A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

ABSTRACT: Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.

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A broad spectrum monoclonal antibody against porcine circovirus type 2 for antigen and antibody detection

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-09715-0 ◽

2019 ◽

Vol 103 (8) ◽

pp. 3453-3464 ◽

Cited By ~ 4

Author(s):

Liping Huang ◽

Yanwu Wei ◽

Deli Xia ◽

Dan Liu ◽

Hongzhen Zhu ◽

...

Keyword(s):

Monoclonal Antibody ◽

Broad Spectrum ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Antibody Detection ◽

Porcine Circovirus

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Complex Links between Natural Tuberculosis and Porcine Circovirus Type 2 Infection in Wild Boar

BioMed Research International ◽

10.1155/2014/765715 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 9

Author(s):

Iratxe Díez-Delgado ◽

Mariana Boadella ◽

MariPaz Martín-Hernando ◽

José Angel Barasona ◽

Beatriz Beltrán-Beck ◽

...

Keyword(s):

Wild Boar ◽

Body Condition ◽

Natural Infection ◽

Natural Populations ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Frequent Event ◽

Free Ranging

Individuals in natural populations are exposed to a diversity of pathogens which results in coinfections. The aim of this study was to investigate the relation between natural infection with tuberculosis (TB) due to infection by bacteria of theMycobacterium tuberculosiscomplex and porcine circovirus type 2 (PCV2) in free-ranging Eurasian wild boar (Sus scrofa). Apparent prevalence for TB lesions and PCV2 infection was extremely high in all age classes, including piglets (51% for TB; 85.7% for PCV2). Modeling results revealed that the relative risk of young (less than 2 years old) wild boar to test positive to PCV2 PCR was negatively associated with TB lesion presence. Also, an interaction between TB, PCV2, and body condition was evidenced: in wild boar with TB lesions probability of being PCV2 PCR positive increased with body condition, whereas this relation was negative for wild boar without TB lesions. This study provides insight into the coinfections occurring in free-ranging host populations that are naturally exposed to several pathogens at an early age. Using TB and PCV2 as a case study, we showed that coinfection is a frequent event among natural populations that takes place early in life with complex effects on the infections and the hosts.

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Antibody Recognition of Porcine Circovirus Type 2 Capsid Protein Epitopes after Vaccination, Infection, and Disease

Clinical and Vaccine Immunology ◽

10.1128/cvi.00418-10 ◽

2011 ◽

Vol 18 (5) ◽

pp. 749-757 ◽

Cited By ~ 47

Author(s):

Benjamin R. Trible ◽

Maureen Kerrigan ◽

Nicholas Crossland ◽

Megan Potter ◽

Kay Faaberg ◽

...

Keyword(s):

Capsid Protein ◽

Natural Infection ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Reading Frame ◽

Antibody Recognition ◽

Wasting Syndrome ◽

Study Support

ABSTRACTOpen reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (PCVAD), including porcine multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The approach was to react porcine sera with CP polypeptide fragments followed by finer mapping studies using overlapping oligopeptides that covered amino acids 141 to 200. The results showed that vaccinated pigs preferentially recognized only the largest polypeptide fragment, CP(43-233). A subset of experimentally infected pigs and pigs with PDNS showed strong reactivity against a CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175, and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The recognition of CP(169-180) and other polypeptides provides opportunities to devise diagnostic tests for monitoring the immunological effectiveness of vaccination.

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Thermal stability of porcine circovirus type 2 in cell culture

Journal of Virological Methods ◽

10.1016/j.jviromet.2007.07.029 ◽

2008 ◽

Vol 147 (1) ◽

pp. 61-66 ◽

Cited By ~ 24

Author(s):

Mark A. O’Dea ◽

Andrew P. Hughes ◽

Linda J. Davies ◽

Jillian Muhling ◽

Ross Buddle ◽

...

Keyword(s):

Thermal Stability ◽

Cell Culture ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Thermal Stability Of

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

Journal of Virology ◽

10.1128/jvi.00042-20 ◽

2020 ◽

Vol 94 (9) ◽

Cited By ~ 1

Author(s):

Yang Zhan ◽

Wanting Yu ◽

Xiong Cai ◽

Xinnuo Lei ◽

Hongyu Lei ◽

...

Keyword(s):

Cell Culture ◽

Capsid Protein ◽

Molecular Mechanisms ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Cell Entry ◽

Porcine Circovirus ◽

Carboxyl Terminus

ABSTRACT 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 vitro. IMPORTANCE 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.

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Environmental distribution of Porcine Circovirus Type 2 (PCV2) in swine herds with natural infection

Scientific Reports ◽

10.1038/s41598-019-51473-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Gonzalo López-Lorenzo ◽

José Manuel Díaz-Cao ◽

Alberto Prieto ◽

Cynthia López-Novo ◽

Ceferino Manuel López ◽

...

Keyword(s):

Natural Infection ◽

Systemic Disease ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Environmental Distribution ◽

Viral Loads ◽

Swine Farms ◽

Individual Farm

Abstract Porcine circovirus type 2 (PCV2) is the aetiological agent of PCV2-Systemic Disease (PCV2-SD) and PCV2-Subclinical Infection (PCV2-SI). PCV2 is highly resistant to environmental conditions, being able to remain in the farm environment and thus represent a risk for infection maintenance. The aim of this study was to identify, under field conditions, the possible critical points in the environment of non-vaccinated farrow-to-weaning swine farms where PCV2 could accumulate and persist. For that, environmental samples from five swine farms with PCV2-SD or PCV2-SI were taken and analysed by qPCR, including different farm areas, farm personnel and management implements. PCV2 DNA was detected in the environment of all farms (42.9% of positive samples). Overall, the PCV2-SD herd seemed to present more positive samples and higher viral loads than the PCV2-SI herds. At individual farm level, weaning areas appeared to be the most contaminated facilities. In addition, PCV2 was found at high levels in most samples from farm workers, especially work boots, suggesting that they may play a role in within-farm transmission. In addition, PCV2 was detected in areas without animals the like warehouses, offices and farm perimeter. Therefore, this study is helpful to improve measures to reduce within-farm PCV2 dissemination.

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