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

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

First molecular detection of porcine circovirus type 4 (PCV4) in Malaysia.

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.

Characterization of a novel reporter system for beak and feather disease virus.

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 (Na3VO4). When BFDV-infected birds were treated with Na3VO4, 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.

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

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

Detection of beak and feather disease virus in India and its implications.

Beak and feather disease virus (BFDV) has been found in Oceania, Africa, Asia and Europe, but the virus has not yet been detected in India. Here we are reporting the detection of BFDV in exotic rainbow lorikeets (Trichoglossus haematodus) in India. In the phylogenetic analysis, India's witnessed BFDV complete genome, replication (Rep) and capsid (Cap) sequences were displayed close to previously reported T. haematodus infecting BFDV from Australia. Further, we observed that the Indian and exotic Psittaciformes except T. haematodus housed together with the BFDV infected rainbow lorikeets did not display clinical signs and were negative for 4-month genome detection. This observation raised the suspicion that BFDV could cause host-specific infections. In addition, our phylogenetic analysis using 361 BFDV complete genome sequences from various bird species revealed that they were mainly grouped according to the specific species. Likewise, similarity plot analysis shows that the BFDV complete genome sequences found in T. haematodus are significantly different in areas such as the origin of Rep, the intergenic region between the 3' ends of the Rep and capsid (Cap) genes, and the Cap gene, compared to the BFDVs found in other birds. Furthermore, the BFDV-host coevolution analysis clarifies that the TimeTree of the evolution of various Psittaciformes bird species is the coevolution of the BFDV complete genome/Rep gene/Rep protein/Cap gene/Cap protein sequences found in the respective bird species. To our best knowledge, it is essential to note that no research has yet provided conclusive scientific evidence or experimental evidence that BFDVs detected from Trichoglossus sp. can infect other bird species. Therefore, it can be expected that the BFDVs found in the exotic bird in India will not infect Indian Psittaciformes. However, we hope that large-scale surveillance of BFDV in Indian birds will help determine the BFDV genome present in Indian birds and take further action.

Genetic Variation Analysis of Porcine Circovirus Type 4 in South China in 2019 to 2021.

Porcine circovirus type 4 (PCV4) is a novel virus associated with porcine dermatitis and nephropathy syndrome (PDNS)-like signs identified firstly in China in 2019. However, the details of the molecular epidemiology of PCV4 are unclear at this time. A total of forty-two related sequences were selected from the GenBank database to explore the spread of PCV4 and its rule in genetic evolution. Of the selected strains, 41 were from south China in 2019 to 2021 and the other was a foreign representative strain. Phylogenetic tree construction, nucleotide and amino acid (aa) sequence alignment, gene recombination and antigen structure prediction were performed on the collected sequences using bioinformatics softwares. The 42 PCV4 strains were divided into two subgenotypes: PCV4a (35/42) and PCV4b (7/42), according to the constructed genetic evolution tree. PCV4a is the main epidemic strain, and it can be further divided into two different gene clusters: PCV4a-1 (22/35) and PCV4a-2 (13/35). The pairwise comparison analysis showed that the complete genome sequence similarity of the 42 PCV4 strains ranged between 97.9% and 100%, and the aa sequences of the Cap proteins of 42 PCV4 strains had three major heterogenic or hypervariable regions-27-28, 96 and 212-all located near the antigenic epitope of the Cap protein. The results of this study can provide some basis for further studying the spread and epidemic growth of PCV4, and the prevention and control of PCV4 infection in China.

Comparative transcriptomic analysis reveals that TPX2 and AURXA are involved in porcine PCV2 infection.

Porcine circovirus type 2 (PCV2) has been a notorious killer for the pig industry, causing substantial economic losses worldwide. However, its pathogenesis is still poorly understood. Comparative transcriptomic analysis and weighted gene co-expression network analysis (WGCNA) were performed in different porcine tissues after PCV2 infection. Our comparative transcriptomic analysis obtained 40 key differentially expressed genes (DEGs), and our WGCNA identified 458 hub genes. Significantly, both TPX2 microtubule nucleation factor (TPX2) and Aurora kinase A (AURKA) are included in these key DEGs and hubs genes. Our gene ontology (GO) analysis indicated that the key DEGs and hub genes participated in cell cycle regulation and immune response. The expressive levels of TPX2 and AURKA went down in the spleen but up in the kidneys after infection with PCV2. We conclude that TPX2 and AURKA played an essential role in PCV2 infection.

