Rapid adaptation of SARS-CoV-2 in BALB/c mice: Novel mouse model for vaccine efficacy

Coronavirus disease 2019 (COVID-19) threatens global public health and economy. In order to develop safe and effective vaccines, suitable animal models must be established. Here we report the rapid adaption of SARS-CoV-2 in BALB/c mice, based on which a convenient, economical and effective animal model was developed. Specifically, we found that mouse-adapted SARS-CoV-2 at passage 6 (MACSp6) efficiently infected both aged and young wild-type BALB/c mice, resulting in moderate pneumonia as well as inflammatory responses. The elevated infectivity of MACSp6 in mice could be attributed to the substitution of a key residue (N501Y) in the receptorbinding domain (RBD). Using this novel animal model, we further evaluated the in vivo protective efficacy of an RBD-based SARS-CoV-2 subunit vaccine, which elicited highly potent neutralizing antibodies and conferred full protection against SARS-CoV-2 MACSp6 challenge. This novel mouse model is convenient and effective in evaluating the in vivo protective efficacy of SARS-CoV-2 vaccine. SummaryThis study describes a unique mouse model for SARS-CoV-2 infection and confirms protective efficacy of a SARS-CoV-2 RBD subunit vaccine.

Oncolytic Activity of a Novel Influenza A Virus Carrying Granulocyte-Macrophage Colony-Stimulating Factor in Hepatocellular Carcinoma.

Oncolytic virotherapy is a promising strategy for the treatment of cancer. Influenza A virus has shown potential as an oncolytic agent. In this study, a recombinant PR8 influenza viral vector, called delNS1-GM-CSF, was generated with a partial deletion in NS and the granulocyte-macrophage colony-stimulating factor (GM-CSF) coding sequence inserted into the influenza nonstructural protein 1 gene. The morphological characteristics of delNS1-GM-CSF were examined. The delNS1-GM-CSF virus replicated well in various cell lines, including MDCK, A549, SMCC7721, and HepG2 cells. Moreover, selective cytotoxicity of the virus was observed in various hepatocellular carcinoma (HCC) cell lines, while no effect was demonstrated in the normal liver cell line LO2, as indicated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet assays. Importantly, using a model based on the growth of HepG2 cells as a xenograft in nude mice, it was found that a reassortant delNS1-GM-CSF virus inhibited tumor growth significantly following intratumoral injection in a dose-dependent manner. Ex vivo results showed that the tumor inhibition efficacy of delNS1-GM-CSF was observed in HCC clinical samples. Taken together, these results are the first to demonstrate that influenza A viruses may have potential as oncolytic virotherapeutic agents against HCC.

Progress in Chinese medicine-probiotics compound microecological preparations for livestock and poultry / 生物工程学报

Chinese medicine-probiotics compound microecological preparation for livestock and poultry is a new type of animal microecological preparations that combine probiotics with traditional Chinese medicine by modern fermentation technology. It could exert synergistic effects of both probiotics and traditional Chinese medicine, with the purpose of improving immune function of livestock and poultry and protect their health. By investigating the literature on probiotics and Chinese medicine microecological preparations in recent years, we summarized the background and strain characteristics of Chinese medicine-probiotics compound microecological preparations (CPCMP) for livestock and poultry in this paper. Furthermore, we elaborated the mechanisms of CPCMP for livestock and poultry and introduced the application of CPCMP in livestock and poultry breeding. Finally, we pointed out the existing problems and proposed the suggestions on the development of CPCMP. This review is expected to provide reference and basis for further research on CPCMP for livestock and poultry.

