A Reverse-transcription Recombinase-aided Amplification Assay for the Rapid Detection of the Far-Eastern Subtype of Tick-borne Encephalitis Virus / 生物医学与环境科学（英文）

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.

Detection of the Siberian Tick-borne Encephalitis Virus in the Xinjiang Uygur Autonomous Region, northwestern China / 病毒学报

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.

Investigation on the endemic foci of new emerged tick-borne encephalitis in Charles Hilary, Xinjiang / 中华流行病学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Using 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.</p><p><b>RESULTS</b>1760 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%).</p><p><b>CONCLUSION</b>In 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.</p>

Prevalence of tick-borne encephalitis virus in ticks from southern Korea

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.

Characteristic analysis of E protein genes of new strains of tick-borne encephalitis virus isolated from China / 病毒学报

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.

Expression of tick-borne encephalitis virus prM-E protein in insect cells and studies on its antigenicity / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Recombinant 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.</p><p><b>CONCLUSION</b>The prM-E protein expressed in insect cells retains good antigenicity.</p>

Serological screening of Tick-borne encephalitis (TBE) among Malaysian encephalitis patients

Tick-borne encephalitis (TBE) is a viral infection of the central nervous system and is caused by tick bites, usually after travel to rural or forested areas. The disease is prevalent in Scandinavia, Western Europe, Central Europe and the former Soviet Union and East Asia including Japan. In Malaysia, so far there are no reported cases of TBE. In the present time, many illnesses have been attributed to traveling to other parts of the world. Thus it is important to carry out TBE prevalence study to determine whether the virus is present among Malaysian population. Samples (sera and CSF) from patients admitted to major MOH hospitals in Peninsular Malaysia and Sabah with a clinical diagnosis of encephalitis but is IgM negative for JE, were tested for TBEV IgM ELISA and TBEV IgG ELISA (DRG, Germany). Out of the 600 samples screened for TBEV IgG, all were non-reactive. In addition, out of the 100 samples screened for TBEV IgM, all the samples were also non-reactive. Our results indicate that currently TBE is not present in the Malaysian population. Among the reasons for this could be lack of the infection agent, absence of the suitable vector or subjects selected for the study did not fit the criteria of possible exposure to TBE infections. Hence we recommend that for any future study, the selection of subjects should include those who returned from tick-infested forested areas.

A serological survey of arboviral diseases among the human population of the Andaman and Nicobar Islands, India.

In an attempt to determine the prevalence of certain arthropod-borne viruses of public health importance amongst the human population of the Andaman and Nicobar Islands of India, 2,401 sera were collected from six major localities. The sera were analysed by the hemagglutination inhibition (HI) and neutralization (N) tests, using Chikungunya (CHIK), Japanese encephalitis (JE), West Nile (WN), dengue (DEN-2), Langat (TP-21) and Kyasanur Forest disease (KFD) viral antigens. The highest prevalence of HI antibodies was detected against KFD virus (22.4%), followed by Langat (20.2%), JE (5.9%), DEN-2 (3.1%), CHIK (2.9%) and WN (0.8%) viruses. Cross-reactions to the viral antigens were also noted. The results of N tests indicated a high prevalence of DEN-2 (25.4%) virus, followed by Langat (17.5%), CHIK (15.3%), KFD (12%), JE (2.19%) and WN (1.8%). These results are discussed in relation to important epidemiological parameters like age, sex and geographical location. To our knowledge, this is the first report of an extensive serosurvey of arthropod-borne viruses on these islands.

Analysis of the Three Dimensional Structure of Envelope Protein of the Japnes Encephalitis virus Isolated in Korea

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.

Field evaluation of formalin inactivated Kyasanur forest disease virus tissue culture vaccine in three districts of Karnataka state.

A formalin inactivated Kyasanur forest disease (KFD) virus tissue culture vaccine produced by the health department of the State Government of Karnataka at Shimoga was administered in Shimoga, Uttar Kannada and Chikmangalur districts during 1990-92 KFD epidemic seasons. The selection of places for vaccination was based on the prevalence of KFD activity in previous years; villages adjacent to KFD affected areas and the villages from which mortality in monkeys was reported. A total of 284 villages was covered under vaccination; 26850 individuals received one dose whereas, 61302 received two doses of vaccine. No untoward reaction was observed in any of the vaccinees. In the 72 KFD affected villages there were 14 patients among 9072 and 10 among 21083 vaccinees receiving one and two doses respectively, whereas 325 patients were reported among 37373 unvaccinated individuals during the same period. In 1990-91 the number of males patients was more than females whereas, in 1991-92 the ratio was reserved. On analysis indicated that the vaccine has a highly significant protective effect.