Establishment and application of a surrogate model for human Ebola virus disease in BSL-2 laboratory.

The Ebola virus (EBOV) is a member of the Orthoebolavirus genus, Filoviridae family, which causes severe hemorrhagic diseases in humans and non-human primates (NHPs), with a case fatality rate of up to 90%. The development of countermeasures against EBOV has been hindered by the lack of ideal animal models, as EBOV requires handling in biosafety level (BSL)-4 facilities. Therefore, accessible and convenient animal models are urgently needed to promote prophylactic and therapeutic approaches against EBOV. In this study, a recombinant vesicular stomatitis virus expressing Ebola virus glycoprotein (VSV-EBOV/GP) was constructed and applied as a surrogate virus, establishing a lethal infection in hamsters. Following infection with VSV-EBOV/GP, 3-week-old female Syrian hamsters exhibited disease signs such as weight loss, multi-organ failure, severe uveitis, high viral loads, and developed severe systemic diseases similar to those observed in human EBOV patients. All animals succumbed at 2-3 days post-infection (dpi). Histopathological changes indicated that VSV-EBOV/GP targeted liver cells, suggesting that the tissue tropism of VSV-EBOV/GP was comparable to wild-type EBOV (WT EBOV). Notably, the pathogenicity of the VSV-EBOV/GP was found to be species-specific, age-related, gender-associated, and challenge route-dependent. Subsequently, equine anti-EBOV immunoglobulins and a subunit vaccine were validated using this model. Overall, this surrogate model represents a safe, effective, and economical tool for rapid preclinical evaluation of medical countermeasures against EBOV under BSL-2 conditions, which would accelerate technological advances and breakthroughs in confronting Ebola virus disease.

Oral delivery of a chitosan adjuvanted COVID-19 vaccine provides long-lasting and broad-spectrum protection against SARS-CoV-2 variants of concern in golden hamsters.

Coronavirus disease 2019 (COVID-19) seriously threatens public health safety and the global economy, which warrant effective prophylactic and therapeutic approaches. Currently, vaccination and establishment of immunity have significantly reduced the severity and mortality of COVID-19. However, in regard to COVID-19 vaccines, the broad-spectrum protective efficacy against SARS-CoV-2 variants and the blocking of virus transmission need to be further improved. In this study, an optimum oral COVID-19 vaccine candidate, rVSVΔG-Sdelta, was selected from a panel of vesicular stomatitis virus (VSV)-based constructs bearing spike proteins from different SARS-CoV-2 strains. After chitosan modification, rVSVΔG-Sdelta induced both local and peripheral antibody response, particularly, broad-spectrum and long-lasting neutralizing antibodies against SARS-CoV-2 persisted for 1 year. Cross-protection against SARS-CoV-2 WT, Beta, Delta, BA.1, and BA.2 strains was achieved in golden hamsters, which presented as significantly reduced viral replication in the respiratory tract and alleviated pulmonary pathology post SARS-CoV-2 challenge. Overall, this study provides a convenient, oral-delivered, and effective oral mucosal vaccine against COVID-19, which would supplement pools and facilitate the distribution of COVID-19 vaccines.

Bif-1c Attenuates Viral Proliferation by Regulating Autophagic Flux Blockade Induced by the Rabies Virus CVS-11 Strain in N2a Cells.

Bax-interacting factor-1 (Bif-1) is a multifunctional protein involved in apoptosis, autophagy, and mitochondrial morphology. However, the associations between Bif-1 and viruses are poorly understood. As discrete Bif-1 isoforms are selectively expressed and exert corresponding effects, we evaluated the effects of neuron-specific/ubiquitous Bif-1 isoforms on rabies virus (RABV) proliferation. First, infection with the RABV CVS-11 strain significantly altered Bif-1 expression in mouse neuroblastoma (N2a) cells, and Bif-1 knockdown in turn promoted RABV replication. Overexpression of neuron-specific Bif-1 isoforms (Bif-1b/c/e) suppressed RABV replication. Moreover, our study showed that Bif-1c colocalized with LC3 and partially alleviated the incomplete autophagic flux induced by RABV. Taken together, our data reveal that neuron-specific Bif-1 isoforms impair the RABV replication process by abolishing autophagosome accumulation and blocking autophagic flux induced by the RABV CVS-11 strain in N2a cells. IMPORTANCE Autophagy can be triggered by viral infection and replication. Autophagosomes are generated and affect RABV replication, which differs by viral strain and infected cell type. Bax-interacting factor-1 (Bif-1) mainly has a proapoptotic function but is also involved in autophagosome formation. However, the association between Bif-1-involved autophagy and RABV infection remains unclear. In this study, our data reveal that a neuron-specific Bif-1 isoform, Bif-1c, impaired viral replication by unchoking autophagosome accumulation induced by RABV in N2a cells to a certain extent. Our study reveals for the first time that Bif-1 is involved in modulating autophagic flux and plays a crucial role in RABV replication, establishing Bif-1 as a potential therapeutic target for rabies.

Characterization of a Vesicular Stomatitis Virus-Vectored Recombinant Virus Bearing Spike Protein of SARS-CoV-2 Delta Variant.

