Classical swine fever virus (CSFV) is the causative agent of Classical swine fever which is a highly contagious disease affecting swine and resulting in severe economic losses. In this study, we developed reverse transcription loopmediated isothermal amplification (RT-LAMP) assay targeting the 5'UTR gene for the detection of CSFV. This amplification method can be obtained in 1 h under isothermal conditions (65°C) employing a set of six specific primers mixtures. Amplification product was visualized by using hydroxynaphthol blue (HNB) dye and agarose gel electrophoresis. The sensitivity was 100 copy numbers. No cross-reactivity related to Japanese encephalitis virus (JEV) and porcine reproductive and respiratory syndrome virus (PR-RSV) was demonstrated. The results demonstrated that the RT-LAMP assay is a useful tool for the rapid and sensitive for CSFV detection in swine.
The aim of this study is to evaluate cellular interaction between free-living amoebae Naegleria fowleri strains and mammalian target cells in vitro. Two Thai strains of N. fowleri; Khon Kaen strain from the environment and Siriraj strain from the patient's cerebrospinal fluid and the Center of Disease Control VO 3081 strain from Atlanta (US) were studied. Human neuroblastoma (SK-N-MC) and African Green monkey Kidney (Vero) cells were used as target cells. Each cell line was inoculated with each strain of N. fowleri at a ratio of 1:1 and observed for 7 days. The uninoculated target cells and each strain of N. fowleri were used as control. The numbers of the challenged and unchallenged cells as well as the free-living amoebae were counted three times by trypan blue exclusion method. The inoculation began when the amoebae attached to the cell membrane and ingested the target cells. In this study, extensive cytopathogenesis with many floating inoculated cells and abundant number of amoebae were observed. The destruction pattern of both inoculated SK-N-MC and Vero target cells were similar. Interestingly, SK-N-MC was more susceptible to N. fowleri strains than the Vero cell. In addition, N. fowleri Siriraj strain showed the highest destruction pattern for each target cell. Our findings suggest that the SK-N-MC should be used as a base model for studying the neuropathogenesis in primary amoebic meningoencephalitis patients.
Dengue viruses cause 50-100 million cases of acute febrile disease every year, including more than 500000 reported cases of the severe forms of the disease-dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Attempts to create conventional vaccines have been hampered by the lack of suitable experimental models, the need to provide protection against all four serotypes simultaneously and the possible involvement of virus-specific immune responses in severe disease. Live attenuated D2 16681-PDK53 vaccine was first developed from Mahidol University, Thailand. This vaccine induced both humoral and cell-mediated immunity and lack of reactogeneticity in humans. Infectious cDNA clones of the virulent D2 16681 virus and its attenuated D2 16681-PDK53 were constructed. The attenuated virus elicited neutralizing antibodies in mice and monkeys and developed viremia in monkeys. At molecular level, patterns of cytokines which are immunological mediators released from human mononuclear cells obtained from dengue naïve and immune donors infected with this attenuated virus compared with virulent virus were studied. In dengue naïve PBMC, the virulent and attenuated clones induced alternation in expression of 25 and 24 versus 13 and 18 genes out of 268 genes on day 1 and 3. In dengue immune PBMC, the virulent and attenuated clones induced alternation in expression of 33 and 38 versus 25 and 29 genes on days 1 and 3. Up-regulation of IL-1beta, IL-6, IL-8, IL-10, IFN-alpha, IFNgammaR, MIP-1alpha, MIP-1beta, MIP-2alpha, VEGF and down-regulation of IL-4, IL-4R, IL-RII, MIF, RANTES, IGF-1, GM-CSF-2 were shown. This review pointed out the infectious clones of the attenuated D2 16681-PDK53 was safe and induced both neutralizing antibodies in vivo and cytokine gene expression in vitro at molecular level. Furthermore, the phenotypic markers of ideal dengue vaccine could be included the alteration of cytokine gene expression and cytokine production in human mononuclear cells.
Dermatophagoides farinae and D. pteronyssinus are the prevalent house dust mites (HDM) in tropical countries and are associated with allergic diseases. This investigation developed a time- resolved immunofluorometric assay (TR-IFMA) for the first time to detect specific IgE antibody in patients with skin prick test positive to HDM but no detectable IgE by other means. Levels of IgE to natural and recombinant HDM allergens were measured by TR-IFMA in 50 HDM-allergic patients and 19 healthy participants compared to sandwich enzyme-linked immunosorbent assay (ELISA). A recombinant allergen, rDerf2, showed a 14 kDa band corresponding to broad range proteins of natural HDM.TR-IFMA showed sensitivity lower than 0.35 kUA/l. TR-IFMA employing three HDM antigens showed good correlations with sandwich ELISA at R2 0.93-0.96. TR-IFMA detected HDM IgE in 62, 62, 25 percent of allergic patient serum sample compared to 28, 32, and 22 percent detected by ELISA result using three HDM allergen. TR-IFMA also detected 26.3, 31.6, and 5.3 percent positive samples from 19 healthy participants while ELISA showed 0, 5.3, and 0 percents IgE positive samples. The use of rDerf2 as an HDM allergen for the assay was verified with no statistically different from other HDM allergens. TR-IFMA showed lower detection limit than ELISA and yielded higher sensitivity for serum of people with allergic symptoms with no detectable HDM IgE. It is anticipated that TR-IFMA for HDM-specific IgE detection will play an important role in future diagnosis of HDM allergy in clinical laboratories and for different research purposes
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