An examination of the nucleic acid sequence alignment of 48 full-length rubella virus genomes revealed that the 5′ terminus of the genome is more conserved than the commonly used detection windows ...for rubella virus RNA located in the E1 protein coding region, suggesting that the 5′ terminus could be a target for improving detection of all rubella virus genotypes. Two candidate primer sets were tested and the window between nucleotides (nts) 98 and 251 was found to have the greatest analytical sensitivity for detection of different genotypes. The new method had a limit of detection of four copies of rubella RNA per reaction with high specificity. The average coefficient variation of Ct was 2.2%. Concordance between the new method and currently used method, based on testing 251 clinical specimens collected from a rubella outbreak, was 99.4%. The assay was further improved upon by the incorporation of detection of both rubella virus RNA and mRNA from a cellular reference gene in a multiplex format. The multiplex format did not reduce the sensitivity or the reproducibility of rubella RNA detection and, of 60 specimens tested, the concordance between the single target and multiplex assays was 85.0%. To assess the utility of the multiplex assay for molecular surveillance, 62 rubella IgM positive serum samples from a rubella outbreak were tested, and eleven tested positive using the multiplex method while none were positive using the method targeting E1. These results show that the assay based on the new detection window near the 5′ terminus of the genome can improve the detection of rubella virus for the purpose of molecular surveillance and case confirmation, with the added benefit of improved efficiency due to multiplexing.
•A new and improved RT-qPCR method targeting the conserved region in the rubella virus genome can detect as few as 3–5 copies of RNA of circulating genotypes in the clinical specimens.•The method allows multiplex detection of rubella and RNase P RNA without losing sensitivity on the detection of rubella virus RNA.•This method can be used to identify cases of rubella virus persistent infection.
Rubella viruses (RV) have been found in an association with granulomas in children with primary immune deficiencies (PID). Here, we report the recovery and characterization of infectious ...immunodeficiency-related vaccine-derived rubella viruses (iVDRV) from diagnostic skin biopsies of four patients. Sequence evolution within PID hosts was studied by comparison of the complete genomic sequences of the iVDRVs with the genome of the vaccine virus RA27/3. The degree of divergence of each iVDRV correlated with the duration of persistence indicating continuous intrahost evolution. The evolution rates for synonymous and nonsynonymous substitutions were estimated to be 5.7 x 10-3 subs/site/year and 8.9 x 10-4 subs/site/year, respectively. Mutational spectra and signatures indicated a major role for APOBEC cytidine deaminases and a secondary role for ADAR adenosine deaminases in generating diversity of iVDRVs. The distributions of mutations across the genes and 3D hotspots for amino acid substitutions in the E1 glycoprotein identified regions that may be under positive selective pressure. Quasispecies diversity was higher in granulomas than in recovered infectious iVDRVs. Growth properties of iVDRVs were assessed in WI-38 fibroblast cultures. None of the iVDRV isolates showed complete reversion to wild type phenotype but the replicative and persistence characteristics of iVDRVs were different from those of the RA27/3 vaccine strain, making predictions of iVDRV transmissibility and teratogenicity difficult. However, detection of iVDRV RNA in nasopharyngeal specimen and poor neutralization of some iVDRV strains by sera from vaccinated persons suggests possible public health risks associated with iVDRV carriers. Detection of IgM antibody to RV in sera of two out of three patients may be a marker of virus persistence, potentially useful for identifying patients with iVDRV before development of lesions. Studies of the evolutionary dynamics of iVDRV during persistence will contribute to development of infection control strategies and antiviral therapies.
