Knockin of the G275E mutation of the nicotinic acetylcholine receptor (nAChR) α6 confers high levels of resistance to spinosyns in Spodoptera exigua.

Spinosyns, including spinosad and spinetoram, act on the insect central nervous system, gradually paralyzing or destroying the target insect. Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit in a number of agricultural pests. Using gene editing, nAChR α6 has been verified as a target for spinosyns in five insect species. Recently, a point mutation (G275E) in exon 9 of nAChR α6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta. To date, no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests. In this study, the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) gene-editing technology. Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain. The results of bioassays showed that the 275E strain was highly resistant to spinosad (230-fold) and spinetoram (792-fold) compared to the unedited background strain, directly confirming that the G275E mutation of the nAChR α6 subunit confers high levels of spinosyn resistance in S. exigua. Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive. This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChR α6 of S. exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.

The functional motions and related key residues behind the uncoating of coxsackievirus A16.

The coxsackievirus A16 (CVA16) is a highly contagious virus that causes the hand, foot, and mouth disease, which seriously threatens the health of children. At present, there are still no available antiviral drugs or effective treatments against the infection of CVA16, and thus it is of great significance to develop anti-CVA16 vaccines. However, the intrinsic uncoating property of the capsid may destroy the neutralizing epitopes and influence its immunogenicity, which hinders the vaccine developments. In the present work, the functional-quantity-based elastic network model analysis method developed by our group was extended to combine with group theory to investigate the uncoating motions of the CVA16 capsid, and then the functionally key residues controlling the uncoating motions were identified by our functional-quantity-based perturbation method. Several motion modes encoded in the topological structure of the capsid were revealed to be responsible for the uncoating of CVA16 particle. These modes predominantly contribute to the fluctuation of the gyration radius of the capsid. Then, by using the perturbation method, four clusters of key sites involved in the uncoating motions were identified, whose perturbations induce significant changes in the fluctuation of the gyration radius. These key residues are mainly located at the 2-fold channels, the quasi 3-fold channels, the bottom of the canyons, and the inter-subunit interfaces around the 3-fold axes. Our studies are helpful for better understanding the uncoating mechanism of the CVA16 capsid and provide potential target sites to prevent the uncoating motions, which is valuable for the vaccine design against CVA16.

Knockout of three aminopeptidase N genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins.

Bacillus thuringiensis (Bt) insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops. Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action. Previous studies suggested aminopeptidase N1 (APN1) as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence-based gene silencing approaches. In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible strain (SCD) of H. armigera to establish three homozygous knockout strains. Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera.

Identification of the ryanodine receptor mutation I4743M and its contribution to diamide insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae).

Insect ryanodine receptors (RyRs) are the targets of diamide insecticides. Two point mutations G4946E and I4790M (numbering according to Plutella xylostella, PxRyR) in the transmembrane domain of the insect RyRs associated with diamide resistance have so far been identified in three lepidopteran pests, P. xylostella, Tuta absoluta and Chilo suppressalis. In this study, we identified one of the known RyR target site resistance mutations (I4790M) in a field-collected population of Spodoptera exigua. The field-collected WF population of S. exigua exhibited 154 fold resistance to chlorantraniliprole when compared with the susceptible WH-S strain. Sequencing the transmembrane domains of S. exigua RyR (SeRyR) revealed that the resistant WF strain was homozygous for the I4743M mutation (corresponding to I4790M in PxRyR), whereas the G4900E allele (corresponding to G4946E of PxRyR) was not detected. The 4743M allele was introgressed into the susceptible WH-S strain by crossing WF with WH-S, followed by three rounds of backcrossing with WH-S. The introgressed strain 4743M was homozygous for the mutant 4743M allele and shared about 94% of its genetic background with that of the recipient WH-S strain. Compared with WH-S, the near-isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole (21 fold), cyantraniliprole (25 fold) and flubendiamide (22 fold), suggesting that the I4743M mutation confers medium levels of resistance to all three diamides. Genetic analysis showed diamide resistance in the 4743M strain was inherited as an autosomal and recessive trait. Results from this study have direct implications for the design of appropriate resistance monitoring and management practices to sustainably control S. exigua.

