Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope.

Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.

Structural roles of PCV2 capsid protein N-terminus in PCV2 particle assembly and identification of PCV2 type-specific neutralizing epitope.

Postweaning multisystemic wasting disease (PMWS) in piglets caused by porcine circovirus type 2 (PCV2) is one of the major threats to most pig farms worldwide. Among all the PCV types, PCV2 is the dominant genotype causing PMWS and associated diseases. Considerable efforts were made to study the virus-like-particle (VLP) assembly and the specific PCV2-associated epitope(s) in order to establish the solid foundation for engineered PCV2 vaccine development. Although the N-terminal fragment including Nuclear Localization Signal (NLS) sequence seems important for recombinant PCV2 capsid protein expression and VLP assembly, the detailed structural and functional information regarding this important fragment are largely unknown. In this study, we report crystal structure of PCV2 VLP assembled from N-terminal NLS truncated PCV2 capsid protein at 2.8 Å resolution and cryo-EM structure of PCV2 VLP assembled from full-length PCV2 capsid protein at 4.1Å resolution. Our in vitro PCV2 VLP assembly results show that NLS-truncated PCV2 capsid protein only forms instable VLPs which were easily disassembled in solution, whereas full-length PCV2 capsid protein forms stable VLPs due to interaction between 15PRSHLGQILRRRP27 (α-helix) and 33RHRYRWRRKN42 (NLS-B) in a repeated manner. In addition, our results also showed that N-terminal truncation of PCV2 capsid protein up to 27 residues still forms PCV2 particles in solution with similar size and immunogenicity, while N-terminal truncation of PCV2 capsid protein with more than 30 residues is not able to form stable PCV2 particles in solution, demonstrating the importance of interaction between the α-helix at N-terminal and NLS-B in PCV2 VLP formation. Moreover, we also report the cryo-EM structure of PCV2 VLP in complex with 3H11-Fab, a PCV2 type-specific neutralizing antibody, at 15 Å resolution. MAb-3H11 specifically recognizes one exposed epitope located on the VLP surface EF-loop (residues 128-143), which is further confirmed by PCV1-PCV2 epitope swapping assay. Hence, our results have revealed the structural roles of N-terminal fragment of PCV2 capsid protein in PCV2 particle assembly and pinpointed one PCV2 type-specific neutralizing epitope for the first time, which could provide clear clue for next generation PCV2 vaccine and diagnostic kits development.

Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana.

BACKGROUND: Beak and feather disease virus (BFDV) is an important disease causing agent affecting psittacines. BFDV is highly infectious and can present as acute, chronic or subclinical disease. The virus causes immunodeficiency and is often associated with secondary infections. No commercial vaccine is available and yields of recombinant BFDV capsid protein (CP) expressed in insect cells and bacteria are yet to be seen as commercially viable, although both systems produced BFDV CP that could successfully assemble into virus-like particles (VLPs). Plants as expression systems are increasingly becoming favourable for the production of region-specific and niche market products. The aim of this study was to investigate the formation and potential for purification of BFDV VLPs in Nicotiana benthamiana. METHODS: The BFDV CP was transiently expressed in N. benthamiana using an Agrobacterium-mediated system and plant expression vectors that included a bean yellow dwarf virus (BeYDV)-based replicating DNA vector. Plant-produced BFDV CP was detected using immunoblotting. VLPs were purified using sucrose cushion and CsCl density gradient centrifugation and visualised using transmission electron microscopy. RESULTS: In this study we demonstrate that the BFDV CP can be successfully expressed in N. benthamiana, albeit at relatively low yield. Using a purification strategy based on centrifugation we demonstrated that the expressed CP can self-assemble into VLPs that can be detected using electron microscopy. These plant-produced BFDV VLPs resemble those produced in established recombinant expression systems and infectious virions. It is possible that the VLPs are spontaneously incorporating amplicon DNA produced from the replicating BeYDV plant vector. CONCLUSIONS: This is the first report of plant-made full-length BFDV CP assembling into VLPs. The putative pseudovirions could be used to further the efficacy of vaccines against BFDV.

Structural insight into the type-specific epitope of porcine circovirus type 3.

