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Abstract Porcine circovirus type 2 (PCV2) is the major swine pathogen associated with Porcine circovirus associated disease (PCVAD) including post-weaning multisystemic wasting syndrome (PMWS). Currently, there are 4 subtypes of PCV2 (PCV2a, b, c and d) and some epidemiological evidences demonstrated that virulence of PCV2 may relate to its subtypes. Recently, PMWS was observed more frequently in swine farms in Thailand; however, the information regarding to PCV2 subtype involved was limited. Therefore, this study was aimed to determine the association between occurrence of PMWS and PCV2 subtypes as well as genetically characterize PCV2 in Thailand. PCV2 DNA was isolated from faecal swabs and whole blood of piglets from PMWS-affected and -negative farms. The full length ORF2 sequences were compared using multiple alignment. The results showed that PCV2 DNA was detected more frequently in PMWS-affected farms. The nucleotide identities of the ORF2 from 9 PCV2 isolates representing each PMWS-affected farm and one from the negative farm ranged from 92.4 to 99.5% suggesting that there is some genetic variation of PCV2 in Thai swine. The 10 PCV2 isolates were classified into 2 clusters, in which the 7 isolates from PMWS-positive farms were in PCV2b cluster 1 A/B. The remaining isolates were separated in the new subtype called PCV2e. The results suggest the presence of new PCV2 subtypes in addition to PCV2a and PCV2b in Asian swine population. However, correlation between subtypes and virulence of PCV2 infection is not conclusive due to limited number of the PCV2 sequences from PMWS negative farms.

Foot-and-Mouth Disease (FMD) is a contagious, blistering disease caused by the Foot-and-Mouth Disease virus (FMDV), which affects livestock globally. Currently, no commercial antiviral agent is available for effective disease control. This study investigated the antiviral potential of natural-derived alkaloids against FMDV in BHK-21 cells. Twelve alkaloids were assessed for their antiviral activities at various stages of FMDV infection, including pre-viral entry, post-viral entry, and prophylactic assays, as well as attachment and penetration assays by evaluating cytopathic effect reduction and directed-virucidal effects. The results showed that ipecac alkaloids, cephaeline (CPL) and emetine (EMT), exhibited dual effects with robust antiviral efficacy by reducing cytopathic effect and inhibiting FMDV replication in a dose-dependent manner. Evaluation through immunoperoxidase monolayer assay and RT-PCR indicated effectiveness at post-viral entry stage, with sub-micromolar EC 50 values for CPL and EMT at 0.05 and 0.24 µM, respectively, and high selective indices. Prophylactic effects prevented infection with EC 50 values of 0.23 and 0.64 µM, respectively. Directed-virucidal effects demonstrated significant reduction of extracellular FMDV, with CPL exhibiting a dose-dependent effect. Furthermore, the replicase (3Dpol) inhibition activity was identified using the FMDV minigenome assay, which revealed strong inhibition with IC 50 values of 0.15 µM for CPL and 4.20 µM for EMT, consistent with the decreased negative-stranded RNA production. Molecular docking confirmed the interaction of CPL and EMT with residues in the active site of FMDV 3Dpol. In conclusion, CPL and EMT exhibited promising efficacy through their dual effects and provide an alternative approach for controlling FMD in livestock., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Foot-and-mouth disease (FMD) is a highly infectious disease of cloven-hoofed animals with a significant economic impact. Early diagnosis and effective prevention and control could reduce the spread of the disease which could possibly minimize economic losses. Epitope characterization based on monoclonal antibodies provide essential information for developing diagnostic assays and vaccine designs. In this study, monoclonal antibodies raised against FMD virus (FMDV) were produced. Sixty-six monoclonal antibodies demonstrated strong reactivity and specificity to FMDV. The purified monoclonal antibodies were further used for bio-panning to select phage expressing specific epitopes from phage-displayed 12 mer-peptide library. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. Two hybridoma clones secreted monoclonal antibodies recognizing linear epitopes on VP2 of FMDV serotype O. The non-neutralizing monoclonal antibody 6F4.D11.B6 recognized the residues 67-78 on antigenic site 2 resinding in VP2, while the neutralizing monoclonal antibody 8D6.B9.C3 recognized a novel linear epitope encompassing residues 115-126 on VP2. This information and the FMDV-specific monoclonal antibodies provide valuable sources for further study and application in diagnosis, therapeutics and vaccine designs to strengthen the disease prevention and control measures.

