Investigation of adenovirus infection in hospitalized children with diarrhea during 2010 in Beijing, China / 中华儿科杂志

<p><b>OBJECTIVE</b>The study was designed to evaluate adenovirus infection in hospitalized children with diarrhea.</p><p><b>METHOD</b>Stool specimens were collected from 519 hospitalized children with diarrhea during 2010, including those defined as community-acquired diarrhea (CAD) who developed diarrhea symptoms within 48 hours after admission, and those defined as hospital-acquired diarrhea (HAD) whose symptoms of diarrhea occurred beyond 48 hours after admission. PCR was employed to identify adenovirus in fecal samples by using universal primers for adenoviruses of all types, and specific primers for adenovirus group F. PCR products with expected size were sequenced for adenovirus typing. Clinical data for children with adenovirus positive specimens were analyzed.</p><p><b>RESULT</b>A total of 519 hospitalized children, including 289 with CAD and 230 with HAD, were enrolled in the study. Out of 519 stool specimens, 76 showed PCR products with expected 301 bp and identified as adenovirus by sequencing, and the overall positive rate was 14.6%. Out of 289 CAD samples, 43 were positive (positive rate was 14.9%). Of them, 20 were identified as enteric adenovirus infection (adenovirus type 41, Ad41). Thirty-three out of 230 HAD samples were positive (positive rate was 14.3%). Of them, 13 were characterized as enteric adenovirus infection (one was Ad40 and others were Ad41). Ad41 in this study could be divided into two genotypes by phylogenetic tree analysis. Non-enteric adenoviruses were identified in 43 specimens (43/76, 56.6%) including 5 of serotype 1, 8 of serotype 2, 15 of serotype 3, 10 of serotype 7, 1 of serotype 12, and 4 of serotype 31. In this study, the positive rate of adenovirus between CAD children and HAD children did not differ (χ(2) = 0.03, P > 0.05), while the positive rate of enteric adenovirus was high in CAD children.</p><p><b>CONCLUSION</b>Adenovirus infection was the main cause of diarrhea in hospitalized children. In this study, the positive rate of adenovirus was similar between children with CAD and with HAD. Enteric adenovirus (adenovirus group F) was the most common adenovirus serotype detected in 2010 in Beijing, and Ad41 was the dominant type.</p>

Detection and genotyping of human bocavirus 2 in children with acute diarrhea / 病毒学报

To investigate the prevalence of HBoV2 in pediatric patients with acute diarrhea in Beijing and the characteristic of the genome of the virus, 553 stool specimens were collected from pediatric outpatients with acute diarrhea in Affiliated Children's Hospital of Capital Institute of Pediatrics during Nov. 2010 to Oct. 2011. TaqMan-based Real-time polymerase chain reaction was performed to detect HBoV2 DNA from these specimens. Two positive specimens with high viral loads were selected for segmented amplification and then the amplified fragments were cloned into the plasmid vector pGEM-T, transformed into Escherichia coli DH5alpha and sequenced. Then genomic sequences assembled from those DNA fragments were compared with other parvovirus genomic sequences in the GenBank. Among these 553 specimens tested, 15 (2.7%) were HBoV2 DNA positive. The highest positive rate was shown in July (7.0%) through the whole year and in 3-6 month age group (4.1%) among different age groups. All these 15 specimens positive for HBoV2 DNA were collected from patients younger than 2 years old, including 4 simultaneously positive for norovirus, 3 positive for rotavirus and 1 positive for adenovirus. By sequence analysis, 2 almost complete HBoV2 genomic sequences assembled from gene fragments amplified from specimens BJQ19 and BJQ390 were typical HBoV2. And they shared high homology with each other (99.2%), while they shared the highest homology with FJ375129 from Shanghai China (99.1% and 99.2%) among other parvoviruses. These data suggest that some of acute diarrhea in pediatric patients in Beijing were associated with HBoV2, and infants and young children aged from 3 months to 2 years, are more likely to be infected by HBoV2.