Genetic analysis and molecular detection of resistance to chlorpyrifos mediated by the A216S substitution in acetylcholinesterase-1 in the plant bug Apolygus lucorum.

The green plant bug Apolygus lucorum is a major pest of Bacillus thuringiensis cotton in China. Previously, we reported that chlorpyrifos resistance in a laboratory-selected strain of A. lucorum (BZ-R) is associated with the homozygosis of an allele in the ace-1 gene encoding an alanine to serine substitution at position 216 of acetylcholinesterase-1. Here we describe the results of crosses between the resistant BZ-R strain (41-fold to chlorpyrifos) and the unselected susceptible BZ-S strain homozygous for the wild type alanine allele at position 216. Resistance to chlorpyrifos was inherited as a semi-dominant trait mainly controlled by a single autosomal gene and co-segregates strongly but not completely with the serine substitution in ace-1. Synergism bioassays and enzyme assays showed that minor contributions to resistance are also made by enhanced cytochrome P450 and carboxylesterase activities. A survey of 25 field populations from five Chinese provinces showed strong positive correlations between 50% lethal concentration against chlorpyrifos and S216 allele and genotype frequencies, although the most tolerant populations still only show 40%-50% S216 allele frequencies. The results above provide important information for designing effective resistance monitoring and management strategies for A. lucorum in China.

Energy transport pathway in proteins: Insights from non-equilibrium molecular dynamics with elastic network model.

Intra-molecular energy transport between distant functional sites plays important roles in allosterically regulating the biochemical activity of proteins. How to identify the specific intra-molecular signaling pathway from protein tertiary structure remains a challenging problem. In the present work, a non-equilibrium dynamics method based on the elastic network model (ENM) was proposed to simulate the energy propagation process and identify the specific signaling pathways within proteins. In this method, a given residue was perturbed and the propagation of energy was simulated by non-equilibrium dynamics in the normal modes space of ENM. After that, the simulation results were transformed from the normal modes space to the Cartesian coordinate space to identify the intra-protein energy transduction pathways. The proposed method was applied to myosin and the third PDZ domain (PDZ3) of PSD-95 as case studies. For myosin, two signaling pathways were identified, which mediate the energy transductions form the nucleotide binding site to the 50 kDa cleft and the converter subdomain, respectively. For PDZ3, one specific signaling pathway was identified, through which the intra-protein energy was transduced from ligand binding site to the distant opposite side of the protein. It is also found that comparing with the commonly used cross-correlation analysis method, the proposed method can identify the anisotropic energy transduction pathways more effectively.

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth, Plutella xylostella.

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field-evolved indoxacarb resistance has been reported in several lepidopteran pests, including the diamondback moth Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong Province of China in 2012, was 750-fold more resistant to indoxacarb and 70-fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of complementary DNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the sixth segment of domain IV of the PxNav protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among 10 field-collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34- to 870-fold) and metaflumizone (1- to 70-fold). The two mutations were never found to co-exist in the same allele of PxNav , suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species.

Analysis of conformational motions and related key residue interactions responsible for a specific function of proteins with elastic network model.

Protein collective motions play a critical role in many biochemical processes. How to predict the functional motions and the related key residue interactions in proteins is important for our understanding in the mechanism of the biochemical processes. Normal mode analysis (NMA) of the elastic network model (ENM) is one of the effective approaches to investigate the structure-encoded motions in proteins. However, the motion modes revealed by the conventional NMA approach do not necessarily correspond to a specific function of protein. In the present work, a new analysis method was proposed to identify the motion modes responsible for a specific function of proteins and then predict the key residue interactions involved in the functional motions by using a perturbation approach. In our method, an internal coordinate that accounts for the specific function was introduced, and the Cartesian coordinate space was transformed into the internal/Cartesian space by using linear approximation, where the introduced internal coordinate serves as one of the axes of the coordinate space. NMA of ENM in this internal/Cartesian space was performed and the function-relevant motion modes were identified according to their contributions to the specific function of proteins. Then the key residue interactions important for the functional motions of the protein were predicted as the interactions whose perturbation largely influences the fluctuation along the internal coordinate. Using our proposed methods, the maltose transporter (MalFGK2) from E. Coli was studied. The functional motions and the key residue interactions that are related to the channel-gating function of this protein were successfully identified.

