We tried to prevent the mitochondrial and DNA damage caused by mechanical stress-associated reactive oxygen species (ROS), and to improve the reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos by antioxidant treatment during the manipulation procedures of SCNT. Bovine recipient oocytes and reconstituted oocytes were treated with antioxidants during manipulation procedures. The H2O2 level, mitochondrial morphology, membrane potential and apoptosis at the one-cell stage, and in vitro development and DNA methylation status of blastocysts were evaluated. Antioxidant treatment during manipulation procedures reduced the H2O2 level of SCNT embryos. Antioxidant-treated SCNT embryos normally formed mitochondrial clumps, similar to IVF embryos, and showed higher mitochondrial membrane potential versus the SCNT control (P < 0.05). Apoptosis and DNA fragmentation were reduced by antioxidant treatment. The development rate to the blastocyst stage was higher (P < 0.05) in the antioxidant treatment groups (30.5 ± 2.5 to 30.6 ± 1.6%) versus the control (23.0 ± 1.9%). The DNA methylation status of blastocysts in the antioxidant treatment groups was lower (P < 0.05) than that of the control and similar to that of IVF embryos. These results indicate that antioxidant treatment during manipulation procedures can prevent cellular damage that may be caused by mechanical stress-associated ROS, and improve nuclear reprogramming.

Virus-like particle (VLP) technology is considered one of the most promising approaches in animal vaccines, due to the intrinsic immunogenic properties as well as high safety profile of VLPs. In this study, we developed a VLP vaccine against infectious bursal disease virus (IBDV), which causes morbidity and mortality in chickens, by expressing a baculovirus in insect cells. To improve the self-proteolytic processing of precursor polyprotein (PP), we constructed a recombinant baculovirus transfer vector that co-expresses PP and the VP4 protease gene of IBDV. Expression and VLP assembly of recombinant proteins and antigenicity of the VLP were examined by Western blotting, ELISA, and transmission electron microscopy. In animal experiments, vaccination with the recombinant VLP induced strong and uniform humoral immunity and provided complete protection against challenge with very virulent (vv) IBDV in SPF chickens (n = 12). As determined by the bursa of Fabricius (BF)/body weight (B/BW) ratio, the protection against post-challenge bursal atrophy was significantly higher (P < 0.001) in VLP-vaccinated birds than in non-vaccinated controls. Since the protective efficacy of the VLP vaccine was comparable to that of a commercially available inactivated vaccine, the recombinant VLP merits further investigation as an alternative means of protection against vvIBD.

Newcastle disease (ND) is a highly pathogenic viral infection of poultry with significant economic impacts worldwide. Despite the widespread use of vaccines, ND outbreaks continue to occur even within vaccinated poultry farms. Furthermore, novel Newcastle disease virus (NDV) genotypes are emerging in poultry, increasing the need for the development of rapid, accurate, and simple diagnostic methods. We therefore developed two novel sets of visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays based on highly conserved regions of the HN and F genes. The limits of detection of the NDV-Common-LAMP assay, for all the NDV strains, were 10^3.0 EID₅₀/0.1 mL for Kr005 and 10^2.0 EID₅₀/0.1 mL for Lasota within 35 min. The sensitivity of the NDV-Patho-LAMP assay, used for the strain differentiation of virulent NDV, was 10^2.0 EID₅₀/0.1 mL for Kr005. No amplification was detected for the non-NDV templates. Next, we probed 95 clinical strains and 7 reference strains with the RT-LAMP assays to assess the feasibility of their use in diagnostics. We observed no cross-reactivity across the 102 strains. Furthermore, there was 100% congruence between the RT-LAMP assays and full-length sequencing of the target genes, indicating the potential for visual RT-LAMP in the identification and differentiation of NDV. These novel RT-LAMP assays are ideally suited for the field or resource-limited environments to facilitate the faster detection and differentiation of NDV, which can reduce or avoid further spread.

Hong SM, et al. J Vet Sci. 2023 Jun;24:e51. https://doi.org/10.4142/jvs.23039

Foot-and-mouth disease (FMD) is considered one of the highly contagious viral infections affecting livestock. In Korea, an FMD vaccination policy has been implemented nationwide since 2010 for the prevention and control of FMD. Since the vaccines are imported from various countries, standardized quality control measures are critical. In this study, we aimed to validate a high-performance liquid chromatography (HPLC) device in the Animal and Plant Quarantine Agency lab and identify an appropriate FMD vaccine pretreatment method for HPLC—a simple, reliable, and practical method to measure antigen content. Based on the analyses of specificity, linearity, accuracy, repeatability, intermediate precision, limits of detection, and limits of quantification using FMD standard samples, we validated the method using a standard material. Overall, we confirmed that the HPLC technique is effective for the quantitative assessment of the FMD virus 146S antigen in Korea. Using commercial FMD vaccines, we evaluated three separation methods and identified the method using n-pentanol and trichloroethylene as optimal for HPLC analysis. Our HPLC method was effective for the analytical detection of the antigen content in FMD vaccine, and it may be useful as a reference method for national lot-release testing.

Infectious bronchitis virus (IBV) causes a highly contagious respiratory disease in chickens, leading to significant economic losses in the poultry industry worldwide. IBV exhibits a high mutation rate, resulting in the continuous emergence of new variants and strains. A complete genome analysis of IBV is crucial for understanding its characteristics. However, it is challenging to obtain whole-genome sequences from IBV-infected clinical samples due to the low abundance of IBV relative to the host genome. Here, we present a novel approach employing next-generation sequencing (NGS) to directly sequence the complete genome of IBV. Through in silico analysis, six primer pairs were designed to match various genotypes, including the GI-19 lineage of IBV. The primer sets successfully amplified six overlapping fragments by long-range PCR and the size of the amplicons ranged from 3.7 to 6.4 kb, resulting in full coverage of the IBV genome. Furthermore, utilizing Illumina sequencing, we obtained the complete genome sequences of two strains belonging to the GI-19 lineage (QX genotype) from clinical samples, with 100% coverage rates, over 1000 × mean depth coverage, and a high percentage of mapped reads to the reference genomes (96.63% and 97.66%). The reported method significantly improves the whole-genome sequencing of IBVs from clinical samples; thus, it can improve understanding of the epidemiology and evolution of IBVs.