Taweewat Deemagarn

Journal of Animal Science and Technology (JAST) is a peer-reviewed, open access journal publishing original research, review articles and notes in all fields of animal science. Topics covered by the journal include: genetics and breeding, physiology, nutrition of monogastric animals, nutrition of ruminants, animal products (milk, meat, eggs and their by-products) and their processing, grasslands and roughages, livestock environment, animal biotechnology, animal behavior and welfare.

Lumpy skin disease virus (LSDV) affects cattle and causes significant economic damage. The live vaccine derived from an attenuated strain is effective but is associated with mild disease and skin lesions in some vaccinated cattle. Moreover, recombinant LSDV strains, particularly one with wild-type field and vaccine strains, have recently emerged and spread throughout Asian countries. A cost-effective LSDV typing method is required. We developed a multi-locus real-time PCR with a high-resolution melting (HRM) assay to differentiate between the wild-type, vaccine, and dominant Asian recombinant strains. Based on a multiple alignment analysis, we selected three target genes for the HRM assay, ORF095, ORF126, and ORF145, in which there are insertions/deletions and nucleotide substitutions between wild-type and vaccine strains, and designed primer sets for the assay. Using the synthetic DNA encoding these genes for the two strains, it was shown that the PCR amplicons intercalated with a saturating fluorescent dye could clearly differentiate between wild-type and vaccine strains in the HRM analysis for all three target genes. Further, using clinical samples, our method was able to identify recombinant strains harboring the wild-type ORF095 and ORF145 and the vaccine strain ORF126 genes. Thus, our HRM assay may provide rapid LSDV typing.

African swine fever virus (ASFV) has been responsible for the globally devastating epidemics in wild and domesticated pigs. Of the 24 identified ASFV genotypes, genotype II is the primary cause for the pandemic occurring in Europe and Asia since its emergence in Georgia in 2007. The current study aimed to characterize the full-length genomic pattern of the ASFV strain from Thailand, TH1_22/CR (Accession No. PP915735), which was then compared with genomic diversity across other Asian isolates using Georgia 2007/1 (Accession No. FR682468) as the reference. Viral DNA was isolated from the pig spleen sample following library preparation and paired-end sequencing using the MiSeq Illumina platform. The sequenced TH1_22/CR isolate spanned 189,395 nucleotides encoding 193 open reading frames (ORFs), exhibiting maximum nucleotide similarity (99.99%) with Georgian (Georgia 2007/1) and Chinese (Wuhan 2019-1 and China HLJ) isolates. Based on phylogenetic analysis, the TH1_22/CR isolate (Accession No. PP915735) was characterized as genotype II, serogroup 8, and IGR-II due to the presence of three tandem repeat sequences (TRSs). Genetic variations including SNPs and single and polynucleotide indels were identified in TH1_22/CR in agreement with other Asian isolates. For comprehensive analysis, the genome was divided into four regions (I–IV) based on gene location. Overall, the TH1_22/CR isolate demonstrated eight SNPs and indels in its genome. Two unique SNPs were reported in the coding regions of the TH1_22/CR isolate, out of which, a C-591-T substitution was seen in MGF 360-4L and a C-297-T was found in A238L, and four unique SNPs were reported in non-coding regions (NCRs). Furthermore, a 29 bp deletion was observed in the IGR between MGF 110-13La and MGF 110-13Lb, as well as 52 bp deletion in the ASFV G ACD 00350 gene. This comparative analysis establishes the foundational information for future studies on the diversity and phylogeography of this regionally significant genetic sub-group of ASFV.

Lumpy skin disease (LSD) is a viral infection that impacts the cattle industry. The most efficient approach to prevent disease involves the utilization of live-attenuated LSD vaccines (LAVs), which stands out as the most successful method. However, LAVs might be subjected to changes to their genomes during replication that increase viral infectivity or virulence. The objective of this study was to monitor alterations in the genetic characteristics of the lumpy skin disease virus (LSDV) in beef cattle following the administration of LAVs in Lopburi Province of Central Thailand. A total of four skin samples from LSD cases were collected from non-vaccinated animals that exhibited LSD clinical symptoms from two distinct districts, spanning three subdistricts within the region. The samples of cattle were analyzed using real-time PCR targeting the LSDV074 p32 gene, the virus was isolated, and the entire genome sequences were evaluated through a single nucleotide polymorphisms (SNPs) analysis, and phylogenetic trees were assembled. The investigations revealed that LSDVs from two isolates from Chai Badan district exhibited significant mutations in the open reading frame (ORF) 023 putative protein, while another two isolates from Lam Sonthi district had a change in the untranslated region (UTR). For a result, the most proficient disease diagnosis and control should be evaluated on viral genetics on a regular basis.

