Molecular characterization of the Tobacco rattle virus RNA2 genome isolated from Gladiolus.

Tobacco rattle virus (TRV-K) was first identified in a symptomatic Gladiolus plant cultivated in Korea. We analyzed the TRV-K genome and compared its phylogeny with other TRV isolates. After constructing of a full-length genomic RNA2 strand clone, a complete sequence was generated from several overlapping clones. The cloned genome was 3261 bases in length, identical to TRV-K, and had three open reading frames. TRV-K had the highest sequence identity with the American isolate TRV-ORY. Sequence analysis of the RNA2 genome showed that TRV-K contains an intact 2a, 2b, and 2c coding sequence and an RNA1-related 3' terminus, which is typical of TRV RNA2. Phylogenetic analysis revealed that TRV-K is in the same cluster as the American isolates and another Korean isolate, TRV-SK; however, it was in a different cluster than the European isolates.

Immune responses of mice intraduodenally infected with Toxoplasma gondii KI-1 tachyzoites.

Toxoplasma gondii Korean isolate (KI-1) tachyzoites were inoculated intraduodenally to BALB/c mice using a silicon tube, and the course of infection and immune responses of mice were studied. Whereas control mice, that were infected intraperitoneally, died within day 7 post-infection (PI), the intraduodenally infected mice survived until day 9 PI (infection with 1 × 10(5) tachyzoites) or day 11 PI (with 1 × 10(6) tachyzoites). Based on histopathologic (Giemsa stain) and PCR (B1 gene) studies, it was suggested that tachyzoites, after entering the small intestine, invaded into endothelial cells, divided there, and propagated to other organs. PCR appeared to be more sensitive than histopathology to detect infected organs and tissues. The organisms spread over multiple organs by day 6 PI. However, proliferative responses of splenocytes and mesenteric lymph node (MLN) cells in response to con A or Toxoplasma lysate antigen decreased significantly, suggesting immunosuppression. Splenic CD4(+) and CD8(+) T-lymphocytes showed decreases in number until day 9 PI, whereas IFN-γ and IL-10 decreased slightly at day 6 PI and returned to normal levels by day 9 PI. No TNF-α was detected throughout the experimental period. The results showed that intraduodenal infection with KI-1 tachyzoites was successful but did not elicit significant mucosal immunity in mice and allowed dissemination of T. gondii organisms to systemic organs. The immunosuppression of mice included reduced lymphoproliferative responses to splenocytes and MLN cells to mitogen and low production of cytokines, such as IFN-γ, TNF-α, and IL-10, in response to T. gondii infection.

Proteomic analysis of Toxoplasma gondii KI-1 tachyzoites.

We studied on the proteomic characteristics of Toxoplasma gondii KI-1 tachyzoites which were originally isolated from a Korean patient, and compared with those of the well-known virulent RH strain using 2-dimensional electrophoresis (2-DE), mass spectrometry, and quantitative real-time PCR. Two-dimensional separation of the total proteins isolated from KI-1 tachyzoites revealed up to 150 spots, of which 121 were consistent with those of RH tachyzoites. Of the remaining 29 spots, 14 showed greater than 5-fold difference in density between the KI-1 and RH tachyzoites at a pH of 5.0-8.0. Among the 14 spots, 5 from the KI-1 isolate and 7 from the RH strain were identified using MALDI-TOF mass spectrometry and database searches. The spots from the KI-1 tachyzoites were dense granule proteins (GRA 2, 3, 6, and 7), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGRPTase), and uracil phosphoribosyltransferase (UPRTase). The spots from the RH strain were surface antigen 1 (SAG 1), L-lactate dehydrogenase (LDH), actin, chorismate synthase, peroximal catalase, hexokinase, bifunctional dihydrofolate reductase-thymidylate synthase (DHTR-TS), and nucleoside-triphosphatases (NTPases). Quantitative real-time PCR supported our mass spectrometric results by showing the elevated expression of the genes encoding GRA 2, 3, and 6 and UPRTase in the KI-1 tachyzoites and those encoding GRA 7, SAG 1, NTPase, and chorismate synthase in the RH tachyzoites. These observations demonstrate that the protein compositions of KI-1 and RH tachyzoites are similar but differential protein expression is involved in virulence.

Suppression of CD4 T-Cells in the spleen of mice infected with Toxoplasma gondii KI-1 tachyzoites.