Background and Aim: Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a global issue that affects Thai swine as well. In Thailand, PRRSV-2 predominates over PRRSV-1. The origin of PRRSV-1 transmission remains undiscovered. This study traced the source of infected pigs responsible for disease transmission among three pig-fattening farms and analyzed the spread of PRRSV-1., Materials and Methods: A total of 696 swine samples from breeding and pig-fattening farms in Thailand were screened for PRRSV using open reading frames (ORF7) reverse transcription polymerase chain reaction (RT-PCR). Positive samples were identified as PRRSV-1 using ORF5 RT-PCR. The analysis included the study of nucleotide homology, GP5 amino acid sequences, and N-linked glycosylation patterns to assess the spread of PRRSV-1 across these farms., Results: Genetic examination identified 28 PRRSV-1-positive samples, of which 13 were chosen as representatives. These strains were categorized into three groups based on breeding farm pig houses and showed distinct distribution patterns across pig-fattening farms. Group 1 included piglets transferred from pig house A to Nakhon Pathom, Chonburi, and Sa Kaeo. Groups 2 and 3 showed transfers from pig houses F and H to Chonburi and Sa Kaeo farms. All 13 PRRSV-1 strains were categorized into PRRSV-1 subtype 1/clade H. N-linked glycosylation analysis revealed that nearly all PRRSV-1 strains exhibited a conserved glycosylation pattern at amino acid positions N37, N46, and N53. This pattern is consistent with the glycosylation profile of the previous Thai PRRSV-1 subtype 1/clade H., Conclusion: The present study highlights the persistent presence of PRRSV-1 in Thai swine, which leads to sporadic outbreaks. The molecular genetic analysis identified three primary strain groups dispersed throughout the pig production system, emphasizing the importance of regular monitoring for new PRRSV strains in this herd. Understanding the PRRSV-1 distribution in swine farms is vital for veterinarians. This knowledge supports strategies for eradicating the virus and managing swine health effectively in Thailand., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Jantafong, et al.)

Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and is an important pathogen affecting cloven-hoof livestock. However, neither effective vaccines covering all serotypes nor specific antivirals against FMDV infections are currently available. In this study, we employed virtual screening to screen for secondary metabolite terpenoids targeting the RNA-dependent RNA polymerase (RdRp), or 3D pol , of FMDV. Subsequently, we identified the potential antiviral activity of the 32 top-ranked terpenoids, revealing that continentalic acid, dehydroabietic acid (abietic diterpenoids), brusatol, bruceine D, and bruceine E (tetracyclic triterpenoids) significantly reduced cytopathic effects and viral infection in the terpenoid-treated, FMDV-infected BHK-21 cells in a dose-dependent manner, with nanomolar to low micromolar levels. The FMDV minigenome assay demonstrated that brusatol and bruceine D, in particular, effectively blocked FMDV 3D pol activity, exhibiting IC50 values in the range of 0.37-0.39 µM and surpassing the efficacy of the antiviral drug control, ribavirin. Continentalic acid and bruceine E exhibited moderate inhibition of FMDV 3D pol . The predicted protein-ligand interaction confirmed that these potential terpenoids interacted with the main catalytic and bystander residues of FMDV 3D pol . Additionally, brusatol and bruceine D exhibited additive effects when combined with ribavirin. In conclusion, terpenoids from natural resources show promise for the development of anti-FMD agents.

Background: Colibacillosis caused by Escherichia coli is one of the main problems in the swine industry. In addition, the emergence of antimicrobial resistance and the combination of virulence genes among pathotypes have led to the emergence of more virulent pathogenic E. coli strains. Phage therapy has become a promising approach to address these issues., Materials and Methods: Virulence genes for intestinal pathogenic E. coli (IPEC) and extraintestinal pathogenic E. coli (ExPEC) were investigated in pathogenic E. coli isolated from pigs. In addition, two potential phages, vB_EcoM-RPN187 and vB_EcoM-RPN226, isolated in our previous study, were further characterized in this study., Results: Both phages were lytic and were highly effective at 20-37°C. Interestingly, they infected the hybrid IPEC/ExPEC strains. vB_EcoM-RPN187 and vB_EcoM-RPN226 possess 167 kbp of linear double-stranded DNA without virulence or antibiotic resistance genes and may be classified as new phage species in the genera Mosigvirus and Tequatrovirus , respectively., Conclusion: Both phages could be promising candidates for phage therapy against pathogenic E. coli ., (Copyright 2024, Mary Ann Liebert, Inc., publishers.)

Background and Aim: African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process., Materials and Methods: SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli . SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA)., Results: The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001)., Conclusion: This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Chootip, et al.)

