Corrigendum to “Evaluation of Potency on Diphtheria and Tetanus Toxoid for Adult Vaccines by In Vivo Toxin Neutralization Assay Using National Reference Standards”Osong Public Health Res Perspect 2018;9(5):278–82

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Evaluation of Potency on Diphtheria and Tetanus Toxoid for Adult Vaccines by In Vivo Toxin Neutralization Assay Using National Reference Standards

OBJECTIVES: Vaccinations against diphtheria and tetanus are essential in providing immunity against these bacterial infections. The potency of diphtheria and tetanus toxoid vaccines can be measured using the in vivo toxin neutralization assay. The limit of potency of this assay was determined only for children. Therefore, we assessed the potency of adult vaccines using this assay to identify the feasibility of limit for adult vaccines. METHODS: Fifteen lots of tetanus-reduced diphtheria and tetanus-diphtheria-acellular pertussis vaccines were used. In vivo toxin neutralization and lethal challenge assays were conducted on each vaccine to calculate the potencies of the toxoids. National reference standards for toxins and antitoxins were used for in vivo toxin neutralization assay. RESULTS: All 15 lots satisfied the limits of potency for lethal challenge assay. The potency of diphtheria and tetanus toxoids exceeded 1 and 8 units/mL, respectively, for in vivo toxin neutralization assay. CONCLUSION: Although additional studies are required for new assays and limits, the current level of potency for adult vaccines as determined by in vivo toxin neutralization assay, was demonstrated in this study. Such efforts to improve assays are expected to promote the development of diphtheria and tetanus vaccines for adults and to contribute to vaccine self-sufficiency.

Development and application of quantitative detection method for nervous necrosis virus (NNV) isolated from sevenband grouper Hyporthodus septemfasciatus

Objective To develop the rapid and efficient quantitative detection tool for nervous necrosis virus isolated from sevenband grouper Hyporhodus septemfasciatus. Methods The viral genes of the NNV (SGYeosu08) isolated from sevenband grouper were phylogenetically analyzed. In addition, novel quantitative PCR primers based on the genomic sequence of SGYeosu08 isolate were designed and compared it with the conventional bio-assay method (TCID

Development and application of quantitative detection method for nervous necrosis virus (NNV) isolated from sevenband grouper Hyporthodus septemfasciatus

OBJECTIVE@#To develop the rapid and efficient quantitative detection tool for nervous necrosis virus isolated from sevenband grouper Hyporhodus septemfasciatus.@*METHODS@#The viral genes of the NNV (SGYeosu08) isolated from sevenband grouper were phylogenetically analyzed. In addition, novel quantitative PCR primers based on the genomic sequence of SGYeosu08 isolate were designed and compared it with the conventional bio-assay method (TCID50) using in vitro and in vivo samples.@*RESULTS@#The phylogenetic analysis of viral genes demonstrated the relationship of SGYeosu08 with members of red-spotted grouper nervous necrosis virus (RGNNV). The qNNV_R1 primer set (R1_F and R1_R) and the qNNV_R2 primer set (R2_F and R2_R) revealed 93% primer efficiency (regression: y = -0.2861x + 9.9401, R(2) = 0.9976) and the revealed 108% primer efficiency (regression: y = -0.3172x + 10.0611, R(2) = 0.9982), respectively. Its comparison with viral infectivity calculated by TCID50 method showed similar kinetic pattern at in vitro and NNV challenged fish (in vivo) samples.@*CONCLUSIONS@#Result show that this method is rapid and efficient to diagnose NNV infection compare to traditional bioassay method (TCID50).

Management of Split Thickness Skin Graft Donor Sites: Comparison of Different Dressing Materials

PURPOSE: The purpose of management of the donor is to maintain a moist condition that promotes healing process and prevents pain, infection. We have performed a prospective study to compare the usefulness between Aquacel Ag(R) and Mepitel(R). METHODS: 36 consecutive patients, in whom STSG was performed, were included into the study. STSG are harvested as a usual manner and the donor site are dressed with Aquacel Ag(R) or Mepitel(R), alternatively. The usefulness are compared with re-epithelialization, pain, frequency to change the second dressing, and ease of application. RESULTS: There are no differences in the days of re-epithelialization, pain perception of patients, but significantly differences in frequency to change the second dressing, and ease of application. Aquacel Ag(R) is better than Mepitel(R). CONCLUSION: We concluded that Aquacel Ag(R) dressing is better than Mepitel(R) for STSG donor site just in the frequency to change the second dressing and ease of application.

