A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc (N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II (Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients. Methods: The full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system. Results: The native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively. Conclusions: The rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations

Polymorphisms of Plasmodium falciparum k13-propeller gene among migrant workers returning to Henan Province, China from Africa

Abstract Background Henan Province has been in the malaria elimination stage, with all reports of the disease being imported since 2012 and over 90% coming from Africa. Surveillance and population studies are essential for the early detection and subsequent prevention of the spread of drug resistance. The K13-propeller gene was recently identified as a proposed molecular marker of artemisinin (ART) resistance. In this study, we detected mutations of the K13-propeller gene in samples taken from imported malaria cases in Henan Province from 2012 to 2015. Methods There were 483 samples that were obtained from Plasmodium falciparum–infected malaria migrant workers who returned to Henan Province from Africa between 2012 and 2015. The single nucleotide polymorphisms in the K13-propeller gene were assessed by nested PCR with DNA sequencing. Frequency and geographic difference of K13-propeller gene mutant types were analyzed. Results Of 483 patients, 476 were cured and 7 died. There were no K13-propeller mutations in the blood samples from the 7 patients who died, but there were 23 different genotypes of the K13-propeller that were observed in 24 (4.97%) of the samples. C580Y, which was the predominant one in the resistance of ART, was not detected in the samples, but R539T and P574L which have also been associated with ART resistance, were observed in two samples from Angola and Equatorial Guinea. No mutations were detected in 11 samples from North Africa. The frequency of the K13-propeller was 6.50% (8/123) in Central Africa, followed by East Africa (1/19, 5.26%), West Africa (9/198, 4.55%) and South Africa (6/132, 4.55%). There was no significant difference among these four areas (P = 0.795). Conclusion R539T and P574L were found in migrant workers who traveled from Africa to Henan Province, although the frequency of the K13-propeller mutants was low. These data may enrich the molecular surveillance of antimalarial resistance and will be helpful for developing and updating the antimalarial policy in Henan Province

Seroprevalance of antibodies specific for severe fever with thrombocytopenia syndrome virus and the discovery of asymptomatic infections in Henan Province, China.

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is a severe emerging disease caused by SFTS virus (SFTSV), and the geographical distribution of SFTS has been increasing throughout China in recent years. To assess SFTSV-specific antibody seroprevalence, a cross-sectional study was conducted for healthy people in high SFTS endemic areas of Henan province in 2016.MethodsThis study used a stratified random sampling method to select 14 natural villages as the investigation sites. From April to May 2016, participants completed a questionnaire survey and serum samples were collected. All serum samples were subjected to ELISA to detect SFTSV-specific IgM and IgG. All IgM-positive samples were further tested by real-time RT-PCR, and isolation of virus from serum was attempted. Any participant who was IgM-positive was followed up with a month later to confirm health status.ResultsIn total, 1463 healthy people participated in this study. The average seropositive rates for SFTSV-specific IgG and IgM were 10.46% (153/1463) and 0.82% (12/1463), respectively. IgM was detected in 12 individuals, and SFTSV RNA was detected in six of them. Virus was isolated from five of the six SFTSV RNA-positive individuals, and phylogenetic analyses revealed that all five isolates belonged to SFTSV group A. No IgM-positive participants exhibited any symptoms or other signs of illness at the one-month follow up.ConclusionsThis study identified a relatively high incidence of SFTSV-specific antibody seropositivity in healthy people in Xinyang city. Moreover, our data provide the first evidence for asymptomatic SFTSV infections, which may have significant implications for SFTS outbreak control

Polymorphisms analysis of the Plasmodium ovale tryptophan-rich antigen gene (potra) from imported malaria cases in Henan Province

