key: cord-1050654-cnsz4ff3 authors: Anantharaj, A.; Gujjar, S.; Kumar, S.; Jain, N.; Wangchuk, J.; Khan, N. A.; Panwar, A.; Kanakan, A.; A, V.; Vasudevan, J. S.; Das, A.; Pandey, A. K.; Pandey, R.; Medigeshi, G. R. title: Kinetics of viral load, immunological mediators and characterization 1 of a SARS-CoV-2 isolate in mild COVID-19 patients during acute phase of infection date: 2020-11-07 journal: nan DOI: 10.1101/2020.11.05.20226621 sha: c7e21d5f5e178c2e4177e4a7d8f62224e8b9730e doc_id: 1050654 cord_uid: cnsz4ff3 Over 95% of the COVID-19 cases are mild-to-asymptomatic who contribute to disease transmission whereas most of the severe manifestations of the disease are observed in elderly and in patients with comorbidities and dysregulation of immune response has been implicated in severe clinical outcomes. However, it is unclear whether asymptomatic or mild infections are due to low viral load or lack of inflammation. We have measured the kinetics of SARS-CoV-2 viral load in the respiratory samples and serum markers of inflammation in hospitalized COVID-19 patients with mild symptoms. We observed a bi-phasic pattern of virus load which was eventually cleared in most patients at the time of discharge. Viral load in saliva samples from a subset of patients showed good correlation with nasopharyngeal samples. Serum interferon levels were downregulated during early stages of infection but peaked at later stages correlating with elevated levels of T-cell cytokines and other inflammatory mediators such as IL-6 and TNF-alpha which showed a bi-phasic pattern. The clinical recovery of patients correlated with decrease in viral load and increase in interferons and other cytokines which indicates an effective innate and adaptive immune function in mild infections. We further characterized one of the SARS-CoV-2 isolate by plaque purification and show that infection of lung epithelial cells (Calu-3) with this isolate led to cytopathic effect disrupting epithelial barrier function and tight junctions. Finally we showed that zinc was capable of inhibiting SARS-CoV-2 infection in this model suggesting a beneficial effect of zinc supplementation in COVID-19 infection. Coronavirus disease of 2019 has infected over 35 million people worldwide leading 61 to over a million deaths (as on 10th October 2020). Severe acute respiratory syndrome 62 coronavirus-2 (SARS-CoV-2), a positive-sense RNA virus from the family Coronaviridae is the 63 causative agent of COVID-19. The RNA genome of SARS-CoV-2 ranges from 26 to 32 64 kilobases in length and similar to other coronaviruses that have jumped species to infect humans, 65 SARS-CoV-2 is believed to have been in circulation in animals such as bats for several decades 66 before infecting humans (1). The genome of SARS-CoV-2 shares about 96% identity with that of 67 bat coronavirus and is about 80% identical to the SARS-CoV-1 (2). Angiotensin converting 68 enzyme-2 (ACE2) has been reported as a primary entry receptor for SARS-CoV-2 (3). ACE2, a 69 type I membrane protein, is expressed in lungs, kidneys, intestine and heart. The spike Most of the patients with COVID-19 exhibit mild or no symptoms and severe clinical symptoms 78 are observed in patients in the older age group (>65 years) or patients with underlying 79 comorbidities such as hypertension and diabetes. Impaired immune response and cytokine storm 80 has been implicated in clinical manifestations observed in severe disease (7-9). Increased viral 81 load has been shown to correlate with disease severity (7, 10) and some of the antivirals such as 82 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi. org/10.1101 org/10. /2020 Remdesivir which inhibit the viral RNA-dependent-RNA-polymerase of SARS-CoV-1, Middle 83 East Respiratory Syndrome Coronavirus (MERS-CoV) and SARS-CoV-2 has been shown to 84 reduce time to recovery and mortality in . As is the case with RNA 85 viruses, coronaviruses accumulate mutations and evolve at a rate similar to other RNA viruses 86 (12, 13) . Variations in the viral genome and emergence of novel clades has been implicated in 87 increased fitness and transmission (14) CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 amplified using the forward primer 5'-GACCCCAAAATCAGCGAAAT-3' and the reverse 152 primer 5'-GCGCGACATTCCGAAGAA-3' and cloned into pGEM®-T-Easy vector (Promega). This clone was linearized using Sac II enzyme and in vitro transcribed using the SP6 RNA 154 polymerase (Promega). The transcript was purified and used as a template for generating 155 standard curve to estimate the copy number. For virus isolates, RNA was isolated from 4 different plaque-purified isolates using duplicates along with quality controls. The amount of each analyte was estimated by standard 171 curve generated using known amount of analyte provided in the assay kit. Assay plates were read 172 using MAGPIX system and data was analysed by xPONENT software using five parametric 173 logistic fit model. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10. 