Foot-and-mouth disease (FMD) is a highly contagious disease in cloven-hoofed animals, caused by the foot-and-mouth disease virus (FMDV). It is endemic in Asia and Africa but spreads sporadically throughout the world, resulting in significant losses in the livestock industry. Effective anti-FMDV therapeutics could be a supportive control strategy. Herein, we utilized computer-aided, structure-based virtual screening to filter lead compounds from the National Cancer Institute (NCI) diversity and mechanical libraries using FMDV 3C protease (3C pro ) as the target. Seven hit compounds were further examined via cell-based antiviral and intracellular protease assays, in which two compounds (NSC116640 and NSC332670) strongly inhibited FMDV, with EC50 values at the micromolar level of 2.88 µM (SI = 73.15) and 5.92 µM (SI = 11.11), respectively. These compounds could inactivate extracellular virus directly in a virucidal assay by reducing 1.00 to 2.27 log TCID50 of the viral titers in 0-60 min. In addition, the time-of-addition assay revealed that NSC116640 inhibited FMDV at the early stage of infection (0-8 h), while NSC332670 diminished virus titers when added simultaneously at infection (0 h). Both compounds showed good FMDV 3C pro inhibition with IC50 values of 10.85 µM (NSC116640) and 4.21 µM (NSC332670). The molecular docking of the compounds on FMDV 3C pro showed their specific interactions with amino acids in the catalytic triad of FMDV 3C pro . Both preferentially reacted with enzymes and proteases in physicochemical and ADME analysis studies. The results revealed two novel small molecules with antiviral activities against FMDV and probably related picornaviruses.

Background: Rabies is a highly fatal infectious disease that poses a significant threat to human health in developing countries. In vitro study-based understanding of pathogenesis and tropism of different strains of rabies virus (RABV) in the central nervous system (CNS) is limited due to the lack of suitable culture models that recapitulate the complex communication pathways among host cells, extracellular matrices, and viruses. Therefore, a three-dimensional (3D) cell culture that mimics cell-matrix interactions, resembling in vivo microenvironment, is necessary to discover relevant underlying mechanisms of RABV infection and host responses., Methods: The 3D collagen-Matrigel hydrogel encapsulating hiPSC-derived neurons for RABV infection was developed and characterized based on cell viability, morphology, and gene expression analysis of neuronal markers. The replication kinetics of two different strains of RABV [wild-type Thai (TH) and Challenge Virus Standard (CVS)-11 strains] in both 2D and 3D neuronal cultures were examined. Differential gene expression analysis (DEG) of the neuropathological pathway of RABV-infected 2D and 3D models was also investigated via NanoString analysis., Results: The 3D hiPSC-derived neurons revealed a more physiologically interconnected neuronal network as well as more robust and prolonged maturation and differentiation than the conventional 2D monolayer model. TH and CVS-11 exhibited distinct growth kinetics in 3D neuronal model. Additionally, gene expression analysis of the neuropathological pathway observed during RABV infection demonstrated a vast number of differentially expressed genes (DEGs) in 3D model. Unlike 2D neuronal model, 3D model displayed more pronounced cellular responses upon infection with CVS-11 when compared to the TH-infected group, highlighting the influence of the cell environment on RABV-host interactions. Gene ontology (GO) enrichment of DEGs in the infected 3D neuronal culture showed alterations of genes associated with the inflammatory response, apoptotic signaling pathway, glutamatergic synapse, and trans-synaptic signaling which did not significantly change in 2D culture., Conclusion: We demonstrated the use of a hydrogel-based 3D hiPSC-derived neuronal model, a highly promising technology, to study RABV infection in a more physiological environment, which will broaden our understanding of RABV-host interactions in the CNS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Muangsanit, Chailangkarn, Tanwattana, Wongwanakul, Lekcharoensuk and Kaewborisuth.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The construction of a full-length infectious clone, essential for molecular virological study and vaccine development, is quite a challenge for viruses with long genomes or possessing complex nucleotide sequence structures. Herein, we have constructed infectious clones of foot-and-mouth disease virus (FMDV) types O and A by joining each viral coding region with our pKLS3 vector in a single isothermal reaction using Gibson Assembly (GA). pKLS3 is a 4.3-kb FMDV minigenome. To achieve optimal conditions for the DNA joining, each FMDV coding sequence was divided into two overlapping fragments of approximately 3.8 and 3.2 kb, respectively. Both DNA fragments contain the introduced linker sequences for assembly with the linearized pKLS3 vector. FMDV infectious clones were produced upon directly transfecting the GA reaction into baby hamster kidney-21 (BHK-21) cells. After passing in BHK-21 cells, both rescued FMDVs (rO189 and rNP05) demonstrated growth kinetics and antigenicity similar to their parental viruses. Thus far, this is the first report on GA-derived, full-length infectious FMDV cDNA clones. This simple DNA assembly method and the FMDV minigenome would facilitate the construction of FMDV infectious clones and enable genetic manipulation for FMDV research and custom-made FMDV vaccine production.