Toxoplasma gondii KI-1, a recent new isolate from Korea, shows similar pathogenicity and infectivity to mice compared to the virulent RH strain. To understand characteristics of host immunity, including immune enhancement or suppression, we investigated proliferative responses and phenotypes of spleen cells. In addition, kinetics of IFN-γ, a Th1 cytokine, was examined in BALB/c mice up to day 6 post-infection (PI). Intraperitoneal injection of mice with 10(3) KI-1 tachyzoites induced significant decreases (P < 0.05) in proliferative responses of spleen cells. This occurred at days 2-6 PI even when concanavalin A (con A) was added and when stimulated with KI-1 antigen, suggesting suppression of the immunity. CD4(+) T-cells decreased markedly at day 2 PI (P < 0.05), whereas CD8(+) T-cells, NK cells, and macrophages did not show significant changes, except a slight, but significant, increase of CD8(+) T-cells at day 6 PI. The capacity of splenocytes to produce IFN-γ by con A stimulation dropped significantly at days 2-6 PI. These results demonstrate that intraperitoneal injection of KI-1 tachyzoites can induce immunosuppression during the early stage of infection, as revealed by the decrease of CD4(+) T-cells and IFN-γ.

Development of a neutralization assay based on the pseudotyped chikungunya virus of a Korean isolate.

The Chikungunya virus (CHIKV) belongs to the Alphavirus genus of Togaviridae family and contains a positive-sense single stranded RNA genome. Infection by this virus mainly causes sudden high fever, rashes, headache, and severe joint pain that can last for several months or years. CHIKV, a mosquito-borne arbovirus, is considered a re-emerging pathogen that has become one of the most pressing global health concerns due to a rapid increase in epidemics. Because handling of CHIKV is restricted to Biosafety Level 3 (BSL-3) facilities, the evaluation of prophylactic vaccines or antivirals has been substantially hampered. In this study, we first iden-tified the whole structural polyprotein sequence of a CHIKV strain isolated in South Korea (KNIH/2009/77). Phylogenetic analysis showed that this sequence clustered within the East/ Central/South African CHIKV genotype. Using this sequence information, we constructed a CHIKV-pseudotyped lenti-virus expressing the structural polyprotein of the Korean CHIKV isolate (CHIKVpseudo) and dual reporter genes of green fluorescence protein and luciferase. We then developed a pseudovirus-based neutralization assay (PBNA) using CHIKVpseudo. Results from this assay compared to those from the conventional plaque reduction neutralization test showed that our PBNA was a reliable and rapid method to evaluate the efficacy of neutralizing antibodies. More importantly, the neutralizing activities of human sera from CHIKV-infected individuals were quantitated by PBNA using CHIKVpseudo. Taken together, these results suggest that our PBNA for CHIKV may serve as a useful and safe method for testing the neutralizing activity of antibodies against CHIKV in BSL-2 facilities.

An infectious cDNA clone of a growth attenuated Korean isolate of MERS coronavirus KNIH002 in clade B.

The MERS-CoV isolated during the 2015 nosocomial outbreak in Korea showed distinctive differences in mortality and transmission patterns compared to the prototype MERS-CoV EMC strain belonging to clade A. We established a BAC-based reverse genetics system for a Korean isolate of MERS-CoV KNIH002 in the clade B phylogenetically far from the EMC strain, and generated a recombinant MERS-CoV expressing red fluorescent protein. The virus rescued from the infectious clone and KNIH002 strain displayed growth attenuation compared to the EMC strain. Consecutive passages of the rescued virus rapidly generated various ORF5 variants, highlighting its genetic instability and calling for caution in the use of repeatedly passaged virus in pathogenesis studies and for evaluation of control measures against MERS-CoV. The infectious clone for the KNIH002 in contemporary epidemic clade B would be useful for better understanding of a functional link between molecular evolution and pathophysiology of MERS-CoV by comparative studies with EMC strain.