Synergistic Pathogenicity by Coinfection and Sequential Infection with NADC30-like PRRSV and PCV2 in Post-Weaned Pigs.

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.

Phylogenetic analysis of porcine circovirus 4 in Henan Province of China: A retrospective study from 2011 to 2021.

Porcine circovirus 4 (PCV4), a novel circovirus, was first discovered in April 2019 in Hunan Province of China. At present, PCV4 infection has been detected in China and South Korea. However, until 2019, there was little information about its circulating status and genetic characteristics. To further clarify the origin and prevalence of PCV4, a total of 152 clinical samples collected from 49 different swine farms of 15 cities in Henan Province of China from 2011 to 2021 were tested for the presence of PCV4 by qPCR, and the complete genome of PCV4 strains was amplified from the positive samples and sequenced. Among these samples, 45.39% (69/152) were positive for PCV4 and 86.67% (13/15) of the cities and 67.35% (33/49) of the swine farms were positive for PCV4. The genome sequences of 15 PCV4 strains were obtained, of which two PCV4 strains (HN-ZMD-201212 and HN-XX-201212) were achieved from archival samples in 2012, indicating that PCV4 has been circulating for at least 10 years in Henan Province of China. The phylogenetic analysis showed that 15 PCV4 strains in our study together with PCV4 strain HNU-AHG1-2019 were clustered into an identical but separate evolutionary branch, with genomic identity ranging from 98.2% to 98.8%. Our research further provides significant epidemiological information on PCV4 in China, which will help understand the origin and genetic characteristics of this new virus.

Molecular-based detection, genetic characterization and phylogenetic analysis of porcine circovirus 4 from Korean domestic swine farms.

Porcine circovirus 4 (PCV4), a novel and unclassified member of the genus Circovirus, was first reported in China in 2019. Aiming to provide more evidence about the active circulation of PCV4, this study screened 335 pooled internal organs and detected the virus (i) at a rate of 3.28%, (ii) from both clinically healthy and clinically sick pigs of various age groups, and (iii) in six out of nine provinces of Korea. The complete genomic sequence of the Korean PCV4 strain (E115) was 1,770 nucleotides in length and had 98.5%-98.9% identity to three PCV4 strains currently available at GenBank. Utilizing a set of bioinformatic programs, it was revealed that the Korean PCV4 strain contained several genomic features of (i) a palindrome stem-loop structure with a conserved nonanucleotide, (ii) packed overlapping ORFs oriented in different directions and (iii) two intergenic regions in between genes encoding the putative replication-associated protein (Rep) and capsid (Cap) proteins. This study also predicted the presence of essential elements for the replication of circoviruses in all PCV4 strains, for example the origin of DNA replication, endonuclease and helicase domains of Rep, and the nuclear localization signal on the putative Cap protein. Finally, based on the phylogeny inferred from sequences of the putative Rep protein, this study further clarified the genetic relationships between PCV4 and other CRESS DNA viruses in general and circoviruses in particular.

NOD2 Genotypes Affect the Symptoms and Mortality in the Porcine Circovirus 2-Spreading Pig Population.

The nucleotide oligomerization domain (NOD)-like receptor 2 (NOD2) is an intracellular pattern recognition receptor that detects components of peptidoglycans from bacterial cell walls. NOD2 regulates bowel microorganisms, provides resistance against infections such as diarrhea, and reduces the risk of inflammatory bowel diseases in humans and mice. We previously demonstrated that a specific porcine NOD2 polymorphism (NOD2-2197A > C) augments the recognition of peptidoglycan components. In this study, the relationships between porcine NOD2-2197A/C genotypes affecting molecular functions and symptoms in a porcine circovirus 2b (PCV2b)-spreading Duroc pig population were investigated. The NOD2 allele (NOD2-2197A) with reduced recognition of the peptidoglycan components augmented the mortality of pigs at the growing stage in the PCV2b-spreading population. Comparison of NOD2 allele frequencies in the piglets before and after invasion of PCV2b indicated that the ratio of NOD2-2197A decreased in the population after the PCV2b epidemic. This data indicated that functional differences caused by NOD2-2197 polymorphisms have a marked impact on pig health and livestock productivity. We suggest that NOD2-2197CC is a PCV2 disease resistant polymorphism, which is useful for selective breeding by reducing mortality and increasing productivity.

