Plasma Heat Shock Protein 90 Alpha: A Valuable Predictor of Early Chemotherapy Effectiveness in Advanced Non-Small-Cell Lung Cancer.

BACKGROUND Heat shock protein-90 alpha (HSP90a) is more abundant in non-small-cell lung cancer (NSCLC) patients than in control individuals. However, whether it can reflect chemotherapy efficacy remains unknown. This study aimed to investigate the association of HSP90a with chemotherapy in advanced NSCLC. MATERIAL AND METHODS We retrospectively evaluated data from patients admitted to the Department of Respiratory Medicine, Shaoxing People's Hospital, from September 2016 to September 2018 with stage IIIB or IV NSCLC and administered 4 cycles of third-generation platinum-based combination chemotherapy (2 drugs simultaneously). Based on the RECIST1.1 criteria, complete remission (CR), partial response (PR), and stable disease (SD) in 60 cases were determined before and after chemotherapy. Before chemotherapy and after 1, 2, and 4 cycles of chemotherapy, plasma HSP90alpha levels were quantitated by ELISA. Chest CT was performed before and after 2 and 4 cycles of chemotherapy. RESULTS After 1-4 cycles of chemotherapy, plasma HSP90alpha levels were significantly lower than pre-chemotherapy levels (P<0.05). The sums of the longest tumor diameters after 2 and 4 cycles of chemotherapy were decreased compared with pre-chemotherapy values (P<0.05). Plasma HSP90alpha levels and tumor size showed no significant correlation before and after chemotherapy (r=0.244, P=0.06). CONCLUSIONS Plasma HSP90alpha can be considered a valuable predictor of early chemotherapy effectiveness in advanced NSCLC, and is positively correlated with tumor remission after chemotherapy. However, plasma HSP90alpha level is not correlated with tumor diameter and pathological type.

[Effects of different drugs on bronchial stenosis by TGF-ß/mTOR signaling pathway in rabbit model].

Objective: To investigate the effect of different drugs on tracheal stenosis caused by transforming growth factor-ß/rapamycin target protein (TGF-ß/mTOR) signaling pathway. Methods: Thirty rabbits were randomly divided into normal control group, normal saline group, penicillin group, budesonide group and erythromycin group. The normal control group was not treated,and tracheal stenosis models were established in the other groups. From the 1st to 10th day after modeling, each group was respectively administered with normal saline (0.75 ml/kg, 2 times/d), intramuscular injection of penicillin (40 000 U/kg, 2 times/d), gastric administration of erythromycin (12.5 mg/kg, 2 times/d), inhalation of budesonide (0.05 mg/kg, 2 times/d). Rabbits were sacrificed on the 11th day after surgery, and tracheal specimens were collected to measure the degree of tracheal stenosis. Relative mRNA expression level of interleukin-6 (IL-6), transforming growth factor-ß (TGF-ß), Type Ⅰ collagen (COL-1), Type Ⅲ collagen (COL-3), and Sirtuin 1 (SIRT-1) were detected by Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR); protein expression of mTOR, phosphorylated protein kinase B (p-AKT), vascular endothelial growth factor (VEGF),SIRT-1 were detected by immunohistochemical analysis; protein expression of nuclear factor κB (NF-κB),phosphorylated nuclear factor κB (p-NF-κB),protein kinase B (AKT),p-AKT,mTOR were detected by Western blotting. Results: The degree of stenosis of normal control group was (14.02±2.86)%, saline group was (64.14±3.21)%, penicillin group was (49.11±2.96)%, budesonide group was (39.52±2.09)%, erythromycin group was (32.60±4.27)%. The differences between any two groups were statistically significant (all P<0.05). Except between erythromycin group and normal control group, the differences in relative expression of IL-6 mRNA between any two groups (1.00±0.00, 9.02±1.50, 4.25±0.87, 2.53±0.17, 1.31±0.56) was statistically significant (all P<0.05), and the differences in relative expression of TGF-ß mRNA among all groups (1.00±0.00, 6.92±0.84, 3.83±0.44, 2.13±0.25, 1.40±0.15) were statistically significant (all P<0.05). The relative expression of SIRT-1 mRNA among all the groups (1.000±0.000, 0.209±0.042, 0.375±0.034, 0.555±0.028, 0.667±0.032) was statistically significant different (all P<0.05); except between erythromycin group and budesonide group,the protein levels of SIRT-1 among all other groups (16.93±2.28, 4.77±1.45, 7.70±0.61, 10.76±1.04, 11.03±1.10) were statistically significant different (all P<0.05). The protein levels of mTOR (9.28±4.56, 58.18±8.12, 44.75±5.56, 32.82±5.99, 24.73±3.56) and p-AKT (16.57±4.86, 61.79±6.66, 42.98±5.99, 32.79±5.34, 24.00±4.40) determined through immunohistochemistry of all groups were statistically significant different (all P<0.05). The protein levels of NF-κB, p-NF-κB, AKT, p-AKT and mTOR determined through Western blotting had the same trend as that of determined through immunohistochemistry. The protein expression of NF-κB,AKT and mTOR in saline group were significantly higher than other groups; those protein expression of erythromycin group was lower than budesonide group and penicillin group. Except between the erythromycin group and the normal control group, the protein expression of mTOR in other groups was statistically significant different (all P<0.05). Conclusion: Penicillin,erythromycin and budesonide can alleviate inflammation by increasing SIRT-1, alleviate tracheal scar hyperplasia induced by TGF-beta/mTOR pathway, and reduce the degree of tracheal stenosis in rabbits.

