The administration route affects the biodistribution of a gene transfer vector and the expression of a transgene. A simian adenovirus 1 vector carrying firefly luciferase and GFP reporter genes (SAdV1-GFluc) were constructed, and its biodistribution was investigated in a mouse model by bioluminescence imaging and virus DNA tracking with real-time PCR. Luciferase activity and virus DNA were mainly found in the liver and spleen after the intravenous administration of SAdV1-GFluc. The results of flow cytometry illustrated that macrophages in the liver and spleen as well as hepatocytes were the target cells. Repeated inoculation was noneffective because of the stimulated serum neutralizing antibodies (NAbs) against SAdV-1. A transient, local expression of low-level luciferase was detected after intragastric administration, and the administration could be repeated without compromising the expression of the reporter gene. Intranasal administration led to a moderate, constant expression of a transgene in the whole respiratory tract and could be repeated one more time without a significant increase in the NAb titer. An immunohistochemistry assay showed that respiratory epithelial cells and macrophages in the lungs were transduced. High luciferase activity was restricted at the injection site and sustained for a week after intramuscular administration. A compromised transgene expression was observed after a repeated injection. When these mice were intramuscularly injected for a third time with the human adenovirus 5 (HAdV-5) vector carrying a luciferase gene, the luciferase activity recovered and reached the initial level, suggesting that the sequential use of SAdV-1 and HAdV-5 vectors was practicable. In short, the intranasal inoculation or intramuscular injection may be the preferred administration routes for the novel SAdV-1 vector in vaccine development.

Computational thinking is a crucial skill for students to analyze and solve problems, and programming education serves as an important pathway to cultivate computational thinking. This study combines relevant literature and utilizes Scratch, a visual programming software, to construct a gamified teaching model for Scratch courses. A quasi-experimental research design was employed, involving two fifth-grade classes from a primary school in K city. The study employed methods such as analysis of student works, SPSS scale data analysis, and student interviews to investigate the impact of gamified teaching in Scratch courses on developing elementary students' computational thinking. The results indicate that gamified teaching in Scratch courses significantly enhances the computational thinking level of elementary students, particularly in the dimensions of critical thinking, problem-solving ability, and algorithmic thinking. Based on the research findings, it is recommended that gamified teaching in Scratch courses should involve appropriate teaching tools, incorporate game-based learning, emphasize problem-solving, encourage collaborative learning, and provide training for educators competent in programming education.

The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.

Twenty-eight patients suffering acute pain following operative removal of impacted third molars took part in the present study. In 20 patients who reported pain reduction exceeding 25% of the initial pain intensity during vibratory stimulation (100 Hz) or TENS (2 or 100 Hz), only 1 patient (given 2 Hz TENS) reported pain increase after injection of 0.8 mg naloxone (i.v.). In 8 patients, not treated with afferent stimulation, 2 experienced increase in pain intensity after naloxone injection. The results show that pain relief using TENS or vibration is not influenced by naloxone.

A novel fowl adenovirus 4 (FAdV-4) has caused significant economic losses to the poultry industry in China since 2015. We established an easy-to-use reverse genetics system for modification of the whole right and partial left ends of the novel FAdV-4 genome, which worked through cell-free reactions of restriction digestion and Gibson assembly. Three recombinant viruses were constructed to test the assumption that species-specific viral genes of ORF4 and ORF19A might be responsible for the enhanced virulence: viral genes of ORF1, ORF1b and ORF2 were replaced with GFP to generate FAdV4-GFP, ORF4 was replaced with mCherry in FAdV4-GFP to generate FAdV4-GX4C, and ORF19A was deleted in FAdV4-GFP to generate FAdV4-CX19A. Deletion of ORF4 made FAdV4-GX4C form smaller plaques while ORF19A deletion made FAdV4-CX19A form larger ones on chicken LMH cells. Coding sequence (CDS) replacement with reporter mCherry demonstrated that ORF4 had a weak promoter. Survival analysis showed that FAdV4-CX19A-infected chicken embryos survived one more day than FAdV4-GFP- or FAdV4-GX4C-infected ones. The results illustrated that ORF4 and ORF19A were non-essential genes for FAdV-4 replication although deletion of either gene influenced virus growth. This work would help function study of genes on the right end of FAdV-4 genome and facilitate development of attenuated vaccines.

Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene-harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, single plasmid-based adenoviral vector systems were constructed where a unique PmeI site was located at the position for insertion of the exogenous gene. The polymerase chain reaction (PCR) amplified transgene could be cloned into PmeI-linearized starting plasmids using one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was termed restriction assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded starting plasmid pKAd5f11pABR-EPG. The modified fiber gene, amplified by overlap extension PCR, could be used to substitute the original fiber in pKAd5f11pABR-EPG to generate an adenoviral plasmid with a new fiber by restriction assembly. On the other hand, pKAd5f11pABR-EPG was also a starting adenoviral plasmid for expressing other transgenes. In conclusion, easy-to-use and upgradable adenoviral vector systems are introduced here, which offer extensive versatility and can serve as a basic platform and functional component library for the synthetic biology of adenoviral vectors.

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.

Objective To establish a method of correlative light and electron microscopy (CLEM) to study the intracellular location of adenovirus protein Ⅸ.Methods MiniSOG (mini singlet oxygen generator) is a recently-invented genetically-encoded tag for CLEM.MiniSOG-fused adenovirus Ⅸ gene (ⅨSOG) was cloned by PCR,and inserted into pcDNA3 plasmid to form pTPL-ⅨSOG,which was used to transfect 293 cells.ⅨSOG expressing cells could be distinguished under fluorescence microscope due to the emission of green fluorescence of miniSOG.The transfected cells were fixed in 2.5％ glutaraldehyde in situ,stained with diaminobenzidine(DAB) through the photooxidation activity of miniSOG,and used to prepare uhrathin sections.Intracellular location of ⅨSOG was studied by observing the sections under transmission electron microscope.Results Eukaryotic expression plasmid carrying ⅨSOG fusion gene was constructed.ⅨSOG expressing cells were selected for DAB photooxidation and preparation of ultrathin sections.ⅨSOG fusion mainly formed punctate aggregations or inclusions in the nucleus.Conclusions The correlative light and electron microscopy method based on miniSOG was successfully established,and it could be used to study the intracellular localization of viral proteins.

Human adenovirus type 41 (HAdV-41) is considered to be a "fastidious adenovirus". E1-deleted HAdV-41 cannot be rescued or amplified in 293 cells. To propagate recombinant HAdV-41 in 293 cells, the backbone plasmid pAdbone41 was reconstructed. That is, the E3 coding sequence of HAdV-41 was deleted and replaced with the HAdV-5 E4orf6 gene; and the E1A enhancer of HAdV-5 was inserted upstream of the E4 promoter of HAdV-41. Novel adenoviral plasmid pAd41E4EE-GFP was generated by homologous recombination of the shuttle plasmid pSh41-GFP with the modified backbone plasmid in the Escherichia coli BJ5183 strain. Adenovirus HAdV-41-E4EE-GFP was rescued by transfecting 293 cells with linearized pAd41E4EE-GFP. After seven rounds of propagation, viruses were purified by the CsCl ultracentrifugation method. HAdV-41-E4EE-GFP in 1.0 ml with a particle titer of 8 x 10(10) vp/mL was obtained which had a particle-to-infectious ratio of 50 : 1. The genome of HAdV-41-E4EE-GFP was confirmed by restriction analyses and polymerase chain reaction. These results showed that a novel HAdV-41 vector system was established in which recombinant HAdV-41 could be constructed and packaged in 293 cells.

To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.

The human adenovirus (HAdV) early protein E1B55K interacts with E4orf6 to form an E3 ubiquitin ligase complex, which plays key roles in virus replication. To illustrate the reason for the fastidiousness of HAdV-41 in 293 cells, interaction between heterotypic E1B55K and E4orf6 proteins was investigated. HAdV-5 E1B55K could interact with HAdV-41 E4orf6, and vice versa. To form E1B55K/E4orf6 E3 ubiquitin ligase, HAdV-41 E4orf6 recruited Cul2 while HAdV-5 E4orf6 interacted with Cul5. The ligase complex formed by HAdV-5 E1B55K and HAdV-41 E4orf6 could cause the degradation of p53 and Mre11. However, in E1-deleted HAdV-41-infected 293TE7 cells, which expressed HAdV-41 E1B55K, viral late mRNAs were exported from nucleus more efficiently and accumulated to a higher concentration in cytoplasm when compared with that in infected 293 cells. These results suggested that interaction between homotypic E1B55K and E4orf6 was indispensable for efficient export of viral late mRNAs.

