Bovine prion diseases are composed of two types of bovine spongiform encephalopathy (BSE), classical BSE and atypical BSE. Recent studies have identified one case of atypical BSE with an E211K mutation. E211K is homologous to the human E200K mutation, which is related to familial Creutzfeldt-Jakob disease (CJD), one of the familial forms of human prion diseases. To date, familial forms of prion diseases have not been reported in non-human animals. Because the familial forms of human prion diseases account for more than 10% of all human prion disease cases, the detection of the E211K mutation in healthy cattle is very important for verifying the role of this mutation as a familial form of BSE. To detect putative mutations related to familial BSE, specifically E211K in Korean native cattle (Hanwoo) and Korean dairy cattle (Holstein), we performed direct sequencing targeting codon 211 and the adjacent regions of the bovine prion protein (PRNP) gene in 384 Hanwoo and 152 Holstein cattle. We did not find the E211K mutation in any of the Korean cattle. Although we did not find the E211K mutation in Korean native cattle, E211K is a postulated mutation; therefore, further screening in other countries and larger samples is highly desirable.
Prion protein is encoded by the prion protein gene (PRNP). Polymorphisms of several members of the prion gene family have shown association with prion diseases in several species. Recent studies on a novel member of the prion gene family in rams have shown that prion-related protein gene (PRNT) has a linkage with codon 26 of prion-like protein (PRND). In a previous study, codon 26 polymorphism of PRND has shown connection with PRNP haplotype which is strongly associated with scrapie vulnerability. In addition, the genotype of a single nucleotide polymorphism (SNP) at codon 26 of PRND is related to fertilisation capacity. These findings necessitate studies on the SNP of PRNT gene which is connected with PRND. In goat, several polymorphism studies have been performed for PRNP, PRND, and shadow of prion protein gene (SPRN). However, polymorphism on PRNT has not been reported. Hence, the objective of this study was to determine the genotype and allelic distribution of SNPs of PRNT in 238 Korean native goats and compare PRNT DNA sequences between Korean native goats and several ruminant species. A total of five SNPs, including PRNT c.-114G > T, PRNT c.-58A > G in the upstream of PRNT gene, PRNT c.71C > T (p.Ala24Val) and PRNT c.102G > A in the open reading frame (ORF) and c.321C > T in the downstream of PRNT gene, were found in this study. All five SNPs of caprine PRNT gene in Korean native goat are in complete linkage disequilibrium (LD) with a D’ value of 1.0. Interestingly, comparative sequence analysis of the PRNT gene revealed five mismatches between DNA sequences of Korean native goats and those of goats deposited in the GenBank. Korean native black goats also showed 5 mismatches in PRNT ORF with cattle. To the best of our knowledge, this is the first genetic research of the PRNT gene in goat.
Interferon-induced transmembrane protein 3 (IFITM3) plays a pivotal role in antiviral capacity in several species. However, to date, investigations of the IFITM3 protein in cattle have been rare. According to recent studies, interspecific differences in the IFITM3 protein result in several unique features of the IFITM3 protein relative to primates and birds. Thus, in the present study, we investigated the bovine IFITM3 protein based on nucleotide and amino acid sequences to find its distinct features. We found that the bovine IFITM3 gene showed a significantly different length and homology relative to other species, including primates, rodents and birds. Phylogenetic analyses indicated that the bovine IFITM3 gene and IFITM3 protein showed closer evolutionary distance with primates than with rodents. However, cattle showed an independent clade among primates, rodents and birds. Multiple sequence alignment of the IFITM3 protein indicated that the bovine IFITM3 protein contains 36 bovine-specific amino acids. Notably, the bovine IFITM3 protein was predicted to prefer inside-to-outside topology of intramembrane domain 1 (IMD1) and inside-to-outside topology of transmembrane domain 2 by TMpred and three membrane embedding domains according to the SOSUI system.
Prion disease is a fatal neurodegenerative disease with a broad host range in humans and animals. It is caused by proteinase K-resistant prion protein (PrPres). In previous studies, a heterogeneous infection in Cervidae and Caprinae was reported. Chronic wasting disease (CWD) has been frequently reported as the only prion disease in Korea that occurs in livestock. Thus, there is a possibility of transmission of CWD to Korean native black goats. However, PrPres has not been investigated thus far in Korean native black goats. We found strong linkage disequilibrium between c.126G>A and c.414T>C (r2 = 1) and between c.718C>T and c.126G>A (r2 = 0.638). In addition, the haplotype GTGTAAAC (representing codons 42, 102, 127, 138, 143, 146, 218 and 240) showed the highest frequency with 45.1%. Among 41 Korean native black goats, 20 animals (48.78%) were homozygous for the susceptible haplotypes (histidine at codon 143, asparagine at codon 146 and arginine at codon 154). Interestingly, we did not detect PrPres bands in any of the tested animals, including the 20 animals carrying potential scrapie susceptible haplotypes.
Interferon-induced transmembrane protein 3 (IFITM3) has potent antiviral activity against several viruses. Recent studies have reported that the chicken IFITM3 gene also plays a pivotal role in blocking viral replication, but these studies are considerably limited due to being conducted at the RNA level only. Thus, the development of a chicken IFITM3 protein-specific antibody is needed to validate the function of IFITM3 at the protein level. Epitope prediction was performed with the immune epitope database analysis resource (IEDB-AR) program. The epitope was validated by four in silico programs, Jped4, Clustal Omega, TMpred and SOSUI. Chicken IFITM3 protein-specific monoclonal antibodies were screened by enzyme-linked immunosorbent assay through affinity between recombinant IFITM3 protein and phage-displayed candidate antibodies. Validation of the reactivity of the chicken IFITM3 protein-specific antibody to chicken tissues was carried out using western blotting. We developed a chicken IFITM3 protein-specific monoclonal antibody using phage display. The reactivity of the antibody with peripheral chicken tissues was confirmed using western blotting. To the best of our knowledge, this was the first development of a chicken IFITM3 protein-specific monoclonal antibody using phage display.