Development of a dual-labeled, hydrolysis probe-based, real-time quantitative PCR assay for detection of both genotypes of duck circovirus-1 (DuCV-1) and DuCV-2.

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.

Combined Effect of Deoxynivalenol (DON) and Porcine Circovirus Type 2 (Pcv2) on Inflammatory Cytokine mRNA Expression.

A host's immune system can be invaded by mycotoxin deoxynivalenol (DON) poisoning and porcine circovirus type 2 (PCV2) infections, which affect the host's natural immune function. Pro-inflammatory cytokines, IL-1ß and IL-6, are important regulators in the process of natural immune response, which participate in inflammatory response and enhance immune-mediated tissue damage. Preliminary studies have shown that DON promotes PCV2 infection by activating the MAPK signaling pathway. Here, we explored whether the mRNA expression of IL-1ß and IL-6, induced by the combination of DON and PCV2, would depend on the MAPK signaling pathway. Specific pharmacological antagonists U0126, SP600125 and SB203580, were used to inhibit the activities of ERK, JNK and p38 in the MAPK signaling pathway, respectively. Then, the mRNA expression of IL-1ß and IL-6 in PK-15 cells was detected to explore the effect of the MAPK signaling pathway on IL-1ß and IL-6 mRNA induced by DON and PCV2. The results showed that PK-15 cells treated with DON or PCV2 induced the mRNA expression of IL-1ß and IL-6 in a time- and dose-dependent manner. The combination of DON and PCV2 has an additive effect on inducing the mRNA expression of IL-1ß and IL-6. Additionally, both DON and PCV2 could induce the mRNA expression of IL-1ß and IL-6 via the ERK and the p38 MAPK signal pathways, while PCV2 could induce it via the JNK signal pathway. Taken together, our results suggest that MAPKs play a contributory role in IL-1ß and IL-6 mRNA expression when induced by both DON and PCV2.

A 14 bp indel polymorphism in the promoter region is associated with different responses to porcine circovirus type 2 infection by regulating MRC1 transcription.

Mannose receptor, C type 1 (MRC1) is a key factor in regulating the body's immune response to resist pathogen invasions. In this study, mRNA expressions of MRC1 gene in nine porcine organs/tissues were compared between Laiwu (LW) and Yorkshire × Landrace crossbred (YL) pigs prior to and post PCV2 infection. We found that, for pigs uninfected with PCV2, MRC1 mRNA expressions in the lung, spleen, large intestine, small intestine and mesenteric lymph node tissues of LW were significantly higher than those of YL pigs (P < 0.05). After PCV2 infection, MRC1 mRNA levels in the liver, kidney and mesenteric lymph node were significantly increased in LW pigs (P < 0.05); while, significantly decreased in the heart and lung tissues of YL pigs (P < 0.05). The transcriptional activity of porcine MRC1 promoter was further analyzed to investigate the molecular mechanism underlying these expressional differences in response to PCV2 infection. Luciferase assay indicated that a 14 bp indel polymorphism "GTTTTTTTTTTTTT" at the site -864 of MRC1 promoter contributed to the transcriptional activity. The frequency of 14 bp insertion in LW and Dapulian pigs, generally resistant to PCV2 infection, was higher than that in Duroc, Landrace and Yorkshire pigs, which were sensitive to PCV2 infection. The promoter with 14 bp insertion displayed higher MRC1 transcription level both prior to and post PCV2 infection compared with that carrying no insertion in PK15 cells (P < 0.01). The results suggest that this 14 bp indel polymorphism is associated with different responses to PCV2 infection by regulating MRC1 transcription.

