Search

A recombinant Ross River virus (RRV) that contains the fluorescent protein mCherry fused to the non-structural protein 3 (nsP3) was constructed, which allowed real-time imaging of viral replication. RRV-mCherry contained either the natural opal stop codon after the nsP3 gene or was constructed without a stop codon. The mCherry fusion protein did not interfere with the viral life cycle and deletion of the stop codon did not change the replication capacity of RRV-mCherry. Comparison of RRV-mCherry and chikungunya virus-mCherry infections, however, showed a cell type-dependent delay in RRV-mCherry replication in HEK 293T cells. This delay was not caused by differences in cell entry, but rather by an impeded nsP expression caused by the RRV inhibitor ZAP (zinc finger CCCH-Type, antiviral 1). The data indicate that viral replication of alphaviruses is cell-type dependent, and might be unique for each alphavirus.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has now been continuing for more than two years. The infection causes COVID-19, a disease of the respiratory and cardiovascular system of variable severity. Here, the humoral immune response of 80 COVID-19 patients from the University Hospital Frankfurt/Main, Germany, was characterized longitudinally. The SARS-CoV-2 neutralization activity of serum waned over time. The neutralizing potential of serum directed towards the human alpha-coronavirus NL-63 (NL63) also waned, indicating that no cross-priming against alpha-coronaviruses occurred. A subset of the recovered patients ( n = 13) was additionally vaccinated with the mRNA vaccine Comirnaty. Vaccination increased neutralization activity against SARS-CoV-2 wild-type (WT), Delta, and Omicron, although Omicron-specific neutralization was not detectable prior to vaccination. In addition, the vaccination induced neutralizing antibodies against the more distantly related SARS-CoV-1 but not against NL63. The results indicate that although SARS-CoV-2 humoral immune responses induced by infection wane, vaccination induces a broad neutralizing activity against multiple SARS-CoVs, but not to the common cold alpha-coronavirus NL63.

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen-antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera-as in cases of non-ARDS sera-combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen-antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.