The synergic effects of CTLA-4/Foxp3-related genotypes and chromosomal aberrations on the risk of recurrent spontaneous abortion among a Chinese Han population.

The current study was aimed to investigate the association of CLTA-4/Foxp3 polymorphisms and chromosomal abnormalities with recurrent spontaneous abortion (RSA) risk in a Chinese Han population. Altogether, 1284 RSA women and 1046 women with normal pregnancy were incorporated in this study. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was implemented to genotype the single-nucleotide polymorphisms (SNPs) located within CTLA4 and Foxp3. Moreover, the cytogenetic diagnosis was performed in line with the standards of G banding karyotype. As a consequence, rs231775 and rs3087243 of CTLA4, as well as rs2232365 and rs2232368 of Foxp3, all appeared to modify the risk of RSA. Besides, significant differences were found between the ratio of structural abnormality and that of numerical abnormality (P < 0.038), and chromosome abnormality was associated with higher miscarriage frequency (>3) than normal karyotypes. Of note, the synergic effects of the genotypes and chromosomal abnormality all tallied with the sub-multiplication model (ORchromosome × ORSNP > ORchromosome+SNP), while rs2232365 GG and chromosomal aberration impacted the RSA risk in a super-multiplicative way that ORchromosome × ORSNP < ORchromosome+SNP. In conclusion, susceptibility to RSA was subject to the synthetic regulation of chromosomal aberrations and genetic mutations within CLTA-4 and Foxp3, suggesting that the conduction of karyotype analysis and genetic detection for RSA patients could effectively guide effective RSA counseling and sound child rearing.

[Clinical features of community-acquired bloodstream infection due to Gram-negative bacilli in patients with liver cirrhosis].

