Distinct evolution of SARS-CoV-2 Omicron XBB and BA.2.86 lineages combining increased fitness and antibody evasion (preprint)

The unceasing circulation of SARS-CoV-2 leads to the continuous emergence of novel viral sublineages. Here, we isolated and characterized XBB.1, XBB.1.5, XBB.1.9.1, XBB.1.16.1, EG.5.1.1, EG.5.1.3, XBF, BA.2.86.1 and JN.1 variants, representing >80% of circulating variants in November 2023. The XBB subvariants carry few but recurrent mutations in the spike, whereas BA.2.86.1 and JN.1 harbor >30 additional changes. These variants replicated in IGROV-1 but no longer in Vero E6 and were not markedly fusogenic. They potently infected nasal epithelial cells, with EG.5.1.3 exhibiting the highest fitness. Antivirals remained active. Neutralizing antibody (NAb) responses from vaccinees and BA.1/BA.2-infected individuals were markedly lower compared to BA.1, without major differences between variants. An XBB breakthrough infection enhanced NAb responses against both XBB and BA.2.86 variants. JN.1 displayed lower affinity to ACE2 and higher immune evasion properties compared to BA.2.86.1. Thus, while distinct, the evolutionary trajectory of these variants combines increased fitness and antibody evasion.

A third vaccine dose equalizes the levels of effectiveness and immunogenicity of heterologous or homologous COVID-19 vaccine regimens (preprint)

Background: To cope with the persistence of the Covid-19 epidemic and the decrease in antibody levels following vaccination, a third dose of vaccine has been recommended in the general population. However, several vaccine regimens had been used initially, and the heterologous ChadOx1-S/BNT162b2 regimen had shown better efficacy and immunogenicity than the homologous BNT162b2/BNT162b2 regimen. Aim : We wanted to determine if this benefit was retained after the third dose. Methods: We combined an observational study of SARS-COV-2 infections among vaccinated healthcare workers at the University-Hospital of Lyon, France, with an analysis of immunological parameters before and after the third mRNA vaccine dose. Results: Following the second vaccine dose, heterologous vaccination regimens were more protective against infection than homologous regimens, but this was no longer the case after the third dose. RBD-specific IgG levels and serum neutralization capacity against different SARS-CoV-2 variants were higher after the third dose than after the second dose in the homologous regimen group, but not in the heterologous group. Conclusion: The advantage conferred by heterologous vaccination is lost after the third dose in terms of both protection and immunogenicity. Immunological measurements one month after vaccination suggest that heterologous vaccination induces maximal immunity after the second dose, whereas the third dose is required to reach the same level in individuals with a homologous regimen.

Determinants of protection against SARS-CoV-2 Omicron BA.1 and Delta infections in fully vaccinated outpatients (preprint)

We aimed to evaluate the association between the humoral and cellular immune responses and symptomatic SARS-CoV-2 infection with Delta or Omicron BA.1 variants in fully vaccinated outpatients. Anti-RBD IgG levels and IFN-{gamma} release were evaluated at PCR-diagnosis of SARS-CoV-2 in 636 samples from negative and positive patients during Delta and Omicron BA.1 periods. Median levels of anti-RBD IgG in positive patients were significantly lower than in negative patients for both variants (p < 0.05). The risk of Delta infection was inversely correlated with anti-RBD IgG titres (aOR = 0.63, 95% CI [0.41; 0.95], p = 0.03) and it was lower in the hybrid immunity group compared to the homologous vaccination group (aOR = 0.22, 95% CI [0.05; 0.62], p = 0.01). In contrast, neither the vaccination scheme nor anti-RBD IgG titers were associated with the risk of BA.1 infection in multivariable analysis. IFN-{gamma} release post-SARS-CoV-2 peptide stimulation was not different between samples from patients infected (either with Delta or Omicron BA.1 variant) or not (p = 0.77). Our results show that high circulating levels of anti-RBD IgG and hybrid immunity were independently associated with a lower risk of symptomatic SARS-CoV-2 infection in outpatients with differences according to the infecting variant.

Long-term humoral response following simultaneous Delta and Omicron BA.1 co-infection (preprint)

To provide insight into the long-term immune response following bivalent vaccines, we sampled vaccinated patients simultaneously co-infected with Delta and BA.1. We reported that simultaneous exposure to the Delta and BA.1 S protein does not confer an additional immune advantage compared to exposure to the Omicron BA.1 S protein alone.

