Diversity of Francisella tularensis subsp. holarctica lineages, China.

We analyzed 10 isolates of Francisella tularensis subspecies holarctica from China and assigned them to known clades by using canonical single-nucleotide polymorphisms. We found 4 diverse subtypes, including 3 from the most basal lineage, biovar japonica. This result indicates unprecedented levels of diversity from a single region and suggests new models for emergence.

Melt-spun polylactide/ethylene vinyl alcohol copolymer fiber.

Polylactide/ethylene vinyl alcohol copolymer (PLA/EVOH) blends and fibers with different weight ratios were prepared by melt blending, and two-step melt spinning, respectively. PLA and EVOH in PLA/EVOH blends were immiscible. When EVOH content was ≤60 %, EVOH with the average diameter of about 3 µm was dispersed in PLA matrix uniformly. The dual continuous phases could be observed in PLA/EVOH blend with 70 wt% EVOH. When the EVOH content was ≥80 %, the spherical PLA phase with the diameter of 0.25 to 1 µm was dispersed in EVOH matrix. The introduction of EVOH as nucleating agent could promote the crystallization of PLA. Both PLA and EVOH components in PLA/EVOH blends formed individual crystal phases. The viscosity of PLA/EVOH blend with 5 % EVOH was lower than that of neat PLA. The viscosity of PLA/EVOH blends with the EVOH content of ≥10 % was much higher than that of neat PLA, which showed obvious shear thinning behavior. With the increase of EVOH content, the shear thinning behavior became obvious and the critical shear rate decreased gradually. The drawn PLA/EVOH fibers with the tensile strength of ≥16 cN/tex exhibited good mechanical properties. In addition, the introduction of EVOH could improve the hydrophilicity of PLA fibers.

Structure and properties of chlorogenic acid-loaded polylactide fiber prepared by melt spinning.

Polylactide/chlorogenic acid (PLA/CGA) blends with different weight ratios were prepared by melt mixing, and corresponding PLA/CGA fibers were produced via a two-step melt spinning process. For PLA/CGA blends, CGA was distributed uniformly in the PLA matrix. The intermolecular interactions between CGA and PLA existed. The viscosity of PLA/CGA blends was much lower than that of neat PLA. With the increase of CGA content, the viscosity of PLA/CGA blends decreased. As the CGA content increased, the crystallinity of both PLA/CGA blends and fibers decreased. In addition, the tensile strength of PLA/CGA fibers was slightly lower than that of neat PLA fiber. For PLA/CGA fibers, the 6-fold drawn PLA/CGA fiber with 3 % CGA owned the highest tensile strength of 420 MPa. The ultraviolet (UV) resistance of PLA/CGA fibers were enhanced significantly by the introduction of CGA. When the CGA content was not <3 %, the UV transmittance of PLA/CGA fibers was <8 %. Moreover, PLA/CGA fibers exhibited good antioxidant properties. PLA/CGA fibers with 10 % CGA owned the highest antioxidant rate of >90 %. In addition, the 6-fold drawn PLA/CGA fiber with 10 % CGA presented excellent release performance with a 7-day cumulative CGA release rate of 19 %.

Structure and properties of polylactic acid/butenediol vinyl alcohol copolymer blend fibers.

Polylactic acid/butenediol vinyl alcohol copolymer (PLA/BVOH) blends with different weight ratios were prepared by melt mixing. PLA and BVOH in PLA/BVOH blends were immiscible while the weak interaction between PLA and BVOH existed. The introduction of BVOH facilitated the crystallization of PLA. Moreover, the crystallization of PLA hindered the crystallization of BVOH. Due to introduction of BVOH, PLA/BVOH blends exhibited shear thinning characteristic except that PLA/BVOH blends with 5-10 % BVOH showed similar rheological property to neat PLA. With the increase of BVOH content, the contact angle of PLA/BVOH blends decreased from 79.75° to 67.33° at 120 s. The hydrophilicity of PLA/BVOH blends was improved. In addition, PLA/BVOH fibers with 5-40 % BVOH and PLA/BVOH/rutin fibers with 3 % rutin were manufactured by melt spinning. The effect of BVOH on the mechanical property of PLA/BVOH fibers was small. However, BVOH improved significantly the rutin release rate and antioxidant properties of PLA/BVOH/rutin fibers.

Smart photochromic materials based on polylactic acid.