1101 Cell Viability assay 176 Cell viability assay was performed using CellTiter-Glo® Assay kit (Promega) according to user CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint glutamine mix and non-essential amino acids). At 24 h pi, cells were washed twice with PBS and 198 once with cold PBS and fixed by using ice cold methanol and kept at -20ºC for 20 min. Methanol 199 was removed, cells were washed once with cold PBS and then once with PBS at room 200 temperature (RT), followed by blocking with 0.2% BSA-PBS for 10 min at RT. Cells were 201 incubated with human chimeric SARS-CoV-2 S1 spike antibody (1: 100 dilution; Genescript -202 HC2001) in 0.2% BSA-PBS for 1h at RT. Cells were then washed three times with 0.2% BSA-203 PBS. This was followed by incubation with secondary antibody conjugated with anti-human 204 Alexa flour 488 (1:500; Life Technologies-A11013) for 30 min at room temperature in dark. Cells were washed with PBS three times and stained with 4′,6-diamidino-2-phenylindole (DAPI) 206 (Molecular probes) at 1:10,000 dilution for 10 min. Cells were washed with PBS and images 207 were captured using Olympus DP80 microscope at 20X magnification. Images were processed 208 by background subtraction using cellSens software. Bright field images were captured using 209 Nikon Eclipse Ti-S at 10X magnification. Whole genome Sequencing and analysis 218 The protocol followed for the sample preparation, sequencing, and data analysis has been 219 described earlier (18). In brief, double-stranded cDNA was synthesized from 50ng of total RNA 220 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint from virus stocks for all the four plaque-purified SARS-CoV-2 isolates. The first strand of 221 cDNA was synthesized using Superscript IV followed by RNA digestion with RNase H for 222 second-strand synthesis using DNA Polymerase I Large fragment (Klenow fragment). 100ng of 223 purified double-stranded cDNA was taken for forward using ARTIC tiling PCRprotocol (V3 The consensus genomes obtained from the whole genome sequencing of 10 Indian isolates and 241 four plaque-purified Indian isolates along with 86 other global SARS-CoV-2 sequences were 242 used for constructing the phylogeny. The sequences were selected from a diverse geographical 243 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint appropriate secondary antibodies tagged with Alexa fluor 488/568/633 (Molecular probes) for 30 267 min at RT by avoiding exposure to light. Cells were washed with IMF buffer three times and 268 stained with DAPI at 1:10,000 dilution for 10 min. Cells were washed with PBS, mounted on 269 glass slide using antifade solution (Molecular probes) and images were captured at 100X 270 magnification using FV3000 confocal microscope (Olympus). Images were processed by 271 background subtraction using cellSens software (Olympus). CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint the morning for estimation of viral load in saliva and compare with the NP/OP sample. Viral 290 load in NP/OP swab samples peaked between day 2 to 4 in most patients and we observed a 291 biphasic increase in viral RNA levels in few patients with a second peak on day 5 ( Figure 1A -292 1F). The peak viral RNA levels ranged from 10 6 to 10 10 copies per ml of virus transport medium. Except for one patient, viral load in saliva correlated with viral load in VTM samples (Figure 294 1H-1K). In one patient, virus persisted in saliva till day 10 while the VTM sample was RT-PCR 295 negative by day 6 of enrolment ( Figure 1G ). In most cases, the viral RNA was below the level 296 of detection by RT-PCR by day 7-9 but two patients showed increasing trend in viral RNA levels 297 although the last sample in these patients were on day 5 and day 7 ( Figure 1D and 1H). These 298 data suggest that the mild or asymptomatic patients harbour high viral load and salivary viral 299 load can be a good alternate for the NP/OP swabs. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint namely the pro-inflammatory IL-12p40 homodimer or IL-12p70, the active IL-12 heterodimer 313 secreted by dendritic cells and macrophages that promotes Th1 response and leads to IFN-g and 314 TNF-α production. We observed a clear increase in IL-12p70 levels from day 5 onwards 315 suggesting priming of T cells by the innate immune system through IL-12p70 secretion (Figure 316 2B). This increase coincided with increased IFN-g levels observed in the same samples (Figure 317 1A). IL-7, a cytokine produced by epithelial cells and DCs and promotes biogenesis and 318 proliferation of lymphocytes also showed an increasing trend coinciding with IL-12p70. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint 336 Increased viral loads have been associated with severe disease by previous reports (7, 15). We 337 were interested in understanding whether the virus isolates from mild or asymptomatic infection 338 have altered ability to evade immune response and cause cellular damage. In order to isolate and 339 characterize an Indian isolate of SARS-CoV-2, we used NP/OP swab samples from six COVID- CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint infectivity of THSTI-BL2010-D in human cell lines, we infected Vero E6, Calu-3 and Caco-2 359 cells with increasing MOIs of the virus and stained for the presence of spike protein by 360 immunofluorescence assay at 24 h pi. As expected, Vero E6 cells showed the maximum 361 infection followed by Calu-3 cells and Caco-2 cells ( Figure 4A ). We next compared growth (Table 1) . THSTI-BL-2010-C sample had one additional mutation 374 G27806T resulting in a synonymous change in ORF7b. Two non-synonymous changes were also 375 observed in ORF1b and N regions (Table 1) Table S1 ) revealed clustering of sequences 381 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint into five major clades according to the GISAID nomenclature system (https://www.gisaid.org). Sequences from all the 14 genomes from our study were subjected to phylogenetic analysis and 383 were assigned to clade 19A which along with the 19B clade forms the earliest clade which 384 originated from Wuhan and was prevalent in Asia during the initial months of the outbreak 385 ( Figure 5 ). This suggests that the SARS-CoV-2 isolates reported in our study are genetically 386 more similar to the reference Wuhan sequence (GenBank: MN908947.3). . