Etiological and epidemiological features of acute meningitis or encephalitis in China: a nationwide active surveillance study.

BACKGROUND: Acute meningitis or encephalitis (AME) results from a neurological infection causing high case fatality and severe sequelae. AME lacked comprehensive surveillance in China. METHODS: Nation-wide surveillance of all-age patients with AME syndromes was conducted in 144 sentinel hospitals of 29 provinces in China. Eleven AME-causative viral and bacterial pathogens were tested with multiple diagnostic methods. FINDINGS: Between 2009 and 2018, 20,454 AME patients were recruited for tests. Based on 9,079 patients with all-four-virus tested, 28.43% (95% CI: 27.50%‒29.36%) of them had at least one virus-positive detection. Enterovirus was the most frequently determined virus in children <18 years, herpes simplex virus and Japanese encephalitis virus were the most frequently determined in 18-59 and ≥60 years age groups, respectively. Based on 6,802 patients with all-seven-bacteria tested, 4.43% (95% CI: 3.94%‒4.91%) had at least one bacteria-positive detection, Streptococcus pneumoniae and Neisseria meningitidis were the leading bacterium in children aged <5 years and 5-17 years, respectively. Staphylococcus aureus was the most frequently detected in adults aged 18-59 and ≥60 years. The pathogen spectrum also differed statistically significantly between northern and southern China. Joinpoint analysis revealed age-specific positive rates, with enterovirus, herpes simplex virus and mumps virus peaking at 3-6 years old, while Japanese encephalitis virus peaked in the ≥60 years old. As age increased, the positive rate for Streptococcus pneumoniae and Escherichia coli statistically significantly decreased, while for Staphylococcus aureus and Streptococcus suis it increased. INTERPRETATION: The current findings allow enhanced identification of the predominant AME-related pathogen candidates for diagnosis in clinical practice and more targeted application of prevention and control measures in China, and a possible reassessment of vaccination strategy. FUNDING: China Mega-Project on Infectious Disease Prevention and the National Natural Science Funds.

Etiological and epidemiological features of acute respiratory infections in China.

Nationwide prospective surveillance of all-age patients with acute respiratory infections was conducted in China between 2009‒2019. Here we report the etiological and epidemiological features of the 231,107 eligible patients enrolled in this analysis. Children <5 years old and school-age children have the highest viral positivity rate (46.9%) and bacterial positivity rate (30.9%). Influenza virus, respiratory syncytial virus and human rhinovirus are the three leading viral pathogens with proportions of 28.5%, 16.8% and 16.7%, and Streptococcus pneumoniae, Mycoplasma pneumoniae and Klebsiella pneumoniae are the three leading bacterial pathogens (29.9%, 18.6% and 15.8%). Negative interactions between viruses and positive interactions between viral and bacterial pathogens are common. A Join-Point analysis reveals the age-specific positivity rate and how this varied for individual pathogens. These data indicate that differential priorities for diagnosis, prevention and control should be highlighted in terms of acute respiratory tract infection patients' demography, geographic locations and season of illness in China.

Etiological, epidemiological, and clinical features of acute diarrhea in China.

National-based prospective surveillance of all-age patients with acute diarrhea was conducted in China between 2009‒2018. Here we report the etiological, epidemiological, and clinical features of the 152,792 eligible patients enrolled in this analysis. Rotavirus A and norovirus are the two leading viral pathogens detected in the patients, followed by adenovirus and astrovirus. Diarrheagenic Escherichia coli and nontyphoidal Salmonella are the two leading bacterial pathogens, followed by Shigella and Vibrio parahaemolyticus. Patients aged <5 years had higher overall positive rate of viral pathogens, while bacterial pathogens were more common in patients aged 18‒45 years. A joinpoint analysis revealed the age-specific positivity rate and how this varied for individual pathogens. Our findings fill crucial gaps of how the distributions of enteropathogens change across China in patients with diarrhea. This allows enhanced identification of the predominant diarrheal pathogen candidates for diagnosis in clinical practice and more targeted application of prevention and control measures.

Combined interventions for mitigation of an influenza A (H1N1) 2009 outbreak in a physical training camp in Beijing, China.

