Rong Hai

Molecular Evolution Analysis and Geographic Investigation of Severe Acute Respiratory Syndrome Coronavirus-Like Virus in Palm Civets at an Animal Market and on Farms

ABSTRACT Massive numbers of palm civets were culled to remove sources for the reemergence of severe acute respiratory syndrome (SARS) in Guangdong Province, China, in January 2004, following SARS coronavirus detection in market animals. The virus was identified in all 91 palm civets and 15 raccoon dogs of animal market origin sampled prior to culling, but not in 1,107 palm civets later sampled at 25 farms, spread over 12 provinces, which were claimed to be the source of traded animals. Twenty-seven novel signature variation residues (SNVs) were identified on the spike gene and were analyzed for their phylogenetic relationships, based on 17 sequences obtained from animals in our study and from other published studies. Analysis indicated that the virus in palm civets at the live-animal market had evolved to infect humans. The evolutionary starting point was a prototype group consisting of three viral sequences of animal origin. Initially, seven SNV sites caused six amino acid changes, at positions 147, 228, 240, 479, 821, and 1080 of the spike protein, to generate low-pathogenicity viruses. One of these was linked to the first SARS patient in the 2003-2004 period. A further 14 SNVs caused 11 amino acid residue changes, at positions 360, 462, 472, 480, 487, 609, 613, 665, 743, 765, and 1163. The resulting high-pathogenicity groups were responsible for infections during the so-called early-phase epidemic of 2003. Finally, the remaining six SNVs caused four amino acid changes, at positions 227, 244, 344, and 778, which resulted in the group of viruses responsible for the global epidemic.

SARS-CoV infection in a restaurant from palm civet.

Contact with food animals was associated with SARS-CoV infection in the People’s Republic of China.

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

BackgroundYersinia 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.ResultsTwo 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 Neis 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.ConclusionThe 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.

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.

Homology Analysis of Pathogenic Yersinia Species Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis Based on Multilocus Sequence Typing

ABSTRACT We developed a multilocus sequence typing (MLST) scheme and used it to study the population structure and evolutionary relationships of three pathogenic Yersinia species. MLST of these three Yersinia species showed a complex of two clusters, one composed of Yersinia pseudotuberculosis and Yersinia pestis and the other composed of Yersinia enterocolitica. Within the first cluster, the predominant Y. pestis sequence type 90 (ST90) was linked to Y. pseudotuberculosis ST43 by one locus difference, and 81.25% of the ST43 strains were from serotype O:1b, supporting the hypothesis that Y. pestis descended from the O:1b serotype of Y. pseudotuberculosis. We also found that the worldwide-prevalent serotypes O:1a, O:1b, and O:3 were predominated by specific STs. The second cluster consisted of pathogenic and nonpathogenic Y. enterocolitica strains, two of which may not have identical STs. The pathogenic Y. enterocolitica strains formed a relatively conserved group; most strains clustered within ST186 and ST187. Serotypes O:3, O:8, and O:9 were separated into three distinct blocks. Nonpathogenic Y. enterocolitica STs were more heterogeneous, reflecting genetic diversity through evolution. By providing a better and effective MLST procedure for use with the Yersinia community, valuable information and insights into the genetic evolutionary differences of these pathogens were obtained.

Genome Rearrangements of Completely Sequenced Strains of Yersinia pestis

ABSTRACT 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.

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.

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.

Burkholderia pseudomallei sequence type 562 in China and Australia.

