Population Variability Generated during Rescue Process and Passaging of Recombinant Mumps Viruses

Recombinant mumps viruses (MuVs) based on established vaccine strains represent attractive vector candidates as they have known track records for high efficacy and the viral genome does not integrate in the host cells. We developed a rescue system based on the consensus sequence of the L- Zagreb vaccine and generated seven different recombinant MuVs by (a) insertion of one or two additional transcription units (ATUs), (b) lengthening of a noncoding region to the extent that the longest noncoding region in MuV genome is created, or (c) replacement of original L-Zagreb sequences with sequences rich in CG and AT dinucleotides. All viruses were successfully rescued and faithfully matched sequences of input plasmids. In primary rescued stocks, low percentages of heterogeneous positions were found (maximum 0.12%) and substitutions were predominantly obtained in minor variants, with maximally four substitutions seen in consensus. ATUs did not accumulate more mutations than the natural MuV genes. Six substitutions characteristic for recombinant viruses generated in our system were defined, as they repetitively occurred during rescue processes. In subsequent passaging of primary rescue stocks in Vero cells, different inconsistencies within quasispecies structures were observed. In order to assure that unwanted mutations did not emerge and accumulate, sub-consensus variability should be closely monitored. As we show for Pro408Leu mutation in L gene and a stop codon in one of ATUs, positively selected variants can rise to frequencies over 85% in only few passages

Targeted attenuation of mumps virus by viral factors that modulate the innate immune response

Cilj ovog istraživanja bio je karakterizirati indukciju mehanizama urođene imunosti na viruse mumpsa različitog stupnja atenuacije. Kao virusni faktori koji mogu modulirati te mehanizme ispitane su defektne intereferirajuće čestice prisutne u virusnoj suspenziji te ekspresija virusnog proteina V. Proizašla saznanja primijenjena su za ciljanu atenuaciju divljeg soja virusa mumpsa kao modela za razvoj novog atenuiranog cjepiva.The aim of this study was to characterize the innate immune responses to mumps viruses of different attenuation level. Defective interfering particles present in the viral suspensions and expression of the viral V protein were examined as viral factors critical for modulation of these responses. The gained knowledge was further applied for targeted attenuation of a wild-type mumps virus as a model for development of a new attenuated vaccine

Targeted attenuation of mumps virus by viral factors that modulate the innate immune response

Cilj ovog istraživanja bio je karakterizirati indukciju mehanizama urođene imunosti na viruse mumpsa različitog stupnja atenuacije. Kao virusni faktori koji mogu modulirati te mehanizme ispitane su defektne intereferirajuće čestice prisutne u virusnoj suspenziji te ekspresija virusnog proteina V. Proizašla saznanja primijenjena su za ciljanu atenuaciju divljeg soja virusa mumpsa kao modela za razvoj novog atenuiranog cjepiva.The aim of this study was to characterize the innate immune responses to mumps viruses of different attenuation level. Defective interfering particles present in the viral suspensions and expression of the viral V protein were examined as viral factors critical for modulation of these responses. The gained knowledge was further applied for targeted attenuation of a wild-type mumps virus as a model for development of a new attenuated vaccine

Genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in Croatia

Human metapneumovirus (HMPV) is recognized as a global and frequent cause of acute respiratory tract infections among people of all ages. The objectives of this study were molecular epidemiology and evolutionary analysis of HMPV strains which produced moderate and severe acute respiratory tract infections in children in Croatia during four consecutive seasons (2011-2014). A total of 117 HMPV-positive samples collected from hospitalized pediatric patients presenting with acute respiratory tract infections and tested by direct immunofluorescence assay were first analyzed by amplifying a part of the F gene. Sixteen samples were further analyzed based on complete F, G, and SH gene sequences. HMPV genome was identified in 92 of 117 samples (78%) and the circulation of multiple lineages of HMPV was confirmed. In 2011, 2012, and 2014, subgroups A2 and B2 co-circulated, while B1 gained prevalence in 2013 and 2014. The study established the presence of a novel subcluster A2c in Croatia. This subcluster has only recently been detected in East and Southeast Asia. This study provides new insights into epidemiology and genetic diversity of HMPV in this part of Europe

Influence of Ribavirin on Mumps Virus Population Diversity

Frequent mumps outbreaks in vaccinated populations and the occurrence of neurological complications (e.g., aseptic meningitis or encephalitis) in patients with mumps indicate the need for the development of more efficient vaccines as well as specific antiviral therapies. RNA viruses are genetically highly heterogeneous populations that exist on the edge of an error threshold, such that additional increases in mutational burden can lead to extinction of the virus population. Deliberate modulation of their natural mutation rate is being exploited as an antiviral strategy and a possibility for rational vaccine design. The aim of this study was to examine the ability of ribavirin, a broad-spectrum antiviral agent, to introduce mutations in the mumps virus (MuV) genome and to investigate if resistance develops during long-term in vitro exposure to ribavirin. An increase in MuV population heterogeneity in the presence of ribavirin has been observed after one passage in cell culture, as well as a bias toward C-to-U and G-to-A transitions, which have previously been defined as ribavirin-related. At higher ribavirin concentration, MuV loses its infectivity during serial passaging and does not recover. At low ribavirin concentration, serial passaging leads to a more significant increase in population diversity and a stronger bias towards ribavirin-related transitions, independently of viral strain or cell culture. In these conditions, the virus retains its initial growth capacity, without development of resistance at a whole-virus population level

Additional file 1: Table S1. of Identification of mumps virus protein and lipid composition by mass spectrometry

Identification of protein bands shown in Figs. 1 & 2. Table S2. List of peptides found in spectra corresponding to NP bands shown in Fig. 1 and possible matching NP and P peptides. Table S3. List of peptides found in spectra corresponding to NP bands shown in Fig. 2 and possible matching NP and P peptides. Figure S1. Peptide mass fingerprint by means of positive ion MALDI reflectron MS of NP forms (depicted in Fig. 2b, reduced sample). Green (bottom) spectrum – NP apparent molecular weight 48 kDa; blue (middle) spectrum – NP apparent molecular weight 55 kDa; red (top) spectrum – NP apparent molecular weight 61 kDa. Full peptide list is given in Table S3. Figure S2. Western blot of mumps virus samples; L-Zg/Vero, JL/Vero, JL/CEF (from left to right) subjected to electrophoresis under reducing denaturating conditions detected by anti-mumps serum. (PDF 612 kb