OBJECTIVES: Many studies have suggested the effectiveness of single control measures in the containment and mitigation of pandemic influenza A (H1N1) 2009. The effects of combined interventions by multiple control measures in reducing the impact of an influenza A (H1N1) 2009 outbreak in a closed physical training camp in Beijing, China were evaluated. METHODS: Oseltamivir was prescribed for the treatment of confirmed cases and possible cases and as prophylaxis for all other participants in this training camp. Public health control measures were applied simultaneously, including the isolation of patients and possible cases, personal protection and hygiene, and social distancing measures. Symptom surveillance of all participants was initiated, and the actual attack rate was calculated. For comparison, the theoretical attack rate for this outbreak was projected using the Newton-Raphson numerical method. RESULTS: A total of 3256 persons were present at the physical training camp. During the outbreak, 405 (68.3%) possible cases and 26 (4.4%) confirmed cases were reported before the intervention and completed oseltamivir treatment; 162 (27.3%) possible cases were reported after the intervention and received part treatment and part prophylaxis. The other 2663 participants completed oseltamivir prophylaxis. Of the possible cases, 181 with fever ≥38.5°C were isolated. The actual attack rate for this outbreak of pandemic influenza A (H1N1) 2009 was 18.2%, which is much lower than the theoretical attack rate of 80% projected. CONCLUSIONS: Combined interventions of large-scale antiviral ring prophylaxis and treatment and public health control measures could be applied to reduce the magnitude of influenza A (H1N1) 2009 outbreaks in closed settings.

Modeling the transmission dynamics of Ebola virus disease in Liberia.

Ebola virus disease (EVD) has erupted many times in some zones since it was first found in 1976. The 2014 EVD outbreak in West Africa is the largest ever, which has caused a large number of deaths and the most serious country is Liberia during the outbreak period. Based on the data released by World Health Organization and the actual transmission situations, we investigate the impact of different transmission routes on the EVD outbreak in Liberia and estimate the basic reproduction number R0 = 2.012 in the absence of effective control measures. Through sensitivity and uncertainty analysis, we reveal that the transmission coefficients of suspected and probable cases have stronger correlations on the basic reproduction number. Furthermore, we study the influence of control measures (isolation and safe burial measures) on EVD outbreak. It is found that if combined control measures are taken, the basic reproduction number will be less than one and thus EVD in Liberia may be well contained. The obtained results may provide new guidance to prevent and control the spread of disease.

Association between hemorrhagic fever with renal syndrome epidemic and climate factors in Heilongjiang Province, China.

The purpose of this study was to quantify the relationship between climate variation and transmission of hemorrhagic fever with renal syndrome (HFRS) in Heilongjiang Province, a highly endemic area for HFRS in China. Monthly notified HFRS cases and climatic data for 2001-2009 in Heilongjiang Province were collected. Using a seasonal autoregressive integrated moving average model, we found that relative humidity with a one-month lag (ß = -0.010, P = 0.003) and a three-month lag (ß = 0.008, P = 0.003), maximum temperature with a two-month lag (ß = 0.082, P = 0.028), and southern oscillation index with a two-month lag (ß = -0.048, P = 0.019) were significantly associated with HFRS transmission. Our study also showed that predicted values expected under the seasonal autoregressive integrated moving average model were highly consistent with observed values (Adjusted R(2) = 83%, root mean squared error = 108). Thus, findings may help add to the knowledge gap of the role of climate factors in HFRS transmission in China and also assist national local health authorities in the development/refinement of a better strategy to prevent HFRS transmission.

Spatiotemporal patterns of Japanese encephalitis in China, 2002-2010.

