key: cord-0907988-mhzgndz7
authors: Schulze, Jessica; Mache, Christin; Balázs, Anita; Frey, Doris; Niemeyer, Daniela; Olze, Heidi; Dommerich, Steffen; Drosten, Christian; Hocke, Andreas C; Mall, Marcus A; Hippenstiel, Stefan; Wolff, Thorsten
title: Analysis of SARS-CoV-2 replication in explant cultures of the human upper respiratory tract reveals broad tissue tropism of wild-type and B.1.1.7 variant viruses
date: 2021-10-15
journal: J Infect Dis
DOI: 10.1093/infdis/jiab523
sha: bf0d7755c5a921ff53a50ade043f0210d9d83452
doc_id: 907988
cord_uid: mhzgndz7

The upper respiratory tract (URT) is the primary entry site for SARS-CoV-2 and other respiratory viruses, but its involvement in viral amplification and pathogenesis remains incompletely understood. Here we investigated primary nasal epithelial cultures, as well as vital explanted tissues to scrutinize the tropism of wild-type SARS-CoV-2 and the recently emerged B.1.1.7 variant. Our analyses revealed a widespread replication competence of SARS-CoV-2 in polarized nasal epithelium as well as in the examined URT and salivary gland tissues, which was also shared by the B.1.1.7 virus thereby highlighting the active role of these anatomic sites in COVID-19.

M a n u s c r i p t 4

The emergence of the highly pathogenic human Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) lead to a global pandemic which caused more than 4.6 million deaths worldwide so far.

SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19) and is transmitted via infected droplets and aerosols [1] .

The upper respiratory tract (URT) is believed to have essential roles in person-to-person spread of SARS-CoV-2 and its dissemination to the lung, but its precise role in COVID-19 pathogenesis remains incompletely understood. A variety of clinical symptoms including ageusia and anosmia, dry mouth, oral lesions, sore throat, dry cough, sinusitis or rhinorrhea indicate that cells of the oropharynx and the URT are targeted by SARS-CoV-2 [2] . High SARS-CoV-2 viral loads in clinical specimens of URT fluids and in saliva of a majority of COVID-19 patients consistently detected at illness onset suggest active viral replication, which is further supported by isolation of infectious virus and detection of viral replicative RNA intermediates from throat-swabs of COVID-19 patients and in salivary glands.

This indicates that tissues of the URT and salivary glands are not just sites of infection but also of viral replication [2] [3] [4] However, knowledge on the precise tissue source of transmissible SARS-CoV-2 remains limited. Productive viral replication has been demonstrated for nasal turbinates [5] , but the permissiveness of other tissues lining the surface of the URT has yet to be investigated.

To date, a number of single cell RNA sequencing studies confirmed expression of SARS-CoV-2 entry factors including angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2) in different cell types of the URT [4, 6, 7] . Here we report an ex vivo approach to characterize SARS-CoV-2 replication and extended tropism in available tissues of the human URT and salivary glands. We utilized surgical specimens freshly derived from paranasal sinuses, adenoids, tonsils as well as salivary glands to establish short-term tissue cultures, which were infected with SARS-CoV-2. For a comparison, we employed seasonal H3N2 influenza A virus (IAV), that is also shed from the URT in naïve individuals over a period of 5-7 days [8] . The newly established models were further used to address the hypothesis that increased transmission and rapid worldwide spread of A c c e p t e d M a n u s c r i p t 5 the recently emerged SARS-CoV-2 lineage B.1.1.7 is caused by more productive replication compared to earlier variants. The B.1.1.7 viruses were first detected in the UK in September 2020 and accumulate higher viral RNA loads in nasopharyngeal swabs of COVID-19 patients, which may also be associated with increased mortality [9, 10] . A c c e p t e d M a n u s c r i p t

We used Vero E6 cells to demonstrate comparable replication capabilities of SARS-CoV-2 and seasonal IAV in a common cell culture model, in which both viruses replicated to titers of about 10 8 pfu/ml (Fig. 1a) . Differentiated ALI cultures of human Calu-3 cell line and phNECs were employed to evaluate infectivity of SARS-CoV-2 in models more closely mimicking the conditions of the URT (Fig.   1a ). In both settings, SARS-CoV-2 isolated early in 2020 (lineage B.1.153) replicated to high viral titers about 10 8 pfu/ml (Calu-3) and 10 7 pfu/ml (phNEC), respectively. Propagation of SARS-CoV-2 proceeded with a slightly delayed kinetic compared to IAV reaching peak titers at 72h p.i.

