirus. Among 243 samples tested, a total of 37 fecal samples from 14 sampling sites were PCR-positive for circoviruses at a frequency rate of 15.23%. We obtained 36 partial rep gene sequences of circoviruses and one complete genome sequence of bat-associated circovirus 12, encompassing a genome size of 2097 nt containing two inversely arranged open reading frames and a conserved nonamer sequence in the apex of a stem-loop structure. In addition, we found four bat species that were harboring circoviruses in Korea based on species identification PCR of circovirus-positive bat fecal samples. Detailed sequence analysis indicated that the bat-associated circovirus sequences identified in this study were related to those of known bat and avian groups of circoviruses. Herein, we report evidence for the presence of bat-associated circoviruses in Korean bats., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Circoviridae is a family of circular single-stranded DNA viruses whose members infect a wide variety of hosts. While well characterized in avian and mammalian hosts, little is known about circoviruses associated with Antarctic animals. From 48 Weddell seal (Leptonychotes weddellii) fecal samples collected on the sea ice in McMurdo between Nov 2014 and Dec 2014, we identified and determined the genomes of novel viruses that fall within two genera of the family Circoviridae, i.e. Circovirus (n = 7) and Cyclovirus (n = 45). We named these viruses as werosea circovirus (WerCV) and werosea cyclovirus (WerCyV). The genomes of WerCV and WerCyV share ~63-64% genome-wide pairwise identity with classified circoviruses and cycloviruses, respectively. Based on the species demarcation threshold of 80% for members of the Circoviridae, the genomes of WerCV and WerCyV represent new species in their respective genera. Evidence indicated recombination in five of the 45 WerCyV genomes identified in this study. These are the first circoviruses found associated with Antarctic pinnipeds, adding to those recently identified associated with Adélie (Pygoscelis adeliae) and chinstrap penguins (P. antarcticus)., (Copyright © 2021. Published by Elsevier B.V.)

Porcine circovirus type 2 (PCV2) is the etiological agent of post-weaning multisystemic wasting syndrome (PMWS). The original prevalent genotype, PCV2a, has been replaced by genotypes 2b and 2d in the swine population worldwide. The Rep protein is critical for viral replication. Comparison of a large number of Rep protein amino acid (aa) sequences showed that three sites distinguish genotype 2b from genotype 2d. In order to analyze the effect of exchanging the amino acids (asparagine and serine) at position 6 in the Rep proteins of PCV2b and PCV2d, two wild-type and two mutant viruses were rescued. Real-time quantitative PCR and a one-step growth curve were used to determine the viral load to assess the replication ability of the rescued viruses. The results showed that there was no significant difference in in vitro performance between the wild-type PCV2b and the mutated virus, while the mutation of PCV2d enhanced viral replication., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The members of the family Circoviridae are considered to be one of the smallest autonomously replicating viruses that are classified into two genera, Circovirus and Cyclovirus. Circoviruses have been found in a variety of vertebrates, but whether they infect endangered protected animals has not been studied in much detail. Here, viral metagenomics and PCR methods were used to detect and verify viral nucleic acid in the blood sample from giant pandas. According to these methods, the complete genome sequence of a novel circovirus, the giant panda associated circovirus (GPCV) from the blood sample of three giant pandas was identified. The GPCV genome is 2090 bp in size and reveals two putative ambisense open-reading frames, encoding the major structural capsid protein and the replication associated protein, respectively, the latter having two predicted introns. Pairwise sequence comparison and phylogenetic analyses indicated GPCV was a putative new species within genus Circovirus based on the species demarcation criteria of the International Committee on the Taxonomy of Viruses. It is the first time that circovirus has been identified from blood sample of giant pandas. These efforts will contribute to future analyses to illuminate the evolutionary relationships between classified and newly identified members of the family Circoviridae., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gyrovirus 3 (GyV3) has been identified in humans and other hosts, suggesting its cross-species pathogenicity, which poses an increased public health risk. In the current study, we established chicken and mouse models of GyV3 infection. We found that GyV3 induced persistent infections, characterized by viremia, aplastic anemia, immunosuppression, and systematic lymphocytic inflammation, in both species. Kinetic viral loads and antigen expression demonstrated rapid viral replication and broad tissue tropism of GyV3 in both models. The highest viral loads and the strongest antigen immunostaining were present in bone marrow and cerebrum in both chickens and mice, indicating that these are target tissues for GyV3. Genetic diversity analysis of VP1 in infected chickens and mice showed that GyV3 adapts to new hosts via rapid evolution of the hypervariable region of the gene encoding the structural protein VP1. Overall, our results indicate that GyV3 is a cross-species pathogenic virus; therefore, more attention needs to be paid to high levels of GyV3-induced neurotropism and aplastic anemia as a public health risk., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVAD), causing substantial economic losses to the swine industry worldwide. PCV3, as a recently discovered virus, is associated with porcine dermatitis, nephropathy syndrome, reproductive failure, congenital tremors, and other clinical symptoms. To further investigate the epidemic profile and genetic diversity of the two viruses, a total of 198 samples from swine at various growth stages suspected for PCVAD on 55 different pig farms between 2018 and 2020 were analyzed for presence of PCV2 and PCV3 by using a multiplex real-time PCR assay. Among the 198 samples, 113 (57.07%) and 72 (36.36%) were positive for PCV2 and PCV3 respectively, and 39 (19.7%) were positive for PCV2 and PCV3 co-infection. Subsequently, whole genome sequences of 34 PCV2 and 19 PCV3 strains were obtained from 30 and 19 clinical samples, respectively. Of these, 8 PCV2 strains belonged to PCV2a, 10 belonged to PCV2b and 16 belonged to PCV2d, indicating PCV2d was the predominant PCV2 genotype circulating in central China. Furthermore, co-infection of different PCV2 genotype strains was identified in three samples (JZ-4, KF-2 and JY-1), and a cross-recombination was found in the ORF2 region of the sequenced 13 PCV2d strains whose putative parental strains were LN6/1999 (MF278777) and MEX/41238/2014 (KT795287) strains. The phylogenetic analysis of PCV3 showed high nucleotide identity (>98%) among sequences obtained in this study and reference sequences. These data will aid our understanding of the molecular epidemiology and evolution of PCV2 and PCV3., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) assay based on a dual-labeled hydrolysis probe to simultaneously detect both duck circovirus (DuCV) 1 and DuCV-2. The reproducibility, sensitivity and specificity of the primer set and probe were evaluated using other duck pathogens. The detection limit was 20 copies per µL. The intra-assay coefficients of variation (CVs) were ≤ 0.73% and the inter-assay CVs were ≤ 1.89%. No cross-reaction occurred with other duck pathogens. In addition, the qPCR assay was successfully applied to the simultaneous detection of DuCV-1 and DuCV-2 in clinical field samples. Therefore, this assay will be useful for laboratory diagnosis and epidemiological field studies of DuCV., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus ( Iridoviridea ) and circovirus ( Circoviridea ) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Background: PCV3 is a pathogen associated with porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, and cardiac and multiorgan inflammation, which was newly identified in 2016 in sows in USA. Recently, PCV3 has also been identified from several non-porcine species like (cattle, dog, wild boar, deer, mice and ticks). However, PCV3 infection in donkey is not well established. Since 2019, 300 blood samples were collected from female donkey, which was characterized by abortion and sterility, in Liaocheng city of China., Results: In the present study, an investigation of PCV3 in donkey blood samples was undertaken employing by real time PCR. Positive rates of PCV3 in donkeys reach to 21.0 %. In addition, one full-length PCV3 genome sequence was obtained, and it had a highest identity with porcine circovirus 3 PCV3/CN/Nanjing2017 strain and is clustered to PCV3a genotype based on ORF2 sequences., Conclusions: This is the first report of detection of PCV3 from female donkeys presenting reproductive failure in large-scale donkey farms, China. In addition, the PCV3 strain identified in this study shared the closest relationship with those from porcine, suggesting that PCV3 may be transmitted from pigs to donkeys. Totally, PCV3 infection in donkey should be concerned although the association between it and reproductive failure are not better understood., (© 2021. The Author(s).)

