Local adaptation of Mycobacterium tuberculosis on the Tibetan Plateau.

During its global dispersal, Mycobacterium tuberculosis (Mtb) has encountered varied geographic environments and host populations. Although local adaptation seems to be a plausible model for describing long-term host-pathogen interactions, genetic evidence for this model is lacking. Here, we analyzed 576 whole-genome sequences of Mtb strains sampled from different regions of high-altitude Tibet. Our results show that, after sequential introduction of a few ancestral strains, the Tibetan Mtb population diversified locally while maintaining strict separation from the Mtb populations on the lower altitude plain regions of China. The current population structure and estimated past population dynamics suggest that the modern Beijing sublineage strains, which expanded over most of China and other global regions, did not show an expansion advantage in Tibet. The mutations in the Tibetan strains showed a higher proportion of A > G/T > C transitions than strains from the plain regions, and genes encoding DNA repair enzymes showed evidence of positive selection. Moreover, the long-term Tibetan exclusive selection for truncating mutations in the thiol-oxidoreductase encoding sseA gene suggests that Mtb was subjected to local selective pressures associated with oxidative stress. Collectively, the population genomics of Mtb strains in the relatively isolated population of Tibet provides genetic evidence that Mtb has adapted to local environments.

Deep N-terminomics of Mycobacterium tuberculosis H37Rv extensively correct annotated encoding genes.

Mycobacterium tuberculosis (MTB) is a severe causing agent of tuberculosis (TB). Although H37Rv, the type strain of M. tuberculosis was sequenced in 1998, annotation errors of encoding genes have been frequently reported in hundreds of papers. This phenomenon is particularly severe at the 5' end of the genes. Here, we applied a TMPP [(N-Succinimidyloxycarbonylmethyl) tris (2,4,6-trimethoxyphenyl) phosphonium bromide] labeling combined with StageTip separating strategy on M. tuberculosis H37Rv to characterize the N-terminal start sites of its annotated encoding genes. Totally, 1047 proteins were identified with 2058 TMPP labeled N-terminal peptides from all the 2625 mass spectrometer (MS) sequenced proteins. Comparative genomics analysis allowed the re-annotation of 43 proteins' N-termini in H37Rv and 762 proteins in Mycobacteriaceae. All revised N-termini start sites were distributed in 5'-UTR of annotated genes due to over-annotation of previous N-terminal initiation codon, especially the ATG. In addition, we identified and verified a novel gene Rv1078A in +3 frame different from the annotated gene Rv1078 in +2 frame. Altogether, our findings contribute to the better understanding of N-terminal of H37Rv and other species from Mycobacteriaceae that can assist future studies on biological study.

Discovery of novel DprE1 inhibitors via computational bioactivity fingerprints and structure-based virtual screening.

Decaprenylphosphoryl-ß-D-ribose oxidase (DprE1) plays important roles in the biosynthesis of mycobacterium cell wall. DprE1 inhibitors have shown great potentials in the development of new regimens for tuberculosis (TB) treatment. In this study, an integrated molecular modeling strategy, which combined computational bioactivity fingerprints and structure-based virtual screening, was employed to identify potential DprE1 inhibitors. Two lead compounds (B2 and H3) that could inhibit DprE1 and thus kill Mycobacterium smegmatis in vitro were identified. Moreover, compound H3 showed potent inhibitory activity against Mycobacterium tuberculosis in vitro (MICMtb = 1.25 µM) and low cytotoxicity against mouse embryo fibroblast NIH-3T3 cells. Our research provided an effective strategy to discover novel anti-TB lead compounds.

Detecting Mycobacterium tuberculosis complex and rifampicin resistance via a new rapid multienzyme isothermal point mutation assay.

