Packaging Quantum Dots in Viral Particles via a Strep-tag II/Streptavidin System for Single-Virus Tracking.

Single-virus tracking provides a powerful tool for studying virus infection with high spatiotemporal resolution. Quantum dots (QDs) are used to label and track viral particles due to their brightness and photostability. However, labeling viral particles with QDs is not easy. We developed a new method for labeling viral particles with QDs by using the Strep-tag II/streptavidin system. In this method, QDs were site-specifically ligated to viral proteins in live cells and then packaged into viral-like particles (VLPs) of tick-borne encephalitis virus (TBEV) and Ebola virus during viral assembly. With TBEV VLP-QDs, we tracked the clathrin-mediated endocytic entry of TBEV and studied its intracellular dynamics at the single-particle level. Our Strep-tag II/streptavidin labeling procedure eliminates the need for BirA protein expression or biotin addition, providing a simple and general method for site-specifically labeling viral particles with QDs for single-virus tracking.

T-Cell Immunoglobulin and Mucin Domain 1 (TIM-1) Is a Functional Entry Factor for Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection affecting humans. The host factors required for viral entry have yet to be described. Here, we found that T-cell immunoglobulin and mucin domain 1 (TIM-1) acted as the cellular entry factor for TBEV. Using a virus overlay protein binding assay, TIM-1 was identified as a virion-interacting protein. Cells that were relatively resistant to TBEV infection became highly susceptible to infection when TIM-1 was ectopically expressed. TIM-1 knockout and viral RNA bypass assays showed that TIM-1 functioned in the entry phase of TBEV infection. TIM-1 mediated TBEV uptake and was cointernalized with virus particles into the cell. Antibodies for TIM-1, soluble TIM-1, or TIM-1 knockdown significantly inhibited TBEV infection in permissive cells. Furthermore, in TIM-1 knockout mice, TIM-1 deficiency markedly lowered viral burden and reduced mortality and morbidity, highlighting the functional relevance of TIM-1 in vivo. With TIM-1, we have identified a key host factor for TBEV entry and a potential target for antiviral intervention. IMPORTANCE TBEV is a tick-transmitted flavivirus that causes serious diseases in the human central nervous system in Eurasia. The host determinants required for viral entry remain poorly understood. Here, we found that TIM-1 is a cellular entry factor for TBEV. Antibodies directed at TIM-1 or soluble TIM-1 treatment decreased virus infection in cell cultures. TIM-1 was cointernalized with virus particles into cells. TIM-1 deficiency significantly lowered viral burden and attenuated pathogenesis in the murine TBEV infection model. The demonstration of TIM-1 as a cellular entry factor for TBEV will improve understanding of virus infection and provide a target for antiviral development.

Nasal delivery of broadly neutralizing antibodies protects mice from lethal challenge with SARS-CoV-2 delta and omicron variants.

Multiple new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have constantly emerged, as the delta and omicron variants, which have developed resistance to currently gained neutralizing antibodies. This highlights a critical need to discover new therapeutic agents to overcome the variants mutations. Despite the availability of vaccines against coronavirus disease 2019 (COVID-19), the use of broadly neutralizing antibodies has been considered as an alternative way for the prevention or treatment of SARS-CoV-2 variants infection. Here, we show that the nasal delivery of two previously characterized broadly neutralizing antibodies (F61 and H121) protected K18-hACE2 mice against lethal challenge with SARS-CoV-2 variants. The broadly protective efficacy of the F61 or F61/F121 cocktail antibodies was evaluated by lethal challenge with the wild strain (WIV04) and multiple variants, including beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) at 200 or 1000 TCID50, and the minimum antibody administration doses (5-1.25 â€‹mg/kg body weight) were also evaluated with delta and omicron challenge. Fully prophylactic protections were found in all challenged groups with both F61 and F61/H121 combination at the administration dose of 20 â€‹mg/kg body weight, and corresponding mice lung viral RNA showed negative, with almost all alveolar septa and cavities remaining normal. Furthermore, low-dose antibody treatment induced significant prophylactic protection against lethal challenge with delta and omicron variants, whereas the F61/H121 combination showed excellent results against omicron infection. Our findings indicated the potential use of broadly neutralizing monoclonal antibodies as prophylactic and therapeutic agent for protection of current emerged SARS-CoV-2 variants infection.

Case Report: A Re-Positive Case of SARS-CoV-2 Associated With Glaucoma.

The current pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has already become a global threat to the human population. Infection with SARS-CoV-2 leads to a wide spectrum of clinical manifestations. Ocular abnormalities have been reported in association with COVID-19, but the nature of the impairments was not specified. Here, we report a case of a female patient diagnosed with glaucoma on re-hospitalization for ocular complications two months after being discharged from the hospital upon recovery from COVID-19. Meanwhile, the patient was found re-positive for SARS-CoV-2 in the upper respiratory tract. The infection was also diagnosed in the aqueous humor through immunostaining with antibodies against the N protein and S protein of SARS-CoV-2. Considering the eye is an immune-privileged site, we speculate that SARS-CoV-2 survived in the eye and resulted in the patient testing re-positive for SARS-CoV-2.

In Vitro Coinfection and Replication of Classical Swine Fever Virus and Porcine Circovirus Type 2 in PK15 Cells.

Increasing clinical lines of evidence have shown the coinfection/superinfection of porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV). Here, we investigated whether PCV2 and CSFV could infect the same cell productively by constructing an in vitro coinfection model. Our results indicated that PCV2-free PK15 cells but not ST cells were more sensitive to PCV2, and the PK15 cell line could stably harbor replicating CSFV (PK15-CSFV cells) with a high infection rate. Confocal and super-resolution microscopic analysis showed that PCV2 and CSFV colocalized in the same PK15-CSFV cell, and the CSFV E2 protein translocated from the cytoplasm to the nucleus in PK15-CSFV cells infected with PCV2. Moreover, PCV2-CSFV dual-positive cells increased gradually in PK15-CSFV cells in a PCV2 dose-dependent manner. In PK15-CSFV cells, PCV2 replicated well, and the production of PCV2 progeny was not influenced by CSFV infection. However, CSFV reproduction decreased in a PCV2 dose-dependent manner. In addition, cellular apoptosis was not strengthened in PK15-CSFV cells infected with PCV2 in comparison with PCV2-infected PK15 cells. Moreover, using this coinfection model we further demonstrated PCV2-induced apoptosis might contribute to the impairment of CSFV HCLV strain replication in coinfected cells. Taken together, our results demonstrate for the first time the coinfection/superinfection of PCV2 and CSFV within the same cell, providing an in vitro model to facilitate further investigation of the underlying mechanism of CSFV and PCV2 coinfection.