Gyrovirus 3 (GyV3), the third novel emerging species of the genus Gyrovirus of the Anelloviridae family, has been described in multiple hosts. Epidemiologically, there are suggestions that GyV3 is associated with diarrhea/proventriculitis, however, no direct causal evidence exists between GyV3 infection and specific clinical diseases. Herein, we infected special pathogen-free (SPF) chickens with GyV3, and then assessed the pathogenicity and tissue tropism. The results revealed that GyV3 induced persistent infection characterized by diarrhea, aplastic anemia, immunosuppression, and persistent systemic lymphocytic inflammation. Clinically, the infected chickens presented ruffled feathers, diarrhea, anemia, and weight loss. Aplastic anemia was characterized by progressive depletion of hematopoietic cells in the bone marrow, immunosuppression was associated with atrophy of the thymus, spleen, and bursa of Fabricious, progressive lymphocytic inflammations were characterized by proventriculitis, adrenalitis, pancreatitis, hepatitis, nephritis, and bronchitis. Viral loads of GyV3 in tissues exhibited "M", "N", "W" or "V" type dynamic changes. The highest level of viral loads was reported in bone marrow at 7dpi, followed by the adrenal gland at 2 dpi, the sciatic nerve at 7 dpi, and bile at 35 dpi. The bone marrow and kidney demonstrate the strongest immunostaining of GyV3-VP1 antigen and were suggested as the target tissues of GyV3. Collectively, GyV3 is an immunosuppressive pathogenic virus that targets the bone marrow and kidney in chickens. Exploring the pathogenicity and tissue tropism of GyV3 will guide the basic understanding of the biology of GyV3 and its pathogenesis in chickens., (© 2021. The Author(s).)

Immunosuppression can increase the susceptibility of chickens to other disease-causing pathogens and interfere with the efficacy of vaccination against those pathogens. Chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) are common causes of immunosuppression in chickens. Immunosuppression was induced by experimental infection with either CAV or IBDV to assess the effect of immunosuppression on the efficacy of vaccination with Mycoplasma gallisepticum strain ts-304 against infection with virulent M. gallisepticum, a common bacterial pathogen of chickens worldwide. Birds were experimentally infected with either CAV or IBDV at 1 week of age, before vaccination and challenge with M. gallisepticum to examine the effect of immunosuppression at the time of vaccination, or at 6 weeks of age, after vaccination against M. gallisepticum but before challenge with virulent M. gallisepticum, to investigate the effect of immunosuppression at the time of challenge. All birds were vaccinated with a single dose of the ts-304 vaccine at 3 weeks of age and experimentally challenged with the virulent M. gallisepticum strain Ap3AS at 8 weeks of age. In immunosuppressed chickens there was a reduction in protection offered by the ts-304 vaccine at two weeks after challenge, as measured by tracheal mucosal thicknesses, serum antibody levels against M. gallisepticum, air sac lesion scores and virulent M. gallisepticum load in the trachea. Immunosuppressed birds with detectable serum antibodies against M. gallisepticum were less likely to have tracheal lesions. This study has shown that immunosuppression caused by infection with CAV or IBDV can interfere with vaccination against mycoplasmosis in chickens., (Copyright © 2021 Elsevier B.V. All rights reserved.)