Interactions between Severe Acute Respiratory Syndrome Coronavirus 2 Replication and Major Respiratory Viruses in Human nasal Epithelium.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), along with extensive non-pharmacological interventions, have profoundly altered the epidemiology of major respiratory viruses. Some studies have described virus-virus interactions, particularly manifested by viral interference mechanisms at different scales. Still, our knowledge of the mutual interactions between SARS-CoV-2 and other respiratory viruses remains incomplete. Here, we studied the interactions between SARS-CoV-2 and several respiratory viruses (influenza, RSV, hMPV, and hRV) in a reconstituted human epithelial airway model, exploring different scenarios affecting the sequence and timing of co-infections. We show that the virus type and the sequence of infections are key parameters of virus-virus interactions, having the impact of primary infections on the regulation of the immune response a determinant role in the outcome of secondary infections.

The risk of COVID-19 death is much greater and age-dependent with type I IFN autoantibodies (preprint)

SARS-CoV-2 infection fatality rate (IFR) doubles with every five years of age from childhood onward. Circulating autoantibodies neutralizing IFN-α, IFN-ω, and/or IFN-β are found in ~20% of deceased patients across age groups. In the general population, they are found in ~1% of individuals aged 20-70 years and in >4% of those >70 years old. With a sample of 1,261 deceased patients and 34,159 uninfected individuals, we estimated both IFR and relative risk of death (RRD) across age groups for individuals carrying autoantibodies neutralizing type I IFNs, relative to non-carriers. For autoantibodies neutralizing IFN-α2 or IFN-ω, the RRD was 17.0[95% CI:11.7-24.7] for individuals under 70 years old and 5.8[4.5-7.4] for individuals aged 70 and over, whereas, for autoantibodies neutralizing both molecules, the RRD was 188.3[44.8-774.4] and 7.2[5.0-10.3], respectively. IFRs increased with age, from 0.17%[0.12-0.31] for individuals <40 years old to 26.7%[20.3-35.2] for those ≥80 years old for autoantibodies neutralizing IFN-α2 or IFN-ω, and from 0.84%[0.31-8.28] to 40.5%[27.82-61.20] for the same two age groups, for autoantibodies neutralizing both molecules. Autoantibodies against type I IFNs increase IFRs, and are associated with high RRDs, particularly those neutralizing both IFN-α2 and -ω. Remarkably, IFR increases with age, whereas RRD decreases with age. Autoimmunity to type I IFNs appears to be second only to age among common predictors of COVID-19 death.

Evaluation of Commercial Anti-SARS-CoV-2 Neutralizing Antibody Assays in seropositive subjects (preprint)

The virus neutralization test (VNT) is the reference for the assessment of the functional ability of neutralizing antibodies (NAb) to block SARS-CoV-2 entry into cells. New competitive immunoassays measuring antibodies preventing interaction between the spike protein and its cellular receptor are proposed as surrogate VNT (sVNT). We tested three commercial sVNT (a qualitative immunochromatographic test and two quantitative immunoassays named YHLO and TECO) together with a conventional anti-spike IgG assay (bioMerieux) in comparison with an in-house plaque reduction neutralization test (PRNT50) using the original 19A strain and different variants of concern (VOC), on a panel of 306 sera from naturally-infected or vaccinated patients. The qualitative test was rapidly discarded because of poor sensitivity and specificity. Areas under the curve of YHLO and TECO assays were, respectively, 85.83 and 84.07 (p-value >0.05) using a positivity threshold of 20 for PRNT50, and 95.63 and 90.35 (p-value =0.02) using a threshold of 80. However, the performances of YHLO and bioMerieux were very close for both thresholds, demonstrating the absence of added value of sVNT compared to a conventional assay for the evaluation of the presence of NAb in seropositive subjects. In addition, the PRNT50 assay showed a reduction of NAb titers towards different VOC in comparison to the 19A strain that could not be appreciated by the commercial tests. Despite the good correlation between the anti-spike antibody titer and the titer of NAb by PRNT50, our results highlight the difficulty to distinguish true NAb among the anti-RBD antibodies with commercial user-friendly immunoassays.