Simple, rapid, and accurate detection of the Mycobacterium tuberculosis complex (MTBC) and drug resistance is critical for improving patient care and decreasing the spread of tuberculosis. To this end, we have developed a new simple and rapid molecular method, which combines multienzyme isothermal rapid amplification and a lateral flow strip, to detect MTBC and simultaneously detect rifampin (RIF) resistance. Our findings showed that it has sufficient sensitivity and specificity for discriminating 118 MTBC strains from 51 non-tuberculosis mycobacteria strains and 11 of the most common respiratory tract bacteria. Further, compared to drug susceptibility testing, the assay has a sensitivity, specificity, and accuracy of 54.1%, 100.0%, and 75.2%, respectively, for detection of RIF resistance. Some of the advantages of this assay are that no special instrumentation is required, a constant low temperature of 39 °C is sufficient for the reaction, the turnaround time is less than 20 min from the start of the reaction to read out and the result can be seen with the naked eye and does not require specialized training. These characteristics of the new assay make it particularly useful for detecting MTBC and RIF resistance in resource-limited settings.

Mycolicibacterium baixiangningiae sp. nov. and Mycolicibacterium mengxianglii sp. nov., two new rapidly growing mycobacterial species.

Four bacterial strains (LJ126T/S18 and Z-34T/S20) recovered from faecal samples of Tibetan antelopes on the Qinghai-Tibet Plateau of China were analysed using a polyphasic approach. All four isolates were aerobic, short rod-shaped, non-motile, Gram-stain-positive, acid-fast and fast-growing. Phylogenetic analyses based upon 16S rRNA and whole-genome sequences showed that the two pair of strains formed two distinct branches within the evolutionary radiation of the genus Mycolicibacterium. Strains LJ126T/S18 and Z-34T/S20 were most closely related to Mycolicibacterium austroafricanum CCUG 37667T, Mycobacterium aurum NCTC 10437T, Mycobacterium pyrenivorans DSM 44605T, Mycobacterium monacense JCM 15658T, Mycolicibacterium sarraceniae JCM 30395T, Mycolicibacterium tokaiense JCM 6373T and Mycobacterium murale JCM 13392T, but readily distinguished from the known species by a combination of chemotaxonomic and phenotypic features and by low average nucleotide identity values (74.4-84.9 %). Consequently, the two strain pairs are considered to represent different novel species of Mycolicibacterium for which the names Mycolicibacterium baixiangningiae sp. nov. and Mycolicibacterium mengxianglii sp. nov. are proposed, with LJ126T (=CGMCC 1.1992T=KCTC 49535T) and Z-34T (=CGMCC 1.1993T=DSM 106172T) as the respective type strains.

Expression and purification of Rv2654 recombinant protein from Mycobacterium tuberculosis and its characteristics of immune response. / ç»“æ ¸åˆ†æžæ†èŒæŠ—åŽŸRv2654é‡ç»„è›‹ç™½çš„è¡¨è¾¾å’Œçº¯åŒ–åŠå ¶è¯±å¯¼çš„å ç–«åº”ç­”ç‰¹å¾.

OBJECTIVES: With the decreased protective effect of Bacille Calmette-Guérin (BCG) vaccine, widespread drug-resistant strains of tuberculosis as well as the lack of effective vaccines, global morbidity and mortality of tuberculosis remains high. The purpose of this study is to evaluate the potential of Mycobacterium tuberculosis antigen Rv2654 as a candidate vaccine against tuberculosis, and to verify the characteristics of cellular and humoral immune responses in mice induced by this protein. METHODS: Isopropyl-beta-D-thiogalactoside (IPTG) was added to induce the expression of Rv2654 recombinant protein in the logarithmic growth stage of bacteria. The recombinant protein was purified by affinity chromatography and identified by Western blotting. The immunoreactivity of Rv2654 recombinant protein with human sera was detected by ELISA. After immunization with Rv2654 recombinant protein, the levels of Th1/Th2 cytokines in peripheral blood of mice was measured using cytokine magnetic bead arrays, and the T and B lymphocyte subsets in spleen of mice was analyzed by flow cytometry. RESULTS: The recombinant protein Rv2654 was successfully induced and expressed. The ELISA data showed that the recombinant protein Rv2654 was responsive to the serum of BCG-inoculated patients or active pulmonary tuberculosis patients. In immunized mice with recombinant protein Rv2654, the expression of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and other cytokines in peripheral blood was elevated (all P<0.05). Flow cytometry analysis showed that the recombinant protein significantly stimulated the differentiation of CD4+T cells and CD8+T cells into effector T cells, and this effect was more obvious when combined with BCG. The recombinant protein Rv2654 also stimulated the activation and proliferation of B cells and their differentiation into memory cells. However, less plasma cells were produced. CONCLUSIONS: Rv2654 protein could induce the cell immune responses and it has good binding ability with serum of BCG-inoculated patients and active pulmonary tuberculosis patients, suggesting that it may serve as a good candidate antigen for tuberculosis immunological prophylaxis and diagnostic vaccines.