The recently identified pathogenic Porcine circovirus type 3 (PCV3) may threaten to reduce the pig population dramatically worldwide. In our previous study, a PCV3-specific monoclonal antibody (mAb-1H11) was successfully applied in immune-histochemistry staining and ELISA, which specifically recognize PCV3 capsid protein in PCV3-positive pig tissues. In the present study, we expressed and purified the soluble sole capsid protein of PCV3. The purified capsid protein was capable of self-assembly into virus-like-particles (VLPs), which is validated by transmission electron microscopy and dynamic light scattering assays. Moreover, the epitope of mAb-1H11 was identified in the CD-loop region (a.a. 72-79) on the VLP surface, which is confirmed by PCV2-PCV3 epitope swapping assay. For the first time, we determined the cryo-EM structure of PCV3-VLP at 8.5 Å resolution that reveals the detailed structural information of PCV3-VLP. In our cryo-EM structure, PCV3-VLP is composed of 60 capsid protein subunits assembled with T = 1 icosahedral symmetry. Consistent to our bio-dot Western blot assay, the structural comparison between PCV3 and PCV2 revealed significant structural differences in the surface-exposed loops, including the CD-loop (a.a. 72-79) and the EF-loop (a.a. 109-131). Our work provides a structural framework for engineering future PCV3 vaccine and diagnosis kits development.

First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells.

Porcine circovirus type 3 (PCV3) was first detected in aborted fetuses in 2015 when sows displaying clinical signs that looked like porcine dermatitis and nephropathy syndrome died suddenly. Primary porcine kidney cells were selected for both the isolation and propagation of PCV3 strain SNUVR181115 (GenBank accession number MK503331) as these cells were permissive to PCV3 infection. PCV3 did not produce cytopathic effect on infected monolayers, therefore PCV3 infection was confirmed by in situ hybridization with a PCV3 specific DNA probe. Electron microscopy was used to analyze cell culture for the presence of virus. The intracytoplasmic inclusion bodies contained virus-like particles arranged in paracrystallline arrays on PCV3-infected primary porcine kidney cell. Virus replication peaked at 6th passage yielding titers close to 106 genomic copies of PCV3 per mL. PCV3 strain SNUVR181115 isolated from primary porcine kidney cells was highly conservative and was clustered with the Korean and Chinese strains. These results demonstrated that primary porcine kidney cells are useful for PCV3 isolation and replication.

Evaluation of a novel inactivated vaccine against duck circovirus in muscovy ducks.

Duck circovirus (DuCV), an immunosuppressive pathogen, causes serious damage to waterfowls worldwide. A highly efficient vaccine would play a crucial role in preventing DuCV infections in the waterfowl breeding industry. However, to date, there is a dearth of commercial vaccines owing to the lack of a cell culture system for propagating the requisite virus amounts in vitro. In this study, we isolated DuCVs from Muscovy ducks, helped them proliferate using peripheral blood mononuclear cells (PBMCs), and developed an inactivated vaccine. Muscovy ducks vaccinated with the inactivated vaccine had higher neutralizing antibody titers than the control ducks and higher protection in the challenge experiment (as assessed by weight measurement). Moreover, the inactivated vaccine did not cause feather abnormalities, growth repression, and dwarf syndrome; likewise, lesions and lymphocyte apoptosis in the bursa of Fabricius, spleen, and thymus were not observed. Significantly lower virus shedding from the inactivated vaccine was detected up to 42 days post-inoculation. Together, these results suggest that the inactivated DuCV vaccine can induce a high immune response, is relatively safer for Muscovy ducks, and thus it is a protective vaccine candidates against DuCV infection.

Structural characterization of the PCV2d virus-like particle at 3.3 Šresolution reveals differences to PCV2a and PCV2b capsids, a tetranucleotide, and an N-terminus near the icosahedral 3-fold axes.

Porcine circovirus 2 (PCV2) has a major impact on the swine industry. Eight PCV2 genotypes (a-h) have been identified using capsid sequence analysis. PCV2d has been designated as the emerging genotype. The cryo-electron microscopy molecular envelope of PCV2d virus-like particles identifies differences between PCV2a, b and d genotypes that accompany the emergence of PCV2b from PCV2a, and PCV2d from PCV2b. These differences indicate that sequence analysis of genotypes is insufficient, and that it is important to determine the PCV2 capsid structure as the virus evolves. Structure-based sequence comparison demonstrate that each genotype possesses a unique combination of amino acids located on the surface of the capsid that undergo substitution. We also demonstrate that the capsid N-terminus moves in response to increasing amount of nucleic acid packaged into the capsid. Furthermore, we model a tetranucleotide between the 5- and 2-fold axes of symmetry that appears to be responsible for capsid stability.

Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus.

The capsid protein of PCV2 was expressed by using a recombinant baculovirus with insect Tn5 cells. A large amount of 28-kDa protein was released into the culture medium and self-assembled into PCV2-like particles (PCV2-LPs) with a buoyant density of 1.365 g/cm(3) and a diameter of 20 nm. PCV2-LPs were efficiently expressed, yielding 1 mg of purified particles per 10(7) Tn5 cells. The PCV2-LPs have antigenicity similar to that of authentic PCV2 particles, allowing us to develop a method for sensitively detecting PCV2-specific IgG antibodies. In addition, the PCV2-LPs appeared to be the most promising PCV2 vaccine candidate, by virtue of their potent immunogenicity.

In vitro and in silico studies reveal capsid-mutant Porcine circovirus 2b with novel cytopathogenic and structural characteristics.

Porcine circovirus 2 (PCV2) is an icosahedral, non-enveloped, and single-stranded circular DNA virus that belongs to the family Circoviridae, genus Circovirus, and is responsible for a complex of different diseases defined as porcine circovirus diseases (PCVDs). These diseases - including postweaning multisystemic wasting syndrome (PMWS), enteric disease, respiratory disease, porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure - are responsible for large economic losses in the pig industry. After serial passages in swine testicle (ST) cells of a wild-type virus isolated from an animal with PMWS, we identified three PCV2b viruses with capsid protein (known as Cap protein) cumulative mutations, including two novel mutants. The mutant viruses were introduced into new ST cell cultures for reisolation and showed, in comparison to the wild-type PCV2b, remarkable viral replication efficiency (> 1011 DNA copies/ml) and cell death via necrosis, which were clearly related to the accretion of capsid protein mutations. The analysis of a Cap protein/capsid model showed that the mutated residues were located in solvent-accessible positions on the external PCV2b surface. Additionally, the mutated residues were found in linear epitopes and participated in pockets on the capsid surface, indicating that these residues could also be involved in antibody recognition. Taking into account the likely natural emergence of PCV2b variants, it is possible to consider that the results of this work increase knowledge of Circovirus biology and could help to prevent future serious cases of vaccine failure that could lead to heavy losses to the swine industry.

Optimized conditions for preserving stability and integrity of porcine circovirus type2 virus-like particles during long-term storage.

Although porcine circovirus type 2 (PCV2) virus-like particles (VLPs) have been successfully harvested from various protein expression systems, conditions to promote their stability and integrity during long-term storage have not been well defined since only the intact VLPs, instead of the monomeric capsid protein (Cap), can induce neutralizing antibodies in pigs in previous studies. In this study, freshly prepared PCV2 VLPs were stored in several media (various concentrations of NaCl, sorbitol, sucrose and trehalose) at three temperatures (4°C, -20°C and -80°C) and their stability and integration were evaluated after 7 month. Addition of 15% trehalose in storage buffer promoted long-term preservation of PCV2 VLPs. In contrast, storage buffer with 5% osmolytes (sucrose, trehalose and sorbitol) did not confer stabilization for long-term storage. These refined storage conditions for stabilization of PCV2 VLPs should enhance their use in vaccines.

Renal tubular necrosis and interstitial hemorrhage ("turkey-egg kidney") in a circovirus-infected Yorkshire cross pig.

A juvenile Yorkshire cross pig with rapidly progressive acute renal failure was submitted for necropsy. There was marked edema and disseminated petechiation of both kidneys, producing the "turkey-egg" appearance that is characteristic of exotic diseases such as African and classical swine fever. Microscopic findings included renal tubular epithelial necrosis with extensive interstitial edema and hemorrhage; lymphoplasmacytic, eosinophilic, and histiocytic tubulointerstitial nephritis; and numerous botryoid intracytoplasmic inclusions within the renal tubular epithelium and interstitial macrophages. Porcine circovirus 2 (PCV2) was readily identified within these lesions by both PCV2-specific immunohistochemistical staining and electron microscopy. Tests for African and classical swine fever viruses, as well as bacterial cultures, were negative. The striking renal lesions in this pig were attributed to PCV2 infection and are distinct from those that are typical of other PCV2-associated diseases.

A retrospective study of circovirus infection in pigeons: nine cases (1986-1993).

