Three dimensional structures of envelope protein from Korean isolates and Nakayama-NIH strain of Japanese encephalitis virus (JEV) were deduced by a computer program (HyperChem 4.0 Chemplus 1.0) based on the data of the three dimentional structure of Tick-borne encephalitis virus. In the three dimensional structure of envelope protein, neutralizing epitope and T-helper cell recognition site of C-terminal region of Korean isolates were structually similar to those of Nakayama-NIH but the N-terminal region was not. Korean JE isolates were compared with Nakayama-NIH strain by using cross-neutralization antibody test. Neutralizing activities of Korean isolates derived from guinea pigs were higher than those of Nakayama-NIH strain against Korean isolates, although the polyclonal antibody titers of Nakayama-NIH showed 1:160 to 1:640 against Korean isolates. According to the results from three dimentional structures and cross-neutralization analyses, the antigenic difference between Korean JE isolates and Nakayama-NIH strain may be dependent on structural difference of envelope protein.
Animals ; Encephalitis Virus, Japanese ; Encephalitis Viruses* ; Encephalitis Viruses, Tick-Borne ; Encephalitis* ; Guinea Pigs ; Korea*

OBJECTIVETo investigate the recent emerged endemic region of tick-borne encephalitis (TBE) regarding its natural reserves, in Charles Hilary, northern Xinjiang and to isolate and characterize the viral geographic strain.

METHODSUsing indirect fluorescent assay to detect tick-borne encephalitis virus (TBEV) specific IgG antibodies from serum of local residents including 2 unconfirmed viral encephalitis patients in 2011 spring-summer. Viruses were isolated from tick samples by inoculating BALB/c mice and BHK-21 cells. For phylogenetic analysis. TBEV NS1 gene fragments were detected by RT-PCR and then subjected to sequence alignment.

RESULTS1760 ticks were captured from the fields to have found that Ixodes persulcatus were dominated among the tick population. Two viral encephalitis patients were diagnosed as TBEV infection. In addition, 35.4% (23/65) local residents were detected positive for presence of TBEV specific-IgG antibodies in serum. After inoculation, morbidity and mortality of BALB/c mice were 72.9% (70/96) and 55.7% (44/79), respectively. TBEV specific-fragments were amplified from brain tissue of dead mice and cells culture supernatant. NS1 sequence alignment showed that the viral isolates were clustered into TBEV far-eastern sub-type, phylogenetically, and were mostly close to the isolates from northeastern China (99%) and Russian strain (98%).

CONCLUSIONIn this study, a new endemic loci of TBE was firstly described in Charles Hilary natural reserve, northern Xinjiang. TBEV geographic isolates belonged to TBEV far-eastern subtype while Ixodes persulcatus and Dermacentor silvarum played crucial roles for disease transition.

Adult ; Animals ; China ; epidemiology ; Encephalitis Viruses, Tick-Borne ; genetics ; immunology ; isolation & purification ; Encephalitis, Tick-Borne ; epidemiology ; transmission ; virology ; Female ; Humans ; Male ; Mice ; RNA, Viral ; genetics

Until the recent emergence/re-emergence of human-pathogenic viruses in ticks, tick-borne viruses have been neglected as causative agents of human disease (particularly in China). To gain insight into the diversity of tick-borne viruses in Xinjiang Uygur Autonomous Region (northwestern China), we conducted illumina deep sequencing-based screening for virus-derived small RNAs in field-collected Ixodes persulcatus ticks. We found 32, 631 unique virus-matched reads. In particular, 77 reads mapped to the tick-borne group within the genus of Flavivirus, and covered 3.8%-2.4% viral genomes. In addition, 32 unique reads were specific to the Siberian subtype of tick-borne encephalitis viruses (TBEV-Sib) which have never been reported in Chinese TBE loci. We confirmed the potential existence of TBEV-Sib by amplification (using reverse transcription-polymerase chain reaction) of genomic fragments from the envelope gene or 3' genomic terminus from the pools of examined ticks. Both sequences demonstrated high homology to TBEV-Sib strains attached geographically to southern Siberia with nucleotide identity of 97.2%-95.5% and aminoacid identity of 99.4%-98.3%, respectively. In conclusion, we report, for the first time, detection of TBEV-Sib in the natural TBE loci of China. These novel data may provide genetic information for further isolation and epidemiologic investigation of TBEV-Sib.
Animals ; Arachnid Vectors ; virology ; China ; Encephalitis Viruses, Tick-Borne ; classification ; genetics ; isolation & purification ; Encephalitis, Tick-Borne ; transmission ; virology ; Genome, Viral ; Humans ; Ixodes ; virology ; Molecular Sequence Data ; Phylogeny

