key: cord-0965395-ni0c8fl7
authors: Zahn, Tobias; Mhedhbi, Ines; Hein, Sascha; Raupach, Jan; Miskey, Csaba; Husria, Younes; Bayanga, Kathrin; Bartel, Detlef; Vieths, Stefan; Ivics, Zoltan; Oberle, Doris; Keller‐Stanislawski, Brigitte; Herrlein, Marie‐Luise; Maier, Thorsten Jürgen; Hildt, Eberhard
title: Persistence of infectious SARS‐CoV‐2 particles for up to 37 days in patients with mild COVID‐19
date: 2021-10-24
journal: Allergy
DOI: 10.1111/all.15138
sha: 46590dca4ab69b767a423eab16ef9a59a916a5e6
doc_id: 965395
cord_uid: ni0c8fl7

BACKGROUND: People suffering from COVID‐19 are typically considered non‐infectious 14 days after diagnosis if symptoms have disappeared for at least 48 h. We describe three patients who independently acquired their infection. These three patients experienced mild COVID‐19 and completely recovered symptomatically within 10 days, but remained PCR‐positive in deep pharyngeal samples for at least 38 days. We attempted to isolate virus from pharyngeal swabs to investigate whether these patients still carried infectious virus. METHODS: Infectious virus was amplified in Vero E6 cells and characterized by electron microscopy and WGS. The immune response was investigated by ELISA and peptide arrays. RESULTS: In all three cases, infectious and replication‐competent virus was isolated and amplified in Vero E6 cells. Virus replication was detected by RT‐PCR and immunofluorescence microscopy. Electron microscopy confirmed the formation of intact SARS‐CoV‐2 particles. For a more detailed analysis, all three isolates were characterized by whole‐genome sequencing (WGS). The sequence data revealed that the isolates belonged to the 20A or 20C clade, and two mutations in ORF8 were identified among other mutations that could be relevant for establishing a long‐term infection. Characterization of the humoral immune response in comparison to patients that had fully recovered from mild COVID‐19 revealed a lack of antibodies binding to sequential epitopes of the receptor‐binding domain (RBD) for the long‐term infected patients. CONCLUSION: Thus, a small portion of COVID‐19 patients displays long‐term infectivity and termination of quarantine periods after 14 days, without PCR‐based testing, should be reconsidered critically.

samples of moderately or asymptomatically infected patients taken after day 10, although significant loads of viral genomes were detected. [8] [9] [10] [11] Based on these findings, a common practice is to isolate COVID-19 patients for 14 days and to discontinue the quarantine without further testing given the patient is asymptomatic for at least 48 h. There are also reports that describe infectivity for at least 24 days after disease onset in patients with severe COVID -19 but showing no infectivity at later stages of a prolonged COVID-19 infection despite positivity in RNA testing. 12 Hence, because of the discrepancy of this finding to the experiments aimed at isolating active virus and the current time periods for quarantine, the timeline for discontinuation of transmission-based precautions is extensively debated.

This study aimed at addressing the question whether persistence of infectious virus particles for prolonged periods of more than 4 weeks is restricted to patients with severe COVID-19 or might also occur in patients with mild symptoms as well. In the present study, we show that in three patients who already had overcome a mild symptomatic COVID-19 disease active virus particles were still present in deep pharyngeal area for up to 37 days.

Thus, there is an urgent medical need to understand the pathophysiology of the prolonged stay of active SARS-CoV-2 particles in some patients and to re-evaluate the procedures for discontinuation of transmission-based precautions of formerly infected COVID-19 patients.

This study shows three long-term SARS-CoV-2 PCR-positive patients persistently producing infectious virus for up to 38 days without lasting symptoms. Patients displayed low antibody titers and decreased binding of IgG against sequential epitopes of spike protein in comparison to patients that fully recovered from mild COVID- 19 . WGS revealed that isolates belong to the 20A or 20C clade and viral eradication might be affected by two mutations in ORF8. Abbreviations: COVID-19, coronavirus disease 2019; M, membrane protein; ORF, open reading frame; qPCR, quantitative polymerase chain reactions; S, spike protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; WGS, whole genome sequencing.

For this study, Vero E6 cells (ATCC ® CRL-1586 ™ ) were used. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM from BioWest) and supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, and 5% v/v fetal bovine serum (FBS superior; Sigma-Aldrich) under atmospheric conditions of 95% relative humidity and 5% CO 2 at 37°C.

Contents of SARS-CoV-2 positive or negative patients' swabs were resuspended in 1 ml of Vero E6 culture medium immediately after collection. The swab suspension was sterile-filtered through a 0.22 µm pore size sterile filter and added to one well of a 12-well cell culture plate with 1.5 × 10 5 Vero E6 cells per well. After 48 h, supernatant and cells were visually checked for indications of infections, such as color change of medium and especially microscopic examination of generated plaques, which reflect the cytopathic effect of SARS-CoV-2. Supernatant was removed and added to one well of a 6-well cell culture plate containing 3 × 10 5 Vero E6 cells per well.

