[Personality and psychological characteristics of premature ejaculation patients].

OBJECTIVE: To investigate the personality and psychological characteristics of premature ejaculation (PE) patients and the correlation between them two. METHODS: Using Eysenck Personality Questionnaire (EPQ) and Symptom Checklist 90 (SCL-90), we conducted an investigation among 94 PE patients seeking medical advice in Drum Tower Hospital from October 2018 to February 2019. RESULTS: The neuroticism score of the PE patients on EPQ was significantly higher than the national norm of adult males (t = 12.010, P < 0.01), and so was their introversion-extroversion score (t = 2.557, P < 0.05), while their concealment score was markedly lower (t = －8.736, P < 0.01). The coercion score of the patients on SCL-90 was remarkably higher than the national norm of adult males (t = 2.787, P < 0.01), and so were their psychosis score (t = 3.944, P < 0.01) and anxiety score (t = 2.512, P < 0.05). There was a significant correlation between the EPQ and SCL-90 scores of the patients. Psychoticism was found highly positively correlated with terror (r = 0.455, P < 0.01), interpersonal relationship (r = 0.295, P < 0.01), hostility (r = 0.375, P < 0.01), psychosis (r = 0.363, P < 0.01), compulsion (r = 0.284, P < 0.01), depression (r = 0.294, P < 0.01), paranoia (r = 0.336, P < 0.01), somatization (r = 0.400, P < 0.01) and anxiety (r = 0.358, P < 0.01), and so was neuroticism with terror (r = 0.466, P < 0.01), interpersonal relationship (r = 0.611, P < 0.01), hostility (r = 0.509, P < 0.01), psychosis (r = 0.593, P < 0.01), compulsion (r = 0.573, P < 0.01), depression (r = 0.560, P < 0.01), paranoia (r = 0.550, P < 0.01), somatization (r = 0.465, P < 0.01) and anxiety (r = 0.572, P < 0.01). Introversion-extroversion, however, was highly negatively correlated with interpersonal relationship (r = －0.226, P < 0.05) and depression (r = －0.228, P < 0.05), and so was concealment with terror (r= － 0.351, P < 0.01), interpersonal relationship (r = －0.433, P < 0.01), hostility (r = －0.347, P < 0.01), psychosis (r = －0.427, P < 0.01), compulsion (r = －0.345, P < 0.01), depression (r = －0.379, P < 0.01) , paranoia (r = －0.393, P < 0.01), somatization (r = －0.204, P < 0.05) and anxiety (r =－0.237, P < 0.05). CONCLUSIONS: The personality and psychological status of PE patients are different from those of normal males, and some personality characteristics of the patients are correlated with their psychological status, especially with high neuroticism.

[Application of fast-track surgery concept in the perioperative nursing care of andrological patients: A randomized controlled study].

OBJECTIVE: To evaluate the application of the fast-track surgery (FTS) concept in the nursing care of andrological patients during the perioperative period. METHODS: A total of 200 males to be treated by andrological surgery were included in a control group and another 200 in an observation group, the former received conventional perioperative nursing care, while the latter underwent an FTS nursing care procedure including a variety of proven effective methods to reduce surgical stress and achieve a quick recovery during the perioperative period. Comparisons were made between the two groups of patients in the postoperative enterokinesia time, anal exhaust time, eating time, off-bed time, defecating time, bowel preparation complications, and degree of comfort and satisfaction. RESULTS: Compared with the controls, the patients in the observation group showed significantly earlier postoperative enterokinesia time (ï¼»5.8±0.9ï¼½ vs ï¼»4.4±1.4ï¼½ h, P<0.01), anal exhaust time (ï¼»10.8±1.8ï¼½ vs ï¼»7.7±2.0ï¼½ h, P<0.01), eating time (ï¼»12.9±0.7ï¼½ vs ï¼»6.3±0.7ï¼½ h, P<0.01), off-bed time ï¼»14.3±2.7ï¼½ vs ï¼»8.2±1.4ï¼½ h, P<0.01), and defecating time (ï¼»49.2±2.6ï¼½ vs ï¼»39.6±2.5ï¼½ h, P<0.01), a lower incidence of bowel preparation complications (P<0.01), and a higher degree of comfort (P<0.01) and satisfaction (ï¼»97.5±0.7ï¼½% vs ï¼»99.4±+0.3ï¼½ %, P<0.01). CONCLUSIONS: The FTS concept can be safely and effectively applied to the perioperative nursing care of andrological patients to achieve a faster recovery and higher degree of comfort and satisfaction postoperatively.

