ACE2-independent infection of T lymphocytes by SARS-CoV-2.

SARS-CoV-2 induced marked lymphopenia in severe patients with COVID-19. However, whether lymphocytes are targets of viral infection is yet to be determined, although SARS-CoV-2 RNA or antigen has been identified in T cells from patients. Here, we confirmed that SARS-CoV-2 viral antigen could be detected in patient peripheral blood cells (PBCs) or postmortem lung T cells, and the infectious virus could also be detected from viral antigen-positive PBCs. We next prove that SARS-CoV-2 infects T lymphocytes, preferably activated CD4 + T cells in vitro. Upon infection, viral RNA, subgenomic RNA, viral protein or viral particle can be detected in the T cells. Furthermore, we show that the infection is spike-ACE2/TMPRSS2-independent through using ACE2 knockdown or receptor blocking experiments. Next, we demonstrate that viral antigen-positive T cells from patient undergone pronounced apoptosis. In vitro infection of T cells induced cell death that is likely in mitochondria ROS-HIF-1a-dependent pathways. Finally, we demonstrated that LFA-1, the protein exclusively expresses in multiple leukocytes, is more likely the entry molecule that mediated SARS-CoV-2 infection in T cells, compared to a list of other known receptors. Collectively, this work confirmed a SARS-CoV-2 infection of T cells, in a spike-ACE2-independent manner, which shed novel insights into the underlying mechanisms of SARS-CoV-2-induced lymphopenia in COVID-19 patients.

Tracking single baculovirus retrograde transportation in host cell via quantum dot-labeling of virus internal component.

BACKGROUND: Quantum dot (QD)-based single virus tracking has become a powerful tool for dissecting virus infection mechanism. However, only virus behaviors at the early stage of retrograde trafficking have been dynamically tracked so far. Monitoring of comprehensive virus retrograde transportation remains a challenge. RESULTS: Based on the superior fluorescence properties of QDs and their labeling of virus internal component, the dynamic interactions between baculoviruses and all key transportation-related cellular structures, including vesicles, acidic endosomes, actins, nuclear pores and nuclei, were visualized at the single-virus level. Detailed scenarios and dynamic information were provided for these critical interaction processes. CONCLUSIONS: A comprehensive model of baculovirus retrograde trafficking involving virus endocytosis, fusion with acidic endosome, translocation to nuclear periphery, internalization into nucleus, and arriving at the destination in nucleus was proposed. Thus the whole retrograde transportation of baculovirus in live host cells was elucidated at the single-virus level for the first time.

Labeling the nucleocapsid of enveloped baculovirus with quantum dots for single-virus tracking.

Utilization of quantum dots (QDs) for single-virus tracking is highly important for understanding virus infection mechanism. However, QD labeling site of real enveloped viruses has been confined to the external envelope so far, causing the impossibility to monitor the late infection events after the loss of envelope. Herein, a strategy to label the internal nucleocapsid of enveloped virus with QDs was proposed. The nucleocapsid of enveloped baculovirus was self-biotinylated during virus replication process in host cells and subsequently labeled with streptavidin-conjugated QDs (SA-QDs). Such host cell-assisted QD labeling was proved to be reliable, specific, efficient and capable of maintaining virus infectivity. Based on such labeling, critical infection events before and after the envelope loss were monitored in real time, including single virus interacting with late endosomes and the subsequent nucleocapsid transporting into cell nucleus. Thus our established QD labeling of enveloped virus nucleocapsid with QDs enables the comprehensive single-virus tracking for deeply understanding virus infection mechanism.

Site-specific labeling of baculovirus in an integrated microfluidic device.

Labeling of viruses can be used to reveal viral infection pathways and screen potential anti-viral drugs. Complex procedures, including virus cultivation, purification and labeling are involved in traditional virus labeling methods. And the manipulation of living virus brings risk to researcher health. In this work, we report a general method for site-specific labeling of the envelope virus in an integrated microfluidic device with simple procedures and high security. Site-specific labeling of virus was achieved by fusing the biotin acceptor peptide (AP-tag) and the biotin ligase enzyme (BirA enzyme) with the envelope protein GP64 of baculovirus. The AP-tag could be modified by BirA enzyme to introduce the biotin moiety onto the viral envelope. Western blots and fluorescence colocalization analysis proved that the baculoviruses were biotinylated and labeled with high efficiency. The integrated device incorporated several operation steps including cell seeding, cell culture, cell transfection, virus culture and virus labeling. Since virus biotinylation was achieved during the process of virus cultivation, the complex procedures of virus labeling were simplified in our device. Furthermore the whole process could be completed in the integrated microfluidic device, and direct contact between viruses and researchers could be eliminated in our method, which could greatly reduce the risk to researcher health during living virus labeling.

