[Genetic and phenotypic characterization of different measles virus strains and their interaction with the innate immune response]. (Doctoral thesis)
- Clinical and Applied Virology
The innate immunity is critical to control viral infections during the development of the adaptive immune response. Cytokines are key players in the early immune response to viral infections. Therefore phenotypic differences between measles virus (MV) strains, relating to cytokine induction, may influence virus spread and severity of disease.
We investigated how 22 different MV strains of 14 circulating genotypes interfere with the early immune response. Virus proliferation of vaccine and wild-type (wt) strains was compared, after IFN-alpha treatment. Wt production was 2 to 47-fold lower in IFN-treated cells, whereas vaccine production was reduced only 2 to 3- fold. Furthermore, we compared the cytokine induction of IFN-beta, RANTES, interleukin 5 (IL-5), IL-6, IL-8, IL-10, TNF-alpha and IL-1 beta and mRNA quantification of IFN-alpha/beta response genes (MxA, 2-5AS). While most of the wt strains induced 71-99% less cytokines than the Schwarz vaccine strain, three wt strains induced similar levels of cytokines. These three wt strains were positive for defective interfering RNAs (diRNA). DiRNA emerged only in virus cultures during multiple passaging and was not detectable in clinical samples of measles patients.
Previous studies showed that proteins encoded by the MV-P gene inhibit IFNalpha/ beta signaling. However, sequence analysis of those gene of all used strains, did not display specific amino acid mutations that correlated with the different phenotypes.
The present data show that MV wt strains differ in their sensitivity and their ability to temper with the innate immune response. In addition our proteomic analyses highlight variations in the cellular response induced by different wt strains. These phenotypic characteristics may result in differences in virulence.
Recently it was shown that macaques infected with two different wt strains (genotype C2 or B3) display variations in clinical parameters, MV replication and antibody responses. In our in vitro study the used C2 strain was one of the most sensitive wt strains, whereas the B3 strain was the least sensitive one to IFNalpha treatment. Since our in vitro findings correlated with the latter in vivo date we compared also the immune response in humans. Thus, we investigated the cytokine levels of IL-5, IL-6, IL-8 and IL-10 in sera collected from patients during MV outbreaks of genotype B3 in Nigeria and Spain and from a C2 outbreak in Luxembourg. In great contrast to B3 infected patients the cytokine response in C2 infected patients seems to be alleviated. Additionally during eight days after onset of rash cytokine levels decreased in C2 patients, whereas they elevated in B3 infected patients. Thus, our findings also force the hypothesis of differences in pathogenicity among various wt strains.
Since 1998 the WHO recommends sequencing the hypervariable region of the MV nucleoprotein (MV-NP HVR) for MV genotyping. Genotyping is an important tool of measles surveillance to document chains of transmission, discriminate between imported or indigenous viruses and monitor elimination programs.
However, with the enhanced vaccination control the genetic variability of circulating strains continues to decrease and identical MV-NP HVR sequences have been found for several years.
Analyzing the variability of the MV-P and H genes, we showed for four different outbreaks in Europe and Africa that phylogenetic analysis of the MV-P/H-pseudogene sequences provides a more refined picture of MV circulation. Identical MVNP HVR sequences found in Belarus and Germany in 2006, may have suggested that strains belong to the same outbreak. However, the MV-P/H pseudo-gene sequences clearly identified both cases as part of two distinct outbreaks. For strains collected throughout Russia 2003 to 2007 the MV-P/H pseudo-gene provides more insights into the time course of strains, indicating rather the circulation and importation of independent variants, than a single major outbreak lasting for several years, like suggested by identical MV-NP HVR sequences. Also in the DR-Congo our findings suggested an independent evolution of variants and multiple independent importations into the country.
By extending the sequencing window recommended by the WHO for molecular epidemiology of MV, links between outbreaks and transmission chains became more clearly defined.