The frequent emergence of SARS-CoV-2 variants thwarts the prophylactic and therapeutic countermeasures confronting COVID-19. Among them, the Delta variant attracts widespread attention due to its high pathogenicity and fatality rate compared with other variants. However, with the emergence of new variants, studies on Delta variants have been gradually weakened and ignored. In this study, a replication-competent recombinant virus carrying the S protein of the SARS-CoV-2 Delta variant was established based on the vesicular stomatitis virus (VSV), which presented a safe alternative model for studying the Delta variant. The recombinant virus showed a replication advantage in Vero E6 cells, and the viral titers reach 107.3 TCID50/mL at 36 h post-inoculation. In the VSV-vectored recombinant platform, the spike proteins of the Delta variant mediated higher fusion activity and syncytium formation than the wild-type strain. Notably, the recombinant virus was avirulent in BALB/c mice, Syrian hamsters, 3-day ICR suckling mice, and IFNAR/GR-/- mice. It induced protective neutralizing antibodies in rodents, and protected the Syrian hamsters against the SARS-CoV-2 Delta variant infection. Meanwhile, the eGFP reporter of recombinant virus enabled the visual assay of neutralizing antibodies. Therefore, the recombinant virus could be a safe and convenient surrogate tool for authentic SARS-CoV-2. This efficient and reliable model has significant potential for research on viral-host interactions, epidemiological investigation of serum-neutralizing antibodies, and vaccine development.

[Application of augmented reality technique in repairing soft tissue defects of lower limbs with posterior tibial artery perforator flap].

Objective: To investigate the accuracy and reliability of augmented reality (AR) technique in locating the perforating vessels of the posterior tibial artery during the repair of soft tissue defects of the lower limbs with the posterior tibial artery perforator flap. Methods: Between June 2019 and June 2022, the posterior tibial artery perforator flap was used to repair the skin and soft tissue defects around the ankle in 10 cases. There were 7 males and 3 females with an average age of 53.7 years (mean, 33-69 years). The injury was caused by traffic accident in 5 cases, bruising by heavy weight in 4 cases, and machine injury in 1 case. The size of wound ranged from 5 cm×3 cm to 14 cm×7 cm. The interval between injury and operation was 7-24 days (mean, 12.8 days). The CT angiography of lower limbs before operation was performed and the data was used to reconstruct the three-dimensional images of perforating vessels and bones with Mimics software. The above images were projected and superimposed on the surface of the affected limb using AR technology, and the skin flap was designed and resected with precise positioning. The size of the flap ranged from 6 cm×4 cm to 15 cm×8 cm. The donor site was sutured directly or repaired with skin graft. Results: The 1-4 perforator branches of posterior tibial artery (mean, 3.4 perforator branches) in 10 patients were located by AR technique before operation. The location of perforator vessels during operation was basically consistent with that of AR before operation. The distance between the two locations ranged from 0 to 16 mm, with an average of 12.2 mm. The flap was successfully harvested and repaired according to the preoperative design. Nine flaps survived without vascular crisis. The local infection of skin graft occurred in 2 cases and the necrosis of the distal edge of the flap in 1 case, which healed after dressing change. The other skin grafts survived, and the incisions healed by first intention. All patients were followed up 6-12 months, with an average of 10.3 months. The flap was soft without obvious scar hyperplasia and contracture. At last follow-up, according to the American Orthopedic Foot and Ankle Association (AOFAS) score, the ankle function was excellent in 8 cases, good in 1 case, and poor in 1 case. Conclusion: AR technique can be used to determine the location of perforator vessels in the preoperative planning of the posterior tibial artery perforator flap, which can reduce the risk of flap necrosis, and the operation is simple.

Nucleic acid visualization assay for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) by targeting the UpE and N gene.

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2×101 copies/µl viral RNA for the UpE assay and 1.2 copies/µl viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.

A Novel Bacterium-Like Particle-Based Vaccine Displaying the SUDV Glycoprotein Induces Potent Humoral and Cellular Immune Responses in Mice.

Sudan virus (SUDV) causes severe lethal hemorrhagic fever in humans and nonhuman primates. The most effective and economical way to protect against Sudan ebolavirus disease is prophylactic vaccination. However, there are no licensed vaccines to prevent SUDV infections. In this study, a bacterium-like particle (BLP)-based vaccine displaying the extracellular domain of the SUDV glycoprotein (eGP) was developed based on a gram-positive enhancer matrix-protein anchor (GEM-PA) surface display system. Expression of the recombinant GEM-displayed eGP (eGP-PA-GEM) was verified by Western blotting and immunofluorescence assays. The SUDV BLPs (SBLPs), which were mixed with Montanide ISA 201VG plus Poly (I:C) combined adjuvant, could induce high SUDV GP-specific IgG titers of up to 1:40,960 and robust virus-neutralizing antibody titers reached 1:460. The SBLP also elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. These data indicate that the SBLP subunit vaccine has the potential to be developed into a promising candidate vaccine against SUDV infections.

Cytoglobin up-regulated by hydrogen peroxide plays a protective role in oxidative stress.

Cytoglobin (Cygb) is a recently discovered intracellular respiratory globin, which exists in all types of cells. It has been suggested that Cygb has a role in protecting cells against oxidative stress. In the present study we have tested this hypothesis. The N2a neuroblastoma cells were exposed to various kinds of insults, including hydrogen peroxide (H(2)O(2)), hypoxia, kainic acid, high extracellular CaCl(2), high osmolarity, UV irradiation and heat shock. Among them, only H(2)O(2)-treatment induced a significant up-regulation of cytoglobin mRNA level. We stably transfected N2a cells with Cygb-siRNA vectors and successfully knocked down Cygb. The Cygb-siRNA could exacerbate cell death upon H(2)O(2)-treatment, as demonstrated by MTT cell viability assay. Thus, Cygb in neuronal cells might be specifically induced under oxidative stress to protect them from death.