Influenza a Neuraminidase-Based Bivalent mRNA Vaccine Induces Th1-Type Immune Response and Provides Protective Effects in Mice.

Vaccines are one of the most effective means of preventing influenza A, typically containing the hemagglutinin (HA) of the influenza A virus. However, antigenic drift and shift of the influenza A virus can lead to instability in vaccine efficacy. Compared to HA, the antigenic variation rate of neuraminidase (NA) is slower. In traditional inactivated influenza vaccines, although they contain a certain amount of NA, there are significant differences between different batches, which cannot consistently induce NA-based immune responses. Therefore, NA is often overlooked in vaccine development. In this study, we report an mRNA vaccine encoding the NA of two strains of influenza A virus. The experimental results demonstrated that when matched with the viral strain, this mRNA vaccine induced high levels of neutralizing antibodies, providing a protective effect to mice in viral challenge experiments, and this immune response was shown to be biased towards the Th1 type. In summary, this study demonstrates that NA is a promising potential antigen, providing new insights for the development of influenza A virus vaccines.

Immune persistence of an inactivated poliovirus vaccine derived from the Sabin strain: a 10-year follow-up of a phase 3 study.

Background: In a previous phase 3 clinical trial, we showed that an inactivated poliovirus vaccine derived from the Sabin strain (sIPV) can induce neutralising antibodies against currently circulating and reference wild poliovirus strains. However, the immune persistence of sIPV remains to be evaluated. Methods: In this study, 400 participants who were eligible for an early phase 3 clinical trial (Jan 1, 2012-Aug 31, 2014) in Pingle County, GuanXi Province, China, were initially involved in one site. Of the participants in the previous phase 3 clinical trial, sera of 287, 262, 237, and 207 participants were sampled at the ages of 4, 6, 8, and 10 years, respectively, after the prime-boost regimen. Neutralising antibodies against attenuated Sabin strains were detected using these serum samples to determine immune persistence. The serum neutralising antibodies titre of 1:8 against poliovirus types 1, 2, and 3 is considered to be a seroprotection level for polio. The trial is registered at ClinicalTrials.gov, NCT01510366. Findings: The protective rates against poliovirus types 1, 2, and 3 in the sIPV group were all 100% at 10 years after the booster immunisation, compared with 98.1%, 100%, and 97.1%, respectively, in the wIPV control group after 10 years. After the booster at 18 months, the geometric mean titres (GMTs) of neutralising antibodies against poliovirus types 1, 2, and 3 in the sIPV group were 13,265.6, 7856.7, and 6432.2, respectively, and the GMTs in the control group (inoculated with inactivated poliovirus vaccine derived from wild strain (wIPV)) were 3915.6, 2842.6, and 4982.7, respectively. With increasing time after booster immunisation, the GMTs of neutralising antibodies against poliovirus types 1, 2, and 3 gradually decreased in both the sIPV and wIPV groups. At the age of ten years, the GMTs of neutralising antibodies against poliovirus types 1, 2, and 3 in the sIPV group were 452.3, 392.8, and 347.5, respectively, and the GMTs in the wIPV group 108.5, 154.8, and 229.3, respectively, which were still at a higher-than-protective level (1:8). Interpretation: Both sIPV and wIPV maintained sufficiently high immune persistence against poliovirus types 1, 2, and 3 for at least 10 years after booster immunisation. Funding: Yunnan Provincial Science and Technology Department, the Bill and Melinda Gates Foundation, the National High-tech Research and Development Program, the National International Science and Technology Cooperation Project, the Yunnan Application Basic Research Project, the Innovation Team Project of Xie He, the Yunnan International Scientific and Technological Cooperation Project, and the Medical and Technology Innovation Project of Xie He.

An Illustration of FY-3E GNOS-R for Global Soil Moisture Monitoring.