The smart photochromic materials based on polylactic acid (PLA) were prepared by melt-blending and hot-pressing, in which photochromic microcapsules (PM) were used as a functional additive, and poly(vinyl acetate) (PVAc) was introduced into the photochromic PLA blends for the first time to improve their properties. The crystallization and melting behavior, morphology, and photochromic performance of PLA/PVAc/PM blends were characterized by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and spectrophotometer, respectively. The results showed that PVAc significantly improved the photochromic properties of PLA/PM blends. Under 30 s UV irradiation, the blends reached a value of ΔE that could be recognized in 3 s by human eyes. This discriminative ΔE value could be maintained for at least 3 min after removal from UV irradiation. Meanwhile, the blend had outstanding photochromic durability and recyclability. Compared to ΔE for 0.5 h of continuous light irradiation, ΔE for 8 h of continuous light irradiation decreased by only about 1, to 14.1. In 25 cycles of 3 s UV irradiation, the values of ΔE for the first and 25th irradiation were 11.4 and 11.6, respectively. The blend showed different photochromic responses to different light intensities. The ΔE values of 8.6, 14.6, 14.6, and 18.4 for irradiation at 600, 800, 1000, and 1200 W/m2 of solar intensity, respectively.

Genetic relationship between Francisella tularensis strains from China and from other countries.

OBJECTIVE: To study the types of subspecies of Francisella tularensis from China and to investigate the genetic relationships between F. tularensis strains from China and from other countries. METHODS: Ten strains of F. tularensis isolated from China were amplified by using typing primers C1/C4 and RD1. On the basis of the lengths of the polymerase chain reaction (PCR) products, it was concluded that these strains of F. tularensis belonged to the same subspecies. At the same time, the fopA, tul4, and 16S rRNA genes of the 10 strains were amplified, and a three-gene based phylogenetic analysis was performed using the Molecular Evolutionary Genetics Analysis software version 4.0. RESULTS: The 10 strains of F. tularensis from China were all identified as belonging to subspecies holarctica (type B). We found no direct relationship between the genotypes of F. tularensis subsp. holarctica and the geographical area from where they were isolated. CONCLUSION: The F. tularensis strains isolated from North China mainly belong to subspecies holarctica (type B). The strains of F. tularensis subsp. holarctica from China may have evolved earlier than those from Europe and North America.

Complete genome sequences of Yersinia pestis from natural foci in China.

Yersinia pestis, the causative agent of plague, is a deadly bacterium that affects humans. Strain D106004 was isolated from a new plague focus in Yulong County, China, in 2006. To gain insights into the epidemic origin, we have sequenced the genomes of D106004 and strains Z176003 and D182038, isolated from neighboring regions.

Genome rearrangements of completely sequenced strains of Yersinia pestis.

Yersinia pestis has caused three worldwide plagues in human history that have led to innumerable deaths. We have completely sequenced the genomes of two strains (D106004 and D182038) of Y. pestis isolated from Yunnan Province of China. The most striking finding of our study is that large amounts of genome rearrangement events exist between the genomes of two Yunnan strains despite being isolated from two foci only 50 kilometers apart. When we compared the genome sequences of the Yunnan strains with six strains (CO92, KIM, 91001, Antiqua, Nepal516, and Pestoides F) of Y. pestis sequenced previously, we found that the genomes of Y. pestis were divided into 61 relatively independent segments. Pairwise comparisons of all 61 segments among eight strains showed that the Yunnan strains were most closely related to strain CO92. We concluded that Y. pestis genomes consist of segments that can change their positions and directions within the genomes caused by genome rearrangements, and our study confirmed the inference that the third plague pandemic originated in Yunnan since the genome sequences of Yunnan strains were closest to the strain CO92 isolated from the United States.

Scrub typhus in previously unrecognized areas of endemicity in China.

Scrub typhus, caused by Orientia tsutsugamushi, has emerged recently in areas of northern China where the disease had not been known to exist. We analyzed epidemiological, clinical, and laboratory data for 104 patients who were admitted to a hospital in Fuyang City between 26 September and 1 November 2008. We showed that the major clinical manifestations of the patients were fever (100%), headache (82%), myalgias (77%), eschar (67%), rash (52%), and unusual facial flushing (62%). Among the 104 patients, the sera of 98% contained IgM antibodies to O. tsutsugamushi detected by indirect immunofluorescence assays (IFA), and DNA of the O. tsutsugamushi 56-kDa gene was amplified by PCR from the blood of 36 patients. We conclude that 104 patients were infected with scrub typhus in Fuyang City, Anhui Province. Our study indicates that physicians need to consider the diagnosis of scrub typhus for febrile patients living in northern China, where scrub typhus had not been considered to exist in the past.

