key: cord-0713127-5wpq72vy
authors: Hsin, Fu; Chao, Tai-Ling; Chan, Yun-Rui; Kao, Han-Chieh; Liu, Wang-Da; Wang, Jann-Tay; Pang, Yu-Hao; Lin, Chih-Hui; Tsai, Ya-Min; Lin, Jing-Yi; Chang, Sui-Yuan; Liu, Helene Minyi
title: Distinct Inductions of and Responses to Type I and Type III Interferons Promote Infections in Two SARS-CoV-2 Isolates
date: 2020-05-01
journal: bioRxiv
DOI: 10.1101/2020.04.30.071357
sha: 616caae5540c7f524f0e848d2db46786b26f3167
doc_id: 713127
cord_uid: 5wpq72vy

The recent emerging coronavirus, SARS-CoV-2, has been rapidly and widely spread and causing an ongoing viral pneumonia outbreak worldwide. It has been observed that SARS-CoV-2 patients show a rather long and asymptomatic incubation time. We characterized the abilities to induce and to response to IFNβ/IFNλ1 of two or our clinical isolates, SARS-CoV-2/NTU01/TWN/human/2020 and SARS-CoV-2/NTU02/TWN/human/2020, which exhibit only two amino acid differences over the ∼30kb viral genome. We found that both isolates may infect Huh7, A549 and Calu-3 cells, yet the RIG-I-like receptor-dependent antiviral signaling was poorly induced in these cells in the early infections. Unexpectedly, we found that the intracellular vRNA levels of these isolates were sustained upon to type I/III IFN treatments, and this phenotype was more pronounced in the Taiwan/NTU01/2020 isolate. The type I/III IFN responses are antiviral but partially proviral in the case of SARS-CoV-2 infections. Poor induction and response to innate immunity may contribute to destitute neutralization index of the antibody produced, and indeed we found that the patient serum could not efficiently neutralize SARS-CoV-2 virions. With better understandings of the interplay between SARS-CoV-2 and the host antiviral innate immunity, our report may provide new insights for the regimen of therapies for SARS-CoV-2 infected patients.

only two amino acid differences over the ~30kb viral genome. We found that both isolates 23 may infect Huh7, A549 and Calu-3 cells, yet the RIG-I-like receptor-dependent antiviral 24 signaling was poorly induced in these cells in the early infections. Unexpectedly, we found 25 that the intracellular vRNA levels of these isolates were sustained upon to type I/III IFN 26 treatments, and this phenotype was more pronounced in the Taiwan/NTU01/2020 isolate. The 27 type I/III IFN responses are antiviral but partially proviral in the case of SARS-CoV-2 28 infections. Poor induction and response to innate immunity may contribute to destitute 29 neutralization index of the antibody produced, and indeed we found that the patient serum 30 could not efficiently neutralize SARS-CoV-2 virions. With better understandings of the 31 interplay between SARS-CoV-2 and the host antiviral innate immunity, our report may 32 provide new insights for the regimen of therapies for SARS-CoV-2 infected patients. converting enzyme 2 (ACE2) as the major receptor for virus entry 3-5 . However, the 42 transmission of SARS-CoV-2 between human seems more efficiently than that of the original 43 SARS-CoV. In April 2020, more than 2,000,000 cases of SARS-CoV-2-infected patients 44 have been reported in more than 185 countries around the world. 45 The induction and expression of type I and type III interferons (IFNs) are the frontlines to clearance 7 . Particularly, type III IFN plays a critical role at the barrier surfaces, such as the 55 airway and the GI tract 9 . It has been shown that IFNλ1 was more potent than type I IFN in 56 restricting viral propagation with less inflammation and tissue damage 10, 11 . In contrast, 57 3 many viruses have evolved some strategies to escape from the induction of response to type 58 I/III IFN to establish the infection 12,13 .

Asymptomatic SARS-CoV-2-infected individuals has been reported in many studies 14 . 60 Some SARS-CoV-2 infected patients showed long incubation time before the symptoms were 61 developed. Two SARS-CoV-2-infected patients admitted to our unit showed distinct clinical 62 virology and serology progresses (Table 1) . Both patients showed normal complete blood 63 count (CBC) initially with mild thrombocytopenia. Although days taken for seroconversion 64 highly varied between the two patients, the immunoglobulin levels including total IgM, IgG 65 and IgA were comparable (Table 1) (Table 1) 15 . Therefore, it is likely that the anti-SARS-CoV-2 innate immunity was not 69 sufficiently induced in time to confront SARS-CoV-2 infections in Patient NTU1. We then 70 isolated viruses from both patients, SARS-CoV-2/NTU01/TWN/human/2020 (GeneBank: 71 MT066175.1) and SARS-CoV-2/NTU02/TWN/human/2020 (GeneBank: MT066176.1), and 72 assessed how these isolates may induce and response to host innate immunity in cell cultures. 73 We then performed many assays in cell culture, where only the innate immunity of the 74 infected host cells are involved, to rule out the variations among infected individuals and to 75 characterize the virology of SARS-CoV-2 isolates. RNA sequencing results determined that 76 SARS-CoV-2/NTU01/TWN/human/2020 represented clade B with the L84S (28144T>C) 77 polymorphism in ORF8, and SARS-CoV-2/NTU02/TWN/human/2020 was highly related to 78 the Wuhan prototype strain (Table 2) . We hypothesize that the induction levels of type I and 79 type III IFNs may be low during SARS-CoV-2 infection, and this may be due to the lack of 80 PRR recognition and/or a strong suppression of type I/III IFN expressions by the viral 81 proteins of SARS-CoV-2. To address these questions, we utilized two of our clinical isolates 82 4 of SARS-CoV-2 to infect Huh7, A549, and Calu3 cells, and monitored their inductions and 83 responses to type I/III interferons during SARS-CoV-2 infections. 84 We first monitored the intracellular vRNA levels of SARS-CoV-2 in Huh7 hepatoma cells, infected Huh7 cells (Fig 1A) . For both isolates, the vRNA levels was higher at 24 h.p.i than 94 those at 48 h.p.i, suggesting that the replication of SARS-CoV-2 in Huh7 is none or very 95 minimal (Fig. 1A) . Also, NTU1 and NTU2 showed comparable intracellular vRNA levels in 96 the infected Huh7 cells (Fig. 1A) . Compared to the wildtype Huh7 cells, we did not observe 97 any significant differences at the intracellular vRNA levels in the SARS-CoV-2 infected 98 MAVS K/D huh7 cells (Fig. 1B) . The loss of MAVS-dependent antiviral innate immunity did 99 not further support replication of SARS-CoV-2 in Huh7 cells (Fig. 1B) , suggesting that the 

