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In practical classification tasks, the sample distribution of the dataset is often unbalanced; for example, this is the case in a dataset that contains a massive quantity of samples with weak labels and for which concrete identification is unavailable. Even in samples with exact labels, the number of samples corresponding to many labels is small, resulting in difficulties in learning the concepts through a small number of labeled samples. In addition, there is always a small interclass variance and a large intraclass variance among categories. Weak labels, few-shot problems, and fine-grained analysis are the key challenges affecting the performance of the classification model. In this paper, we develop a progressive training technique to address the few-shot challenge, along with a weak-label boosting method, by considering all of the weak IDs as negative samples of every predefined ID in order to take full advantage of the more numerous weak-label data. We introduce an instance-aware hard ID mining strategy in the classification loss and then further develop the global and local feature-mapping loss to expand the decision margin. We entered the proposed method into the Kaggle competition, which aims to build an algorithm to identify individual humpback whales in images. With a few other common training tricks, the proposed approach won first place in the competition. All three problems (weak labels, few-shot problems, and fine-grained analysis) exist in the dataset used in the competition. Additionally, we applied our method to CUB-2011 and Cars-196, which are the most widely-used datasets for fine-grained visual categorization tasks, and achieved respective accuracies of 90.1% and 94.9%. This experiment shows that the proposed method achieves the optimal effect compared with other common baselines, and verifies the effectiveness of our method. Our solution has been made available as an open source project.

For large amounts of transition metal oxides, sulfides and carbon groups (IVA), the pulverization of electrode materials in lithium-ion batteries (LIBs) is always a serious and common problem due to volume expansion and stress accumulation resulting from phase transformation or alloying during the charge–discharge process, which leads to capacity fading and thus limits the cycling performance of LIBs. To solve these problems, conventionally, the rational design of electrode materials is needed. Here in this work, we report the synthesis of a novel anode material, hexagonal H4.5Mo5.25O18·(H2O)1.36 microrods (HMs) via a simple hydrothermal method. During the lithium-ion insertion/desertion process of the HMs, it was found that the HMs are first drastically transformed into Li2MoO4 nanotubes and then Li2MoO4 nanowire clusters embedded in amorphous sphere cages with a Li2O matrix, Mo metal and SEI thin film. Surprisingly, we discovered that the HMs exhibited extraordinary long-life cyclability with an unusual phenomenon: the specific capacity first decreased and then increased. The outstanding electrochemical performance could be explained by the formation of intermediate phase Li2MoO4 nanowires and amorphous sphere cages, which can maintain the lithium-ion paths and electronic transport, and prohibit the mechanical and chemical degradation of the electrode materials. The results show that the pulverization of the HM anode materials induced by lithium-ion insertion–extraction played a trigger role in the formation of a continuous pulverization film. Accordingly, a “damage-reconstruction” model based on ex situ XRD and FESEM analyses combined with ex situ XPS, FTIR and TEM characterizations of the charge–discharge process was proposed to explain such an unusual and intriguing finding. Compared with the conventional method for protecting electrode materials from pulverization, the robust continuous gel-like pulverization film containing a unique combination of intermediate phase and amorphous sphere cages provides a new insight into the mechanism for the extraordinary long-term cyclability of electrode materials.

The plague, which is caused by the Gram-negative coccobacillus bacterium Yersinia pestis, has been classified as a reemerging infectious disease by the World Health Organization. The Qinghai-Tibet Plateau natural plague focus is the largest plague focus in China, and Marmota himalayana is the primary host of the plague. Tibetan sheep (Ovis aries) were first identified as naturally infected hosts of Y. pestis based on etiological evidence in 1975, and activities such as slaughtering or skinning Tibetan sheep that have been infected by Y. pestis or died from Y. pestis infection had caused severe human plague in Qinghai. Tibetan sheep are important domestic livestock in the Qinghai-Tibet Plateau. Knowledge regarding the infection rate of Y. pestis in Tibetan sheep is important for understanding the range of infection and improving measures to control plague epidemics in this area. In this study, a serological survey involving 12,710 Tibetan sheep in all 44 counties in Qinghai Province was conducted. The total positive rate of indirect hemagglutination assay for Y. pestis in Tibetan sheep in Qinghai was 0.68% (86/12,710). Serological positivity to the Y. pestis F1 antibody was found in Tibetan sheep in all prefectures, except the Haidong and Haibei prefectures in Qinghai, with the seropositive rate in different counties ranging from 0.33% to 5.2% and the titers in the positive sera ranging from 1:20 to 1:5120. In addition, the seropositive rates in animal plague focus counties were higher than the rates in non-animal plague counties. Such results indicated a widespread infection of Tibetan sheep with Y. pestis in Qinghai, even though only sporadic epidemics of Tibetan sheep plague have been reported in Qinghai.