Microchip-based immunoassays with application of silicon dioxide nanoparticle film.

Highly sensitive detection of proteins offers the possibility of early and rapid diagnosis of various diseases. Microchip-based immunoassay integrates the benefits from both immunoassays (high specificity of target sample) and microfluidics (fast analysis and low sample consumption). However, direct capture of proteins on bare microchannel surface suffers from low sensitivity due to the low capacity of microsystem. In this study, we demonstrated a microchip-based heterogeneous immunoassay using functionalized SiO(2) nanoparticles which were covalently assembled on the surface of microchannels via a liquid-phase deposition technique. The formation of covalent bonds between SiO(2) nanoparticles and polydimethylsiloxane substrate offered sufficient stability of the microfluidic surface, and furthermore, substantially enhanced the protein capturing capability, mainly due to the increased surface-area-to-volume ratio. IgG antigen and FITC-labeled anti-IgG antibody conjugates were adopted to compare protein-enrichment effect, and the fluorescence signals were increased by ~75-fold after introduction of functionalized SiO(2) nanoparticles film. Finally, a proof-of-concept experiment was performed by highly efficient capture and detection of inactivated H1N1 influenza virus using a microfluidic chip comprising highly ordered SiO(2) nanoparticles coated micropillars array. The detection limit of H1N1 virus antigen was 0.5 ng mL(-1), with a linear range from 20 to 1,000 ng mL(-1) and mean coefficient of variance of 4.71%.

[Identification of peptide mimotopes of an abroad-spectrum neutralizing epitope of highly pathogenic avian influenza hemagglutinin].

A monoclonal antibody (8H5), which showed strong neutralization activity against 33 strains of H5N1 viruses isolated from hosts at various regions from 2002 to 2006, was characterized in our lab recently. This result indicated the presence of highly conserved neutralizing site on hemagglutinin (HA) of various H5N1 subtypes. In the present study, the peptide phage display technique was applied to generate mimotope of the conserved neutralizing epitope recognized by 8H5 mAb. Five peptides displayed on phage were identified to specifically bind to 8H5 mAb. One of the five peptides, 123, was further displayed on the virus-like particle assembled from aa 1-149 fragment of HBcAg. The chimeric particle HBc-T123 conserved the specific binding to 8H5 mAb, and competed with H5N1 viruses for 8H5 mAb. The antiserum induced by HBc-T123 intensively stained on SF21 cells infected by recombinant baculovirus containing HA gene of YU22 virus, indicating the production of cross-reactive antibody to H5N1 HA.

[Experimental study on treatment of acute limb ischemia with vascular endothelial growth factor-121 gene transfer].

OBJECTIVE: To investigate the feasibility of intramuscular gene therapy for acute arterial ischemic diseases by use of plasmid pcDNA3-VEGF121 and to evaluate therapeutic efficiency of vascular endothelial growth factor (VEGF) by different routes of administration. METHODS: Fifty New Zealand White rabbits were randomly assigned to either gelation sponge carrying-pcDNA3-VEGF121 (n = 18), intramuscular injection-pcDNA3-VEGF121 (n = 18), or pcDNA3 (as control group, n = 14). After ligation of the external iliac artery and complete excision of the femoral artery, 500 micrograms of the plasmid pcDNA3-VEGF121 were transfected into the muscles of the ischemic limb by gelation sponge carrying or direct intramuscular-injection. Immediately after gene transfection, blood flow of the internal iliac artery were measured. VEGF121 gene expression was detected by RT-PCR after 2 days, 1 week, 2 weeks, 3 weeks and 4 weeks of transfection. After 30 days, blood flow of the internal iliac artery, angiographic score and histological vessels of ischemic hindlimbs were measured respectively. RESULTS: In the two VEGF-treated groups, VEGF121 mRNA expressed in the transfected ischemic muscles after 2 days and lasted 2 weeks. Immediately after gene transfection, blood flow of the internal iliac artery had no significant difference between three groups. After 30 days, blood flow of the internal iliac artery, angiographic score and capillary density were significantly greater in both VEGF-treated groups than in control group. Complexity of vascular branching and vessel density of gelation sponge-VEGF treated limbs were significantly greater when compared with the intramuscular-injection limbs. CONCLUSION: These findings suggest the feasibility of employing gene therapy of pcDNA3-VEGF121 could augment collateral development and tissue perfusion in an animal model of hindlimb ischemia, and gelation sponge carrying VEGF gene may respect a potential therapy methods.