Objective To compare the coding sequences (CDS) of Yersinia pestis D106004 strain from Yulong County in Yunnan Province and Z176003 strain from Qing-Tibet Plateau in order to find the differences between their genomes and the genetic characteristics. Methods The CDS of Yersinia pestis D106004 strain and Z176003 strain were searched and compared by BLAST. Twenty-two differential CDS were selected to design 22 pairs of primers. PCR amplification was carried out in 119 representative plague strains from different isolation sources (natural foci of Himalayan marmot plague in the Qinghai-Tibet Plateau, natural foci of Apodemus chevrieri and Eothenomys miletus plague in Yunnan), time span of about 50 years, and distribution in six ecological types including Tibet, Qinghai, Sichuan, Gansu and Yunnan, and PCR products were sequenced and verified. The strains were all from the State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Results In 119 representative plague strains of 6 ecological types, the cumulative sequence length of 22 differential CDS PCR amplification products was 2.13 × 106 bp. Among the 119 representative plague strains in the foci of Yulong D106004 strain and Qinghai-Tibet Plateau Z176003 strain, 22 differential CDS had high homology, there was no difference in 78.2％ (2047/2618) sequences of differential CDS, and 21.8％ (571/2618) sequences had three types of gene mutations ( deletion , missense and frameshift mutations). The characteristics of the differences were stable in the 6 ecological plague strains of the foci, and they were divided into 6 geographical distributions. Conclusion Yulong D106004 strain and Qinghai-Tibet Plateau Z176003 strain have high homology, close genetic relationship, and little difference in genome, but the genetic characteristics of different ecotype strains are stable.

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.

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.

Objectives To identify coding sequences in the genome of 2 strains of Yersinia pestis from the Qinghai-Tibet Plateau,which is a natural focus of the Himalayan marmot,in order to ascertain the genetic diversity of Y.pestis.Methods The software BLAST,the Perl programming language,and the software Excel were used to compare coding sequences and statistically analyze the Z176003 strain of Y.pestis from Tibet,the D106004 strain from Yulong,Yunnan,and a "default coding sequence database." Results Preliminary comparison of the Z176003 strain of Y.pestis from Tibet and the D106004 strain from Yulong,Yunnan yielded 418 different coding sequences.The "default coding sequence database" had 135 different coding sequences.Eighty six coding sequences were ultimately determined to differ between the 2 strains and the database of the Y.pestis genome.Conclusion Coding sequences from the 2 strains of Y.pestis from the Qinghai-Tibet Plateau differed.Coding sequences that truly differed were groups of different coding sequences of Y.pestis.This finding should provide reliable information for further study of the genetic characteristics of Y.pestis from the Qinghai-Tibet plateau.

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.

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.

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.

Yersinia pestis is the causative agent of bubonic and pneumonic plagues.Strains of Y.pestis are classified into four biovars:antiqua,mediaevalis,orientalis,and microtus[1].There are two microtus-related plague foci in China:the Microtus brandti plague focus in the Xilin Gol Grassland(focus L)and the Microtus fuscus plague focus in the Qinghai-Tibet Plateau(focus M).Microtus strains are avirulent to humans,and experiments have shown that strains isolated from Microtus brandti in Inner Mongolia are virulent to

To detect Yersinia pestis in the foci surveillance rapidly and specially, we developed a multiplex polymerase chain reaction (PCR) with an internal control technique. The specific segments of Yesinia pestis were co-amplified by PCR directly from the hosts’ organ samples with an internal control to prevent false negative results that might be caused by PCR inhibitors. Comparing the PCR results with that of the bacteria culture, the positive detection rate of PCR method was higher and it was more rapid. So, it is a useful method for the detection of Yesinia pestis in the plague foci surveillance.