The Adaboost vehicle detection algorithm based on Haar feature has a good performance in real-time and accuracy. However, the method has a lot of omission and error detection in the detection of Special vehicle under complicated traffic flow. In this paper, a method of taxi window area detection is proposed to replace vehicle detection. At the same time, a method of sliding color histogram matching is proposed to reduce the error detection. Finally, the traffic surveillance video is used to verify the algorithm, Detection results proves that the algorithm has a good accuracy and real-time performance for the detection of taxi vehicles.

This paper presents a method for automatic detection of taxi drivers beard behavior. First, Haar-Adaboost algorithm is used to locate the special window area of taxi. Secondly, the image is preprocessed by multi-scale retina enhancement algorithm on image value channel, then face feature points and chin area are extracted from the window area, and the beard pixel is segmented by skin tone and gray threshold method. Finally, The method was tested by real traffic surveillance video and the validity was proved

Yep-phi, a lytic phage of Yersinia pestis, was isolated in China and is routinely used as a diagnostic phage for the identification of the plague pathogen. Yep-phi has an isometric hexagonal head containing dsDNA and a short non-contractile conical tail. In this study, we sequenced the Yep-phi genome (GenBank accession no. HQ333270) and performed proteomics analysis. The genome consists of 38 ,616 bp of DNA, including direct terminal repeats of 222 bp, and is predicted to contain 45 ORFs. Most structural proteins were identified by proteomics analysis. Compared with the three available genome sequences of lytic phages for Y. pestis, the phages could be divided into two subgroups. Yep-phi displays marked homology to the bacteriophages Berlin (GenBank accession no. AM183667) and Yepe2 (GenBank accession no. EU734170), and these comprise one subgroup. The other subgroup is represented by bacteriophage ΦA1122 (GenBank accession no. AY247822). Potential recombination was detected among the Yep-phi subgroup.

The Qinghai-Tibet plateau is a natural plague focus and is the largest such focus in China. In this area, while Marmota himalayana is the primary host, a total of 18 human plague outbreaks associated with Tibetan sheep (78 cases with 47 deaths) have been reported on the Qinghai-Tibet plateau since 1956. All of the index infectious cases had an exposure history of slaughtering or skinning diseased or dead Tibetan sheep. In this study, we sequenced and compared 38 strains of Yersinia pestis isolated from different hosts, including humans, Tibetan sheep, and M. himalayana. Phylogenetic relationships were reconstructed based on genome-wide single-nucleotide polymorphisms identified from our isolates and reference strains. The phylogenetic relationships illustrated in our study, together with the finding that the Tibetan sheep plague clearly lagged behind the M. himalayana plague, and a previous study that identified the Tibetan sheep as a plague reservoir with high susceptibility and moderate sensitivity, indicated that the human plague was transmitted from Tibetan sheep, while the Tibetan sheep plague originated from marmots. Tibetan sheep may encounter this infection by contact with dead rodents or through being bitten by fleas originating from M. himalayana during local epizootics., Author summary Plague is mainly a disease of wild rodents, and their parasitic fleas are considered the transmitting vectors. However, human plague originating from Ovis aries (Tibetan sheep) is found in the Qinghai-Tibet plateau in China, where Marmota. himalayana is the primary plague host. Tibetan sheep-related human plague infection is always associated with slaughtering or skinning diseased or dead Tibetan sheep. The plague in Tibetan sheep clearly lags that in M. himalayana. In this study, we performed a genome-wide single nucleotide polymorphism analysis of Tibetan sheep-related plague events, including pathogens isolated from humans, Tibetan sheep, and marmots. Through genomic analysis, together with the epidemiological connections, we confirmed that human plague came from Tibetan sheep, and the Tibetan sheep plague originated from marmots. Tibetan sheep account for about 1/3 of the total number of sheep in China. Tibetan sheep and goats are important domestic livestock on the Qinghai-Tibet plateau. Therefore, the hazards of Tibetan sheep plague should not be underestimated.