An effective soil moisture retrieval method for FY-3E (Fengyun-3E) GNOS-R (GNSS occultation sounder II-reflectometry) is developed in this paper. Here, the LAGRS model, which is totally oriented for GNOS-R, is employed to estimate vegetation and surface roughness effects on surface reflectivity. Since the LAGRS (land surface GNSS reflection simulator) model is a space-borne GNSS-R (GNSS reflectometry) simulator based on the microwave radiative transfer equation model, the method presented in this paper takes more consideration on the physical scattering properties for retrieval. Ancillary information from SMAP (soil moisture active passive) such as the vegetation water content and the roughness coefficient are investigated for the final algorithm's development. At first, the SR (surface reflectivity) data calculated from GNOS-R is calculated and then calibrated, and then the vegetation roughness factor is achieved and used to eliminate the effects on both factors. After receiving the Fresnel reflectivity, the corresponding soil moisture estimated from this method is retrieved. The results demonstrate good consistency between soil moisture derived from GNOS-R data and SMAP soil moisture, with a correlation coefficient of 0.9599 and a root mean square error of 0.0483 cm3/cm3. This method succeeds in providing soil moisture on a global scale and is based on the previously developed physical LAGRS model. In this way, the great potential of GNOS-R for soil moisture estimation is presented.

Long non-coding RNA MCM3AP antisense RNA 1 silencing upregulates microRNA-24-3p to accelerate proliferation and migration of vascular endothelial cells in myocardial infarction rats by reducing EIF4G2.

Long non-coding RNAs (lncRNAs) are crucial drivers in the progression of human diseases such as myocardial infarction (MI). However, the impact of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) on MI remains unknown. This research was determined to explore the effect of MCM3AP-AS1 modulating microRNA-24-3p (miR-24-3p) and eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) on MI. The rat MI models were constructed and, respectively, treated with altered MCM3AP-AS1, miR-24-3p or/and EIF4G2. Subsequently, the cardiac function, myocardial pathological injury, malondialdehyde content and superoxide dismutase activity were determined. The vascular endothelial cells (VECs) were isolated and treated severally, and then proliferation and migration of VECs were measured. MCM3AP-AS1, miR-24-3p, EIF4G2 and vascular endothelial growth factor (VEGF) expressions in myocardial tissues and VECs were assessed. MCM3AP-AS1 and EIF4G2 were upregulated while miR-24-3p and VEGF were downregulated in MI rat myocardial tissues. MCM3AP-AS1 silencing or miR-24-3p elevation improved cardiac function and myocardial pathological injury, suppressed malondialdehyde content, and also enhanced VEGF expression and superoxide dismutase activity in MI rats. In VECs, downregulated MCM3AP-AS1 or upregulated miR-24-3p accelerated cell proliferation and migration. These effects of miR-24-3p upregulation were reversed by overexpressed EIF4G2. Our study summarizes that reduced MCM3AP-AS1 elevates miR-24-3p to promote proliferation and migration of MI rat VECs by inhibiting EIF4G2.

A flow-through chromatography purification process for Vero cell-derived influenza virus (H7N9).

Immunization is the most effective way to respond to an influenza epidemic. To produce Vero cell-derived influenza vaccines, a more efficient, stable and economical purification process is required. In this study, we purified the H7N9 influenza virus grown in Vero cells that were cultured in a serum-free medium by using a combination of anion exchange chromatography (AEC) and ligand-activated core chromatography (LCC), which avoids the virus capture step. After purification, 99.95 % host cell DNA (hcDNA) (final concentration: 28.69 pg/dose) and 98.87 % host cell protein (HCP) (final concentration: 28.28 ng/dose) were removed. The albumin content was 11.36 ng/dose. All these remnants met the current Chinese Pharmacopoeia and WHO requirements. The final virus recovery rate was 58.74 %, with the concentration of hemagglutinin recorded at 132.12 µg/mL. The flow-through chromatography purification process represents an alternative to the existing processes for cell-derived influenza viruses and might be suitable for the purification of other viruses as well.

An Experimental Study on Rapid Localization of the Atrioventricular Node During Open-Heart Surgery.