Conformational Motions and Functionally Key Residues for Vitamin B12 Transporter BtuCD-BtuF Revealed by Elastic Network Model with a Function-Related Internal Coordinate.

BtuCD-BtuF from Escherichia coli is a binding protein-dependent adenosine triphosphate (ATP)-binding cassette (ABC) transporter system that uses the energy of ATP hydrolysis to transmit vitamin B12 across cellular membranes. Experimental studies have showed that during the transport cycle, the transporter undergoes conformational transitions between the "inward-facing" and "outward-facing" states, which results in the open-closed motions of the cytoplasmic gate of the transport channel. The opening-closing of the channel gate play critical roles for the function of the transporter, which enables the substrate vitamin B12 to be translocated into the cell. In the present work, the extent of opening of the cytoplasmic gate was chosen as a function-related internal coordinate. Then the mean-square fluctuation of the internal coordinate, as well as the cross-correlation between the displacement of the internal coordinate and the movement of each residue in the protein, were calculated based on the normal mode analysis of the elastic network model to analyze the function-related motions encoded in the structure of the system. In addition, the key residues important for the functional motions of the transporter were predicted by using a perturbation method. In order to facilitate the calculations, the internal coordinate was introduced as one of the axes of the coordinate space and the conventional Cartesian coordinate space was transformed into the internal/Cartesian space with linear approximation. All the calculations were carried out in this internal/Cartesian space. Our method can successfully identify the functional motions and key residues for the transporter BtuCD-BtuF, which are well consistent with the experimental observations.

Epidemiology of childhood enterovirus infections in Hangzhou, China.

BACKGROUND: There are over 100 serotypes of enterovirus species A-D, which are the common cause of various symptoms in infants, such as meningitis, encephalitis and hand foot mouth disease (HFMD). This study aims to investigate the epidemiological characteristics of enteroviruses in Hangzhou, Zhejiang province, China, and to provide relevant information to guide public health responses and interventions. METHODS: Systematic surveillance was conducted on enterovirus infections. Samples were collected from children admitted to the inpatient wards and outpatient departments between January 2010 and December 2012 in the Children's Hospital, Zhejiang University School of Medicine. Enteroviruses from all specimens were detected by RT-PCR using a commercialized detection kit. RESULTS: From 13026 samples collected and examined, 2673 (21.21%) were found positive for enteroviruses. The annual enterovirus-positive rate decreased from 32.78% in 2010 to 14.23% in 2012. Positivity rate for enteroviruses was highest among children aged less than 5 years. The monthly positivity rate for enterovirus infection ranged from 2.6% to 34.83%, with a peak in June and July. Serotypes causing severe symptoms such as HFMD including EV71 and CA16 were decreasing, while the proportion of unidentified EV serotypes causing herpangina and viral encephalitis were on the rise. CONCLUSIONS: EV infection is highly prevalent among young children in Hangzhou, as it is in the most other parts of the world. Further surveillance using methods that can subtype all EVs is warranted to better monitor these infections and their etiology.

Rapid and Sensitive Identification of Bacterial Infection and Bacteria Gram Types in Pleural Fluid of Children.

