Analysis of SARS-CoV-2 infection dynamic in vivo using reporter-expressing viruses

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

Potent universal-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody

“…SARS-CoV-2 Epsilon was kindly provided by Dr. Charles Chiu, UCSF. The recombinant reporter-expressing SARS-CoV-2 were generated previously (21, 38).…”

Immunization with recombinant accessory protein-deficient SARS-CoV-2 protects against lethal challenge and viral transmission

“…No reuse allowed without permission. The rSARS-CoV-2 WT was generated previously 21 , and the rSARS-CoV-2 expressing mCherryNluc (rSARS-CoV-2 mCherryNluc) was generated using the previously describe strategy 24 .…”

“…This conclusion is based on comprehensive assessment in two different animal models of SARS-CoV-2 infection and transmission, the K18 hACE2 transgenic mouse 22,23 and the golden Syrian hamster models 21 , respectively. The rSARS-CoV-2 WT was generated previously 21 , and the rSARS-CoV-2 expressing mCherryNluc (rSARS-CoV-2 mCherryNluc) was generated using the previously describe strategy 24 .…”

“…The natural isolate SARS-CoV-2 of USA-WA1/2020 (NR-52281), and α (NR-54000), β (NR-54008), δ (NR-55611), and o (NR-56461) VoC were obtained from BEI Resources. The rSARS-CoV-2 WT was generated previously 21 , and the rSARS-CoV-2 expressing mCherryNluc (rSARS-CoV-2 mCherryNluc) was generated using the previously describe strategy 24 .…”

“…No reuse allowed without permission. replication was evaluated using a non-invasive in vivo imaging system (IVIS) in the whole organism (Nluc), as previously described 24 . Strong Nluc signal in the lungs of mockvaccinated mice was detected at 2 and 4 days post-challenge with rSARS-CoV-2 mCherryNluc, whereas no Nluc signal was detected in the lungs of K18 hACE2 transgenic mice vaccinated with rSARS-CoV-2 Δ3a/Δ7b (Figs.…”