Investigation of a novel VP4 subgenotype of rotavirus in children with diarrhea in Beijing during 2009-2010 / 病毒学报

P[8]b is a newly discovered sub-genotype for VP4 gene of group A human rotaviruses (HRV) worldwide. This study was to develop an effective method to identify P[8]a, P[8]b, P[4] and P[6] (sub) genotypes of VP4 genes of HRV and to investigate the prevalence of P[8]b sub-genotype and its G/P combinations of HRV in outpatient and inpatient children with diarrhea in Children's Hospital affiliated to Capital Institute of Pediatrics from 2009 to 2010. By analyzing the collected nucleotide sequences of VP4 gene for all known P genotypes of HRV including P[8]b subtype from GenBank and using softwares of DNAS-tar and MegAlign to align and analyze multiple sequences, probes for P[8]a, P[8]b, P[4] and P[6] (sub) genotypes in the corresponding regions which are highly divergent among genes from different genotypes and conserved within genes of VP4s in same genotypes were designed. Then four sets of primers for PCR amplified DIG labeled probes were designed and corresponding DIG-labeled specific P genotype probes were synthesized with PCR by using VP8* genes of Beijing field HRV strains representing P-genotypes P[8]a, P[8]b, P[4] and P[6], respectively, as templates. Dot-blot hybridization was developed based on cDNA of VP4 genes. The dot-blot hybridization assay for P genotyping was reliable which was confirmed by sequencing of RT-PCR products of VP4 genes amplified from corresponding clinical samples. P genotyping for VP4 genes from 88 HRV positive specimens from the Outpatient Department (55%, 88/160) and 79 HRV positive specimens from the hospitalized (70.5%, 79/112) children with diarrhea indicated that P[8] a subtype was still the most prevalent sub-genotype, which was 96.6% (85/88) and 62.0% (49/79) respectively. The positive rate for P[8]b subtypes in hospitalized children with HRV diarrhea was higher (27.9%, 22/79) than that of in outpatient (2.3%, 2/88) HRV infected children. HRV with P[4] genotype was only found in one of the hospitalized children (1.3%, 1/79), and HRV with P[6] genotype was not detected from specimens either from outpatient or inpatient. G9P[8]b was the predominant combination among the P[8]b subtype of HRV positive specimens in this study. The results in this study indicated that G9P[8]b HRV circulated in children with diarrhea in Beijing.

Inhibition of the cystathionine-gamma-lyase/hydrogen sulfide pathway in rat vascular smooth muscle cells by cobalt-60 gamma radiation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Radiation is a promising treatment for in stent restenosis and restenosis following percutaneous transluminal coronary angioplasty, which has troubled interventional cardiologists for a long time. It inhibits neointima hyperplasia, vascular remodeling, and increases the mean luminal diameter. The mechanism of intracoronary brachytherapy for restenosis is not well understood. Endogenous gaseous transmitters including nitric oxide and carbon monoxide are closely related to restenosis. Hydrogen sulfide, a new endogenous gaseous transmitter, is able to inhibit the proliferation of vascular smooth muscle cells and vascular remodeling. This study aimed to clarify the effect of radiation on cystathionine-gamma-lyase/hydrogen sulfide pathway in rat smooth muscle cells.</p><p><b>METHODS</b>We studied the effect of radiation on the cystathionine-gamma-lyase/hydrogen sulfide pathway. Rat vascular smooth muscle cells were radiated with (60)Co gamma at doses of 14 Gy and 25 Gy respectively. Then the mRNA level of cystathionine-gamma-lyase was studied by quantitative reverse-transcription competitive polymerase chain reaction. Hydrogen sulfide concentration in culture medium was determined by methylene blue spectrophotometry. Cystathionine-gamma-lyase activity in vascular smooth muscle cells was also studied.</p><p><b>RESULTS</b>(60)Co gamma radiation at a dose of 1 Gy did not affect the cystathionine-gamma-lyase/hydrogen sulfide pathway significantly. However, (60)Co gamma radiation at doses of 14 Gy and 25 Gy decreased the hydrogen sulfide synthesis by 21.9% (P<0.05) and 26.8% (P<0.01) respectively. At the same time, they decreased the cystathionine-gamma-lyase activity by 15.1% (P<0.05) and 20.5% (P<0.01) respectively, and cystathionine-gamma-lyase mRNA expression by 29.3% (P<0.01) and 38.2% (P<0.01) respectively.</p><p><b>CONCLUSION</b>Appropriate (60)Co gamma radiation inhibits the H(2)S synthesis by inhibiting the gene expression of cystathionine-gamma-lyase and the cystathionine-gamma-lyase activity.</p>