Construction of A Stable Full-Length cDNA Clone of Japanese Encephalitis Virus Strain SA14-14-2 Using Low Copy Number Plasmid

Recently the reverse genetics system contributed to the progresses in the investigation of positive-stranded RNA viruses. Here, we report the successful construction of a stable full-length infectious cDNA clone of the live attenuated JEV vaccine strain SA14-14-2. The eleven kilobase viral RNA genome was reverse transcribed, amplified as four overlapping DNA fragments and successively ligated into the low copy number plasmid pACYC184, which contains the p15A origin of replication. In vitro-transcribed RNAs had a specific infectivity of approximately 104 PFU/microgram RNA, and the resulting virus exhibited growth kinetics and plaque morphology similar to the parental virus in cell culture. The structural and functional integrity of the cDNA clone was stably maintained even after at least 150 generations in Escherichia coli strain TOP10. The cDNA clone was engineered to contain single nucleotide change to create a XhoI site and knock out a XbaI site (A to C at nt 9134) acting as a genetic marker. This genetic marker was retained in the recovered progeny virus. Our results suggest that the instability of the full-length infectious JEV cDNA clone can be overcome by employing low copy number plasmid pACYC184. This infectious JEV cDNA clone will aid future studies of pathogenesis, virulence, and replication. Furthermore, it will facilitate the development of SA14-14-2 based recombinant vaccines.

Construction of A Stable Full-Length cDNA Clone of Japanese Encephalitis Virus Strain SA14-14-2 Using Low Copy Number Plasmid

Recently the reverse genetics system contributed to the progresses in the investigation of positive-stranded RNA viruses. Here, we report the successful construction of a stable full-length infectious cDNA clone of the live attenuated JEV vaccine strain SA14-14-2. The eleven kilobase viral RNA genome was reverse transcribed, amplified as four overlapping DNA fragments and successively ligated into the low copy number plasmid pACYC184, which contains the p15A origin of replication. In vitro-transcribed RNAs had a specific infectivity of approximately 104 PFU/microgram RNA, and the resulting virus exhibited growth kinetics and plaque morphology similar to the parental virus in cell culture. The structural and functional integrity of the cDNA clone was stably maintained even after at least 150 generations in Escherichia coli strain TOP10. The cDNA clone was engineered to contain single nucleotide change to create a XhoI site and knock out a XbaI site (A to C at nt 9134) acting as a genetic marker. This genetic marker was retained in the recovered progeny virus. Our results suggest that the instability of the full-length infectious JEV cDNA clone can be overcome by employing low copy number plasmid pACYC184. This infectious JEV cDNA clone will aid future studies of pathogenesis, virulence, and replication. Furthermore, it will facilitate the development of SA14-14-2 based recombinant vaccines.

Development of PCR-ELISA for Detection of HIV-1 in Biomedical Products / 대한진단검사의학회지

BACKGROUND: Biomedical products such as viral vaccines can be contaminated with hazardous viruses during manufacturing processes and storage, thus causing harmful side effects. To assure the safety of biomedical products, highly effective and sensitive methods should be available to detect contaminating viruses. In this study, we performed recovery tests to determine the limit of detection of HIV-1. METHODS: An HIV-1 plasmid preparation was serially diluted and spiked into various culture media (DMEM, RPMI-1640, IMDM, GICM, and SDM) containing 10% fetal bovine serum (FBS). The HIV-1 plasmid was detected by PCR alone or a combination of PCR and ELISA (PCR/ELISA). RESULTS: When spiked into DMEM, RPMI, and IMDM, less than 4x10(-2) ng of HIV-1 plasmid was not detectable as HIV-1 PCR products in agarose gel. Intra- and inter-assays (n=6) showed that the PCR-ELISA system could detect PCR products diluted as much as 1, 875 times from HIV-1 plasmid serially spiked in various media. CONCLUSIONS: The PCR/ELISA system can be useful for the detection of trace amounts of hazardous viruses which may be present as contaminants in biological products.