OBJECTIVE: Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. METHODS: A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. RESULTS: The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. CONCLUSION A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.
Encephalitis Viruses, Tick-Borne ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; RNA, Viral ; analysis

The prevalence of tick-borne encephalitis virus (TBEV) in southern Korea was determined by collecting ticks using tick drags. A total of 4,077 of 6,788 ticks collected were pooled (649 pools) according to collection site, species, and developmental stage and assayed for TBEV. The TBEV protein E and NS5 gene fragments were detected using RT-nested PCR in six pools of nymphs collected from Jeju Island (2,491 ticks). The minimum field detection rates for TBEV were 0.17% and 0.14% for Haemaphysalis longicornis and Haemayphysalis. flava nymphs, respectively. The 252 bp NS5 and 477 bp protein E gene amplicons were sequenced. Phylogenetic analysis showed that the NS5 and protein E genes of the Jeju strain were clustered with Western subtype (98.0% and 99.4% identity, respectively). The Western subtype of TBEV is endemic in Korea, including Jeju Island. The study of vector and zoonotic host susceptibility to TBEV is required to better understand its potential impact on public health.
Animals ; Arachnid Vectors/*virology ; Base Sequence ; DNA Primers/genetics ; Encephalitis Viruses, Tick-Borne/classification/*genetics ; Encephalitis, Tick-Borne/*epidemiology ; Molecular Sequence Data ; *Phylogeny ; Prevalence ; Republic of Korea/epidemiology ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Ticks/*virology ; Viral Envelope Proteins/genetics

BACKGROUNDTo express the prM-E protein in Sf9 cells, and lay a basis for further study on the function of the viral proteins and development of specific diagnostic reagents.

METHODSThe recombinant prM-E protein of tick-borne encephalitis virus was expressed in insect cell Sf9 by RT-PCR amplification of prM-E gene, construction of donor plasmid of Bac-to-Bac baculovirus expression system, homologous recombination of donor plasmid with bacmid DNA at the site of Tn7 and transfection of insect cell Sf9.

RESULTSRecombinant subviral particles, about 30 nm in diameter, consisting of prM-E were observed by electron microscope in the supernatant of infected cells, which indicated that infected cells released virus-like particles (VLPs) into the culture medium. The results of Western-blot and the indirect immunofluorescence assay (IFA) showed that the recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins. Using the recombinant prM-E as antigens to detect samples of patient sera by ELISA and IFA, all of 16 sera from patients with tick-borne encephalitis were positive and all of 6 sera from other patients were negative.

CONCLUSIONThe prM-E protein expressed in insect cells retains good antigenicity.

Animals ; Blotting, Western ; Cell Line ; Encephalitis Viruses, Tick-Borne ; genetics ; Fluorescent Antibody Technique, Indirect ; Gene Expression Regulation, Viral ; Reverse Transcriptase Polymerase Chain Reaction ; Spodoptera ; Viral Envelope Proteins ; genetics ; immunology ; metabolism

In order to determine the characteristics and genotypes of E protein genes of tick-borne encephalitis (TBE) virus strains DXAL-5, 12,13,16,18, 21 isolated from Ixodes persulcatus in the Northeast of China, cDNA synthesis of E protein genes of the six DXAL strains was performed using RT-PCR, and the E protein genes were cloned and sequenced. The results showed that the nucleotide sequence of E protein gene of the six DXAL strains was 1488 bp in length respectively and the length of predicted protein was 496 aa respectively. Sequence comparison of E protein genes among the six DXAL strains and the reference TBE virus strains showed that the six DXAL strains were more homologous to Far Eastern subtype strains than to Siberian subtype strains or European subtype strains. And the majority of subtype-determining amino acid sites of the six DXAL strains belonged to TBE virus Far Eastern subtype. Phylogenetic analysis of protein E showed that the six DXAL strains were all within the clade containing Far Eastern subtype strains. The new strains had higher identities and closer phylogenetic relationships with Senzhang strain, so we speculate that this vaccine strain still have good protection against the new TBE virus isolates. In the A, B and C antigenic domains of protein E, the six DXAL strains had different degrees of amino acid changes. These mutations were likely to affect the function of E protein.
Amino Acid Sequence ; Animals ; Base Sequence ; China ; DNA, Complementary ; chemistry ; genetics ; Encephalitis Viruses, Tick-Borne ; classification ; genetics ; isolation & purification ; Mice ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Alignment ; Sequence Analysis, DNA ; Viral Envelope Proteins ; chemistry ; genetics

