Pathogenicity of novel hantavirus isolate and antigenicity and immunogenicity of novel strain-based inactivated vaccine.

BACKGROUND: Hantaan virus (HTNV, Orthohantavirus hantanensae species, Hantaviridae family) is the main etiological agent responsible for hemorrhagic fever with renal syndrome (HFRS). The novel HTNV may pose a potential danger to the control and prevention of HFRS in China, which highlights the importance of vaccine development in public health management. In previous studies, our laboratory discovered and successfully isolated a new HTNV strain, HV004 strain, from Apodemus agrarius captured in an epidemic area in Hubei, China. METHODS: An initial biological and pathogenicity characterization of HTNV 76-118 (standard train), HV114 strain (a clinical isolate from Hubei province in 1986), and the novel isolate HV004 strain from the epidemic areas of Hubei province were performed in susceptible cells and in vivo. An experimental HV004 strain inactivated vaccine was prepared, and its corresponding immunogenicity was analyzed in BALB/c mice. RESULTS: HV004 strain had a similar but higher pathogenicity than HTNV 76-118 and HV114 in suckling mice. A subcutaneous vaccination (s.c.) with the inactivated HTNV vaccine adjuvanted with aluminum, followed by a challenge intraperitoneally with 106 FFU/ml HTNV, afforded full protection against an HTNV challenge. All immunized mice in every group elicited serum neutralizing antibodies with increasing dosages, which may protect mice from HTNV infection. A dose-dependent stimulation index of splenocytes was also observed in immunized mice. The percentage of IFN-γ-producing CD3+CD8+ T cells was significantly higher in the spleens of immunized mice than in those of control mice. CONCLUSIONS: These findings suggest that the inactivated HTNV vaccine may stimulate mice to produce high levels of antibodies with neutralization activity and elicit specific anti-HTNV humoral and cellular immune responses in BALB/c mice against the prevalent strain of HTNV in south central China.

Extraction optimization and identification of four advanced glycation-end products inhibitors from lotus leaves and interaction mechanism analysis.

Previous research indicated lotus leaves extract could effectively inhibit advanced glycation end-products (AGEs) formation, but the optimal extraction condition, bio-active compounds and interaction mechanism remain unclear. The current study was designed to optimize the extraction parameters of AGEs inhibitors from lotus leaves by bio-activity-guided approach. The bio-active compounds were enriched and identified, the interaction mechanisms of inhibitors with ovalbumin (OVA) were investigated by fluorescence spectroscopy and molecular docking. The optimum extraction parameters were solid-liquid ratio of 1:30, ethanol concentration of 70 %, ultrasonic time of 40 min, temperature of 50 °C, and power of 400 W. Isoquercitrin, hyperoside, astragalin, and trifolin were identified from the 80 % ethanol fraction of lotus leaves (80HY). Hyperoside and isoquercitrin were dominant AGEs inhibitors and accounted for 55.97 % of 80HY. Isoquercitrin, hyperoside, trifolin interacted with OVA via the same mechanism, hyperoside exhibited the strongest affinity, trifolin caused the most conformational changes.

The IgE/IgG binding capacity and structural changes of Alaska Pollock parvalbumin glycated with different reducing sugars.

Parvalbumin (PV) is one of the major allergens in fish. The aim of our present work was to research the influence mechanism of glycation with different reducing sugars (glucose, fructose, ribose, lactose, and galactose) on the immunoglobulin E (IgE) and immunoglobulin G (IgG) binding capacity and structure changes of PV in Alaska Pollock. PV glycated with glucose or fructose (PV-Glu/ PV-Fru) exhibited the higher IgE/IgG binding capacities than that of ribose, galactose, or lactose. During glycation, the lysine (Lyr), tyrosine (Tyr), and phenylalanine (Phe) of PV were gradually embed into core area of three-dimensional structure of protein, which reflected in the ultraviolet (UV) spectrum and fluorescence spectra. Moreover, the increase of surface hydrophobicity had confirmed the conformation alteration of glycated PV. These results suggest that there is a specific association among the change of PV in glycation and in potential allergenicity. The types and conformation of reducing sugar greatly influenced the IgE/IgG binding capacity of PV, and glycation with ribose and galactose was a promising approach for reducing the IgE/IgG binding capacity of PV from Alaska Pollock. PRACTICAL APPLICATIONS: Parvalbumin (PV), the major allergen of fish, it can not only maintain the physiological activity of cells, but also cross react with human amyloid protein to alleviate Alzheimer's disease and Parkinson's syndrome. This study revealed that the IgE/IgG binding capacity and structural changes of PV from Alaska Pollock modified by glycation with different reducing sugars. This will help us to understand the sensitization and structural change of the glycated products after the reaction of PV with different reducing sugars. It provides an effective carbonyl source for the preparation of low antigenicity PV based on glycation and lays a foundation for glycation modification of other food allergens.

