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

Semkum, Ploypailin / Thangthamniyom, Nattarat / Chankeeree, Penpitcha / Keawborisuth, Challika / Theerawatanasirikul, Sirin / Lekcharoensuk, Porntippa

Vaccines

2023 Volume 11, Issue 6

Abstract: 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 ... ...

Abstract	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.
Language	English
Publishing date	2023-06-18
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2703319-3
ISSN	2076-393X
ISSN	2076-393X
DOI	10.3390/vaccines11061111
Database	MEDical Literature Analysis and Retrieval System OnLINE

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

Theerawatanasirikul, Sirin / Lueangaramkul, Varanya / Thangthamniyom, Nattarat / Chankeeree, Penpitcha / Semkum, Ploypailin / Lekcharoensuk, Porntippa

Animals. 2022 Aug. 07, v. 12, no. 15

2022

Abstract: 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 ... ...

Abstract	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.
Keywords	Foot-and-mouth disease virus ; active sites ; andrographolide ; antagonists ; antiviral properties ; genes ; inhibitory concentration 50 ; interferons ; livestock ; median effective concentration ; proteinases ; serotypes ; vaccination
Language	English
Dates of publication	2022-0807
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2606558-7
ISSN	2076-2615
ISSN	2076-2615
DOI	10.3390/ani12151995
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Non-Nucleoside Inhibitors Decrease Foot-and-Mouth Disease Virus Replication by Blocking the Viral 3D

Theerawatanasirikul, Sirin / Semkum, Ploypailin / Lueangaramkul, Varanya / Chankeeree, Penpitcha / Thangthamniyom, Nattarat / Lekcharoensuk, Porntippa

Viruses

2022 Volume 15, Issue 1

Abstract: Foot-and-mouth disease virus (FMDV), an economically important pathogen of cloven-hoofed livestock, is a positive-sense, single-stranded RNA virus classified in ... ...

Abstract	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
MeSH term(s)	Animals ; Foot-and-Mouth Disease Virus/genetics ; Antiviral Agents/pharmacology ; Antiviral Agents/metabolism ; RNA-Dependent RNA Polymerase/metabolism ; Foot-and-Mouth Disease ; Virus Replication
Chemical Substances	Antiviral Agents ; RNA-Dependent RNA Polymerase (EC 2.7.7.48)
Language	English
Publishing date	2022-12-30
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v15010124
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Article: Andrographolide and Deoxyandrographolide Inhibit Protease and IFN-Antagonist Activities of Foot-and-Mouth Disease Virus 3C

Theerawatanasirikul, Sirin / Lueangaramkul, Varanya / Thangthamniyom, Nattarat / Chankeeree, Penpitcha / Semkum, Ploypailin / Lekcharoensuk, Porntippa

Animals : an open access journal from MDPI

2022 Volume 12, Issue 15

Abstract	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
Language	English
Publishing date	2022-08-07
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2606558-7
ISSN	2076-2615
ISSN	2076-2615
DOI	10.3390/ani12151995
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Natural Phytochemicals, Luteolin and Isoginkgetin, Inhibit 3C Protease and Infection of FMDV, In Silico and In Vitro

Theerawatanasirikul, Sirin / Thangthamniyom, Nattarat / Kuo, Chih-Jung / Semkum, Ploypailin / Phecharat, Nantawan / Chankeeree, Penpitcha / Lekcharoensuk, Porntippa

Viruses. 2021 Oct. 21, v. 13, no. 11

2021

Abstract: 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 (3Cᵖʳᵒ) is the main protease essential in the picornavirus life cycle, which is ...

Abstract	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 (3Cᵖʳᵒ) 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 3Cᵖʳᵒ 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 3Cᵖʳᵒ. Our findings suggested that luteolin and isoginkgetin are promising antiviral agents for FMDV and other picornaviruses.
Keywords	computer simulation ; energy transfer ; fluorescence ; foot-and-mouth disease ; inhibitory concentration 50 ; luteolin ; median effective concentration ; proteinases ; viral load ; viruses
Language	English
Dates of publication	2021-1021
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v13112118
Database	NAL-Catalogue (AGRICOLA)

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

Semkum, Ploypailin / Kaewborisuth, Challika / Thangthamniyom, Nattarat / Theerawatanasirikul, Sirin / Lekcharoensuk, Chalermpol / Hansoongnern, Payuda / Ramasoota, Pongrama / Lekcharoensuk, Porntippa

Viruses. 2021 June 01, v. 13, no. 6

2021

Abstract: 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 ... ...

Abstract	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.
Keywords	DNA-directed RNA polymerase ; Foot-and-mouth disease virus ; antiviral agents ; foot-and-mouth disease ; genes ; humans ; plasmids ; research and development ; reverse genetics ; transfection
Language	English
Dates of publication	2021-0601
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v13061047
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Natural Phytochemicals, Luteolin and Isoginkgetin, Inhibit 3C Protease and Infection of FMDV, In Silico and In Vitro.