BACKGROUND: To verify the validity and feasibility of using a mechanical compression method to locate the atrioventricular node in open-heart surgery. METHODS: Ten healthy miniature pigs were used to establish an animal model of the beating heart under cardiopulmonary bypass. During the operation, the atrioventricular node and its surrounding areas were stimulated by mechanical compression (mechanical compression method), and the occurrence of complete atrioventricular block was judged by real-time electrocardiograph monitoring and direct observation of the heart rhythm to identify the position of the atrioventricular node. The final localization of the atrioventricular node was determined using the iodine staining method, and the results were used as the "gold standard" to test the effectiveness and feasibility of the mechanical compression method for locating the atrioventricular node. RESULTS: With the beating heart model, complete atrioventricular block occurred after mechanical compression of the "atrioventricular node" area in 10 pigs. Nine pigs regained normal conduction immediately after the compression was released, and one pig failed to recover. No atrioventricular block or other arrhythmias occurred after mechanical compression of the "non-atrioventricular node" area. The sensitivity of the method was 86.6%, specificity was 100.0%, misdiagnosis rate was 0.0%, missed diagnosis rate was 13.4%, positive predictive value was 100.0%, negative predictive value was 97.9%, positive likelihood ratios were +∞, negative likelihood ratios were 13.4%, accuracy was 98.1%, and diagnostic odds ratio was +∞. CONCLUSION: This study innovatively proposes the application of the mechanical compression method to locate the atrioventricular node during operation and preliminarily proves that this method is effective and feasible through animal experiments.

Gambogic Acid Shows Anti-Proliferative Effects on Non-Small Cell Lung Cancer (NSCLC) Cells by Activating Reactive Oxygen Species (ROS)-Induced Endoplasmic Reticulum (ER) Stress-Mediated Apoptosis.

BACKGROUND Gambogic acid (AG) is believed to be a potent anti-cancer agent. ER (endoplasmic reticulum) stress-induced cell apoptosis was identified as one of the anti-proliferative mechanisms of several anti-cancer agents. In this study, we investigated the involvement of ER stress-induced apoptosis in the anti-proliferative effect of GA on NSCLC (non-small cell lung cancer) cells. MATERIAL AND METHODS GA at 0, 0.5, and 1.0 µmol/l was used to treat A549 cells. We also used the ER stress-specific inhibitor 4-PBA (4-phenylbutyric acid) (1 µmol/l) to co-treat the cells incubated with GA. Cell viability was assessed by MTT (methyl thiazolyl tetrazolium) assay. Cell apoptosis was evaluated by MTT (methyl thiazolyl tetrazolium) assay. Intracellular ROS (reactive oxygen species) production was detected by DCFH-DA (2,7- dichloro-dihydrofluorescein diacetate) florescent staining. Western blotting was used to assess the expression and phosphorylation levels of protein. RESULTS GA treatment significantly reduced cell viabilities of NSCLC cells in a concentration-dependent manner. GA treatment increased intracellular ROS level, expression levels of GRP (glucose-regulated protein) 78, CHOP (C/EBP-homologous protein), ATF (activating transcription factor) 6 and caspase 12, as well as the phosphorylation levels of PERK (protein kinase R-like ER kinase) and IRE (inositol-requiring enzyme) 1alpha. Co-treatment of 4-PBA dramatically impaired the inhibitory effect of GA on cell viability. 4PBA co-treatment also decreased expression levels of GRP78, CHOP, ATF6, and caspase12, as well as the phosphorylation levels of PERK and IRE1alpha, in GA-treated NSCLC cells, without affecting ROS levels. CONCLUSIONS GA inhibited NSCLC cell proliferation by inducing ROS-induced ER stress-medicated apoptosis of NSCLC cells.

An adjuvant compound that enhances immunogenicity at fractional doses of the Sabin-inactivated poliovirus vaccine (sIPV) with a long duration of protection in a rat model.