Objective: o investigate the features of pathogenic bacteria for community-acquired bloodstream infection due to Gram-negative bacilli in patients with liver cirrhosis and optimal therapeutic strategy. Methods: A retrospective analysis was performed for the clinical data of patients with liver cirrhosis who were admitted to 302 Hospital of PLA due to community-acquired bloodstream infection from January 2010 to December 2015, and a statistical analysis was performed for their clinical features, pathogenic bacteria, and results of drug sensitivity test. The Pearson chi-square test was used for comparison of rates, and the Wilcoxon rank sum test was used for comparison of ranked data. Results: A total of 240 patients (including 178 male patients) with liver cirrhosis caused by various reasons were enrolled, with a mean age of 51.7 ± 11.1 years, an overall clinical remission rate of 80.42%, and an ineffective/mortality rate of 19.58%. The patients who used sensitive antibiotics within 12 hours after the onset of community-acquired bloodstream infection achieved a significantly higher improvement rate than those who used such drugs at more than 12 hours after onset (88.2% vs 58.1%, P < 0.001). The improvement rate achieved by the application of sensitive antibiotics at more than 12 hours after onset decreased with the increase in the Child-Pugh grade (P < 0.05). A total of 245 strains of Gram-negative bacilli were isolated, among which the six most common ones were 135 strains of Escherichia coli (55.1%), 62 strains of Klebsiella pneumoniae (25.3%), 16 strains of Aeromonas (6.5%), 4 strains of non-typhoidal Salmonella (1.6%), 3 strains of Enterobacter cloacae (1.2%), and 2 strains of Acinetobacter baumannii (0.8%). These Gram-negative bacilli had the highest sensitivity to meropenem (98.5%), followed by imipenem (97.9%), amikacin (97.5%), piperacillin/tazobactam (94.7%), cefmetazole (93.7%), and cefoperazone/sulbactam (93%). Different bacteria had different sensitivities to antibiotics. Conclusion: Once community-acquired bloodstream infection occurs in patients with liver cirrhosis, highly sensitive antibiotics should be used as early as possible. Cefoperazone/sulbactam, piperacillin/tazobactam, imipenem, and meropenem can be used as first-line empirical antibiotics, and drug combination should be considered when necessary.

The Emerging Duck Flavivirus Is Not Pathogenic for Primates and Is Highly Sensitive to Mammalian Interferon Antiviral Signaling.

UNLABELLED: Flaviviruses pose a significant threat to both animals and humans. Recently, a novel flavivirus, duck Tembusu virus (DTMUV), was identified to be the causative agent of a serious duck viral disease in Asia. Its rapid spread, expanding host range, and uncertain transmission routes have raised substantial concerns regarding its potential threats to nonavian hosts, including humans. Here, we demonstrate that DTMUV is not pathogenic for nonhuman primates and is highly sensitive to mammal type I interferon (IFN) signaling. In vitro assays demonstrated that DTMUV infected and replicated efficiently in various mammalian cell lines. Further tests in mice demonstrated high neurovirulence and the age-dependent neuroinvasiveness of the virus. In particular, the inoculation of DTMUV into rhesus monkeys did not result in either viremia or apparent clinical symptoms, although DTMUV-specific humoral immune responses were detected. Furthermore, we revealed that although avian IFN failed to inhibit DTMUV in avian cells, DTMUV was more sensitive to the antiviral effects of type I interferon than other known human-pathogenic flaviviruses. Knockout of the type I IFN receptor in mice caused apparent viremia, viscerotropic disease, and mortality, indicating a vital role of IFN signaling in protection against DTMUV infection. Collectively, we provide direct experimental evidence that this novel avian-origin DTMUV possesses a limited capability to establish infection in immunocompetent primates due to its decreased antagonistic activity in the mammal IFN system. Furthermore, our findings highlight the potential risk of DTMUV infection in immunocompromised individuals and warrant studies on the cross-species transmission and pathogenesis of this novel flavivirus. IMPORTANCE: Mosquito-borne flaviviruses comprise a large group of pathogenic and nonpathogenic members. The pathogenic flaviviruses include dengue, West Nile, and Japanese encephalitis viruses, and the nonpathogenic flaviviruses normally persist in a natural cycle and rarely cause disease in humans. A novel flavivirus, DTMUV (also known as duck egg drop syndrome flavivirus [DEDSV]) was identified in 2012 in ducks and then rapidly spread to several Asian countries. This new flavivirus was then shown to infect multiple avian species, resulting in neurological symptoms with unknown routes of transmission. There is public concern regarding its potential transmission from birds to humans and other nonavian hosts. Our present study shows that the mammalian IFN system can efficiently eliminate DTMUV infection and that the emergence of severe DTMUV-associated disease in mammals, especially humans, is unlikely. Currently, DTMUV infection mostly affects avian species.

A high-performance electrochemical supercapacitor based on a polyaniline/reduced graphene oxide electrode and a copper(ii) ion active electrolyte.