Immune Responses of Mice Intraduodenally Infected with Toxoplasma gondii KI-1 Tachyzoites

Toxoplasma gondii Korean isolate (KI-1) tachyzoites were inoculated intraduodenally to BALB/c mice using a silicon tube, and the course of infection and immune responses of mice were studied. Whereas control mice, that were infected intraperitoneally, died within day 7 post-infection (PI), the intraduodenally infected mice survived until day 9 PI (infection with 1x10(5) tachyzoites) or day 11 PI (with 1x10(6) tachyzoites). Based on histopathologic (Giemsa stain) and PCR (B1 gene) studies, it was suggested that tachyzoites, after entering the small intestine, invaded into endothelial cells, divided there, and propagated to other organs. PCR appeared to be more sensitive than histopathology to detect infected organs and tissues. The organisms spread over multiple organs by day 6 PI. However, proliferative responses of splenocytes and mesenteric lymph node (MLN) cells in response to con A or Toxoplasma lysate antigen decreased significantly, suggesting immunosuppression. Splenic CD4+ and CD8+ T-lymphocytes showed decreases in number until day 9 PI, whereas IFN-gamma and IL-10 decreased slightly at day 6 PI and returned to normal levels by day 9 PI. No TNF-alpha was detected throughout the experimental period. The results showed that intraduodenal infection with KI-1 tachyzoites was successful but did not elicit significant mucosal immunity in mice and allowed dissemination of T. gondii organisms to systemic organs. The immunosuppression of mice included reduced lymphoproliferative responses to splenocytes and MLN cells to mitogen and low production of cytokines, such as IFN-gamma, TNF-alpha, and IL-10, in response to T. gondii infection.

Proteomic Analysis of Toxoplasma gondii KI-1 Tachyzoites

We studied on the proteomic characteristics of Toxoplasma gondii KI-1 tachyzoites which were originally isolated from a Korean patient, and compared with those of the well-known virulent RH strain using 2-dimensional electrophoresis (2-DE), mass spectrometry, and quantitative real-time PCR. Two-dimensional separation of the total proteins isolated from KI-1 tachyzoites revealed up to 150 spots, of which 121 were consistent with those of RH tachyzoites. Of the remaining 29 spots, 14 showed greater than 5-fold difference in density between the KI-1 and RH tachyzoites at a pH of 5.0-8.0. Among the 14 spots, 5 from the KI-1 isolate and 7 from the RH strain were identified using MALDI-TOF mass spectrometry and database searches. The spots from the KI-1 tachyzoites were dense granule proteins (GRA 2, 3, 6, and 7), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGRPTase), and uracil phosphoribosyltransferase (UPRTase). The spots from the RH strain were surface antigen 1 (SAG 1), L-lactate dehydrogenase (LDH), actin, chorismate synthase, peroximal catalase, hexokinase, bifunctional dihydrofolate reductase-thymidylate synthase (DHTR-TS), and nucleoside-triphosphatases (NTPases). Quantitative real-time PCR supported our mass spectrometric results by showing the elevated expression of the genes encoding GRA 2, 3, and 6 and UPRTase in the KI-1 tachyzoites and those encoding GRA 7, SAG 1, NTPase, and chorismate synthase in the RH tachyzoites. These observations demonstrate that the protein compositions of KI-1 and RH tachyzoites are similar but differential protein expression is involved in virulence.

Suppression of CD4+ T-Cells in the Spleen of Mice Infected with Toxoplasma gondii KI-1 Tachyzoites

Toxoplasma gondii KI-1, a recent new isolate from Korea, shows similar pathogenicity and infectivity to mice compared to the virulent RH strain. To understand characteristics of host immunity, including immune enhancement or suppression, we investigated proliferative responses and phenotypes of spleen cells. In addition, kinetics of IFN-gamma, a Th1 cytokine, was examined in BALB/c mice up to day 6 post-infection (PI). Intraperitoneal injection of mice with 103 KI-1 tachyzoites induced significant decreases (P < 0.05) in proliferative responses of spleen cells. This occurred at days 2-6 PI even when concanavalin A (con A) was added and when stimulated with KI-1 antigen, suggesting suppression of the immunity. CD4+ T-cells decreased markedly at day 2 PI (P < 0.05), whereas CD8+ T-cells, NK cells, and macrophages did not show significant changes, except a slight, but significant, increase of CD8+ T-cells at day 6 PI. The capacity of splenocytes to produce IFN-gamma by con A stimulation dropped significantly at days 2-6 PI. These results demonstrate that intraperitoneal injection of KI-1 tachyzoites can induce immunosuppression during the early stage of infection, as revealed by the decrease of CD4+ T-cells and IFN-gamma.