To investigate the components of fibrillous inclusion body (FIB), which was formed in packaging cells during the replication of human adenovirus type 41 (Ad41), Ad41 long fiber knob (LFK) and short fiber knob (SFK) proteins were expressed in prokaryote respectively and then used to immunize BALI mice for preparation of anti-LFK serum and anti-SFK sera. The activity and specificity of anti-LFK and an ti-SFK sera were confirmed with Western blot, indirect immunofluorescence assay (IFA) and immunonegative staining, suggesting these sera could be applied in immuno-colloidal gold labelling electron microscopy (EM). 293TE cells were infected with wild-type Ad41. Ultrathin sections of infected cells were made, and labelled with immuno-colloidal gold technique using anti-Ad41 sera, anti-LFK sera, anti-SFK sera, or anti-fiber monoclonal antibody 4D2, respectively. The labelled sections were observed under EM, and the results demonstrated that both Ad41 long fiber protein and short fiber protein were included FIB.

Human adenovirus type 41 (HAdV-41) is well known for its fastidiousness in cell culture. To construct an infectious clone of HAdV-41, a DNA fragment containing the left and right ends of HAdV-41 as well as a kanamycin resistance gene and a pBR322 replication origin was excised from the previously constructed plasmid pAd41-GFP. Using homologous recombination, the plasmid pKAd41 was generated by co-transformation of the E. coli BJ5183 strain with this fragment and HAdV-41 genomic DNA. Virus was rescued from pKAd41-transfected 293TE7 cells, a HAdV-41 E1B55K-expressing cell line. The genomic integrity of the rescued virus was verified by restriction analysis and sequencing. Two fibers on the virion were confirmed by western blot. Immunofluorescence showed that more expression of the hexon protein could be found in 293TE7 cells than in 293 cells after HAdV-41 infection. The feature of non-lytic replication was preserved in 293TE7 cells, since very few progeny HAdV-41 viruses were released to the culture medium. These results show that pKAd41 is an effective infectious clone and suggest that the combination of pKAd41 and 293TE7 cells is an ideal system for virological study of HAdV-41.

OBJECTIVE To investigate the genetic stability of non-replicating recombinant adenovirus which used Ad41 as vector and could express VP6 gene of group A rotavirus during continous passage, in order to develop the vaccine of rotavirus. METHODS The recombinant adenovirus rvAd41-VP6 (o) was prepared by our laboratory early, it then was continuously propagated on 293TE7 cells for 14 passages. After that samples of the infected cells were collected at every 2 passages for the detection of the integration of the VP6 gene by PCR, and the expression of the target protein was detected by Western Blot analysis. RESULTS Analysis by PCR revealed that, there was stable integration of specific VP6 gene in the rvAd41-VP6 (o), Western Blot analysis confirmed that rvAd41-VP6 (o) could stably expressed the group-specific antigen structural protein VP6 (o), and it had preferable genetic stability. CONCLUSION The recombinant adenovirus rvAd41-VP6 (o) which could stably express the VP6 (o) gene had favorable biological property in vitro, and it has provided a basis for further research of animal immunization.

To determine the ratio of short fiber (sfiber) to long fiber (lfiber) in human adenovirus type 41 (HAdV-41) virions, sfiber and lfiber were expressed in E. coli, quantified, and used as loading standards in Western blot. Densitometric analyses of the standard and target bands indicated that the ratio of sfiber to lfiber in virions was 5.7±0.7. Sfiber-deleted HAdV-41, HAdV-41-DSF-GFP, was constructed, and Western blot analysis showed that the amount of lfiber in HAdV-41-DSF-GFP was about 7.3±1.9 times of that in HAdV-41 virions, confirming a ratio of approximate 6 for sfiber to lfiber in HAdV-41. In HAdV-41-infected cells, mRNAs of the sfiber and lfiber were comparable in quantity, while the expression at protein level was significantly different. Our results suggested an unequal number of short and long fibers, which might result from their differential protein expression during HAdV-41 packaging. The method used here could be extended to quantify other trace proteins.

