Vector Competence and the Susceptibility of Anopheles pullus and Anopheles belenrae to Plasmodium vivax-Infected Blood From Thai Patients.

There are eight Anopheles spp. present in the Republic of Korea (ROK), including five members of the Anopheles Hyrcanus Group that cannot be identified using current morphological methods. The vector competence of only Anopheles sinensis s.s., An. lesteri, and An. kleini have been investigated. As the geographical distribution of Anopheles spp. varies in the ROK, determining the relative vector competence of the Anopheles spp. provides a basis for delineating malaria risks to Korean populations and U.S. military/civilian populations deployed to the ROK. Anopheles belenrae and An. pullus, collected from a malaria high-risk area in the ROK, were evaluated for vector competence of P. vivax. A total of 1,000 each of An. dirus (Thai strain), and Korean strains of An. pullus and An. belenrae were fed on P. vivax infected blood collected from Thai patients via artificial membrane feeding. The overall oocyst infection rates for An. dirus, An. pullus, and An. belenrae dissected on days 8-9 postfeed were 64.1, 12.0, and 11.6%, respectively. The overall sporozoite infection rates for those species dissected on days 14-15 postfeed were 84.5, 3.4, and 5.1% respectively. The salivary gland sporozoite indices for positive females with +4 (>1,000 sporozoites) were observed in An. dirus (72.8%), but not observed for either An. pullus or An. belenrae. Most sporozoite-positive An. pullus (83.3%) and An. belenrae (71.4%) females were observed with only +1 (1-10 sporozoites) salivary glands. These data indicate that both An. belenrae and An. pullus are very poor vectors of P. vivax.

Geographical Distribution and Epidemiologic Factors of Chigger Mites on Apodemus agrarius during Autumn in Korea.

A nationwide survey of chigger mites causing scrub typhus and an investigation of epidemiologic factors for chigger mites was conducted at 16 localities in 8 provinces in Korea during autumn 2009, 2012, and 2013. A total of 233 Apodemus agrarius were captured, and all were infested with chigger mites. The chigger index was highest in Chungcheongbuk-do in 2009 (358.3) and 2012 (290.1) and Chungcheongnam-do in 2013 (294.4). The predominant chigger mite species was Leptotrombidium pallidum in the northern and central parts and L. scutellare in the southern and western parts, Korea. L. pallidum was not found in Jellanam-do and Gyeongsangnam-do and the distribution of L. scutellare had been expanded in the northern parts of Korea. The chigger index of L. pallidum was positively correlated with temperature and negatively correlated with humidity. The incidence of scrub typhus is dependent on L. scutellare index. These findings could be helpful to monitor the distribution of chigger mites and to develop a preventive measures for scrub typhus in Korea.

Comparative Microbiome Analysis of Three Species of Laboratory-Reared Periplaneta Cockroaches.

Cockroaches inhabit various habitats, which will influence their microbiome. Although the microbiome can be influenced by the diet and environmental factors, it can also differ between species. Therefore, we conducted 16S rDNAtargeted high-throughput sequencing to evaluate the overall bacterial composition of the microbiomes of 3 cockroach species, Periplaneta americana, P. japonica, and P. fuliginosa, raised in laboratory for several generations under the same conditions. The experiments were conducted using male adult cockroaches. The number of operational taxonomic units (OTUs) was not significantly different among the 3 species. With regard to the Shannon and Pielou indexes, higher microbiome values were noted in P. americana than in P. japonica and P. fuliginosa. Microbiome composition was also evaluated, with endosymbionts accounting for over half of all OTUs in P. japonica and P. fuliginosa. Beta diversity analysis further showed that P. japonica and P. fuliginosa had similar microbiome composition, which differed from that of P. americana. However, we also identified that P. japonica and P. fuliginosa host distinct OTUs. Thus, although microbiome compositions may vary based on multiple conditions, it is possible to identify distinct microbiome compositions among different Periplaneta cockroach species, even when the individuals are reared under the same conditions.

