The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro

Sissy ThereseSonnleitnerab StefanieSonnleitnera EvaHinterbichlera HannahHalbfurtera Dominik B.C.Kopeckya StephanKoblmüllerc ChristianSturmbauerc WilfriedPoschb GernotWaldera

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discuss the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.

Keywords: SARS-CoV-2Whole genome sequencing (WGS)Mutational dynamicsAdaptationSerial passage in vitro


Adjunctive homeopathic treatment of hospitalized COVID-19 patients (COVIHOM): A retrospective case series

Takacs Michael1Frass Michael2Walder Gernot3Fibert Philippa4Rottensteiner Maria5Glück Walter5Lechleitner Peter1Oberbaum Menachem6Leisser Ilse7Chandak Kavita8Panhofer Peter9Weiermayer Petra10

Complement Ther Clin Pract. 2021 May 11;44:101415. doi: 10.1016/j.ctcp.2021.101415. Online ahead of print.


Background: and purpose. COVID-19 is a novel viral disease causing worldwide pandemia. The aim of this study was to describe the effect of adjunctive individualized homeopathic treatment delivered to hospitalized patients with confirmed symptomatic SARS-CoV-2 infection.

Patient presentation: Thirteen patients with COVID-19 were admitted. Mean age was 73.4 ± 15.0 (SD) years. Twelve (92.3%) were speedily discharged without relevant sequelae after 14.4 ± 8.9 days. A single patient admitted in an advanced stage of septic disease died in hospital. A time-dependent improvement of relevant clinical symptoms was observed in the 12 surviving patients. Six (46.2%) were critically ill and treated in the intensive care unit (ICU). Mean stay at the ICU of the 5 surviving patients was 18.8 ± 6.8 days. In six patients (46.2%) gastrointestinal disorders accompanied COVID-19.

Conclusion: The observations suggest that adjunctive homeopathic treatment may be helpful to treat patients with confirmed COVID-19 even in high – risk patients especially since there is no conventional treatment of COVID-19 available at present.

Keywords: COVID-19; Complementary therapies; Homeopathy; Intensive care unit; SARS-CoV-2.

Links: PMID: 33989861; PMCID: PMC8110319; DOI: 10.1016/j.ctcp.2021.101415

Maintenance of neutralizing antibodies over ten months in convalescent SARS-CoV-2 afflicted patients

Sissy Therese SonnleitnerMartina PrelogBianca JansenChantal Rodgarkia-DaraSarah GietlCarmen Maria SchöneggerStephan KoblmüllerChristian SturmbauerWilfried Posch … See all authors 

Transbound Emerg Dis. 2021 May 7. doi: 10.1111/tbed.14130. Online ahead of print.


Knowledge of the level and duration of protective immunity against SARS-CoV-2 after primary infection is of crucial importance for preventive approaches. Currently, there is a lack of evidence on the persistence of specific antibodies. We investigated the generation and maintenance of neutralizing antibodies of convalescent SARS-CoV-2-afflicted patients over a ten-month period post-primary infection using an immunofluorescence assay, a commercial chemiluminescent immunoassay and an in-house enzyme-linked neutralization assay. We present the successful application of an improved version of the plaque-reduction neutralization assay which can be analysed optometrically to simplify data interpretation. Based on the results of the enzyme-linked neutralization assay, neutralizing antibodies were maintained in 77.4% of convalescent individuals without relevant decay over ten months. Furthermore, a positive correlation between severity of infection and antibody titre was observed. In conclusion, SARS-CoV-2-afflicted individuals have been proven to be able to develop and maintain neutralizing antibodies over a period of ten months after primary infection. Findings suggest long-lasting presumably protective humoral immune responses after wild-type infection.


An in vitro model for assessment of SARS-CoV-2 infectivity by defining the correlation between virus isolation and quantitative PCR value: isolation success of SARS-CoV-2 from oropharyngeal swabs correlates negatively with Cq value

Sissy Therese Sonnleitner12Julian Dorighi3Bianca Jansen3Carmen Schönegger3Sarah Gietl3Stephan Koblmüller4Christian Sturmbauer4Wilfried Posch5Gernot Walder3

Virol J. 2021 Apr 7;18(1):71. doi: 10.1186/s12985-021-01542-y.


Background: At the beginning of the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), little was known about its actual rate of infectivity and any COVID-19 patient positive in laboratory testing was supposed to be highly infective and a public health risk factor.