Additionally, 1 ml of Vero E6 cell culture medium was added to the well for a final volume of 2 ml. After 48 h (96 h after initial incubation), supernatant (2 ml) of one well of a 6-well plate was split into six wells of a 6-well plate containing 3 × 10 5 cells per well and filled with medium for a total volume of 1 ml per well. An additional 48 h later, supernatant was removed and inactivated by addition of TRIzol ™ LS Reagent (ThermoFisher Scientific) for further sequencing analysis. Cells were washed with PBS and treated with TRIzol ™ Reagent (ThermoFisher Scientific) for quantitative RT-PCR or washed with PBS and fixed with 3.7% formaldehyde for immunofluorescence staining and confocal laser scanning microscopy (CLSM).

To determine viral titers in the supernatant of Vero E6 cells after 

The total RNA isolated from the cell culture supernatant was used for Illumina library preparation using a modified NNSR priming method. 14 We removed the rRNA from the samples with a QIAseq The overall quality of the reads were checked with the FastQ C tool. 15 Raw reads were trimmed and aligned against the reference sequence of SARS-COV-2 Wuhan-Hu-1 isolate (Accession number: NC_045512)

with Trimmomatic (v0.39) and SNAP, respectively. 16, 17 After sorting, the consensus call was performed with Rsamtools and SAMtools. 18, 19 The variant and phylogenetic analysis was performed with Nextclade (https://clades.nexts train.org/). 20 The phylogenetic tree was visualized with auspice, which is part of the Nextstrain project. 20 of these peptide arrays showed much stronger binding to defined epitopes in case of the adjusted convalescent sera as compared to the sera derived from the long-term infected patients.

Incubated cells were fixed with 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Afterward, cells were blocked with 1% Nuclei were stained with 4′,6-Diamidin-2-phenylindole (DAPI; diluted 1:1000 in PBS). CLSM analysis was performed using a Leica SP8 confocal laser scanning microscope (Leica).

The study (PEI-SARS-CoV2) was approved by the local ethics committee (Ethik-Kommission, Landesärztekammer Hessen 60314 Frankfurt am Main) and written informed consent was obtained from all patients (2020-1664_2-evBO). Patients provided a written agreement to publish their medical data. The study was performed in accordance with the provisions of the Declaration of Helsinki and good clinical practice guidelines. As control, three documentably SARS-Cov-2-negative, healthy volunteers P4-6 were included in this study and material from swabs was subjected to in vitro replication following the identical procedure as described for patients 1-3.

To 

To further confirm the formation of intact viral particles, virions were isolated by ultracentrifugation and analyzed by TEM after 

To characterize the isolates in more detail and classify the isolates, Next-Generation Sequencing (NGS) analysis was performed. For this purpose, RNA was isolated after a single passage to minimize the risk of in vitro mutations. Based on the sequence analysis, the isolates derived from P1 and P3 could be identified as belonging to the 20C clade, whereas that of P2 belongs to the 20A clade ( Figure 5A ). The detailed analysis of the whole-genome revealed eleven mutations in P1 and P3 and ten mutations in P2 compared to the aligned strain NC_045512.2 (Table 1 ). These mutations include the common mutations D614G of the spike protein as well as the often cooccurring mutations P314L in ORF1b/RdRP and T265I in ORF1a. [23] [24] [25] [26] Additionally, the also already well-known Q57H mutation in ORF3a

(not detected in isolate of P2) and the S686G mutation in the spike that was originally described in virus isolates of ferrets were identified. 24 

To study the humoral immune response of the long-term infected patients, the amount of SARS-CoV-2 RBD antibodies were deter- For a more detailed analysis of the antibody response, peptide arrays covering the S, N, M, and E protein were established, and the binding pattern of the sera derived from patients 1-3, from three negative patients, and from three convalescent plasma samples were determined (Figure 7) . It should be emphasized that this technique mainly reflects binding to sequential epitopes. To identify specifically recognized peptides, the signals obtained for sera from SARS-CoV-2 negative samples were considered as background ( Figure 7A ).

If the background signals were subtracted from the specific signals ( Figure 7C) , there was only one sequential epitope specifically detected in the N protein by serum derived from patient 1.