Active electrolyte enhanced supercapacitors (AEESCs) have received increasing attention because of their large specific capacitance and easy fabrication process. The better matching between the active electrolyte and the counter electrode and the slow self-discharge rate are the challenges of this type of supercapacitor. In this paper, a novel AEESC with polyaniline/reduced graphene oxide hydrogel (PANI/RGOHG) as the anode and Cu(ii) ions as the cathodic active electrolyte is constructed. Experimental results demonstrate that the electrode potentials of PANI and Cu(ii) can match perfectly, thus the device has a wide working voltage range. Because of the large specific capacitance of both PANI and Cu(ii), a high average specific capacitance of a single electrode of 1120 F g-1 at 2.6 A g-1 is achieved. Meanwhile, self-discharge is also suppressed because the reduction product of Cu(ii) is immobilized on the electrode. These results demonstrate that the performance of AEESCs strongly depends on the choice of a suitable electrode material, and also reveal that Cu(ii) is a promising cathodic active electrolyte for AEESCs.

[Expression and significance of autophagy in rabbit model of tracheal stenosis].

Objective: To investigate the expression and significance of autophagy in rabbit model of tracheal stenosis. Methods: A total of 18 rabbits were equally divided into 3 groups (blank control group, saline group, erythromycin group) in accordance with the random number table. After rabbit model of tracheal stenosis was established, no treatment was done with blank control group. Saline group was atomized with saline (0.54 mg/kg, 2 times/day), and erythromycin group was fed on erythromycin (7.5 mg/kg, 2 times/day) for 7 days before and 10 days after the operation. On the eleventh day, rabbits were executed, and their trachea were collected. The proportion of collagen fiber area of tracheal lamina propria (LP) and epithelium (EP) was assessed by Masson staining. The mRNA of autophagy associated gene-3 (ATG3) and autophagy associated gene-5 (ATG5) of tracheal mucosa were assessed by Real-Time Polymerase Chain Reaction (RT-PCR). The protein of microtubule-associated protein 1 light chain ß(3) (LC3B), ATG3 and ATG5 were assessed by Western blot. Results: The proportion of collagen fiber area of tracheal LP and EP of blank control group was (6.79±0.67)%, saline group was (40.55±5.40)%, erythromycin group was (27.48±0.43)%. The differences between any two groups was all statistically significant (all P<0.01). The relative value of ATG3 mRNA and ATG5 mRNA in saline group were significantly lower than blank control group (all P<0.01). Those value in erythromycin group were significantly higher than the saline group (all P<0.01). The protein levels of LC3B-Ⅱ/Ⅰ, ATG3 and ATG5 in saline group were significantly lower than blank control group (all P<0.01). After low dose of erythromycin intervention, all the protein levels were significantly higher than the saline group (all P<0.01). Conclusions: The expression of autophagy is decreased in rabbit model of trachea stenosis. Low dose of erythromycin could increase the expression of autophagy and at the same time alleviate the degree of fibrosis of the tracheal mucosa. Autophagy may alleviate tracheal fibrosis through up-regulating its expression level and play a protective role.

[Effect of low dose erythromycin on the proliferation of granulation tissue after tracheal injury].

