Library Dissertation Showcase

Construction of a transfer bacmid to express the elephant endotheliotropic herpesvirus (EEHV1) portal capsid protein (U76) in insect cells using recombinant baculoviruses

  • Year of Publication:
  • 2022

Elephant endotheliotropic herpesvirus (EEHV) causes highly fatal haemorrhagic disease in juvenile elephants, with cases reported worldwide. Since its discovery in 1995, more than 100 juvenile elephants have died from EEHV. An efficacious treatment and vaccination strategy is severely lacking as In vitro isolation and propagation of the virus have been unsuccessful, which make it hard to understand the biology of EEHV and to develop treatments. There is a lack of studies available on the molecular mechanisms of EEHV. Using a Baculovirus Expression System to study EEHV would potentially reduce the number of deaths in the elephant population by helping to develop new control strategies. The portal protein U76 of elephant endotheliotropic herpesvirus type 1 (EEHV1) is believed to play an essential role in capsid assembly, which is a potential target for antiviral drugs. This study aimed to amplify and clone the U76 gene into a pFastBac1 plasmid using a NEBuilder HIFI DNA assembly cloning kit to in the future express the protein in vitro without the need for a competent cell replication system. After generation of the recombinant plasmid, the pFastBac1-U76 EEHV1 clone was sequenced to identify the DNA template genotype and correct insertion of U76 within the bacmid. PCR confirmed correct amplification and cloning of the U76 gene. Sequencing identified that the viral template was from an EEHV1A genotype source and that there was a mutation in the inserted U76 gene, which had an impact on the amino acid sequence (D314N). Despite a mutation, this report presents an efficient strategy for EEHV gene expression. Future studies on the expression and the assembly of different capsid proteins such as U76, using a Baculovirus Expression System, are needed to investigate EEHV capsid formation and molecular biology. It would help identify the potential use of these proteins in developing future treatments.

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