An acute norovirus gastroenteritis outbreak in a hospital / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate and analyze a case of acute norovirus gastroenteritis outbreak in a hospital.</p><p><b>METHODS</b>Data were collected through the on-the-spot investigation and faecal specimens were tested by polymerase chain reaction-reverse transcription (RT-PCR). Serum specimens were tested by Western blot.</p><p><b>RESULTS</b>The outbreak lasted 26 days. A total of 87 cases were reported, including 65 inpatients, 15 healthcare workers, 6 accompanies, and 1 pantryman. Twelve (60%) of 20 stool specimens were norovirus-positive by RT-PCR and 19 of 24 blood samples were norovirus-positive by Western blot. The outbreak was effectively controlled by isolating and treating the patients, extensive disinfection, and health education.</p><p><b>CONCLUSION</b>The gastroenteritis outbreak was caused by norovirus with unkown infection source.</p>

Norovirus associated outbreaks of acute gastroenteritis in hospitals in Beijing in late 2006 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To identify the etiological agent of acute gastroenteritis outbreaks occurred in 6 hospitals in Beijing from November to the end of 2006 and to characterize the virus on molecular biology.</p><p><b>METHODS</b>Rota-strip was used to detect rotavirus antigen and PAGE for rotavirus RNA in stool specimens collected from patients with gastroenteritis at the first stage. Reverse transcription-polymerase chain reaction(RT-PCR) was performed to detect Norovirus in stool specimens. Full length of Norovirus VP1 genes were amplified from Norovirus positive samples by RT-PCR and sequenced after the gene was cloned into vector pUCm-T. The sequences of partial VP1 gene at the 5' end of full-length gene were obtained from 2 of the Norovirus positive samples to analyze the relationship of these viruses at molecular level.</p><p><b>RESULTS</b>Norovirus was detected in 17 of the 30 stool specimens with an overall positive rate of 56.7%. Positive rates were all above 42.9% in specimens collected from each of the hospital. Sequence analysis on one of the full-length of VP1 amplified by RT-PCR from one of the positive specimens NV8-Beijing indicated that it belonged to G II-4 genotype. This strain shared 94.8% and 95.2% identities over the complete major capsid gene to Lanzhou strain form Lanzhou China and Farmington Hill strain from USA which belonged to G II-4 genotype, but only 91.1% identity to the CR2905 from Beijing China identified from specimen collected in late 2004. The sequences of 5' end of VP1 genes amplified from other two samples collected from different hospitals showed only one nucleotide mutation compared to NV8-Beijing in the correspondence part, indicating that these two strains also belonged to G II-4 genotype.</p><p><b>CONCLUSION</b>Norovirus was the etiological agent causing outbreaks of acute gastroenteritis in hospitals in Beijing and the strain with G II-4 genotype which was closer to the Lanzhou and Farmington strains than Beijing strains identified in 2004.</p>

Comparison of methods for Norovirus detection in stool specimens collected from hospitalized patients with hospital acquired diarrhea / 中华流行病学杂志