Objective: To investigate the effect of low dose erythromycin on the proliferation of granulation tissue after tracheal injury. Methods: Forty-two rabbits were randomly divided into 7 groups (n=6 each), group A (saline control group), group B (penicillin group), group C (low dose erythromycin group), group D (low dose erythromycin and penicillin group), group E (budesonide group), group F (low dose erythromycin and budesonide group), group G (low dose erythromycin, penicillin and budesonide group). All rabbits received tracheotomy, and the tracheal mucosa was scraped with a nylon brush 20 times for tracheal stenosis model. Rabbits were treated with corresponding drugs from a week before operation to 9 days after operation. The serum concentrations of transforming growth factor - beta 1 (TGF-ß(1)), vascular endothelial growth factor (VEGF), interleukin (IL) -6, IL-8 were determined and the tracheal specimens were harvested for measuring degree of stenosis on the 10th day after operation. Results: Serum concentrations of TGF-ß(1) in group A, B, C, D, E, F and G were (17.6±1.3), (18.2±3.1), (13.0±1.1), (14.0±1.0), (21.0±6.1), (13.6± 3.5), (8.2±1.3) ng/L; VEGF were (88.1±4.1), (85.8±4.3), (58.1±6.3), (56.5±2.4), (87.8±2.8), (57.0±3.7), (34.3±6.7) ng/L; IL-6 were (67.8±4.0), (66.1±3.5), (54.1±4.8), (52.1±3.2), (64.6±4.9), (49.4±4.2), (35.9±3.7) ng/L; IL-8 were (112.8±5.2), (116.6±4.1), (88.0±6.2), (85.5±3.5), (114.4±4.6), (82.6±3.8), (55.9±6.0) ng/L, respectively. The serum concentrations of TGF-ß(1), VEGF, IL-6 and IL-8 in group C, D, F and G were significantly lower than those in group A, B and E (all P<0.05). Compared with the other groups, the serum concentrations in group G were the lowest (all P<0.05). All 42 rabbits had tracheal stenosis with different degrees of proliferation of granulation tissue. The degree of tracheal stenosis in Group A, B, C, D, E, F and G were (53.3±4.4)%, (48.2±5.0)%, (24.3±4.4)%, (29.5±3.2)%, (47.8±6.5)%, (27.9±3.1)%, (15.6±2.0)%, respectively. The degree of tracheal stenosis in group C, D, F and G was significantly lower than that in group A, B and E, which had statistical differences (all P<0.05). Compared with the other groups, the degree of tracheal stenosis in group G was the lowest (all P<0.05). Conclusions: Low dose of erythromycin can effectively inhibit the proliferation of granulation tissue after tracheal injury in rabbits. And it has better effectiveness when combined with other antibiotics and hormone.

In vitro and in vivo characterization of chimeric duck Tembusu virus based on Japanese encephalitis live vaccine strain SA14-14-2.

Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate.

Clinical and immunological analysis of measles patients admitted to a Beijing hospital in 2014 during an outbreak in China.

At the end of 2013, China reported a countrywide outbreak of measles. From January to May 2014, we investigated the clinical and immunological features of the cases of the outbreak admitted to our hospital. In this study, all 112 inpatients with clinically diagnosed measles were recruited from the 302 Military Hospital of China. The virus was isolated from throat swabs from these patients, and cytokine profiles were examined. By detecting the measles virus of 30 of the 112 patients, we found that this measles outbreak was of the H1 genotype, which is the major strain in China. The rates of complications, specifically pneumonia and liver injury, differed significantly in patients aged 18 years: pneumonia was more common in children, while liver injury was more common in adults. Pneumonia was a significant independent risk factor affecting measles duration. Compared to healthy subjects, measles patients had fewer CD4+IL-17+, CD4+IFN-γ +, and CD8+IFN-γ + cells in both the acute and recovery phases. In contrast, measles patients in the acute phase had more CD8+IL-22+ cells than those in recovery or healthy subjects. We recommend that future studies focus on the age-related distribution of pneumonia and liver injury as measles-related complications as well as the association between immunological markers and measles prognosis.

Rational design of thermostable vaccines by engineered peptide-induced virus self-biomineralization under physiological conditions.

The development of vaccines against infectious diseases represents one of the most important contributions to medical science. However, vaccine-preventable diseases still cause millions of deaths each year due to the thermal instability and poor efficacy of vaccines. Using the human enterovirus type 71 vaccine strain as a model, we suggest a combined, rational design approach to improve the thermostability and immunogenicity of live vaccines by self-biomineralization. The biomimetic nucleating peptides are rationally integrated onto the capsid of enterovirus type 71 by reverse genetics so that calcium phosphate mineralization can be biologically induced onto vaccine surfaces under physiological conditions, generating a mineral exterior. This engineered self-biomineralized virus was characterized in detail for its unique structural, virological, and chemical properties. Analogous to many exteriors, the mineral coating confers some new properties on enclosed vaccines. The self-biomineralized vaccine can be stored at 26 °C for more than 9 d and at 37 °C for approximately 1 wk. Both in vitro and in vivo experiments demonstrate that this engineered vaccine can be used efficiently after heat treatment or ambient temperature storage, which reduces the dependence on a cold chain. Such a combination of genetic technology and biomineralization provides an economic solution for current vaccination programs, especially in developing countries that lack expensive refrigeration infrastructures.

