Adenoviral vectors are widely used for gene therapy and vaccine development. To circumvent pre-existing immunity against commonly used human adenovirus type 5, vectors based on rare human serotype or animal adenoviruses have attracted increasing interest. Previously, we constructed vectors based on fowl adenovirus 4 (FAdV-4) and replaced the knob of FAdV-4 fiber2 with that of FAdV-1 fiber1 to generate FAdV4-CF1K vectors with enhanced transduction efficiency in human cells. In this study, we aimed to modify the packaging system to efficiently produce FAdV-4 vectors carrying transgenes toxic to viral replication. Chicken LMH cells failed to form colonies at low seeding densities. We collected used medium from LMH cell cultures and used it as a supplement to adapt LMH cells, generating the colony-competent subclone LMH-C3532. A lentiviral vector encoding a codon-optimized tetracycline repressor (tetR) was transduced into LMH-C3532 to establish a tetR-integrated cell line, LMH-tetR24. An adenoviral plasmid, pKFAV4-CF1K-CtG, was constructed in which a tetracycline operator (tetO)-bearing CMV promoter controlled GFP expression. The SwaI-flanked GFP in this plasmid was replaced with the HA gene from an H5N1 influenza virus to generate pKFAV4-CF1K-CtHA. Linearized adenoviral plasmids were transfected into LMH-tetR24 cells, and recombinant FAdV4-CF1K-CtG and FAdV4-CF1K-CtHA viruses were successfully rescued, amplified, and purified. When infected with FAdV4-CF1K-CtG at various multiplicities of infection (MOI), the progeny virus yield from LMH-tetR24 cells was 4-10 times higher than that from LMH-C3532 cells. For FAdV4-CF1K-CtHA, the yield difference between the two cell lines was even more pronounced, reaching 3-4 orders of magnitude. Overexpression of HA in LMH-C3532 cells negatively affected FAdV4-CF1K-CtHA replication, resulting in smaller and fewer plaques. In conclusion, by separately integrating tetR into packaging cells and TetO into the adenoviral plasmid, we established a system that can be routinely used to package FAdV-4 vectors. Notably, this system facilitates the propagation of FAdV-4 vectors carrying toxic transgenes.

West Nile virus (WNV) and chikungunya virus (CHIKV) are zoonotic pathogens transmitted by mosquitoes, which cause significant morbidity and mortality globally. Currently, no human vaccine is available against both WNV and CHIKV. In this study, we developed a novel bivalent recombinant human adenovirus type 5 (Ad5)-based vaccine, designated Ad5-WNV-CHIKV, which encodes WNV prM-E and CHIKV E3-E2-6 K-E1 antigens. The expression of the target antigens for WNV and CHIKV was validated through western blotting and an immunofluorescence assay. We subsequently assessed the humoral and cellular immune response in C57BL/6 mice following intramuscular administration of a single dose of Ad5-WNV-CHIKV. Both low-dose (2 × 108 viral particles [vp] per mouse) and high-dose (1 × 109 vp per mouse) administration of Ad5-WNV-CHIKV elicited robust immune responses against the viral targets, with specific immunoglobulin G production against WNV-E, CHIKV E1, and CHIKV E2 proteins, and generation of neutralizing antibodies against WNV and CHIKV, in a dose-dependent manner. Moreover, the vaccine induced strong cellular immunity, characterized by multifunctional antigen-reactive CD4+ and CD8+ T-cell populations, as determined by ELISpot and intracellular cytokine staining assays. A single dose of the bivalent Ad5-WNV-CHIKV vaccine induced strong neutralizing antibody and cellular immune responses against both WNV and CHIKV in mice. These findings provide a foundation for the development of vaccines targeting WNV and CHIKV. A single dose of vaccine could potentially prevent both diseases.

The development of broad-spectrum universal influenza vaccines and optimization of vaccination strategies to address the threats posed by pandemics and emerging influenza viruses are critical for public health. In this study, an adenovirus type 5 vector-based influenza vaccine carrying the hemagglutinin (HA) stem of H1, HA stem of H3, and neuraminidase (NA) of N1 from the influenza virus was constructed. Immune responses were evaluated in mice using various vaccination strategies: prime-only (intramuscular [IM] or intranasal [IN]) and prime-boost (IM + IN). Compared with the prime-only strategy, the prime-boost strategy significantly enhanced the systemic immune response, inducing higher levels of antigen-specific IgG, mucosal IgA, and T cell immunity in the spleen and lungs. Furthermore, the IN boosting strategy provided complete protection in mice challenged with the H1N1-PR8, rgH3N2-X31, and rgH5N1-Vietnam viruses, significantly reducing viral loads in the lungs and alleviating lung tissue pathologies. In conclusion, this study elucidates potential avenues for the development and application of universal influenza vaccines using customized mucosal boosting strategies.

