This study was aimed to explore the features of clustered regularly interspaced short palindromic repeats (CRISPR) structures in Shigella by using bioinformatics. We used bioinformatics methods, including BLAST, alignment and RNA structure prediction, to analyze the CRISPR structures of Shigella genomes. The results showed that the CRISPRs existed in the four groups of Shigella, and the flanking sequences of upstream CRISPRs could be classified into the same group with those of the downstream. We also found some relatively conserved palindromic motifs in the leader sequences. Repeat sequences had the same group with corresponding flanking sequences, and could be classified into two different types by their RNA secondary structures, which contain "stem" and "ring". Some spacers were found to homologize with part sequences of plasmids or phages. The study indicated that there were correlations between repeat sequences and flanking sequences, and the repeats might act as a kind of recognition mechanism to mediate the interaction between foreign genetic elements and Cas proteins.
Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; Genome, Bacterial ; Plasmids ; Shigella ; genetics

Objective To detect the distribution of clustered regularly interspaced short palindromic repeat(CRISPR)associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance. Methods CRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR,with its products sequenced and compared.Results The CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appepared as follows:cas2,cas1 (a) and cas1(b)were 96.44%,97.61%and 96.97%,respectively. There were two mutations including 3177129 site(C→G)and 3177126 site(G→C)of cas1(b)gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113. Results showed that in terms of both susceptibility and antibiotic-resistance,strain 2003135 was stronger than Z23 and 2008113. Conclusion CRISPR system widely existed in Shigella,with the level of drug resistance in cas1(b) gene mutant strains higher than in wild strains. Cas1(b)gene mutation might be one of the reasons causing the different levels of resistance.

Objective:To understand the composition of the pathogen spectrum of hand, foot and mouth disease(HFMD)in Xi′an from 2017 to 2018 and the genetic characteristics of enterovirus 71 (EV-A71).Methods:From 2017 to 2018, 1735 anal swab specimens from HFMD patients in Xi′an HFMD sentinel hospitals were collected, and qPCR was used to identify EV-A71, Coxsackie virus A 16 (CV-A16) and other enteroviruses. Selected EV-A71 strains were isolated, and VP1 region was amplified and sequenced, then phylogenetic tree and homologic comparison were conducted.Results:A total of 1 565 positive samples of enterovirus RNA were detected, positive rate was 90.20%(1565/1735), of which 24.79%(388/1 565)were positive for EV-A71, 21.85%(342/1 565)were positive for CV-A16, and 53.36%(835/1 565)were positive for other enterovirus. The main pathogen in 2017 was EV-A71(45.08%) and in 2018 was non-EV-A71-non-CV-A16 enteroviruses (73.38%), and the difference in pathogen composition was statistically significant ( χ2=370.57, P<0.001). HFMD caused by EV-A71 was concentrated in April to July, accounting for 73.97% (287/388) of the total cases. The homology analysis of the EV-A71 VP1 region showed that the 20 isolated EV-A71 strains belonged to the C4a subtype, with nucleotide and amino acid homology of 94.4%-96.1% and 99.4%-100% respectively, which were separated from those of the previous Xi′an City, the homology of EV-A71 obtained was above 91.5%. The result of the genetic evolution analysis of the EV-A71 VP1 region showed that the EV-A71 strain and the representative strain of the C4a subtype in Xi′an from 2008 to 2018 were in the same branch, forming multiple epidemic clusters, the variation degree of VP1 region increased with time. Conclusions:Non-EV-A71-non-CV-A16 enteroviruses were the main pathogens of HFMD in Xi′an from 2017 to 2018, and C4a subtype EV-A71 is still circulating in Xi′an. The monitoring of the pathogen spectrum and molecular epidemiology of HFMD should be strengthened to provide work for further effective prevention and treatment of HFMD.

