key: cord-300423-q2i328sz
authors: Bai, Lei; Zhao, Yongliang; Dong, Jiazhen; Liang, Simeng; Guo, Ming; Liu, Xinjin; Wang, Xin; Huang, Zhixiang; Sun, Xiaoyi; Zhang, Zhen; Dong, Lianghui; Liu, Qianyun; Zheng, Yucheng; Niu, Danping; Xiang, Min; Song, Kun; Ye, Jiajie; Zheng, Wenchao; Tang, Zhidong; Tang, Mingliang; Zhou, Yu; Shen, Chao; Dai, Ming; Zhou, Li; Chen, Yu; Yan, Huan; Lan, Ke; Xu, Ke
title: Co-infection of influenza A virus enhances SARS-CoV-2 infectivity
date: 2020-10-14
journal: bioRxiv
DOI: 10.1101/2020.10.14.335893
sha: 
doc_id: 300423
cord_uid: q2i328sz

The upcoming flu season in the northern hemisphere merging with the current COVID-19 pandemic raises a potentially severe threat to public health. Through experimental co-infection of IAV with either pseudotyped or SARS-CoV-2 live virus, we found that IAV pre-infection significantly promoted the infectivity of SARS-CoV-2 in a broad range of cell types. Remarkably, increased SARS-CoV-2 viral load and more severe lung damage were observed in mice co-infected with IAV in vivo. Moreover, such enhancement of SARS-CoV-2 infectivity was not seen with several other viruses probably due to a unique IAV segment as an inducer to elevate ACE2 expression. This study illustrates that IAV has a special nature to aggravate SARS-CoV-2 infection, and prevention of IAV is of great significance during the COVID-19 pandemic.

The outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) at 31 the end of 2019 has become pandemic worldwide. Up to date, there had been more than 36 32 million confirmed infected cases and 1 million deaths globally (https://covid19.who.int/). 33 The ending time and the final severity of the current COVID-19 pandemic wave are still 34 uncertain. Meanwhile, the upcoming seasonal influenza merging with the current pandemic 35 might bring more challenges and pose a bigger threat to public health. There are many 36 debates on whether seasonal flu would impact the severity of the COVID-19 pandemic and 37 whether massive influenza vaccination is necessary for the coming winter. However, no 38 experimental evidence is available concerning IAV and SARS-CoV-2 co-infection. 39 It is well known that disease symptoms from SARS-CoV-2 and IAV infections are quite 40 similar, such as fever, cough, pneumonia, acute respiratory distress syndrome, etc(1, 2). 41 Moreover, both SARS-CoV-2 and IAV are airborne transmitted pathogens that infect the 42 same human tissues such as the respiratory tract, nasal, bronchial, and alveolar epithelial 43 cultures (3, 4) . Besides, alveolar type II cells (AT2 pneumocytes) appeared to be 44 preferentially infected by SARS-CoV-2, which were also the primary site of IAV 45 replication(5, 6). Therefore, the overlap of the COVID-19 pandemic and seasonal influenza 46 would pose a large population under the high risks of co-occurrent infection by these two 47 viruses(7). 48 Unfortunately, during the last winter flu season in the southern hemisphere, there was 49 little epidemiological evidence about the interaction between COVID-19 and flu, probably 50 due to a low IAV infection rate resulted from social distancing (8, 9) . A case report showed 51 that three out of four SARS-CoV-2 and IAV co-infected patients rapidly develop to 52 respiratory deterioration(10). On the contrary, other reports only observed mild symptoms 53 in limited co-infection outpatients(11). Thus, the clinical co-infection outcomes are still 54 unclear when a large population will face the threats of both viruses. 55 In this study, we tested whether IAV infection could affect the subsequent SARS-CoV-2 56 infection in both infected cells and mice. The results demonstrate that the pre-infection of 57 IAV strongly enhances the infectivity of SARS-CoV-2 by boosting viral entry in the cells 58 and by elevating viral load plus more severe lung damage in infected mice. These data 59 suggest a clear auxo-action of IAV on SARS-CoV-2 infection, which implies the great 60 importance of influenza virus and SARS-CoV-2 co-infection to public health.