In this study, a new subunit vaccine that comprised native F1 and recombinant rV270 was evaluated for protective efficacy using mouse, guinea pig and rabbit models in comparison with the live attenuated vaccine EV76. Complete protection against challenging with 10 6 colony-forming units (CFU) of virulent Yersinia pestis strain 141 was observed for mice immunized with the subunit vaccines and EV76 vaccine. In contrast, the subunit vaccine recipes VII (F1-20 μg + rV270-10 μg) and IX (F1-40 μg + rV270-20 μg) and EV76 vaccine provided 86%, 79% and 93% protection against the same level of challenge in guinea pigs and 100%, 83% and 100% protection in rabbits, respectively. The immunized mice with the vaccines had significantly higher IgG titres than the guinea pigs and rabbits, and the immunized guinea pigs developed significantly higher IgG titres than the rabbits, but the anti-F1 response in guinea pigs was more variable than in the mice and rabbits, indicating that guinea pig is not an ideal model for evaluating protective efficacy of plague subunit vaccine, instead the rabbits could be used as an alternative model. All the immunized animals with EV76 developed a negligible IgG titre to rV270 antigen. Furthermore, analysis of IgG subclasses in the immunized animals showed a strong response for IgG1, whereas those receiving EV76 immunization demonstrated predominant production of IgG1 and IgG2a isotypes. The subunit vaccine and EV76 vaccine are able to provide protection for animals against Y. pestis challenge, but the subunit vaccines have obvious advantages over EV76 in terms of safety of use.

Application of the PCR-derived technology in gene identification and genotypes of different ecotype Yersinia pestis to make the high-throughput experimental results can reflect the epidemic history and compare the diversity in genome, pathogenicity, so that results from these experiments provide an important basis for clinical diagnosis, treatment and origin. But the experiment should be considered typing ability, practicality, budget and other experimental factors or conditions, because each PCR-derivative technology has advantages and disadvantages.

To analyze the plasmid features and geographical distribution characteristics of Yersinia pestis of different plague foci in China.A total of 2 213 Yersinia pestis strains were colected from 11 Chinese plague foci separated during 1943 to 2012, and plasmid DNA according to alkali cracking method, and measured the relative molecular mass (Mr) of plasmid DNA based on the standard plasmid contrast method, then analyzed the plasmid profiles by agar gel electrophoresis.A total of 2 213 strains had 16 kinds of plasmids with different Mr, including 4×10(6), 6×10(6), 7×10(6), 13×10(6), 16×10(6), 20×10(6), 22×10(6), 23×10(6), 27×10(6), 30×10(6), 36×10(6), 45×10(6), 52×10(6), 65×10(6), 72×10(6) and 90×10(6). Plasmid were classified into 26 kinds of plasmid profiles. A total of 2 213 Yersinia pestis strains contained 4 large plasmids, 52×10(6), 65×10(6), 72×10(6) and 90×10(6), whose ratio was 22.10% (589/2 213), 75.60% (1 672/2 213), 0.17% (4/2 213), 2.12% (47/2 213), respectively. Among which, strains with plasmid 52×10(6), 65×10(6), 90×10(6) distributed in Qinghai-Tibet plateau Himalayan Marmot natural plague foci, strains with 72×10(6) plasmid only distributed in Inner Mongolia Meriones unguiculatus natural plague foci and Junggar Basin R. opimus natural plague foci, and 65×10(6) plasmid distributed in all the other foci.Strains in Chinese 11 plague foci contained 4 kinds of large plasmid, the Mr respectively were 52×10(6), 65×10(6), 72×10(6), 90×10(6), which were classified into 26 kinds of plasmid profiles with other plasmid. These plasmid profiles distributed in relatively independent epidemic focus.

The serum levels of interleukin-2 (IL-2), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-4, IL-6, and IL-10 of pneumonic plague patients were determined by enzyme-linked immunosorbent assay. IL-6 was the only elevated cytokine in the patients, and its level increased with a clear time course, indicating that IL-6 might be a prognostic marker for predicting the progression of plague.

This article documents the public availability of transcriptome sequence data and assembled, annotated unigenes for the marmot flea, Oropsylla silantiewi.(1.)

