Genomic Evolution and Variation of SARS-CoV-2 in the Early Phase of COVID-19 Pandemic in Guangdong Province, China.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) with unknown origin spread rapidly to 222 countries, areas or territories. To investigate the genomic evolution and variation in the early phase of COVID-19 pandemic in Guangdong, 60 specimens of SARS-CoV-2 were used to perform whole genome sequencing, and genomics, amino acid variation and Spike protein structure modeling analyses. Phylogenetic analysis suggested that the early variation in the SARS-CoV-2 genome was still intra-species, with no evolution to other coronaviruses. There were one to seven nucleotide variations (SNVs) in each genome and all SNVs were distributed in various fragments of the genome. The Spike protein bound with human receptor, an amino acid salt bridge and a potential furin cleavage site were found in the SARS-CoV-2 using molecular modeling. Our study clarified the characteristics of SARS-CoV-2 genomic evolution, variation and Spike protein structure in the early phase of local cases in Guangdong, which provided reference for generating prevention and control strategies and tracing the source of new outbreaks.

Low-dose cadmium exposure acts on rat mesenchymal stem cells via RANKL/OPG and downregulate osteogenic differentiation genes.

Chronic cadmium (Cd) toxicity is a significant health concern, and the mechanism of long-term low-dose Cd exposure on bone has not been fully elucidated till date. This study aimed to assess the association between rat mesenchymal stem cells (MSCs) and long-term Cd exposure through 38-week intake of CdCl2 at 1 and 2 mg/kg body weight (bw). Increased gene expression of receptor activator of NF-κB ligand (RANKL) and decreased gene expression of osteoprotegerin (OPG) were observed. Fold change of RANKL gene expression (fold change = 1.97) and OPG gene expression (fold change = 1.72) showed statistically significant differences at dose 2 mg/kg bw. Decreased expression of key genes was observed during the early osteogenic differentiation of MSCs. The gene expression of Osterix in 1 mg/kg bw group was decreased by 3.70-fold, and the gene expressions of Osterix, Osteopontin, collagen type I alpha 2 chain (COL1a2) and runt-related transcription factor 2 (RUNX2) in 2 mg/kg bw group were decreased by 1.79, 1.67, 1.45 and 1.35-folds, respectively. Exposure to CdCl2 induced an increase in the renal Cd load, but only an adaptive response was observed, including increased expression of autophagy-related proteins LC3B and Beclin-1, autophagy receptor p62, and heme oxygenase 1 (HO-1), which is an inducible isoform that releases in response to stress. There were no significant changes in the urinary low molecular weight proteins including N-acetyl-b-D-glucosaminidase (NAG), ß2-microglobulin and albumin (U-Alb). Urinary calcium (Ca) excretion showed no increase, and no obvious renal histological changes. Taken together, these results indicated that the chronic CdCl2 exposure directly act on MSCs through RANKL/OPG pathway and downregulate the key genes involved in osteogenic differentiation of MSCs. The toxic effect of Cd on bone may occur in parallel to nephrotoxicity.

[Etiologic characteristics of Vibrio parahaemolyticus strains causing outbreaks and sporadic cases in Guangdong, 2009].

OBJECTIVE: To study the serotypes, virulence features and molecular characteristics of Vibrio parahaemolyticus isolated in food poisoning cases and surveillance program on diarrhea patients in Guangdong, 2009. METHODS: 95 Vibrio parahaemolyticus strains from food poisoning cases and 15 strains from surveillance program on diarrhea patients were serotyped and detected for tdh (thermostable direct hemolysin, tdh) and trh (tdh-related hemolysin gene, trh) by PCR. 81 sero-variant Vibrio parahaemolyticus strains were selected through PFGE subtyping. RESULTS: There were 15 Vibrio parahaemolyticus strains isolated from surveillance program on diarrhea patients and 95 strains were isolated from 11 Vibrio parahaemolyticus-caused food poisoning cases in 2009. Among these strains, O3:K6 (46.67% and 44.21%) and O4:K8 (33.33% and 28.42%) were the dominant serotypes, but not the 7 food-borne strains. There were 93 (84.54%) tdh(+)trh(-), 13 (11.81%) tdh(-)trh(-), and 3 (3.65%) tdh(+)trh(+) strains. The similarity value was between 57.7% to 100.0% of the 81 strains after PFGE sub-typing method and 36 PFGE subtypes were identified. PFGE001 and PFGE029 appeared to be the dominant subtypes. CONCLUSION: O3:K6 and O4:K8 were the most dominant serotypes in Vibrio parahaemolyticus-caused diarrhea and food poisoning cases in Guangdong and tdh were detected in most of the strains. Dominant PFGE subtypes were causing both sporadic and outbreak cases in different areas in Guangdong province.

[Surveillance program on and the distribution related to the virulence-associated genes of Vibrio cholerae in estuary of Pearl River].

OBJECTIVE: To understand the distribution, molecular characteristics and virulence genes of the O1 and O139 Vibrio cholerae isolates from the Pearl River Estuary water. METHODS: Vibrio cholerae isolates collected from the Pearl River estuary waters from January 2009 to December 2010, were tested by PCR for eight virulence-related genes, including cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and the regulatory protein genes (tcpI, toxR). Genetic relation was assessed by pulsed-field gel electrophoresis (PFGE) and the patterns were clustered by BioNumerics. RESULTS: From 1152 aquatic samples, 69 isolates were identified, including 41 Inaba, 18 Ogawa and 10 O139. All the isolates showed ctxA negative, while the hlyA and toxR genes were positive in all the isolates. 34.15% (14/41) of the Inaba strains were hlyA(+) toxR(+) ompU(+) ace(+) zot(+) tcpI(+), while 66.67% (12/18) belonged to Ogawa strains and 70% (7/10) of the O139 strains were hlyA(+) toxR(+). Through PFGE analysis, the O1 isolates formed three clusters in this study. The patterns of O1 isolates differed widely, with the similarity as 72.8% - 100.0%, while the patterns of O139 isolates having the similarity of 69.9% - 95.5%. CONCLUSION: The non-toxigenic O1 and O139 V. cholerae had a wide distribution in the environment of Pearl River estuary water during the non-epidemic period of cholera. All the aquatic isolates presented diversities on the related virulent genes.

