Porntippa Lekcharoensuk

Publications (20) View all

• Article: Development of an inactivated 3C(pro)-3ABC (mu3ABC) ELISA to differentiate cattle infected with Foot and mouth disease virus from vaccinated cattle.

  Vasinee Srisombundit, Nattarat Tungthumniyom, Wilai Linchongsubongkoch, Chalermpol Lekcharoensuk, Ladawan Sariya, Pongrama Ramasoota, Porntippa Lekcharoensuk
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  ABSTRACT: Foot and mouth disease, a highly contagious disease of cloven-hoofed animals, is still endemic in Asia, Africa, and a few countries in South America. Subclinical and persistent infections usually occur in vaccinated cattle exposed to FMDV. Successful control and eradication measures need a diagnostic assay that can distinguish between immune responses to infection and vaccination. The non-structural 3ABC ELISA is the most reliable differential diagnostic assay. However, expression of the native 3ABC gene in insect cells yielded truncated versions of the proteins; thus, a monoclonal antibody to capture digested proteins is needed to develop the assay. The purpose of this study was to develop a simple indirect 3ABC ELISA using complete 3ABC protein. The full-length mutated 3ABC protein with inactive 3C(pro) (mu3ABC) gene was constructed. The histidine-tagged mu3ABC protein was produced in insect cells for easy purification and measuring. This permits simple assay design and reproducible assay development. mu3ABC ELISA had diagnostic specificity and sensitivity of 96.6% and 84%, respectively, compared to Ceditest(®) FMDV-NS. Agreement of both assays was excellent with κ value of 0.823 (p<0.05). The mu3ABC ELISA could distinguish infected from vaccinated animals.These factors are necessary for the successful development of an in-house NSP-based ELISA. Availability of a reliable assay with acceptable costs would facilitate successful disease control and the establishment of disease-free zones. Expansion of such zones may ultimately decrease the risk of introducing FMDV into disease-free countries, thus accelerating global FMD control.
  Journal of virological methods 01/2013; · 2.13 Impact Factor
• Article: Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses.

  Porntippa Lekcharoensuk, Witthawat Wiriyarat, Nantawan Petcharat, Chalermpol Lekcharoensuk, Prasert Auewarakul, Juergen A Richt
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  ABSTRACT: Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems.
  Vaccine 02/2012; 30(8):1453-9. · 3.77 Impact Factor
• Article: Expression of foot and mouth disease virus nonstructural polyprotein 3ABC with inactive 3C(pro) in Escherichia coli.

  Ladawan Sariya, Nattarat Thangthumniyom, Worawidh Wajjwalku, Wilairat Chumsing, Pongrama Ramasoota, Porntippa Lekcharoensuk
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  ABSTRACT: Nonstructural 3ABC protein of foot and mouth disease virus (FMDV) was widely used to differentiate vaccinated from natural FMDV-infected animals. 3ABC is a polyprotein which is auto-processed to 3A, three copies of 3B and 3C(pro) by 3C(pro) protease. The 3ABC gene was cloned and expressed in Escherichia coli as native or mutated 3ABC (mu3ABC) forms. Cysteine residues 142 and 163 of the catalytic triad within the 3C(pro) of mu3ABC were changed to serine and glycine, respectively, to inhibit its protease activity. Both native and mutated 3ABC ORFs were cloned into BamHI and HindIII restriction sites of an expression vector, pQE80L. The expression of the recombinant native 3ABC and mu3ABC genes in E. coli BL21 was induced with 0.2mM isopropyl-beta-d-thiogalactopyranoside at 37 °C for 5h. SDS-PAGE and Western blot analysis revealed that the full length 3ABC was present in the lysate from mu3ABC but not native 3ABC transformed cells. The recombinant mu3ABC was expressed mainly in the inclusion body and presented as monomer and dimer. In addition, the mu3ABC reacted strongly with a convalescent serum from a natural FMDV-infected cattle but very weakly with a serum from vaccinated cattle. This study clearly demonstrates that successful expression of the full length 3ABC occurs only when the protease active sites within the 3C(pro) were completely abolished. This information would accelerate in house development of the 3ABC-based diagnostic test that can distinguish between vaccinated and FMDV-infected animals.
  Protein Expression and Purification 07/2011; 80(1):17-21. · 1.59 Impact Factor
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  Available from: Pariwat Poolperm

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

  Tippawan Jantafong, Alongkot Boonsoongnern, Pariwat Poolperm, Kitcha Urairong, Chalermpol Lekcharoensuk, Porntippa Lekcharoensuk
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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.
  Virology Journal 02/2011; 8:88. · 2.34 Impact Factor
• Article: Pathogenicity of swine influenza viruses possessing an avian or swine-origin PB2 polymerase gene evaluated in mouse and pig models.

  Wenjun Ma, Kelly M Lager, Xi Li, Bruce H Janke, Derek A Mosier, Laura E Painter, Eva S Ulery, Jingqun Ma, Porntippa Lekcharoensuk, Richard J Webby, Jürgen A Richt
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  ABSTRACT: PB2 627K is a determinant of influenza host range and contributes to the pathogenicity of human-, avian-, and mouse-adapted influenza viruses in the mouse model. Here we used mouse and pig models to analyze the contribution of a swine-origin and avian-origin PB2 carrying either 627K or 627E in the background of the classical swine H1N1 (A/Swine/Iowa/15/30; 1930) virus. The results showed PB2 627K is crucial for virulence in the mouse model, independent of whether PB2 is derived from an avian or swine influenza virus (SIV). In the pig model, PB2 627E decreases pathogenicity of the classical 1930 SIV when it contains the swine-origin PB2, but not when it possesses the avian-origin PB2. Our study suggests the pathogenicity of SIVs with different PB2 genes and mutation of codon 627 in mice does not correlate with the pathogenicity of the same SIVs in the natural host, the pig.
  Virology 11/2010; 410(1):1-6. · 3.35 Impact Factor