OBJECTIVE: The aim of the study is to examine the spatiotemporal pattern of Japanese Encephalitis (JE) in mainland China during 2002-2010. Specific objectives of the study were to quantify the temporal variation in incidence of JE cases, to determine if clustering of JE cases exists, to detect high risk spatiotemporal clusters of JE cases and to provide evidence-based preventive suggestions to relevant stakeholders. METHODS: Monthly JE cases at the county level in mainland China during 2002-2010 were obtained from the China Information System for Diseases Control and Prevention (CISDCP). For the purpose of the analysis, JE case counts for nine years were aggregated into four temporal periods (2002; 2003-2005; 2006; and 2007-2010). Local Indicators of Spatial Association and spatial scan statistics were performed to detect and evaluate local high risk space-time clusters. RESULTS: JE incidence showed a decreasing trend from 2002 to 2005 but peaked in 2006, then fluctuated over the study period. Spatial cluster analysis detected high value clusters, mainly located in Southwestern China. Similarly, we identified a primary spatiotemporal cluster of JE in Southwestern China between July and August, with the geographical range of JE transmission increasing over the past years. CONCLUSION: JE in China is geographically clustered and its spatial extent dynamically changed during the last nine years in mainland China. This indicates that risk factors for JE infection are likely to be spatially heterogeneous. The results may assist national and local health authorities in the development/refinement of a better preventive strategy and increase the effectiveness of public health interventions against JE transmission.

[Expression profile analysis of host HeLa cells invasived by Shigella flexneri 2a].

The changes of genes expression in HeLa cell during the invasion with Shigella species for 1h and 3h were analyzed by cDNA microarrays. The data showed that the expression levels of 752 genes were altered twice or greater as compared with the control 509 of them were up-regulated, and 306 were down-regulated. It was supposed that some signal pathways in HeLa cell were activated, then many genes were induced, and at last comprehensive cell responses were produced, so that HeLa cell could prevent against Shigella species infection. Two striking difference cDNA fragments TNFR 1B and ERBB2, which were up-regulated in the host epithelial cell during Shigella infection, analyzed expression by real time RT-PCR as determined by cDNA arrays. We suggested they play important roles in response to the invasive S. flexneri 2457T. Based on the results of gene expression alterations, the molecular pathogenic mechanism of Shigella species could be greatly and deeply understood, and the strategy for prevention against and treatment for shigellosis would be formed.

[Screen in vivo induced gene of Mycobacterium tuberculosis by IVIAT].

In order to search new candidates of pharmaceutical target, in vivo induced antigen technology (IVIAT) was used to screen in vivo induced (ivi) genes of Mycobacterium tuberculosis (M. TB). Genomic expression library of M. TB was first constructed with an inducible plasmid pKK223-8; the titer of the library was 1.02 x 10(5) CFU. Sera from ten tuberculosis patients were pooled and absorbed against in vitro-grown M. TB and Escherichia coli, and used to probe the genomic expression library. 16 positive clones were identified by immunological screen, including 22 ORF: two encoding lipid metabolism proteins, five information pathways proteins, two PE/PPE proteins, six intermediary metabolism and respiration proteins, one cell wall and cell processes protein, four conserved hypothetical proteins and two conserved hypothetical proteins with an orthologue in Mycobacterium bovis. Parts of these genes can be used as candidates of pharmaceutical target because they may be relate with virulence.

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T.

AIM: To screen the immunogenic membrane proteins of Shigella flexneri 2a 2457T. METHODS: The routine two-dimensional polyacrylamide gel electrophoresis (2-DE) and Western blotting were combined to screen immunogenic proteins of S. flexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was performed to determine the molecular weight of peptides. Electrospray ionization (ESI-MS/MS) was performed to determine the sequences of the interesting peptides. RESULTS: A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein (YaeT) was detected, which might be a candidate target of vaccine. CONCLUSION: Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.

[Identification of a protein interacting with Shigella flexneri IpaC invasin by yeast two-hybrid system].

Two-hybrid system was applied to screen proteins interacting with IpaC in the host cell. By using two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKT-IpaC. A human HeLa cDNA library was screened to isolate protein factors that might interact with IpaC. Among the 2 x 10(6) clones screened, 22 positive clones were picked out. Sequence analysis revealed that two of them contained cDNA fragments from collagenase. Subsequently the domain of IpaC interacting with collagenase fragment was identified. These results suggest that IpaC might play a role in some biological processes where collagenase is involved.

[Knockout of the ptsG gene in Escherichia coli and cultural characterization of the mutants].