Effect and Mechanism of Elaeagnus angustifolia Flower and Its Major Flavonoid Tiliroside on Inhibiting Non-enzymatic Glycosylation.

This study aimed to evaluate the antiglycation ability of Elaeagnus angustifolia flower extract and to elucidate the mechanism with its major compound. The results indicated that E. angustifolia flower extract and its major compound tiliroside (24.2 mg/g of extract) exhibited excellent antiglycation ability with inhibition rates of 92.1 and 78.9% at 37.5 µg/mL, which are much higher than that of aminoguanidine (55.3% at 37.5 µg/mL). The stable tiliroside-ovalbumin (OVA) complexes were formed through a spontaneous exothermic progress in an equimolar manner, and hydrophobic interaction, hydrogen bond, and van der Waals forces were the major driving forces. Tiliroside could significantly ameliorate the conformation changes of OVA induced by the glycation reaction, quench its fluorescence by a static mechanism, and change the microenvironment adjacent to tryptophan and tyrosine. Molecular docking revealed that tiliroside inserted into the OVA hydrophobic pocket resulted in the formation of five hydrogen bonds. Orbitrap tandem mass spectrometry showed that tiliroside significantly suppressed the glycation of OVA, and the number of glycation sites was reduced from 9 to 5 after tiliroside was added. The above results indicated that E. angustifolia flowers and tiliroside have a good antiglycation effect and can be used as food additives to suppress the undesired glycation reaction during food processing.

Reduced IgE/IgG binding capacities of bovine α-Lactalbumin by glycation after dynamic high-pressure microfluidization pretreatment evaluated by high resolution mass spectrometry.

Dynamic high-pressure microfluidization (DHPM) pretreatment and glycation with lactose were employed to modify α-Lactalbumin (α-LA) with respect to the IgE/IgG binding capacities. No significant difference on incorporation ratio value of glycated α-LA was observed with and without DHPM pretreatment. However, IgE/IgG binding capacities of α-LA were decreased after glycation and DHPM pretreatment promoted the reduction. The lowest IgE/IgG binding capacities of glycated α-LA were obtained by DHPM pretreatment at 110 MPa. Native α-LA was mainly glycated at K62, K94, K98, whereas glycation sites and degree of substitution per peptide (DSP) were added after DHPM treatment. Therefore, the reduced IgE/IgG binding capacities of α-LA was attributed to the characteristics of glycated sites, including the amount, location, and DSP values. Interestingly, K98 played the most important role in decreasing IgE/IgG binding capacities of α-LA. The study revealed that glycation combined with DHPM was a promising way to decrease the allergenicity of proteins.

The effect of ginger and garlic addition during cooking on the volatile profile of grass carp (Ctenopharyngodon idella) soup.

Ginger and garlic have long been used in Asian countries to enhance the flavor and to neutralize any unpleasant odors present in fish soup. The purpose of this study was to evaluate the change in the amount of volatile components present in fish soup compared to boiled water solutions of ginger and garlic. The fish soup was prepared by boiling oil-fried grass carp (Ctenopharyngodon idella) with or without ginger and/or garlic. Generally, boiling garlic and ginger in water led to a decrease in the amount of the principal volatile constituents of these spices, together with the formation of some new volatiles such as pentanal, hexanal, and nonanal. The results showed that 16 terpenes present in raw ginger, predominantly camphene, ß-phellandrene, ß-citral, α-zingiberene, and (E)-neral, were detected in fish soup with added ginger and thus remained in the solution even after boiling. Similarly, 2-propen-1-ol and three sulfur compounds (allyl sulfide, diallyl disulfide, and diallyl trisulfide) present in raw garlic, were present in trace amounts in the boiled garlic solution, but were present in considerably larger amounts in the boiled fish solution with garlic or garlic plus ginger. In conclusion, the effect of adding spices on the volatile profile of grass carp soup can be attributed to the dissolution of flavor volatiles mainly derived from raw spices into the solution, with few additional volatiles being formed during boiling. In addition, boiling previously fried grass carp with spices led to enhanced volatile levels compared to boiled spice solutions.