Objective:To analyze the etiological and epidemiological characteristics of hand, foot and mouth disease (HFMD) in Xi′an from 2019 to 2021, so as to provide evidence for the prevention and control of HFMD.Methods:Stool specimens and anal swabs were collected from patients with HFMD. Enteroviruses (EVs) including enterovirus 71 (EV71), coxsackievirus A16 (CVA16), CVA6 and CVA10 were detected by RT-PCR. Excel 2007 and SPSS18.0 software were used for data collection and statistical analysis, respectively. The epidemiological data of HFMD cases were analyzed by descriptive epidemiology method. The VP1 gene sequence of the representative strain of each CVA6 genotype was downloaded. Phylogenetic trees were constructed using MEGA X software and the genetic characteristics were analyzed.Results:A total of 1 531 HFMD cases were involved and 1 365 were positive for EVs with a positive rate of 89.16%. The detection rates of EV71, CVA16, CVA6, CVA10 and other EVs were 1.31% (20/1 531), 32.46% (497/1 531), 38.47% (589/1 531), 5.09% (78/1 531) and 11.23% (172/1 531), respectively. There were significant differences in the pathogen composition in HFMD cases of different clinical types (χ 2=46.14, P<0.01) and occupations (χ 2=34.65, P<0.01) as well as in different years (χ 2=462.86, P<0.01). The average age was greater in patients with CVA16 infection than in those with CVA6 or CVA10 infection ( F=6.00, P<0.01). In 2019, the HFMD cases were mainly caused by CVA16, while in 2020 and 2021, the main pathogen was CVA6. Enterovirus-positive cases showed a bimodal distribution with the main peak from May to July and the secondary peak from September to November. CVA16 was the predominant pathogen in spring and summer, and CVA6 was the predominant pathogen in autumn. CVA6 was the dominant pathogen in eight districts and counties of Xi′an; CVA16 was the dominant pathogen in six districts and counties; CVA6 and CVA16 co-circulated in one district. The CVA6 isolates belonged to two evolutionary branches of D3a subtype. Conclusions:CVA6 and CVA16 were the prevalent pathogens of HFMD and CVA6 subtype D3a circulated in Xi′an from 2019 to 2021. The pathogen composition of HFMD cases showed obvious differences in population, time and regional distribution.

Objective:To characterize the epidemiology of hand, foot and mouth disease (HFMD) cases caused by coxsackievirus group A type 6 (CV-A6) in Xi’an from 2016 to 2020 and provide evidence for the prevention and control of HFMD.Methods:The enterovirus (EV) types were identified using real-time RT-PCR. The data of HFMD cases were collected from the National Notifiable Infectious Diseases Reporting System of China Center for Disease Control and Prevention. Descriptive epidemiological method were used to analyze the distributions and the data were statistically analyzed with Excel 2007 and SPSS 18.0.Results:In the 4 034 HFMD cases, 17.85% had enterovirus group A type 71 (EV-A71) infections, 23.92% had coxsackievirus group A type 16 (CV-A16) infections, and 47.79% had other EV infections. 2 571 HFMD cases were randomly selected, including 1 268 other EV positive cases. The detection rate of CV-A6 in HFMD cases was 34.73% (893/2 571), and the constituent ratio of CV-A6 in other EV positive cases was 70.43% (893/1 268). The cases mainly occurred in children aged≤5 years (95.18%), more boys were affected than girls (1.47∶1). HFMD caused by CV-A6 was concentrated in April to June. Compared with EV-A71 and CV-A16, the clinical classification had significant difference in CV-A6 group ( χ2=139.55, P<0.001), but the ratio of sex and age-group had no significant difference ( F=2.74, P=0.065; χ2=2.43, P=0.297). Conclusions:The predominant pathogen of HFMD in Xi’an from 2016 to 2020 were other EV, among which CV-A6 accounted for the highest proportion in other EV positive cases. It is necessary to strengthen the prevention and control of HFMD caused by CV-A6 and carry out the surveillance for various pathogens of HFMD.

Objective:To investigate the positive rates of 2019-nCoV nucleic acid in different specimens from confirmed COVID-19 cases during hospitalization and after discharge.Methods:Patients with confirmed COVID-19 were enrolled from designated hospitals. Nasal swabs, throat swabs, and specimens of stool, urine and blood were collected during hospitalization. After the patients were discharged, nasal swabs, throat swabs and stool specimens were collected during follow-up. Real-time RT-PCR was used to detect 2019-nCoV nucleic acid.Results:This study involved 25 confirmed COVID-19 cases. During hospitalization, all patients tested positive in both nasal and throat swab 2019-nCoV nucleic acid tests, and nine of them (36.00%) were positive in stool specimen test. Urine and blood specimen test results were all negative. Nasal swabs, throat swabs and stool specimens were collected from each patient 7 d and 14 d after discharge. Two patients (8.00%) tested positive for 2019-nCoV nucleic acid again in nasal and throat swab tests on 7 d, while all stool specimen tests were negative. No 2019-nCoV nucleic acid was detected in nasal swabs, throat swabs or stool samples on 14 d.Conclusions:2019-nCoV nucleic acid was detected in stool samples of confirmed COVID-19 cases during hospitalization. Nasal and throat swab nucleic acid tests turned positive again in some patients after discharge.

