Within the last two decades, the emergence and global impact of three highly pathogenic coronaviruses of zoonotic origin have compelled rapid attention to anthropogenic drivers of disease evolution and the prevention of potential future zoonotic outbreaks. Numerous zoonotic viruses could lead to the next pandemic after the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic.
Camelids are the sole survivors of the mammalian suborder Tylopoda, which first appeared 40 million years ago. The Camelidae are a family of superbly adapted animals that live in particular ecological niches with harsh temperatures, little foliage, food, and water. Camelini (Old World Camels) is comprised of only one genus and two species. Llama (Lama glama), Guanaco (Lama guanicoe), Vicugna (Vicugna vicugna), and Alpaca (Alpaca vicugna) are the four species of the Lamini (New World camelids). The Camelidae family has a remarkable level of conservation, with all extant individuals having the same chromosome number and possessing distinct heavy-chain antibodies.
In a recent study published in the Developmental & Comparative Immunology journal, the discovery of a novel form of mammalian interferon-Beta (IFN-β) with four cysteine residues from the dromedary camel and the five remaining members of the mammalian suborder Tylopoda is described. Following infection with the camelpox virus and stimulation with the viral mimic poly, the expression of IFN-β mRNA was examined.
In vitro, recombinant dromedary IFN-β generated in a prokaryotic expression system increased the expression of IFN-responsive genes in camel kidney cells and provided antiviral resistance to camelpox virus. Camelid IFNs are anticipated to be important in the fight against new zoonotic diseases found in camelids.
Study: Beta interferons from the extant camelids: Unique among eutherian mammals. Image Credit: Rita_Kochmarjova / Shutterstock
The authors developed polymerase chain reaction (PCR) primers around the potential IFNB gene coding region. They cloned and sequenced a 646 basepair (bp) fragment from dromedary camel genomic DNA using CdIFNbNF and CdIFNbNR primers. A 561-bp Open Reading Frame (ORF) encoding a 186-amino-acid protein was found in the cloned fragment. The dromedary camel IFN-β, when compared to other mammalian models, was shown to share identity with porcine (83.2%), human (77.4%), and murine (62%) orthologs.
The authors amplified and cloned a 1274 bp fragment from a DraI-digested genomic DNA library using gene walking. The IFNB CDS was 108 bp long, while the 5' regulatory region was 1166 bp long, upstream of the start codon. A TATA box was discovered 110 kilobases (kb) upstream of the start codon in this upstream regulatory region. The Virus-Responsive Elements (VRE) containing all four positive regulatory domains (PRDs) I, II, III, and IV were also discovered in the camel IFNB promoter region upstream of the TATA box.
To express the protein as an N-terminal His6-tagged protein, the mature protein-coding sequence of the dromedary IFN- was cloned into the pET28a vector. The coding sequence of the mature IFN protein includes sixteen E.coli uncommon codons, which could inhibit recombinant expression in E.coli. To augment the limiting uncommon codons, the authors performed recombinant expression in both BL21 and Rosetta 2 E.coli that contained a plasmid. With uncommon codon supplementation, the recombinant protein was expressed better in the Rosetta 2 strain of E.coli.
While inclusion bodies generated the most recombinant protein, the recombinant protein was determined to be functionally inert following purification and in vitro refolding. As a result, the authors used a lower temperature to induce the recombinant protein in Rosetta2 E.coli. In addition, the authors used affinity chromatography to obtain purified protein which was confirmed via Western blot prior to its use in in vitro assays, even though the amount of protein expressed in the sample extract was lower even in low-temperature induction.
The authors used the Camelpox virus (CMLV) and camel kidney cell lines to test the antiviral effectiveness of dromedary IFN-β in vitro. IFN-β treated cells were resistant to infection from CMLV. When compared to the control protein treated or untreated control cells infected with CLMV, the IFN-β and IFN-α1 treated cells displayed no cytopathic effects. They employed real-time qPCR to assess the decrease of CMLV DNA after IFN-β treatment to measure the antiviral activity. In comparison to untreated cells or cells treated with His6-tagged control protein, cells pre-treated with IFN-β had less CMLV DNA.
The IFNB gene was cloned and identified in this study from the dromedary camel and all other existing Camelidae members. Camelid IFN-β is the only eutherian mammal with two pairs of cysteines in the mature proteins. Antiviral resistance to the cytopathic effects of the camelpox virus was established in camel kidney cells treated with prokaryotically generated recombinant dromedary IFN-β. IFN-β from this distinct cohort of mammals will aid in the understanding of antiviral immunity mechanisms and the development of antivirals against diseases that could cause the next zoonotic pandemic.