<p><b>OBJECTIVE</b>In order to find out a more convenient, rapid and efficient way in detecting human Norovirus infections in specimens collected from hospitalized patients with acute non-bacterial diarrhea in Beijing.</p><p><b>METHODS</b>Two kits for enzyme immunoassay (EIA) were used to detect human Noroviruses in stool specimens collected from 69 infants and young children as well as 15 adults who were all diagnosed as acute non-bacterial diarrhea in 4 different hospitals. Reverse transcription-polymerase chain reaction(RT-PCR) was performed to evaluate the data from two kits in this study. Data were statistically analyzed by SPSS 11.5 software. chi2 test was used to test categorical variables.</p><p><b>RESULTS</b>Out of 84 stool specimens collected from infants and young children or adults with acute non-bacterial diarrhea, 17 (20.2%) were Norovirus positive determined by EIA kit A and 31 (36.9%) were Norovirus positive determined by EIA kit B. chi2 test used to test categorical variables showed significant differences (P < 0.01), suggesting that the EIA kit B was superior to the EIA kit A. Among these 84 stool specimens, 20 were tested by RT-PCR simultaneously. Out of those 20 specimens, 11 (55.0%) were Norovirus positive as determined by RT-PCR, which was higher than that from 2 EIA kits.</p><p><b>RESULTS</b>from 10 (50.0%) samples detected by EIA kit A were consistent with those detected by RT-PCR. Through chi2 test, the categorical variables showed significant differences with P < 0.05, suggesting that RT-PCR was superior to the EIA kit A. Results from 14 (70.0%) samples detected by EIA kit B were consistent with those detected by RT-PCR while chi2 test showed that the differences were not significant (P > 0.05), among categorical variables suggesting that EIA kit B was as sensitive as RT-PCR in detecting Norovirus. The were hospital acquired diarrhea outbreaks in these three hospitals since at least 2 Norovirus positive specimens were detected in each of the hospitals.</p><p><b>CONCLUSION</b>To detect human Noroviruses infection, EIA seemed to be more convenient and time saving than RT-PCR which had been used worldwide. The EIA kit B in this study was comparable to RT-PCR for detecting Norovirus in stool specimens. Norovirus was a pathogen causing hospital acquired diarrhea outbreaks in these three hospitals.</p>

A study on viral gastroenteritis attributed to noroviruses in hospitals / 中华流行病学杂志

<p><b>OBJECTIVE</b>To better understand the clinical feature of viral gastroenteritis attributed to noroviruses and to summarize the experience on an outbreak of acute gastroenteritis through rapidly colleting and confirmation of related information regarding to noroviruses in hospitals.</p><p><b>METHODS</b>Information on an outbreaks involving 18 patients with acute gastroenteritis in one hospital regarding its epidemiological and clinical features and to perform bacteria culture for stool specimens on every patient. On 7 patients, rotavirus antigen were RNA tested together with norovirus nucleic acid were examined by ELISA and PAGE and RT-PCR.</p><p><b>RESULTS</b>(1) Most of the patients were elderly with several chronic diseases. (2) Watery diarrhea (12/18, 66.67%) and few with mucous (3/18, 16.67%) were seen. Most stool examination was normal (10/18, 55.56%) but few stool specimen could be found with some leucocytes (3/18, 16.67%) and little occult blood (4/18, 22.22%). (3) All bacteria culture in stools showed negative. There was no rotavirus RNA identified but 3 specimen showed norovirus nucleic acid positive as 42.86% (3/7).</p><p><b>CONCLUSION</b>Norovirus was one of the important pathogens causing acute gastroenteritis outbreaks in hospitals attacking elderly with several chronic diseases in particular. Surveillance program targeting elderly inpatient with diarrhea should be enhanced, especially in autumn and winter.</p>