Background and Aim: Feline infectious peritonitis (FIP), one of the most important infectious diseases in cats is caused by FIP virus (FIPV), a mutated variant of feline coronavirus. Feline infectious peritonitis has a negative impact on feline health, with extremely high mortality in clinical FIP-infected cats, particularly young cats. There are no approved drugs for FIP treatment, and therapeutic possibilities for FIP treatment are limited. This study aimed to utilize nature-derived bioactive flavonoids with antiviral properties to inhibit FIPV infection in Crandell-Rees feline kidney (CRFK) cells., Materials and Methods: The cytotoxicity of 16 flavonoids was evaluated on CRFK cells using a colorimetric method (MTS) assay. Viral kinetics of FIPV at 50 tissue culture infectious dose (TCID 50 )/well was determined during the first 24-h post-infection (HPI). Antiviral activity was evaluated based on the replication steps of the virus life cycle, including pre-compound, attachment, penetration, post-viral entry, and virucidal assays. The antiviral efficacy of flavonoids against FIPV was determined based on positive FIPV-infected cells with the immunoperoxidase monolayer assay and viral load quantification using reverse transcription-quantitative polymerase chain reaction., Results: Two flavonoids, namely, isoginkgetin and luteolin, inhibited FIPV replication during post-viral entry in a dose-dependent manner, with 50% maximal effective concentrations = 4.77 ± 0.09 and 36.28 ± 0.03 μM, respectively. Based on viral kinetics, both flavonoids could inhibit FIPV replication at the early stage of infection at 0-6-HPI for isoginkgetin and 2-6-HPI for luteolin using a time-of-addition assay. Isoginkgetin exerted a direct virucidal effect that reduced the viral titers by 2 and 1.89 log 10 TCID 50 /mL at 60 and 120 min, respectively., Conclusion: Isoginkgetin interfered with FIPV replication during both post-viral infection and virucidal experiments on CRFK cells, whereas luteolin inhibited the virus after infection. These results demonstrate the potential of herbal medicine for treating FIP., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Triratapiban, et al.)

Foot-and-mouth disease virus (FMDV), an economically important pathogen of cloven-hoofed livestock, is a positive-sense, single-stranded RNA virus classified in the Picornaviridae family. RNA-dependent RNA polymerase (RdRp) of RNA viruses is highly conserved. Compounds that bind to the RdRp active site can block viral replication. Herein, we combined double virtual screenings and cell-based antiviral approaches to screen and identify potential inhibitors targeting FMDV RdRp (3D pol ). From 5596 compounds, the blind- followed by focus-docking filtered 21 candidates fitting in the 3D pol active sites. Using the BHK-21 cell-based assay, we found that four compounds-NSC217697 (quinoline), NSC670283 (spiro compound), NSC292567 (nigericin), and NSC65850-demonstrated dose-dependent antiviral actions in vitro with the EC50 ranging from 0.78 to 3.49 µM. These compounds could significantly block FMDV 3D pol activity in the cell-based 3D pol inhibition assay with small IC50 values ranging from 0.8 nM to 0.22 µM without an effect on FMDV's main protease, 3C pro . The 3D pol inhibition activities of the compounds were consistent with the decreased viral load and negative-stranded RNA production in a dose-dependent manner. Conclusively, we have identified potential FMDV 3D pol inhibitors that bound within the enzyme active sites and blocked viral replication. These compounds might be beneficial for FMDV or other picornavirus treatment.

Escherichia coli is the most common cause of economic loss in swine industry. Nowadays, bacteriophages have been proven as good candidates for controlling bacterial infections. In this study, 6 phages were isolated and selected based on their high efficacy against 11 stains of E. coli isolated from diarrheal pigs. Six groups of weaned piglets were assigned (control, bacterial control (BC), two phage control (PC) and two phage treatment (PT) groups). Two titers (2 × 10 9 PFU/animal and 2 × 10 10 PFU/animal) of phage cocktails consisting of these phages were tested in the PC and PT groups via oral gavage at 24, 48, and 72 h against an E. coli cocktail (2 × 10 9  CFU/animal) that was given to the piglets at 0, 12, 24, and 48 h of the trial. A significant reduction of fecal E. coli counts was observed in both PT groups from day 1 to 7 following the final phage dosage when compared to those of the BC group. Microbiomes in feces obtained 24 h after the final phage administration revealed phage therapy with both dosages could restore the gut's bacterial composition. Moreover, the given phage cocktails resulted in a significantly higher average daily gain of piglets during the first few weeks in both PC groups and the PT group receiving a higher phage dosage. These findings suggest that bacteriophages might be a potential alternative to antibiotics in the treatment of pathogens. In addition, they could also be utilized to improve pig growth performance., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A DNA fragment containing CpG motifs (CpG ODN) is one of the potent immunopotentiators used to improve vaccine efficacy. It can enhance a protective immunity by stimulating both innate and adaptive immune responses. In this study, we designed and constructed a recombinant plasmid carrying the combined CpG ODN to generate an immunopotentiator for boosting the immunogenicity of porcine circovirus type 2 (PCV2) virus-like particles (VLPs). The capsid protein of PCV2b was expressed in insect cells and purified by affinity chromatography. The purified capsid protein was incubated with the CpG ODN in the reaction that allowed VLPs formation and encapsidation of the CpG ODN to occur simultaneously. Morphology of the reassembled VLPs was similar to the PCV2 virions as observed using an electron microscope. When the CpG ODN-encapcidated VLPs was treated with DNase I, the VLPs could protect the packaged CpG ODN from the enzyme digestion. Moreover, we immunized mice subcutaneously with VLPs, CpG ODN-loaded VLPs, or phosphate buffer saline for three times at two-week intervals. The results showed that the CpG ODN-loaded VLPs could elicit significantly higher levels of PCV2-specific neutralizing antibodies and interferon gamma (IFN-γ) expression in the immunized mice compared to those conferred by the VLPs alone. Conclusively, we have proved that the CpG ODN incorporated in VLPs can serve as a potent immunopotentiator for PCV2 vaccine development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Osteoarthritis (OA) is mostly incurable and non-regenerative with long-term complications. Autologous conditioned serum (ACS), which is enriched in Interleukin 1 receptor antagonists (IL-1RA) and growth factors, could be an alternative treatment to accelerate the positive therapeutic effects. ACS is proposed to alleviate inflammation by blocking IL-1 receptors. However, to date, there is no report focusing on the cell-mediated anti-inflammation and regenerative effect caused by ACS, especially the ACS from patients. Therefore, this study aims to investigate the therapeutic potential of ACS generated from dogs with spontaneous OA, focusing on its promising anti-inflammatory and regenerative properties in vitro compared to the matched plasma. We found that ACS prepared from ten OA dogs contained significant concentrations of IL-1RA, vascular endothelial growth factor, and transforming growth factor beta, which are key cytokines in anti-inflammation and angiogenesis. Furthermore, we found that ACS suppressed T cell activity by reducing proliferation of effector T cells and simultaneously expanding numbers of immune suppressive FOXP3 + T cells. Lastly, we showed that ACS enhanced the proliferation of osteocytes and fibroblasts and promoted extracellular matrix gene expression in primary chondrocyte culture. Therefore, these studies indicate that ACS prepared from dogs with OA is active as an immunomodulatory and regenerative strategy for use in OA management.