Analysis of the beak and feather disease viral genome indicates evidence of multiple introduction events into Saudi Arabia.

Psittacine beak and feather disease (PBFD), caused by beak and feather disease virus (BFDV) is a highly contagious disease in wild and captive psittacine populations and has an almost global presence. However, the BFDV infection in Saudi Arabia remains largely unknown. In the present study, we report the full genome sequence of BFDV strains from Saudi Arabia and its genetic diversity. The complete genome sequences were analyzed for 14 BFDV-infected birds representing 6 psittacine species. The complete genome sequence of BFDV strains was compared with 201 previously reported sequences to evaluate their diversity and possible recombination events, if any. Our analysis revealed that newly sequenced BFDV genomes from Saudi Arabia belonged to six different strains. Phylogenetic analysis suggested that the isolated BFDV genomes were highly recombinant with a high degree of diversity. It is evident from the study that psittacine species in Saudi Arabia are at risk from the spread of BFDV. As per the CITES trade database, about 190,000 parrots have been imported to Saudi Arabia since 1975 over a thousand instances. Presumably, during any of these trade events or unregulated trade of birds has predisposed the introduction of BFDV to Saudi Arabia. Understanding the epidemiology of BFDV is necessitated to address the threat posed by the virus to the psittacine population of Saudi Arabia.

Detection and genetic characterization of porcine circovirus 4 (PCV4) in Guangxi, China.

Porcine circovirus type 4 (PCV4), a novel circovirus, was identified in pigs with serious symptoms, including porcine dermatitis and nephropathy syndrome (PDNS)-like signs, in China in 2019. This study investigated the prevalence and genome diversity of PCV4 in pigs from Guangxi Province, China, between 2015 and 2019. Thirteen of 257 (5.1%) samples were positive for PCV4, 9 of these (69.2%) PCV4-positive samples were coinfected with PCV2 or PCV3, and one PCV4-positive sample was coinfected with both PCV2 and PCV3. Three complete PCV4 genomes shared 36.9-73.8% nucleotide similarity with other representative circovirus genomes. Phylogenetic analysis indicated that PCV4 was most closely related to bat-associated circovirus and mink circovirus. In summary, this is the first epidemiological investigation and evolutionary analysis of PCV4 in Guangxi Province, China, and the results provide insight into the molecular epidemiology of PCV4.

Molecular characteristics of a novel duck circovirus subtype 1d emerging in Anhui, China.

The frequency of infection of duck circovirus (DuCV) in Anhui province, China is not well-characterized. Therefore, in this study, we collected 69 samples from sick ducks and tested them for the presence of DuCV by conventional polymerase chain reaction (PCR) analysis. The complete viral genomes of five DuCV strains from five different cities were randomly selected, amplified via PCR, sequenced, and subjected to recombination analysis. The five DuCV genomes were named as AHAU9, AHAU25, AHAU28, AHAU37, and AHAUHQ. We found that 36.2 % of the ducks were infected with DuCV. The five DuCV strains had genome lengths ranging from 1987 to 1995 nucleotides, with a sequence similarity of 81.8-98.2 %. Among them, AHAU28, AHAU37, and AHAUHQ were closely related to the reference strain YF180403, GX1105 strain, and wd2015028 of DuCV, respectively. AHAU9 and AHAU25 were found to belong to a new DuCV subtype, DuCV-1d. Moreover, recombination analysis showed that the DuCV-1d subtype strains had the same recombination pattern. These results improve the understanding of the frequency of DuCV infection in Anhui province. Our findings may be useful for preventing and controlling the spread of DuCV.

Nuclear transporter karyopherin subunit alpha 3 levels modulate Porcine circovirus type 2 replication in PK-15 cells.