Real-time polymerase chain reaction (RT-PCR) techniques have been increasingly used to detect microbial DNA in clinic for the diagnosis of bacterial infection. This study aims to developing an RT-PCR method to detect bacteria in pleural fluid (PF). We performed a method to simultaneously detect and classify the clinically relevant bacterial pathogens in hydrothorax with Gram probe RT-PCR (GRT-PCR), which targets the conserved region of the 16S rRNA gene. Our results showed this method could specifically and correctly identify 14 clinically important bacterial strains in hydrothorax including 7 gram-positive and 7 gram-negative bacteria. And the sensitivity of this GRT-PCR method in serial dilution can reach 10 CFU/mL. In clinical trial, 180 PF samples from children who were clinically suspected to suffer from bacterial pneumonia and empyema were collected. These samples were detected by GRT-PCR, standard culture, and biochemical routine analysis. The positive rate of the GRT-PCR array was 17.78% (32/180), significantly higher than that of PF culture (11.67%; 21/180; P = .003). When PF culture was used as control, the sensitivity of GRT-PCR was 95.24% (95% confidence interval = 74.13-99.75), and the specificity was 92.45% (95% confidence interval = 86.89-95.86). Our study showed that GRT-PCR is a more effective method for rapid, sensitive, and specific diagnosis of bacterial infection in hydrothorax compared with other traditional methods.

[Epidemiological investigation of hand-foot-and-mouth disease in children and exposed population in Hangzhou city].

OBJECTIVE: To investigate the epidemic characteristics of hand-foot-and-mouth disease (HFMD) in children and exposed population in Hangzhou city. METHODS: The throat swab or stool samples from children with HFMD admitted in Hangzhou Children's Hospital were collected. The HFMD pathogens were detected by real-time fluorescent quantitative PCR. The distribution of different HFMD pathogens in HFMD patients was subsequently determined. Human enteric virus type-71 (HEV71) in stool samples from subjects, who had close or general contact to 54 severe HFMD children with positive HEV71, was detected, and these contact persons were followed-up for one month. The diversity of predominant pathogens of HFMD in the area during 2011-2013 was investigated. RESULTS: In 641 HFMD children, the male/female ratio was 1.4:1 and 80.3% was 1-3 years old. HEV71 was detected in 24.3% HFMD children (156/641), while coxsackievirus group-A type-16 (CVA16) and other enteroviruses were detected in 4.7% (30/641) and 71.0% (455/641) of the cases, respectively. 75.6% (118/156) of HEV71-infected cases were diagnosed as severe HFMD cases, while those for CVA16-infected and other HFMD viruses-infected were 13.3% (4/30) and 6.2% (28/455) respectively (Χ(2)=43.28, P<0.05). HEV71 was the predominant HFMD pathogens during 2011-2012, while the predominant HFMD pathogens in 2013 were the other HFMD viruses. In the 54 close contact persons or 54 general contact persons, 9 or 10 persons were detectable for HEV71, but no clinical symptoms of HFMD were presented. CONCLUSION: There are no marked changes of epidemic seasons, favorable age and gender ratio of HFMD in Hangzhou area in 2013. The infection of HEV71 tends to cause the severe HFMD but the other enteroviruses have substituted HEV71 as the predominant pathogens of HFMD.

Polymorphism analysis of the ABCA3 gene: association with neonatal respiratory distress syndrome in preterm infants.

BACKGROUND: Previous reports indicated that mutations in the adenosine triphosphate (ATP)-binding cassette transporter A3 (ABCA3) cause fatal respiratory failure in term infants, and common ABCA3 gene polymorphisms have been characterized at the population level in Caucasians. But the role of ABCA3 in relation to respiratory distress syndrome (RDS) in newborns has not been evaluated within a Chinese population. The aim of this study was to analyze eight single-nucleotide polymorphisms (SNPs) of the ABCA3 gene, and to assess the ABCA3 gene as a candidate gene for susceptibility to RDS in newborns. METHODS: Eight SNPs were selected and genotyped in 203 newborns. The data analysis and statistical tests were used for allele frequencies, haplotype and Hardy-Weinberg equilibrium pairwise linkage disequilibrium measures. RESULTS: There was a haplotype association with SNP rs313909 and SNP rs170447, but no haplotype association was observed among the newborns with and without RDS (P > 0.05). The minor allele frequency (G) of the coding SNP (cSNP) rs323043 (P585P) was significantly increased in preterm infants with RDS. CONCLUSION: There is an association between a synonymous cSNP rs323043 and the development of RDS.

[Detection of human papillomavirus in the upper respiratory tract in children without recurrent respiratory papillomatosis].