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi. org/10.1101 org/10. /2020 Addition of zinc along with the zinc ionophore pyrithione, which stimulates zinc uptake, was 404 shown to inhibit SARS-CoV infection in Vero E6 cells (28). We were interested to use our cell 405 culture system to assess whether zinc has an inhibitory effect on SARS-CoV-2 infection. We CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint symptoms (7) and viral genetics was not found to correlate with severe disease (15). We 427 focussed our efforts to monitor the kinetics of viral load and inflammatory mediators in the 428 serum samples of hospitalized COVID-19 patients who displayed mild to no symptoms but were able to clear the virus and recover from the disease. This clearly suggests that a robust immune 441 response in younger adults is capable to clearing the infection and age and imbalance in immune 442 response could contribute to severe disease in older individuals and patients with co-morbidities. 443 We observed that decrease in viral loads coincided with increase in IFN responses which is in CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint cases (7) whereas we observed an increase in both IFN-a and IFN-g in all the patients who CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 Vero E6 monolayers compared to the Wuhan strain where the plaques were very heterogeneous 473 in size. As robust and consistent plaque reduction neutralization titers (PRNT) depend on high 474 quality plaque assays, we believe our virus strain will help in establishing PRNT assays that CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 1. Boni MF, Lemey P, Jiang X, Lam TT, Perry BW, Castoe TA, Rambaut A, Robertson DL. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint This article has supplementary information. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint SARS-CoV-2 RNA levels were measured by RT-PCR using total RNA isolated from VTM 658 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint samples every day after enrolment. G-K) SARS-CoV-2 RNA levels were measured by RT-PCR 659 using total RNA isolated from saliva and VTM samples every day after enrolment. Data points 660 indicate geometric mean normalized to RNASe P levels which was used as endogenous controls 661 for extraction efficiency. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10. 1101 Variant analysis of SARS-CoV-2 genomes Viral and host factors related to the clinical outcome of COVID-568 19 Adaptor protein complexes-1 and 3 are involved Evidence that D614G Increases Infectivity of the COVID-19 Virus. 610 Cell Zn(2+) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc 613 ionophores block the replication of these viruses in cell culture Comparative Replication and Immune Activation Profiles of SARS-CoV-617 2 and SARS-CoV in Human Lungs: An Ex Vivo Study With Implications for the 618 Pathogenesis of COVID-19 Inhibition of SARS-CoV-2 by type I and type III interferons A Central Role of Interleukin-8 in the Pathogenesis of ARDS Neutrophils in viral infections: Current concepts and 629 caveats International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity First isolation 633 of SARS-CoV-2 from clinical samples in India Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-638 2 and SARS-CoV with implications for clinical manifestations, transmissibility, and 639 laboratory studies of COVID-19: an observational study Analysis of ACE2 in polarized epithelial 642 cells: surface expression and function as receptor for severe acute respiratory syndrome-643 associated coronavirus Zinc supplementation for the prevention of pneumonia 645 in children aged 2 months to 59 months cells were treated with 25 and 50 µM of ZnSO4 or as mock treated control. Viral 705 titres were determined as represented as pfu/ml. (n=3). Error bars represent mean ± SD Vero E6 cells were infected with nasopharyngeal 711 swab samples for 1 h and cultured in growth medium thereafter. CPE was evident at 48 h post-712 infection. Images were captured using Nikon Eclipse Ti-S microscope at 20X magnification Zinc uptake in Calu-3 cells. Calu-3 cells after incubation with indicated 715 concentrations of zinc sulfate for 4 h and labile zinc levels were measured by staining with 716 The relative change in the mean fluorescence intensities of ZP-1 is 717 shown in the graph. Error bars represent Mean with SD. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity The authors declare that they have no conflict of interest to disclose. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10.1101/2020.11.05.20226621 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 7, 2020. ; https://doi.org/10. 1101