4.6 Article

Neuromasts and Olfactory Organs of Zebrafish Larvae Represent Possible Sites of SARS-CoV-2 Pseudovirus Host Cell Entry

期刊

JOURNAL OF VIROLOGY
卷 96, 期 24, 页码 -

出版社

AMER SOC MICROBIOLOGY
DOI: 10.1128/jvi.01418-22

关键词

SARS-CoV-2; ACE2 receptor; endocytosis; two-pore channel type 2; zebrafish

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资金

  1. Health and Medical Research Fund (COVID-19) from the Hong Kong Government Food and Health Bureau [HMRF20SC07(COVID190213)]
  2. Hong Kong Innovation Technology Fund [PRP/073/20FX, UIM/385]
  3. Hong Kong Research Grants Council General Research Fund [16100719]
  4. Hong Kong Innovation and Technology Commission [ITCPD/17-9]

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Using zebrafish embryos/larvae as an in vivo model, this study explored the mechanism of host cell entry of SARS-CoV-2. The endolysosomal system was found to be involved in viral entry, and TPC2 played a key role in the translocation of the virus through this system. The peripheral sensory organs were identified as a likely site for viral host cell entry.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease coronavirus disease 2019 (COVID-19), which has resulted in millions of deaths globally. Here, we explored the mechanism of host cell entry of a luciferase-ZsGreen spike (SARS-CoV-2)-pseudotyped lentivirus using zebrafish embryos/larvae as an in vivo model. Successful pseudovirus entry was demonstrated via the expression of the luciferase (luc) gene, which was validated by reverse transcription-PCR (RT-PCR). Treatment of larvae with chloroquine (a broad-spectrum viral inhibitor that blocks membrane fusion) or bafilomycin A1 (a specific inhibitor of vacuolar proton ATPases, which blocks endolysosomal trafficking) significantly reduced luc expression, indicating the possible involvement of the endolysosomal system in the viral entry mechanism. The pharmacological inhibition of two-pore channel (TPC) activity or use of the tpcn2(dhkz1a) mutant zebrafish line also led to diminished luc expression. The localized expression of ACE2 and TPC2 in the anterior neuromasts and the forming olfactory organs was demonstrated, and the occurrence of endocytosis in both locations was confirmed. Together, our data indicate that zebrafish embryos/larvae are a viable and tractable model to explore the mechanism of SARS-CoV-2 host cell entry, that the peripheral sense organs are a likely site for viral host cell entry, and that TPC2 plays a key role in the translocation of the virus through the endolysosomal system.IMPORTANCE Despite the development of effective vaccines to combat the COVID-19 pandemic, which help prevent the most life-threatening symptoms, full protection cannot be guaranteed, especially with the emergence of new viral variants. Moreover, some resistance to vaccination remains in certain age groups and cultures. As such, there is an urgent need for the development of new strategies and therapies to help combat this deadly disease. Here, we provide compelling evidence that the peripheral sensory organs of zebrafish possess several key components required for SARS-CoV-2 host cell entry. The nearly transparent larvae provide a most amenable complementary platform to investigate the key steps of viral entry into host cells, as well as its spread through the tissues and organs. This will help in the identification of key viral entry steps for therapeutic intervention, provide an inexpensive model for screening novel antiviral compounds, and assist in the development of new and more effective vaccines. Despite the development of effective vaccines to combat the COVID-19 pandemic, which help prevent the most life-threatening symptoms, full protection cannot be guaranteed, especially with the emergence of new viral variants. Moreover, some resistance to vaccination remains in certain age groups and cultures.

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