Abstract
Bovine herpesvirus 1 (BoHV-1) is an important pathogen of domestic and wild cattle responsible for major economic losses in dairy and beef industries throughout the world. Inhibition of viral entry plays a crucial role in the control of BoHV-1 infection and aptamers have been reported to inhibit viral replication. In this study, nine DNA aptamers that target BoHV-1 were generated using systemic evolution of ligands by exponential enrichment. Of the nine candidates, aptamer IBRV-A4 exhibited the highest affinity and specificity for BoHV-1, which bound to BoHV-1 with a Kd value of 3.519 nM and demonstrated the greatest virus binding as shown by fluorescence imaging. The neutralizing ability of aptamer IBRV-A4 was determined using neutralization assays and real time PCR in BoHV-1 infected Madin-darby bovine kidney cells. Virus titration, immunofluorescence and confocal laser scanning microscopy showed virus replication significantly decreased when aptamer IBRV-A4 was added to BoHV-1 infected MDBK cells at 0 and 0.5 hours post-infection, whereas no change was seen when IBRV-A4 was added 2 hours post-infection. This concludes that aptamer IBRV-A4 efficiently inhibits viral entry of BoHV-1 in MDBK cells and is therefore a novel tool for diagnosis and treatment of BoHV-1 infection in cattle.
Highlights
Bovine herpesvirus 1 (BoHV-1) primarily causes infection in the upper respiratory tract (IBR) which is clinically characterized by dyspnea, hyperpyrexia, nasitis and conjunctivitis[1]
In order to maximize the enrichment of ssDNA aptamers binding to BoHV-1, BoHV-1 virus was purified through density gradient centrifugation
The ssDNA aptamers were selected from a random DNA library by systematic evolution of ligands by exponential enrichment (SELEX) using purified BoHV-1 as an immobilized target[13]
Summary
Bovine herpesvirus 1 (BoHV-1) primarily causes infection in the upper respiratory tract (IBR) which is clinically characterized by dyspnea, hyperpyrexia, nasitis and conjunctivitis[1]. Vaccination is the most commonly used method of BoHV-1 control and is effective at reducing clinical symptoms and limiting virus shedding but does not provide complete protection against BoHV-1 transmission within cattle herds[6] This is largely due to the virus’s ability to evade the host immune system and establish latent infection[6,7]. We isolated nine DNA aptamers that target BoHV-1 from a pool of random DNA sequences using SELEX From these nine candidate aptamers, a single aptamer referred to as IBRV-A4, was identified which binds BoHV-1 with high affinity and specificity. Our investigation demonstrated that aptamer IBRV-A4 efficiently inhibits the infectivity of BoHV-1 by blocking viral entry
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