Development of One-step Real-time Reverse Transcription-polymerase Chain Reaction in Combination with Automated RNA Extraction for Detection and Quantitation of Hepatitis A Virus

One-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using the MagNA Pure LC and LightCycler(TM) system was developed and validated for the detection and quantitation of hepatitis A virus (HAV) RNA. The assay was evaluated using in-house synthetic HAV RNA standard. The real-time RT-PCR assay could quantitate a dynamic range of HAV RNA standard between 10(2) and 10(8) copies per reaction. The regression coefficient of the standard curve was an 0.99. The detection limit of the assay was 31.3 RNA copies per reaction. The coefficient variations (CVs) of the assay in combination with automated RNA extraction were less than 1.91% in both intra- and inter-assay. The real-time RT-PCR assay for quantitative detection of HAV would serve a useful method for improving the safety of biological products.

Evaluation of Limit of Detection and Range of Quantitation for RT-PCR, Real-Time RT-PCR and RT-PCR-ELISA Detection of Bovine Viral Diarrhoea Virus Contamination in Biologics Derived from Cell Cultures

Risk of viral contamination is one of major concerns common to all biologics derived from cultivated cells. Bovine viral diarrhoea virus (BVDV) has widely been known as a contaminant of cell culture-derived vaccines. The objective of the study was to assess the limit of detection and range of quantitation of the detection methods for BVDV using a reverse transcription-polymerase chain reaction (RT-PCR) assay, real-time RT-PCR assay, and RT-PCR-ELISA. One milliliter of cell culture supernatant containing 106.5+/-0.2 median tissue culture infectious dose (TCID50)/ml of BVDV NADL strain was subjected to RNA isolation. The isolated RNA was 10-fold serially diluted and each diluted sample (10-1 to 10-6) was subjected to RT-PCR on a GeneAmpR PCR System 9700 and/or LightCycler(TM). The amplified products were analyzedly (1) agarose gel electrophoresis for RT-PCR assay, (2) melting curve analysis for real-time RT-PCR assay (in this case a program is automatically linked to amplification step), and (3) ELISA using capture and detection probes for RT-PCR-ELISA. The limit of detection of the 3 assay methods was equally estimated to be 316 TCID50/ml of starting virus culture supernatant subjected to the assay. The quantitation range of real-time RT-PCR assay and RT-PCR-ELISA was estimated to be from 3.16x105 to 3.16x102 TCID50/ml of starting virus culture supernatant. The overall results suggested that the 3 assay methods for BVDV detection can be reliably applied to evaluate BVDV contamination in biologics derived from cell cultures.

Infantile Solitary Eosinophilic Granuloma of the Lymph Node: A case report

Infantile form of histiocytosis X is commonly presented as multiorgan desseminated form such as Letterer-Siwe disease. Lymph node involvement of histiocytosis X is usually accompanied by adjacent bone or skin lesion. Solitary nodal eosinophilic granuloma without evidence of other organ involvement is very rare. A case herein report is a 11 month-old female infant presented with fever and palpable both inguinal lymph nodes. There was neither skin lesion nor hepatosplenomegaly. Laboratory evaluation was within normal range except increased alkaline phosphatase and many neutrophils in urine. Radiologic examination revealed no remarkable bone lesions. And she showed good clinical outcome without evidence of other organ involvements. On microscopic examination of inguinal lymph node it was replaced by infiltration of histiocytes mainly along the sinusoid. Some of histiocytes showed morphologic features of "histiocytosis X cell" having nuclear grooves or multilobulation. Multinulceated giant cells were frequently see. Numerous eosinphils were also infiltrated and showed multifocal microabscess formation. Immunohistochemical staining revealed that majority of histiocytes were postitive for S-100 protein but multinucleated histriocytes, phagocytic histiocytes and those around the abscess were positive for macrophage marker, suck as CD68 and alpha-1-antichymotrypsin. Interestingly some histiocytes showed positivity for both S-100 protein and macrophage marker. These results suggest that histiocytosis X is proliferative disorder of phenotypically heterogenous population of histiocytes in contrast to the theory that it is a proliferative disorder of Langerhans cells.