title: Influence of population diversity on neurovirulence potential of plaque purified L-Zagreb variants.
abstract: BACKGROUND
  Despite continuing research efforts, determinants of mumps virus virulence are still largely unknown. One of consequences of this is difficulty in striking a balance between efficacy and safety of live attenuated mumps vaccines. Among mumps vaccine strains associated with occurrence of postvaccinal aseptic meningitis is L-Zagreb, developed by further attenuation of vaccine strain L-3. Starting from an archived L-Zagreb sample with suboptimal neuroattenuation score, we isolated different viral variants and compared their genetic and phenotypic properties, in investigation of neurovirulence markers.
  
  
  METHODS
  Six different L-Zagreb variants were isolated by plaque purification. Their neurovirulent status was determined by rat-based neurovirulence test; population structure was determined by deep sequencing.
  
  
  RESULTS
  We isolated one well neuroattenuated viral variant, two marginally neuroattenuated, and three insufficiently neuroattenuated. No genetic markers of neurovirulence could be identified. None of variants had detectable amounts of defective interfering particles. Two characteristics set insufficiently neuroattenuated variants apart from less-neurovirulent ones: elevated variability level in regions 1293-3314, 5363-7773 and 9382-11657, and/or elevated number of mutations present in frequencies ≥ 1%. The most neurovirulent variants possessed both of these features.
  
  
  CONCLUSIONS
  Distinctive heterogeneity profiles were obtained for insufficiently neuroattenuated L-Zagreb variants. No markers that would discriminate between marginally and well neuroattenuated variants were identified. The findings of this study may serve as a guideline during development of an improved L3/L-Zagreb vaccine strain.
authors.affiliation: Centre for research and knowledge transfer in biotechnology, University of Zagreb, Rockefellerova 10, 10 000 Zagreb, Croatia; Center of Excellence for Viral Immunology and Vaccines, CERVirVac, Croatia. Electronic address: jivancic@unizg.hr.
authors.forename: Jelena
authors.initials: J
authors.lastname: Ivancic-Jelecki
authors.affiliation: Centre for research and knowledge transfer in biotechnology, University of Zagreb, Rockefellerova 10, 10 000 Zagreb, Croatia; Center of Excellence for Viral Immunology and Vaccines, CERVirVac, Croatia. Electronic address: dforcic@unizg.hr.
authors.forename: Dubravko
authors.initials: D
authors.lastname: Forcic
authors.affiliation: Centre for research and knowledge transfer in biotechnology, University of Zagreb, Rockefellerova 10, 10 000 Zagreb, Croatia; Center of Excellence for Viral Immunology and Vaccines, CERVirVac, Croatia. Electronic address: mmarkusi@unizg.hr.
authors.forename: Maja
authors.initials: M
authors.lastname: Jagusic
authors.affiliation: Centre for research and knowledge transfer in biotechnology, University of Zagreb, Rockefellerova 10, 10 000 Zagreb, Croatia; Center of Excellence for Viral Immunology and Vaccines, CERVirVac, Croatia. Electronic address: tkgulija@unizg.hr.
authors.forename: Tanja
authors.initials: T
authors.lastname: Kosutic-Gulija
authors.affiliation: Research and development department, Institute of Immunology Inc., Rockefellerova 10, 10 000 Zagreb, Croatia.
authors.forename: Renata
authors.initials: R
authors.lastname: Mazuran
authors.affiliation: Centre for research and knowledge transfer in biotechnology, University of Zagreb, Rockefellerova 10, 10 000 Zagreb, Croatia; Center of Excellence for Viral Immunology and Vaccines, CERVirVac, Croatia. Electronic address: maja.langbalija@gmail.com.
authors.forename: Maja Lang
authors.initials: ML
authors.lastname: Balija
authors.affiliation: Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Electronic address: doctoroots@gmail.com.
authors.forename: Ofer
authors.initials: O
authors.lastname: Isakov
authors.affiliation: Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Electronic address: nshomron@gmail.com.
authors.forename: Noam
authors.initials: N
authors.lastname: Shomron
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D003673:Defective Viruses / Q000472:pathogenicity; D009108:Mumps Vaccine; D009109:Mumps virus / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity*; D012367:RNA, Viral / Q000235:genetics; D051381:Rats; D011917:Rats, Inbred Lew; D017423:Sequence Analysis, RNA; D014613:Vaccines, Attenuated; D014709:Vero Cells; D010948:Viral Plaque Assay; D014774:Virulence
chemical_list: D009108:Mumps Vaccine; D012367:RNA, Viral; D014613:Vaccines, Attenuated
keywords: L-Zagreb; Mumps; Mumps vaccine; Rat-based neurovirulence test; Virus population diversity; Virus variants