Assessment of comparative genomic hybridization experiment by an in situ synthesized CombiMatrix microarray with Yersinia pestis vaccine strain EV76 DNA.

OBJECTIVE: The quality of microarray data influences the accuracy of comparative genomic analyses to a large extent. To ensure that the results obtained by using an in situ synthesized microarray are accurate, data quality is to be assessed by evaluating the melting temperature (Tm) of probes, probability of false synthesis rates, and fragmentation of labeled targets. METHODS: DNA from the Yersinia pestis vaccine strain EV76 was used for microarray analyses. Microarray results were confirmed by PCR. Statistical and bioinformatics methods were employed to perform microarray data analyses and evaluation. RESULTS: Correlation coefficients of the three datasets were above 0.95 after two-time stripping and hybridization with a labeled DNA with the size of fragmentation being 200 bp - 2 kb, which showed that the hybridization results were highly reproducible. Correlation coefficients were lower with the values ranging from 0.87 to 0.92 between the datasets generated from hybridization with different sizes of the labeled DNA fragment. For the relationship between Tm and signal intensity, there was a different distribution of Tm in the lowest 300 or 3,000 probes with a range of 70 °C-72 °C and the highest 300 or 3,000 probes with a range of 72 °C-74 °C. CONCLUSION: The results of this study suggest that the initial microarray design may affect the accuracy of final analyses and that the probe Tm and the size of the labeled fragment may be the two factors of the greatest importance.

MLVA distribution characteristics of Yersinia pestis in China and the correlation analysis.

BACKGROUND: Yersinia pestis, the aetiological agent of plague, has been well defined genotypically on local and worldwide scales. In November 2005, five cases of severe pneumonia of unknown causes, resulting in two deaths, were reported in Yulong, Yunnan province. In this study, we compared Y. pestis isolated from the Yulong focus to strains from other areas. RESULTS: Two hundred and thirteen Y. pestis strains collected from different plague foci in China and a live attenuated vaccine strain of Y. pestis (EV76) were genotyped using multiple-locus variable-number tandem repeat analysis (MLVA) on 14 loci. A total of 214 Y. pestis strains were divided into 85 MLVA types, and Nei's genetic diversity indices of the various loci ranged between 0.02 - 0.76. Minimum spanning tree analysis showed that Y. pestis in China could be divided into six complexes. It was observed that Microtus strains were different from the other three biovar strains. Each plague focus had its own unique MLVA types. CONCLUSION: The strains isolated from Yulong, Yunnan province had a unique MLVA type, indicating a new clone group. Our results suggest that Yulong strains may have a close relationship with strains from the Qinghai-Tibet Plateau plague focus.

Identification of Yersinia Pestis of Xilingele Plateau Ecotype Isolated from China Using Insertion Sequences as Target.

OBJECTIVE: This study aims to develop a strategy for distinguishing Yersinia pestis of the Xilingele Plateau ecotype from other biovar of Yersinia pestis using insertion sequences. METHODS: Genome computational analysis was used to discover the positions of insertion sequences unique to Yersinia pestis 91001. PCR was used to confirm that these positions were exclusively present in the Xilingele Plateau ecotype of Yersinia pestis, while it was absent in other Yersinia species. RESULTS: It was validated that the amplicons of IS100-45-1 IS100-51-1, IS285-40 and IS285-41 were exclusive to Yersinia pestis of the Xilingele Plateau ecotype. CONCLUSIONS: Insertion sequences could be reliable candidate biomarkers for Yersinia pestis strains of Xilingele Plateau ecotype. We can differentiate Yersinia pestis strains of Xilingele Plateau ecotype from other Yersinia pestis using simple PCR methods.

Ten years of surveillance of the Yulong plague focus in China and the molecular typing and source tracing of the isolates.