In 2007, the Korean Society of Infectious Diseases recommended an immunization schedule for adults residing in Korea. This recommendation includes four categories: standard immunization for healthy persons, supplementary immunization for persons with underlying illnesses, recommendations for health-care personnel and for international travelers. The Standard Immunization Schedule consists of universal vaccination (tetanus-diphtheria/tetanus-diphtheria-pertussis (Td/Tdap); hepatitis A vaccine for persons aged 20s; human papilloma virus vaccine for women under 26 years old; meningococcal vaccine for military recruits and college students in dormitories; influenza vaccination for persons aged 50 years or more and pregnant women; and pneumococcal vaccination for persons aged 65 years or more) and catch-up vaccination (hepatitis A vaccine for persons aged 30s; hepatitis B vaccine, varicella and MMR vaccines for persons who have uncertain specific immunities). The Supplementary Immunization Schedule recommends 4 vaccines, i.e., pneumococcal, influenza, meningococcal, and Haemophilus influenzae type b, appropriate to the underlying illnesses, and confirmation of the presence of antibodies to hepatitis A and hepatitis B. The Recommendation for Health-Care Personnel includes universal vaccination (influenza and pertussis) and catch-up vaccination (hepatitis B, measles-mumps-rubella and varicella for seronegative health-care personnel). The Recommended Immunization for International Travelers focuses reinforcement of vaccinations for healthy persons and includes certain immunizations necessary for international travel (yellow fever, meningococcal, plaque, tick-borne encephalitis, and cholera). These guidelines can be seen at www.ksid.or.kr.
Adult ; Aged ; Antibodies ; Chickenpox ; Collodion ; Communicable Diseases ; Encephalitis, Tick-Borne ; Female ; Fever ; Haemophilus influenzae type b ; Hepatitis A ; Hepatitis A Vaccines ; Hepatitis B ; Hepatitis B Vaccines ; Humans ; Immunization ; Immunization Schedule ; Influenza, Human ; Korea ; Meningococcal Vaccines ; Military Personnel ; Papilloma ; Reinforcement (Psychology) ; Vaccination ; Vaccines ; Viruses

In 2007, the Korean Society of Infectious Diseases recommended an immunization schedule for adults residing in Korea. This recommendation includes four categories: standard immunization for healthy persons, supplementary immunization for persons with underlying illnesses, recommendations for health-care personnel and for international travelers. The Standard Immunization Schedule consists of universal vaccination (tetanus-diphtheria/tetanus-diphtheria-pertussis (Td/Tdap); hepatitis A vaccine for persons aged 20s; human papilloma virus vaccine for women under 26 years old; meningococcal vaccine for military recruits and college students in dormitories; influenza vaccination for persons aged 50 years or more and pregnant women; and pneumococcal vaccination for persons aged 65 years or more) and catch-up vaccination (hepatitis A vaccine for persons aged 30s; hepatitis B vaccine, varicella and MMR vaccines for persons who have uncertain specific immunities). The Supplementary Immunization Schedule recommends 4 vaccines, i.e., pneumococcal, influenza, meningococcal, and Haemophilus influenzae type b, appropriate to the underlying illnesses, and confirmation of the presence of antibodies to hepatitis A and hepatitis B. The Recommendation for Health-Care Personnel includes universal vaccination (influenza and pertussis) and catch-up vaccination (hepatitis B, measles-mumps-rubella and varicella for seronegative health-care personnel). The Recommended Immunization for International Travelers focuses reinforcement of vaccinations for healthy persons and includes certain immunizations necessary for international travel (yellow fever, meningococcal, plaque, tick-borne encephalitis, and cholera). These guidelines can be seen at www.ksid.or.kr.
Adult ; Aged ; Antibodies ; Chickenpox ; Collodion ; Communicable Diseases ; Encephalitis, Tick-Borne ; Female ; Fever ; Haemophilus influenzae type b ; Hepatitis A ; Hepatitis A Vaccines ; Hepatitis B ; Hepatitis B Vaccines ; Humans ; Immunization ; Immunization Schedule ; Influenza, Human ; Korea ; Meningococcal Vaccines ; Military Personnel ; Papilloma ; Reinforcement (Psychology) ; Vaccination ; Vaccines ; Viruses