The epidemic characteristics and changing trend of hemorrhagic fever with renal syndrome in Hubei Province, China.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) is caused by different hantaviruses within the Bunyaviridae family. HFRS is a fulminant, infectious disease that occurs worldwide and is endemic in all 31 provinces of China. Since the first HFRS case in Hubei Province was reported in 1957, the disease has spread across the province and Hubei has become one of the seriously affected areas in China with the greatest number of reported HFRS cases in the 1980's. However, the epidemic characteristics of HFRS in Hubei are still not entirely clear and long-term, systematic investigations of this epidemic area have been very limited. METHODS: The spatiotemporal distribution of HFRS was investigated using data spanning the years 1980 to 2009. The annual HFRS incidence, fatality rate and seasonal incidence between 1980 and 2009 were calculated and plotted. GIS-based spatial analyses were conducted to detect the spatial distribution and seasonal pattern of HFRS. A spatial statistical analysis, using Kulldorff's spatial scan statistic, was performed to identify clustering of HFRS. RESULTS: A total of 104,467 HFRS cases were reported in Hubei Province between 1980 and 2009. Incidence of and mortality due to HFRS declined after the outbreak in 1980s and HFRS cases have been sporadic in recent years. The locations and scale of disease clusters have changed during the three decades. The seasonal epidemic pattern of HFRS was characterized by the shift from the unimodal type (autumn/winter peak) to the bimodal type. CONCLUSIONS: Socioeconomic development has great influence on the transmission of hantaviruses to humans and new epidemic characteristics have emerged in Hubei Province. It is necessary to reinforce preventative measures against HFRS according to the newly-presented seasonal variation and to intensify these efforts especially in the urban areas of Hubei Province.

Establishment of SYBR green-based qPCR assay for rapid evaluation and quantification for anti-Hantaan virus compounds in vitro and in suckling mice.

Hantaan viruses cause two severe diseases lacking efficient treatment, yet no effective prophylactic vaccines are available. Continued exploration of alternative antiviral agents to treat hantavirus-related syndromes remains compulsory. The fluorescence-based quantitative real-time PCR (qPCR) has become the touchstone for target gene quantification. In the present study, standard curves for Hantaan virus (HTNV), mouse, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were generated by serial 10-fold dilutions of the constructed recombinant plasmid pGEM-T/HTNV, pGEM-T/mouse-GAPDH, and pGEM-T/human-GAPDH, respectively. Comparisons between the indirect immunofluorescence assay and qPCR assay in the detection of HTNV-infected Vero E6 cells showed improved detection limit and sensitivity of latter method. To characterize the inhibitory effect of several conventional antivirals (arbidol and ribavirin) and unconventional antivirals (indomethacin and curcumin) on HTNV, the levels of viral RNAs were measured for 4 days post-treatment of HTNV-infected Vero E6 cells and 18 days post-inoculation of HTNV-infected suckling mice. Our results validated that HTNV was sensitive to ribavirin and arbidol treatment, while indomethacin and curcumin may also be therapeutically effective in treating HTNV infection. As a result, the establishment and application of qPCR may be a useful tool for the evaluation of potential antivirals for Hantaan virus infection in vitro and in vivo.

Amelioration of influenza virus-induced reactive oxygen species formation by epigallocatechin gallate derived from green tea.