In case of the serum derived from patient 2 two linear epitopes in the S1 domain of the spike, one epitope derived from the N protein and one from the M protein were identified. In contrast to this, the linear epitopes recognized by serum derived from patient 3 were completely different from the patterns observed for the other patients. Here, we identified one epitope at the C-terminal part of the spike S2 domain and one in the N protein ( Figure 7A ,C; Table 2 ). In contrast, a wide variety of sequential epitopes was recognized with high intensity by convalescent sera ( Figure 7B ) derived from patients who rapidly eliminated the virus ( Figure 7B ). These epitopes were not recognized by sera derived from long-term patients (Table 3 ). In the case of the convalescent sera, epitopes localized within the RBD were recognized. This finding is in clear contrast to the binding pattern obtained for the long-term patients; here, no specific binding to linear epitopes covering the RBD was observed. Taken together, these data indicate that the pattern of sequential epitopes differs for sera derived from long-term infected patients and from patients that eliminated the infection within 10 days.

To 

The data presented in this study demonstrate that in non- with increasing outbreaks in Europe, followed by displacement of other lineages in North and South America. 28 It is particularly characterized by its D614G mutation in the spike protein, caused by A23403G nucleotide change. 29 Clade 20C on the other hand represents a daughter clade of 20A, also harboring the D614G mutation. 30 This mutation had already been described as part of a widespread variation, leading to increased infectivity due to facilitated ACE2-binding characteristics. 23, 24 This D614G mutation is known to be frequently accompanied by the P314L in ORF1b/RdRP that had also been identified in the patient 1 isolate (Table 1) . 25 Another common mutation that could be detected in these isolates was T265I

in ORF1a. The threonine substitution by isoleucine at position 265

leads to the addition of a beta-sheet structure. 26 We could identify the Q57H mutation in ORF3a in the isolates of P1 and P3, which was first described in Singapore in February 2020. This mutation coexists in many cases with the mutation T85I in non-structural protein 2 (NSP2) that, in contrast, could not be identified in this isolate. 24 One mutation that was originally described in ferrets and was immune evasion, but it seems to be a mutation with globally increasing incidence. 31 Furthermore, next to the well-recognized mutations D614G and P314L in ORF1b a new and so far unknown mutation V818A in ORF1b, was detected. Because ORF8 is known to play a crucial role in inhibiting the type I interferon signaling pathway as well as major histocompatibility complex I (MHC I) degradation, it was interesting to detect two mutations in ORF8. 32, 33 As MHC I degradation facilitates immune evasion, it was an unexpected finding that mutation D35Y that we identified in ORF8 was formerly described as providing protein stability and decreasing disease severity. 34, 35 The second ORF8 mutation A51S, in contrast, is located within two possible CD4+ T cell epitopes. 36 cellular immune response. In addition to these mutations affecting T cell epitopes, two further mutations in the spike protein were identified that could affect the T cell response. This includes T95I, a mutation occurring within a predicted CD8+ T cell epitope, 37 and mutation H245R that might be located both within a T cell and/or B cell epitope. 38 Therefore, it can be hypothesized that these mutations contribute to impaired virus elimination, as reflected by a productive long-term SARS-CoV-2 infection. Moreover, we found distinct differences in relative antibody concentrations in serum between the three long-term patients as compared to reconvalescent patients. In the majority of SARS-CoV-2 patients, IgG and IgM is detectable 2-3 weeks after onset of symptoms. 39 For patients with no obvious emerging symptoms or who remained completely asymptomatic, IgG and IgM levels are consistently less pronounced compared to patients with noticeable mild, moderate, or severe symptoms. 40 

The hypothesis of a potentially constrained efficiency of viral neutralization in all three patients is supported by characterization of linear epitopes recognized by antibodies of these patients ( Figure 7 ; Table 2 ). No binding to linear epitopes within the RBD of the spike that is assumed to be crucial for neutralization was found. 44 Instead, linear epitopes derived from the nucleocapsid protein were recognized by sera from all three patients.

Plaque reduction neutralization tests revealed a potential neutralizing effect against infection with the three isolates similar to an infection with the alpha-variant (B.1.1.7) by sera of vaccinated subjects, indicating a protection by vaccination. Both the three isolates as well as the alpha-variant are characterized by a D614G mutation in the spike protein that was described as enhancing viral loads and improving transmission. 45, 46 However, we observed differences in the efficiency of neutralization by sera of vaccinated individuals (Figure 8 ).

The isolate P1 required less diluted sera to be blocked compared to the alpha-variant and the other isolates, although we detected no differences in gene sequences between P1 and P3. As the same PFU/ml was used in this approach, these results were unexpected.

Based on the data presented in this study, the assumption that genomic material detected by PCR in long-term positive patients is essentially remaining genome RNA fragments has to be critically reconsidered. Our findings provide strong evidence that long-term PCR-positive patients are possible carriers of still intact and infectious virus. Considering the long positivity of these patients, stopping quarantine periods after 10 days without PCR-based testing must be critically reconsidered because there might be a risk of virus transmission.

We would like to thank Gert Carra and Robin Oliver Murra for their excellent technical support. Jacomine Krijnse-Locker for her support concerning electron microscopy.

The authors declare no conflict of interest. 

Informed consent was obtained from all subjects involved in the study. 

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