Development of Differentiating Infected from Vaccinated Animals (DIVA) Real-Time PCR for African Horse Sickness Virus Serotype 1.

African horse sickness (AHS) is a highly infectious and often fatal disease caused by 9 serotypes of the orbivirus African horse sickness virus (AHSV). In March 2020, an AHS outbreak was reported in Thailand in which AHSV serotype 1 was identified as the causative agent. Trivalent live attenuated vaccines serotype 1, 3, and 4 were used in a targeted vaccination campaign within a 50-km radius surrounding the infected cases, which promptly controlled the spread of the disease. However, AHS-like symptoms in vaccinated horses required laboratory diagnostic methods to differentiate infected horses from vaccinated horses, especially for postvaccination surveillance. We describe a real-time reverse transcription PCR-based assay for rapid characterization of the affecting field strain. The development and validation of this assay should imbue confidence in differentiating AHS-vaccinated horses from nonvaccinated horses. This method should be applied to determining the epidemiology of AHSV in future outbreaks.

Infection, dissemination, and transmission of lumpy skin disease virus in Aedes aegypti (Linnaeus), Culex tritaeniorhynchus (Giles), and Culex quinquefasciatus (Say) mosquitoes.

Lumpy skin disease virus (LSDV) is a transboundary viral disease in cattle and water buffaloes. Although this Poxvirus is supposedly transmitted by mechanical vectors, only a few studies have investigated the role of local vectors in the transmission of LSDV. This study examined the infection, dissemination, and transmission rates of LSDV in Aedes aegypti, Culex tritaeniorhynchus, and Culex quinquefasciatus following artificial membrane feeding of 102.7, 103.7, 104.7 TCID50/mL LSDV in sheep blood. The results demonstrated that these mosquito species were susceptible to LSDV, with Cx tritaeniorhynchus exhibiting significantly different characteristics from Ae. aegypti and Cx. quinquefasciatus. These three mosquito species were susceptible to LSDV. Ae. aegypti showed it as early as 2 days post-infection (dpi), indicating swift dissemination in this particular species. The extrinsic incubation period (EIP) of LSDV in Cx. tritaeniorhynchus and Cx. quinquefasciatus was 8 and 14 dpi, respectively. Ingestion of different viral titers in blood did not affect the infection, dissemination, or transmission rates of Cx. tritaeniorhynchus and Cx. quinquefasciatus. All rates remained consistently high at 8-14 dpi for Cx. tritaeniorhynchus. In all three species, LSDV remained detectable until 14 dpi. The present findings indicate that, Ae. aegypti, Cx. tritaeniorhynchus, and Cx. quinquefasciatus may act as vectors during the LSDV outbreak; their involvement may extend beyond being solely mechanical vectors.

Long-term monitoring of immune response to recombinant lumpy skin disease virus in dairy cattle from small-household farms in western Thailand.

Lumpy skin disease (LSD) was firstly reported in Thailand in 2021 which affected the cattle industry. However, there is limited information on the immune response of LSDV infection in Thailand where recombinant vaccine strain circulated. The aim of this research was to study the duration of LSD immune response of subclinical and clinical animals after natural infection in dairy cattle. Sixty-six dairy cattle from ten farms in central and western regions of Thailand were investigated. Antibody was detected by virus neutralization test and ELISA. Cell mediated immunity (CMI)-related cytokine gene expressions were evaluated. Antibody was detected until at least 15 months after the noticeable symptom. Cattle with subclinical disease had lower antibody levels compared to animals which had clinical disease. IFN-γ and TNF-α levels were increased, while IL-10 level was decreased in the infected animals compared to the controls. This study elucidated immune responses in dairy cattle herd affected by recombinant LSDV.

Development of a real-time RT-PCR system applicable for rapid and pen-side diagnosis of foot-and-mouth disease using a portable device, PicoGene® PCR1100.

Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease, causing devastating losses to the livestock industry. A diagnostic method that enables quick decisions is required to control the disease, especially in FMD-free countries. Although conventional real-time reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive method widely used for the diagnosis of FMD, a time lag caused by the transport of samples to a laboratory may allow the spread of FMD. Here, we evaluated a real-time RT-PCR system using a portable PicoGene PCR1100 device for FMD diagnosis. This system could detect the synthetic FMD viral RNA within 20 min with high sensitivity compared to a conventional real-time RT-PCR. Furthermore, the Lysis Buffer S for crude nucleic extraction improved the viral RNA detection of this system in a homogenate of vesicular epithelium samples collected from FMD virus-infected animals. Furthermore, this system could detect the viral RNA in crude extracts prepared using the Lysis Buffer S from the vesicular epithelium samples homogenized using a Finger Masher tube, which allows easy homogenization without any equipment, with a high correlation compared to the standard method. Thus, the PicoGene device system can be utilized for the rapid and pen-side diagnosis of FMD.

Complete Genomic Characterization of Lumpy Skin Disease Virus Isolates from Beef Cattle in Lopburi Province, Central Thailand, during 2021-2022.

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.

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.

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 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.

Use of nanopore sequencing to characterize african horse sickness virus (AHSV) from the African horse sickness outbreak in thailand in 2020.

African horse sickness (AHS) is a highly infectious and deadly disease despite availability of vaccines. Molecular characterization of African horse sickness virus (AHSV) detected from the March 2020 Thailand outbreak was carried out by whole-genome sequencing using Nanopore with a Sequence-Independent Single Primer Amplification (SISPA) approach. Nucleotide sequence of the whole genome was compared with closest matching AHSV strains using phylogenetic analyses and the AHSV-1 virus shared high sequence identity with isolates from the same outbreak. Substitution analysis revealed non-synonymous and synonymous substitutions in the VP2 gene as compared to circulating South African strains. The use of sequencing technologies, such as Nanopore with SISPA, has enabled rapid detection, identification and detailed genetic characterization of the AHS virus for informed decision-making and implementation of disease control measures. Active genetic information sharing has also allowed emergence of AHSV to be better monitored on a global basis.

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.