AIM: To study whether epigallocatechin gallate (EGCG), a green tea-derived polyphenol, exerted anti-influenza A virus activity in vitro and in vivo. METHODS: Madin-Darby canine kidney (MDCK) cells were tested. The antiviral activity of EGCG in the cells was determined using hemagglutination assay and qPCR. Time of addition assay was performed to determine the kinetics of inhibition of influenza A by EGCG. The level of reactive oxygen species (ROS) were determined with confocal microscopy and flow cytometry. BALB/c mice were treated with EGCG (10, 20 or 40 mg·kg(-1)·d(-1), po) for 5 d. On the 3rd d of the treatment, the mice were infected with influenza A virus. Histopathological changes, lung index and virus titers in the lungs were determined. RESULTS: Treatment of influenza A-infected MDCK cells with EGCG (1.25-100 nmol/L) inhibited influenza A replication in a concentration-dependent manner (the ED(50) value was 8.71±1.11 nmol/L). Treatment with EGCG (20 nmol/L) significantly suppressed the increased ROS level in MDCK cells following influenza A infection. In BALB/c mice infected with influenza virus, oral administration of EGCG (40 mg·kg(-1)·d(-1)) dramatically improved the survival rate, decreased the mean virus yields and mitigated viral pneumonia in the lungs, which was equivalent to oral administration of oseltamivir (40 mg·kg(-1)·d(-1)), a positive control drug. CONCLUSION: The results provide a molecular basis for development of EGCG as a novel and safe chemopreventive agent for influenza A infection.

[Genetic analysis on S segment of hantaviruses in rodent hosts from Wuhan area, Hubei province].

OBJECTIVE: To investigate the infection and genotype of hantaviruses in rodents from Wuhan area, Hubei province. METHODS: Rodents were trapped in fields and residential areas of Xinzhou and Jiangxia districts of Wuhan in autumn and winter seasons, from 2000 to 2003 and from 2009 to 2011. Trapped rodents were identified, and hantavirus antigens were detected in the lung tissues with indirect immunofluorescence assay (IFA). Partial S segment sequences were amplified with RT-PCR in hantavirus antigen positive samples and then sequenced. Phylogenetic tree was constructed to analyze the genetic characteristics of hantaviruses. RESULTS: From 2000 to 2003, 437 rodents were trapped, with 24 (5.49%) lung tissues showed hantavirus antigen positive. From 2009 to 2011, 173 rodents were trapped and 7 (4.05%) were hantavirus antigen positive. Rattus norvegicus were the dominant species of rodents. Partial S segment sequences were amplified from 22 samples with Hantaan and Seoul viruses specific primers and sequenced. Partial S segments of Seoul viruses (nucleotide 588 - 1147) were amplified from 17 rodents (13 R. norvegicus and 4 Apodemus agrarius). Seven of these sequences belonged to 3 genetic lineage, while two novel genetic lineages were formed by 9 and 1 sequences, respectively. Partial S segments of Hantaan viruses (nucleotide 615 - 1141) were amplified from 5 A. agrarius. One of these sequences belonged to 7 genetic lineages, and 4 sequences formed one novel genetic subtype. CONCLUSION: Hantaan and Seoul viruses co-circulated in Wuhan area, Hubei province. Novel genetic lineages were identified in this study and Seoul virus might have caused spillover infection in A. agrarius.

Genetic characterization of a new subtype of Hantaan virus isolated from a hemorrhagic fever with renal syndrome (HFRS) epidemic area in Hubei Province, China.

To characterize hantaviruses currently circulating in the hemorrhagic fever with renal syndrome (HFRS) epidemic area of Hubei Province, rodents were captured and serum samples were collected from several HFRS patients. The partial S segment of the hantaviruses amplified from two serum samples had a high degree of sequence identity to the corresponding hantavirus strain isolated from Apodemus agrarius (designated as HV004). The complete S, M, and L segment sequences of HV004 were determined. The sequence identities between strain HV004 and other Hantaan viruses (HTNVs) were 83 %-90 % at the nucleotide level and 95 %-99 % at the amino acid level. Phylogenetic analysis showed that HV004 belonged to a new HTNV lineage. These data suggest the presence of a new HTNV subtype, which probably caused the HFRS cases in the endemic area of Hubei Province.

Genetic analysis of hantaviruses and their rodent hosts in central-south China.

Hantaan virus (HTNV) and Seoul virus (SEOV) are two major zoonotic pathogens of hemorrhagic fever with renal syndrome (HFRS) in Asia. Hubei province, which is located in the central-south China, had been one of the most severe epidemic areas of HFRS. To investigate phylogenetic relationships, genetic diversity and geographic distribution of HTNV and SEOV in their reservoir hosts, a total of 687 rodents were trapped in this area between 2000 and 2009. Sequences of partial S- and M-segments of hantaviruses and mitochondrial D-loop gene from 30 positive samples were determined. Our data indicated that SEOV and HTNV were co-circulating in Hubei. Phylogenetic analysis based on partial S- and M-segment sequences revealed two and three previously undefined lineages of SEOV, and a novel genetic lineage of HTNV, respectively. Four inter-lineage reassortment SEOVs carried by Rattus norvegicus and Apodemus agrarius were observed. It suggests that SEOV may cause spillover infections to A. agrarius naturally. The abundance of the phylogenetic lineages of SEOV suggested that central-south China was a radiation center for SEOVs.