Objective Protein coding sequences in the origin tectonics in Yersinia pestis genome were annotated according to the existence of the ribosome binding sites.Methods Select a characteristic plate in Y.pestis,count the quantity of CDS with tranditional and RBS methods,compare their consistency.Find the location of mutations and their affect on the CDS by blast 10 Y.pestis,determine the reliability of the RBS methods.Results In the annotation of the CO92 sequence,47 CDS existed in this section.By the method in this research,74 CDS could be identified,24 were completely identical to the original annotation,and 8 were partially identical.Nine CDS were found to have multiple starting codons.Most of the 42 new discovered CDS encoded short peptides less than 100 amino acids.The influence of mutation to the CDS was checked in 10 strains of Yersinia pestis.In this section of genome,36 mutation loci were identified.According to the original CO92 annotation,12 CDS were influenced,but according to the RBS annotation,the influenced CDS decreased to 9,and other 3 reduced the lethality.Conclusion The result of the research suggested that RBS could be the most important index in the CDS annotation,but could not be used to replace the method based on the Markove model at present.

Objective To determine the optimal distance between ribosomal binding site(RBS) and translation initiation site(TIS) by statistical analysis of RBS-TIS distances in Yersinia pestis CO92.Methods All copies of 3'-terminal 16S rRNA and 177 annotated RBS sequences in CO92 were analyzed to identify the characteristics of Y.pestis RBS sequences.The positions and number of all RBS sequences in CO92 and their distances from the TIS ATG(GTG,TTG,CTG) were determined by statistical analysis.The characteristics of the 20 upstream bases in the annotated coding DNA sequences(CDSs) in CO92 were observed.Results A total of 5081 potential RBS sequences were found in CO92 genome.Of the 5081 RBS sequences,2909 had downstream open reading frames(ORFs);of the 2909 RBS sequences,1541 had the ORFs identical to the annotated CDSs,and 535 had the same termination sites as the annotated CDSs but had different initiation sites.In the 20 upstream bases of 3887 annotated CO92 CDSs,57.7% contained RBS sequences.Conclusion The most frequent distances between RBS and TIS were 7 and 8 bases in CO92 genome.RBS can be an important gene index for identification.

Objective To study the single nucleotide polymorphisms (SNP) characteristics of Yersinia pestis strains from different natural foci in China.Methods Genome-wide comparison was done to find SNP sites by the Mummer program among 9 Yersinia pestis genome which was downloaded from NCBI.Then 13 genic fragments including 19 SNP sites were amplified by PCR and sequenced in 133 Yersinia pestis strains,and the results were cluster analyzed with the BioNumerics software.Results Three thousand seven hundred and eighty sequence variation sites were found by genome-wide comparison.Using the different combinations of SNP sites,UPGMA cluster analysis revealed obvious geographic regional and eco-aggregation characteristics of Yersinia pestis strains isolated from China.Conclusions As relatively stable genetic markers,SNP can better reflect the genome characteristics of Yersinia pestis in different plague natural foci of China.

For plague surveillance in the silence periods,the whole genome sequences of 10 Yersinia pestis strains and 3 Yersinia pseudotuberculosis strains downloaded from Internet were compared.In the 68 Y.pestis Tectonics divided according to CO92 chromosome sequence,67 appeared in Y.paeudotuberculosis genomes in whole or in most part,the arrangement of these tectonics were determined.Y.pseudotuberculosis has the same 7 copies of rRNA gene clusters as Y.pestis,and they caused the earliest genome rearrangement in Y.pseudotuberculosis.Before Y.pestis evolved from Y.pseudotuberculosis,there were 16 positions inserted with 4 species of insertion sequences,and no rearrangement caused by these insertion sequences was found in Y.pseudotuberculosis.Totally 20 Pestis-Islands(PeI) specifying for Y.pestis and 39 Pseudo-Islands(PsI) specifying for Y.pseudotuberculosis were identified,and their sequences could be used as differentiation characters when atypical strains appeared in the surveillance.

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.

Single nucleotide polymorphisms(SNPs) mainly refer to the polymorphism of DNA sequence caused by a single nucleotide mutation,including the synonymous SNPs and non-synonymous SNPs.With the rapid development of sequencing technology,a large number of bacterial genome sequences are available.So,it's possible to identify potential SNPs sites by sequencing technology and bioinformatics methods.Also,SNPs,because of their own characteristics,have been widely used as a new molecular marker in bacterial genotyping,evolution and epidemiology research.In this paper,advances in the research on the genome-wide search of SNPs sites and analysis of the Yersinia pestis microevolution based on SNPs data are reviewed.