Theerawatanasirikul, Sirin / Thangthamniyom, Nattarat / Kuo, Chih-Jung / Semkum, Ploypailin / Phecharat, Nantawan / Chankeeree, Penpitcha / Lekcharoensuk, Porntippa

Viruses

2021 Volume 13, Issue 11

Abstract: 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 ( ... ...

Abstract	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
MeSH term(s)	3C Viral Proteases/antagonists & inhibitors ; 3C Viral Proteases/chemistry ; 3C Viral Proteases/genetics ; 3C Viral Proteases/metabolism ; Animals ; Antiviral Agents/chemistry ; Antiviral Agents/pharmacology ; Biflavonoids/chemistry ; Biflavonoids/pharmacology ; Computer Simulation ; Enzyme Inhibitors/chemistry ; Enzyme Inhibitors/pharmacology ; Foot-and-Mouth Disease/virology ; Foot-and-Mouth Disease Virus/chemistry ; Foot-and-Mouth Disease Virus/drug effects ; Foot-and-Mouth Disease Virus/enzymology ; Foot-and-Mouth Disease Virus/genetics ; Humans ; Luteolin/chemistry ; Luteolin/pharmacology ; Phytochemicals/chemistry ; Phytochemicals/pharmacology
Chemical Substances	Antiviral Agents ; Biflavonoids ; Enzyme Inhibitors ; Phytochemicals ; isoginkgetin ; 3C Viral Proteases (EC 3.4.22.28) ; Luteolin (KUX1ZNC9J2)
Language	English
Publishing date	2021-10-21
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v13112118
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production.

Semkum, Ploypailin / Kaewborisuth, Challika / Thangthamniyom, Nattarat / Theerawatanasirikul, Sirin / Lekcharoensuk, Chalermpol / Hansoongnern, Payuda / Ramasoota, Pongrama / Lekcharoensuk, Porntippa

Viruses

2021 Volume 13, Issue 6

Abstract	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.
MeSH term(s)	Animals ; Antiviral Agents/chemistry ; Antiviral Agents/pharmacology ; Cell Line ; Foot-and-Mouth Disease/drug therapy ; Foot-and-Mouth Disease/virology ; Foot-and-Mouth Disease Virus/drug effects ; Foot-and-Mouth Disease Virus/genetics ; Gene Expression Regulation, Viral ; Gene Order ; Genome, Viral ; Humans ; Models, Molecular ; Molecular Structure ; Plasmids/genetics ; RNA, Messenger/chemistry ; RNA, Messenger/genetics ; Structure-Activity Relationship ; Transfection ; Virus Replication/drug effects
Chemical Substances	Antiviral Agents ; RNA, Messenger
Language	English
Publishing date	2021-06-01
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v13061047
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Molecular characterization of bovine ephemeral fever virus in Thailand between 2013 and 2017.

Chaisirirat, Thanawat / Sangthong, Pradit / Arunvipas, Pipat / Petcharat, Nantawan / Thangthamniyom, Nattarat / Chumsing, Wilairat / Lekcharoensuk, Porntippa

Veterinary microbiology

2018 Volume 227, Page(s) 1–7

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Animals ; Antibodies, Viral/blood ; Asia, Southeastern/epidemiology ; Cattle ; Cattle Diseases/epidemiology ; Cattle Diseases/virology ; Ephemeral Fever/blood ; Ephemeral Fever/epidemiology ; Ephemeral Fever/virology ; Ephemeral Fever Virus, Bovine/genetics ; Ephemeral Fever Virus, Bovine/isolation & purification ; Genome, Viral ; Mutation ; Open Reading Frames/genetics ; Phylogeny ; RNA, Viral/genetics ; Thailand/epidemiology ; Viral Proteins/genetics ; Whole Genome Sequencing
Chemical Substances	Antibodies, Viral ; RNA, Viral ; Viral Proteins
Language	English
Publishing date	2018-10-15
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	753154-0
ISSN	1873-2542 ; 0378-1135
ISSN (online)	1873-2542
ISSN	0378-1135
DOI	10.1016/j.vetmic.2018.10.013
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Genetic diversity of porcine circovirus type 2 (PCV2) in Thailand during 2009–2015

Thangthamniyom, Nattarat / Alongkot Boonsoongnern / Nantawan Petcharat / Narut Thanantong / Pariwat Poolperm / Payuda Hansoongnern / Ploypailin Semkum / Porntippa Lekcharoensuk / Pradit Sangthong

Veterinary microbiology. 2017 Sept., v. 208

2017

Abstract: 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 ...

Abstract	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.
Keywords	Bayesian theory ; farms ; genetic drift ; genetic similarity ; genetic variation ; haplotypes ; phylogeny ; polymerase chain reaction ; Porcine circovirus ; Porcine circovirus-2 ; topology ; viruses ; Thailand
Language	English
Dates of publication	2017-09
Size	p. 239-246.
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	753154-0
ISSN	1873-2542 ; 0378-1135
ISSN (online)	1873-2542
ISSN	0378-1135
DOI	10.1016/j.vetmic.2017.08.006
Database	NAL-Catalogue (AGRICOLA)

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