A real-time PCR assay based on a specific mutation of PstS1 gene for detection of M. bovis strains.

The Mycobacterium tuberculosis complex (MTBC) is composed of several genetically related and pathogenic mycobacterial species, including M. tuberculosis, M. bovis and M.africanum et al. In our previous study, we found that M. bovis strains had a unique SNP located in position 1055 in the sequence of the pstS1 gene in which a T was substituted by a C. In this study, specific primers and MGB probes were designed according to the mutation in PstS1 gene, and a sensitive, specific and rapid real-time PCR assay for M. bovis was established. Then the assay was used to detect M. bovis in simulation samples. The minimum detectable concentration is 101 copies for M. bovis DNA. The standard curve showed correlation coefficient between threshold cycle and PstS1 gene fragment copy number was 0.997 and slope is -3.144. The minimum detectable concentration is 101 cells/ml for simulation sample. In addition, M.bovis strain 93006, 14 clinical BCG stains and 7 clinical M.bovis strain showed positive while the other strains showed negative results, which proved good specificity. This assay had high sensitivity and specificity for identification of M. bovis from the simulation specimens. The assay can be applied for epidemiological and ecological surveillance of M. bovis strains.

Ribokinase screened from T7 phage displayed Mycobacterium tuberculosis genomic DNA library had good potential for the serodiagnosis of tuberculosis.

Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is the leading cause of death among infectious diseases in the worldwide. Lack of more sensitive and effective diagnostic reagents has increased the awareness of rapid diagnosis for tuberculosis. In this study, T7 phage displayed genomic DNA library of M. tuberculosis was constructed to screen the antigens that specially bind with TB-positive serum from the whole genome of M. tuberculosis and to improve the sensitivity and specificity of tuberculosis serological diagnosis. After three rounds of biopanning, results of DNA sequencing and BLAST analysis showed that 19 positive phages displayed four different proteins and the occurrence frequency of the phage which displayed ribokinase was the highest. The results of indirect ELISA and dot immunoblotting indicated that representative phages could specifically bind to tuberculosis-positive serum. The prokaryotic expression vector containing the DNA sequence of ribokinase gene was then constructed and the recombinant protein was expressed and purified to evaluate the serodiagnosis value of ribokinase. The reactivity of the recombinant ribokinase with different clinical serum was detected and the sensitivities and specificities in tuberculosis serodiagnosis were 90% and 86%, respectively by screening serum from tuberculosis patients (n = 90) and uninfected individuals (n = 90) based on ELISA. Therefore, this study demonstrated that ribokinase had good potential for the serodiagnosis of tuberculosis.

Mutations within embCAB Are Associated with Variable Level of Ethambutol Resistance in Mycobacterium tuberculosis Isolates from China.

