Jiufeng Sun

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis.

BackgroundClonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome.ResultsWe combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines.ConclusionsThis study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control.

The Carcinogenic Liver Fluke, Clonorchis sinensis: New Assembly, Reannotation and Analysis of the Genome and Characterization of Tissue Transcriptomes

Clonorchis sinensis (C. sinensis), an important food-borne parasite that inhabits the intrahepatic bile duct and causes clonorchiasis, is of interest to both the public health field and the scientific research community. To learn more about the migration, parasitism and pathogenesis of C. sinensis at the molecular level, the present study developed an upgraded genomic assembly and annotation by sequencing paired-end and mate-paired libraries. We also performed transcriptome sequence analyses on multiple C. sinensis tissues (sucker, muscle, ovary and testis). Genes encoding molecules involved in responses to stimuli and muscle-related development were abundantly expressed in the oral sucker. Compared with other species, genes encoding molecules that facilitate the recognition and transport of cholesterol were observed in high copy numbers in the genome and were highly expressed in the oral sucker. Genes encoding transporters for fatty acids, glucose, amino acids and oxygen were also highly expressed, along with other molecules involved in metabolizing these substrates. All genes involved in energy metabolism pathways, including the β-oxidation of fatty acids, the citrate cycle, oxidative phosphorylation, and fumarate reduction, were expressed in the adults. Finally, we also provide valuable insights into the mechanism underlying the process of pathogenesis by characterizing the secretome of C. sinensis. The characterization and elaborate analysis of the upgraded genome and the tissue transcriptomes not only form a detailed and fundamental C. sinensis resource but also provide novel insights into the physiology and pathogenesis of C. sinensis. We anticipate that this work will aid the development of innovative strategies for the prevention and control of clonorchiasis.

Clonorchis sinensis enolase: Identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products

Enolase plays a key role in energy metabolism and development of most organisms. We isolated a gene encoding enolase from Clonorchis sinensis (C. sinensis) adult cDNA library and expressed the recombinant protein in Escherichia coli. C. sinensis enolase (Csenolase) was identified as both an excretory/secretory product and a tegumental component of C. sinensis by western blot analysis. The transcriptional level of Csenolase was examined at adult worm, metacercaria, cercaria and egg of C. sinensis, and results showed that Csenolase is transcribed at the four life stages of C. sinensis while showing a significant higher expression level at the stage of adult worm. Immunohistochemical localization indicated that Csenolase was specifically deposited on the tegument of adult worm and cyst wall of metacercaria. Ligand blot assay revealed a specific characteristic of dose-dependent plasminogen-binding activity of Csenolase and kinetic parameters were explored using 2-phospho-D-glycerate (2-PGA) as the primary substrate by monitoring the conversion of nicotinamide-adenine dinucleotide (NADH) into nicotinamide adenine dinucleotide (NAD). In addition, Csenolase exhibited active enzyme activity in catalytic reactions while the anti-Csenolase serum inhibited the enzyme activity. In vitro incubation experiments revealed that Csenolase might play key roles in the growth of the parasites. In conclusion, Csenolase is an important glycolytic enzyme required for the development of C. sinensis, and may be a potential vaccine candidate and drug target against C. sinensis infection.

Stage-specific expression, immunolocalization of Clonorchis sinensis lysophospholipase and its potential role in hepatic fibrosis

Lysophospholipase, belonging to the complex family of phospholipases, is supposed to play a vital role in virulence and pathogenesis of parasites and fungi. In the current study, the potential role of Clonorchis sinensis lysophospholipase (CslysoPLA) in hepatic fibrosis induced by C. sinensis was explored for the first time. In the liver of the cat infected with C. sinensis, CslysoPLA was recognized in the lumen between adult worms and surrounding bile duct epithelia together with some inside the cells by means of immunolocalization. Both Cell Counting Kit-8 (CCK-8 assay) and cell cycle analysis of human hepatic stellate cell line LX-2 showed that a higher percentage of cells were at proliferation phase after incubation with lower concentrations of recombinant CslysoPLA (rCslysoPLA). Quantitative real-time polymerase chain reaction (RT-PCR) demonstrated an upregulation in fibrogenic genes of smooth muscle α-actin, collagen III, matrix metalloproteinase 2 and tissue inhibitors of metalloproteinase II in LX-2 treated with rCslysoPLA. Moreover, human biliary epithelial cell line 5100 proliferated significantly in response to rCslysoPLA. Notably, CslysoPLA was localized in the adenomatoid hyperplastic tissue within the intrahepatic bile duct of experimentally infected rats by immunolocalization analysis. In addition, quantitative RT-PCR implied that CslysoPLA was differentially expressed at the developmental stages of C. sinensis (metacercariae, adult worms and eggs), with the highest level at metacercariae stage. Immunolocalization analysis showed that CslysoPLA was distributed in the intestine, vitelline gland, tegument and eggs in the adult worms and in the tegument and vitelline gland in the metacercariae, respectively. Collectively, it suggests that CslysoPLA might be involved in the initiation and promotion of C. sinensis-related human hepatic fibrosis and advance future studies on its promotion to C. sinensis-induced cholangiocarcinogenesis.