Circovirus infections were diagnosed in 12 pigeons from the United States 4 pigeons from Australia, and 1 pigeon from Canada (1986-1993). Circovirus was identified by electron microscopic examination of basophilic botryoid cytoplasmic inclusions that had a histologic appearance similar to that of psittacine beak and feather disease virus inclusions. Inclusions were seen in splenic, bursal, gut-associated, and bronchus-associated lymphoid tissue macrophages and in bursal epithelial cells. Inclusions were composed of paracrystalline arrays of tightly packed, nonenveloped icosahedral virions 14-17 nm in diameter. Histologic changes in the spleens ranged from lymphofollicular hyperplasia with mild discrete lymphocellular necrosis to lymphoid depletion and diffuse histiocytosis. Lesions in the bursa of Fabricius ranged from mild lymphocellular necrosis to severe cystic bursal atrophy. Remaining histologic findings coincided with concurrent bacterial, viral, fungal, and parasitic infections. Immunoperoxidase staining and DNA in situ hybridization demonstrated that pigeon circovirus is distinct from psittacine beak and feather disease virus; however both viruses apparently share some homologous DNA sequences. Clinical and diagnostic findings indicate that pigeon circovirus may be similar to psittacine beak and feather disease virus with respect to acquired immunodeficiency and subsequent multiple secondary infections.

Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe.

Samples of lung, liver, kidney, pancreas, spleen, and lymph node from pigs with postweaning multisystemic wasting syndrome from California (USA) and samples of mesenteric lymph nodes from similarly diseased pigs from Brittany (France) were examined by light microscopy, in situ hybridization (ISH), and/or virus isolation. Whole genomic probes for porcine circovirus (PCV) and chicken anemia virus (CAV) were used for ISH. Tissue homogenate supernatants were inoculated onto PK/15 cells for virus isolation, and the presence of viral antigen and viral particles was verified by indirect immunofluorescence, ISH, and electron microscopy. Histologic examination of lung from pigs from California revealed interstitial pneumonia, alveolar epithelial hyperplasia, and basophilic nuclear and cytoplasmic inclusions in mononuclear cell infiltrates and various pulmonary epithelial cells. Granulomatous lymphadenitis with syncytial cells typified the lesions seen in the pigs from France. PCV-like nucleic acid was detected by ISH in lung, pancreas, lymph node, kidney, and liver in pigs from California. Positive signal was also obtained in lymph node sections from pigs from France. Probes for CAV were consistently negative. PK/15 cell cultures inoculated with lung preparations from diseased California pigs and mesenteric lymph node preparations from pigs from France had positive fluorescence by indirect staining for PCV using pooled polyclonal pig sera and hyperimmune rabbit serum and had variable staining with a panel of 7 monoclonal antibodies specific for cell culture contaminant PCV. PCV-like nucleic acid was also detected by ISH in cell cultures. Cytopathic effect was not observed. Electron microscopic examination of inoculated cell cultures revealed 17-nm viral particles morphologically consistent with PCV. No other virus particles were observed. Although genomic analysis for the definitive identification of these viral isolates remains to be done, the evidence provided strongly suggests that these tissue isolates are closely related to, although antigenically distinct from, the original PCV cell culture contaminant.

Particles resembling circovirus in the bursa of Fabricius of pigeons.

Histological examination of the bursae from 12 pigeons under 4 months old revealed basophilic globular inclusion bodies, 5 to 25 microns in diameter, in the cytoplasm and the nuclei of the various bursal follicular cells. Electron microscopy of these inclusions revealed large electron-dense areas containing non-enveloped icosahedral viral particles, 14-19 nm in diameter, either loosely arranged or in paracrystalline array. Similar basophilic globular inclusion bodies were seen in the spleen and cecal tonsils of a few pigeons and in the duodenum of one pigeon. There were various degrees of lymphoid depletion in the bursa, spleen, and bone marrow. The morphology of the inclusions in the bursa and size of the viral particles are most consistent with circovirus. Preliminary studies on the bursae of two pigeons were negative for psittacine beak and feather disease (PBFD) viral antigen and nucleic acid by immunoperoxidase staining, DNA in situ hybridization, and polymerase chain reaction techniques, suggesting that this virus differs from PBFD virus. Most of the pigeons had concurrent infections such as paramyxovirus-1, salmonellosis, herpesvirus, and hepatic and cerebral trichomoniasis associated with adenovirus.