In eukaryotic systems, three major types of cell junctions have been well characterized. While bacterial adhesion mechanisms also exhibit remarkable diversity, the molecular processes that regulate the dynamic modulation of binding strength between elongated bacterial cells and host cells remain poorly understood. Fusobacterium nucleatum (F. nucleatum) utilizes the surface adhesin CbpF to interact with the highly expressed host receptors CEACAM1 and CEACAM5 on cancer cells to facilitate tumor colonization. By elucidating the structural details of CbpF binding to human CEACAM1/CEACAM5 receptors, and through mechanistic investigations, we identified that the prominent EFNGQYQ loop on CbpF and the key Q78 residue of CEACAM1/CEACAM5 constitute the molecular linchpin of this pathogen-host interface. Furthermore, we found a distinct type of binding particle and proposed a Velcro-like adhesion model. In this model, CbpF mediates robust attachment through the simultaneous interaction of multiple binding sites, akin to the interlocking mechanism of Velcro. This multivalent interaction allows F. nucleatum to dynamically switch between firm anchoring and easy detachment, adapting to varying physiological microenvironments. Our study elucidates the dynamic modulation of bacterial adhesion strength and lays the foundation for developing therapeutic interventions to disrupt the bacterium-host interface.

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.

Mouse adenoviruses (MAdV) play important roles in studying host–adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes.

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.

The transcriptome of fowl adenovirus has not been comprehensively revealed. Here, we attempted to analyze the fowl adenovirus 4 (FAdV-4) transcriptome by deep sequencing. RNA samples were extracted from chicken LMH cells at 12, 18 or 26 h post-FAdV-4 infection, and subjected to Illumina strand-specific RNA-seq or nanopore full-length PCR-cDNA sequencing. After removing the reads of host cells, the data of FAdV-4 nanopore full-length cDNAs (transcripts) were corrected with reads from the Illumina RNA-seq, mapped to the viral genome and then used to predict viral open reading frames (ORFs). Other than 42 known ORFs, 39 novel ORFs were annotated to the FAdV-4 genome. Different from human adenovirus 5, one FAdV-4 ORF was often encoded by several transcripts, and more FAdV-4 ORFs were located on two exons. With these data, 18 major transcription start sites and 15 major transcription termination sites were defined, implying 18 viral promoters and 15 polyadenylation signals. The temporal cascade of viral gene transcription was observed in FAdV-4-infected cells, with six promoters possessing considerable activity in the early phase. Unexpectedly, four promoters, instead of one major late promoter, were engaged in the transcription of the viral genus-common genes on the forward strand. The clarification of the FAdV-4 transcriptome laid a solid foundation for the study of viral gene function, virulence and virus evolution, and it would help construct FAdV-4 as a gene transfer vehicle. The strategy of de novo ORF prediction could be used to parse the transcriptome of other novel adenoviruses.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified phlebovirus associated with severe hemorrhagic fever in humans. Studies have shown that SFTSV nucleoprotein (N) induces BECN1-dependent autophagy to promote viral assembly and release. However, the function of other SFTSV proteins in regulating autophagy has not been reported. In this study, we identify SFTSV NSs, a nonstructural protein that forms viroplasm-like structures in the cytoplasm of infected cells as the virus component mediating SFTSV-induced autophagy. We found that SFTSV NSs-induced autophagy was inclusion body independent, and most phenuivirus NSs had autophagy-inducing effects. Unlike N protein-induced autophagy, SFTSV NSs was key in regulating autophagy by interacting with the host's vimentin in an inclusion body-independent manner. NSs interacted with vimentin and induced vimentin degradation through the K48-linked ubiquitin-proteasome pathway. This negatively regulating Beclin1-vimentin complex formed and promoted autophagy. Furthermore, we identified the NSs-binding domain of vimentin and found that overexpression of wild-type vimentin antagonized the induced effect of NSs on autophagy and inhibited viral replication, suggesting that vimentin is a potential antiviral target. The present study shows a novel mechanism through which SFTSV nonstructural protein activates autophagy, which provides new insights into the role of NSs in SFTSV infection and pathogenesis. IMPORTANCE Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerging tick-borne pathogen that causes multifunctional organ failure and even death in humans. As a housekeeping mechanism for cells to maintain steady state, autophagy plays a dual role in viral infection and the host's immune response. However, the relationship between SFTSV infection and autophagy has not been described in detail yet. Here, we demonstrated that SFTSV infection induced complete autophagic flux and facilitated viral proliferation. We also identified a key mechanism underlying NSs-induced autophagy, in which NSs interacted with vimentin to inhibit the formation of the Beclin1-vimentin complex and induced vimentin degradation through K48-linked ubiquitination modification. These findings may help us understand the new functions and mechanisms of NSs and may aid in the identification of new antiviral targets.