To analyze the results of etiology and serology of plague among human and infected animals in Qinghai province from 2001 to 2010.Thirty-seven cases of human infected with plague, 53 541 different animal samples, 5 685 sets of vector insects flea and 49 039 different animal serum samples were obtained between 2001 and 2010. A total of 7 811 samples of serum from healthy farmers and herdsmen in 14 counties in Qinghai from 2005 to 2007 were collected. Yersinia pestis (Y. pestis) were detected in visceral and secretions from human, infected animals and vector insects, respectively. Plague antigen was detected by reverse indirect hemagglutination assay (RIHA) in those samples. Indirect hemagglutination assay (IHA) was used to test plague FI antibody in serum of human and infected animals.37 human plague cases were confirmed, 21 strains of plague Y. pestis were isolated from human cases and 14 positive were detected out. 133 of 7 811 samples of human serum were IHA positive, with the positive rate at 1.7%. A total of 146 strains of plague were isolated from infected animals and vector insects, 99 out of which were from infected animals, with a ratio of Marmota himalayan at 72.7% (72/99) and the other 47 were from vector insects, with a ratio of callopsylla solaris at 68.1% (32/47). The number of IHA and PIHA positive were 300 and 10, respectively. A total of 3 animals and 3 insects species were identified as new epidemic hosts for plague. The natural plague focus of Microtus fuscus was discovered and confirmed and coexisted with natural focus of Marmota himalayan in Chengduo county, Yushu prefecture. The epidemic situation of plague is distributed mainly in Haixi, Yushu and Hainan prefectures.From 2001 to 2010, animal infected with plague was detected in successive years and human plague was very common in Qinghai. New infected animals and vector insects species and new epidemic areas were confirmed, hence the trend of plague prevalence for humans and animals is very active in Qinghai province.

Source tracing of pathogens is critical for the control and prevention of infectious diseases. Genome sequencing by high throughput technologies is currently feasible and popular, leading to the burst of deciphered bacterial genome sequences. Utilizing the flooding genomic data for source tracing of pathogens in outbreaks is promising, and challenging as well. Here, we employed Yersinia pestis genomes from a plague outbreak at Xinghai county of China in 2009 as an example, to develop a simple two-step strategy for rapid source tracing of the outbreak. The first step was to define the phylogenetic position of the outbreak strains in a whole species tree, and the next step was to provide a detailed relationship across the outbreak strains and their suspected relatives. Through this strategy, we observed that the Xinghai plague outbreak was caused by Y. pestis that circulated in the local plague focus, where the majority of historical plague epidemics in the Qinghai-Tibet Plateau may originate from. The analytical strategy developed here will be of great help in fighting against the outbreaks of emerging infectious diseases, by pinpointing the source of pathogens rapidly with genomic epidemiological data and microbial forensics information.

To the Editor: Plague is one of the oldest identifiable diseases. Modern public health measures and effective antimicrobial treatments have led to a decrease in plague cases worldwide. However, plague remains endemic in many natural foci. Since the early 1990s, the World Health Organization (WHO) has reported a steadily increasing trend in human plague cases, which has resulted in the recognition of plague as a reemerging disease (1). The emergence of antimicrobial drug–resistant strains of Yersinia pestis, along with an increasing number of plague cases, remind us that plague still poses a serious public health threat (2,3). In China, human cases of plague have been reported to WHO nearly every year from 1989 to 2003; these account for 9.5% of cases and 15.5% of deaths from this disease in Asia (1). Human cases of plague in China are usually caused by contact with plague-infected rodents. Here, we report the results of a serologic survey by using 3 methods (passive hemagglutination assay, Western blot, and protein microarray analysis) in marmot hunters in Qinghai Province, China. One hundred twenty serum samples were collected in 2 villages in Huangyuan County, Qinghai Province, from marmot hunters (63 samples) and their family members (57 samples); none had a history of fever in the past 2 years. One hundred nineteen serum samples were collected from persons with no history of marmot hunting in 2 nearby counties in Qinghai Province in which plague was not endemic. Thirty serum samples were collected from persons in Beijing and used as negative controls. All serum samples were initially screened with a passive hemagglutination assay to detect immunoglobulin (Ig) G antibody against F1 antigen of Y. pestis, by using a standard protocol (4). We then used an F1 antigen–based Western blot to analyze all serum samples. The protein microarray analysis was performed with 149 purified recombinant proteins of Y. pestis (5). The results of the serologic survey are summarized in the Table. The passive hemagglutination assay showed 17 positive samples in the marmot hunter population. None of the control serum samples were positive for F1 antigen in this assay. Western blot identified 9 additional positive samples in the marmot hunter population, resulting in a seropositivity rate of 21.7% (26/120). We also found positive samples in 4 (3.4%) of 119 serum samples by using Western blot in persons from areas in which plague was not endemic. Identical results were also obtained by using protein microarray analysis, which validated the results of Western blot. Table Analysis of sera for plague antibody by 3 methods Previous studies have shown that plague antibodies were more prevalent in males in the exposed population, and differences in the age, sex, or ethnic group of plague patients are the result of variations in exposure to the pathogen, not intrinsic factors (6,7). Our study showed that in the marmot hunter population, the plague seropositivity rate was significantly higher in males (36.8%, 25/68) than in females (2.0%, 1/52, p