Metabolic engineering provide powerful tools for the systematic manipulation of cellular metabolic activities. The ptsG gene for glucose-specific transporter Enzyme II CBGlc of the phosphotransferase system was knock-out so as to reduce the accumulation of acetic acid in the high cell-density culture of Escherichia coli on excess glucose. The chloramphenicol-resistant cassette with short shared sequences on both ends generated by PCR was electroporated into Escherichia coli DH5alpha and JM109. Recombination between linear DNA cassettes and Escherichia coli chromosomes took place by Red recombinase functions. Therefore, the ptsG gene was disrupted to construct the mutants called DH5alphaP and JM109P. There was no difference between the mutants and parent strains in LB media.However, in LB media supplemented with glucose, the mutants of Escherichia coli deficient in ptsG showed greater biomass, together with exploiting more glucose. The maximal cell density obtained with DH5alphaP was approximately 3 times more than that of DH5alpha, then the result of JM109P increased fourfold. The products of recombinant protein TNF respectively accounted for 24.3% of total cellular protein in DH5alphaP with A600 8.28 and 20.8% of total cellular protein in JM109P with A600 7.62. The specific volume expression amount of TNF was greater in the ptsG mutant than in its parent strain. These results demonstrate that the ptsG-mutant strains will be available for high cell-density culture.

[Expression of hydantoin hydrolase gene in Escherichia coli].

Hydantoin hydrolase with responsibility for the ring opening of hydantoin is one of the components of hydantoin utility enzymes of Arthrobacter BT801 which can convert 5-benzylhydantoin into L-phenylalanine. The expression of hydantoin hydrolase gene (hyuH) is very important in elucidation of mechanisms of bio-catalysis and its application in asymmetry synthesis of amino acids. To improve the production and activity of the enzyme, the hydantoin hydrolase gene was amplified by PCR and cloned into E. coli by using vector pT221. The hydantoin hydrolase gene was highly expressed in E. coli BL21 (DE3) under the control of T7 promoter. A protein band about 50kD was detected by SDS-PAGE in the recombinant cell lysate. The objective protein in BL21 (DE3)/pT221-hyuH accounted for 40% of total cellular protein, mostly in soluble form. The products in the recombinant strain showed biological activity.

[Construction of SW480 cell model identifying Shigella virulent genes].

Signature-tagged mutagenesis (STM) is a novel technology with high throughput screening ability to identify virulent genes of pathogen in vivo. An appropriate animal or cell line model is one of prerequisites by exploiting this technique. In order to apply STM to Shigella flexneri, RC426 was constructed as an attenuated mutant with chloramphenicol resistance and aroA and virG genes inactivated by homologous recombination; another attenuated strain T32 was used as an oral S. flexneri 2a vaccine due to a spontaneous deletion in three loci (ipaBCDA, invA and virG) on the virulence plasmid. The wild type strain 2457T had the invasion ability into host cells. The three strains, RC426, T32 and 2457T, were mixed together to invade colon cancer cell line SW480, and the distinct strains were recovered and counted from cell lysates of invaded SW480 in different time. The results showed that there were statistically significant differences between the amounts of two attenuated strains recovered and that of virulent strain within 12h invasion, indicating SW480 was a suitable cell model for applying STM to screen virulent genes of Shigella flexneri.

Identification of alkA gene related to virulence of Shigella flexneri 2a by mutational analysis.

AIM: In vivo induced genes are thought to play an important role during infection of host. AlkA was identified as an in vivo-induced gene by in vivo expression technology (IVET), but its virulence in Shigella flexneri was not reported. The purpose of this study was to identify the role of alkA gene in the pathogenesis of S. flexneri. METHODS: PCR was used to amplify alkA gene of S. flexneri 2a and fragment 028pKm. The fragment was then transformed into 2457T05 strain, a S flexneri 2a strain containing Red recombination system, which was constructed with a recombinant suicide plasmid pXLkd46. By in vivo homologous recombination, alkA mutants were obtained and verified by PCR and sequencing. Intracellular survival assay and virulence assay were used to test the intracellular survival ability in HeLa cell model and the virulence in mice lung infection model respectively. RESULTS: Deletion mutant of S. flexneri 2a alkA was successfully constructed by gamma Red recombination system. The mutant exhibited significant survival defects and much significant virulence defects in mice infection assay. CONCLUSION: AlkA gene plays an important role in the infection of epithelial cells and is a virulent gene of Shigella spp.