The EmbCAB proteins have been considered a target for ethambutol (EMB). Mutations in embCAB are known to confer most EMB resistance. However, the knowledge about the effects of embCAB mutations on the EMB resistance level and about the role of mutation-mutation interactions is limited in China. Here, we sequenced embCAB among 125 Mycobacterium tuberculosis isolates from China and quantified their EMB MICs by testing growth at 10 concentrations. Furthermore, a multivariate regression model was established to assess the effects of both individual mutations and multiple mutations. Our results revealed that in China, 82.6% of EMB-resistant isolates (71/86 isolates) harbored at least one mutation within embCAB Most of the mutations were located in the embB and embA upstream region. Several individual mutations and multiple mutations within this region contributed to the different levels of EMB resistance. Their effects were statistically significant. Additionally, there was an association between high-level EMB resistance and multiple mutations.

Southern East Asian origin and coexpansion of Mycobacterium tuberculosis Beijing family with Han Chinese.

The Beijing family is the most successful genotype of Mycobacterium tuberculosis and responsible for more than a quarter of the global tuberculosis epidemic. As the predominant genotype in East Asia, the Beijing family has been emerging in various areas of the world and is often associated with disease outbreaks and antibiotic resistance. Revealing the origin and historical dissemination of this strain family is important for understanding its current global success. Here we characterized the global diversity of this family based on whole-genome sequences of 358 Beijing strains. We show that the Beijing strains endemic in East Asia are genetically diverse, whereas the globally emerging strains mostly belong to a more homogenous subtype known as "modern" Beijing. Phylogeographic and coalescent analyses indicate that the Beijing family most likely emerged around 30,000 y ago in southern East Asia, and accompanied the early colonization by modern humans in this area. By combining the genomic data and genotyping result of 1,793 strains from across China, we found the "modern" Beijing sublineage experienced massive expansions in northern China during the Neolithic era and subsequently spread to other regions following the migration of Han Chinese. Our results support a parallel evolution of the Beijing family and modern humans in East Asia. The dominance of the "modern" Beijing sublineage in East Asia and its recent global emergence are most likely driven by its hypervirulence, which might reflect adaption to increased human population densities linked to the agricultural transition in northern China.

Isolation and identification of multiple drug resistant nontuberculous mycobacteria from organs of cattle produced typical granuloma lesions.

Nontuberculosis mycobacteria are widespread in the environment and some are zoonotic. 320 tissue samples with visible lesions were obtained from dairy cows and examined by histopathology. Eleven samples showed typical granulomatous lesions and a total of 8 strains were cultured. Three genes (16S rRNA, hsp65 and rpoB) were sequenced for species identification. All mycobacterial isolates were tested for rifampicin, isoniazid, ethambutol, streptomycin, capreomycin, kanamycin, para-aminosalicylic acid susceptibility. Six strains were identified as M. fortuitum, 1 was M. avium, 1 was M. conceptionense, isolated from cattle for the first time. Seven of the 8 isolated strains showed multiple drug resistance.

Molecular Characteristics of Mycobacterium tuberculosis Strains Isolated from Cutaneous Tuberculosis Patients in China.

Cutaneous tuberculosis (CTB) is probably underreported due to difficulties in detection and diagnosis. To address this issue, genotypes of Mycobacterium tuberculosis strains isolated from 30 patients with CTB were mapped at multiple loci, namely, RD105 deletions, spacer oligonucleotides, and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTRs). Fifty-eight strains of pulmonary tuberculosis (PTB) were mapped as experimental controls. Drug resistance-associated gene mutations were determined by amplicon sequencing of target regions within 7 genes. Beijing family isolates were the most prevalent strains in CTB and PTB. MIRU-VNTR typing separated the Beijing strains from the non-Beijing strains, and the majority of CTB could be separated from PTB counterparts. Drug resistance determining regions showed only one CTB strain expressing isomazid resistance. Thus, while the CTB strains belonged to the same phylogenetic lineages and sub-lineages as the PTB strains, they differed at the level of several MIRU-VNTRs and in the proportion of drug resistance.

Characterization of Mycobacterium tuberculosis isolates from Hebei, China: genotypes and drug susceptibility phenotypes.