Cardiovascular abnormalities are the leading cause of neonatal death among patients with congenital rubella syndrome (CRS). Although persistence of rubella virus (RV) in fetal endothelium has been ...repeatedly suggested as a possible cause of cardiovascular birth defects, evidence of the permissiveness of fetal endothelial cells to RV is lacking. In this study we evaluated the ability of RV to infect and persist in primary fetal endothelial cells derived from human umbilical vein (HUVEC). We found that wild type (wt) low passage clinical RV productively infected HUVEC cultures without producing cytopathology or ultrastructural changes. RV did not inhibit host cell protein synthesis, cell proliferation, or interfere with the cell cycle. Persistently infected cultures were easily established at low and high multiplicities of infection (MOI) with both laboratory and wt clinical RV strains. However, synchronous infections of entire HUVEC monolayers were only observed with clinical RV strains. The release of infectious virions into media remained at consistently high levels for several subcultures of infected HUVEC. The results indicate that macrovascular fetal endothelial cells are highly permissive to RV and allow slow persistent RV replication. The findings provide more evidence for the suggestion that vascular pathologies in CRS are triggered by persistent rubella virus infection of the endothelium.
...current data have clearly shown an association between defects in T-cell immunity, granulomas, and RV. A role for vaccine virus in Fuchs' uveitis is also suspected.7,8 Granulomas have been ...reported only once in CRS.9 We hypothesize that the immune deficiency allows persistence of the attenuated virus, polarization of macrophages to M2 occurs, compromising viral clearance, mutations accrue, and complete viral escape occurs upon acquisition of CD8 epitope mutations.\nFin/22.15/PID Epitope Epitope sequence in vaccine virus Substitutions in patients' virus Neutralizing B-cell epitopes E1214-233low * QQSRWGLGSPNCHGPDWASP None E1245-251 LVGATPE None E1260-266 ADDPLLR None E1274-285 VWVTPVIGSQAR I280arrow rightT CD8+ T-cell epitopes C9-22 MEDLQKALETQSRA T18arrow rightA C11-29 DLQKALETQSRALRAELAA T18arrow rightA C264-272 RIETRSARH T267arrow rightI Table E4 Changes in the antigenic structure of RV antigens of RVs/Oulu.FIN/22.15/PID relative to RA27/3 vaccine Antigen Type Vendor Catalog no.
Both wild type (WT) and vaccine rubella virus (RV) can pass through the placenta to infect a human fetus, but only wtRV routinely causes pathology. To investigate possible reasons for this, we ...compared establishment of persistence of wtRV and RA27/3 vaccine strains in fetal endothelial cells. We showed that yields of RA27/3 and wtRV were similar after the first round of replication, but then only vaccine-infected cultures went through a crisis characterized by partial cell loss and gradual decline of virus titer followed by recovery and establishment of persistent cultures with low levels of RA27/3 secretion. We compared various steps of virus replication, but we were unable to identify changes, which might explain the 2-log difference in RA27/3 and wtRV yields in persistently infected cultures. Whole genome sequencing did not reveal selection of virus variants in either the wtRV or RA27/3 cultures. Quantitative single-cell analysis of RV replication by in situ hybridization detected, on average, 1-4 copies of negative-strand RNA and ~50 copies of positive-strand genomic RNA in cells infected with both vaccine and WT viruses. The distinct characteristics of RA27/3 replication were the presence of large amounts of negative-strand RV RNA and RV dsRNA at the beginning of the crisis and the accumulation of high amounts of genomic RNA in a subpopulation of infected cells during crisis and persistence. These results suggest that RA27/3 can persist in fetal endothelial cells, but the characteristics of persistence and mechanisms for the establishment and maintenance of persistence are different from wtRV.