A novel assessment method for COVID-19 humoral immunity duration using serial measurements in naturally-infected and vaccinated subjects. (preprint)

Background: It is crucial for medical decision-making and vaccination strategies to collect information on sustainability of immune responses after infection or vaccination, and how long-lasting antibodies against SARS-COV-2 could provide a humoral and protective immunity, preventing reinfection with SARS-CoV-2 or its variants. The aim of this study is to present a novel method to quantitatively measure and monitor the diversity of SARS-CoV-2 specific antibody profiles over time. Methods: Two collections of serum samples were used in this study: A collection from 20 naturally-infected subjects (follow-ups to 1 year) and a collection from 83 subjects vaccinated with one or two doses of Pfizer BioNtech vaccine (BNT162b2/BNT162b2) (follow-ups to 6 months). The Multi-SARS-CoV-2 assay, a multiparameter serology test, developed for the serological confirmation of past-infections was used to determine the reactivity of six different SARS-CoV-2 antigens. For each patient sample, 3 dilutions (1/50, 1/400 and 1/3200) were defined as an optimal set over the six antigens and their respective linear ranges, allowing accurate quantitation of the corresponding six specific antibodies. Nonlinear mixed-effects modelling was applied to convert intensity readings from 3 determined dilutions to a single quantification value for each antibody. Results: Median half-life for the 20 naturally infected vs 74 vaccinated subjects (two doses) was respectively 120 vs 50 days for RBD, 127 vs 53 days for S1 and 187 vs 86 days for S2 antibodies. Respectively, 90% of the antibody concentration wanes after 158 vs 398 days for RBD, 171 vs 420 days for S1, and 225 vs 620 days for S2 after the second vaccine shot. Conclusion: The newly proposed method, based on a series of a limited number of dilutions, can convert a conventional qualitative assay into a quantitative assay. This conversion helps define the sustainability of specific immune responses against each relevant viral antigen and can help in defining the protection characteristics after an infection or a vaccination.

SARS-CoV-2 infected cells trigger an acute antiviral response mediated by Plasmacytoid dendritic cells in mild but not severe COVID-19 patients (preprint)

Type I and III interferons (IFN-I/{lambda}) are key antiviral mediators against SARS-CoV-2 infection. Here, we demonstrate that the plasmacytoid dendritic cells (pDCs) are predominant IFN-I/{lambda} source by sensing SARS-CoV-2-infected cells. We show that sensing of viral RNA by pDCs requires sustained cell adhesion with infected cells. In turn, the pDCs restrict viral spread by a local IFN-I/{lambda} response directed toward SARS-CoV-2-infected cells. This specialized function enables pDCs to efficiently turn-off viral replication, likely by a concentrated flux of antiviral effectors at the contact site with infected cells. Therefore, we propose that pDC activation is essential to locally control SARS-CoV-2-infection. By exploring the pDC response in patients, we further demonstrate that pDC responsiveness correlates with the severity of the disease and in particular that it is impaired in severe COVID-19 patients. Thus, the ability of pDCs to respond to SARS-CoV-2-infected cells could be a key to understand severe cases of COVID-19. HighlightsO_LIpDCs are immune cells against SARS-CoV-2-infected cells C_LIO_LIpDC-mediated IFN-I/{lambda} response against SARS-CoV-2 infected cells control COVID- 19 progression C_LIO_LIpDC response by SARS-CoV-2 is restricted to IRF7-prioritized signaling leading to antiviral control C_LIO_LIpDC antiviral response directed toward contacting SARS-CoV-2-infected cells C_LI

Evaluation of commercial anti-SARS-CoV-2 antibody assays and comparison of standardized titers in vaccinated healthcare workers. (preprint)