Entering the nucleus is important for Porcine circovirus type 2 (PCV2) replication. Karyopherins (KPNs) mediate the nuclear import of many cytoplasmic proteins. Our previous study showed that KPNA3 is involved in interferon production during PCV2 infection induced by Poly I:C and ISD (Interferon stimulatory DNA). However, it remains unclear whether PCV2 replication is associated with KPNA3. In the present study, knockdown of KPNA3 promoted the replication of PCV2, whereas overexpression of KPNA3 inhibited PCV2 replication in PK-15 cells. Furthermore, KPNA3 knockdown inhibited IRF3 and reduced the expression of antiviral genes including IFN-ß, ISG54, Mx1 and ISG56, while the opposite results were obtained after KPNA3 overexpression. KPNA3 knockdown also promoted p65 nuclear translocation and increased the mRNA expression of IL-10 and IL-1ß. These results suggested that KPNA3 facilitates IRF3 entry into the nucleus and the production of an antiviral response, resulting in PCV2 replication inhibition and blockage of NF-κB signal activation.

The Carboxyl Terminus of the Porcine Circovirus Type 2 Capsid Protein Is Critical to Virus-Like Particle Assembly, Cell Entry, and Propagation.

The capsid protein (Cap) is the sole structural protein and the main antigen of porcine circovirus type 2 (PCV2). Structural loops of the Cap play crucial roles in viral genome packaging, capsid assembly, and virus-host interactions. Although the molecular mechanisms are yet unknown, the carboxyl terminus (CT) of the PCV2 Cap is known to play critical roles in the evolution, pathogenesis, and proliferation of this virus. In this study, we investigated functions of CT. Removal of this loop leads to abrogation of the in vitro Cap self-assembly into virus-like particles (VLPs). Likewise, the mutated virus resists rescue from PK15 cell culture. A conserved PXXP motif in the CT is dispensable for VLP assembly and subsequent cell entry. However, its removal leads to the subsequent failure of virus rescued from PK15 cells. Furthermore, substituting either the PCV1 counterpart or an AXXA for the PXXP motif still supports virus rescue from cell culture but results in a dramatic decrease in viral titers compared with wild type. In particular, a strictly conserved residue (227K) in the CT is essential for VLP entry into PK15 cells, and its mutation to alanine greatly attenuates cell entry of the VLPs, supporting a mechanism for the failure to rescue a mutated PCV2 infectious DNA clone (K227A) from PK15 cell culture. These results suggest the CT of the PCV2 Cap plays critical roles in virus assembly, viral-host cell interaction(s), and virus propagation in vitroIMPORTANCE The carboxyl terminus (CT) of porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to be associated with immunorecognition, alterations of viral titer in swine sera, and pathogenicity. However, the molecular mechanisms underlying these effects remain unknown. In this study, roles of the critical residues and motifs of the CT are investigated with respect to virus-like particle (VLP) assembly, cell entry, and viral proliferation. The results revealed that the positively charged 227K of the CT is essential for both cell entry of PCV2 VLPs and virus proliferation. Our findings, therefore, suggest that the CT should be considered one of the key epitopes, recognized by neutralizing antibodies, for vaccine design and a target for drug development to prevent PCV2-associated diseases (PCVADs). Furthermore, it is important to respect the function of 227K for its role in cell entry if using either PCV2 VLPs for nanoscale DNA/drug cell delivery or using PCV2 VLPs to display a variety of foreign epitopes for immunization.

Assessing circovirus gene flow in multiple spill-over events.

The establishment of viral pathogens in new host environments following spillover events probably requires adaptive changes within both the new host and pathogen. After many generations, signals for ancient cross-species transmission may become lost and a strictly host-adapted phylogeny may mimic true co-divergence while the virus may retain an inherent ability to jump host species. The mechanistic basis for such processes remains poorly understood. To study the dynamics of virus-host co-divergence and the arbitrary chances of spillover in various reservoir hosts with equal ecological opportunity, we examined structural constraints of capsid protein in extant populations of Beak and feather disease virus (BFDV) during known spillover events. By assessing reservoir-based genotype stratification, we identified co-divergence defying signatures in the evolution BFDV which highlighted primordial processes of cryptic host adaptation and competing forces of host co-divergence and cross-species transmission. We demonstrate that, despite extensive surface plasticity gathered over a longer span of evolution, structural constraints of the capsid protein allow opportunistic host switching in host-adapted populations. This study provides new insights into how small populations of endangered psittacine species may face multidirectional forces of infection from reservoirs with apparently co-diverging genotypes.