[Aging-related change of erectile function and the expression of SIRT1].

OBJECTIVE: To explore aging-related changes in erectile function and the expressions of SIRT1 and other relevant factors in rats. METHODS: We divided 40 male SD rats into four age groups of equal number: 2-month-old (2 mo), 8-month-old (8 mo), 14-month-old (14 mo), and 20-month-old (20 mo), measured the intracavernous pressure (ICP), mean arterial pressure (MAP), and ICP/MAP ratio by electrostimulation of the cavernous nerve, evaluated fibrosis in the corpus cavernosum by Masson's trichrome staining, detected the expressions of SIRT1, P53, and FOXO3a by Western blot, and determined the levels of NO and cGMP using the NO/cGMP kit. RESULTS: Both the ICP/MAP ratio and the cGMP level were elevated with aging, reaching the peak at 8 months and then gradually decreased. Masson staining showed an aging-related increase of collagen fibers in the corpus cavernosum.The expression of SIRT1 was reduced while those of P53 and FOXO3a increased with aging. CONCLUSIONS: Aging-related erectile dysfunction may be attributed to the reduced activity of the NO/cGMP pathway, apoptosis and oxidative stress, and SIRT1 may play a role in aging-related erectile dysfunction.

Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans.

A unique and conserved neutralization epitope in H5N1 influenza viruses identified by an antibody against the A/Goose/Guangdong/1/96 hemagglutinin.

Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab' fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.

[Combination of acupressure and magnetic sticker improved the quality of life in patients with advanced gastroenteric tumor: a clinical observation].

OBJECTIVE: To explore the clinical effect of combination of acupressure and magnetic sticker on the quality of life (QOL) including appetite, defecation, and sleep in patients with advanced gastroenteric tumor. METHODS: Totally 147 patients with advanced gastroenteric tumor were assigned to 4 groups according to different treatment methods, i.e., the supportive treatment group (A, 20 cases), the acupressure treatment group (B, 41 cases), the magnetic sticker treatment group (C, 40 cases), and a combination of acupressure and magnetic sticker treatment group (D, 46 cases). They were respectively treated with different methods, supportive treatment for group A, acupressure for group B, magnetic sticker for group C, and a combination of acupressure and magnetic sticker for group D. The scores of food intake, defecation frequency, sleep time, Karnofsky, and QOL were compared before treatment and at day 14 after treatment. RESULTS: After treatment, the scores of food intake, defecation frequency, and sleep time were obviously improved in B, C and D groups (P < 0.01). There was statistical difference between group D and group A (P < 0.01). In addition, in comparison with A group, both Karnofsky score and QOL score increased in B, C and D groups (P < 0.01). CONCLUSION: The assisted therapy of the combination of acupressure and magnetic sticker could ameliorate QOL such as the digestive functions and sleep in patients with advanced gastroenteric tumor.

Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation.

The emerging novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) was recently isolated from patients with severe pneumonia and renal failure and was associated with an unexplained high crude fatality rate of 56%. We performed a cell line susceptibility study with 28 cell lines. HCoV-EMC was found to infect the human respiratory tract (polarized airway epithelium cell line Calu-3, embryonic fibroblast cell line HFL, and lung adenocarcinoma cell line A549), kidney (embryonic kidney cell line HEK), intestinal tract (colorectal adenocarcinoma cell line Caco-2), liver cells (hepatocellular carcinoma cell line Huh-7), and histiocytes (malignant histiocytoma cell line His-1), as evident by detection of high or increasing viral load in culture supernatants, detection of viral nucleoprotein expression by immunostaining, and/or detection of cytopathic effects. Although an infected human neuronal cell line (NT2) and infected monocyte and T lymphocyte cell lines (THP-1, U937, and H9) had increased viral loads, their relatively lower viral production corroborated with absent nucleoprotein expression and cytopathic effects. This range of human tissue tropism is broader than that for all other HCoVs, including SARS coronavirus, HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63, which may explain the high mortality associated with this disease. A recent cell line susceptibility study showed that HCoV-EMC can infect primate, porcine, and bat cells and therefore may jump interspecies barriers. We found that HCoV-EMC can also infect civet lung fibroblast and rabbit kidney cell lines. These findings have important implications for the diagnosis, pathogenesis, and transmission of HCoV-EMC.

