Rapid Spread and Genetic Characterisation of a Recently Emerged Recombinant Lumpy Skin Disease Virus in Thailand.

The emergence of the lumpy skin disease virus (LSDV) was first detected in north-eastern Thailand in March 2021. Since then, the abrupt increase of LSD cases was observed throughout the country as outbreaks have spread rapidly to 64 out of a total of 77 provinces within four months. Blood, milk, and nodular skin samples collected from affected animals have been diagnosed by real-time PCR targeting the p32 gene. LSDV was isolated by primary lamb testis (PLT) cells, followed by Madin-Darby bovine kidney (MDBK) cells, and confirmed by immunoperoxidase monolayer assay (IPMA). Histopathology and immunohistochemistry (IHC) of a skin lesion showed inclusion bodies in keratinocytes and skin epithelial cells. Phylogenetic analyses of RPO30 and GPCR genes, and the whole genome revealed that Thai viruses were closely related to the vaccine-derived recombinant LSDV strains found previously in China and Vietnam. Recombination analysis confirmed that the Thai LSDV possesses a mosaic hybrid genome containing the vaccine virus DNA as the backbone and a field strain DNA as the minor donor. This is an inclusive report on the disease distributions, complete diagnoses, and genetic characterisation of LSDV during the first wave of LSD outbreaks in Thailand.

Thermal inactivation of African swine fever virus in feed ingredients.

African swine fever virus (ASFV) causes a fatal infectious disease affecting domestic pigs and wild boars. ASFV is highly stable and easily transmitted by consumption of contaminated swine feed and pork products. Heat treatment of feed ingredients is a means to minimize the risk of contamination through swine feed consumption. The objectives of this study were to determine the thermal inactivation of ASFV in non-animal and animal origin feed ingredients. The rate of thermal inactivation is represented by decimal reduction time (DT) or time required to reduce ASFV per 1 log at temperature T. The mean D60, D70, D80 and D85 of meat and bone meal (MBM), soybean meal (SBM), and maize grain (MZ) are in the ranges 5.11-6.78, 2.19-3.01, 0.99-2.02, and 0.16-0.99 min, respectively. DT is used to compare the heat resistance of ASFV in the feed ingredient matrices. The mean DT of ASFV in MBM, SBM and MZ was not statistically significant, and the heat resistance of ASFV in MBM, SBM, and MZ was not different at 60, 70, 80, or 85 °C. The multiple DT was used to develop a DT model to predict DT at various inactivation temperatures. The DT models for MBM, SBM, and MZ are log DT = - [Formula: see text] + 2.69, log DT = - [Formula: see text] + 2.55, and log DT = - [Formula: see text] + 4.01. To expand and ease the field applications, a spreadsheet predicting the DT and the inactivation time (with 95% confidence interval) from these DT models is available to download.

Persistence of African swine fever virus on porous and non-porous fomites at environmental temperatures.

BACKGROUND: African swine fever (ASF) is a lethal contagious disease affecting both domestic pigs and wild boars. Even though it is a non-zoonotic disease, ASF causes economic loss in swine industries across continents. ASF control and eradication are almost impossible since effective vaccines and direct antiviral treatment are not available. The persistence of ASFV on fomites plays an important role in the indirect transmission of ASFV to pigs encountering ASFV-contaminated fomites. ASFV persistence on porous and non-porous fomites (glass, metal, rubber, and cellulose paper) at different environmental temperatures was determined. The persistence of ASFV of fomites was determined by the rate of ASFV inactivation in terms of DT, or the time required to reduce ASFV per 1 log at each selected environmental temperature (T). DT is used to compare the persistence of ASFV on the fomites. RESULTS: The mean D25, D33, and D42, of dried infectious ASFV on glass, metal, rubber, and paper were in the ranges 1.42-2.42, 0.72-1.94, and 0.07-0.23 days, respectively. The multiple DT were used to develop a DT model to predict the DT for some other environmental temperatures. The DT models to predict the persistence of dried infectious ASFV on glass, metal, rubber, and paper are log DT = (- T/21.51) + 1.34, log DT = (- T/20.42) + 1.47, log DT = (- T/14.91) + 2.03, and log DT = (- T/10.91) + 2.84, respectively. A spreadsheet as a quick and handy tool predicting the persistence time of dried infectious ASFV on fomites at various environmental temperatures based on these DT models is available for public to download. CONCLUSION: Persistence of dried infectious ASFV on paper are significantly the longest at lower environmental temperatures whereas that of dried infectious ASFV on paper is significantly the shortest at higher environmental temperature.

Thermal Inactivation of African Swine Fever Virus in Swill.

