| WooYoung Choi | 5 Articles |
The rotavirus vaccine is a live vaccine, and there is a possibility of infection by the virus strain used in the vaccine. We investigated the process of determining whether an infection was caused by the vaccine strain in a severe complex immunodeficiency (SCID) patient with rotavirus infection. The patient was vaccinated with RotaTeq prior to being diagnosed with SCID. The testing process was conducted in the following order: confirming rotavirus infection, determining its genotype, and confirming the vaccine strain. Rotavirus infection was confirmed through enzyme immunoassay and VP6 gene detection. G1 and P[8] were identified by multiplex polymerase chain reaction for the genotype, and G3 was further identified using a single primer. By detecting the fingerprint gene (WC3) of RotaTeq, it was confirmed that the detected virus was the vaccine strain. Genotypes G1 and P[8] were identified, and the infection was suspected of having been caused by rotavirus G1P[8]. G1P[8] is the most commonly detected genotype worldwide and is not included in the recombinant strains used in vaccines. Therefore, the infection was confirmed to have been caused by the vaccine strain by analyzing the genetic relationship between VP4 and VP7. Rotavirus infection by the vaccine strain can be identified through genotyping and fingerprint gene detection. However, genetic linkage analysis will also help to identify vaccine strains.
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Ebola and Marburg viruses (EBOVs and MARVs, respectively) are causative agents of severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. In 2014, there was a major Ebola outbreak in various countries in West Africa, including Guinea, Liberia, Republic of Sierra Leone, and Nigeria. EBOV and MARV are clinically difficult to diagnose and distinguish from other African epidemic diseases. Therefore, in this study, we aimed to develop a method for rapid identification of the virus to prevent the spread of infection. Methods We established a conventional one-step reverse transcription-polymerase chain reaction (RT-PCR) assay for these pathogens based on the Superscript Reverse Transcriptase-Platinum <i>Taq</i> polymerase enzyme mixture. All assays were thoroughly optimized using <i>in vitro</i>-transcribed RNA. Results We designed seven primer sets of nucleocapsid protein (NP) genes based on sequences from seven filoviruses, including five EBOVs and two MARVs. To evaluate the sensitivity of the RT-PCR assay for each filovirus, 10-fold serial dilutions of synthetic viral RNA transcripts of EBOV or MARV NP genes were used to assess detection limits of viral RNA copies. The potential for these primers to cross react with other filoviruses was also examined. The results showed that the primers were specific for individual genotype detection in the examined filoviruses. Conclusion The assay established in this study may facilitate rapid, reliable laboratory diagnosis in suspected cases of Ebola and Marburg hemorrhagic fevers. Citations
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The purpose of this study was to verify the feasibility of using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promotor based <i>Pichia pastoris</i> expression system to produce tick-borne encephalitis virus (TBEV) virus-like particles (VLPs). Methods The complementary DNA encoding the TBEV prM signal peptide, prM, and E proteins of TBEV Korean strain (KrM 93) was cloned into the plasmid vector pGAPZɑA, then integrated into the genome of <i>P. pastoris</i>, under the control of the GAP promoter. Expression of TBEV VLPs was determined by Western blotting using monoclonal antibody against TBEV envelope (E) protein. Results Recombinant TBEV VLPs consisting of prM and E protein were successfully expressed using the GAP promoter-based <i>P. pastoris</i> expression system. The results of Western blotting showed that the recombinant proteins were secreted into the culture supernatant from the <i>P. pastoris</i> and glycosylated. Conclusion This study suggests that recombinant TBEV VLPs from <i>P. pastoris</i> offer a promising approach to the production of VLPs for use as vaccines and diagnostic antigens. Citations
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To examine the effect of neuraminidase (NA) mutations on the NA inhibitor (NAI) resistance phenotype, the recombinant influenza A/Chungbuk/4448/2008(H1N1) virus isolated in South Korea during the 2008–2009 season was generated by reverse genetics. Methods Site-directed mutagenesis was introduced on the <i>NA</i> gene of A/Chungbuk/4448/2008(H1N1) virus, and a total of 23 single, double, and triple mutants were generated. Resistance phenotype of these recombinant viruses was determined by NA-inhibition (NAI) assays based on a fluorometric method using two NAIs (oseltamivir and zanamivir). Results NA-inhibition assays showed that all the single and double mutants containing the Y275 except the single Y275-E119V mutant conferred important levels of resistance to oseltamivir, whereas all the single, double, and triple mutants containing the E119V mutation were associated with the resistance to zanamivir. Conclusion Considering the effect of mutations in <i>NA</i> gene on the resistance to NAIs, it is important to monitor the possible emergence and dissemination of multidrug-resistant variants in the human population due to amino acid changes at <i>NA</i> gene as well as to develop novel NAIs. Citations
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In this study, we demonstrated that TBEV-infected ticks have been distributed in the ROK, combined with our previous results. These results suggest that TBEV may exist in the ROK, and <i>H. longicornis, H. flava</i>, and <i>I. nipponensis</i> may be potential vectors of TBEV. In addition, these results emphasize the need for further epidemiological research of TBEV. Methods We examined for the presence of RNA of TBEV by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) using ixodid ticks captured in 25 localities of 10 provinces. Ticks were collected by the flagging and dragging method or using sentinel BG traps at forests, grass thickets, and grassland. A total of 13,053 ticks belonging to two genera and four species were collected and pooled (1292 pools), according to collection site, species of tick, and developmental stage. Results Among 1292 pools, the envelope (E) protein gene of TBEV was detected using RT-nested PCR in 10 pools (3 pools of the 1,331 adult ticks and 7 pools of the 11,169 nymph ticks) collected from Gangwon-do province, Jeonrabuk-do province, and Jeju Island. The minimum infection rates for TBEV of <i>Haemaphysalis longicornis, Haemaphysalis flava</i>, and <i>Ixodes nipponensis</i> were 0.06%, 0.17%, and 2.38%, respectively. Phylogenetic analysis based on the partial E protein gene was performed to identify relationships between the TBEV strains. This showed that 10 Korean strains clustered with the Western subtype. Conclusion In this study, we investigated the prevalence of tick-borne encephalitis virus (TBEV) in ixodid ticks from various regions of the Republic of Korea (ROK) during 2011–2012 to identify whether TBEV is circulating and to determine the endemic regions of TBEV. Citations
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