Dengue virus envelope domain III immunization elicits predominantly cross-reactive, poorly neutralizing antibodies localized to the AB loop: implications for dengue vaccine design.

Dengue virus (DENV) is a mosquito-borne virus that causes severe health problems. An effective tetravalent dengue vaccine candidate that can provide life-long protection simultaneously against all four DENV serotypes is highly anticipated. A better understanding of the antibody response to DENV envelope protein domain III (EDIII) may offer insights into vaccine development. Here, we identified 25 DENV cross-reactive mAbs from immunization with Pichia pastoris-expressed EDIII of a single or all four serotype(s) using a prime-boost protocol, and through pepscan analysis found that 60 % of them (15/25) specifically recognized the same highly conserved linear epitope aa 309-320 of EDIII. All 15 complex-reactive mAbs exhibited significant cross-reactivity with recombinant EDIII from all DENV serotypes and also with C6/36 cells infected with DENV-1, -2, -3 and -4. However, neutralization assays indicated that the majority of these 15 mAbs were either moderately or weakly neutralizing. Through further epitope mapping by yeast surface display, two residues in the AB loop, Q316 and H317, were discovered to be critical. Three-dimensional modelling analysis suggests that this epitope is surface exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially on the surface of the mature virion. It is concluded that EDIII as an immunogen may elicit cross-reactive mAbs toward an epitope that is not exposed on the virion surface, therefore contributing inefficiently to the mAbs neutralization potency. Therefore, the prime-boost strategy of EDIII from a single serotype or four serotypes mainly elicited a poorly neutralizing, cross-reactive antibody response to the conserved AB loop of EDIII.

Isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14, from domestic rabbits.

We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (RbCoV HKU14), from domestic rabbits. The virus was detected in 11 (8.1%) of 136 rabbit fecal samples by reverse transcriptase PCR (RT-PCR), with a viral load of up to 10(8) copies/ml. RbCoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects. Northern blotting confirmed the production of subgenomic mRNAs coding for the HE, S, NS5a, E, M, and N proteins. Subgenomic mRNA analysis revealed a transcription regulatory sequence, 5'-UCUAAAC-3'. Phylogenetic analysis showed that RbCoV HKU14 formed a distinct branch among Betacoronavirus subgroup A coronaviruses, being most closely related to but separate from the species Betacoronavirus 1. A comparison of the conserved replicase domains showed that RbCoV HKU14 possessed <90% amino acid identities to most members of Betacoronavirus 1 in ADP-ribose 1″-phosphatase (ADRP) and nidoviral uridylate-specific endoribonuclease (NendoU), indicating that RbCoV HKU14 should represent a separate species. RbCoV HKU14 also possessed genomic features distinct from those of other Betacoronavirus subgroup A coronaviruses, including a unique NS2a region with a variable number of small open reading frames (ORFs). Recombination analysis revealed possible recombination events during the evolution of RbCoV HKU14 and members of Betacoronavirus 1, which may have occurred during cross-species transmission. Molecular clock analysis using RNA-dependent RNA polymerase (RdRp) genes dated the most recent common ancestor of RbCoV HKU14 to around 2002, suggesting that this virus has emerged relatively recently. Antibody against RbCoV was detected in 20 (67%) of 30 rabbit sera tested by an N-protein-based Western blot assay, whereas neutralizing antibody was detected in 1 of these 20 rabbits.

Evaluation and analysis of dengue virus enhancing and neutralizing activities using simple high-throughput assays.

The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose-response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.

[Establishment and preliminary application of dengue virus envelope domain III IgG antibody capture enzyme-linked immuno-absorbent assay].

OBJECTIVE: To establish a highly sensitive and specific assay to detect dengue virus (DENV) envelope protein domain III (EDIII) IgG antibody, and to explore its value in the diagnosis and seroepidemiological survey of dengue. METHODS: The DENV EDIII IgG antibody capture ELISA was developed using the recombinant full-length DENV EDIII, which was prepared by Pichia yeast expression system as the capture antigen. The serum samples were collected from the same group of 35 DENV-1 patients of primary infection during disease period in 2006 and their follow-up phase in 2010; and the sensitivity of the assay was compared to that of the commercial Panbio DENV IgG ELISA. RESULTS: The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from disease period and follow-up phase was 87% (20/23) and 94% (33/35), respectively; whereas the sensitivity of Panbio DENV IgG ELISA was 71% (25/35) and 0, respectively. The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from both periods was similar, without statistical significance (χ(2) = 0.946, P = 0.331). For serum samples from disease period, the sensitivity of DENV EDIII IgG ELISA was comparable with that of Panbio DENV IgG ELISA (χ(2) = 1.924, P = 0.165). However, DENV EDIII IgG ELISA demonstrated a significantly higher sensitivity than Panbio DENV IgG ELISA in detecting the serum samples from follow-up phase (χ(2) = 62.432, P = 0.000). CONCLUSION: DENV EDIII IgG capture ELISA is highly sensitive in detecting IgG in the serum samples from either disease period or follow-up phase. This method might be a promising alternative for diagnosis and seroepidemiologic survey of dengue.

