Sequence duplication in 3' UTR modulates virus replication and virulence of Japanese encephalitis virus.

Japanese encephalitis virus (JEV), an important neurotropic pathogen, belongs to the genus Flavivirus of the family Flaviviridae and has caused huge threat to public health. It is still obscure regarding the functions of stem-loop (SL) and dumbbell (DB) domains of JEV 3' UTR in viral replication and virulence. In the current study, using the infectious clone of JEV SA14 strain as a backbone, we constructed a series of deletion mutants of 3' UTR to investigate their effects on virus replication. The results showed that partial deletions within SL or DB domain had no apparent effects on virus replication in both mammalian (BHK-21) and mosquito (C6/36) cells, suggesting that they were not involved in viral host-specific replication. However, the entire SL domain deletion (ΔVR) significantly reduced virus replication in both cell lines, indicating the important role of the complete SL domain in virus replication. The revertant of ΔVR mutant virus was obtained by serial passage in BHK-21 cells that acquired a duplication of DB domain (DB-dup) in the 3' UTR, which greatly restored virus replication as well as the capability to produce the subgenomic flavivirus RNAs (sfRNAs). Interestingly, the DB-dup mutant virus was highly attenuated in C57BL/6 mice despite replicating similar to WT JEV. These findings demonstrate the significant roles of the duplicated structures in 3' UTR in JEV replication and provide a novel strategy for the design of live-attenuated vaccines.

Clinical Characteristics of H1N1 Influenza A-Associated Mild Encephalopathy with Reversible Splenial Lesion: 4 Pediatric Cases.

OBJECTIVE: Mild encephalopathy with reversible splenial lesion (MERS) is associated with a variety of infections and anti-epileptic drug withdrawal. Here we report the clinical characteristics of H1N1 influenza A-associated MERS based on our experience of four pediatric cases. METHODS: A detailed retrospective analysis of four patients with H1N1 influenza A-associated MERS was performed at Guangzhou Women and Children's Medical Center. RESULTS: All patients exhibited mild influenza-like illness and seizures. Three patients presented with a new-onset seizure with fever after 5 years of age. 75% patients had altered mental status. For all four patients, influenza A (H1N1) viral RNA was detected in throat swab specimens at least twice. Brain magnetic resonance images revealed similar ovoid lesions in the corpus callosum, mainly in the splenium and for one patient in the splenium and genu of the corpus callosum. Only one patient had an abnormal electroencephalogram tracing. Cells and protein in the cerebrospinal fluid were normal in all patients. All patients received oseltamivir and one patient received intravenous immunoglobulin. As a result, all patients fully recovered after 2 months and showed no neurologic sequelae at discharge. CONCLUSION: This case series provides insight towards clinical features of H1N1 influenza A-associated MERS.

[Incidence of late-onset sepsis in very low birth weight and extremely low birth weight infants and risk factors for late-onset sepsis].

OBJECTIVE: To investigate the incidence of late-onset sepsis (LOS) in very low birth weight (VLBW) and extremely low birth weight (ELBW) infants in the neonatal intensive care unit (NICU) and the risk factors for LOS. METHODS: A retrospective analysis was performed for the clinical data of all VLBW and ELBW infants who were hospitalized in the NICU between January 2011 and December 2013. According to the presence or absence of LOS, these infants were divided into LOS group and non-LOS group. The incidence and mortality rates of LOS, common pathogenic bacteria, and risk factors for LOS were analyzed. RESULTS: Of the 226 VLBW and ELBW infants, 117 (51.8%) developed LOS, among whom 45 had a confirmed diagnosis of LOS and 72 had a clinical diagnosis of LOS. The LOS group had a significantly higher mortality rate than the non-LOS group [13.7% (16/117) vs 4.6% (5/109); P<0.05]. Bacterial culture found 51 strains of pathogenic bacteria, among which 32 (63%) were Gram-negative bacteria, 16 (31%) were Gram-positive bacteria, and 3 (6%) were fungi. The multivariate logistic regression analysis showed that gestational age, small for gestational age (SGA), duration of parenteral nutrition, peripherally inserted central catheter (PICC) placement, and mechanical ventilation were independent risk factors for LOS in VLBW and ELBW infants (OR=0.84, 1.59, 1.34, 3.11, and 4.55 respectively; P<0.05). CONCLUSIONS: LOS has high incidence and mortality rates in VLBW and ELBW infants. Common pathogenic bacteria of LOS are Gram-negative bacteria. Low gestational age, long duration of parenteral nutrition, SGA, PICC placement, and mechanical ventilation may increase the risk of LOS in VLBW and ELBW infants.