OBJECTIVEBoth the 2, 6 linkage and its topology on target cells are critical for the recognition by human influenza virus. The binding preference of avian flu virus H5N1 HA to the 2, 3-linked sialylated glycans is considered the major factor limiting its efficient infection and transmission in humans. To monitor potential adaptation of H5N1 virus in human population, the surveillance of receptor-binding specificity was undertaken in China.METHODSThe binding specificity of 32 human H5N1 virus strains isolated from 2003 to 2009 was tested by 2, 3-specific sialidase-treated chicken red blood cell (CRBC) agglutination assay and a solid-phase direct binding assay with synthetic sialylglycopolymers.RESULTSDual binding preference to 2, 3 and 2, 6-glycans were found in two strains: A/Guangdong/1/06 (A/GD/1/06) and A/Guangxi/1/08 (A/GX/1/08). Though minor effect of short-2, 6-binding was detected in A/GX/1/08 at a low virus titer, both showed high affinity to the oligosaccharide at a high load. Notably both are of the long-2, 6-recognition, with the same topology as that of human H1N1 and H3N2 viruses.CONCLUSIONThe findings suggest that human H5N1 virus in China likely acquired the potential human-adaptation ability. Further research and surveillance on receptor-binding specificity of H5N1 viruses are required.

Background During the 2009 pandemic influenza H1N1 (2009) virus (pH1N1) outbreak, school students were at an increased risk of infection by the pH1N1 virus. However, the estimation of the attack rate showed significant variability. Methods Two school outbreaks were investigated in this study. A questionnaire was designed to collect information by interview. Throat samples were collected from all the subjects in this study 6 times and sero samples 3 times to confirm the infection and to determine viral shedding. Data analysis was performed using the software STATA 9.0. Findings The attack rate of the pH1N1 outbreak was 58.3% for the primary school, and 52.9% for the middle school. The asymptomatic infection rates of the two schools were 35.8% and 37.6% respectively. Peak virus shedding occurred on the day of ARI symptoms onset, followed by a steady decrease over subsequent days (p = 0.026). No difference was found either in viral shedding or HI titer between the symptomatic and the asymptomatic infectious groups. Conclusions School children were found to be at a high risk of infection by the novel virus. This may be because of a heightened risk of transmission owing to increased mixing at boarding school, or a lack of immunity owing to socio-economic status. We conclude that asymptomatically infectious cases may play an important role in transmission of the pH1N1 virus.

OBJECTIVE:To observe the serum immune responses and protection in mice model of the recombinant adenovirus vector mediated human rotavirus VP6 gene expression through coden optimization (rvAdVP6(o)) in comparison with the wild type (rvAdVP6).METHODS:6-8 week female BALB/c mice were randomly grouped and immunized three times intranasally with 10(8) TCID50 rvAdVP6(o) and rvAdVP6, respectively, then detect the serum IgG level against rotavirus induced by rvAdVP6(o) and rvAdVP6. The amount of sheding viral antigens in feces was detectd after mice rotavirus was taked orally.RESULTS:The serum IgG level against rotavirus induced by rvAdVP6(o) was higher than that of rvAdVP6 after three times of immunization. The immunized mice shed lower amount of viral antigens in feces as compared with the rvAdVP6.CONCLUSION:The recombinant adenovirus which encode optimized human rotavirus VP6 proteins (rvAdVP6(o)) could induce stronger serum immune and protective responses against the challenge of the rotavirus than the wild type (rvAdVP6) at the same immunizing dosage.

This study aimed to develop an effective experimental vaccine against highly pathogenic H5N1 Avian Influenza (HPAI) virus and to optimize their immunization programs. As reported previously, various DNA-based or recombinant vaccinia viral(Tiantan)-based H5N1 vaccine candidates, which containing a single cistronic construct (HAop, or NAop) or a bicistronic construct (HAop/M2 or NAop/M1) of H5N1 influenza virus (Anhui strain) were constructed and characterized in our lab. In this study, we further analysed the immunogenicity in mice of these vaccine candidates by various protocols (single or combined immunization). Our results showed that: comparing with immunization with DNA-based or rTTV-based H5N1 vaccine only, combined DNA-based with rTTV-based H5N1 vaccine immunization via prime-boost strategy enhanced immune response significantly against multi-H5N1 antigens detected by hemagglutination inhibition (HI) assay, NA- or M1- or M2-specific antibody detection, and micro-neutralizing antibody test and IFN-gamma ELISpot assay. Priming with DNA-based vaccine induced higher level of humoral response against HA or NA antigen than priming with rTTV-based vaccine; In contract, M1 and M2-specific antibody levels were higher among that of priming with rTTV -based vaccine. These findings provide a basis for further development of novel H5N1 vaccines and for the optimization of the immunization programs of combined multi-antigens vaccine candidates.