Genetic Characterization of Japanese Encephalitis Virus Genotype 5 Isolated from Patient, South Korea, 2015.

We isolated Japanese encephalitis virus genotype 5 from human specimens in South Korea. Whole-genome analysis showed 90.4% identity with other genotype 5 viruses from humans. This virus had a unique insertion in the NS4A gene. However, the envelope protein contained Lys 84, which was specific to strains of genotype 5 viruses from South Korea.

Genetic and pathogenic diversity of severe fever with thrombocytopenia syndrome virus (SFTSV) in South Korea.

To investigate nationwide severe fever with thrombocytopenia syndrome virus (SFTSV) infection status, we isolated SFTSVs from patients with suspected severe fever with thrombocytopenia syndrome (SFTS) in 207 hospitals throughout South Korea between 2013 and April 2017. A total of 116 SFTSVs were isolated from 3137 SFTS-suspected patients, with an overall 21.6% case fatality rate. Genetic characterization revealed that at least 6 genotypes of SFTSVs were co-circulating in South Korea, with multiple reassortments among them. Of these, the genotype B-2 strains were the most prevalent, followed by the A and F genotypes. Clinical and epidemiologic investigations revealed that genotype B strains were associated with the highest case fatality rate, while genotype A caused only one fatality among 10 patients. Further, ferret infection studies demonstrated varying clinical manifestations and case mortality rates with different strains of SFTSV, which suggests this virus could exhibit genotype-dependent pathogenicity.

Severe fever with thrombocytopenia syndrome phlebovirus non-structural protein activates TPL2 signalling pathway for viral immunopathogenesis.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV), listed in the World Health Organization Prioritized Pathogens, is an emerging phlebovirus with a high fatality1-4. Owing to the lack of therapies and vaccines5,6, there is a pressing need to understand SFTSV pathogenesis. SFSTV non-structural protein (NSs) has been shown to block type I interferon induction7-11 and facilitate disease progression12,13. Here, we report that SFTSV-NSs targets the tumour progression locus 2 (TPL2)-A20-binding inhibitor of NF-κB activation 2 (ABIN2)-p105 complex to induce the expression of interleukin-10 (IL-10) for viral pathogenesis. Using a combination of reverse genetics, a TPL2 kinase inhibitor and Tpl2-/- mice showed that NSs interacted with ABIN2 and promoted TPL2 complex formation and signalling activity, resulting in the marked upregulation of Il10 expression. Whereas SFTSV infection of wild-type mice led to rapid weight loss and death, Tpl2-/- mice or Il10-/- mice survived an infection. Furthermore, SFTSV-NSs P102A and SFTSV-NSs K211R that lost the ability to induce TPL2 signalling and IL-10 production showed drastically reduced pathogenesis. Remarkably, the exogenous administration of recombinant IL-10 effectively rescued the attenuated pathogenic activity of SFTSV-NSs P102A, resulting in a lethal infection. Our study demonstrates that SFTSV-NSs targets the TPL2 signalling pathway to induce immune-suppressive IL-10 cytokine production as a means to dampen the host defence and promote viral pathogenesis.

Production of Ebola virus-like particles in Drosophila melanogaster Schneider 2 cells.

In this study, we generated recombinant virus-like particles (VLPs) against family Filoviridae, genus Ebolavirus, species Zaire ebolavirus, strain Makona (EBOV) in Drosophila melanogaster Schneider 2 (S2) cells using the EBOV Makona. S2 cells were cotransfected with four viral plasmids encoding EBOV Makona proteins and protein expression was analyzed by immunoblotting. We confirmed that EBOV Makona proteins were successfully expressed in S2 cells. Additionally, we further examined the formation of intracellular and extracellular VLPs by electron microscopy. eVLPs were produced by sucrose gradient ultracentrifugation of S2 cells transfected with EBOV Makona genes, and production of VLPs was confirmed by immunoblot analysis. Collectively, our findings showed that the S2 cell system could be a promising tool for efficient production of eVLPs.

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus Antibodies in Rural Areas, South Korea.

