Five methods for the RNA detection of rabies virus were directly compared in this study. These in... more Five methods for the RNA detection of rabies virus were directly compared in this study. These included conventional nucleic acid sequence-based amplification with electrochemiluminescence (NASBA-ECL) assay, reverse transcription (RT)-heminested (hn) polymerase chain reaction (PCR) and TaqMan real-time RT-PCR using protocols as described previously. The first two methods have been routinely utilised for ante-mortem diagnosis of human rabies in Thailand and other rabies-endemic Asian and African countries. In addition, two real-time NASBA assays based on the use of a NucliSens EasyQ analyser (NASBA-Beacon-EQ) and LightCycler real-time PCR machine (NASBA-Beacon-LC) were studied in parallel. All methods target the N gene, whereas the L gene is used for RT-hnPCR. Using serial dilutions of purified RNA from rabies-infected dog brain tissue to assess sensitivity, all five methods had comparable degrees of sensitivities of detection. However, both real-time NASBA assays had slightly lower sensitivities by 10-fold than the other three assays. This finding was also true (except for TaqMan real-time RT-PCR due to a mismatch between the target and probe sequences) when laboratory-adapted (challenge virus standard-11) virus was used in the assays. Testing on previously NASBA-ECL positive clinical samples from 10 rabies patients (saliva [6] and brain [4]) and 10 rabies-infected dog brain tissues, similar results were obtained among the five methods; real-time NASBA assays yielded false-negative results on 2 saliva samples. None of the assays showed positive results on cerebrospinal fluid specimens of 10 patients without rabies encephalitis. Due to the unavailability of the NASBA-ECL assay, the results show that TaqMan real-time RT-PCR and RT-hnPCR can be useful for ante- and post-mortem diagnosis of rabies.
Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors ... more Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors studied magnetic resonance imaging (MRI) and cytokine and virus distribution in rabies-infected dogs of both clinical types. MRI examination of the brain and upper spinal cord was performed in two furious and two paralytic dogs during the early clinical stage. Rabies viral nucleoprotein RNA and 18 cytokine mRNAs at 12 different brain regions were studied. Rabies viral RNA was examined in four furious and four paralytic dogs during the early stage, and in one each during the late stage. Cytokine mRNAs were examined in two furious and two paralytic dogs during the early stage and in one each during the late stage. Larger quantities of rabies viral RNA were found in the brains of furious than in paralytic dogs. Interleukin-1beta and interferon-gamma mRNAs were found exclusively in the brains of paralytic dogs during the early stage. Abnormal hypersignal T2 changes were found at hippocampus, hypothalamus, brainstem, and spinal cord of paralytic dogs. More widespread changes of less intensity were seen in furious dog brains. During the late stage of infection, brains from furious and paralytic rabid dogs were similarly infected and there were less detectable cytokine mRNAs. These results suggest that the early stage of furious dog rabies is characterized by a moderate inflammation (as indicated by MRI lesions and brain cytokine detection) and a severe virus neuroinvasiveness. Paralytic rabies is characterized by delayed viral neuroinvasion and a more intense inflammation than furious rabies. Dogs may be a good model for study of the host inflammatory responses that may modulate rabies virus neuroinvasiveness.
SIRRabies surveillance and reporting are poor in developing countries, and most governments fail... more SIRRabies surveillance and reporting are poor in developing countries, and most governments fail to appreciate either the public health or the economic burden imposed by human and animal rabies [1]. Although postmortem diagnosis of rabies can easily be made by fluorescent ...
Standard nucleic acid sequence based amplification (NASBA) method without internal control RNA (I... more Standard nucleic acid sequence based amplification (NASBA) method without internal control RNA (IC RNA), although uncertain, it may not avoid false negative. The objective of this study as to develop and to evaluate whether NASBA with internal control RNA (IC-NASBA) ...
Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors ... more Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors studied magnetic resonance imaging (MRI) and cytokine and virus distribution in rabies-infected dogs of both clinical types. MRI examination of the brain and upper spinal cord was performed in two furious and two paralytic dogs during the early clinical stage. Rabies viral nucleoprotein RNA and 18 cytokine mRNAs at 12 different brain regions were studied. Rabies viral RNA was examined in four furious and four paralytic dogs during the early stage, and in one each during the late stage. Cytokine mRNAs were examined in two furious and two paralytic dogs during the early stage and in one each during the late stage. Larger quantities of rabies viral RNA were found in the brains of furious than in paralytic dogs. Interleukin-1beta and interferon-gamma mRNAs were found exclusively in the brains of paralytic dogs during the early stage. Abnormal hypersignal T2 changes were found at hippocampus,...