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.

Foot-and mouth-disease (FMD) caused by the FMD virus (FMDV) is highly contagious and negatively affects livestock worldwide. The control of the disease requires a combination of measures, including vaccination; however, there is no specific treatment available. Several studies have shown that plant-derived products with antiviral properties were effective on viral diseases. Herein, antiviral activities of andrographolide (AGL), deoxyandrographolide (DAG), and neoandrographolide (NEO) against FMDV serotype A were investigated using an in vitro cell-based assay. The results showed that AGL and DAG inhibited FMDV in BHK-21 cells. The inhibitory effects of AGL and DAG were evaluated by RT-qPCR and exhibited EC50 values of 52.18 ± 0.01 µM (SI = 2.23) and 36.47 ± 0.07 µM (SI = 9.22), respectively. The intracellular protease assay revealed that AGL and DAG inhibited FMDV 3C pro with IC50 of 67.43 ± 0.81 and 25.58 ± 1.41 µM, respectively. Additionally, AGL and DAG significantly interfered with interferon (IFN) antagonist activity of the 3C pro by derepressing interferon-stimulating gene (ISGs) expression. The molecular docking confirmed that the andrographolides preferentially interacted with the 3C pro active site. However, NEO had no antiviral effect in any of the assays. Conclusively, AGL and DAG inhibited FMDV serotype A by interacting with the 3C pro and hindered its protease and IFN antagonist activities.

Porcine epidemic diarrhea virus (PEDV) causes devastating enteric disease that inflicts huge economic damage on the swine industry worldwide. A safe and highly effective PEDV vaccine that contains only the virus-neutralizing epitopes (not enhancing epitope), as well as a ready-to-use PEDV neutralizing antibody for the passive immunization of PEDV vulnerable piglets (during the first week of life) are needed, particularly for PEDV-endemic farms. In this study, we generated monoclonal antibodies (mAbs) to the recombinant S1 domain of PEDV spike (S) protein and tested their PEDV neutralizing activity by CPE-reduction assay. The mAb secreted by one hybrodoma clone (A3), that also bound to the native S1 counterpart from PEDV-infected cells (tested by combined co-immunoprecipitation and Western blotting), neutralized PEDV infectivity. Epitope of the neutralizing mAb (mAbA3) locates in the S1A subdomain of the spike protein, as identified by phage mimotope search and multiple sequence alignment, and peptide binding-ELISA. The newly identified epitope is shared by PEDV G1 and G2 strains and other alphacoronaviruses. In summary, mAbA3 may be useful as a ready-to-use antibody for passive immunization of PEDV-susceptible piglets, while the novel neutralizing epitope, together with other, previously known protective epitopes, have potential as an immunogenic cocktail for a safe, next-generation PEDV vaccine.