BACKGROUND: At the same dosage, the new generation of Sabin-inactivated poliovirus vaccine (sIPV) is less immunogenic than the traditional oral polio vaccine (OPV) dosage in China. The useful adjuvant might be a necessary strategy to strengthen the immune protective effects. METHODS: In this study, we produced an adjuvant compound (named KML05) that could promote immunogenicity and fractional doses of sIPV with a long duration of protection in a rat model. The compound adjuvant had both advantages and a function of MF59 and carbopol971P. RESULTS: The effect seroconversion of experimental animals immunized with KML05 could be extended to one-eighth of the dose. According to the result of the geometric mean titers (GMTs), KML05 adjuvant could save eight times the amount of sIPV D-antigen usage, but aluminum hydroxide adjuvant could save twice at the same titers. Additionally, it was found that there was a significant difference in the GMT titer of poliovirus type 2 between animals immunized by KML05 and alum adjuvant (P < 0.05). At 12th-month postvaccination, the neutralization titers stimulated by IPV-KML05 were maintained over a longer time period in immunized animals. CONCLUSION: Our research team developed KML05 adjuvant, which combined carbopol971P with MF59, increased antibody responses to sIPV for a longer duration of protection in a rat model.

Gambogic Acid Induces Apoptosis of Non-Small Cell Lung Cancer (NSCLC) Cells by Suppressing Notch Signaling.

BACKGROUND Activation of Notch signaling was found to be associated with cancer. Gambogic acid (GA) was reported to be an anti-cancer agent. This study investigated the anti-cancer effect of GA on human non-small cell lung cancer (NSCLC) cells. Involvement of the Notch pathway was also studied. MATERIAL AND METHODS GA at 0, 0.5, 0.75, and 1.0 µmol/l was used to incubate A549 and SPC-A1 cells. MTT assay was used to determine the cell viability. TUNEL assay was used to detect the apoptosis. Western blotting was used to evaluate protein expression levels, protein phosphorylation levels, and nuclear translocation levels. RESULTS Notch signaling pathway was activated in NSCLC cells. GA treatment significantly inhibited NSCLC cell viability and increased cell apoptosis. GA treatment significantly decreased the expression levels of DLL1, DLL3, DLL4, Jagged1, Jagged2, Bcl2, and PK3K, inhibited NICD nuclear translocation and Akt phosphorylation, and increased expression level of active caspase3. CONCLUSIONS GA inhibited NSCLC cell viability by inducing apoptosis. Inhibition of the Notch signaling pathway was the mechanism involved in the anti-proliferation effect of GA on NSCLC.

Analysis of the dose-sparing effect of adjuvanted Sabin-inactivated poliovirus vaccine (sIPV).

Sabin-based inactivated poliovirus vaccine(sIPV) is gradually replacing live-attenuated oral polio vaccine(OPV). Sabin-inactivated poliovirus vaccine(sIPV) has played a vital role in reducing economic burden of poliomyelitis and maintaining appropriate antibody levels in the population. However, due to its high cost and limited manufacturing capacity, sIPV cannot reach its full potential for global poliovirus eradication in developing countries. Therefore, to address this situation, we designed this study to evaluate the dose-sparing effects of AS03, CpG oligodeoxynucleotides (CpG-ODN) and polyinosinic:polycytidylic acid (PolyI:C) admixed with sIPV in rats. Our results showed that a combination of 1/4-dose sIPV adjuvanted with AS03 or AS03 with BW006 provides a seroconversion rate similar to that of full-dose sIPV without adjuvant and that, this rate is 5-fold higher than that of 1/4-dose sIPV without adjuvant after the first immunization. The combination of AS03 or AS03 with BW006 as an adjuvant effectively reduced sIPV dose by at least 4-fold and induced both humoral and cellular immune responses. Therefore, our study revealed that the combination of AS03 or AS03 with BW006 is a promising adjuvant for sIPV development.

Preparation and evaluation of a novel, live, attenuated influenza H1N1 vaccine strain produced by a modified classical reassortment method.