Dengue virus (DENV) infection is a worldwide public health problem, which can cause severe dengue... more Dengue virus (DENV) infection is a worldwide public health problem, which can cause severe dengue hemorrhagic fever (DHF) and life-threatening dengue shock syndrome (DSS). There are currently no anti-DENV drugs available, and there has been an intensive search for effective anti-DENV agents that can inhibit all four DENV serotypes. In this study, we tested whether vivo-morpholino oligomers (vivo-MOs), whose effect on DENV infection has not previously been studied, can inhibit DENV infection. Vivo-MOs were designed to target the top of 3' stem-loop (3' SL) in the 3' UTR of the DENV genome and tested for inhibition of DENV infection in monkey kidney epithelial (Vero) cells and human lung epithelial carcinoma (A549) cells. The results showed that vivo-MOs could bind to a DENV RNA sequence and markedly reduce DENV-RNA, protein, and virus production in infected Vero and A549 cells. Vivo-MOs at a concentration of 4 µM could inhibit DENV production by more than 10-fold when com...
Noninvasive molecular tests of urine cells have been developed to monitor the activity of kidney ... more Noninvasive molecular tests of urine cells have been developed to monitor the activity of kidney diseases. We evaluate whether measurement of urinary messenger RNA (mRNA) levels of chemokine and growth factor genes could distinguish between diffuse proliferative lupus nephritis (class IV LN) and others and whether it is able to predict the response to therapy. Prebiopsy urine samples were collected
Diagnosis of rabies relies on the fluorescent antibody test (FAT) from brain impression smears. T... more Diagnosis of rabies relies on the fluorescent antibody test (FAT) from brain impression smears. The mouse brain inoculation test is used to confirm FAT but requires weeks until the result is known. TaqMan real-time PCR has been described for rabies viral RNA detection; however, this is burdened by primer and probe binding site mismatches. The purpose of this study was to develop a TaqMan real-time RT-PCR assay as an adjunct to FAT, based on national data of 239 rabies nucleoprotein sequences from rabies-infected brain specimens collected between 1998 and 2003. Two showed as many as 3 mismatches. However, mismatches on primer and/or probe binding sites did not affect amplification or detection. One hundred and forty-three brain samples submitted for rabies diagnosis from all over the country between 2005 and 2007 were also tested. Results were concordant with FAT. Thirteen rabies proven samples from Myanmar, Cambodia, Indonesia and India; 3 of which had up to 7 mismatches at primer/probe binding sites, could be detectable. Challenge Virus Standard, a fixed virus strain with 4 mismatches at probe binding site, could not be detected but remained amplified. This assay could be used as an adjunct to FAT and may serve as a rabies surveillance tool.
Five methods for the RNA detection of rabies virus were directly compared in this study. These in... more Five methods for the RNA detection of rabies virus were directly compared in this study. These included conventional nucleic acid sequence-based amplification with electrochemiluminescence (NASBA-ECL) assay, reverse transcription (RT)-heminested (hn) polymerase chain reaction (PCR) and TaqMan real-time RT-PCR using protocols as described previously. The first two methods have been routinely utilised for ante-mortem diagnosis of human rabies in Thailand and other rabies-endemic Asian and African countries. In addition, two real-time NASBA assays based on the use of a NucliSens EasyQ analyser (NASBA-Beacon-EQ) and LightCycler real-time PCR machine (NASBA-Beacon-LC) were studied in parallel. All methods target the N gene, whereas the L gene is used for RT-hnPCR. Using serial dilutions of purified RNA from rabies-infected dog brain tissue to assess sensitivity, all five methods had comparable degrees of sensitivities of detection. However, both real-time NASBA assays had slightly lower sensitivities by 10-fold than the other three assays. This finding was also true (except for TaqMan real-time RT-PCR due to a mismatch between the target and probe sequences) when laboratory-adapted (challenge virus standard-11) virus was used in the assays. Testing on previously NASBA-ECL positive clinical samples from 10 rabies patients (saliva [6] and brain [4]) and 10 rabies-infected dog brain tissues, similar results were obtained among the five methods; real-time NASBA assays yielded false-negative results on 2 saliva samples. None of the assays showed positive results on cerebrospinal fluid specimens of 10 patients without rabies encephalitis. Due to the unavailability of the NASBA-ECL assay, the results show that TaqMan real-time RT-PCR and RT-hnPCR can be useful for ante- and post-mortem diagnosis of rabies.
Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors ... more Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors studied magnetic resonance imaging (MRI) and cytokine and virus distribution in rabies-infected dogs of both clinical types. MRI examination of the brain and upper spinal cord was performed in two furious and two paralytic dogs during the early clinical stage. Rabies viral nucleoprotein RNA and 18 cytokine mRNAs at 12 different brain regions were studied. Rabies viral RNA was examined in four furious and four paralytic dogs during the early stage, and in one each during the late stage. Cytokine mRNAs were examined in two furious and two paralytic dogs during the early stage and in one each during the late stage. Larger quantities of rabies viral RNA were found in the brains of furious than in paralytic dogs. Interleukin-1beta and interferon-gamma mRNAs were found exclusively in the brains of paralytic dogs during the early stage. Abnormal hypersignal T2 changes were found at hippocampus, hypothalamus, brainstem, and spinal cord of paralytic dogs. More widespread changes of less intensity were seen in furious dog brains. During the late stage of infection, brains from furious and paralytic rabid dogs were similarly infected and there were less detectable cytokine mRNAs. These results suggest that the early stage of furious dog rabies is characterized by a moderate inflammation (as indicated by MRI lesions and brain cytokine detection) and a severe virus neuroinvasiveness. Paralytic rabies is characterized by delayed viral neuroinvasion and a more intense inflammation than furious rabies. Dogs may be a good model for study of the host inflammatory responses that may modulate rabies virus neuroinvasiveness.
SIRRabies surveillance and reporting are poor in developing countries, and most governments fail... more SIRRabies surveillance and reporting are poor in developing countries, and most governments fail to appreciate either the public health or the economic burden imposed by human and animal rabies [1]. Although postmortem diagnosis of rabies can easily be made by fluorescent ...
Standard nucleic acid sequence based amplification (NASBA) method without internal control RNA (I... more Standard nucleic acid sequence based amplification (NASBA) method without internal control RNA (IC RNA), although uncertain, it may not avoid false negative. The objective of this study as to develop and to evaluate whether NASBA with internal control RNA (IC-NASBA) ...
Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors ... more Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors studied magnetic resonance imaging (MRI) and cytokine and virus distribution in rabies-infected dogs of both clinical types. MRI examination of the brain and upper spinal cord was performed in two furious and two paralytic dogs during the early clinical stage. Rabies viral nucleoprotein RNA and 18 cytokine mRNAs at 12 different brain regions were studied. Rabies viral RNA was examined in four furious and four paralytic dogs during the early stage, and in one each during the late stage. Cytokine mRNAs were examined in two furious and two paralytic dogs during the early stage and in one each during the late stage. Larger quantities of rabies viral RNA were found in the brains of furious than in paralytic dogs. Interleukin-1beta and interferon-gamma mRNAs were found exclusively in the brains of paralytic dogs during the early stage. Abnormal hypersignal T2 changes were found at hippocampus,...
Dengue virus (DENV) infection is a worldwide public health problem, which can cause severe dengue... more Dengue virus (DENV) infection is a worldwide public health problem, which can cause severe dengue hemorrhagic fever (DHF) and life-threatening dengue shock syndrome (DSS). There are currently no anti-DENV drugs available, and there has been an intensive search for effective anti-DENV agents that can inhibit all four DENV serotypes. In this study, we tested whether vivo-morpholino oligomers (vivo-MOs), whose effect on DENV infection has not previously been studied, can inhibit DENV infection. Vivo-MOs were designed to target the top of 3' stem-loop (3' SL) in the 3' UTR of the DENV genome and tested for inhibition of DENV infection in monkey kidney epithelial (Vero) cells and human lung epithelial carcinoma (A549) cells. The results showed that vivo-MOs could bind to a DENV RNA sequence and markedly reduce DENV-RNA, protein, and virus production in infected Vero and A549 cells. Vivo-MOs at a concentration of 4 µM could inhibit DENV production by more than 10-fold when com...
Noninvasive molecular tests of urine cells have been developed to monitor the activity of kidney ... more Noninvasive molecular tests of urine cells have been developed to monitor the activity of kidney diseases. We evaluate whether measurement of urinary messenger RNA (mRNA) levels of chemokine and growth factor genes could distinguish between diffuse proliferative lupus nephritis (class IV LN) and others and whether it is able to predict the response to therapy. Prebiopsy urine samples were collected
Diagnosis of rabies relies on the fluorescent antibody test (FAT) from brain impression smears. T... more Diagnosis of rabies relies on the fluorescent antibody test (FAT) from brain impression smears. The mouse brain inoculation test is used to confirm FAT but requires weeks until the result is known. TaqMan real-time PCR has been described for rabies viral RNA detection; however, this is burdened by primer and probe binding site mismatches. The purpose of this study was to develop a TaqMan real-time RT-PCR assay as an adjunct to FAT, based on national data of 239 rabies nucleoprotein sequences from rabies-infected brain specimens collected between 1998 and 2003. Two showed as many as 3 mismatches. However, mismatches on primer and/or probe binding sites did not affect amplification or detection. One hundred and forty-three brain samples submitted for rabies diagnosis from all over the country between 2005 and 2007 were also tested. Results were concordant with FAT. Thirteen rabies proven samples from Myanmar, Cambodia, Indonesia and India; 3 of which had up to 7 mismatches at primer/probe binding sites, could be detectable. Challenge Virus Standard, a fixed virus strain with 4 mismatches at probe binding site, could not be detected but remained amplified. This assay could be used as an adjunct to FAT and may serve as a rabies surveillance tool.
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