Genotype-specific neutralization determinants in envelope protein: implications for the improvement of Japanese encephalitis vaccine.

Japanese encephalitis remains the leading cause of viral encephalitis in children in Asia and is expanding its geographical range to larger areas in Asia and Australasia. Five genotypes of Japanese encephalitis virus (JEV) co-circulate in the geographically affected areas. In particular, the emergence of genotype I (GI) JEV has displaced genotype III (GIII) as the dominant circulating genotype in many Asian regions. However, all approved vaccine products are derived from GIII strains. In the present study, bioinformatic analysis revealed that GI and GIII JEV strains shared two distinct amino acid residues within the envelope (E) protein (E222 and E327). By using reverse genetics approaches, A222S and S327T mutations were demonstrated to decrease live-attenuated vaccine (LAV) SA14-14-2-induced neutralizing antibodies in humans, without altering viral replication. A222S or S327T mutations were then rationally engineered into the infectious clone of SA14-14-2, and the resulting mutant strains retained the same genetic stability and attenuation characteristics as the parent strain. More importantly, immunization of mice with LAV-A222S or LAV-S327T elicited increased neutralizing antibodies against GI strains. Together, these results demonstrated that E222 and E327 are potential genotype-related neutralization determinants and are critical in determining the protective efficacy of live Japanese encephalitis vaccine SA14-14-2 against circulating GI strains. Our findings will aid in the rational design of the next generation of Japanese encephalitis LAVs capable of providing broad protection against all JEV strains belonging to different genotypes.

Clinical characteristics of 154 patients suspected of having Ebola virus disease in the Ebola holding center of Jui Government Hospital in Sierra Leone during the 2014 Ebola outbreak.

This article sought to analyze the clinical features of 154 patients suspected of having Ebola virus disease (EVD) in an Ebola holding center in Sierra Leone from October 1 through November 9, 2014. We found that 108 of the 154 patients were confirmed with EVD. Eighty-five had known outcomes. Forty-nine of the 85 patients had been exposed to EVD. The average mortality rate was 60%. The mean interval between the onset of symptoms and hospitalization was 5.8 ± 3.3 days. The mean incubation period was 9.2 ± 6.7 days. Common symptoms of the EVD patients on admission were fatigue (85.2%), anorexia (84.3%), fever (75.9%), and headache (72.2%). Our data showed that the total symptoms of confirmed EVD patients were significantly higher than those of non-EVD patients (9 vs. 5.5; p < 0.001). The likelihood of EVD was 87.6% when a patient presented more than 6 out of 21 symptoms on admission. The survivors were significantly younger than non-survivors (24.0 ± 10.0 years vs. 31.3 ± 15.3 years; p = 0.016). The real-time polymerase chain reaction (PCR) analysis showed that, in the survivors, the virus load was significantly lower (Ct value: 25.2 ± 4.1 vs. 28.7 ± 5.7; p = 0.002). Multivariate analysis showed that age, fever, and viral load were independent predictors of mortality. Taken together, our data suggested that a cutoff of six symptoms could be used to predict patients with high or low risk of EVD. It seemed that age, fever, and viral load were the main risk factors associated with EVD mortality.

Generation and characterization of a protective mouse monoclonal antibody against human enterovirus 71.