The indirect transmission of the African swine fever virus (ASFV) is through contaminated fomite, feed ingredients, pork- and pig-derived products, including swill, as ASFV is highly stable within suitable organic material. Some previous studies have indicated that ASFV outbreaks were associated with swill feeding, particularly in smallholder pig farms. These outbreaks emphasize the significance of the appropriate heat treatment of swill to eliminate ASFV residual titer. The World Organization for Animal Health (OIE) recommended the heat treatment of swill at a temperature of at least 90°C for at least 60 min, with continuous stirring, while the Food and Agriculture Organization (FAO) recommended heat treatment at 70°C for 30 min. The lack of scientific evidence regarding ASFV inactivation by heat treatment of swill leads to such inconsistent recommendations. Therefore, the objectives of this study were to assess the thermal inactivation of ASFV in three swill formulae and to develop a D T model to predict D T at some other inactivation temperatures. The significant reduction of ASFV in swill occurred at temperatures as low as 60°C. D T or decimal reduction time (DRT) is defined as the time required to reduce the virus titer by 1 log, and this was also used as a comparative index of heat resistance. The mean D 60, D 70, D 75, and D 80 of ASFV in three swill formulae were in the ranges 23.21-33.47, 5.83-10.91, 2.15-2.22, and 1.36-1.47 min, respectively. These D T could be widely used for any nutritive composition of swill other than the three swill formulae in this study since there was no statistical difference of all D T of ASFV across three swill formulae. Based on D 70 and the predicted D 90 from the D T model in this study, including the highest ASFV titer in pork products, the calculated inactivation times at 70 and 90°C were 119 and 4 min, respectively.

A molecular hybrid comprising AS1411 and PDGF-BB aptamer, cholesterol, and doxorubicin for inhibiting proliferation of SW480 cells.

Cancer treatment commonly relies on chemotherapy. This treatment faces many challenges, including treatment specificity and undesired side effects. To address these, a Dox-loaded Chol-aptamer molecular hybrid (Dox-CAH) was developed. This multivalent interaction system combines the key function of each integrated species: doxorubicin, cholesterol, and two aptamers binding to nucleolin and platelet-derived growth factor BB (PDGF-BB). The study has four stages: preparation of CAH via oligonucleotide hybridization, intercalation of doxorubicin into CAH, verification of CAH binding on SW480 by fluorescence microscopy and flow cytometry, and investigation of effect of Dox-CAH on SW480 proliferation. CAH was successfully prepared, as confirmed by electrophoresis. Flow cytometry and fluorescence microscopy demonstrated CAH binding to SW480, due to the presence of the AS1411 aptamer. This molecular hybrid exhibited specific binding because it did not bind to CCD 841 CoN. CAH binding to PDGF-BB compromises its function, as shown by enzyme-linked immunosorbent assay (ELISA) and cell assay. The DNA duplex in this molecular hybrid reduces the cytotoxicity of the Dox-CAH. Binding and the reduction of Dox-CAH toxicity may improve treatment specificity and minimize side effects. Dox-CAH is a model for more effective anticancer therapy, allowing incorporation of chemotherapeutic drugs and recognition elements.

Anti-Proliferative Effect of Doxorubicin-Loaded AS1411 Aptamer on Colorectal Cancer Cell.

BACKGROUND: Doxorubicin (Dox) inhibits DNA replication and causes DNA damage resulting in cell death. It is a common drug for treatment of many cancers. Treatment efficacy and side effects of Dox are critical issues in using it because the drug lacks of specificity. The objective of this study was to improve the specificity of Dox by the incorporation of this drug with AS1411 aptamer (ASA). METHODS: Dox was intercalated into the duplex sites of ASA, a recognition molecule for a number of cancer cells, and formed Dox-loaded ASA. The recognition ability proceeded through specific binding between the aptamer and nucleolin overexpressed in the cancer cells. The tested cells were human colorectal adenocarcinoma cell line (SW480) and human normal colon cell CCD841 CoN (CCD841). Binding of ASA to the cells was tested using flow cytometer and fluorescence microscope. Intercalation of Dox into DNA duplex was confirmed by fluorescence spectrometry. Effect of ASA, Dox, and Dox-loaded ASA on cell viability was examined by cell proliferation assay. Caspase-3 activation was analyzed by western blotting. RESULTS: ASA bound specifically to SW480 cells via interaction between the aptamer and nucleolin because the nucleolin was highly expressed in SW480 cells. ASA decreased the viability of SW480 cells in a dose-dependent manner. Dox was more toxic than ASA. Fluorescence quenching revealed that Dox was able to intercalate in base pairing sites of the aptamer. Dox-loaded ASA inhibited the proliferation of SW480 cells, because the aptamer facilitated the Dox uptake into these cells which caused the cell apoptosis, indicated by the significant decrease in procaspase-3, apoptosis marker protein. CONCLUSION: This study succeeded to prepare Dox-loaded ASA by intercalation of the drug that inherited the binding function from the aptamer and anti-cancer activity from Dox. Dox-loaded ASA showed promise for effective cancer treatment with lower level of side effects.