We investigated 1,228 residents of 3 rural areas in South Korea and determined that 50 (4.1%) were positive for severe fever with thrombocytopenia syndrome virus antibodies. Fever and gastrointestinal symptoms in the previous 3 years and career duration were associated with virus seropositivity.

Prevalence and Density of Digenetic Trematode Metacercariae in Clams and Oysters from Western Coastal Regions of the Republic of Korea.

A survey was performed to know the recent infection status of digenetic trematode metacercariae in clams and oysters from 4 sites in western coastal regions of the Republic of Korea (=Korea). Four species of clams (Mactra veneriformis, Ruditapes philippinarum, Cyclina sinensis, and Saxidomus purpuratus) were collected from Taean-gun, Chungcheongnam-do (Province), Buan-gun (County) and Gochang-gun, Jeollabuk-do, and oysters, Crassostrea gigas, from Shinan-gun, Jeollanam-do were transferred to our laboratory on ice and examined by the artificial digestion method. The metacercariae of Himasthla alincia were detected in 3 species of clams, M. veneriformis, R. philippinarum, and C. sinensis from the 3 surveyed areas. The positive rate and the mean density per clam infected were 98.9% (30.8 metacercariae) in M. veneriformis, 60.0% (5.0) in R. philippinarum, and 96.0% (28.4) in C. sinensis. The positive rate (mean density) of Acanthoparyphium tyosenense metacercariae in M. veneriformis was 50.0% (2.1) from Taean-gun and 70.0% (2.8) from Gochang-gun. The metacercariae of Parvatrema spp. were detected in M. veneriformis and R. philippinarum from Taean-gun and Gochang-gun; the positive rate (mean density) was 63.3% (4,123) and 50.0% (19) in M. veneriformis, and 6.7% (126) and 100% (238) in R. philippinarum from the 2 regions, respectively. The metacercariae of Gymnophalloides seoi were detected in all 30 oysters from Shinan-gun, and their average density per oyster was 646. From the above results, it has been confirmed that more than 3 species of metacercariae are prevalent in clams from the western coastal regions, and G. seoi metacercariae are still prevalent in oysters from Shinan-gun, Jeollanam-do, Korea.

Molecular genomic characterization of tick- and human-derived severe fever with thrombocytopenia syndrome virus isolates from South Korea.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral disease caused by the SFTS virus (SFTSV) from Bunyaviridae that is endemic in East Asia. However, the genetic and evolutionary characteristics shared between tick- and human-derived Korean SFTSV strains are still limited. METHODOLOGY/PRINCIPAL FINDINGS: In this study we identify, for the first time, the genome sequence of a tick (Haemaphysalis longicornis)-derived Korean SFTSV strain (designated as KAGWT) and compare this virus with recent human SFTSV isolates to identify the genetic variations and relationships among SFTSV strains. The genome of the KAGWT strain is consistent with the described genome of other members of the genus Phlebovirus with 6,368 nucleotides (nt), 3,378 nt, and 1,746 nt in the Large (L), Medium (M) and Small (S) segments, respectively. Compared with other completely sequenced human-derived Korean SFTSV strains, the KAGWT strain had highest sequence identities at the nucleotide and deduced amino acid level in each segment with the KAGWH3 strain which was isolated from SFTS patient within the same region, although there is one unique amino acid substitution in the Gn protein (A66S). Phylogenetic analyses of complete genome sequences revealed that at least four different genotypes of SFTSV are co-circulating in South Korea, and that the tick- and human-derived Korean SFTSV strains (genotype B) are closely related to one another. Although we could not detect reassortant, which are commonly observed in segmented viruses, further large-scale surveillance and detailed genomic analysis studies are needed to better understand the molecular epidemiology, genetic diversity, and evolution of SFTSV. CONCLUSIONS/SIGNIFICANCE: Full-length sequence analysis revealed a clear association between the genetic origins of tick- and human-derived SFTSV strains. While the most prevalent Korean SFTSV is genotype B, at least four different genotypes of SFTSV strains are co-circulating in South Korea. These findings provide information regarding the molecular epidemiology, genetic diversity, and evolution of SFTSV in East Asia.