Objective Measurement and analysis of the complete genome sequences of Yersinia Pestis from a new plague natural foci and adjacent foci in China, to know the genetic relationship among the epidemic strain isolated in Yulong (D 106004) and Jianchuan strains (D 182038) and the Tibetan strain ( Z 176003 ). Methods Three complete genome sequences were sequenced using the whole-genome shotgun and Solexa method and comparative genomics analysis was done among the three sequences. Genome comparative analysis among the coding sequences was done by BLAST software, SNPs finding was done by the program, genome rearrangements were analyzed using MAUVE software. Results All of the genomes of Yersinia pestis strains D182038, D106004 and Z176003 consist of a single circular chromosome and three virulence plasmids, pMT1, pCD1 and pPCP1. They had similar characteristics in chromosome and plasmid features, and there were no significant difference in coming sequence (CDS) of the cluster of orthologous groups of proteins (COG) functional classification and the number of insertion sequence in the three strains (x2 =3.03, 0.257, all P ＞ 0.05). The comparative genomics results showed that the three bacteria had 2882 genes with 100％ homology, of 3636 genes predicted in D106004, 2994 were identical with D182038's and 3113 with Z176003's, and of which 240 had 90％ homology with D182038's and 200 with Z176003 's. Synonymous single nucleotide polymorphisms(sSNPs) were 59 and 68, and non-synonymous SNPs(nsSNPs) were 104 and 203 between strains D106004 and Z176003/D182038. There were 11 segments rearrangements between D106004 and Z176003, which was less than 16 segments rearrangements between D106004 and D182038. Conclusions The three strains are highly homologous, the Yulong strain has more similarity with Tibet strain than with Jianchuan strain, the strain from Yulong foci may be evolved from Tibet foci.

云南省在研究证明家鼠型鼠疫流行病学特征的基础上,探索适合我国国情的家鼠型鼠疫监控模式,达到控制家鼠鼠疫流行的目的,采取鼠疫监控基础研究与鼠疫监控模式研究相结合、实验室研究与现场研究相结合的方法.该项目研究证明了家鼠鼠疫流行病学特征;经系统试验筛选了毒杀黄胸鼠的适合云南的化学药物,筛选出配制毒饵用高效、廉价基饵;制定了云南省鼠疫应急处理预案;家鼠型鼠疫监控模式研究取得多项成果.通过该项目的研究,云南省家鼠型鼠疫的基本情况已经了解,适合我国国情的家鼠型鼠疫监控模式已经找到,推广应用该模式,以达到有效控制家鼠型鼠疫流行的目标.

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 165 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.

Objective To study the subspecies of Francisella tularensis in China and the genetic relationships among the various strains of them. Methods Ten strains of F. tularensis from North China were subject to PCR using two specific primers C1C4 and RD1. Their subspecies were identified based on the length of the amplification products. At the same time, PCR on three specific genes was performed using fopA, tul4 and 16S rRNA primers, followed by sequencing. Based on the three specific genes, phylogenetic analysis was conducted using MEGA 4 software to involve the 10 strains of F. tularensis and the three strains of F. tularensis type B and one strain of subsp. novicida published on the NCBI website. Results The 10 strains of F. tularensis were identified as type B based on the PCR results using two specific primers C1C4 and RD1. According to the phylogenetic tree structured by MEGA 4, the 10 strains of F. tularensis from China can be classified into two types: B1 type, including 410108, 410109 and 410111, and B2 type including the other seven strains. In contrast, the three foreign strains were of type B3. Conclusion The F. tularensis isolated in North China may be predominated by type B. As for the origin of F. tularensis type B, the F. tularensis in China has probably emerged earlier than those in Europe and America. Phylogenetic analysis based on the three specific genes can be used as a reliable genotyping tool for F. tularensis.

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.