OBJECTIVE: The purpose of this prospective study was to investigate the presence of human papillomavirus (HPV) in tonsillectomy and adenoidectomy specimens from pediatric patients without juvenile-onset recurrent respiratory papillomatosis (JORRP), so as to understand the effect of HPV infection in the upper respiratory tract in children. METHODS: Two hundred and forty-one pediatric patients without known JORRP or other HPV-related diseases undergoing tonsillectomy and/or adenoidectomy for hypertrophy or chronic tonsillitis were enrolled in this prospective study. One hundred and seventy-seven fresh samples of tonsillar tissues and 195 samples of adenoid tissues were collected and then examined for the presence of HPV DNA with the polymerase chain reaction (PCR) technique and typing. Laryngeal papilloma specimens from 17 patients obtained during routine debulking procedures were also analyzed and served as positive controls. RESULTS: All 17 papilloma specimens were positive for HPV DNA and the type was 6 or 11. This result confirmed that the methods used were valid for detecting HPV infection. HPV DNA was detected in 2 of the 177 tonsillar specimens and zero of the 195 adenoid specimens. The two positive samples were confirmed with typing. One was positive for HPV6 and the other for HPV11. Review of the medical records of these two cases confirmed that there were no history of HPV-related diseases. Histologic analysis of their specimens showed lymphoid hyperplasia, no specific changes suggesting HPV infection and no signs of malignancy. The HPV infection rate in upper respiratory tract was 0.8% (2/241). CONCLUSION: There is HPV infection in upper respiratory tract in Chinese children without JORRP, but maybe is not sufficient for the formation of JORRP.

Ghrelin ameliorates hypoxia-induced pulmonary hypertension via phospho-GSK3 ß/ß-catenin signaling in neonatal rats.

Effective treatment and/or prevention strategies for neonatal persistent pulmonary hypertension of the newborn (PPHN) have been an important topic in neonatal medicine. However, mechanisms of impaired pulmonary vascular structure in hypoxia-induced PPHN are poorly understood and consequently limit the development of effective treatment. In this study, we aimed to explore the molecular signaling cascades in the lungs of a PPHN animal model and used primary cultured rat pulmonary microvascular endothelial cells to analyze the physiological benefits of ghrelin during the pathogenesis of PPHN. Randomly selected newborn rats were exposed to hypoxia (10-12%) or room air and received daily s.c. injections of ghrelin (150 µg/kg) or saline. After 2 weeks, pulmonary hemodynamics and morphometry were assessed in the rats. Compared with the control, hypoxia increased pulmonary arterial pressure, right ventricle (RV) hypertrophy, and arteriolar wall thickness. Ghrelin treatment reduced both the magnitude of PH and the RV/(left ventricle+septum (Sep)) weight ratio. Ghrelin protected neonatal rats from hypoxia-induced PH via the upregulation of phosphorylation of glycogen synthase kinase 3ß (p-GSK3ß)/ß-catenin signaling and associated with ß-catenin translocation to the nucleus in the presence of growth hormone secretagogue receptor-1a. Our findings suggest that s.c. administration of ghrelin improved PH and attenuated pulmonary vascular remodeling after PPHN. These beneficial effects may be mediated by the regulation of p-GSK3ß/ß-catenin expression. We propose ghrelin as a novel potential therapeutic agent for PPHN.

Epidemiology and burden of rotavirus infection among children in Hangzhou, China.