With the availability of vaccines, commercial assays detecting anti-SARS-CoV-2 antibodies (Ab) evolved towards quantitative assays directed to the spike glycoprotein or its receptor binding domain (RBD). The main objective of the present study was to compare the Ab titers obtained with quantitative commercial binding Ab assays, after 1 dose (convalescent individuals) or 2 doses (naive individuals) of vaccine, in healthcare workers (HCW). Antibody titers were measured in 263 sera (from 150 HCW) with 5 quantitative immunoassays (Abbott RBD IgG II quant, bioMerieux RBD IgG, DiaSorin Trimeric spike IgG, Siemens Healthineers RBD IgG, Wantai RBD IgG). One qualitative total antibody anti RBD detection assay (Wantai) was used to detect previous infection before vaccination. The results are presented in binding Ab units (BAU)/mL after application, when possible, of a conversion factor provided by the manufacturers and established from a World Health Organization (WHO) internal standard. There was a 100% seroconversion with all assays evaluated after two doses of vaccine. With assays allowing BAU/ml correction, Ab titers were correlated ({rho}= 0.84-0.99). However, a significant difference between values persisted. The titer differences varied by a mean 3.04% between Siemens and bioMerieux assays to 50.54% between Siemens and DiaSorin assays. Titer harmonization is still to be improved despite better results were obtained between assays detecting the same Ab against the same antigen. The next step towards a true standardization of the assays would be to include the International Standard in the calibration of each assays to express the results in IU/mL.

Immunogenicity and efficacy of heterologous ChadOx1/BNT162b2 vaccination (preprint)

Following severe adverse reactions in patients vaccinated with the AstraZeneca ChadOx1 (Chad) vaccine, European health authorities have recommended that patients under the age of 55 who received one dose of Chad vaccine receive a second dose of Pfizer BNT162b2 (BNT) vaccine as a booster. However, the effectiveness and the immunogenicity of this vaccination regimen have not been formally tested. Here, we show that the heterologous Chad/BNT combination confers better protection against SARS-CoV-2 infection than the homologous BNT/BNT combination in a population of health care workers. To understand the underlying mechanism, we monitored in a longitudinal way the anti-spike immunity conferred by each vaccinal combination. Both combinations induced strong anti-spike antibody responses after boost in all vaccinated individuals. However, sera from heterologous vaccinated individuals displayed a stronger neutralizing activity, regardless of the SARS-CoV-2 variant analyzed, and this was associated with more switched memory RBD-specific B cells with an activated phenotype and less IgA. The Chad vaccine induced a stronger T cell response than the BNT vaccine after the priming dose, and the reciprocal was true for the IgG response, which could explain the complementarity of both vaccines when used in an heterologous setting. This strongly protective vaccination regimen could be therefore particularly suitable for immunocompromised individuals.

Live virus neutralization testing in convalescent and vaccinated subjects against 19A, 20B, 20I/501Y.V1 and 20H/501.V2 isolates of SARS-CoV-2 (preprint)

Background: SARS-CoV-2 mutations appeared recently and can lead to conformational changes in the spike protein and probably induce modifications in antigenicity. In this study, we wanted to assess the neutralizing capacity of antibodies to prevent cell infection, using a live virus neutralisation test. Methods: Sera samples were collected from different populations: two-dose vaccinated COVID-19-naive healthcare workers (HCWs; Pfizer-BioNTech BNT161b2), 6-months post mild COVID-19 HCWs, and critical COVID-19 patients. We tested various clades such as 19A (initial one), 20B (B.1.1.241 lineage), 20I/501Y.V1 (B.1.1.7 lineage), and 20H/501Y.V2 (B.1.351 lineage). Results: No significant difference was observed between the 20B and 19A isolates for HCWs with mild COVID-19 and critical patients. However, a significant decrease in neutralisation ability was found for 20I/501Y.V1 in comparison with 19A isolate for critical patients and HCWs 6-months post infection. Concerning 20H/501Y.V2, all populations had a significant reduction in neutralising antibody titres in comparison with the 19A isolate. Interestingly, a significant difference in neutralisation capacity was observed for vaccinated HCWs between the two variants whereas it was not significant for the convalescent groups. Conclusion: Neutralisation capacity was slightly reduced for critical patients and HCWs 6-months post infection. No neutralisation escape could be feared concerning the two variants of concern in both populations. The reduced neutralising response observed towards the 20H/501Y.V2 in comparison with the 19A and 20I/501Y.V1 isolates in fully immunized subjects with the BNT162b2 vaccine is a striking finding of the study.