Psittacine beak and feather disease in three captive sulphur-crested cockatoos (Cacatua galerita) in Thailand.

Three sulphur-crested cockatoos (Cacatua galerita) were diagnosed as psittacine beak and feather disease (PBFD). Histopathology of the feather pulp and follicles showed intracytoplasmic botryoid clusters or granular inclusion bodies in epithelial cells and macrophages. Electron microscopy revealed multiple cytoplasmic clusters of electron dense viral particles corresponding to the inclusions. PBFD virus (circovirus) DNA-specific product was detected from formalin-fixed paraffin-embedded feathers by nested polymerase chain reaction (PCR) method.

Electron microscopical observations of psittacine beak and feather disease in an Umbrella cockatoo (Cacatua alba).

Psittacine beak and feather disease (PBFD) was diagnosed in an umbrella cockatoo (Cacatua alba) with severe feather dystrophy and loss. Electron microscopically, the intranuclear and intracytoplasmic inclusion bodies observed by light microscopy were composed of viral particles forming paracrystalline arrays, whorls, semicircles or concentric circles. Recovered viral particles from the skin and feather follicle tracts were icosahedral and 15 to 20 nm in diameter.

Characterization of the recombinant proteins of porcine circovirus type2 field isolate expressed in the baculovirus system.

Porcine circovirus (PCV) type2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS. The presence of the virus was identified by PCR using primers specific to PCV type2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immunoblotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells.

A novel porcine circovirus-like agent P1 is associated with wasting syndromes in pigs.

A novel porcine pathogen tentatively named P1, which was obtained from the sera of the pigs exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS) experimentally caused the classical clinic signs and pathologic lesions of the disease in pigs by direct in vivo injection with P1 DNA plasmids. Twenty colostrum-fed (CF) pigs that were free of PCV2 and P1 at 1 month of age were randomly designated equally to two groups. Group 1 pigs were each injected with 400 µg of the cloned P1 plasmid DNA into the superficial inguinal lymph nodes and Group 2 were injected with same amount of the empty pSK vector DNA and served as controls. Viremias were positively detected in 8 of 10 P1 infected pigs from 14-21 days post-inoculation (dpi). The 8 infected animals showed pallor of skin and diarrhea. Gross lesions in the pigs euthanized on 35 dpi were similarly characterized by encephalemia, haemorrhage of the bladder mucosa, haemorrhage of the superficial inguinal lymph nodes, lung atrophy and haemorrhage. Histopathological lesions were arteriectasis and telangiectasia of the cavitas subarachnoidealis, interstitial pneumonia, mild atrophy of the cardiac muscle cells, histiocytic hyperplasia of the follicles in the tonsils, and haemorrhage of the inguinal lymph nodes. P1 DNA and antigens were confirmed by PCR and immunohistochemistry in the tissues and organs of the infected pigs, including the pancreas, bladders, testicles/ovaries, brains, lungs and liver. There were no obvious clinical signs and pathological lesions in the control pigs. This study demonstrated that P1 infection is one of the important pathologic agents on pig farms.

Porcine circovirus type 2 morphogenesis in a clone derived from the l35 lymphoblastoid cell line.

Porcine circovirus type 2 (PCV2) is the essential infectious agent of post-weaning multisystemic wasting syndrome (PMWS), one of the most important diseases of swine. Although several studies have described different biological properties of the virus, some aspects of its replication cycle, including ultrastructural alterations, remain unknown. The aim of the present study was to describe for the first time a complete morphogenesis study of PCV2 in a clone of the lymphoblastoid L35 cell line at the ultrastructural level using electron microscopy techniques. Cells were infected with PCV2 at a multiplicity of infection of 10 and examined at 0, 6, 12, 24, 48, 60 and 72h post-infection. PCV2 was internalized by endocytosis, after which the virus aggregated in intracytoplasmic inclusion bodies (ICIs). Subsequently, PCV2 was closely associated with mitochondria, completing a first cytoplasmic phase. The virus entered the nucleus for replication and virus assembly and encapsidation occurred with the participation of the nuclear membrane. Immature virions left the nucleus and formed ICIs in a second cytoplasmic phase. The results suggest that at the end of the replication cycle (between 24 and 48h), PCV2 was released either by budding of mature virion clusters or by lysis of apoptotic or dead cells. In conclusion, the L35-derived clone represents a suitable in-vitro model for PCV2 morphogenesis studies and characterization of the PCV2 replication cycle.