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Identification and characterization of DNA aptamers specific to VP2 protein of canine parvovirus.

ABSTRACT: Canine parvovirus-2 (CPV-2) is ubiquitously distributed in dog population worldwide causing a severe and often fatal gastroenteritis. Owing to its highly contagious nature, rapid detection of CPV is crucial in effective control of the disease. Aptamers have emerged as potential alternative to antibodies as affinity reagents in diagnostic field. Present study was aimed to select and validate ssDNA aptamers specific to CPV. Systematic evolution of ligands through exponential enrichment (SELEX) method was employed for selection of CPV structural protein (VP2) specific DNA aptamers. SELEX was performed using a pool of ssDNA library comprising 30 random nucleotide region. A total of seven rounds of SELEX were performed using VP2 protein as target antigen which yielded ten aptamers (1A-10A) with distinct sequences. Target binding of all ten aptamers was assessed by dot blot and enzyme-linked oligonucleotide assay (ELONA) which revealed that 5A, 6A, 9A, and 10A were superior binders. In silico analysis of the aptamers revealed the existence of binding site on VP2 protein, and binding pattern was similar to in vitro findings. The affinity (K_D) of all these four binders against CPV was evaluated by ELONA indicating relatively higher affinity of 6A and 10A than remaining two DNA sequences. Out of which, aptamer 6A displayed cross-reactivity with canine distemper virus and canine corona virus. Hence, aptamer 10A was considered as better binding sequence having high specificity and affinity for CPV. The study confirms the future utility of selected aptamers in development of a reliable diagnostic for rapid detection of CPV. KEY POINTS: • Canine parvovirus-specific ssDNA aptamers were identified with nanomolar affinity (100-150 nM). • Three aptamers displayed negligible cross-reactivity with other related viruses. • Aptamer 10A displayed high binding affinity and specificity to CPV.

SUBMITTER: Singh M

PROVIDER: S-EPMC8553593 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Identification and characterization of DNA aptamers specific to VP2 protein of canine parvovirus.

Singh Mithilesh M Tripathi Pranav P Singh Smriti S Sachan Manisha M Chander Vishal V Sharma Gaurav Kumar GK De Ujjwal Kumar UK Kota Sathish S Putty Kalyani K Singh Raj Kumar RK Nara Seema S

Applied microbiology and biotechnology 20211029 23

Canine parvovirus-2 (CPV-2) is ubiquitously distributed in dog population worldwide causing a severe and often fatal gastroenteritis. Owing to its highly contagious nature, rapid detection of CPV is crucial in effective control of the disease. Aptamers have emerged as potential alternative to antibodies as affinity reagents in diagnostic field. Present study was aimed to select and validate ssDNA aptamers specific to CPV. Systematic evolution of ligands through exponential enrichment (SELEX) met ...[more]

PMID: 34714365

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Project description:BackgroundPorcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. Interleukin-6 (IL-6) has the roles to support host immune response to infections as a pleiotropic cytokine. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV.MethodsThe recombinant plasmid, pGVP2-IL6, was constructed by porcine IL-6 gene substituting for EGFP gene of the PRV transfer plasmid pGVP2-EGFP containing VP2 gene of PPV. Plasmid pGVP2-IL6 was transfected into swine testicle cells pre-infected with the virus rPRV-VP2-EGFP strain through homologous recombination and plaque purification to generate a recombinant virus rPRV-VP2-IL6. The recombinant PRV was further identified by PCR and DNA sequencing, and the expression of the VP2 protein and porcine IL-6 was analyzed by reverse transcription-PCR (RT-PCR) and Western blot. The virus titer was calculated according to Reed and Muench method. The immunogenicity of the recombinant virus was preliminarily evaluated in mice by intramuscular administration twice with the rPRV-VP2-IL6 at 4-week intervals.ResultsA recombinant virus rPRV-VP2-IL6 was successfully constructed and confirmed in this study. The properties of rPRV-VP2-IL6 were similar to the parental virus HB98 in terms of growth curve, morphogenesis and virus plaque sizes, and rPRV-VP2-IL6 was proliferated in different cell types. It induced specific antibodies against PPV as well as a strong increase of PPV-specific lymphocyte proliferation responses in mice immunized with rPRV-VP2-IL6, and provided partial protection against the virulent PPV challenge. rPRV-VP2-IL6 also induced a high level of neutralizing antibodies against PRV, and significantly reduced the mortality rate of (1 of 10) following virulent PRV challenge compared with the control (10 of 10).ConclusionsThe recombinant rPRV-VP2-IL6 might be a potential candidate vaccine against PRV and PPV infections in pigs.

Dataset Information

Identification and characterization of DNA aptamers specific to VP2 protein of canine parvovirus.

Publications

Identification and characterization of DNA aptamers specific to VP2 protein of canine parvovirus.

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