Determination of platelet-bound glycoprotein-specific autoantibodies by flow cytometric immunobead assay in primary immune thrombocytopenia.

OBJECTIVES: Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by premature platelet destruction induced by autoantibodies directed against platelet glycoproteins (GPs). Despite being a clinically important disorder, ITP lacks a feasible diagnostic assay for routine clinical use. This study was meant to evaluate a newly developed flow cytometric immunobead assay for determination of platelet-bound GP-specific autoantibodies in comparison with indirect monoclonal antibody-specific immobilization of platelet antigen (MAIPA) in the diagnosis of ITP. METHODS: Platelet-bound and plasma GPIIb/IIIa and GPIb/IX autoantibodies were determined by flow cytometric immunobead assay and indirect modified MAIPA, respectively. The average fluorescence level for platelet-bound, GP-specific autoantibodies was given as a ratio to three normal controls tested simultaneously. RESULTS: The median value of platelet-bound GPIIb/IIIa and GPIb/IX autoantibodies in ITP group were 3.09 (range 0.78, 30.2) and 3.09 (range 0.72, 19.2), respectively, which were significantly higher than non-ITP group [1.01 (0.67, 5.59) and 1.01 (0.79, 5.56), respectively, P<0.001] and normal controls [1.02 (0.72, 1.76) and 1.03 (0.79, 1.73), respectively, P<0.001]. The receiver-operating characteristics curve analysis showed an area under the curve of 0.895 for GPIIb/IIIa autoantibody and 0.859 for GPIb/IX autoantibody, respectively. Combined detection of GPIIb/IIIa or GPIb/IX autoantibodies by flow cytometric immunobead assay showed a sensitivity of 82.11% for ITP diagnosis. CONCLUSION: This study demonstrated that determination of platelet-bound, GP-specific autoantibodies by flow cytometric immunobead assay was a convenient, sensitive, and specific test for the differential diagnosis of thrombocytopenic patients.

[Study of intracerebral focus changes on CADASIL by using MR imaging].

OBJECTIVE: To investigate dynamic changes of intracerebral focus on cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). METHODS: Using the modified Scheltens scale, the magnetic resonance (MR) changes of lesion distribution, size and shape in 7 patients from a CADASIL family were retrospectively analyzed during 3 years observed. RESULTS: In 6 of 7 patients, the number and volume of lesion areas in the white matter were increased (parietal lobe, n = 6; temporal lobe, n = 5; frontal lobe, n = 3; occipital lobe, n = 2) and some areas even became confluent as a mass or chain. New lacunar infarcts (n = 1 - 5) appeared in 5 patients and the arcuate fiber were involved in 1 patients. Slight enlargement could be seen in lateral ventricle (n = 1) or lateral ventricle with third ventricle (n = 1). CONCLUSION: MR imaging can help us to reveal dynamic changes of brain lesions and prognosis in patients with CADASIL.

Application of MRI in indirect temporomandibular joint injury without condylar fracture.

OBJECTIVE: To discuss the application of MRI in indirect temporomandibular joint injury without condylar fracture. METHODS: MRI examination on temporomandibular joint was conducted in 28 patients with indirect injury to temporomandibular joint without condylar fracture. The scanning sequence included T(1)WI, PDWI on oblique sagittal section at both open and closed mouth positions, and T(1)WI, T(2)WI on oblique coronal section. The MRI appearance was analyzed by 2 senior radiologists. RESULTS: Among the 56 temporomandibular joints of 28 patients, 35 joints exhibited pathological changes on MRI, in which there were 9 bone injuries, 21 articular disc dislocation, 24 intracapsular hematocele and hydrops. CONCLUSIONS: MRI can clearly reveal bone injury, articular disc dislocation as well as articular capsule abnormality in the indirect injury of temporomandibular joint without condylar fracture. It is highly advocated in clinical use.