Serological and Molecular Detection of Toxoplasma gondii and Babesia microti in the Blood of Rescued Wild Animals in Gangwon-do (Province), Korea.

Infections of Toxoplasma gondii and Babesia microti are reported in many wild animals worldwide, but information on their incidence and molecular detection in Korean wild fields is limited. In this study, the prevalence of T. gondii and B. microti infection in blood samples of 5 animal species (37 Chinese water deer, 23 raccoon dogs, 6 roe deer, 1 wild boar, and 3 Eurasian badgers) was examined during 2008-2009 in Gangwon-do (Province), the Republic of Korea (=Korea) by using serological and molecular tests. The overall seropositivity of T. gondii was 8.6% (6/70); 10.8% in Chinese water deer, 4.3% in raccoon dogs, and 16.7% in roe deer. PCR revealed only 1 case of T. gondii infection in Chinese water deer, and phylogenic analysis showed that the positive isolate was practically identical to the highly pathogenetic strain type I. In B. microti PCR, the positive rate was 5.7% (4/70), including 2 Chinese water deer and 2 Eurasian badgers. Phylogenetic analysis results of 18S rRNA and the ß-tubulin gene showed that all positive isolates were US-type B. microti. To our knowledge, this is the first report of B. microti detected in Chinese water deer and Eurasian badger from Korea. These results indicate a potentially high prevalence of T. gondii and B. microti in wild animals of Gangwon-do, Korea. Furthermore, Chinese water deer might act as a reservoir for parasite infections of domestic animals.

Viral and serological kinetics in Zika virus-infected patients in South Korea.

Zika virus is a mosquito-borne flavivirus that causes clinical symptoms similar to those observed in dengue and chikungunya virus infections. The Korea Centers for Disease Control and Prevention initiated laboratory testing using a real-time reverse transcription-polymerase chain reaction in January 2016. More than 1,000 suspected cases of infection were tested and nine were confirmed as imported cases of Zika virus infection from January to July 2016. The travel destinations of the infected individuals were Brazil, Philippines, Viet Nam, Guatemala, Puerto Rico, and the Dominican Republic. Phylogenetic analysis based on the partial envelope gene indicated that the viruses belonged to the Asian genotype circulating in South America. We further investigated the duration for which the viral RNA and virus-specific antibodies were detectable after the symptom onset. After the day of symptom onset, Zika virus was detectable until 6 days in serum, 14 days in urine and saliva, and 58 days in semen. Immunoglobulin M against Zika virus was detected as early as 2 days after the symptom onset and was maintained at these levels until 41 days, whereas Immunoglobulin G was detectable from 8 days after the symptom onset and was maintained until 52 days. These findings would help diagnostic laboratories improve their testing programs for Zika virus infection.

Heat shock proteins 70 and 90 from Clonorchis sinensis induce Th1 response and stimulate antibody production.