Genotyping of a Korean isolate of Toxoplasma gondii by multilocus PCR-RFLP and microsatellite analysis

Although the Korean isolate KI-1 of Toxoplasma gondii has been considered to be a virulent type I lineage because of its virulent clinical manifestations, its genotype is unclear. In the present study, genotyping of the KI-1 was performed by multilocus PCR-RFLP and microsatellite sequencing. For 9 genetic markers (c22-8, c29-2, L358, PK1, SAG2, SAG3, GRA6, BTUB, and Apico), the KI-1 and RH strains exhibited typical PCR-RFLP patterns identical to the type I strains. DNA sequencing of tandem repeats in 5 microsatellite markers (B17, B18, TUB2, W35, and TgM-A) of the KI-1 also revealed patterns characteristic of the type I. These results provide strong genetic evidence that KI-1 is a type I lineage of T. gondii.

Evaluation of the Korean Isolate-1 Tachyzoite Antigen for Serodiagnosis of Toxoplasmosis

To evaluate the usefulness of the Korean Isolate-1 (KI-1) antigen for serodiagnosis of toxoplasmosis, antigen profiles of KI-1 tachyzoites were analyzed in comparison with RH tachyzoites by SDS-PAGE and immunoblotting. ELISA was performed on latex agglutination (LA)-positive and negative serum samples using KI-1 and RH antigens. Immunoblotting of the KI-1 antigen showed multiple antigen bands with molecular sizes of 22-105 kDa. Among them, 1 and 6 common bands were noted against a KI-1-infected and a RH-infected human serum, respectively, which represented differences in antigenic profiles between KI-1 and RH tachyzoites. However, all 9 LA-positive human sera were found positive by ELISA, and all 12 LA-negative sera were negative by ELISA; the correlation between the ELISA titers and LA titers was high (r = 0.749). Our results suggest that tachyzoites of KI-1 may be useful for serodiagnosis of human toxoplasmosis.

Evaluation of the Korean Isolate-1 Tachyzoite Antigen for Serodiagnosis of Toxoplasmosis

To evaluate the usefulness of the Korean Isolate-1 (KI-1) antigen for serodiagnosis of toxoplasmosis, antigen profiles of KI-1 tachyzoites were analyzed in comparison with RH tachyzoites by SDS-PAGE and immunoblotting. ELISA was performed on latex agglutination (LA)-positive and negative serum samples using KI-1 and RH antigens. Immunoblotting of the KI-1 antigen showed multiple antigen bands with molecular sizes of 22-105 kDa. Among them, 1 and 6 common bands were noted against a KI-1-infected and a RH-infected human serum, respectively, which represented differences in antigenic profiles between KI-1 and RH tachyzoites. However, all 9 LA-positive human sera were found positive by ELISA, and all 12 LA-negative sera were negative by ELISA; the correlation between the ELISA titers and LA titers was high (r = 0.749). Our results suggest that tachyzoites of KI-1 may be useful for serodiagnosis of human toxoplasmosis.

Genetic characteristics of the Korean isolate KI-1 of Toxoplasma gondii

Toxoplasma gondii tachyzoites were isolated from an ocular patient in the Republic of Korea and maintained in the laboratory (designated KI-1). In the present study, its genotype was determined by analyzing dense granule antigen 6 (GRA6) gene and surface antigen 2 (SAG2) gene as typing markers. Digestion of the amplification products of GRA6 and of the 5' and 3' ends of SAG2, respectively, with Mse I, Sau3A I, and Hha I, revealed that KI-1 is included in the genotype I, which includes the worldwide virulent RH strain. In addition, when the whole sequences of the coding regions of SAG1, rhoptry antigen 1 (ROP1), and GRA8 genes of KI-1 were compared with those of RH, minor nucleotide polymorphisms and amino acid substitutions were identified. These results show that KI-1 is a new geographical strain of T. gondii that can be included in the genotype I.

Laboratory passage and characterization of an isolate of Toxoplasma gondii from an ocular patient in Korea

Toxoplasma gondii tachyzoites were isolated from the blood of an ocular patient, and have been successfully passaged in the laboratory, for over a year, by peritoneal inoculation in mice. The isolated parasite was designated the Korean Isolate-1 (KI-1) and its characteristics were compared with those of the RH strain, a wellknown virulent strain originating from a child who suffered from encephalitis. The morphology, pathogenicity, infectivity and cell culture characteristics of the KI-1 were similar to those of the RH strain. Both RH and KI-1 antigens were detected by an anti-T. gondii monoclonal antibody (mAb), Tg563, against the major surface protein SAG1 (30 kDa), whereas no reaction was observed against an anti-Neospora caninum mAb, 12B4. The KI-1 was confirmed as an isolate of T. gondii. A long-term laboratory maintenance and characterization of a local T. gondii isolate is reported for the first time in the Republic of Korea.