The genetic diversity of Yersinia pestis , the etiologic agent of plague, is extremely limited because of its recent origin coupled with a slow clock rate. Here we identified 2,326 SNPs from 133 genomes of Y. pestis strains that were isolated in China and elsewhere. These SNPs define the genealogy of Y. pestis since its most recent common ancestor. All but 28 of these SNPs represented mutations that happened only once within the genealogy, and they were distributed essentially at random among individual genes. Only seven genes contained a significant excess of nonsynonymous SNP, suggesting that the fixation of SNPs mainly arises via neutral processes, such as genetic drift, rather than Darwinian selection. However, the rate of fixation varies dramatically over the genealogy: the number of SNPs accumulated by different lineages was highly variable and the genealogy contains multiple polytomies, one of which resulted in four branches near the time of the Black Death. We suggest that demographic changes can affect the speed of evolution in epidemic pathogens even in the absence of natural selection, and hypothesize that neutral SNPs are fixed rapidly during intermittent epidemics and outbreaks.

Yersinia pestis is a bacterium that is transmitted between fleas, which have a body temperature of 26 °C, and mammalian hosts, which have a body temperature of 37 °C. To adapt to the temperature shift, phenotype variations, including virulence, occur. In this study, an antigen microarray including 218 proteins of Y. pestis was used to evaluate antibody responses in a pooled plague serum that was unadsorbed, adsorbed by Y. pestis cultivated at 26 °C, or adsorbed by Y. pestis cultivated at 26 and 37 °C to identify protein expression changes during the temperature shift. We identified 12 proteins as being expressed at 37 °C but not at 26 °C, or expressed at significantly higher levels at 37 °C than at 26 °C. The antibodies against 7 proteins in the serum adsorbed by Y. pestis cultivated at 26 and 37 °C remained positive, suggesting that they were not expressed on the surface of Y. pestis in LB broth in vitro or specifically expressed in vivo. This study proved that protein microarray and antibody profiling comprise a promising technique for monitoring gene expression at the protein level and for better understanding pathogenicity, to find new vaccine targets against plague.

Background. Primarypneumonicplague(PPP)causedbyYersiniapestisisthemostthreateningclinicalformofplague. An outbreak was reported in July 2009 in Qinghai Province, China.Methods. This outbreak was investigated by clinical, epidemiological, bacteriological, and immunologicalmethods. Multilocus variablenumber tandem repeat analysis (MLVA) was used to track the source of the outbreak.Results. The index case, a patient with PPP, contaminated 11 close contacts. All the 12 cases, including theindexpatient, experiencedsuddenonsetof fever, headache,and productivecoughingwithbloody sputum.Threeofthem died. Nevertheless, another 61 direct and 256 indirect contacts were not infected during the 2-weekquarantine. Antibodies to F1 antigen were detected in 9 survival cases, with a 4-fold increase in titers in serumsamples collected at different periods. Seven strains of Y. pestis were isolated from dogs and patients. Fieldinvestigation and MLVA of the isolated strains revealed that this outbreak was started by a deceased dog.Conclusion. Dogs are believed to be an indicator animal for plague surveillance, but their association with PPPis rare. Our results provide evidence for this possibility, which suggests the public health significance of dogs asa source of plague.Plague, caused by Yersinia pestis, was classified asa reemerging infectious disease by the World HealthOrganization (WHO) in the early 1980s, because thereported cases around the world were increasing atthat time. There are different forms of plague, in-cluding bubonic and pneumonic, and the latter is themost threatening clinical form. Primary and second-arypneumonicplagueshavebeenwell-documentedinhistory.Theprimarypneumonicplague(PPP)out-breaks in Oakland in 1919, Los Angeles in 1924 [1],Manchuria during 1910–1911 [2], and Madagascar in1957 are the most frequently cited ones. PPP was alsoreported recently in the United States, India, Uganda,Zambia, Ecuador, and Madagascar [3–11]. BecausePPP progresses so rapidly that the patients often re-ceived a diasgnosis or were suspected to have plagueonly in the late stage of the disease, most of the PPPcases were reported on the basis of retrospectiveepidemiological investigations with only some ofthem confirmed by bacterial isolation and antibodydetection.On29July2009,asuspectedPPPoutbreakinXinghaiCounty, Qinghai provinceof China,was reportedtotheQinghai Ministry of Health by China’s public healthemergency reporting system. The next day, a group ofplague experts, including epidemiologists, bacteriolo-gists, doctors, and management officers, were sentout for further investigation in the Xinghai CountyTibetan (XCT) hospital, where the suspected patientshad been admitted.