BACKGROUND: Heat shock proteins (HSPs) are found in all prokaryotes and most compartments of eukaryotic cells. Members of the HSP family mediate immune responses to tissue damage or cellular stress. However, little is known about the immune response induced by the oriental liver fluke, Clonorchis sinensis, even though this organism is carcinogenic to humans. We address this issue in the present study in mouse bone marrow dendritic cells (mBMDCs), using recombinant HSP70 and 90 from C. sinensis (rCsHSP70 and rCsHSP90). METHODS: rCsHSP70 and rCsHSP90 were produced in an E. coli system. Purified recombinant proteins were treated in BMDCs isolated from C57BL/6 mice. T cells were isolated from Balb/c mice and co-cultured with activated mBMDCs. Expression of surface molecules was measured by flow cytometry and cytokine secretion was quantified using ELISA. C57BL/6 mice were divided into four groups, including peptide alone, peptide/Freund's adjuvant, peptide/CsHSP70, peptide/CsHSP90, and were immunized intraperitoneally three times. Two weeks after final immunization, antibodies against peptide were measured using ELISA. RESULTS: Both proteins induced a dose-dependent upregulation in major histocompatibility complex and co-stimulatory molecule expression and increased secretion of pro-inflammatory cytokines including interleukin (IL)-1ß, -6, and -12p70 and tumor necrosis factor-α in mBMDCs. Furthermore, when allogenic T cells were incubated with mBMDCs activated by rCsHSP70 and rCsHSP90, the helper T cell (Th)1 cytokine interferon-γ was up-regulated whereas the level of the Th2 cytokine IL-4 was unchanged. These results indicate that rCsHSPs predominantly induce a Th1 response. Over and above these results, we also demonstrated that the production of peptide-specific antibodies can be activated after immunization via in vitro peptide binding with rCsHSP70 or rCsHSP90. CONCLUSION: This study showed for the first time that the HSP or HSP/peptide complexes of C. sinensis could be considered as a more effective vaccine against C. sinensis infection as results of the activator of host immune response as well as the adjuvant for antigenic peptide conjugate to induce peptide-specific antibody response in mice.

Epidemiological and Clinical Features of Severe Fever with Thrombocytopenia Syndrome During an Outbreak in South Korea, 2013-2015.

Since the first reported case of severe fever with thrombocytopenia syndrome (SFTS) in South Korea in 2013, between 2013 and 2015, we collected 1,697 serum samples from suspected patients who experienced symptoms of SFTS. We performed reverse transcriptase polymerase chain reaction using total RNA extracted from the patients' sera. When viral RNA was detected in the sera, SFTS was diagnosed. Among the 1,697 samples, 170 were positive for SFTS virus. We then analyzed the epidemiologic features of these 170 cases. As a result, we found that the annual number of cases increased steadily. However, the annual case fatality rate exhibited a downward trend. The majority of patients were aged ≥ 60 years, and most cases occurred during May-October in the eastern and southern parts of the country. These results may be useful for effective SFTS control by describing the clinical and epidemiologic features of the disease in South Korea.

Severe Fever with Thrombocytopenia Syndrome Presenting with Hemophagocytic Lymphohistiocytosis.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the newly discovered SFTS Bunyavirus, and there have been no case reports of SFTS patients presenting with hemophagocytic lymphohistiocytosis (HLH) in the English literature. We report a case of SFTS presenting with HLH in a 73-year-old immunocompetent male farmer. Although the patient had poor prognostic factors for SFTS, such as old age and central nervous system symptoms, he recovered fully with supportive care.

Pygidiopsis summa (Digenea: Heterophyidae): Status of Metacercarial Infection in Mullets from Coastal Areas in the Republic of Korea.

To know the infection status of zoonotic trematode metacercariae in brackish water fish, we surveyed mullets collected from 18 coastal areas in the Republic of Korea. The metacercariae of Pygidiopsis summa were detected in 236 (68.2%) out of 346 mullets examined. They were found in mullets from 15 areas except for those from Boseong-gun (Jeollanam-do), Pohang-si, and Uljin-gun (Gyeongsangbuk-do). Especially in mullets from Taean-gun (Chungcheongnam-do) and Geoje-si (Gyeongsangnam-do), their prevalences were 100% and 95.5%, and the average metacercarial density was more than 1,000 per fish. They were also detected in mullets from 3 coastal lakes, Gyeongpoho, Songjiho, and Hwajinpoho, in Gangwon-do, and their average densities were 419, 147, and 672 per infected fish, respectively. The metacercariae of 5 other heterophyid species, including Heterophyes nocens, Heterophyopsis continua, Metagonimus sp., Stictodora fuscata, and Stictodora lari, were found in the mullets examined. The metacercariae of H. nocens were detected in 66.7, 100, 28.6, 81.6, 3.9, 61.5, and 27.3% of mullets from Muan-gun, Shinan-gun, Haenam-gun, Gangjin-gun, and Boseong-gun (Jeollanam-do), Hadong-gun, and Geoje-si (Gyeongsangnam-do), and their metacercarial intensities were 64, 84, 119, 99, 1, 24, and 24 per fish infected, respectively. From the above results, it has been confirmed that P. summa metacercariae are heavily infected in mullets from coastal areas of Korea. It is suggested that residents who frequently consume raw mullet dish can be easily infected with heterophyid flukes.