Plague, caused by Yersinia pestis, was classified as a reemerging infectious disease by the World Health Organization. The five human pneumonic plague cases in Yulong County in 2005 gave rise to the discovery of a Yulong plague focus in Yunnan province, China. Thereafter, continuous wild rodent plague (sylvatic plague) was identified as the main plague reservoir of this focus. In this study, the epizootics in Yulong focus were described, and three molecular typing methods, including the different region (DFR) analysis, clustered regularly interspaced short palindromic repeats (CRISPRs), and the multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) (14+12), were used for the molecular typing and source tracing of Y. pestis isolates in the Yulong plague focus. Simultaneously, several isolates from the vicinity of Yunnan were used as controls. The results showed that during the 10-year period from 2006 to 2016, an animal plague epidemic occurred in 6 of those years, and 5 villages underwent an animal plague epidemic within a 30-km2 area of the Yulong plague focus. Searching for dead mice was the most effective monitoring method in this plague focus. No positive sample has been found in 6937 captured live rodents thus far, suggesting that the virulence of strains in the Yulong plague focus is stronger and the survival time of mice is shorter after infection. Strains from Lijiang, Sichuan and Tibet were of the same complex based on a typing analysis of DFR and CRISPR. The genetic relationship of Y. pestis illustrated by MLVA "14+12" demonstrates that Tibet and Sichuan strains evolved from the strains 1.IN2 (Qinghai, 1970 and Tibet, 1976), and Lijiang strains are closer to Batang strains (Batang County in Sichuan province, 2011, Himalaya marmot plague foci) in terms of genetic or phylogenic relationships. In conclusion, we have a deeper understanding of this new plague focus throughout this study, which provides a basis for effective prevention and control.

Safety and immunogenicity from a phase I trial of inactivated severe acute respiratory syndrome coronavirus vaccine.

BACKGROUND: Emergence of severe acute respiratory syndrome (SARS) from the winter of 2002 to the spring of 2003 has caused a serious threat to public health. METHODS: To evaluate the safety and immunogenicity of the inactivated SARS coronavirus (SARS-CoV) vaccine, 36 subjects received two doses of 16 SARS-CoV units (SU) or 32 SU inactivated SARS-CoV vaccine, or placebo control. RESULTS: On day 42, the seroconversion reached 100% for both vaccine groups. On day 56, 100% of participants in the group receiving 16 SU and 91.1% in the group receiving 32 SU had seroconverted. The geometric mean titre of neutralizing antibody peaked 2 weeks after the second vaccination, but decreased 4 weeks later. CONCLUSION: The inactivated vaccine was safe and well tolerated and can elicit SARS-CoV-specific neutralizing antibodies.

[Establishment and evaluation of identification method for Yersinia pestis and Yersinia pseudotuberculosis].

OBJECTIVE: To establish a gene identification method of Yersinia pestis and Yersinia pseudotuberculosis for plague surveillance. METHODS: According to the specific genomic sequences of Y. pestis and Y. pseudotuberculosis, i.e. "pestis Island (PeI)" and "pseudotuberculosis Island (PsI)" and the published genomic sequences of 12 strains of Y. pestis and 4 strains of Y. pseudotuberculosis, the specific identification primers of these sequences were designed. RESULTS: A total of 52 strains of Y. pestis and 57 strains of Y. pseudotuberculosis and other intestinal bacteria strains were tested with PCR. Of the 5 pairs of Y. pestis identification primers, PeI2 and PeI11 were specific for Y. pestis. Besides Y. pestis, the primers PeI1, PeI3 and PeI12 could detect part of 57 Y. pseudotuberculosis strains. Of the 5 pairs of Y. pseudotuberculosis identification primers, PsI1 could detect all the 52 strains of Y. pestis and 57 strains of Y. pseudotuberculosis. PsI7, PsI16, PsI18 and PsI19 were specific for Y. pseudotuberculosis. CONCLUSION: The primers PsI1, PeI 2 and PeI11, PsI7, PsI16, PsI18 and PsI19 can be used in the rapid identification of Y. pestis and Y. pseudotuberculosis, which can be also used to explore the circulation of atypical Y. pestis in quiescent plague foci.

Chromosomal rearrangement features of Yersinia pestis strains from natural plague foci in China.

The Yersinia pestis chromosome contains a large variety and number of insert sequences that have resulted in frequent chromosome rearrangement events. To identify the chromosomal rearrangement features of Y. pestis strains from five typical plague foci in China and study spontaneous DNA rearrangements potentially stabilized in certain lineages of Y. pestis genomes, we examined the linking mode of locally collinear blocks (LCBs) in 30 Y. pestis strains by a polymerase chain reaction-based method. Our results suggest most strains have relatively stable chromosomal arrangement patterns, and these rearrangement characteristics also have a very close relationship with the geographical origin. In addition, some LCB linking modes are only present in specific strains. We conclude Y. pestis chromosome rearrangement patterns may reflect the genetic features of specific geographical areas and can be applied to distinguish Y. pestis isolates; furthermore, most of the rearrangement events are stable in certain lineages of Y. pestis genomes.