Evaluation of human enterovirus 71 and coxsackievirus A16 specific immunoglobulin M antibodies for diagnosis of hand-foot-and-mouth disease.

BACKGROUND: Hand-foot-and-mouth disease (HFMD) is caused mainly by the human enterovirus type 71 (HEV71) and the Coxsackievirus A group type 16 (CVA16). Large outbreaks of disease have occurred frequently in the Asia-Pacific region. Reliable methods are needed for diagnosis of HFMD in children. IgM-capture ELISA, with its notable advantages of convenience and low cost, provides a potentially frontline assay. We aimed to evaluate the newly developed IgM-capture ELISAs for HEV71 and CVA16 in the diagnosis of HFMD, and to measure the kinetics of IgM over the course of HEV71 or CVA16 infections. RESULTS: We mapped, for the first time, the kinetics of IgM in HEV71 and CVA16 infection. HEV71- and CVA16-IgM were both detectable in some patients on day 1 of illness, and in 100% of patients by day 5 (HEV71) and day 8 (CVA16) respectively; both IgMs persisted for several weeks. The IgM detection rates were 90.2% (138 of 153 sera) and 68.0% (66 of 97 sera) for HEV71 and CVA16 infections, respectively, during the first 7 days of diseases. During the first 90 days after onset these values were 93.6% (233 of 249 sera) and 72.8% (91 of 125 sera) for HEV71 and CVA16 infections, respectively. Some cross-reactivity was observed between HEV71- and CVA16-IgM ELISAs. HEV71-IgM was positive in 38 of 122 (31.1%) CVA16 infections, 14 of 49 (28.6%) other enteroviral infections and 2 of 105 (1.9%) for other respiratory virus infected sera. Similarly, CVA16-IgM was apparently positive in 58 of 211 (27.5%) HEV71 infections, 16 of 48 (33.3%) other enterovirus infections and 3 of 105 (2.9%) other respiratory virus infected sera. Nevertheless, the ELISA yielded the higher OD450 value of main antibody than that of cross-reaction antibody, successfully identifying the enteroviral infection in 96.6% (HEV71) and 91.7% (CVA16) cases. When blood and rectal swabs were collected on the same day, the data showed that the agreement between IgM-capture ELISA and real-time RT-PCR in HEV71 was high (Kappa value = 0.729) while CVA16 somewhat lower (Kappa value = 0.300). CONCLUSIONS: HEV71- and CVA16-IgM ELISAs can be deployed successfully as a convenient and cost-effective diagnostic tool for HFMD in clinical laboratories.

[Characterization and secreted expression of dengue virus type I-IV envelope glycoprotein domain III in Pichia pastoris].

OBJECTIVE: To achieve secretory and extracellular production of recombinant dengue virus serotypes I-IV envelope glycoprotein domain III (DENV-1-4 EDIII) in Pichia pastoris. METHODS: EDIII genes of DENVI-IV were amplified and cloned into vector pPIC9K, respectively. These recombinant plasmids were then linearized and transferred into Pichia pastoris strain GS115. Clones highly produced in 4.0 mg/ml G418 were amplified and induced by methanol to achieve the secreted recombinant proteins. Ni-NTA agarose beads were used for purification, while SDS-PAGE and Western blotting were used for identification. RESULTS: The recombinant plasmids pPIC9K-DENV-1-4 EDIII were constructed and successfully transferred into Pichia pastoris strain GS115. The recombinant EDIII proteins were expressed in a secretory way with the molecular weight about 12 × 10(3) and specifically identified by anti-His monoclonal antibody and anti-DENVI-IV mice sera. CONCLUSION: DENVI-IV EDIII proteins are successfully achieved from Pichia pastoris expression system and could be used for development of dengue vaccines, diagnostic reagents and study of biological function of the E protein.

