Genetic characterization of porcine circovirus type 2 in piglets from PMWS-affected and -negative farms in Thailand

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

Novel constructs and 1-step chromatography protocols for the production of Porcine Circovirus 2d (PCV2d) and Circovirus 3 (PCV3) subunit vaccine candidates

Porcine circovirus type 2 (PCV2) has been a major problem for the pig production industry worldwide for decades. While the majority of commercially available vaccines are based on the original PCV2a genotype, the current dominant genotype is PCV2d. The notable differences between genotypes could lead to incomplete cross-protection. Moreover, most current subunit PCV2 vaccines are generated from expensive insect cell culture technology. In this work, we present a new workflow for production of an updated and relatively inexpensive PCV2d vaccine candidate. After expression in fed-batch Escherichia coli fermentation systems with a simple one-step ion-exchange chromatography purification protocol, the yield of purified PCV2d-based antigen reached over 1 g per litre bacterial culture. Using similar procedures, we also demonstrated even higher PCV2d-based antigen yields from a chimeric PCV2d-PCV3 capsid construct, which is cleaved during fermentation to release PCV2d- and PCV3-related polypeptides. Although the PCV2d-based recombinant protein from this protocol did not form viral-like particles as analysed by size-exclusion chromatography, it could effectively induce capsid-specific and PCV2d-neutralising antibodies in immunised animals, indicating significant potential as a new vaccine candidate that can be easily manufactured at commercial scale

Novel Swine Influenza Virus Subtype H3N1, United States

A new subtype H3N1 may have arisen from reassortment of a H3N2 turkey isolate, a human H1N1 isolate, and currently circulating swine viruses

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses

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 (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro 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 3CLpro activities with IC50 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 EC50 (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 3CLpro-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

A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production

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

In silico and in vitro analysis of small molecules and natural compounds targeting the 3CL protease of feline infectious peritonitis virus

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 (3CLpro) 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 3CLpro was performed to screen the inhibitory effect of the candidate compounds with IC50 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 EC50 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 3CLpro 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

Non-Nucleoside Inhibitors Decrease Foot-and-Mouth Disease Virus Replication by Blocking the Viral 3D^pol

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 (3Dpol). From 5596 compounds, the blind- followed by focus-docking filtered 21 candidates fitting in the 3Dpol 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 3Dpol activity in the cell-based 3Dpol inhibition assay with small IC50 values ranging from 0.8 nM to 0.22 µM without an effect on FMDV’s main protease, 3Cpro. The 3Dpol 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 3Dpol inhibitors that bound within the enzyme active sites and blocked viral replication. These compounds might be beneficial for FMDV or other picornavirus treatment

Andrographolide and Deoxyandrographolide Inhibit Protease and IFN-Antagonist Activities of Foot-and-Mouth Disease Virus 3Cpro

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 3Cpro 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 3Cpro by derepressing interferon-stimulating gene (ISGs) expression. The molecular docking confirmed that the andrographolides preferentially interacted with the 3Cpro 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 3Cpro and hindered its protease and IFN antagonist activities

The Application of the Gibson Assembly Method in the Production of Two pKLS3 Vector-Derived Infectious Clones of Foot-and-Mouth Disease Virus

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

Small Molecules Targeting 3C Protease Inhibit FMDV Replication and Exhibit Virucidal Effect in Cell-Based Assays

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 (3Cpro) 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 3Cpro inhibition with IC50 values of 10.85 µM (NSC116640) and 4.21 µM (NSC332670). The molecular docking of the compounds on FMDV 3Cpro showed their specific interactions with amino acids in the catalytic triad of FMDV 3Cpro. 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