Effects of different surfactant administrations on cerebral autoregulation in preterm infants with respiratory distress syndrome.

To treat respiratory distress syndrome, surfactant is currently delivered via less invasive surfactant administration (LISA) or INtubation SURfactant Extubation (INSURE). The aim of this study was to compare the effect of the two delivery methods of surfactant on cerebral autoregulation. Near infrared spectroscopy monitoring was carried out to detect cerebral oxygen saturation (ScO2), and the mean arterial blood pressure (MABP) was simultaneously recorded. Of 44 preterm infants included, the surfactant was administrated to 22 via LISA and 22 via INSURE. The clinical characteristics, treatments and outcomes of the infants showed no significant differences between the two groups. The correlation coefficient of ScO2 and MABP (r ScO2-MABP) 5 min before administration was similar in the two groups. During surfactant administration, r ScO2-MABP increased in both groups (0.44±0.10 to 0.54±0.12 in LISA, 0.45±0.11 to 0.69±0.09 in INSURE). In the first and second 5 min after instillation, r ScO2-MABP was not significantly different from baseline in the LISA group, but increased in the first 5 min after instillation (0.59±0.13, P=0.000 compared with the baseline in the same group) and recovered in the second 5 min after instillation (0.48±0.10, P=0.321) in the INSURE group. There were significant differences in the change rates of r ScO2-MABP between the two groups during and after surfactant administration. Our results suggest that cerebral autoregulation may be affected transiently by surfactant administration. The effect duration of LISA is shorter than that of INSURE (<5 min in LISA vs. 5-10 min in INSURE).

Newborns' sleep-wake cycle development on amplitude integrated electroencephalography.

BACKGROUND: To observe the development of neonatal sleep among healthy infants of different conceptional age (CA) by analyzing the amplitude-integrated electroencephalography (aEEG) of their sleep-wake cycles (SWC). METHODS: Bedside aEEG monitoring was carried out for healthy newborns from 32 to 46 weeks CA between September 1, 2011 and August 30, 2012. For each aEEG tracing, mean duration of every complete SWC, number of SWC repetition within 12 hours, mean duration of each narrow and broadband of SWC, mean voltage of the upper edge and lower edge of SWC, mean bandwidth of SWC were counted and calculated. Analysis of the correlations between voltages or bandwidth of SWC and CA was performed to assess the developmental changes of central nervous system of newborns with different CA. RESULTS: The SWC of different CA on aEEG showed clearly identifiable trend after 32 weeks of CA. The occurrence of SWC gradually increases from preterm to post-term infants; term infants had longer SWC duration. The voltage of upper edge of the broadband decreased at 39 weeks, while the lower edge voltage increases and the bandwidth of broadband declined along with the growing CA. The upper edge of the narrowband dropped while the lower edge rised gradually, especially in preterm stage. The width of the narrowband narrowed down while CA increased. CONCLUSIONS: The SWC on aEEG of 32-46 weeks infants showed a continuous, dynamic and developmental progress. The appearance of SWC and the narrowing bandwidth of narrowband is the main indicator to identify the CA-dependent SWC from the preterm to the late preterm period. The lower edge of the broadband identifies the term to post-term period.

Inhibitory effects of autologous γ-irradiated cell conditioned medium on osteoblasts in vitro.