限制性酶切-组装是构建和改造腺病毒载体的一种简便方法,本研究用该方法构建同时携带GFP和萤火虫荧光素酶(Firefly Luciferase,Fluc)报告基因的猴1型腺病毒(Simian Adenovirus 1,SAdV-1)载体.PCR分别扩增GFP和Fluc基因,重叠延伸PCR将两个目的基因通过T2A短肽编码序列串联得到融合基因GFluc.限制性内切酶SpeI酶切腺病毒质粒pKSAV1-EG,去除原有的外源基因,将载体片段与GFluc片段进行限制性酶切-组装,产物转化大肠杆菌感受态细胞,获得腺病毒质粒pKSAV1-GFluc.通过限制性内切酶酶切分析、PCR产物测序证实质粒构建正确.pKSAV1-GFluc质粒经SwaI酶切线性化后转染SAdV-1包装细胞系293SE13,4 d后GFP+细胞形成荧光灶,并持续增大,表明重组病毒拯救成功;对重组病毒基因组进行了限制性酶切鉴定.扩增病毒并进行纯化,在细胞水平和动物模型中检测到目的蛋白GFP和Fluc的表达.本研究成功构建同时携带GFP和Fluc报告基因的SAdV-1载体,显示了限制性酶切-组装方法的易用性和可靠性,并为研究SAdV-1载体在小鼠模型的组织分布奠定基础.

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.

评价一种SARS-CoV-2 Beta变异株和甲型流感病毒H3N2新型重组双价疫苗在小鼠模型中的免疫保护效果.本研究构建了表达SARS-CoV-2南非变异株(B.1.351)棘突蛋白1(S1)和H3N2柬埔寨分离株(A/Cambodia/e0826360/2020)血凝素(HA)的重组双价非复制Ad5载体疫苗,命名为HAdV5-S1-2A-HA,经单针肌肉注射免疫BALB/c雌鼠后,采用ELISA、血凝抑制实验、假病毒中和实验与Elispot实验进行体液与细胞免疫学检测,免后3～6周采用H3N2-X31病毒、H1N1-PR8与SARS-CoV-2(B.1.351)进行攻毒保护实验.HAdV5-S1-2A-HA免疫两周后,低剂量组(1×108vp/只)免疫小鼠后可检出HA特异体液免疫与S1特异的细胞免疫应答;而高剂量组(5X 109vp/只)诱导小鼠产生了较强的双抗原(S1,HA)特异的体液和细胞免疫应答,并能完全保护小鼠对H3N2-X31攻击,降低SARS-CoV-2(B.1.351)感染后小鼠肺部病毒载量,延迟H1N1-PR8病毒感染后小鼠死亡发生.本研究制备的重组双价疫苗HAdV5-S1-2A-HA在小鼠体内诱导抗原特异的体液与细胞免疫应答.同时,它还可以在小鼠中诱导对SARS-CoV-2和H3N2感染的免疫保护,具有较好的研发与应用前景.

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

The novel coronavirus pandemic has stimulated the scientific activity of virology and interdisciplinary sciences: medical cybernetics and bioinformatics. The article is focused on the study of algorithms for processing bioinformatic data of genomic origin predominantly for the purposes of predominantly immunoinformatics and computational vaccinology. The schemes of algorithms developed by the authors for the analysis of bioinformatic data are presented. The algorithms for processing genomic information developed by the authors based on the analysis of the available literature and many years of experience in computational and laboratory experiments can be used not only for the design and analysis of epitope vaccine components, but also for the other tasks of computational virology and microbiology. In silico experiments on the analysis of bioinformatic data are relatively low-cost and multi-informative, but they require highly qualified scientists with extensive experience, interdisciplinary training, and, accordingly, a wide range of knowledge and skills. However, for the complete analysis and implementation of, for example, the epitope vaccines, subsequent validation by the laboratory and in vivo experiments are required.