[To evaluate the neutralizing abilities of anti-dengue virus antibodies with nonstructural protein 1 antigen capture enzyme-linked immunosorbent assay].

OBJECTIVE: To produce neutralizing antibodies against envelope protein domain III (EDIII) of dengue virus serotype I (DENV-1) and evaluate the nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA) for identification of antibody neutralizing abilities. METHODS: Five BALB/c mice and one New Zealand Rabbit were immunized with recombinant EDIII protein of DENV-1 for the production of hybridomas and hyperimmune sera. Indirect ELISA, immunofluorescence assay (IFA) and Western Blot analyses were applied to identify specificity of antibodies. Comparing to plaque reduction neutralization test (PRNT), the new established DENV-1 specific NS1 antigen capture ELISA was used for detecting the neutralizing abilities of these antibody. RESULTS: Four strains of monoclonal antibodies (mAbs) named 1A1, 1B3, 3D3 and 9D6 and one hyperimmune serum of rabbit were obtained, all of which were approved to have neutralizing abilities to DENV-1 with the PRNT titer of 1:1024, 1:512, 1:256, 1:4096 and 1:4096. MAb 3D3 with the lowest neutralization titer in PRNT had not shown neutralizing ability to DENV-1 in NS1 antigen capture ELISA, while MAbs 1A1, 1B3 and 9D6 and the rabbit hyperimmune serum could protect the C6/36 from being infected by DENV-1 with the neutralization titer of 1:32, 1:32, 1:128 and 1:128 in this assay. CONCLUSION: NS1 antigen capture ELISA could be used to identify antibody neutralizing abilities to DENV, it was a faster and more convenient way to screen antibodies with high neutralization titer and might also be used as one of the methods to evaluate the effects of vaccines.

Structural and Functional Characterization of a Cross-Reactive Dengue Virus Neutralizing Antibody that Recognizes a Cryptic Epitope.

Understanding the molecular basis of the neutralizing antibody response to dengue virus (DENV) is an essential component in the design and development of effective vaccines and immunotherapeutics. Here we present the structure of a cross-reactive, neutralizing antibody, 3E31, in complex with domain III (DIII) of the DENV envelope (E) protein and reveal a conserved, temperature-sensitive, cryptic epitope on DIII that is not available in any of the known conformations of E on the dengue virion. We observed that 3E31 inhibits E-mediated membrane fusion, suggesting that the antibody is able to neutralize virus through binding an as-yet uncharacterized intermediate conformation of DENV E and sterically block trimer formation. Finally, we show that, unlike cross-reactive fusion peptide-specific antibodies, 3E31 does not promote antibody-dependent enhancement of infection at sub-neutralizing concentrations. Our results highlight the 3E31 epitope on the E protein DIII as a promising target for immunotherapeutics or vaccine design.

A patient with asymptomatic severe acute respiratory syndrome (SARS) and antigenemia from the 2003-2004 community outbreak of SARS in Guangzhou, China.

An asymptomatic case of severe acute respiratory syndrome (SARS) occurred early in 2004, during a community outbreak of SARS in Guangzhou, China. This was the first time that a case of asymptomatic SARS was noted in an individual with antigenemia and seroconversion. The asymptomatic case patient and the second index case patient with SARS in the 2003-2004 outbreak both worked in the same restaurant, where they served palm civets, which were found to carry SARS-associated coronaviruses. Epidemiological information and laboratory findings suggested that the findings for the patient with asymptomatic infection, together with the findings from previously reported serological analyses of handlers of wild animals and the 4 index case patients from the 2004 community outbreak, reflected a likely intermediate phase of animal-to-human transmission of infection, rather than a case of human-to-human transmission. This intermediate phase may be a critical stage for virus evolution and disease prevention.

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells.

AIM: To study the selective killing of human umbilical vein endothelial cells (HUVECs) by a double suicide gene under the regulation of a kinase domain insert containing receptor (KDR) promoter and mediated by an adenoviral gene vector. METHODS: Human KDR promoter was cloned by polymerase chain reaction (PCR), and two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, and the infection rates were estimated by detection of green fluorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured. RESULTS: The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs: HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMV-CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK (P < 0.001). CONCLUSION: Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Functional properties of DENV EDIII­reactive antibodies in human DENV­1­infected sera and rabbit antiserum to EDIII.