Human enterovirus 71 (EV71) infection has emerged as a major threat to children; however, no effective antiviral treatment or vaccine is currently available. Antibody-based treatment shows promises to control this growing public health problem of EV71 infection, and a few potent monoclonal antibodies (mAbs) targeting viral capsid protein have been well described. Here, we generated an EV71-specific mouse mAb 2G8 that conferred full protection against lethal EV71 challenge in a suckling mouse model. 2G8 belonged to IgM isotype and neutralized EV71 at the attachment stage. Biochemical assays mapped the binding epitope of 2G8 to the SP70 peptide, which spanning amino acid residues 208-222 on the VP1 protein. Alanine scanning mutagenesis defined the essential roles of multiple residues, including Y208, T210, G212, K215, K218, L220, E221, and Y222, for 2G8 binding. Then, a panel of single mutation was individually introduced into the EV71 infectious clone by reverse genetics, and three mutant viruses, K215A, K218A, and L220A, were successfully recovered and characterized. Biochemical and neutralization assays revealed that K218A mutant partially escaped 2G8 neutralization, while L220A completely abolished 2G8 binding and neutralization. In particular, neutralization assays with human sera demonstrated that K218A and L220A substitutions are also critical for antibody neutralization in natural infection population. These findings not only generate a protective mAb candidate with therapeutic potential but also provide insights into antibody-mediated EV71 neutralization mechanism.

Novel cis-acting element within the capsid-coding region enhances flavivirus viral-RNA replication by regulating genome cyclization.

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5' cyclization sequence (5'CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5'CS, and the presence of DCS-PK facilitates the formation of 5'-3' RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.

Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation.

Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'-O methylation is replicative, but its viral RNA lacks 2'-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-O methyltransferases.

A chimeric dengue virus vaccine using Japanese encephalitis virus vaccine strain SA14-14-2 as backbone is immunogenic and protective against either parental virus in mice and nonhuman primates.

The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.

Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection of novel avian influenza A (H7N9) virus.

BACKGROUND: The emerged human infection with avian influenza A (H7N9) virus in China since 2013 has aroused global concerns. There is great demand for simple and rapid diagnostic method for early detection of H7N9 to provide timely treatment and disease control. The aim of the current study was to develop a rapid, accurate and feasible reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of H7N9 virus. RESULTS: The detection limits of the H7- and N9-specific RT-LAMP assay were both approximately 0.2 PFU per reaction. No cross-reactivity was observed with other subtype of influenza viruses or common respiratory viral pathogens. The assay worked well with clinical specimens from patients and chickens, and exhibited high specificity and sensitivity. CONCLUSIONS: The H7/N9 specific RT-LAMP assay was sensitive and accurate, which could be a useful alternative in clinical diagnostics of influenza A (H7N9) virus, especially in the hospitals and laboratories without sophisticated diagnostic systems.

A novel reporter system for neutralizing and enhancing antibody assay against dengue virus.

BACKGROUND: Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. RESULTS: In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals. CONCLUSIONS: This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies.

Recombinant tandem multi-linear neutralizing epitopes of human enterovirus 71 elicited protective immunity in mice.

BACKGROUND: Human Enterovirus 71 (EV71) has emerged as the leading cause of viral encephalitis in children, especially in the Asia-Pacific regions. EV71 vaccine development is of high priority at present, and neutralization antibodies have been documented to play critical roles during in vitro and in vivo protection against EV71 infection. RESULTS: In this study, a novel strategy to produce EV71 vaccine candidate based on recombinant multiple tandem linear neutralizing epitopes (mTLNE) was proposed. The three well identified EV71 linear neutralizing epitopes in capsid proteins, VP1-SP55, VP1-SP70 and VP2-SP28, were sequentially linked by a Gly-Ser linker ((G4S)3), and expressed in E.coli in fusion with the Trx and His tag at either terminal. The recombinant protein mTLNE was soluble and could be purified by standard affinity chromatography. Following three dosage of immunization in adult mice, EV71-specific IgG and neutralization antibodies were readily induced by recombinant mTLNE. IgG subtyping demonstrated that lgG1 antibodies dominated the mTLNE-induced humoral immune response. Especially, cytokine profiling in spleen cells from the mTLNE-immunized mice revealed high production of IL-4 and IL-6. Finally, in vivo challenge experiments showed that passive transfer with anti-mTLNE sera conferred full protection against lethal EV71 challenge in neonatal mice. CONCLUSION: Our results demonstrated that this rational designed recombinant mTLNE might have the potential to be further developed as an EV71 vaccine in the future.