Vector Competence of Anopheles kleini and Anopheles sinensis (Diptera: Culicidae) From the Republic of Korea to Vivax Malaria-Infected Blood From Patients From Thailand.

In total, 1,300 each of Anopheles kleini Rueda and Anopheles sinensis Wiedemann sensu stricto (s.s.) females (colonized from the Republic of Korea) and Anopheles dirus Peyton & Harrison (Thai strain) were allowed to feed on blood from Thai malaria patients naturally infected with Plasmodium vivax The overall oocyst infection rates for An. dirus, An. kleini, and An. sinensis s.s. were 77.4, 46.1, and 45.9%, respectively. The mean number of oocysts was significantly higher for An. dirus (82.7) compared with An. kleini (6.1) and An. sinensis s.s. (8.6), whereas the mean number of oocysts for An. kleini and An. sinensis s.s. was similar. The overall sporozoite infection rates for An. dirus, An. kleini, and An. sinensis s.s. dissected on days 14-15, 21, and 28 days post-feed were significantly higher for An. dirus (90.0%) than An. kleini (5.4%), whereas An. kleini sporozoite rates were significantly higher than An. sinensis s.s. (<0.1%). The overall sporozoite indices for positive females with +3 (100-1,000 sporozoites) and +4 (>1,000 sporozoites) salivary gland indices were significantly higher for An. dirus (85.7%), compared with An. kleini (47.1%). Only one An. sinensis s.s. had sporozoites (+2; >10-100 sporozoites). These results indicate that An. kleini is a competent vector of vivax malaria. Although An. sinensis s.s. develops relatively high numbers of oocysts, it is considered a very poor vector of vivax malaria due to a salivary gland barrier.

One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses.

OBJECTIVES: 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 Taq polymerase enzyme mixture. All assays were thoroughly optimized using in vitro-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.

Molecular detection of severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses in ixodid ticks collected from vegetation, Republic of Korea, 2014.

Ticks play an important role in transmission of arboviruses responsible for emerging infectious diseases, and have a significant impact on human, veterinary, and wildlife health. In the Republic of Korea (ROK), little is known about information regarding the presence of tick-borne viruses and their vectors. A total of 21,158 ticks belonging to 3 genera and 6 species collected at 6 provinces and 4 metropolitan areas in the ROK from March to October 2014 were assayed for selected tick-borne pathogens. Haemaphysalis longicornis (n=17,570) was the most numerously collected, followed by Haemaphysalis flava (n=3317), Ixodes nipponensis (n=249), Amblyomma testudinarium (n=11), Haemaphysalis phasiana (n=8), and Ixodes turdus (n=3). Ticks were pooled (adults 1-5, nymphs 1-30, and larvae 1-50) and tested by one-step reverse transcription polymerase chain reaction (RT-PCR) or nested RT-PCR for the detection of severe fever with thrombocytopenia virus (SFTSV), tick-borne encephalitis virus (TBEV), Powassan virus (POWV), Omsk hemorrhagic fever virus (OHFV), and Langat virus (LGTV). The overall maximum likelihood estimation (MLE) [estimated numbers of viral RNA positive ticks/1000 ticks] for SFTSV and TBEV was 0.95 and 0.43, respectively, while, all pools were negative for POWV, OHFV, and LGTV. The purpose of this study was to determine the prevalence of SFTSV, TBEV, POWV, OHFV, and LGTV in ixodid ticks collected from vegetation in the ROK to aid our understanding of the epidemiology of tick-borne viral diseases. Results from this study emphasize the need for continuous tick-based arbovirus surveillance to monitor the emergence of tick-borne diseases in the ROK.