Seroprevalence of antibody against human metapneumovirus in Beijing / 中华儿科杂志

<p><b>OBJECTIVE</b>To understand the seroprevalence of antibody against the newly identified human metapneumovirus (hMPV) in Beijing.</p><p><b>METHODS</b>The antigenic specificity of hMPV N protein cloned into vector pET30a and then expressed in E coli was verified by using SDS-PAGE and Western blotting in 116 serum specimens. The plasmid pET30a without insert was used as control. Totally 710 serum specimens collected from non-respiratory infection patients visited the Outpatient Departments of Children's Hospital affiliated to Capital Institute of Pediatrics and Xuanwu Hospital, Beijing from April 1996 to March 1997 were tested for specific IgG antibody against hMPV N protein.</p><p><b>RESULTS</b>The bands with expected molecular weight showed only on the membranes transferred by the expressed hMPV N protein and incubated with rabbit hyperimmune serum against hMPV N protein polypeptides as well as the collected human sera, indicating the specificity of the expressed hMPV N protein. Out of 710 specimens tested, 17.2% (122/710) were positive for antibody to N protein. Antibody positive rate was the lowest in 2 to 6 months old infants (3.1%); the rate declined from 13.2% in newborns to 6.1% in 1 to 2 months old infants, then to 3.1% in the 2 to 6 months group, and sustained at about 3.0% from 6 months group to 30 years of age, then increased to 28.1% in 30 to 39-year-old adults, 32.3% in 40 to 49-year-old adults and to 38.5% in the group over age of 50 years.</p><p><b>CONCLUSION</b>The expressed hMPV N protein is reliable when it was used as antigen for testing specific IgG antibody against hMPV in human sera. The high seroprevalence of antibody against hMPV N protein and early age antibody acquisition suggest that hMPV has been circulating in Beijing and the importance of the virus as pathogen should be further analyzed.</p>

Study on the inhibitive effect of Astragalus Injection solution on hepatic fibrosis in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of Astragalus Injection solution on rat hepatic stellate cells (HSC) and hepatic fibrosis.</p><p><b>METHODS</b>HSCs of rats were incubated with various concentrations of Astragalus Injection solution (0 mg/ml, 25 mg/ml, 50 mg/ml, 100 mg/ml, 200 mg/ml, 400 mg/ml) for 24, 48 and 72 hours. Cell proliferation was detected with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphennyltetrazolium bromide (MTT) colorimetric assay. Cell cycle was detected with flow cytometry. Cell apoptosis was detected with acridine orange/ethidium bromide (AO/EB) fluorescent staining and flow cytometry. In vivo, rats were randomly allocated into a normal control group, a model control group and an Astragalus Injection group. Astragalus Injection (800 mg.kg-1.d-1) was administered to rats of the Astragalus Injection group. Rats of the model control group received saline. Serum concentrations of hyaluronic acid (HA) and laminin (LN), hepatic tissue activity of superoxide dismutase (SOD), and hepatic tissue contents of malondialdehyde (MDA) were measured in these groups at 8 weeks. Hepatic tissue expression of LN was assessed by using immunohistochemistry. The pathological changes of hepatic tissues were examined by hematoxylin-eosin (HE) and van Gieson (VG) staining of their histological slides.</p><p><b>RESULTS</b>In vitro, compared with the 0 mg/ml group, the proliferation of HSCs in other concentration groups was significantly inhibited by Astragalus Injection solution in a dose and time dependent manner, the cell proliferation cycle of HSCs was blocked in the G2-M phase, there was no apoptosis of HSCs in AO/EB fluorescent staining and flow cytometry. In vivo, compared with rats of the model control group, the rats of the Astragalus Injection solution treated group had remarkably decreased serum HA and LN levels (114.3+/-25.6) microg/L vs (85.6+/-37.3) microg/L and (78.8+/-11.7) microg/L vs (66.8+/-17.6) microg/L, P < 0.05, and liver MDA level (3.7+/-0.4) micromol/g protein vs (2.4+/-0.2) micromol/g protein, P < 0.01, but had increased activity of liver SOD (49.6+/-5.7) NU/mg protein vs (75.9+/-5.9) NU/mg protein, P < 0.01. Microscopic studies revealed that the livers of rats receiving Astragalus Injection solution showed decreases in fibrosis and in expression of LN.</p><p><b>CONCLUSIONS</b>Astragalus Injection solution has an inhibitive effect on experimental hepatic fibrogenesis. The mechanisms of its effects might possibly be associated with its antioxidant activity, expression of decreasing LN and its inhibition of HSCs proliferation.</p>