Refining “long-COVID” by a prospective multimodal evaluation of patients with long-term symptoms related to SARS-CoV-2 infection (preprint)

Background COVID-19 long-haulers or “long-COVID” represent 10% of COVID-19 patients and remain understudied. Methods In this prospective study, we recruited 30 consecutive patients seeking medical help for persistent symptoms (> 30 days) attributed to COVID-19. All reported a viral illness compatible with COVID-19. The patients underwent a multi-modal evaluation including clinical, psychological, virological, specific immunological assays and were followed longitudinally. Results The median age was 40 [interquartile range: 35-54] and 18 (60%) were female. After a median time of 152 [102-164] days after symptom onset, fever, cough and dyspnea were less frequently reported as compared with the initial presentation, but paresthesia and burning pain emerged in 18 (60%) and 13 (43%) patients, respectively. The clinical examination was unremarkable in all patients although the median fatigue and pain visual analogic scales were 7 [5-8] and 5 [2-6], respectively. Extensive biological studies were unremarkable, as were multiplex cytokine and ultra-sensitive interferon-a2 measurements. At this time, nasopharyngeal swab and stool RT-PCR were negative for all tested patients. Using SARS-CoV-2 serology and IFN-γ ELISPOT, we found evidence of a previous SARS-CoV-2 infection in 50% (15/30) of patients, with objective evidence of lack or waning of immune response in two. Finally, psychiatric evaluation showed that 11 (36.7%), 13 (43.3%) and 9 (30%) patients had a positive screening for anxiety, depression and post-traumatic stress disorder, respectively. Conclusions Half of patients seeking medical help for long-COVID lack SARS-CoV-2 immunity. The presence of SARS-CoV-2 immunity did not cluster clinically or biologically long haulers, who reported severe fatigue, altered quality of life, and exhibited psychological distress. Key points Among 30 consecutive patients reporting persistent symptoms (median 6 months) self-attributed to COVID-19, pain, fatigue and disability were reported in virtually all patients. More than one third of patients suffer from psychological disorders such as anxiety, depression and/or post-traumatic stress disorder, regardless of SARS-CoV-2 immunity. At the time of evaluation, only 50% of patients had cellular and/or humoral sign of a past SARS-CoV-2, and serology positivity varied depending of the kit used. Exhaustive clinical, biological and immunological evaluations failed to find an alternative diagnosis, or to identify specific cytokine signature including type I interferon.

Antibodies against type-I Interferon: detection and association with severe clinical outcome in COVID-19 patients (preprint)

Objectives Impairment of type I interferon (IFN-I) immunity has been reported in critically ill COVID-19 patients. This defect can be explained by the presence of circulating autoantibodies against IFN-I. We set out to improve the detection and the quantification of such antibodies (Abs) in a cohort of severe Covid-19 patients, in an effort to better document the prevalence of these Abs as the pandemics evolves and how they correlate with the clinical course of the disease. Methods Anti-IFN-a Abs was investigated 84 critical COVID-19 patients who were admitted to ICU at the Lyon University Hospital, France with a commercially available kit (Thermo-Fisher). Results Twenty-one patients out of 84 (25%) had anti-IFNa2 Ab above cut-off (>34ng/mL) in sera. A neutralizing activity against IFN-a2 was evidenced in 15 of them, suggesting that 18% of patients were positive for neutralizing anti-IFN-a and -w auto-Abs. In addition, in most of patients with neutralizing IFN-I Abs, we noticed an impairment of the IFN-I response. However, we did not find any difference in terms of clinical characteristics or outcome between critical COVID-19 patients with or without neutralizing anti-IFN-a2 auto-Abs in these conditions. Finally, we detected anti-type I IFN auto-Abs in sera of COVID-19 patients were detected throughout the ICU stay. Conclusions We report that 18% of severe COVID-19 patients were positive for these Anti-Type-I IFN Abs, confirming the detrimental role of these Abs on the antiviral response. Our results further support the use of recombinant type I IFNs not targeted by the auto-Abs (e.g., IFN-b) in COVID-19 patients with an impaired IFN-I response.