IAV promotes SARS-CoV-2 virus infectivity. 63 To study the interaction between IAV and SARS-CoV-2, A549 (a hypotriploid alveolar A549 was converted to be highly sensitive (up to 10,000-fold) against the pSARS-CoV-2 72 virus after different doses of IAV infections (from low MOI of 0.01 to high MOI of 1, also 73 shown by pSARS-CoV-2 with mCherry reporter in Fig. S1 ). In contrast, the pre-infection 74 of IAV had no impacts on pseudotyped VSV particles bearing VSV-G protein (Fig.1C ). We 75 further tested more cell lines to show that the enhancement of the pSARS-CoV-2 infectivity 76 by IAV was a general effect although the increased folds were different (lower basal level 77 of infectivity, higher enhancement fold) (Fig.1D ).

To validate the above results, we substituted the pSARS-CoV-2 with the SARS-CoV-2 79 live (experimental scheme shown in Fig.1E ). We found that the pre-infection of IAV 80 strongly increased the copy numbers of the SARS-CoV-2 genome (E and N genes) in both 81 cell lysates and supernatants of A549 (~15 folds) (Fig.1F) . Notably, in Calu-3 (Fig.1G ) and 82 NHBE ( Fig.1H ) cells that are initially susceptible to SARS-CoV-2, IAV pre-infection could 83 further increase >5 folds of SARS-CoV-2 infectivity.

Collectively, these data suggest an auxo-action of IAV on SARS-CoV-2 in a broad range 85 of cell types.

87 load and more severe lung damage. 88 The hACE2 transgenic mice were applied to study the interaction between IAV and 89 SARS-CoV-2 in vivo. Mice were infected with 3x10 5 PFU of SARS-CoV-2 with or without 90 2000 PFU of IAV pre-infection and were then sacrificed two days later after SARS-CoV-2 91 infection (the experimental scheme is shown in Fig. 2A) . The viral RNA genome copies 92 from lung homogenates confirmed that SARS-CoV-2 efficiently infected both groups 93 (more than 4x10 8 N gene copies) (Fig. 2B) , while the influenza NP gene was only detected 94 in IAV pre-infection group (Fig. 2B ). Intriguingly, a significant increase in SARS-CoV-2 95 viral load (12.9-fold increase in E gene and 6.6-fold increase in N gene) was observed in 96 lung homogenates from co-infection mice compared to that from SARS-CoV-2 single-97 infected mice (Fig. 2C ). The histological data in Fig. 2D further illustrated that IAV and 98 SARS-CoV-2 co-infection induced more severe lung pathologic changes with massive 99 infiltrating cells and obvious alveolar necrosis as compared to SARS-CoV-2 single 100 infection or mock infection.

IAV components specifically facilitate the entry process of SARS-CoV-2. 102 We further tested if several other viruses on hand had similar effects to promote SARS- CatL were increased around three folds (A549 in Fig. 4A , Calu-3 in Fig. S2 ). An obvious 119 switch of intracellular ACE2 expression was triggered at 12 h post-IAV-infection (Fig. 4C ).

In the meantime, influenza NP, Mx1, and ISG54 increased accordingly confirming a 121 successful infection of IAV (Fig. 4B ). 

The data indicated that IAV permitted increased SARS-CoV-2 infection through the up-133 regulation of ACE2 expression.

Enhanced SARS-CoV-2 infectivity is independent of IFN signaling.

ACE2 was reported to be an interferon-stimulated gene (ISG) in human airway epithelial 136 cells (19) . IAV infection will also stimulate type I IFN signaling. We, therefore, tested 137 whether the augment of ACE2 expression is dependent on IFN or not. For this, cells were 

The A/WSN/33 virus was generated by reverse genetics as previously described (25). All the mRNA levels were normalized by β-actin in the same cell.

The relative number of SARS-CoV-2 viral genome copy number were determined using IFNα for 12 hours. Cells were then infected with pSARS-CoV-2 for another 24 hours 455 followed by measuring luciferase activity and mRNA expression levels of indicated genes.

The data of mRNA levels were expressed as fold changes relative to non-treatment cells. Figure S1 . IAV facilitates the entry process of pSARS-CoV-2 (Fig.1) . Figure S2 . IAV infection induces elevated ACE2 expression (Fig.4) . Figure S3 . Enhanced SARS-CoV-2 infection is independent of IFN signaling (Fig.5) . Figure S4 . IAV facilitates viral entry of WT or mutant SARS-CoV-2.

Mutations in the Spike Protein of Middle East Respiratory Syndrome 333

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 338 receptor

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by 340 a Clinically Proven Protease Inhibitor

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Could an endo-lysosomal ion channel be the Achilles heel of SARS-CoV2? 343

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

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