Objective:To explore the positive rates of 2019-nCoV nucleic acid at different time of courses of COVID-19.Methods:Patients with confirmed COVID-19 were enrolled in this study. Nasal and throat swabs were collected from different courses of disease. Real-time RT-PCR was used to detect 2019-nCoV nucleic acid.Results:From January 23 to February 20, 2020, a total of 120 confirmed cases of COVID-19 were reported in Xi’an, and 85 cases(70.83%) were positive in first detection. The COVID-19 cases included consistently nucleic acid positive and intermittently nucleic acid positive patients. 2019-nCoV nucleic acid could be detected in incubation period, and the longest observed duration of nucleic acid positive in this study was 26 days. The positive rate of 2019-nCoV nucleic acid was up to 84.21% on the 6th day, and the positive rate decreased as time passed during the course of COVID-19. Three patients (2.86%) were tested positive for 2019-nCoV nucleic acid again in nasal and throat swabs after discharge.Conclusions:The positive rate of 2019-nCoV nucleic acid was higher in the early stage of disease. 2019-nCoV nucleic acid can be detected in incubation period, and virus shedding may last for a long period.

OBJECTIVETo detect the distribution of clustered regularly interspaced short palindromic repeat (CRISPR) associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance.

METHODSCRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR, with its products sequenced and compared.

RESULTSThe CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appeared as follows: cas2, cas1 (a) and cas1 (b) were 96.44%, 97.61% and 96.97%, respectively. There were two mutations including 3177129 site(C→G)and 3177126 site (G→C) of cas1 (b) gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113.

RESULTSshowed that in terms of both susceptibility and antibiotic-resistance, strain 2003135 was stronger than Z23 and 2008113.

CONCLUSIONCRISPR system widely existed in Shigella, with the level of drug resistance in cas1 (b) gene mutant strains higher than in wild strains. Cas1 (b) gene mutation might be one of the reasons causing the different levels of resistance.

Bacterial Proteins ; genetics ; CRISPR-Associated Proteins ; genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Bacterial ; genetics ; Mutation ; Shigella ; genetics

Objective: To understand the etiological and clinical characteristics of children with severe hand, foot and mouth disease (HFMD) in Xi′an in 2018, and to provide the evidence for clinical diagnosis and treatment. Methods: The children with severe HFMD admitted at Xi′an Children′s Hospital from January to December 2018 were selected as the research objects.Clinical data were collected, and the anal swab were detected by adopting real time(RT)-polymerase chain reaction(PCR). Results: Ninety-five cases of HFMD were treated in Xi′an Children′s Hospital in 2018, of which 92 cases were severe and 3 cases were critical.Eighty-seven cases were positive for enterovirus nucleic acid, 30 cases were enterovirus 71(EV71)(31.6%), 39 cases were coxsackievirus A6(CA6) (41.0%), 3 cases were CA16(3.2%), 2 cases were CA10(2.1%) and 13 cases were other enteroviruses (13.7%). Among 95 patients, the ratio of male to female was 1.1∶1.0; the peak period of incidence of HFMD was from May to July, and the main age of onset of severe HFMD was under 3 years old.The main clinical manifestations were mental retardation, vomiting, irritability, lethargy and convulsion.Severe cases of CA6 are prone to convulsion.The main form of rash in CA6 cases was bullous rash, and demethylation may occur in recovery period.The rash in EV71 cases was small, thick, hard and few.After active treatment, only one child with EV71 infection died because of severe cerebral dysfunction, frequent convulsions and neurogenic pulmonary edema.The other child was discharged with hemiplegia and language dysfunction.The other severe children were cured and discharged from hospital. Conclusions In 2018, CA6 was the main pathogen of severe HFMD in Xi′an, with bullae was the main manifestation of skin rash, and nail removal could occur during convalescence.Critical and death cases were still caused by EV71.