Rabies is a deadly viral disease caused by the rabies virus (RABV), transmitted through a bite of an infected host, resulting in irreversible neurological symptoms and a 100% fatality rate in humans. Despite many aspects describing rabies neuropathogenesis, numerous hypotheses remain unanswered and concealed. Observations obtained from infected primary neurons or mouse brain samples are more relevant to human clinical rabies than permissive cell lines; however, limitations regarding the ethical issue and sample accessibility become a hurdle for discovering new insights into virus-host interplays. To better understand RABV pathogenesis in humans, we generated human-induced pluripotent stem cell (hiPSC)-derived neurons to offer the opportunity for an inimitable study of RABV infection at a molecular level in a pathologically relevant cell type. This study describes the characteristics and detailed proteomic changes of hiPSC-derived neurons in response to RABV infection using LC-MS/MS quantitative analysis. Gene ontology (GO) enrichment of differentially expressed proteins (DEPs) reveals temporal changes of proteins related to metabolic process, immune response, neurotransmitter transport/synaptic vesicle cycle, cytoskeleton organization, and cell stress response, demonstrating fundamental underlying mechanisms of neuropathogenesis in a time-course dependence. Lastly, we highlighted plausible functions of heat shock cognate protein 70 (HSC70 or HSPA8) that might play a pivotal role in regulating RABV replication and pathogenesis. Our findings acquired from this hiPSC-derived neuron platform help to define novel cellular mechanisms during RABV infection, which could be applicable to further studies to widen views of RABV-host interaction.

Foot-and-mouth-disease virus (FMDV) is a picornavirus that causes a highly contagious disease of cloven-hoofed animals resulting in economic losses worldwide. The 3C protease (3C pro ) is the main protease essential in the picornavirus life cycle, which is an attractive antiviral target. Here, we used computer-aided virtual screening to filter potential anti-FMDV agents from the natural phytochemical compound libraries. The top 23 filtered compounds were examined for anti-FMDV activities by a cell-based assay, two of which possessed antiviral effects. In the viral and post-viral entry experiments, luteolin and isoginkgetin could significantly block FMDV growth with low 50% effective concentrations (EC50). Moreover, these flavonoids could reduce the viral load as determined by RT-qPCR. However, their prophylactic activities were less effective. Both the cell-based and the fluorescence resonance energy transfer (FRET)-based protease assays confirmed that isoginkgetin was a potent FMDV 3C pro inhibitor with a 50% inhibition concentration (IC50) of 39.03 ± 0.05 and 65.3 ± 1.7 μM, respectively, whereas luteolin was less effective. Analyses of the protein-ligand interactions revealed that both compounds fit in the substrate-binding pocket and reacted to the key enzymatic residues of the 3C pro . Our findings suggested that luteolin and isoginkgetin are promising antiviral agents for FMDV and other picornaviruses.

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CL pro ), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CL pro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CL pro activities with IC 50 of 3.57 ± 0.36 μM to 25.90 ± 1.40 μM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 μM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC 50 (6.11 ± 1.90 to 7.75 ± 0.48 μM and 1.99 ± 0.30 to 4.03 ± 0.60 μM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CL pro -ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important porcine viruses worldwide. Recently, severe PRRS outbreaks had occurred in two farms located in eastern and southern Thailand where stringent vaccination had been routinely practiced. Genetic analysis of GP5 identified two highly virulent PRRSVs designated as NA/TH/S001/2015 and NA/TH/E001/2016 from the southern and eastern farms, respectively. Both incidences were the first outbreaks of severe PRRSV since the implementation of the modified live virus (MLV) vaccine, indicating the concurrent emergence of immune-escape viruses. The genetics of the two PRRSV variants, the previous studied sequences from Thailand, and the reference strains were characterized with a focus on the GP5 and NSP2 genes. The results indicated that NA/TH/S001/2015 and NA/TH/E001/2016 shared less than 87% nucleotide similarity to the MLV and PRRSV type 2, lineages 1 and 8.7 (NA), respectively. A comparative analysis of the retrospective GP5 sequences categorized the PRRSVs into five groups based on the clinical outcomes, and both of the novel PRRSV strains were in the same group. Epitope A, T cell epitope, and N-linked glycosylation patterns within GP5 of both PRRSV variants were highly variable and significantly differed from those of MLV. As observed in highly virulent type 2 strains, NA/TH/S001/2015 contained a single amino acid deletion at position 33 in the hypervariable region 1 (HV-1) of GP5. Amino acid analysis of the hypervariable region of NSP2 revealed that NA/TH/E001/2016 had a unique deletion pattern that included two discontinuous deletions: a 127-amino acid deletion from residues 301 to 427 and a single amino acid deletion at position 470. These results indicate the emergence of two novel PRRSV strains and highlight the common genetic characteristics of the immune-escaping PRRSV variants., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The computational search of chemical libraries has been used as a powerful tool for the rapid discovery of candidate compounds. To find small molecules with anti-feline infectious peritonitis virus (FIPV) properties, we utilized a virtual screening technique to identify the active site on the viral protease for the binding of the available natural compounds. The protease 3CL (3CL pro ) plays an important role in the replication cycle of FIPV and other viruses within the family Coronaviridae. The 15 best-ranked candidate consensus compounds, based on three docking tools, were evaluated for further assays. The protease inhibitor assay on recombinant FIPV 3CL pro was performed to screen the inhibitory effect of the candidate compounds with IC 50 ranging from 6.36 ± 2.15 to 78.40 ± 2.60 μM. As determined by the cell-based assay, the compounds NSC345647, NSC87511, and NSC343256 showed better EC 50 values than the broad-spectrum antiviral drug ribavirin and the protease inhibitor lopinavir, under all the test conditions including pre-viral entry, post-viral entry, and prophylactic activity. The NSC87511 particularly yielded the best selective index (>4; range of SI = 13.80-22.90). These results indicated that the natural small-molecular compounds specifically targeted the 3CL pro of FIPV and inhibited its replication. Structural modification of these compounds may generate a higher anti-viral potency for the further development of a novel therapy against FIP., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Generation of stable cell lines is a widely used technique for continuous recombinant protein production. Advantages of the constitutive stable over the transient protein expression are uniformity of the expression across cell populations as well as high quantity and consistency of the protein yields. This data describe step-by-step procedure for the production of glycoprotein without a transmembrane domain (GΔTM) of bovine ephemeral fever virus (BEFV) by mammalian stable cells. LentiX-293T cells were transfected with four plasmid constructs to generate a recombinant lentivirus. Subsequently, 293T cells were transduced by the recombinant virus and the polyclonal stable cell pools were then selected by puromycin. Next, limiting dilution was performed from each cell pool to isolate the monoclonal stable cells expressing GΔTM protein. Western blot analysis showed that all monoclonal cell clones could stably express GΔTM protein. The data confirms that the stable 293T cell line expressing the secretory GΔTM protein is an attractive platform for antigen production., (© 2019 The Author(s).)