Skeletal complications from radiation therapy have been reported in patients with breast, brain and pelvic cancer, and types of blood cancer. However, it remains to be elucidated whether localized radiotherapy may result in systemic adverse effects on the unirradiated skeleton through an abscopal mechanism. The present study investigated the abscopal effect of radiation on osteoblasts mediated by autologous γ-irradiated cell conditioned medium. Osteoblasts obtained from calvarial bones were incubated with irradiated cell conditioned medium (ICCM) and changes in cell viability, alkaline phosphatase (ALP) activity, mineralization ability, cell apoptosis and the gene expression levels of ALP, osteocalcin (BGP), osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) and caspase 3 were observed. Notably, ICCM regulated osteoblast function, inhibiting viability and differentiation, resulting in apoptosis or cell death. ICCM at 10 or 20%, from osteoblasts irradiated with 10 Gy γ-rays, significantly inhibited the proliferation of osteoblastic cells (P<0.001). In addition, an increase in apoptosis was noted in the osteoblasts incubated with ICCM at 40% with increasing doses of radiation, accompanied by an upregulation in the mRNA expression of caspase 3. In addition, ICCM at 20% inhibited the ALP activity in the 5 and 10 Gy groups and osteoblast mineralization, particularly at 10 Gy ICCM. Additionally, the mRNA expression levels of ALP, BGP, OPG and RANKL of the cells treated with ICCM at 20% were downregulated significantly compared with those treated with medium from unirradiated cells. The present study provided novel evidence to elucidate radiation-therapy-associated side effects on the skeleton.

Clinical efficacy and safety of chelation treatment with typical penicillamine in cross combination with DMPS repeatedly for Wilson's disease.

The aim of this study was to assess the clinical efficacy and safety of chelation treatment with penicillamine (PCA) in cross combination with sodium 2, 3-dimercapto-1-propane sulfonate (DMPS) repeatedly in patients with Wilson's disease (WD). Thirty-five patients with WD were enrolled. They were administrated intravenous DMPS in cross combination with oral PCA alternately which was practiced repeatedly, all with Zinc in the meantime. During the treatment, clinical observations and 24-h urine copper excretion as well as adverse effects of medicines were recorded and analyzed. Although the incidence of adverse effects was not significantly different after either intravenous DMPS or oral PCA treatment, levels of 24-h urine copper tended to be higher after short-term intravenous DMPS than that of oral PCA. Adverse effects in the course of intravenous DMPS were mainly neutropenia, thrombocytopenia, allergic reaction and bleeding tendency. As compared with oral PCA alone or intravenous DMPS alone, such repeated cross combination treatment could as much as possible avoid continued drug adverse effects or poor curative effect and had less chance to stop treatment in WD patients. Improved or recovered liver function in 71% of the patients, alleviated neurologic symptoms in 50% of the patients, and disappeared hematuria in 70% of the patients could be observed during the follow-up period of 6 months to 5 years after such combined chelation regimen. Chelation treatment repeatedly with oral penicillamine in cross combination with intravenous DMPS alternately could be more beneficial for WD patients to relieve symptoms, avoid continued drug adverse effects and maintain lifelong therapy.

[Immunopathological mechanism of spleen damage in acute disseminated MCMV infection].