A recent study has reported that the virus-like particle of SARS-CoV-2 could enter Huh7, supported SARS-CoV-2 replication, and that SARS-CoV-2 is likely a weak inducer and also 166 a strong suppressor of type I/III IFNs. 167 Our results indicate that the viral RNA of SARS-CoV-2 was able to be detected by showed that SARS-CoV-2 nsp13 was capable to interact with TBK1, which is a critical 228 kinase to activation IRF3 for type I/III IFN expressions 17 . The detail mechanism remained to 229 be further studied. (Table 1) , was able to effectively neutralize NTU1 246 virions at >1:160 dilution in the PRNT assay ( Supplementary Fig. 4) . However, both serum 247 collected from Patient NTU1 before and after seroconversion did not well-neutralize the 248 virion isolated from the same patient (NTU1) The PRNT50 for NTU1 is about 1:80 before 249 seroconversion and 1:160 after seroconversion (Fig. 4) . These results together indicated that 250 although the total IgM, IgG, and IgA amount between both patients were at the comparable 251 or ever higher levels (Table 1) (Fig 3, E-F) . Intriguingly, a recent report showed that among the top 50 genes 273 which were co-expressed with ACE2, an entry receptor for SARS-CoV2, many of them were 274 ISGs or genes that are involved in the regulation innate immunity, including ASS1, OAS1, 275 MX1, and etc. 22 . In our study, we found that pretreatment of TBK1 inhibitor prior go SARS- Fig. 3B and Fig. 1B) . We examined the mRNA levels of ACE2 in our 279 samples and found that the expression of ACE2 was increased after of IFNβ and/or IFNλ1 report showed that ACE2 may be an ISG in human epithelial cells 23 . Our study together with 284 recent reports suggest that SARS-CoV-2 may utilize certain ISGs to benefit its own life cycle.

This may also explain why patients with chronic inflammation disease are more prone to 286 COVID19. Whether the response to type I/III interferon treatments were purely anti-SARS-

CoV-2 remained debatable.

The two isolates in this study, SARS-CoV-2/NTU01/TWN/human/2020 (NTU1) and (Table 2 ). Tang's group found genetic analysis of 103 SARS-CoV-2 297 genomes by SNP could define the L and S lineages of SARS-CoV-2 in two linked SNPs at 298 sites 8782 and 288144 24 . However, the virulence or pathogenicity of these two lineages of 299 SARS-CoV-2 is still unclear. We tried to subclone and express ORF8 from both isolates in 300 either mammalian cells or in E. coli, and none of the system could support decent expression 301 of ORF8 (data not shown). Therefore, at this point, we are not able to design any molecular 302 biological studies to elucidate whether the genotypic difference in ORF8 between the two 303 isolates might contribute to the phenotypic differences in the response to type I/III IFNs. 304 Lastly, we found that seroconversion in patients did not correlate with their abilities for 

This will be rather useful to determine the regiment for each COVID19 patients as well as to 310 evaluate the vaccine protectivity in the future. TransIT-mRNA transfection kit (Mirus Bio) following manufacturer's protocol. 8 hours post-356 transfection, total RNA was extracted with Trizol and analyzed by qPCR.

Plaque reduction neutralization assay 359 Plaque reduction neutralization (PRNT) assay was performed to determine the SARS-CoV-2 360 titers as previously described with minor modification 28 . Briefly, the VeroE6 cells were 361 seeded at 2 x 10 5 cells/well in the 24-well tissue culture plates with DMEM containing 10% 362 FCS and antibiotics one day before infection. Serial diluted serum was preinteracted with 363 SARS-CoV-2 at 37°C for one hour and then added to the cell monolayer for 1 hour at 37°C.

Subsequently, viruses were removed, and the cell monolayer was washed once with PBS 365 before covering with media containing 1% methylcellulose for 5-7 days. The cells were fixed 366 with 10% formaldehyde overnight. After removal of overlay media, the cells were stained 367 with 0.7% crystal violet and the plaques were counted.

Statistic analysis 370 All data were presented the means ± standard deviation (SD) from two independent 371 experiments, and the two-tailed Student's t-test was used as statistical analysis, *p<0.05, 372 **p<0.01.

Ethical approval statement 375 The study was approved by the Research Ethics Committee or Institutional Review Board of 376 the NTUH Research Ethics Committee (202002002RIND) and informed consent was waived. 

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