Identification of RanBMP interacting with Shigella flexneri IpaC invasin by two-hybrid system of yeast.

AIM: Bacillary dysentery caused by Shigella flexneri is still a threat to human health. Of four invasion plasmid antigen proteins (IpaA,B,C and D), IpaC plays an important role in the pathogenicity of this pathogen. The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease. METHODS: By applying two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKT-ipaC. The bait plasmid was transformed AH109, and proved to express IpaC and then HeLa cDNA library plasmids were introduced into the above transformed AH109. The transformation mixture was plated on medium lacking Trp, Leu, and His in the initial screen, then restreaked on medium lacking Trp, Leu, His and Ade. Colonies growing on the selection medium were further assayed for beta-galactosidase activity. BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid. RESULTS: Among the 2X10(6) transformants, 64 positive clones were obtained as determined by activation of His, Ade and LacZ reporter genes. Sequence analysis revealed that cDNA inserts of two colonies were highly homologous to a known human protein, RanBPM. CONCLUSION: These results provide evidence that IpaC may be involved in the invasion process of S. flexneri by interacting with RanBPM, and RanBPM is most likely to be the downstream target of IpaC in the cascade events of S. flexneri infection.

[The red recombination system and its application to gene knock-out in microorganism].

Since many DNA-sequencing projects of varied microorganisms have been completed,studies on their functional genomics become more important. Inactivation of an interesting gene is a direct method to characterize its function. Though the Escherichia coli RecA recombination system can be used to produce gene mutants,it needs a complex manipulation process. Furthermore, its efficiency is very low. Recently a Red recombination system was developed. This recombination system consists of three proteins:alpha protein (gamma exonuclease), beta protein and Gam protein. In this system, the linear targeting DNA which contains a selectable marker flanked with a homologous region as short as only 35 approximately 60 bp can be directly targeted for gene knock-out with a higher efficiency.

[Cloning and expression of L-N-carbamoylase gene from Arthrobacter BT801 in Escherichia coli].

Hydantoin-utility-enzyme is widely used in enzymic production of various amino acids. One of its component, carbamoylase, is responsible for the conversion of N-carbamylamino acids to corresponding amino acids, which is crucial for the stereoselectivity and rate limiting. To improve the production of the enzyme, an L-N-carbamoylase gene from Arthrobacter BT801, a hydantoinase producting strain being able to convert 5-benzylhydantoin to phenylalanine, was cloned into E. coli. The gene was highly expressed in E. coli M15 under control of T5 promoter. A protein band about 44kD was detected by SDS-PAGE in the recombinant cell lysate. The objective product, which is principally in soluble form, represented 40% of total cell protein. The N-carbamoylase specific activity of the recombinant M15/pQE60- hyuC is 53 times higher than that of Arthrobacter BT801. The total biotransformation activity increased 8.1 times when. M15/pQE60-hyuC was added into the Arthrobacter BT801 reaction system. The successful expression of the enzyme is significant for the application of the hydantoinase producing strain or the enzyme thereof.

[Construction of gene library of Arthrobacter BT801 and isolation & expression of hydantoinase gene].

Hydantoinase can be widely used in enzymic production of various amino acids. In order to obtain the hydantoinase genes in Arthrobacter BT801, its chromatosomal DNA is isolated and partialy digested with Sau3A I to collect fragments of about 30kb. Then, this fragment is inserted into the Hpa I and Pst I site of cosmid pKC505. The genomic library was thus constructed by packing in vitro with lambda phage package protein and transfecting E. coli DH5alpha. A positive transformant was selected from the library using thin layer chromatography and other methods. A DNA fragment containing complete hydantoinase genes was sequenced by sub-cloning into pUC18. The gene can express active protein under control of its own promoter and T5 promoter in E. coli. The isolation of the gene established foundition for research and application of the hydantoinase.