[Comparative study on the phenotypic characteristics and molecular typing of foodborne Vibrio parahaemolyticus in Guangdong province].

OBJECTIVE: To understand the phenotypic characteristics of foodborne Vibrio parahaemolyticus in Guangdong province through carrying out a comprehensive comparison including pulse field gel electrophoresis, ribotyping and serotyping. METHODS: 74 different Vibrio parahaemolyticus strains isolated from seafood and cases due to food poisoning in Guangdong province were under serotyping and susceptibility testing, in addition to the testing of direct heat hemolysin (tdh) and the heat hemolysin-related hemolysin hormone (trh) via PCR. Ribosomal genotyping (ribotyping) with EcoR I restriction enzyme was utilized on 74 different Vibrio parahaemolyticus isolates, whereas pulsed-field gel electrophoresis (PFGE) with the Not I restriction enzyme was used on 74 different Vibrio parahaemolyticus isolates. BioNumerics software was used to compare the isolates from different sources, times and places in order to elicit the correlation between different strains. RESULTS: Although Vibrio parahaemolyticus was 100.00% sensitive to chloramphenicol, it still presented different levels of resistance against 13 other antibiotics. Among the 74 different strains of Vibrio parahaemolyticus under testing, 24.32% showed positive for the tdh virulence gene, whereas 4.05% positive for trh. 74 different Vibrio parahaemolyticus strains were found to belong to 26 serotypes, where the O5:K17 and O2:K28 serotypes were dominant in those isolates that causing seafood-poisoning. The O3:K6 serotype was found to be the dominant of those isolates that causing food-poisoning. Based on ribosomal genotyping, the 74 Vibrio parahaemolyticus isolates were divided into 62 different ribotypes, whereas the 74 strains of Vibrio parahaemolyticus were divided into 67 different PFGE types, thus exhibiting considerable genetic diversities of the strains. CONCLUSION: Majority of the isolates causing food-poisoning carried tdh virulence gene. PFGE was shown to have the highest resolution, followed by ribotyping with serotyping being the lowest, where the combination of the three could improve the resolution.

Phenotypic and genotypic characterization Vibrio cholerae O139 of clinical and aquatic isolates in China.

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.

[Distribution and molecular characteristics of Vibrio cholerae serogroups O1 and O139 isolates in estuary of Pearl River].

OBJECTIVE: Through systematic monitoring of the number and strain types of O1 and O139 Vibrio cholerae in the Pearl River estuary waters to analyze it's relevance with the temperature of environment, and the relevance between strains in water and isolates during outbreaks and epidemics as well as to estimate the methods used for environmental water detection and the potential role in cholera surveillance program. METHODS: Twenty-four stations along the Pearl River were selected and the water samples were collected monthly from March 2006 to February 2007. V. cholerae O1 and O139 strains were isolated from the samples. Real-time PCR established in our laboratory was used to detect V. cholerae O1 and O139. Air temperature and water temperature were collected during sampling. Pulsed field gel electrophoresis (PFGE) was applied in molecular typing of the isolates. RESULTS: 862 water samples were collected during the study period. A total number of 77 O1 and O139 V. cholerae were isolated in 67 water samples and the positive rates were 7.77% for isolation and 26.33% for real-time PCR. Seasonal trend of positive rates by month were approximately coincident with the change of water temperature. The positive rates in the stations in urban area were higher than those in other areas. Toxigenic O139 strains were found in one station located in downstream of a marine market. Most of the O1 and O139 isolates were non-toxigenic. No trend of seasonal variation of the strains was noticed. Within these 75 isolates, 49 PFGE patterns were identified and the patterns differed widely with the similarity of 57.4% - 100%. CONCLUSION: V. cholerae existed as the natural habitat in estuary water of the Pearl River and showed obvious genetic diversity. Data from monitoring waters might show the separation of strains with certain seasonal association. But the crowd did not show the relationship between the infections. Results from water surveillance program might provide indicators on the appearance of cholera pathogen which might be used in assessing the environmental risk of cholera epidemics as well as the alert of cholera.

[Development and application of real-time polymerase chain reaction to detect Vibrio cholerae O1 and O139 in river water].

OBJECTIVE: To develop a real-time SYBR Green polymerase chain reaction (PCR) for detection of Vibrio cholerae serogroups O1 and O139, and to evaluate its reliability through detection of estuary water samples. METHODS: O antigen rfb genes specific for O1 and O139 were used for the design of PCR primers. The real-time SYBR Green PCR system in detecting O1 and O139 specific rfb genes in one tube was developed, and its sensitivity, specificity and reproducibility were evaluated. The ability of the real-time PCR in detection of estuary water samples was compared with the routine PCR and bacteria isolation. RESULTS: The amplification of O1 or O139 specific target gene could be detected according to the melt curve temperature of amplicons. No amplification was observed in the templates of other 10 non-cholerae vibrios. When comparing to the real-time PCR to bacteria isolation in detection of 524 estuary water samples, it showed high sensitivity, plus also positive in real-time PCR detection among all the samples in which bacteria of O1 or O139 were isolated. CONCLUSION: The real-time SYBR Green PCR could be used as the first step of rapid environment screen of V. cholerae in water samples thus might enhance the efficiency of isolation in screening of large amount of water samples.