Digital measurement of anatomical parameters associated with vertebral units fixed by ribs and pedicle fixation of T4-T10 / 中华创伤骨科杂志

Objective To perform digital 3D measurement of anatomic parameters associated with vertebral units fixed by ribs and pedicle fixation of T4-T10,providing evidence for clinical application.Methods Spiral CT scan of T4-T10 vertebrae was conducted in 15 normal adults without spinal disorder.The data were imported into computer to establish units and disc models of thoracic and rib vertebrae T4-T10 using Mimics 16.0 software and Ansys 11.0 finite element software.The following data were measured in the vertebral pedicle-rib unit fixation group (group A) by software:transverse diameter of vertebral pedicle-rib unit,length of the outside pedicle screw,inclination angle of pedicle screw,and maximum and minimum inclination angles of pedicle screw;the following data were also measured in the pedicle fixation group (group B) by software:pedicle transverse diameter,length of pedicle screw,inclination angle of pedicle screw,and maximum and minimum inclination angles of pedicle screw.The corresponding parameters were compared between groups A and B.Results In both groups,the transverse diameters and screw lengths gradually increased with the increase in the vertebral sequence while the inclination angle of pedicle screw,and maximum and minimum inclination angles of pedicle screw decreased with the increase in the vertebral sequence.All the parameters in groups A were significantly larger than the corresponding ones in group B (P ＜ 0.05).Conclusions Of the spinal segment of T4-T10,the rib vertebral unit fixation can provide greater safe screw angles and screw diameters for clinical surgery.This is of vital significance for reducing the surgical complexity and improving screw prehension.

Differentiation of neural stem cells induced by erythropoietin in vitro / 中国组织工程研究

BACKGROUND:In recent years, neural stem cels are considered to be ideal for the treatment of spinal cord injury, but the proportion of its natural differentiation into neurons in the host body is relatively low, which severely restricts the therapeutic effect on spinal cord injury. OBJECTIVE:To investigate the effect of erythropoietin on the differentiation of neural stem cels in vitro. METHODS:Under sterile condition, neural stem cels from the hippocampus of neonatal Wistar rats were isolated, cultured and identified by immunofluorescencein vitro. The third generation of neural stem cels were randomly divided into 0.5, 5, 50 U/mL erythropoietin groups and control group (with no erythropoietin). RESULTS AND CONCLUSION:Compared with the control group, the differentiation rate of neural stem cels was significantly improved in the 0.5, 5, 50 U/mL erythropoietin groups (P 0.05). These findings indicate that erythropoietin can effectively induce the differentiation of neural stem cels into neurons in vitro, and moreover, it can significantly improve the differentiation rate of neural stem cels into neurons.

Molecular and genetic diagnosis of male infertility：practices and challenges / 中华检验医学杂志

Genetic factors are important causes of male infertility and account for about 30%infertility cases.Therefore, it is necessary to carry out molecular and genetic detections in the diagnosis and treatment of male infertility.Here the most commonly used techniques for molecular and genetic diagnosis of male infertility in recent years are discussed , including chromosomal abnormities analysis , Y chromosome microdeletions detection , gene mutation screening and sperm quality and function examination.

Preliminary screening of different permeation enhancers in transcutaneous immunization with inactivated human highly pathogenic avian influenza vaccine / 中华微生物学和免疫学杂志

Objective To screen the potent permeation enhancers used in transcutaneous immunization with inactivated highly pathogenic avian influenza vaccine. Methods Five different permeation enhancers, ethanol, propylene glycol, dimethyl sulfoxide, ratinoic acid, oleic acid, were used to treat the skin of BALB/c mice before transcutaneous immunization. Sera were collected before the flist transcutaneous immunization and every two weeks post immunization. The titers of influenza virus-specific humoral IgG and IgA were assayed in serum, lung and nasal lavages by ELISA. The titers of hemagglutination inhibition ( HAI), IFN-γand IL-4 produced by splenic lymphocytes were also detected. Except that, clinical symptom of the skin in different time points and skin pathological changes were observed. Results The serum IgG titers, HAI titers and the influenza virus-specific lgA and IgG in lung and nasal lavages in the groups of HA +CT + DMSO, HA + CT + RA and HA + CT + OA were significantly higher than those of HA and HA + CT groups( P ＜0.05). Moreover, the numbers of splenic lymphocytes producing IFN-γ and IL-4 were increased in the above three groups than those in control groups. In addition, no evident clinical symptoms were observed, but stratum corneum of the skin in different groups showed different changes. Conclusion DMSO,RA and OA are potent permeation enhancers in mouse model inoculated with inactivated high pathogenic avian influenza vaccine transcutaneously.