In our previous study, complete protection was observed in Chinese-origin rhesus macaques immunized with SV1 (20 µg F1 and 10 µg rV270) and SV2 (200 µg F1 and 100 µg rV270) subunit vaccines and with EV76 live attenuated vaccine against subcutaneous challenge with 6×10(6) CFU of Y. pestis. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological and immunohistochemical techniques. In addition, the glomerular basement membranes (GBMs) of the immunized animals and control animals were checked by electron microscopy. The results show no signs of histopathological lesions in the lungs, livers, kidneys, lymph nodes, spleens and hearts of the immunized animals at Day 14 after the challenge, whereas pathological alterations were seen in the corresponding tissues of the control animals. Giemsa staining, ultrastructural examination, and immunohistochemical staining revealed bacteria in some of the organs of the control animals, whereas no bacterium was observed among the immunized animals. Ultrastructural observation revealed that no glomerular immune deposits on the GBM. These observations suggest that the vaccines can effectively protect animals from any pathologic changes and eliminate Y. pestis from the immunized animals. The control animals died from multi-organ lesions specifically caused by the Y. pestis infection. We also found that subcutaneous infection of animals with Y. pestis results in bubonic plague, followed by pneumonic and septicemic plagues. The histopathologic features of plague in rhesus macaques closely resemble those of rodent and human plagues. Thus, Chinese-origin rhesus macaques serve as useful models in studying Y. pestis pathogenesis, host response and the efficacy of new medical countermeasures against plague.

Objective LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study. Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co2+ affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography. Results Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 106 CFU of Y. pestis virulent strain 141. Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

Long-term protection and antibody response for the subunit vaccine F1-rV270 were determined by using the mouse model. Antibodies to F1 and rV270 were still detectable over a period of 518 days. The complete protection against lethal challenge of Yersinia pestis could be achieved up to day 518 after primary immunization.

Background DFR (different region) analysis has been developed for typing Yesinia pestis in our previous study, and in this study, we extended this method by using 23 DFRs to investigate 909 Chinese Y. pestis strains for validating DFR-based genotyping method and better understanding adaptive microevolution of Y. pestis. Methodology/Principal Findings On the basis of PCR and Bionumerics data analysis, 909 Y. pestis strains were genotyped into 32 genomovars according to their DFR profiles. New terms, Major genomovar and Minor genomovar, were coined for illustrating evolutionary relationship between Y. pestis strains from different plague foci and different hosts. In silico DFR profiling of the completed or draft genomes shed lights on the evolutionary scenario of Y. pestis from Y. pseudotuberculosis. Notably, several sequenced Y. pestis strains share the same DFR profiles with Chinese strains, providing data for revealing the global plague foci expansion. Conclusions/significance Distribution of Y. pestis genomovars is plague focus-specific. Microevolution of biovar Orientalis was deduced according to DFR profiles. DFR analysis turns to be an efficient and inexpensive method to portrait the genome plasticity of Y. pestis based on horizontal gene transfer (HGT). DFR analysis can also be used as a tool in comparative and evolutionary genomic research for other bacteria with similar genome plasticity.

F1 antigen is an attractive candidate for the development of a subunit vaccine against plague. In previous study, the extraction of this antigen from Yersinia pestis is characterized by using organic solvents. In this work, a new purification strategy that produced high-purity F1 antigen from Y. pestis EV76 was developed by the substitution of physical disruption for organic solvent one, followed by a combination of ammonium sulfate fractionation and Sephacryl S-200HR column filtration chromatography. As revealed in this study, this purification procedure is simple and effective, and avoids potential adverse effect on the antigen by organic solvents. Highly purified F1 that adsorbed to 25% (v/v) Al(OH) 3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to F1 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10 4 CFU of Y. pestis virulent strain 141.