Abstract Background Plasmodium ovale has two different subspecies: P. ovale curtisi and P. ovale wallikeri, which may be distinguished by the gene potra encoding P. ovale tryptophan-rich antigen. The sequence and size of potra gene was variable between the two P. ovale spp., and more fragment sizes were found compared to previous studies. Further information about the diversity of potra genes in these two P. ovale spp. will be needed. Methods A total of 110 dried blood samples were collected from the clinical patients infected with P. ovale, who all returned from Africa in Henan Province in 2011–2016. The fragments of potra were amplified by nested PCR. The sizes and species of potra gene were analysed after sequencing, and the difference between the isolates were analysed with the alignment of the amino acid sequences. The phylogenetic tree was constructed by neighbour-joining to determine the genetic relationship among all the isolates. The distribution of the isolates was analysed based on the origin country. Results Totally 67 samples infected with P. o. wallikeri, which included 8 genotypes of potra, while 43 samples infected with P. o. curtisi including 3 genotypes of potra. Combination with the previous studies, P. o. wallikeri had six sizes, 227, 245, 263, 281, 299 and 335 bp, and P. o. curtisi had four sizes, 299, 317, 335 and 353 bp, the fragment sizes of 299 and 335 bp were the overlaps between the two species. Six amino acid as one unit was firstly used to analyse the amino acid sequence of potra. Amino acid sequence alignment revealed that potra of P. o. wallikeri differed in two amino acid units, MANPIN and AITPIN, while potra of P. o. curtisi differed in amino acid units TINPIN and TITPIS. Combination with the previous studies, there were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The phylogenetic tree showed that 11 isolates were divided into two clusters, P. o. wallikeri which was then divided into five sub-clusters, and P. o. curtisi which also formed two sub-clusters with their respective reference sequences. The genetic relationship of the P. ovale spp. mainly based on the number of the dominant amino acid repeats, the number of MANPIN, AITPIN, TINPIN or TITPIS. The genotype of the 245 bp size for P. o. wallikeri and that of the 299 and 317 bp size for P. o. curtisi were commonly exiting in Africa. Conclusion This study further proved that more fragment sizes were found, P. o. wallikeri had six sizes, P. o. curtisi had four sizes. There were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The genetic polymorphisms of potra provided complementary information for the gene tracing of P. ovale spp. in the malaria elimination era

Epidemiological and etiological characteristics of fever, thrombocytopenia and leukopenia syndrome in Henan Province, China, 2011-2012.

The Fever, Thrombocytopenia and Leukopenia Syndrome (FTLS) is caused by a bunyavirus known as the FTLS virus (FTLSV), which was recently discovered in China. We examined the epidemiological and etiological features of 637 laboratory-confirmed cases of FTLS with onset from January 2011 to December 2012 in Henan Province, China. The highest incidence of FTLS occurred between May and August: 76.5% of all laboratory-confirmed cases occurred during those four months. Of the laboratory-confirmed cases, 60.9% were in the 46-69 years old age groups; 96.1% (612/637) occurred in farmers; 98.1% (625/637) were reported from Xinyang Prefecture. During the same time period, 2047 cases were reported in China. The nucleotide and amino acid sequences of FTLSV strains identified during 2011-2012 in Henan Province were ≥ 96% identical. This findings provides insight for developing public-health interventions for the control and prevention of FTLS in epidemic area

The evolutionary history and spatiotemporal dynamics of the fever, thrombocytopenia and leukocytopenia syndrome virus (FTLSV) in China.

In 2007, a novel bunyavirus was found in Henan Province, China and named fever, thrombocytopenia and leukocytopenia syndrome virus (FTLSV); since then, FTLSV has been found in ticks and animals in many Chinese provinces. Human-to-human transmission has been documented, indicating that FTLSV should be considered a potential public health threat. Determining the historical spread of FTLSV could help curtail its spread and prevent future movement of this virus.To examine the pattern of FTLSV evolution and the origin of outbreak strains, as well to examine the rate of evolution, the genome of 12 FTLSV strains were sequenced and a phylogenetic and Bayesian phylogeographic analysis of all available FTLSV sequences in China were performed. Analysis based on the FTLSV L segment suggests that the virus likely originated somewhere in Huaiyangshan circa 1790 (95% highest probability density interval: 1756-1817) and began spreading around 1806 (95% highest probability density interval: 1773-1834). Analysis also indicates that when FTLSV arrived in Jiangsu province from Huaiyangshan, Jiangsu Province became another source for the spread of the disease. Bayesian factor test analysis identified three major transmission routes: Huaiyangshan to Jiangsu, Jiangsu to Liaoning, and Jiangsu to Shandong. The speed of FTLSV movement has increased in recent decades, likely facilitated by modern human activity and ecosystem changes. In addition, evidence of RNA segment reassortment was found in FTLSV; purifying selection appears to have been the dominant force in the evolution of this virus.Results presented in the manuscript suggest that the Huaiyangshan area is likely be the origin of FTLSV in China and identified probable viral migration routes. These results provide new insights into the origin and spread of FTLSV in China, and provide a foundation for future virological surveillance and control