Preparation of the Coxsackievirus A16 VP1 subunit vaccine and detection of its immunogenicity / 中华微生物学和免疫学杂志

Objective To prepare VP1 protein vaccine of Coxsackievirus A16(CA16) and evalu-ate immunngenicity the subunit vaccines of Coxsackievirus (VP1), and to establish foundation for studying CA16 vaccine. Methods CA16 VP1 was amplified by RT-PCR and cloned into pFastBac HT A plasmid, recombinated with Bacmid DNA by transposition reaction and then transfected Sf9 cell, mixed with adjuvant AI(OH)_3. After immunization BALB/c mice, evaluating immune effectiveness after booster injections 2 weeks. Results The expressed protein was analyzed by SDS-PAGE and Western blot, mice immunized with CA16 (VP1) both induced specific IgG antibody and neutralization antibody. The best immunization antigen was 20 μg, IgG antibody was 1: 1600, neutralization antibody was 1:250, typical Th1/Th2 immune response was determined by lymphocyte proliferation assay and cytokine analysis. Conclusion The CA16 VP1 gene was cloned successfully and expressed in Sf9 insect cells, CA16 VP1 protein vaccine induced both humoral and cellular immune response, to lay solid foundation for further study on CA16 vaccine.

Development and Identification of a Live Attenuated Influenza B Virus Vaccine Candidate / 生物化学与生物物理进展

A cold-adapted (ca), temperature sensitive (ts), live attenuated influenza B virus strain B/Ann Arbor/1/66 was chosen for influenza virus rescue research, in which six internal gene segments, PB1, PB2, PA, NP, M, NS, were fully synthesized and nine amino acid substitutions were artificially alter by human intervention. The resultant B/Ann Arbor/1/66 plasmids were named as pAB121-PB1, pAB122-PB2, pAB123-PA, pAB124-HA, pAB125-NP, pAB126-NA, pAB127-M and pAB128-NS, respectively. A recombinant influenza A virus was previously generated entirely from cloned cDNA. An infectious recombinant influenza B virus was generated here, and designated as rMDV-B, by plasmid-based reverse genetics. The rMDV-B virus contained HA and NA genes from an epidemic influenza B vires strain B/Malaysia/2506/2004 in the background of internal genes derived from influenza B virus strain B/Ann Arbor/1/66. HA titer of rMDV-B in MDCK cells and embryonated chicken eggs ranged from 1 : 64 to 1 : 512. The results may allow an effective live influenza B vaccine to be produced from a single master strain, providing a model for the design of future live human influenza vaccines.

Immunogenicity of a novel vaccine with VLP as vector for Wilm's tumor / 中华普通外科杂志

Objective To develop a vaccine for Wilm's tumor in order to lay a foundation for effective treatment and substitute for the radiotherapy and chemotherapy of this tumor. Methods DNA fragments encoding the WT1 antigens (including, HLA-A * 2402 and HLA-A * 2402x), and DNA fragments encoding the couple antigens HLA-A * 2402-HBc and HLA-A * 2402x-HBc were cloned and inserted into the eukaryotic expression vector pcDNA3.1(+) in sense orientation and baculovirus expression pFactbac respectively, then transfected into COS-7 cells and Sf 9 cell respectively. The expression of the target proteins in the cells were identified by CPE, SDS-PAGE and electron microscopy. Virus-like particles were observed under electron microscopy. Results The fusion gene of HLA-A * 2402 and HLA-A * 2402x and HBc express virus-like particle protein. Conclusions This preliminary result shows a hopeful future in developing an effective immunotherapy to Wilm's tumor.

Construction of Attenuated Influenza Virus Vaccines of Vaccines of-2006-2007 / 生物化学与生物物理进展