Live attenuated influenza vaccine (LAIV)-based Vero cells could provide a better choice to control and prevent influenza virus infections. This study used the human influenza virus A/Yunnan/1/2005Vca(H3N2) (YN/05Vca) as a donor strain. YN/05Vca has a double phenotype of cold adaption (ca) and Vero cell adaption (va). The parental virus strain used was the wild-type A/Solomon Islands/3/2006 (H1N1) (SI/06wt). The study employed the modified classical reassortment method to generate a new virus strain. After co-infection of Vero cells, some different sub-types of the reassorted viruses were generated randomly. Then, the specific anti-serum (anti-YN/05Vca) could combine with and neutralize the donor virus, and the original parental virus could not grow in Vero cells at a low temperature until it was re-structured with the meaningful gene fragment from the donor virus in Vero cells. According to the plaques and RT-PCR results, a new monoclonal strain of Vero cell cold adaption virus was screened: SI/06Vca. After immunological and biological identification, this new strain virus could be used as a seed bank for LAIV, which has maintained surface antigenicity with SI/06wt. Consequently, this new Vero cell cold adaption virus SI/06Vca could be used for large-scale vaccine production with sufficient safety and efficacy, as confirmed by animal experiments with mice and ferrets.

Development and identification of a new Vero cell-based live attenuated influenza B vaccine by a modified classical reassortment method.

BACKGROUND: It was to generate a new Vero and cold-adapted live attenuated influenza B vaccine with enough safety and immunogenicity. METHODS: According to modified classical reassortment method, the donor strain was B/Yunnan/2/2005Vca(B), and the parental virus strain was B/Brisbane/60/2008wt. After co-infection in Vero cells, the prepared antibody serum inhibited the donor strain growth, and screening conditions inhibited the parental virus growth, which induced the growth of the new reassortant virus B/Brisbane/60/2008Vca(B) grow. Through intraperitoneal injection (i.j.) and intranasal injection (n.j.) we evaluated the safety and immunogenicity of the vaccine. RESULTS: A high-yield of the reassortant virus was produced in Vero cells at 25°C, similar to the donor strains. After sequencing, it was found that B/Brisbane/60/2008Vca(B) Hemagglutinin (HA) and Neuraminidase (NA) gene fragments were from B/Brisbane/60/2008wt, while the other 6 gene fragments were from B/Yunnan/2/2005Vca(B). The n.j. immune pathway experiments showed no significant differences between the treatment and the PBS control group with respect to weight changes (P > 0.5). Furthermore, the new strain had a sufficient geometric mean titter (GMT) against B/Brisbane/60/2008wt. CONCLUSION: The new reassortant live attenuated influenza B vaccine was safe and having enough immune stimulating ability.

Reassortment of high-yield influenza viruses in vero cells and safety assessment as candidate vaccine strains.

Vaccination is the practiced and accessible measure for preventing influenza infection. Because chicken embryos used for vaccine production have various insufficiencies, more efficient methods are needed. African green monkey kidney (Vero) cells are recommended by the World Health Organization (WHO) as a safe substitute for influenza vaccine production for humans. However, the influenza virus usually had low-yield in Vero cells, which limits the usage of Vero cellular vaccines. This study used 2 high-yield influenza viruses in Vero cells: A/Yunnan/1/2005Va (H3N2) and B/Yunnan/2/2005Va (B) as donor viruses. It used 3 wild strain viruses to reassort new adaptation viruses, including: A/Tianjin/15/2009(H1N1), A/Fujian/196/2009(H3N2), and B/Chongqing/1384/2010(B). These three new viruses could maintain the characteristic of high-yield in Vero cells. Furthermore, they could keep the immunogenic characteristics of the original wild influenza viruses. Importantly, these viruses were shown as safe in chicken embryo and guinea pigs assessment systems. These results provide an alternative method to produce influenza vaccine based on Vero cells.