Methods: One hundred oropharyngeal samples were obtained during routine work flow of testing symptomatic persons by quantitative polymerase chain reaction (qPCR) and were inoculated onto cell culture of VeroB4 cells to study the degree of infectivity of SARS-CoV-2 in vitro. Quantification by virus titration and an external standard using synthetic RNA gave the breaking point of infectivity in SARS-CoV-2 in vitro.

Results: A clear negative correlation (r = – 0.76; p < 0.05) could be asserted between the viral load in quantitative polymerase chain reaction (qPCR) and the probability of a successful isolation in serial isolation experiments of specific oropharyngeal samples positive in qPCR. Quantification by virus titration and an external standard using synthetic RNA indicate a Cq between 27 and 30 in E-gene screening PCR as a breaking point in vitro, where infectivity decreases significantly and isolations become less probable.

Conclusions: This study showed that only the 21% of samples with the highest viral load were infectious enough to transmit the virus in vitro and determined that the dispersion rate in vitro is surprisingly close to those calculated in large retrospective epidemiological studies for SARS-CoV-2. This raises the question of whether this simple in vitro model is suitable to give first insights in dispersion characters of novel or neglected viral pathogens. The statement that SARS-CoV-2 needs at least 40,000 copies to reliably induce infection in vitro is an indication of its transmissibility in Public Health decisions. Applying quantitative PCR systems in diagnosis of SARS-CoV2 can distinguish between patients providing a high risk of transmission and those, where the risk of transmission is probably limited to close and long-lasting contacts.


SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8 + T cell responses

Benedikt Agerer1Maximilian Koblischke2Venugopal Gudipati3Luis Fernando Montaño-Gutierrez4Mark Smyth1Alexandra Popa1Jakob-Wendelin Genger1Lukas Endler1David M Florian2Vanessa Mühlgrabner3Marianne Graninger2Stephan W Aberle2Anna-Maria Husa4Lisa Ellen Shaw5Alexander Lercher1Pia Gattinger6Ricard Torralba-Gombau1Doris Trapin7Thomas Penz1Daniele Barreca1Ingrid Fae8Sabine Wenda8Marianna Traugott9Gernot Walder10Winfried F Pickl711Volker Thiel1213Franz Allerberger14Hannes Stockinger3Elisabeth Puchhammer-Stöckl2Wolfgang Weninger5Gottfried Fischer7Wolfgang Hoepler9Erich Pawelka8Alexander Zoufaly8Rudolf Valenta36111516Christoph Bock117Wolfgang Paster4René Geyeregger4Matthias Farlik5Florian Halbritter4Johannes B Huppa3Judith H Aberle2Andreas Bergthaler18

Sci Immunol. 2021 Mar 4;6(57):eabg6461. doi: 10.1126/sciimmunol.abg6461.

AbstractCD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

Links: PMID: 33664060; DOI: 10.1126/sciimmunol.abg6461

Home quarantine in COVID-19: A study on 50 consecutive patients in Austria

Sarah Gietl1Carmen M Schönegger2Markus Falk3Stefanie Weiler2Simone Obererlacher2Bianca Jansen2Sissy-Therese Sonnleitner2Gernot Walder2

Clin Med (Lond). 2021 Jan;21(1):e9-e13.
doi: 10.7861/clinmed.2020-0787.

AbstractA cohort of the first 50 COVID-19 patients in East Tyrol, a region in the southwest of Austria, were monitored in home quarantine. Specific viral ribonucleic acid was detected in throat swabs and stool samples. Analysis indicated a median virus shedding duration of 13 days; however, statistical outliers highlight the importance of consequent testing. This underlines the need of negative throat swabs prior to removing quarantine. We monitored the disease’s characteristics via an in-house score called Corona Severity Index, in order to predict an aggravation of the disease. Special attention was paid to early symptoms, such as headache, which appeared to be significantly more common in younger patients (p=0.019). Anosmia and ageusia showed a predominance in female patients (p=0.028). Investigation revealed seven relapses and viral shedding fluctuation in four cases. A follow-up examination shed light on seroconversion which could be observed in 35 of 40 participants. This further clarifies the necessity of establishing discharge standards and follow-up management for COVID-19 patients.

Keywords: COVID-19; clinical characteristics,; home quarantine; recurrences; virus shedding.


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