The third plague pandemic originated from Yunnan Province, China in the middle of the 19^th century. The last human plague epidemic in Yunnan occurred from 1986–2005. On June 6, 2016, a case of human plague was reported in the Xishuangbanna Prefecture, Yunnan. The patient suffered from primary septicemic plague after exposure to a dead house rat (Rattus flavipectus), which has been identified as the main plague reservoir in the local epizootic area. Moreover, a retrospective investigation identified another bubonic plague case in this area. Based on these data, human plague reemerged after a silent period of ten years. In this study, three molecular typing methods, including a clustered regularly interspaced short palindromic repeats (CRISPR) analysis, different region analysis (DFR), and multiple-locus variable number of tandem repeats analysis (MLVA), were used to illustrate the molecular characteristics of Yersinia pestis (Y. pestis) strains isolated in Yunnan. The DFR profiles of the strains isolated in Yunnan in 2016 were the same as the strains that had previously been isolated in this Rattus flavipectus plague focus. The c3 spacer present in the previously isolated strains was absent in the spacer arrays of the Ypc CRISPR loci of the strains isolated in 2016. The MLVA analysis using MLVA (14+12) showed that the strains isolated from the human plague case and host animal plague infection in 2016 in Yunnan displayed different molecular patterns than the strains that had previously been isolated from Yunnan and adjacent provinces.

The pathogen of plague is Yersinia pestis (Y. pestis), one of the deadliest pathogens in the world and belonging to the family Enterobacteriaceae. In this work, the biological characteristics and complete genome sequence analysis of a novel lytic Y. pestis-specific phage JC221 isolated from Yunnan Province, China, was studied. JC221 belongs to the Myoviridae family and has a regular icosahedral head and a long contractile tail. The double-stranded DNA genome of JC221 contains 174,931 bp, and the G + C content is 41.23 %. There are 274 predicted genes, of which only 103 hits of genes or gene products are found in database searches, and there are no known virulence-related or antibiotic resistance genes. The genome sequence of JC221 showed <80 % identity to other phages, and evolutionary analysis revealed that bacteriophage JC221 belongs to the Yersinia phage cluster. Furthermore, the bacteriophage could completely lyse most of the tested Y. pestis strains (12/13) at 28 °C and 37 °C, and some Shigella strains could be lysed at 37°C. Morphological and genomic analysis indicated that JC221 is a new Y. pestis phage and a new member of the Tequatrovirus phages. The novel Y. pestis phage JC221 has important reference value for the study of environmental microecology and epidemiology of plague foci.

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. Plague is a type of zoonosis that is highly lethal to humans. The surveillance of animal hosts is critical for the prevention and control of plague. The Yulong plague focus is a newly discovered plague focus in China in recent years. The plague outbreak had attracted widespread attention because 5 people were infected in 2005, 2 of whom died. We have monitored the plague focus for a decade, and isolated strains and DNAs of Yersinia pestis were studied. The structure, origin and evolutionary trend of the Yulong plague focus were clarified, which provides a scientific basis for the effective prevention and control of human plague. This article also provides a set of paradigms for the systematic study of new plague foci, which is a perfect combination of traditional monitoring methods and modern research methods.

The study of carrier state phages challenged the canonical lytic-lysogenic binary, and carrier state appears to be ubiquitous and ecologically important. However, the mechanisms of the carrier state are not well elucidated due to the limited phage models. Herein, we reported phage HQ103, similar to Escherichia coli phage P2. In contrast to the temperate P2 phage, the HQ103 phage does not insert its genome into the bacterial chromosome and displays a dual behavior depending on the temperature. At 37°C, HQ103 lyses the host and forms clear plaques due to the truncation of repressor CI and mutation of promoter Pc. In contrast, HQ103 maintains a carrier state lifestyle with Y. pestis at an environmental temperature (21°C). Mechanistically, we found that the host-encoded histone-like nucleoid-structuring protein H-NS, which is highly expressed at 21°C to silence the Cox promoter Pe and inhibits the phage lytic cycle. Subsequently, the HQ103 carrier state Y. pestis could grow and co-exist with the phage in the soil at 21°C for one month. Thus, this study reveals a novel carrier state lifestyle of phage HQ103 due to the H-NS mediated xenogeneic silencing and demonstrates that the carrier state lifestyle could promote long-term phage-host coexist in nature.

Yunnan Province, China is thought to be the original source of biovar Orientalis of Yersinia pestis, the causative agent of the third plague pandemic that has spread globally since the end of the 19th century. Although encompassing a large area of natural plague foci, Y. pestis strains have rarely been found in live rodents during surveillance in Yunnan, and most isolates are from rodent corpses and their fleas. In 2017, 10 Y. pestis strains were isolated from seven live rodents and three fleas in Heqing County of Yunnan. These strains were supposed to have low virulence to local rodents Eothenomys miletus and Apodemus chevrieri because the rodents were healthy and no dead animals were found in surrounding areas, as had occurred in previous epizootic disease. We performed microscopic and biochemical examinations of the isolates, and compared their whole-genome sequences and transcriptome with those of 10 high virulence Y. pestis strains that were isolated from nine rodents and one parasitic flea in adjacent city (Lijiang). We analyzed the phenotypic, genomic, and transcriptomic characteristics of live rodent isolates. The isolates formed a previously undefined monophyletic branch of Y. pestis that was named 1.IN5. Six SNPs, two indels, and one copy number variation were detected between live rodent isolates and the high virulence neighbors. No obvious functional consequence of these variations was found according to the known annotation information. Among genes which expression differential in the live rodent isolates compared to their high virulent neighbors, we found five iron transfer related ones that were significant up-regulated (| log₂ (FC) | > 1, p.adjust < 0.05), indicating these genes may be related to the low-virulence phenotype. The novel genotype of Y. pestis reported here provides further insights into the evolution and spread of plague as well as clues that may help to decipher the virulence mechanism of this notorious pathogen.

Plague, caused by Yersinia pestis, is a zoonotic disease that can reemerge and cause outbreaks following decades of latency in natural plague foci. However, the genetic diversity and spread pattern of Y. pestis during these epidemic-silent cycles remain unclear. In this study, we analyze 356 Y. pestis genomes isolated between 1952 and 2016 in the Yunnan Rattus tanezumi plague focus, China, covering two epidemic-silent cycles. Through high-resolution genomic epidemiological analysis, we find that 96% of Y. pestis genomes belong to phylogroup 1.ORI2 and are subdivided into two sister clades (Sublineage1 and Sublineage2) characterized by different temporal-spatial distributions and genetic diversity. Most of the Sublineage1 strains are isolated from the first epidemic-silent cycle, while Sublineage2 strains are predominantly from the second cycle and revealing a west to east spread. The two sister clades evolved in parallel from a common ancestor and independently lead to two separate epidemics, confirming that the pathogen responsible for the second epidemic following the silent interval is not a descendant of the causative strain of the first epidemic. Our results provide a mechanism for defining epidemic-silent cycles in natural plague foci, which is valuable in the prevention and control of future plague outbreaks.