Effect and mechanism of Tanshensu on fibrotic rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect and mechanism of Tanshensu on experimental fibrotic rats.</p><p><b>METHODS</b>Pigs serum was used to induce liver fibrosis in Wistar rats. The rats in Tanshensu-treated group were injected peritoneally with Tanshensu solution at the dose of 300 mg x kg(-1) x d(-1), and the rats in model-control group and normal-control group received the same volume of double distill water. At the end of the twelfth week, the hepatic stellate cells (HSCs) were isolated from the liver of one rat in model-control group using in-situ perfusion with pronase and collagenase, then density gradient centrifugation, and the other rats were killed to take the serum and liver samples. MTT colorimetric assay was used for detecting the proliferation of HSCs and flow cytometry was used for observing the cell cycles of HSCs under different concentrations of Tanshensu. The hyaluronic acid (HA) level in serum was detected and the morphological changes of liver tissue were observed.</p><p><b>RESULTS</b>There was a decline of serum HA level in Tanshensu-treated group compared to that of the model-control group (231.4 ng/ml +/- 41.1 ng/ml vs. 398.7 ng/ml +/- 54.5 ng/ml, F =154.796, P < 0.05). Both HE and VG stain showed a decline of liver fibrosis degree in Tanshensu-treated group. And Tanshensu had an inhibition effect on the proliferation of HSCs at the concentrations of 50 mg/L, 100 mg/L, and 200 mg/L (1.60x10(-2) +/- 8.17x10(-4), 1.10x10(-2) +/- 1.41x10(-3), and 6.75x10(-3) +/- 3.30x10(-3) vs. 7.18x10(-2) +/- 1.71x10(-3), F =1154.221, P <0.01).</p><p><b>CONCLUSIONS</b>Tanshensu shows a therapeutic effect on liver fibrosis in rats induced by pig's serum through inhibiting the proliferation of hepatic stellate cells.</p>

Study on expression of cytokines mRNA induced by B7-1-transfected Raji and Jurkat cells / 中国实验血液学杂志

To investigate the function of B7 co-stimulator in activation and differentiation of T cell, B7 gene was transfected into Raji and Jurkat cells by liposome, B7 expression in tumor cells was detected with flow cytometry, and expression of IL-2, IL-4 and IFN-gammamRNA was detected by RT-PCR. Kinetics of secretion of three cytokines was also analyzed at 4, 12, 20 and 48 hours after gene transfection. The results showed that B7(+) Raji cells could induce mRNA expression of IL-2, IL-4 and IFN-gamma on T cell surface; B7(+) Jurkat cells could induce secretion of IL-2 and IFN-gamma. However, B7(-) Raji and B7(-) Jurkat cells could not induce secretion of cytokines. Kinetics of the three cytokines secretion were different, IL-2 and IL-4 were only detectable after 4 hours of T cell activation, whereas IFN-c was detectable after 12 hours of stimulation. The peak levels of IL-2, IL-4 and IFN-gamma were found at 20 hours after activation. It was concluded that tumor cell lines transfected with B7 gene could enhance their immunocompetence, activating T cell efficiently and B7-1 play more critical role in T cell activation and differentiation.

Effects of Karst Environmental Factors on Expression and Activity of Bacterial Extracellular Carbonic Anhydrase / 微生物学通报

Effects of main environmental factors, such as temperature, pH , metal ions and anions, on expression and activity of bacterial extracellular car bonic anhydrase(CA) were studied, exemplified by a bacterium named GLRT102Ca, wh ich was separated from karst ecosystems of Southwest China. The results showed that the tested strain could express different activity of extracellular carboni c anhydrase within the scope of experimental temperature (10℃~50℃) and pH (5. 5~9.0). The activity of extracellular carbonic anhydrase was higher at tempera ture of 20℃~30℃ and at neutral and alkaline trending condition. Moreover, the expression of activity of extracellular carbonic anhydrase could be generally p romoted at the experimental range of concentration of 4 kinds of metal ions such as Ca2+, Mg2+, Zn2+ and Co2+, along with 8 kinds of ani ons such as SO2-_4, H_2PO-_4, NO-_3, NO-_2, Cl-,Br-, I- and HCO-_3. This research provides a cert ain theoretical basis for further study on the role of microbial CA in karst pro cesses.