BACKGROUND: Tuberculosis remains a major public health problem in China. The Hebei province is located in the Beijing-Tianjin-Hebei integration region; however little information about the genetic diversity of Mycobacterium tuberculosis was available in this area. This study describes the first attempt to map the molecular epidemiology of MTB strains isolated from Hebei. METHODS: Spoligotyping and 15-locus MIRU-VNTR were performed in combination to yield specific genetic profiles of 1017 MTB strains isolated from ten cities in the Hebei province in China during 2014. Susceptibility testing to first line anti-TB drugs was also conducted for all strains using the L-J proportion method. RESULTS: Based on the SpolDB4.0 database, the predominant spoligotype belonged to the Beijing family (90.5%), followed by T family (6.3%). Using 15-locus MIRU-VNTR clustering analysis, 846 different patterns were identified, including 84 clusters (2-17 strains per cluster) and 764 individual types. Drug susceptibility pattern showed that 347 strains (34.1%) were resistant to at least one of the first line drugs, including 134 (13.2%) multi-drug resistance strains. Statistical analysis indicated that drug resistance was associated with treatment history. The Beijing family was associated with genetic clustering. However, no significant difference was observed between the Beijing and non-Beijing family in gender, age, treatment history and drug resistance. CONCLUSIONS: The Mycobacterium tuberculosis strains in Hebei exhibit high genetic diversity. The Beijing family is the most prevalent lineage in this area. Spoligotyping in combination with 15-locus MIRU-VNTR is a useful tool to study the molecular epidemiology of the MTB strains in Hebei.

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients' serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions.

Analysis of embCAB mutations associated with ethambutol resistance in multidrug-resistant mycobacterium tuberculosis isolates from China.

Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(-16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306-497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.

Discovery of the disubstituted oxazole analogues as a novel class anti-tuberculotic agents against MDR- and XDR-MTB.

A high-throughput screening effort on 45,000 compounds resulted in the discovery of a disubstituted oxazole as a new structural class inhibitor of Mycobacterium tuberculosis (Mtb). In order to improve the activity and investigate the SAR of this scaffold, a series of disubstituted azole analogues have been designed and synthesized. The newly synthesized compounds 1a-y were evaluated for their in vitro anti-TB activity versus replicating, multi- and extensive drug resistant Mtb strains. All the compounds, except 1o, 1p and 1q, showed potent anti-TB activity with MIC of 1-64 mg/L. The test of broad spectrum panel revealed that this series are specific to Mtb. The cytotoxicity assessment indicated that the compounds were not cytotoxic against HEK 293 cells. The compounds could have a novel mechanism to anti-Mtb as they can inhibit drug sensitive and drug resistant Mtb.

Single nucleotide polymorphism in Ag85 genes of Mycobacterium tuberculosis complex: analysis of 178 clinical isolates from China and 13 BCG strains.

Host immune pressure and associated immune evasion of pathogenic bacteria are key features of host-pathogen co-evolution. Human T-cell epitopes of Mycobacterium tuberculosis (M. tuberculosis) were evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. However, in our previous studies, proteins MPT64, PstS1, Rv0309 and Rv2945c all harbored higher numbers of amino acid substitutions in their T cell epitopes, which suggests their roles in ongoing immune evasion. Here, we used the same set of 180 clinical M. tuberculosis complex (MTBC) isolates from China, amplified the genes encoding Ag85 complex, and compared the sequences. The results showed that Ag85 were hyperconserved in T/B cell epitopes and the genes were more likely to be under purifying selection. The divergence of host immune selection on different proteins may result from different function of the proteins. In addition, A312G of Ag85A and T418C of Ag85B may represent special mutations in BCG strains, which may be used to differentiate M.bovis and BCG strains from MTB strains. Also, C714A in Ag85B seems to be a valuable phylogenetic marker for Beijing strains.

Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Hunan, China.