In all URT and salivary gland explant cultures, SARS-CoV-2 replicated to similar titers up to 10 4 pfu/ml at 24h p.i. (Fig. 1b) . This is in line with comparable expression of host factors in these tissues supporting SARS-CoV-2 growth including ACE2, TMPRSS2, furin and CD147 as analyzed by quantitative real-time PCR (Fig. 1c) . In the URT explant cultures as well as phNECs, expression of the viral entry receptor ACE2 was lower compared to expression levels of host proteases TMPRSS2 and furin and the recently suggested alternate entry receptor CD147 [11] . In contrast to SARS-CoV-2, replication of IAV that uses sialic acid conjugates as receptors was more restricted. This becomes apparent by failure of IAV to productively infect tonsillar and salivary gland tissue, which contrasts with very efficient replication in paranasal sinuses (up to 10 8 pfu/ml) and adenoids (up to 10 5 pfu/ml) (Fig. 1b) .

Having established the URT and phNEC culture as suitable infection models we used them to compare replication of the early reference virus and lineage B.1.1.7 SARS-CoV-2. However, no significant differences in viral growth were identified in either of the primary models (Fig. 2a, b) .

COVID patients upregulate genes for antiviral type I and III IFN in their nasal mucosa [12] and this was recapitulated in the phNEC (Fig. 2c) . Interestingly, we observed a significantly reduced upregulation of IFN-λ transcripts as well as a trend for lesser IFN- induction by the B.1.1.7 virus in comparison to the reference virus, whereas similar levels of IFN- and the IFN-inducible gene IFIT1 were found in cells infected with either virus (Fig. 2c) . high titers in the range of 10 8 PFU/ml in phNEC ALI cultures, which closely reflects high viral loads in nasal tissues in COVID-19 patients [3] .

Tissue of the nasal cavity has been reported to be permissive for SARS-CoV-2 propagation due to expression of ACE2 and TMPRSS2 with highest levels of ACE2 expression detected in ciliated and secretory cells [7, 13] . Interestingly, SARS-CoV-2 accumulated to lower titers in whole tissue samples of paranasal sinuses compared to phNEC cultures, which was unexpected as both tissues originate [5, 13] . Differences in replication efficiency may be caused by different levels of ACE2 expression in freshly excised nasal tissue compared to nasal epithelial cells. The latter were differentiated ex vivo for 28 days under ALI conditions during which they acquire high ACE2 levels throughout the cultivation period [6] . In fact, slightly but significantly enhanced expression of ACE2 in phNECs compared to nasal tissue could be confirmed by qPCR in our study. Our observations show substantial replication of SARS-CoV-2 in cells and tissues of the nasal cavity thereby confirming and extending findings from a study conducted in primary human airway epithelial cultures from the upper and lower airways [13] .

In contrast to tissue samples of paranasal sinuses and adenoids, SARS-CoV-2 viral titers in tonsils and salivary glands exceeded that of IAV. The reason for this discrepancy in the tonsillar tissue is not readily apparent. Previous data suggested that interactions between immune cells and epithelial cells during SARS-CoV-2 infection can increase susceptibility, which may explain the observed robust replication in lymphoid tissues such as adenoids and tonsils [7] . Due to high expression of ACE2 and A c c e p t e d M a n u s c r i p t 9 TMPRSS2 along with detection of viral RNA in COVID-19 autopsies, salivary glands are considered potential amplification sites for the virus [4] which is supported by active replication in parotid salivary glands available in our study. [14] . Hence, our findings give initial support for a model, in which the phenotype of the B.1.1.7 lineage involves the capacity to distinctively target the host innate immune response, which may impact on the efficacy of viral transmission as has been observed for HIV-1 [15] but further analyses is required to substantiate this hypothesis for SARS-CoV-2.

In summary, we show that propagation and shedding of infectious SARS-CoV-2 in body fluids of the URT can be modelled in an ex vivo approach using primary organ cultures and an advanced reconstituted polarized nasal epithelium. Widespread replication competence of SARS-CoV-2 in various URT and salivary gland tissues, as shown in this study, very likely contributes to high viral titers and efficient transmission of SARS-CoV-2 between people. High viral loads in URT further allow seeding of virus to the lower respiratory tract as seen in the pulmonary phase of COVID-19. We expect these newly established infection models to be highly useful in determining the replicative capacity of new emerging viral variants, to investigate their potential virulence in the URT and salivary glands, and to evaluate the effectiveness of new antiviral compounds. 

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CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells

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Global Health (Corona Virus Pre-Exploration Project)

Foundation (DFG) (SFB-TR 84), A.C.H. and S.H. by the Federal Ministry of Education and Research (BMBF) (alvBarriere-COVID-19) and Charité 3 R

Influenza and other Respiratory Viruses

Acknowledgments: We thank Katharina Hellwig for excellent technical assistance.

A c c e p t e d M a n u s c r i p t