BACKGROUND: Rotavirus is the most common cause of acute diarrhea in children younger than 5 years worldwide. However, few data have been collected on children with rotavirus diarrhea basing on outpatient department surveillance. OBJECTIVES: To define the epidemiology of rotavirus diarrhea and to investigate the burden associated with diarrhea and rotavirus infection in Hangzhou, China. STUDY DESIGN: Systematic surveillance of rotavirus diarrhea was conducted in inpatient wards and outpatient department from January 2007 to December 2008 in the Children's Hospital of Zhejiang University School of Medicine. All stool specimens were tested for rotavirus by latex agglutination test. RESULTS: 46,499 stool samples were collected and 15,649 (33.7%) were tested positive for rotavirus. Positive rate for rotavirus was highest among children aged 12-24 months (39.0-39.6%). 92.4% children with rotavirus infection were <2 years, with constitution ratios of 21.8%, 41.8%, 21.8%, 8.4% and 6.2% in children aged 0-6 months, 7-12 months, 13-18 months, 19-24 months and >24 months, respectively. The percentage of children whose samples were tested positive for rotavirus ranged from 22.6% to 44.9% at different months, with a peak in October, November and December. The estimated annual rotavirus-associated outpatient visit and hospitalization incidences were 20.1 episodes/1000 children and 2.1 cases/1000 children for children <5 years of age, and were 39.1/1000 and 4.1/1000 for children <2 years of age in Hangzhou, respectively. CONCLUSIONS: Rotavirus is the leading cause of severe diarrhea of children in Hangzhou, especially for children <2 years, which highlight the need of widespread rotavirus immunization for young children.

[Analysis of the epidemic characteristics of the etiological agents in children with hand, foot and mouth disease and its clinical significance].

OBJECTIVE: To investigate the epidemic characteristics of etiological agents in children with hand, foot and mouth disease (HFMD) and analyze the differences between the severe and mild cases with HFMD seen from 2008 to 2009 in the Children's Hospital. METHODS: A total of 154 patients with HFMD were enrolled from May 2008 to September 2008 and from May 2009 to September 2009, including 28 severe HFMD patients. Data from 80 cases with suspected herpangina were collected as control. Enterovirus universal type, enterovirus type 71 (EV71) and coxsackie virus group A 16 (CA16) were detected by real-time RT-PCR respectively. RESULTS: The positive rate of enterovirus universal type in the 154 patients with HFMD was 81.82%(126/154). EV71 positive rate in these 126 patients with enterovirus universal type infection was 57.14%(72/126). The positive rate of enterovirus universal type in the 80 cases with suspected herpangina was 68.75%(55/80). There was no EV71 infection in these 80 cases with suspected herpangina. EV71 infection was mainly popular in 2008. Both EV71 and CA16 were prevalent in 2009. The epidemic characteristics of enterovirus infection with HFMD between 2008 and 2009 had significant differences (χ(2) = 23.50, P = 0.000) (P < 0.01). The epidemic characteristics of enterovirus infection between severe and mild HFMD patients also had significant differences (χ(2) = 29.85, P < 0.01). There were 28 cases with severe HFMD, in whom the EV71 positive rate was 92.86% (26/28). EV71 positive rate in the mild HFMD was 36.51% (46/126) (χ(2) = 29.22, P < 0.01). There was no significant difference in the gender (χ(2) = 0.135, P = 0.714) and virus load (t = 0.141, P = 0.889) between the mild and severe HFMD cases. But the age of mild and severe HFMD showed a significant difference (t = 2.926, P = 0.009). Patients who were less than 2 years of age had a proportion of 88.89% (8/9) with severe HFMD. The mean age of mild HFMD patients was 3.19 years. CONCLUSION: HFMD showed different epidemic characteristics at different times of enterovirus infection. There was no significant difference in the gender and virus load between the mild and severe cases with HFMD. Children under 3 years of age with EV71 infection were at high risk for severe HFMD.

[Establishment and clinical application of a new real time PCR assay for simultaneous detection of human herpesvirus-6A and human herpesvirus-6B].