Molecular characterization and immune modulation properties of Clonorchis sinensis -derived RNASET2

BackgroundClonorchis sinensis (C. sinensis, Cs) is a trematode parasite that often causes chronic cumulative infections in the hepatobiliary ducts of the host and can lead to pathological changes by continuously released excretory/secretory proteins (ESPs). A T2 ribonuclease in trematode ESPs, has been identified as a potent regulator of dendritic cell (DCs) modulation. We wondered whether there was a counterpart present in Cs ESPs with similar activity. To gain a better understanding of Cs ESPs associated immune responses, we identified and characterized RNASET2 of C. sinensis (Cs RNASET2) in this paper.MethodsWe expressed Cs RNASET2 in Pichia pastoris and identified its molecular characteristics using bioinformatic analysis and experimental approaches. The immune modulation activities of Cs RNASET2 were confirmed by evaluating cytokine production and surface markers of recombinant Cs RNASET2 (rCs RNASET2) co-cultured DCs, and monitoring levels of IgG isotypes from rCs RNASET2 administered BALB/c mice.ResultsCs RNASET2 appeared to be a glycoprotein of T2 ribonuclease family harboring conserved CAS motifs and rich in B-cell epitopes. Furthermore, Cs RNASET2 was present in Cs ESPs and was able to modulate cytokine production of DCs. In addition, rCs RNASET2 could significantly suppress the expression of lipopolysaccharide-induced DCs maturation markers. In addition, when subcutaneously administered with rCs RNASET2 there was a marked effect on IgG isotypes in mouse sera.ConclusionCollectively, we revealed that Cs RNASET2, a T2 ribonuclease present in Cs ESPs, could modulate DCs maturation and might play an important role in C. sinensis associated immune regulation in the host.

The Molecular Epidemiological Characteristics and Genetic Diversity of Salmonella Typhimurium in Guangdong, China, 2007–2011

Background Salmonella enterica serovar Typhimurium is the most important serovar associated with human salmonellosis worldwide. Here we aimed to explore the molecular epidemiology and genetic characteristics of this serovar in Guangdong, China. Methodology We evaluated the molecular epidemiology and genetic characteristics of 294 endemic Salmonella Typhimurium clinical isolates which were collected from 1977 to 2011 in Guangdong, China, and compared them with a global set of isolates of this serovar using epidemiological data and Multilocus Sequence Typing (MLST) analysis. Principal Finding The 294 isolates were assigned to 13 Sequencing types (STs) by MLST, of which ST34 and ST19 were the most common in Guangdong. All the STs were further assigned to two eBurst Groups, eBG1 and eBG138. The eBG1 was the major group endemic in Guangdong. Nucleotide and amino acid variability were comparable for all seven MLST loci. Tajima’s D test suggested positive selection in hisD and thrA genes (p<0.01), but positive selection was rejected for the five other genes (p>0.05). In addition, The Tajima’s D test within each eBG using the global set of isolates showed positive selection in eBG1 and eBG138 (p<0.05), but was rejected in eBG243 (p>0.05). We also analyzed the phylogenetic structure of Salmonella Typhimurium from worldwide sources and found that certain STs are geographically restricted. ACSSuT was the predominant multidrug resistance pattern for this serovar. The resistant profiles ACSSuTTmNaG, ACSSuTTmNa and ACSuTTmNaG seem to be specific for ST34, and ASSuTNa for ST19. Conclusion Here we presented a genotypic characterization of Salmonella Typhimurium isolates using MLST and found two major STs are endemic in Guangdong. Our analyses indicate that genetic selection may have shaped the Salmonella Typhimurium populations. However, further evaluation with additional isolates from various sources will be essential to reveal the scope of the epidemiological characteristics of Salmonella Typhimurium in Guangdong, China.