Six gene segments,PB1,PB2,PA,NP,M and NS,were fully synthesized which derived from the master donor virus (MDV),cold-adapted(ca),temperature sensitive(ts),live attenuated influenza virus strain A/Ann Arbor/6/60(MDV-A).Meanwhile,five amino acid substitutions (PB1-391E,58lG,661T,PB2-265S,NP-34G) were artificially altered by human intervention.HA and NA fragments derived from the 2006-2007 circulating strain A/New Caledonia/20/99 (H1N1).Eight fragments were ligated with modified pAD3000 for rescue plasmid construction.Eiight transcription/expression plasmids were named as pMDV-A-PB2,pMDV-A-PB1,pMDV-A-PA,pMDV-A-NP,pMDV-A-M,pMDV-A-NS,pMDV-A-HA,pMDV-A-NA,respectively.The COS-l cells were co-transfected with eight plasmids representing 6 internal viral backbone of the strain A/AA/6/60 and two plasmids containing the CDNA of the HA and NA segments of the strain A/New Caledonia/20/99 (H1N1),the results showed that cold-adapted,attenuated reassortant influenza A virus Was rescued successfully.Titers of a reassorted influenza A virus in embryonated chicken eggs mnged from 1:29to l:210.The rescue system of six intemal genes used as backbone opens the way for further research on gene function and neotype vaccine candidate of cold-adapted,live attenuated human influenza virus.

Generation of cold-adapted, attenuated reassortant human influenza A virus with eight-plasmid system / 中华微生物学和免疫学杂志

Objective To set up a technical platform of reverse genetics based on the 8 plasmid.virus rescue system of cold-adapted influenza virus strain. Methods The cold-adapted, temperature sensitive, live attenuated influenza virus strain A/AnnArbor/6/60(H2N2)was chosen as the master donor virus(MDV)for rescue research,and its six internal gene fragments PB2,PB1,PA,NP,M and NS were artificially synthesized. Meanwhile, five amino acid mutations have been introduced as tags. Six fragments were ligated with modified pAD3000 for the construction of rescue plasmid. Six transcription/expression plasmids(pMDV-A-PB2,pMDV-A-PB1,pMDV-A-PA,pMDV-A-NP,pMDV-A-M,and pMDV-A-NS)were obtained, and their sequences were accurate. Results The reassorted virus named as rMDV-A contains HA and NA gene segments derived from PR8 strain along with six gene segments,PB2,PB1,PA,NP,M and NS,from MDV. The COS-1 cells were co-transfected with eight recombinant plasmids. The results showed that a cold-adapted, attenuated reassortant influenza A virus with hemagglutination activity was rescued successfullv bv"6+2" combination of MDV and PR8, and the allanotoic fluid of the injected eggs gave a posigenes of A/AA/6/60 used as backbone has provided experimental materials for further research on the gene function and novel vaccine candidate of cold-adapted, attenuated human influenza virus.

Construction of Attenuated Influenza Virus Vaccines of 2006～2007 / 生物化学与生物物理进展

Six gene segments, PB1, PB2,PA, NP, M and NS, were fully synthesized which derived from the master donor virus(MDV), cold-adapted(ca),temperature sensitive(ts), live attenuated influenza virus strain A/Ann Arbor/6/60(MDV-A). Meanwhile, five amino acid substitutions (PB1-391E, 581G, 661T, PB2-265S, NP-34G) were artificially altered by human intervention. HA and NA fragments derived from the 2006～2007 circulating strain A/New Caledonia/20/99 (H1N1). Eight fragments were ligated with modified pAD3000 for rescue plasmid construction. Eight transcription/expression plasmids were named as pMDV-A-PB2, pMDV-A-PB1, pMDV-A-PA, pMDV-A-NP, pMDV-A-M, pMDV-A-NS, pMDV-A-HA, pMDV-A-NA, respectively. The COS-1 cells were co-transfected with eight plasmids representing 6 internal viral backbone of the strain A/AA/6/60 and two plasmids containing the cDNA of the HA and NA segments of the strain A/New Caledonia/20/99 (H1N1), the results showed that cold-adapted, attenuated reassortant influenza A virus was rescued successfully. Titers of a reassorted influenza A virus in embryonated chicken eggs ranged from 1∶29 to 1∶210. The rescue system of six internal genes used as backbone opens the way for further research on gene function and neotype vaccine candidate of cold-adapted, live attenuated human influenza virus.