AIM: To explore the immunopathological mechanism of spleen damage in acute disseminated infection of murine cytomegalovirus (MCMV) in vivo by observing the association of virus titers and the expressions of caspase-1 and pro-inflammatory factors (IL-1ß and IL-18) with the degree of pathological damage of the spleen. METHODS: BALB/c mice (n=24) were randomly divided into 2 groups (n=12 per group), experimental group and control group. The control mice were sham-infected. The experimental mice were infected with MCMV Smith for establishing the models of acute disseminated MCMV infection. Three mice of each group were randomly chosen to be killed on day 3, 7, 14 and 28 after infection, respectively. Viral titers in the spleen tissues were determined using a standard plaque assay; the expression of caspase-1 in the splenocytes was detected by Western blot; the expressions of IL-1ß and IL-18 in the spleen tissues were observed by immunohistochemical staining; the degree of spleen histological damage was observed by HE staining and graded by a semiquantitative method. RESULTS: Viral titers in the spleen peaked on day 3, but quickly diminished on day 7 and virus was not detected in the spleen on day 14 after infection. Compared with the normal control group, the protein levels of caspase-1 in MCMV-infected mice were markedly elevated on day 3 (P<0.01), and then dropped slowly; the expressions of IL-1ß and IL-18 in the spleen tissues gradually increased to the climax on day 7, then decreased on day 14 and turned to the normal on day 28. However, the pathological condition of the spleen in infected mice deteriorated gradually until day 14, and then showed obviously the signs of recovery on day 28. The spleen damage was aggravated following the elevation of IL-1ß and IL-18 expressions and alleviated after they declined. CONCLUSION: MCMV infection would stimulate the increase of caspase-1 expression and the production of pro-inflammatory factors (IL-1ß and IL-18). IL-1ß and IL-18 not only exert antiviral effect, but also might be involved in the immunopathological injury of spleen tissue during the acute disseminated MCMV infection.

[Clinical analysis of 8 cases with acute invasive pulmonary aspergillosis in younger children].

OBJECTIVES: To analyze the clinical features of acute invasive pulmonary aspergillosis in younger children, in order to improve the levels of early recognition, diagnosis and management of this disease. METHOD: Clinical data of 8 patients aged below 15 months who were diagnosed as acute invasive pulmonary aspergillosis from August 2010 to February 2011 in general pediatric wards in our hospital were retrospectively analyzed for the high-risk factors of the hosts, clinical manifestations, laboratory findings and lung CT imaging, the processes of diagnosis and treatment, and the outcomes. RESULT: Five cases were tested for serum GM test absorbent index (GMI) ranged from 1.92 to 3.27; in 2 cases sputum culture was positive for Aspergillus fumigatus for twice, and 1 infant was serum GMI 2.85 and a sputum culture was positive for Aspergillus fumigatus positive, all these findings were accordant with the clinical diagnosis. Seven cases had a history of receiving intravenously broad-spectrum antibiotics or plus corticosteroids (6 hospitalized, 1 out-patient), and one was only 1 month old, whose parents had severe tinea pedis. 4 patients of high-fever type had sustained high temperature, severe changes of lungs without obvious respiratory symptoms and signs in early phase, and significant increase of the rod granulocyte rate (0.25 - 0.68), which was apparently discordant with the normal WBC count and high sensitivity C-reactive protein (hs-CRP) value. Another 4 cases of non-high-fever type were present with normal WBC count, hs-CRP value and the percentage of rod granulocyte. Among them, 3 infants had low-grade fever, with serious respiratory symptoms and signs and changes of lungs CT. Another 1-month-old case only showed lower vigor and response. Lung CT imaging often showed multiple irregular large nodules, patches and streaks of density (6 cases) and unilateral lobar consolidation (1 case), with some involving the pleura; one appeared severe peri-main bronchus lesions with stenoses of bilateral main bronchi. The first case died of multiple organ failure because of severe sepsis complication. Another 7 cases were treated with voriconazole promptly after clinical or suspected diagnosis, and the state of patients relieved rapidly within 1 - 3 d. CONCLUSION: The abuse of broad-spectrum antibiotics and corticosteroids may increase the risk of invasive pulmonary aspergillosis in younger children. There may be the risk of nosocomial infection and spread of aspergillus in general pediatric wards. Cases of high-fever type in early period of disease had two inconsistency: few symptoms and signs, while severe changes of lungs CT; apparent increase of peripheral rod granulocyte, while normal WBC count and hs-CRP value. Preemptive voriconazole therapy could obtain significant effect and reduce the mortality rate.