Fowl adenovirus 4 (FAdV-4) has the potential to be constructed as a gene transfer vector for human gene therapy or vaccine development to avoid the pre-existing immunity to human adenoviruses. To enhance the transduction of FAdV-4 to human cells, CELO fiber1 knob (CF1K) was chosen to replace the fiber2 knob in FAdV-4 to generate recombinant virus F2CF1K-CG. The original FAdV4-CG virus transduced 4% human 293 or 1% HEp-2 cells at the multiplicity of infection of 1000 viral particles per cell. In contrast, F2CF1K-CG could transduce 98% 293 or 60% HEp-2 cells under the same conditions. Prokaryotically expressed CF1K protein blocked 50% transduction of F2CF1K-CG to 293 cells at a concentration of 1.3 µg/mL while it only slightly inhibited the infection of human adenovirus 5 (HAdV-5), suggesting CF1K could bind to human cells in a manner different from HAdV-5 fiber. The incorporation of CF1K had no negative effect on the growth of FAdV-4 in the packaging cells. In addition, CF1K-pseudotyped HAdV-41 could transduce HEp-2 and A549 cells more efficiently. These data indicated that CF1K had the priority to be considered when there is a need to modify adenovirus tropism.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

Background Pre-existing immunities hamper the application of human adenovirus (HAdV) vectors in gene therapy or vaccine development. Fowl adenovirus (FAdV)-based vector might represent an alternative. Methods An intermediate plasmid containing FAdV-4 fiber genes, pMD-FAV4Fs, was separated from FAdV-4 adenoviral plasmid pKFAV4GFP. An overlap extension polymerase chain reaction (PCR) was employed for fiber modification in pMD-FAV4Fs, and the modified fibers were restored to generate new adenoviral plasmids through restriction-assembly. FAdV-4 vectors were rescued and amplified in chicken LMH cells. Fluorescence microscopy and flow cytometry were used to evaluate the gene transfer efficiency. The amount of viruses binding to cells was determined by a real-time PCR. A plaque-forming assay and one-step growth curve were used to evaluate virus growth. Results Four sites in the CD-, DE-, HI- and IJ-loop of fiber1 knob could tolerate the insertion of exogenous peptide. The insertion of RGD4C peptide in the fiber1 knob significantly promoted FAdV-4 transduction to human adherent cells such as 293, A549 and HEp-2, and the insertion to the IJ-loop demonstrated the best performance. The replacement of the fiber2 knob of FAdV-4 with that of HAdV-35 improved the gene transfer to human suspension cells such as Jurkat, K562 and U937. Fiber-modified FAdV-4 vectors could transduce approximately 80% human cells at an acceptable multiplicity of infection. Enhanced gene transfer mainly resulted from increased virus binding. Fiber modifications did not significantly influence the growth of recombinant FAdV-4 in packaging cells. Conclusions As a proof of principle, it was feasible to enhance gene transduction of FAdV-4 vectors to human cells by modifying the fibers.

2020年12月15日,大连市报告了4名码头冷链货物搬运工人SARS-CoV-2核酸检测呈阳性,在此之前,大连市已经连续136天没有报告本土病例.在这次大连COVID-19疫情(简称"大连新冠疫情")中,我们收集了2020年12月15日至2021年1月8日期间大连新冠疫情中全部感染者(83)及部分接触的轮船货物样本,其中确诊病例占61.45％(51/83),无症状感染者占38.55％(32/83).通过高通量测序,共获得76条SARS-CoV-2全基因组序列,其中72条(86.75％,72/83)来自临床样本,4条来自R国籍A货船上的冷链食品外包装样本.基因组分析数据显示,与武汉参考株(NC_045512)相比,76条全基因组分别存在12～16个核苷酸突变位点,共享12个核苷酸突变位点,符合B.1.1进化分支突变特征.结合病毒基因组学和现场流行病学调查结果综合分析表明,大连新冠疫情是一起由SARS-CoV-2污染的进口冷链产品感染码头工人导致的本土疫情,在传播过程中至少形成了3个病毒代际和3个相对独立的传播链.