Bovine ephemeral fever virus (BEFV) causes an acute febrile disease in cattle and water buffalo. The disease has an impact on dairy and beef production in tropical and subtropical countries. Vaccination is used for disease prevention and control. In this study, we developed a recombinant lentivirus to produce mammalian stable cells expressing histidine-tagged BEFV G protein with a deleted transmembrane domain (GΔTM) as a secretory protein. In addition, guinea pigs were immunised with the purified GΔTM protein and booster immunised at a 3-week interval. The mammalian stable cells were able to continuously produce GΔTM protein for a minimum of 25 passages. All of the mammalian stable cells expressing GΔTM protein could react specifically with a BEFV convalescent bovine serum. Serum samples from the immunised guinea pigs could react strongly and specifically with the purified GΔTM protein. Moreover, post-immunised guinea pig sera contained antibodies that could neutralise BEFV. These results indicate that the G protein without a transmembrane domain can be used as a subunit vaccine for the prevention and control of BEFV. The availability of the mammalian stable cells, which constitutively express GΔTM protein, could facilitate the potential use of the secretory protein for BEFV diagnosis and vaccine development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine ephemeral fever (BEF) is an arthropod-borne disease caused by bovine ephemeral fever virus (BEFV), a negative sense, single-stranded RNA virus. BEFV is endemic in tropical and sub-tropical regions including Thailand, a country in mainland Southeast Asia. However, there are few studies on BEFV and no available information regarding molecular characteristics of BEFV in Thailand. Therefore, the aims of this study were to genetically characterize Thai BEFVs and reveal their evolutions by phylogenetic analysis of G gene ectodomain sequences. From 2013 to 2017, blood samples were collected from bovine that matched with BEF case definition from three regions of Thailand. Thai BEFV G genes and a whole genome of an isolate, East Asia/TH/LRI0045/2016, were sequenced and characterized. Additionally, their phylogenies were constructed. This is the first report on genetics of BEFV in Southeast Asia. G ectodomain encoding region of Thai BEFV found during 2013-2017 are closely related to the second and third sub-clades of East Asia lineage. In addition, we observed mutation in the putative P' ORF of all Thai BEFVs which generated a premature stop codon. Thai G gene sequences are closely related to those of mainland Chinese and Taiwanese isolates. The whole genomic sequences of Thai BEFV and East Asia/China/JT02 L/2002 possess common characteristics, suggesting shared evolutionary relationship between East and Southeast Asian strains. Further studies on relationship between animal translocation, circulation of BEFV in Greater Mekong subregion and acquisition of more G gene sequences may improve understanding of BEFV epidemiology in mainland Southeast Asia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Porcine circovirus type 2 (PCV2), the essential cause of porcine circovirus associated disease (PCVAD), has evolved rapidly and it has been reported worldwide. However, genetic information of PCV2 in Thailand has not been available since 2011. Herein, we studied occurrence and genetic diversity of PCV2 in Thailand and their relationships to the global PCV2 based on ORF2 sequences. The results showed that 306 samples (44.09%) from 56 farms (80%) were PCV2 positive by PCR. Phylogenetic trees constructed by both neighbor-joining and Bayesian Inference yielded similar topology of the ORF2 sequences. Thai PCV2 comprise four clusters: PCV2a (5.5%), PCV2b (29.41%), intermediate clade 1 (IM1) PCV2b (11.03%) and PCV2d (54.41%). Genetic shift of PCV2 in Thailand has occurred similarly to the global situation. The shift from PCV2b to PCV2d was clearly observed during 2013-2014. The viruses with genetically similar to the first reported PCV2 in 2004 have still circulated in Thailand. The first Thai PCV2b and PCV2d were closely related to the neighboring countries. The haplotype network analysis revealed the relationship of PCV2 in Thailand and other countries. These results indicate that genetic diversity of PCV2 in Thailand is caused by genetic drift of the local strains and intermittent introduction of new strains or genotypes from other countries. Genetic evolution of PCV2 in Thailand is similar to that occurs globally., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The nonstructural protein 1 (NS1) of influenza A viruses can act as a viral replication enhancer by antagonizing type I interferon (IFN) induction and response in infected cells. We previously reported that A/Puerto Rico/8/1934 (H1N1) (PR8) containing the NS1 gene derived from A/swine/IA/15/1930 (H1N1) (IA30) replicated more efficiently than the wild type virus. Here, we identified amino acids in NS1 critical for enhancing viral replication., Methods: To identify a key amino acid in NS1 which can increase the virus replication, growth kinetics of PR8 viruses encoding single mutation in NS1 were compared in A549 cells. NS1 mutant functions were studied using dsRNA-protein pull down, RIG-I mediated IFNβ-promoter activity assays and growth curve analysis in murine lung epithelial type I (Let1) cells., Results: The G45R mutation in the NS1 of PR8 (G45R/NS1) virus is critical for the enhanced viral replication in A549 cells. G45R/NS1 slightly decreased NS1 binding to dsRNA but did not interfere with its suppression of RIG-I-mediated type I IFN production. Likewise, replication of G45R/NS1 virus was increased in comparison to wild type virus in both wild type and type I interferon receptor null Let1 cells., Conclusions: The non-conserved amino acid, R45, enhances viral replication which is apparently independent of dsRNA binding and suppression of type I IFN, suggesting a non-characterized function of NS1 for the enhanced viral replication. As G45R/NS1 virus induced the type I IFN induction and response in infected A549 cells, it is also interesting to investigate virus virulence for further studies.

