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. 2022 Mar 11;11(3):338.
doi: 10.3390/pathogens11030338.

Drug Sensitivity of Vaccine-Derived Rubella Viruses and Quasispecies Evolution in Granulomatous Lesions of Two Ataxia-Telangiectasia Patients Treated with Nitazoxanide

Raeesa Faisthalab  1 , Suganthi Suppiah  1 , Morna Dorsey  2 , Kathleen E Sullivan  3 , Joseph Icenogle  1 , Ludmila Perelygina  1
Affiliations

Drug Sensitivity of Vaccine-Derived Rubella Viruses and Quasispecies Evolution in Granulomatous Lesions of Two Ataxia-Telangiectasia Patients Treated with Nitazoxanide

Raeesa Faisthalab et al. Pathogens. .

Abstract

A strong association between rubella virus (RuV) and chronic granulomas, in individuals with inborn errors of immunity, has been recently established. Both the RA27/3 vaccine and wild-type RuV strains were highly sensitive to a broad-spectrum antiviral drug, nitazoxanide (NTZ), in vitro. However, NTZ treatment, used as a salvage therapy, resulted in little or no improvements of RuV-associated cutaneous granulomas in patients. Here, we report investigations of possible causes of treatment failures in two ataxia-telangiectasia patients. Although a reduction in RuV RNA in skin lesions was detected by real-time RT-PCR, live immunodeficiency-related vaccine-derived rubella viruses (iVDRV) were recovered from granulomas, before and after the treatments. Tizoxanide, an active NTZ metabolite, inhibited replications of all iVDRVs in cultured A549 cells, but the 50% and 90% inhibitory concentrations were 10-40 times higher than those for the RA27/3 strain. There were no substantial differences in iVDRV sensitivities, neither before nor after treatments. Analysis of quasispecies in the E1 gene, a suspected NTZ target, showed no effect of NTZ treatments on quasispecies' complexity in lesions. Thus, failures of NTZ therapies were likely due to low sensitivities of iVDRVs to the drug, and not related to the emergence of resistance, following long-term NTZ treatments.

Keywords: ataxia-telangiectasia; cutaneous granulomas; immunodeficiency-related vaccine-derived rubella viruses (iVDRV); nitazoxanide; quasispecies.

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Conflict of interest statement

The authors declare no conflict of interest. (The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the United States Centers for Disease Control and Prevention).

Figures

Figure 1
Figure 1
Course of skin granuloma treatments showing the duration of each treatment and oral NTZ dosage. Amounts of iVDRV RNA (the red dots) were determined by RuV RT-qPCR absolute quantification and presented as genomic RNA copies per an entire biopsy. The designations of iVDRV strains isolated from skin lesion biopsies before and at different times after NTZ therapy are shown above the red dots.
Figure 2
Figure 2
Inhibition of replication of iVDRV strains isolated from skin lesions before and after the NTZ therapies. (A). Dose response analysis of A549 cells treated with tizoxanide and infected with iVDRV strains. Data represent the mean of three (RA27/3, CA strains) or four (RI strains) independent experiments +/− SD. (B). Table of IC50 and IC90 of tizoxanide in A459 cells infected with iVDRV strains.
Figure 3
Figure 3
Phylogenetic tree of iVDRV strains. The genetic relationships between the whole genome sequences of iVDRV strains, RA27/3 and the 32 WHO reference viruses were inferred using the Maximum Likelihood method in MEGA10. All taxa are labeled with WHO names with the RI iVDRV sequences marked with red dots and the CA iVDRV sequences marked with blue dots. The scale bar indicates the number of base substitutions per site. RA27/3 and iVDRVs represent a separate branch on the tree with RA27/3 being basal.
Figure 4
Figure 4
Diversity of iVDRV consensus sequences. (A). Nucleotide and (B). amino acid substitutions in iVDRV genomes relative to parental RA27/3 virus (GenBank #FJ211588) are depicted as vertical lines. The positions of the coding sequences (CDS) of the nonstructural and structural precursors (yellow pointed bars) and mature protein CDSs (green pointed bars) in the genomic sequence are indicated below the RA27/3 reference sequence. The total number of substitutions are shown for each iVDRV in the table (C). The numbers of ambiguous disagreements are in parentheses.
Figure 5
Figure 5
iVDRV quasispecies in tissues and viral isolates before and after NTZ therapy. (A). Neighbor-joining tree (non-rooted) for quasispecies within the granuloma samples (triangles) and virus isolates (circles) from the CA and RI cases. The genetic distances were computed using the Maximum Composite Likelihood method and phylogenetic trees were constructed using Mega10 software. (B). Diversity parameters of quasispecies in skin lesions. (C). Quasispecies diversity in virus isolates.
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