OBJECTIVE: Human herpesvirus 6 (HHV-6) isolates are classified into two variants, HHV-6A and HHV-6B, based on distinct genetic, antigenic and biological characteristics. HHV-6 has been associated with encephalitis in children recently. This study aimed to establish a real time PCR assay for simultaneous detection of the two subtypes of HHV-6, and apply this new assay to children with suspected encephalitis, then analyze the relationship between the infection with HHV-6 and encephalitis in children. METHOD: The universal primers and variant-specific TaqMan probes were designed based on the highly conserved sequences of the DNA polymerase gene (U38) of HHV-6. The 5' end of the probes for HHV-6A and HHV-6B was labeled with the fluorescein reporter tetrachloro-6-carboxyfluorescein and 6-carboxyfluorescein (6-FAM), separately, while the 3' end were quenched with 6-carboxy-tetramethylrhodamine. The real time PCR assay for simultaneous detection of HHV-6A and HHV-6B was established. Then, the plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 10(9) to 10(0) copies/microl, together with controls were used for testing both sensitivity and specificity of the real time PCR assay. The cerebrospinal fluid (CSF) specimens from 445 cases of suspected encephalitis were tested with this real time PCR and positive samples were then sequenced. RESULT: Both HHV-6A (strain ZJ-159) and HHV-6B (strain GS) were positive on the real time PCR assay. There were no cross-reaction with herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B virus, Staphylococcus aureus, Mycoplasma pneumoniae and human DNA. A linear regression curve was obtained when plotting Ct values against the log10 of the viral DNA input for both subtypes of HHV-6. The sensitivity threshold was 10 copies/microl for the real time PCR. HHV-6 positive rate by the real time PCR assay was 4.72% (21/445), including 4 cases with HHV-6A infection, 16 cases of HHV-6B infection and 1 case with mixed HHV-6A and HHV-6B infection. The new PCR assay usually took 2 to 3 hours to provide results. CONCLUSION: This new real time PCR assay can simultaneously detect both subtypes of HHV-6, and have high specificity and sensitivity. It will provide an early and sensitive diagnosis of HHV-6 encephalitis in children.

Rapid diagnosis of sepsis and bacterial meningitis in children with real-time fluorescent quantitative polymerase chain reaction amplification in the bacterial 16S rRNA gene.

A method for the detection of bacterial pathogens in sepsis and bacterial meningitis with 16S rRNA gene- based real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) is developed. A total of 190 blood specimens and 5 cerebrospinal fluid specimens from neonates with suspected sepsis or bacterial meningitis were evaluated with 16S rRNA gene-based real-time FQ-PCR assay. The positive rate of the real-time FQ-PCR assay was significantly higher (25/195, 12.82%) than that of bacterial culture (15/195, 7.69%; P = .002). When bacterial culture was used as a control, the sensitivity of the real-time FQ-PCR was 100%, the specificity was 94.4%, and Youden's index was 0.944. This study suggests that 16S rRNA gene-based real-time FQ-PCR assay is an important and accurate method in the detection of bacterial pathogens of sepsis and bacterial meningitis and should have a promising usage in the diagnosis of sepsis and bacterial meningitis.

Erythropoietin attenuates white matter damage, proinflammatory cytokine and chemokine induction in developing rat brain after intra-uterine infection.

To investigate the possible ameliorating effect of recombinant human erythropoietin (rhEPO) on white matter damage, pro-inflammatory cytokine and chemokine induction in developing rat brain after intra-uterine Escherichia coli infection. E. coli was inoculated into uterine cervix of the time-pregnant rats and the control was injected with normal saline. Following maternal E. coli inoculation, the pups received a single intraperitoneal injection of rhEPO at a dose of 5000 IU/kg body weight immediately after birth. Immunohistochemical staining and Western blot analysis for 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), neurofilament (NF) and glial fibrillary acidic protein (GFAP) were performed to assess white matter damage in pup brains at post-natal day 1 (P1), P3 and P7. Pro-inflammatory cytokines and chemokines were detected by real-time quantitative RT-PCR at the mRNA levels to evaluate the inflammatory response in pup brains at P1, P3 and P7. A single dose of rhEPO treatment (5000 IU/kg body weight) attenuated white matter damage in developing rat brain after intra-uterine E. coli infection. The protein levels of CNPase and NF in pup brains at P7 significantly increased after post-natal rhEPO treatment as compared with the intra-uterine E. coli-treated group. Also, post-natal rhEPO injection markedly attenuated the intra-uterine E. coli infection-induced increases in GFAP protein expression and the mRNA levels of pro-inflammatory cytokines and chemokines. Post-natal EPO administration as a single dose may exert a neuroprotective effect on white matter damage by reducing pro-inflammatory cytokine and chemokine induction in developing rat brain after intra-uterine E. coli infection.