Application of flow cytometry in sperm chromatin structure assay / 中华检验医学杂志

Objective To study the factors effecting on flow cytometric sperm chromatin structure assay.Methods The SCSA explores the metachromatic properties of acridine orange (AO) and flow cytometry to monitor the susceptibility of sperm chromatin DNA to acid induced denaturation in situ because of the low pH treatment. Results COMP?t, ?t, SD?t value were different upon on the storage methods (①sperm was preserved in 4℃, ②sperm was cryopreservated after dilution ③sperm was cryopreservated). Results show that the second method above made the least artificial injury to sperm DNA. It did not affect the results if the samples were quickly thawed in a 37℃ water bath and detected immediately and the detect current velocity was lower than 300 cell/sec. The intra -CV was 7 28% and the internal CV was 8 92%. Mean and standard deviations of COMP?t were 8 7?11 0% in 511 healthy men. Because data present right skew distribution, the reference range of COMP?t is

Gene analysis of new isolates HA of influenza A （H1N1） virus / 免疫学杂志

Objective　To obtain genetic characterization of the HA1 of new isolates of influenza A （H1N1） virus. Methods　Virus was amplified in embryonated chicken eggs， the virion DNA was transcribed into cDNA by reverse transcriptase， cDNA amplified by PCR， the products of PCR were purified. Afterward， DNA sequence analysis was performed by the dideoxy-mediated chain termination method， using synthetic oligo nucleotide primers. Results　The HA1 domain of new isolates of influenza A （H1N1） virus showed that their HA1 genes were 981 nucleotide in length coding for a HA1 protein with 327 amino acids， deletion of a glycosylation site and an amino acid. The homology of amino acid sequences of protein molecules on HA1 domains of new influenza A virus when compared with A／Guifang／10／94（H1N1） and A／Bayern ／07／95 （H1N1） viruses，was 92.8％ and 91.3％ respectively； The homology of amino acid sequences of protein molecules on HA1 domains of A／Jingjun／11／98（H1N1） when compared with A／Jingjun／13／98（H1N1） or A／Jingjun／807／97（H1N1） viruses，was as high as 98％； The homology of amino acid sequences of protein molecules on HA1 domains of A／Guifang／10／94（H1N1） when compared with A／Bayern／07／95（H1N1） viruses was as high as 96％. Conclusions　The HA1 gene of　new H1N1 virus strains is　different from those of A／Guifang／10／94 （H1N1） and A／Bayern／07／95（H1N1）， they will probably be new mutation strains.

Study of anti-lipid A antibodies of bacterial endotoxin from phage antibody library / 中华传染病杂志

Objective To study the treatment of sepsis caused by G - bacteria, anti-lipid A antibodies of bacterial endotoxin were screened from phage antibody library. Methods The mRNA was extracted from human B-lymphocytes against lipid A of bacterial endotoxin, reversely transcripted and amplified by polymerase chain reaction using general primers scanning Fd and light chain of IgG. The amplified fragments were inserted into pCOMB3 vector and electrotransfected competent E.coli XL 1-blue cells. Furthermore, the recombinant phage was lysed by coculture with helper VCSM13. Results Fab displayed on the surface as fusion protein with the N terminal of coat protein Ⅲ, and 4.8?10 6 clone library was established. Antibodies against lipid A of bacterial endotoxin were screened. Specific antibodies against lipid A of bacterial endotoxin were enriched by 100 times after three rounds of panning with lipid A.Conclusions Three clones exhibited specific binding to lipid A is identified by direct and competitive ELISA methods. The succcess of isolating anti-lipid A proves the usefulness of phage display system in human McAb preparation. The result shows that we have got the recombinant phage antibody.

Expression of inositol 1,4,5-triphosphate receptor in airway smooth muscle cells of rats and its roles in chronic asthma formation / 第三军医大学学报

Objective To detect the expression of inositol 1,4,5-triphosphate receptor (IP 3R) subtypes in normal rat airway smooth muscle cells (ASMCs) and changes during chronic asthma formation. Methods ASMCs were cultured by collagen enzyme digestion method. The expressions of subtypes of IP 3R were detected by RT-PCR and the purified PCR products were linked with pGEM-T vector for DNA sequencing. Chronic asthma model was established with egg albumin. The changes of IP 3Rs were detected by RT-PCR method. Results All subtypes of IP 3R were expressed in airway smooth muscle cells of normal rats. The expression of IP 3R1 in asthma groups increased obviously as compared with that in the control group (P