Inapparent Streptococcus agalactiae infection in adult/commercial tilapia

We report on inapparent infections in adult/commercial tilapia in major tilapia fish farms in Guangdong. A total of 146 suspected isolates were confirmed to be S. agalactiae using an API 20 Strep system and specific PCR amplification. All isolates were identified as serotype Ia using multiplex serotyping PCR. An MLST assay showed single alleles of adhP (10), atr (2), glcK (2), glnA (1), pheS (1), sdhA (3) and tkt (2), and this profile was designated ‘unique ST 7’. The analysis of virulence genes resulted in 10 clusters, of which dltr-bca-sodA-spb1-cfb-bac (62, 42.47%) was the predominant virulence gene profile. The PFGE analysis of S. agalactiae yielded 6 distinct PFGE types (A, B, C, D, F and G), of which Pattern C (103) was the predominant type, accounting for approximately 70.55% (103/146) of the total S. agalactiae strains. Therefore, unlike what has been found in juvenile tilapia, in which PFGE pattern D/F is the major prevalent pattern, we found that pattern C was the major prevalent pattern in inapparent infected adult/commercial tilapia in Guangdong, China. In conclusion, we close a gap in the current understanding of S. agalactiae epidemiology and propose that researchers should be alert for inapparent S. agalactiae infections in adult/commercial tilapia to prevent a potential threat to food safety.

Returning ex-patriot Chinese to Guangdong, China, increase the risk for local transmission of Zika virus

OBJECTIVES Fast expansion and linkage to microcephaly and Guillain Barre syndrome have made Zika virus (ZIKV) track attention of global health authority concerns. The epidemiology, virological characteristics and genetic evolution of introduced ZIKV to Guangdong, China, were investigated. METHODS Analyses of the epidemiological characteristics and genetic diversity of ZIKV isolates were performed. RESULTS A total of twenty-eight confirmed ZIKV infection cases were imported into China in 2016, of which 19 were imported into Guangdong, China from Venezuela (16), the Samoa Islands (1), Suriname (1) and Guatemala (1). Serial sampling studies of the cases indicated longer shedding times of ZIKV particles from urine and saliva samples than from serum and conjunctiva swab samples. Seven ZIKV strains were successfully isolated from serum, urine and conjunctiva swab samples using cell culture and neonatal mouse injection methods. Genomic analysis indicated that all viruses belonged to the Asian lineage but had different evolutionary transmission routes with different geographic origins. The molecular clock phylogenetic analysis of the ZIKV genomes indicated independent local transmission that appeared to have been previously established in Venezuela and Samoa. Additionally, we found 7 unique non-synonymous mutations in the genomes of ZIKV that were imported to China. The mutations may indicate that ZIKV has undergone independent evolutionary history not caused by sudden adaptation to Chinese hosts. CONCLUSION The increasing number of ex-patriot Chinese returning from ZIKV hyper-endemic areas to Guangdong combined with the presence of a variety of Aedes species indicate the potential for autochthonous transmission of ZIKV in Guangdong.

A family cluster of imported ZIKV cases: Viremia period may be longer than previously reported.

Chan et al., in this Journal, recently summarized the unexpected emerging arboviral disease caused by Zika virus infection. Zika virus (ZIKV) was first isolated from ZIKA forest of Uganda in 1947. Until 2007, the first large outbreak of ZIKV was reported in Yap Island, Western Pacific. Later on, outbreaks and autochthonous transmissions of ZIKV were observed in several Pacific island countries and South America. In 2015, ZIKV first attracted global public authorities’ attention due to Guillain Barre syndrome and microcephaly complications. Zika virus disease is described as a mild, self-limiting, febrile illness with low hospitalization rate. Only 20% of Zika cases are symptomatic infections with maculopapular rash, fever, malaise, fatigue, myalgia, arthralgia and conjunctivitis, and the others have remained asymptomatic cases. In this study, we report an imported family cluster of ZIKV infection cases from Venezuela, South America, in Guangdong, China, comprised of three laboratory confirmed cases with clinical symptoms and one laboratory confirmed asymptomatic infection case. The cases were classified according to the diagnostic criteria for Zika virus disease (WS259-2016) of the Ministry of Health of the People’s Republic of China. Key clinical milestones of each patient and their epidemiological/clinical characteristics were summarized in Fig. 1 and Supplementary Table 1, respectively. Case 1, a 6-year-old boy, immigrated to Caracas, Venezuela, and lived with his parents before flying back to China through the USA on Feb 20. On Feb 25, he showed symptoms of maculopapular rash, fever (38 C), conjunctivitis, pharyngitis and adenopathy upon arrival in Baiyun international airport in Guangzhou, China. However, his parents reported that the boy has showed similar symptoms one day before they arrived at Guangzhou, China. The boy was admitted to the Guangzhou 8th People’s Hospital at Guangzhou as a suspected ZIKV infection. The serum and

Low Divergence of Clonorchis sinensis in China Based on Multilocus Analysis

Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb]) was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of C. sinensis.