The envelope domain III (EDIII) of the dengue virus (DENV) has been confirmed to be involved in receptor binding. It is the target of specific neutralizing antibodies, and is considered to be a promising subunit dengue vaccine candidate. However, several recent studies have shown that anti­EDIII antibodies contribute little to the neutralizing or enhancing ability of human DENV­infected serum. The present study involved an analysis of the neutralization and antibody­dependent enhancement (ADE) activities of EDIII­reactive antibodies in human convalescent sera from patients with primary DENV­1 infection and rabbit antiserum immunized with recombinant DENV­1 EDIII protein. The results indicated that serum neutralization was not associated with titres of EDIII­binding antibodies in the human DENV­1­infected sera. The depletion of anti­EDIII antibodies from these serum samples revealed that the anti­EDIII antibodies of the patients contributed little to neutralization and ADE. However, the EDIII­reactive antibodies from the rabbit antiserum exhibited protective abilities of neutralization at a high dilution (~1:50,000) and ADE at a low dilution (~1:5,000) for the homotypic DENV infection. Notably, the rabbit antiserum displayed ADE activity only at a dilution of 1:40 for the heterotypic virus infection, which suggests that EDIII­reactive antibodies may be safe in secondary infection with heterotypic viruses. These results suggest that DENV EDIII is not the predominant antigen of the DENV infection process; however, purified or recombinant DENV EDIII may be used as a subunit vaccine to provoke an effective and safe antibody response.

Relative rates of non-pneumonic SARS coronavirus infection and SARS coronavirus pneumonia.

BACKGROUND: Although the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) has been sequenced and a possible animal reservoir identified, seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. METHODS: We cloned and purified the nucleocapsid protein and spike polypeptide of SARS-CoV and examined their immunogenicity with serum from patients with SARS-CoV pneumonia. An ELISA based on recombinant nucleocapsid protein for IgG detection was tested with serum from 149 healthy blood donors who donated 3 years previously and with serum positive for antibodies against SARS-CoV (by indirect immunofluorescence assay) from 106 patients with SARS-CoV pneumonia. The seroprevalence of SARS-CoV was studied with the ELISA in healthy blood donors who donated during the SARS outbreak in Hong Kong, non-pneumonic hospital inpatients, and symptom-free health-care workers. All positive samples were confirmed by two separate western-blot assays (with recombinant nucleocapsid protein and recombinant spike polypeptide). FINDINGS: Western-blot analysis showed that the nucleocapsid protein and spike polypeptide of SARS-CoV are highly immunogenic. The specificity of the IgG antibody test (ELISA with positive samples confirmed by the two western-blot assays) was 100%, and the sensitivity was 94.3%. Three of 400 healthy blood donors who donated during the SARS outbreak and one of 131 non-pneumonic paediatric inpatients were positive for IgG antibodies, confirmed by the two western-blot assays (total, 0.48% of our study population). INTERPRETATION: Our findings support the existence of subclinical or non-pneumonic SARS-CoV infections. Such infections are more common than SARS-CoV pneumonia in our locality.

Kinase domain insert containing receptor promotor controlled suicide gene system kills human umbilical vein endothelial cells.

AIM: To evaluate the killing effect of double suicide gene mediated by adenovirus and regulated under kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. METHODS: By PCR technology, human KDR promoter gene, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simple virus-thymidine kinase (TK) gene were cloned. Plasmid pKDR-CDglyTK was constructed with them. Then, a recombinant adenoviral plasmid pAdKDR-CDglyTK was constructed in a "two-step transformation protocol". The newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses, which were further propagated and purified. Human umbilical vein endothelial cells (HUVEC) were infected with a different multiplicity of infection (MOI) of resultant recombinant adenovirus, the infection rate was measured with the aid of (GFP) expression. Infected cells were cultured in culture media containing different concentrations of (GCV) and/or 5-(FC), and the killing effects were measured. RESULTS: Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed, and they infected HUVEC cells efficiently. Our data indicated that the infection rate was relevant to MOI of recombinant adenoviruses. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of recombinant adenoviruses. Our data also indicated that the two prodrugs used in combination were much more effective on killing transgeneic cells than GCV or 5-FC used alone. CONCLUSION: Prodrug/KDR-CDglyTK system is effective on killing HUVEC cells, its killing effect correlates to the concentration of prodrugs and recombinant adenovirus' MOI. Combined use of the two prodrugs confers better killing effects on transgeneic cells.