Analyzed immunogenicity of fractional doses of Sabin-inactivated poliovirus vaccine (sIPV) with intradermal delivery in rats.

OBJECTIVE: The live-attenuated oral polio vaccine (OPV) will be no longer used when wild poliovirus (WPV) eliminating in worldwide, according to GPEI (the Global Polio Eradication Initiative) Reports. It is planning to replace OPV by Sabin-based inactivated poliovirus vaccine (sIPV) in developing countries, with purpose of reducing of the economic burden and maintaining of the appropriate antibody levels in population. It studied serial fractional doses immunized by intradermal injection (ID) in rats, to reduce consume of antigen and financial burden, maintaining sufficient immunogenicity; Methods: Study groups were divided in 4 groups of dose gradient, which were one-tenth (1/10), one-fifth (1/5), one-third (1/3) and one-full dose (1/1), according to the volume of distribution taken from the same batch of vaccine (sIPV). Wistar rats were injected intradermally with the needle and syringe sing the mantoux technique taken once month for 3 times. It was used as positive control that intramuscular inoculation (IM) was injected with one-full dose (1/1) with same batch of sIPV. PBS was used as negative control. Blood samples were collected via tail vein. After 30 d with 3 round of immunization, it analyzed the changes of neutralization antibody titers in the each group by each immunization program end; Results: The results of seroconversion had positive correlation with different doses in ID groups. The higher concentration of D-antigen (D-Ag) could conduct higher seroconversion. Furthermore, different types of viruses had different seroconversion trend. It showed that the geometric mean titers (GMTs) of each fractional-dose ID groups increased by higher concentration of D-Ag, and it got significant lower than the full-dose IM group. At 90th days of immunization, the GMTs for each poliovirus subtypes of fractional doses were almost higher than 1:8, implied that it could be meaning positive seroprotection titer for polio vaccine types, according to WHO suggestion; Conclusions: The fractional dose with one-fifth (1/5) could be used by intradermal injection to prevent poliovirus infection, if there were more human clinical detail research consistent with this findings in rats.

Construction high-yield candidate influenza vaccine viruses in Vero cells by reassortment.

Usage of influenza vaccine is the best choice measure for preventing and conclusion of influenza virus infection. Although it has been used of chicken embryo to produce influenza vaccine, following with WHO recommended vaccine strain, there were uncontrollable factors and its deficiencies, specially, during an influenza pandemic in the world. The Vero cells are used for vaccine production of a few strains including influenza virus, because of its homology with human, recommended by WHO. However, as known most of the influenza viruses strains could not culture by Vero cells. It was used two high-yield influenza viruses adapted in Vero cells as donor viruses, such as A/Yunnan/1/2005Va (H3N2) and B/Yunnan/2/2005Va (B), to construct high-yield wild influenza virus in Vero cells under antibody selection pressure. After reassortment and passages, it obtained the new Vaccine strains with A/Tianjin/15/2009Va (H1N1), A/Fujian/196/2009Va (H3N2) and B/Chongqing/1384/2010Va (B), which was not only completely keeping their original antigenic (HA and NA), but also grown well in Vero cells with high-yield. All results of gene analysis and HA, HI shown that this reassortment method could be used to find new direction to product the influenza vaccine. J. Med. Virol. 88:1914-1921, 2016. © 2016 Wiley Periodicals, Inc.

Reliability of pseudotyped influenza viral particles in neutralizing antibody detection.