A young man with X-linked severe combined immunodeficiency developed a persistent vaccine-derived rubella virus (VDRV) infection, with the emergence of cutaneous granulomas more than fifteen years ...after receipt of two doses of measles-mumps-rubella (MMR) vaccine. Following nasopharyngeal swab (NP) collection, VDRV was detected by real-time polymerase chain reaction (RT-qPCR) and sequencing, and live, replication-competent VDRV was isolated in cell culture. To assess duration and intensity of viral shedding, sequential respiratory samples, one cerebrospinal fluid sample, and two urine samples were collected over 15 months, and VDRV RNA was detected in all samples by RT-qPCR. Live VDRV was cultured from nine of the eleven respiratory specimens and from one urine specimen. To our knowledge, this was the first reported instance of VDRV cultured from respiratory specimens or from urine. To assess potential transmission to close contacts, NP specimens and sera were collected from all household contacts, all of whom were immunocompetent and previously vaccinated with MMR. VDRV RNA was not detected in any NP swabs from the contacts, nor did serologic investigations suggest VDRV transmission to any contacts. This report highlights the need to understand the prevalence and duration of VDRV shedding in granuloma patients and to estimate the risk of VDRV transmission to immune and non-immune contacts.
We report two patients with DNA repair disorders (Artemis deficiency, Ataxia telangiectasia) with destructive skin granulomas, presumably triggered by live-attenuated rubella vaccinations. Both ...patients showed reduced naïve T cells. Rapid resolution of skin lesions was observed following hematopoietic stem cell transplantation. However, the patient with AT died due to complications of severe hepatic veno-occlusive disease 6 month after HSCT. Dried blood spots obtained after birth were available from this patient and showed absent T-cell receptor excision circles (TRECs). Therefore, newborn screening may help to prevent patients with moderate T-cell deficiency from receiving live-attenuated rubella vaccine potentially causing granulomas.
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.
Measles and rubella are vaccine-preventable viral diseases and can be prevented by safe, highly effective vaccination with measles- and rubella-containing vaccines. Given the myriad causes of febrile ...exanthems, laboratory surveillance for both measles and rubella is important to document the incidence of these diseases and to track the progress and maintenance of elimination in near- and post-elimination settings. Diagnostic challenges can hinder effective surveillance and classification challenges can hinder efforts to demonstrate achievement or maintenance of elimination. In this report, we review diagnostic and classification challenges for measles and rubella in near- and post-elimination settings.
An estimated 100,000 cases of congenital rubella syndrome (CRS) occur worldwide each year. The reported mortality rate for infants with CRS is up to 33%. The cellular mechanisms responsible for the ...multiple congenital defects in CRS are presently unknown. Here we identify cell types positive for rubella virus (RV) in CRS infants.
Cells and organs involved in RV replication were identified in paraffin-embedded autopsy tissues from three fatal case-patients by histopathologic examination and immunohistochemical (IHC) staining using a rabbit polyclonal RV antibody. Normal rabbit antisera and RV antisera preabsorbed with highly purified RV served as negative controls.
RV antigen was found in interstitial fibroblasts in the heart, adventitial fibroblasts of large blood vessels, alveolar macrophages, progenitor cells of the outer granular layer of the brain, and in capillary endothelium and basal plate in the placenta. The antibody specificity was verified by IHC staining of multiple tissue sections from other infectious disease cases. RV infection of each cell type is consistent with abnormalities which have been identified in patients with CRS, in the heart, large blood vessels, and brain. Antigen distribution was consistent with inflammatory response to vascular injury and systemic spread of RV.
The identification of RV positive cell types in CRS is important to better understand the pathology and pathogenesis of CRS.
•Autopsy tissues from three fatal CRS cases were analyzed immunohistochemically.•Rigorous negative controls were used to verify the specificity of immunostaining.•Rubella antigen was detected in cardiac fibroblasts, alveolar macrophages, neuronal progenitor cells and endothelial cells.•Rubella antigen localization was consistent with CRS pathology.Wild type rubella virus (RV) transferred from mothers with rubella to their unborn children results in 100,000infants/year worldwide born with birth defects collectively known as congenital rubella syndrome (CRS). Indeed, RV is the most teratogenic infectious agent known. Identifying cell types involved in RV infection of infants with CRS was possible using tissues from three Romanian infants, who unfortunately died, and using a very specific antiserum to RV. The cell types involved in RV infection which were identified were consistent with the pathology of CRS. Additional studies further enumerating the cell types involved in CRS are needed.
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