To determine the prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan province, drug susceptibility testing and spoligotyping methods were performed among 171 M. tuberculosis isolates. In addition, the mutated characteristics of 12 loci, including katG, inhA, rpoB, rpsL, nucleotides 388 to 1084 of the rrs gene [rrs(388-1084)], embB, pncA, tlyA, eis, nucleotides 1158 to 1674 of the rrs gene [rrs(1158-1674)], gyrA, and gyrB, among drug-resistant isolates were also analyzed by DNA sequencing. Our results indicated that the prevalences of isoniazid (INH), rifampin (RIF), streptomycin (SM), ethambutol (EMB), pyrazinamide (PZA), capreomycin (CAP), kanamycin (KAN), amikacin (AKM), and ofloxacin (OFX) resistance in Hunan province were 35.7%, 26.9%, 20.5%, 9.9% 15.2%, 2.3%, 1.8%, 1.2%, and 10.5%, respectively. The previously treated patients presented significantly increased risks for developing drug resistance. The majority of M. tuberculosis isolates belonged to the Beijing family. Almost all the drug resistance results demonstrated no association with genotype. The most frequent mutations of drug-resistant isolates were katG codon 315 (katG315), inhA15, rpoB531, rpoB526, rpoB516, rpsL43, rrs514, embB306, pncA96, rrs1401, gyrA94, and gyrA90. These results contribute to the knowledge of the prevalence of drug resistance in Hunan province and also expand the molecular characteristics of drug resistance in China.

Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from China.

To investigate the molecular characterization of multidrug-resistant tuberculosis (MDR-TB) isolates from China and the association of specific mutations conferring drug resistance with strains of different genotypes, we performed spoligotyping and sequenced nine loci (katG, inhA, the oxyR-ahpC intergenic region, rpoB, tlyA, eis, rrs, gyrA, and gyrB) for 128 MDR-TB isolates. Our results showed that 108 isolates (84.4%) were Beijing family strains, 64 (59.3%) of which were identified as modern Beijing strains. Compared with the phenotypic data, the sensitivity and specificity of DNA sequencing were 89.1% and 100.0%, respectively, for isoniazid (INH) resistance, 93.8% and 100.0% for rifampin (RIF) resistance, 60.0% and 99.4% for capreomycin (CAP) resistance, 84.6% and 99.4% for kanamycin (KAN) resistance, and 90.0% and 100.0% for ofloxacin (OFX) resistance. The most prevalent mutations among the MDR-TB isolates were katG315, inhA15, rpoB531, -526, and -516, rrs1401, eis-10, and gyrA94, -90, and -91. Furthermore, there was no association between specific resistance-conferring mutations and the strain genotype. These findings will be helpful for the establishment of rapid molecular diagnostic methods to be implemented in China.

Fever with thrombocytopenia associated with a novel bunyavirus in China.

BACKGROUND: Heightened surveillance of acute febrile illness in China since 2009 has led to the identification of a severe fever with thrombocytopenia syndrome (SFTS) with an unknown cause. Infection with Anaplasma phagocytophilum has been suggested as a cause, but the pathogen has not been detected in most patients on laboratory testing. METHODS: We obtained blood samples from patients with the case definition of SFTS in six provinces in China. The blood samples were used to isolate the causal pathogen by inoculation of cell culture and for detection of viral RNA on polymerase-chain-reaction assay. The pathogen was characterized on electron microscopy and nucleic acid sequencing. We used enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and neutralization testing to analyze the level of virus-specific antibody in patients' serum samples. RESULTS: We isolated a novel virus, designated SFTS bunyavirus, from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction. RNA sequence analysis revealed that the virus was a newly identified member of the genus phlebovirus in the Bunyaviridae family. Electron-microscopical examination revealed virions with the morphologic characteristics of a bunyavirus. The presence of the virus was confirmed in 171 patients with SFTS from six provinces by detection of viral RNA, specific antibodies to the virus in blood, or both. Serologic assays showed a virus-specific immune response in all 35 pairs of serum samples collected from patients during the acute and convalescent phases of the illness. CONCLUSIONS: A novel phlebovirus was identified in patients with a life-threatening illness associated with fever and thrombocytopenia in China. (Funded by the China Mega-Project for Infectious Diseases and others.).