BACKGROUND: Current influenza control strategies require an active surveillance system. Pseudotyped viral particles (pp) together with the evaluation of pre-existing immunity in a population might satisfy this requirement. However, the reliability of using pp in neutralizing antibody (nAb) detection are undefined. METHODOLOGY/PRINCIPAL FINDINGS: Pseudotyped particles of A(H1N1)pmd09 (A/California/7/2009) and HPAI H5N1 (A/Anhui/1/2005), as well as their reassortants, were generated. The reliability of using these pp in nAb detection were compared concurrently with the corresponding viruses by a hemagglutination inhibition test, as well as ELISA-, cytopathic effect-, and fluorescence-based microneutralization assays. In the qualitative detection on nAbs, the pp and their corresponding viruses were in complete agreement, with an R2 value equal to or near 1 in two different populations. In the quantitative detection on nAbs, although the geometric mean titers (95% confidence interval) differed between the pp and viruses, no significant difference was observed. Furthermore, humoral immunity against the reassortants was evaluated; our results indicated strong consistency between the nAbs against reassortant pp and those against naïve pp harboring the same hemagglutinin. CONCLUSION/SIGNIFICANCE: The pp displayed high reliability in influenza virus nAb detection. The use of reassortant pp is a safe and convenient strategy for characterizing emerging influenza viruses and surveying the disease burden.

High-yield production of a stable Vero cell-based vaccine candidate against the highly pathogenic avian influenza virus H5N1.

Highly pathogenic avian influenza (HPAI) viruses pose a global pandemic threat, for which rapid large-scale vaccine production technology is critical for prevention and control. Because chickens are highly susceptible to HPAI viruses, the supply of chicken embryos for vaccine production might be depleted during a virus outbreak. Therefore, developing HPAI virus vaccines using other technologies is critical. Meeting vaccine demand using the Vero cell-based fermentation process has been hindered by low stability and yield. In this study, a Vero cell-based HPAI H5N1 vaccine candidate (H5N1/YNVa) with stable high yield was achieved by reassortment of the Vero-adapted (Va) high growth A/Yunnan/1/2005(H3N2) (YNVa) virus with the A/Anhui/1/2005(H5N1) attenuated influenza vaccine strain (H5N1delta) using the 6/2 method. The reassorted H5N1/YNVa vaccine maintained a high hemagglutination (HA) titer of 1024. Furthermore, H5N1/YNVa displayed low pathogenicity and uniform immunogenicity compared to that of the parent virus.

[Preparation of vero cell-adapted influenza H5N1 virus strain by genetic reassortment].

OBJECTIVE: To prepare Vero cell-adapted influenza H5N1 virus strain by Genetic Reassortment and produce influenza H5N1 vaccine using Vero cell as a substrate. METHODS: Embryonated specific pathogen-free (SPF) hen's eggs and Vero cells were co-infected with Vero cell-adapted influenza virus A/Yunnan/1/2005 Va(H3N2) and A/Anhui/1/2005 (H5N1) via reverse genetics. The reassortant was screened with goat antibody against strain A/Yunnan/1/2005 Va(H3N2) and identified for subtype by hemagglutination-inhibition (HI) assays and gene analysis of HA and NA. RESULTS: A Vero cell-adapted influenza H5N1 virus strain was obtained, and there was no significant difference in serum antibody titers of monovalent inactivated vaccine reassorted before and after (F = 0.857, P > 0.05). CONCLUSION: The Vero cell-adapted influenza virus of epidemic strain may be reassortment between Vero cell-adapted and epidemic strains.

[Hasubanan type alkaloids in Stephania hernandifolia].

OBJECTIVE: To study the hasubanan type alkaloids in Stephania hernandifolia. METHOD: The dried herbs of S. hernandifolia. were extracted with 95% ethanol. After removal of the solvent, the residue was first partitioned between acid water and petroleum ether, then the aqueous layer was basified and extracted with chloroform to obtain crude alkaloids. Column chromatograghic methods with on silica gel, Rp-18, MCI CHP 20P, Sephadex LH-20 were applied for the isolation and purification of the crude alkaloid fraction. The structures were elucidated by their physicochemical properties and spectral data. RESULT: Nine hasubanan type alkaloids were obtained and identified as aknadinine(1), longanone(2), stephasunoline (3), N-methylstephuline(4), epistephamiersine(5), prostephabyssine(6), aknadilactam(7), dihydroepistephamiersine(8), hasubanonine(9). CONCLUSION: Compounds 2-8 were isolated from this plant for the first time.