Six-month antibody response to SARS-CoV-2 in healthcare workers assessed by virus neutralisation and commercial assays (preprint)

We conducted a prospective study in healthcare workers (n=296) of the University Hospital of Lyon, France. Serum samples (n=296) collected six months after disease onset were tested using three commercial assays: the Wantai Ab assay detecting total antibodies against the receptor binding domain (RBD) of the S protein, the bioMerieux Vidas assay detecting IgG to the RBD and the Abbott Architect assay detecting IgG to the N protein. The neutralising antibody (NAb) titre was also determined for all samples with a virus neutralisation assay (VNA) using live virus. The positivity rate was 100% with the Wantai assay, 84.8% with the bioMerieux assay and 55.4% with the Abbott assay. Only 51% of HCWs were positive for the presence of NAb. Less than 10 % of HCWs had a NAb titre greater than 80. At a neutralising titre of 80, the area under the curves [IC 95%] was 0.71 [0.62-0.81], 0.75 [0.65-0.85] and 0.95 [0.92-0.97] for Wantai, Abbott and Vidas respectively. The data presented herein suggest that commercial assays detecting antibodies against the N protein must not be used in long-term seroprevalence surveys while the Wantai assay could be useful for this purpose. VNA should remain the gold standard to assess the protective antibody response, but some commercial assays could be used as first-line screening of long-term presence of NAb.

Clinical and microbiological assessments of COVID-19 in healthcare workers: a prospective longitudinal study (preprint)

Background A comprehensive assessment of COVID-19 in healthcare workers (HCWs) including the investigation of viral shedding duration is critical. Methods A longitudinal study including 319 HCWs was conducted. After SARS-CoV-2 screening with RT-PCR assay, other respiratory pathogens were tested with a multiplex molecular panel. For SARS-CoV-2 positive HCWs, the normalized viral load was determined weekly; viral culture and virus neutralization assays were also performed. For 190 HCWs tested negative, SARS-CoV-2 serological testing was performed one month after the inclusion. Findings Of the 319 HCWs included, 67 (21.0%) were tested positive for SARS-CoV-2; two of them developed severe COVID-19. The proportion of smell and taste dysfunction was significantly higher in SARS-CoV-2 positive HCWs than in negative ones (38.8% vs 9.5% and 37.3% vs 10.7%, respectively, p<0.001). Of the 67 positive patients, 9.1% were tested positive for at least another respiratory pathogen (vs 19.5%, p=0.07). The proportion of HCWs with a viral load > 5.0 log10 cp/ml (Ct value <25) was less than 15% at 8 days after symptom onset; 12% of them were still positive after 40 days (Ct >37). More than 90% of culturable virus had a viral load > 4.5 log10 cp/ml (Ct < 26) and were collected within 10 days after symptom onset. From HCWs tested negative, 6/190 (3.2%) exhibited seroconversion for IgG antibodies. Interpretation Our data suggest that the determination of normalized viral load (or its estimation through Ct values) can be useful for discontinuing isolation of HCWs and facilitating their safe return to work. HCWs presenting mild COVID-19 are unlikely infectious 10 days after symptom onset.

Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia (preprint)

In the current COVID-19 pandemic context, proposing and validating effective treatments represents a major challenge. However, the lack of biologically relevant pre-clinical experimental models of SARS-CoV-2 infection as a complement of classic cell lines represents a major barrier for scientific and medical progress. Here, we advantageously used human reconstituted airway epithelial models of nasal or bronchial origin to characterize viral infection kinetics, tissue-level remodeling of the cellular ultrastructure and transcriptional immune signatures induced by SARS-CoV-2. Our results underline the relevance of this model for the preclinical evaluation of antiviral candidates. Foremost, we provide evidence on the antiviral efficacy of remdesivir and the therapeutic potential of the remdesivir-diltiazem combination as a rapidly available option to respond to the current unmet medical need imposed by COVID-19. One Sentence SummaryNew insights on SARS-CoV-2 biology and drug combination therapies against COVID-19.

Molecular characterization of SARS-CoV-2 in the first COVID-19 cluster in France reveals an amino-acid deletion in nsp2 (Asp268Del) (preprint)

We present the first genetic characterization of a COVID-19 cluster in Europe using metagenomic next-generation sequencing (mNGS). Despite low viral loads, the mNGS workflow used herein allowed to characterize the whole genome sequences of SARS-CoV2 isolated from an asymptomatic patient, in 2 clinical samples collected 1 day apart. Comparison of these sequences suggests viral evolution with development of quasispecies. In addition, the present workflow identified a new deletion in nsp2 (Asp268Del) which was found in all 3 samples originating from this cluster as well as in 37 other viruses collected in England and in Netherlands, suggesting the spread of this deletion in Europe. The impact of Asp268Del on SARS-CoV-2 transmission and pathogenicity, as well as on PCR performances and anti-viral strategy should be rapidly evaluated in further studies.