Porcine reproductive and respiratory syndrome virus (PRRSV) affects the swine industry worldwide. Annual surveillances taken from 2008 to 2013 revealed a 13.86% prevalence of PRRSVs in swine populations in Thailand. The selected positive samples were genetically characterized based on global systems and phylogenetic trees that were constructed using 967 ORF5 samples from this study, the collective sequences from Thailand and Southeast Asia and reference sequences. The results showed that both types I and II have been circulating in Thai swine and that genotype II was more prevalent than genotype I. Only type II was found in other countries in Southeast Asia. Type I PRRSVs from Thailand are clustered in subtype 1, clades A, D and H. Type II PRRSVs are topologically classified in lineage 1 and sublineages 5.1, 5.2 and 8.7, of which sublineage 8.7 was predominant, especially after 2010. PRRSVs in sublineage 8.7 are divided into two groups: classical NA and HP-PRRSV. An analysis of all HP-PRRSVs in Southeast Asia revealed four separate clades--A (SX2009-like), B (09HEN1-like), JXA1-like and GXFCH08-like--reflecting four different introductions of these viruses into Thailand, Lao PDR, Cambodia and Vietnam. HP-PRRSV first appeared in Thailand and Cambodia in 2008, 2 years before the first epidemic outbreaks. Recently, the genetics of PRRSVs in Southeast Asia have become more diverse. Thus, PRRSV genetics must be continually characterized and phylogenetically analyzed using global systematic classifications to provide annual genetic information for PRRS control and vaccine selection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Foot-and-mouth disease virus (FMDV) is an animal pathogen of great concern. It is contagious to cloven-hoofed animals and affects animals in extensive areas worldwide. In general, the primary eradication strategies for foot-and-mouth disease (FMD) in Japan are stamping out the disease and restriction of movement. It is also important to completely disinfect the infected area to prevent the spread of FMDV, including vehicles and people as well. However, there is no report on the effect of commercially available disinfectants against FMDV in a short contact time. In this study, we evaluated the virucidal effect of thirteen commercially available products, and got the following results: acidic ethanol disinfectants, alkaline cleaners and sodium hypochlorite had great effect (>3.0 log10 reduction in titer) against FMDV. On the other hand, neutral ethanol disinfectants, hand soaps, and quaternary ammonium compound sanitizers did not show great effect against FMDV. Therefore, it is presumed that acidic ethanol disinfectants are effective for human use and alkaline cleaners are effective for use in the infected environment for the control of a FMD outbreak.