To the Editor: Melioidosis is increasingly being recognized in tropical and subtropical areas worldwide; the world’s 2 major endemic foci are Thailand and northern Australia (1,2). Phylogenetic analyses of Burkholderia pseudomallei isolates, performed by using multilocus sequence typing (MLST) (3), have led to phylogeographic associations that can be used to track melioidosis epidemics (4). However, in contrast to the previous separation of B. pseudomallei into 2 phylogenetic groups (Australia and Southeast Asia/rest of the world) (5), we report an MLST sequence type (ST) that seems to be present in northern Australia, Taiwan, and southern China. In mainland China, melioidosis was first reported in 1990 (6) and is now known to be endemic to several tropical provinces, including Hainan, a southern island province close to Southeast Asia. Since 2008, cases of melioidosis in Hainan have escalated; from July 2008 through July 2012, a total of 110 cases were microbiologically diagnosed at 2 general hospitals (Sanya People’s Hospital and Haikou Municipal Hospital). We characterized clinical isolates of B. pseudomallei from the 110 cases by using MLST, pulsed-field gel electrophoresis (PFGE), and 4-locus multilocus variable-number tandem-repeat analysis (MLVA-4) (3,7,8). MLST revealed 40 STs, 39 of which were consistent with STs from Southeast Asia, as evident from the global B. pseudomallei MLST database (http://bpseudomallei.mlst.net/). A single ST, ST562, which accounted for 3 cases in Hainan, was previously described on the global database as being from Australia; the 20 isolates from humans and 10 isolates from the environment deposited until September 1, 2014, all from Australia, had been isolated from 2005 through 2012. Although not deposited in the global MLST database, ST562 has also recently been reported from Taiwan (7). Among the 253 isolates of B. pseudomallei collected in Taiwan during 2004–2010, 1 clinical isolate and 9 environmental isolates were described as being ST562. Moreover, these 10 ST562 isolates displayed a unique PFGE pulsotype, distinct from that of other B. pseudomallei strains from Taiwan (7). Of the 3 patients from Hainan from whom ST562 strains were isolated, 2 resided in the city of Sanya and 1 in the neighboring city of Lingshui (Technical Appendix); all denied a history of foreign travel, they shared no common risk factors, and all survived the infection. Further analysis of ST562, performed by using eBURST-based (http://eburst.mlst.net/) population analysis of the MLST dataset, showed that ST562 is a single-locus variant of ST167, which is represented on the MLST dataset by multiple human and environmental isolates from Thailand and to date by 1 human isolate from Cambodia. ST167 accounted for 1 of the 110 B. pseudomallei strains from Hainan. The narK locus of ST167contains allele 3 instead of allele 29, as seen in ST562; 3 base differences are found in allele 3: C72T (C→T position 72), C126T, and A435G. According to PFGE, the 3 ST562 isolates from Hainan displayed a single pulsotype, and the other 107 isolates from Hainan belonged to distinct and diverse pulsotypes, similar to those observed in Taiwan. The uniformity of PFGE patterns in the Hainan and Taiwan isolates supports the possibility that ST562 might be a recently emerging clone. PFGE patterns of Hainan ST562 exhibited 86% similarity with ST167, differing by 6 bands (Figure). Figure Pulsed-field gel electrophoresis (PFGE) patterns for 3 sequence type (ST) 562 and 1 ST167 Burkholderia pseudomallei strains isolated during 2008–2012, Hainan, China. The isolate source, isolation time, ST, and 4-locus multilocus variable-number ... Hainan ST562 isolates were further analyzed by using MLVA-4 (8), which divided 3 isolates (from patients A, B, and C) into 3 distinct MLVA-4 types (Figure). The 2008 isolate (MLVA-4 profile 11,8,15,7) and one 2012 isolate (profile 11,8,17,7) exhibited close relatedness, whereas another 2012 isolate (profile 10,10,11,5) was divergent from these, indicating that ST562 isolates in Hainan have been present long enough for some divergence into lineages. Two mutually exclusive gene clusters, B. thailandensis–like flagellar gene cluster (BTFC) and Yersinia-like fimbrial gene cluster (YLF), have been linked to geographic origin and have been suggested for differentiating groups of B. pseudomallei (9). By PCR we found that ST562 isolates of Hainan were all YLF positive. BTFC predominates in Australian B. pseudomallei strains, and YLF predominates in Southeast Asia. Presence of YLF was also observed in strains from Papua New Guinea, possibly reflecting that country’s location, intermediate between major foci of melioidosis (10). In conclusion, by using MLST and the online MLST database, we revealed that B. pseudomallei ST562 is present in southern China as well as in Australia and Taiwan. The intercontinental character of this ST raises new questions about the epidemiology and control of melioidosis. Given the usual geographic separation of B. pseudomallei STs, we suggest that this wide-ranging presence of ST562 might result from more recent spread caused by transmission between regions. Increasing farming exchanges and trade of agricultural products between melioidosis-endemic regions might facilitate breaking of the geographic barrier; clonal introduction of B. pseudomallei could potentially occur in new locations. Improved and cooperative surveillance is required for elucidating the current and future global dispersion range of B. pseudomallei and for monitoring the consequent melioidosis infections. Technical Appendix: Geographic distribution of 43 Burkholderia pseudomallei sequence type 562 strains identified in Australia; Taiwan; and Hainan, China, during 2004–2012. Click here to view.(92K, pdf)

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.