[Study on the identification characteristics of rRNA genes on Yersinia pestis].

OBJECTIVE: To study the identification characteristics of rRNA genes on Yersinia (Y.) pestis. METHODS: By means of comparative genomics, we compared the rRNA genome sequences of nine completely sequenced strains of Y. pestis isolated from China and other countries by Clustal W software. We also compared the 2000 bp sequence adjacent to the rRNA genes, rRNA genes and 16S-23S rRNA spacer region respectively to determine the identification features of rRNA genes for Y. pestis. RESULTS: There were 6 rRNA gene clusters in the strains of D182038, D106004, Z176003 and CO92 respectively (6 copies strain). There were 7 rRNA gene clusters in the strains of 91001, KIM, Nepal516, Antiqua and Pestoides F (7 copies strain). According to the 2000 bp sequence, 13 types of rRNA gene clusters could classify the strains between the 6 copies and 7 copies. There were 4 types of tRNA gene among the 16S-23S rRNA spacer region that could classify the strains among the 6 copies and 7 copies strains respectively. The number of point mutation among the 23S rRNA gene was statistically different in some copies under ANOVA analysis (F = 0.548, P = 0.815 > 0.05 among the strains and F = 5.228, P < 0.01 among the copies). CONCLUSION: The 2000 bp sequence adjacent to the rRNA genes, tRNA gene and 23S rRNA gene sequence could serve as the identification sign of rRNA genes for classifing the strains of Y. pestis.

[Clone expression of the caf1 gene of Yersinia pestis and immunological evaluation on recombinant F1 antigen].

OBJECTIVE: To get recombinant F1 antigen (rF1) and to construct the detection dipstick of plague antibody. METHODS: The caf1 gene removing the signal peptide coding sequence was cloned into plasmid pET32a(+) by double-digested sites of BamHI and Not I. Recombinant plasmid caf1-pET32a(+) was transformed into BL21 (DE3) and the rF1 was expressed. Expression products were purified by affinity chromatography. Dual detection dipstick of plague antibody was constructed with purified rF1 and natural F1, and evaluated with 528 human serum samples of Zhejiang province. RESULTS: The fusion protein rF1 of 35.5 KD was expressed by BL21 strains containing caf1-pET32a(+). The sensitivity of rF1 showed equivalent to or higher than the natural F1 antigen in detecting plague antibody. It seemed that there was a better consistency of 97.9% (kappa = 0.466) when 528 human sera was detected by rF1 and natural F1. CONCLUSION: We successfully extracted the rF1 with good immunological activity that might be used to detecting Yersinia pestis.

Spatial variation of Yersinia pestis from Yunnan Province of China.

Yunnan Province of China is considered the site of origin for modern plague. We analyzed the genotypes of eight Yersinia pestis strains isolated from three counties in Yunnan Province by pulse field gel electrophoresis (PFGE). PFGE showed that strains isolated from the same site were identical regardless of hosts or year of isolation. However, Y. pestis strains isolated from geographically distinct loci were genetically divergent. Whole genome sequences of two strains from two foci in Yunnan showed that the genetic variation of Y. pestis strains was caused by genome rearrangement. We concluded that Y. pestis strains in each epidemic focus in Yunnan were a clonal population and selected by host environments. The genomic variability of the Y. pestis strains from different foci were caused by genome rearrangement, which may provide a positive selective advantage for Y. pestis to adapt to its host environments.

Seroprevalence of human granulocytotropic anaplasmosis in central and southeastern China.

Human granulocytic anaplasmosis (HGA) is an emerging tick-borne infectious disease. To determine the prevalence of HGA in central and southeastern China, a total of 323 human sera were collected from individuals at high risk for exposure to ticks and animals. The IgG antibody against the etiologic agent of HGA, Anaplasma phagocytophilum was detected with indirect immunofluorescence assay. The results showed that 20% of the tested individuals (64/323) were positive to A. phagocytophilum and that the incidence was higher in male (22%) than female (16%). We concluded that A. phagocytophilum infection was prevalent in central and southeastern China.