Five residues in the multifunctional catalase–peroxidase KatG of Burkholderia pesudomallei are essential for catalase, but not peroxidase, activity. Asp141 is the only one of these catalase-specific residues not related with the covalent adduct found in KatGs that when replaced with a nonacidic residue reduces catalase activity to 5% of native levels. Replacing the nearby catalytic residue Arg108 causes a reduction in catalase activity to 35% of native levels, whereas a variant with both Asp141 and Arg108 replaced exhibits near normal catalase activity (82% of native), suggesting a synergism in the roles of the two residues in support of catalase activity in the enzyme. Among the Asp141 variants, D141E is unique in retaining normal catalase activity but with modified kinetics, suggesting more favorable compound I formation and less favorable compound I reduction. The crystal structure of the D141E variant has been determined at 1.8-Å resolution, revealing that the carboxylate of Glu141 is moved only slightly compared with Asp141, but retains its hydrogen bond interaction with the main chain nitrogen of Ile237. In contrast, the low temperature ferric Electron Paramagnetic Resonance spectra of the D141A, R108A, and R108A/D141A variants are consistent with modifications of the water matrix and/or the relative positioning of the distal residue side chains. Such changes explain the reduction in catalase activity in all but the double variant R108A/D141A. Two pathways of hydrogen bonded solvent lead from the entrance channel into the heme active site, one running between Asp141 and Arg108 and the second between Asp141 and the main chain atoms of residues 237–239. It is proposed that binding of substrate H₂O₂ to Asp141 and Arg108 controls H₂O₂ access to the heme active site, thereby modulating the catalase reaction. Proteins 2007.

Crystals of Burkholderia pseudomallei KatG retain their ability to diffract X-rays at high resolution after adjustment of the pH from 5.6 to 4.5, 6.5, 7.5, and 8.5, providing a unique view of the effect of pH on protein structure. One significant pH-sensitive change lies in the appearance of a perhydroxy group attached to the indole nitrogen of the active site Trp111 above pH 7, similar to a modification originally observed in the Ser324Thr variant of the enzyme at pH 5.6. The modification forms rapidly from molecular oxygen in the buffer with 100% occupancy after one minute of soaking of the crystal at room temperature and pH 8.5. The low temperature (4 K) ferric EPR spectra of the resting enzyme, being very sensitive to changes in the heme iron microenvironment, confirm the presence of the modification above pH 7 in native enzyme and variants lacking Arg426 or Met264 and its absence in variants lacking Trp111 or Tyr238. The indole-perhydroxy group is very likely the reactive intermediate of molecular oxygen in the NADH oxidase reaction, and Arg426 is required for its reduction. The second significant pH-sensitive change involves the buried side chain of Arg426 that changes from one predominant conformation at low pH to a second at high pH. The pH profiles of the peroxidase, catalase, and NADH oxidase reactions can be correlated with the distribution of Arg426 conformations. Other pH-induced structural changes include a number of surface-situated side chains, but there is only one change involving a displacement of main chain atoms triggered by the protonation of His53 in a deep pocket in the vicinity of the molecular 2-fold axis.

The catalase reaction of catalase‐peroxidases involves catalase‐specific features built into a peroxidase core. An arginine, 20 Å from the active‐site heme, acts as a molecular switch moving between two conformations, one that activates heme oxidation and one that activates oxoferryl heme reduction by H2O2, facilitating the catalatic pathway in a peroxidase. The influence of the arginine is imparted to the heme through its association with or dissociation from a tyrosinate that modulates reactivity through a Met‐Tyr‐Trp crosslinked adduct and a π electron interaction of the heme with the adduct Trp.