Jelena Ivancic-Jelecki

Concentration and purification of rubella virus using monolithic chromatographic support.

The production of economically acceptable viral vaccines of high quality requires simple and efficient methods for purification and concentration of viral particles. Ion-exchange chromatography (IEC) has become one of commonly used methods for large-scale downstream purification of viruses. Viruses possess different biological and/or biochemical properties and therefore IEC conditions must be established specifically for each virus. Live attenuated rubella virus vaccines have been manufactured and successfully used widely to protect people from rubella and congenital rubella syndrome for almost 40 years. The aim of this study was to search for an efficient method for concentration and purification of rubella virus using IEC. The selected operating conditions using quaternary amine monolithic supports enabled highly efficient binding, purification and concentration of rubella virus from complex biological suspension without additional procedures. Eluted viral particles maintained their infectivity and viral recovery was almost 100%. At the same time, viral preparation was successfully depleted from host cell protein and DNA. This work indicates the possibility of using monoliths to improve the rubella virus yields in productions where high virus titers during cultivation can hardly be achieved.

Comparisons of mumps virus potency estimates obtained by 50% cell culture infective dose assay and plaque assay

The two most commonly used methods for the determination of a virus potency are plaque assay and 50% cell culture infective dose (CCID(50)) assay, both based on cytopathic effect observation. We compared the potency estimates obtained by plaque and CCID(50) assays for nine mumps virus strains that produce different cytopathic effects in Vero cells. The ratios of CCID(50) and plaque assay quantification results differed for different strains and were in a range of 0.66-10, indicating that quantification results for some mumps virus strains are almost identical regardless of whether CCID(50) or plaque method is used, while the potency estimates of other strains strongly depend on the choice of the assay.

A Study of the Genetic Variability of Human Respiratory Syncytial Virus in Croatia, 2006–2008

Human respiratory syncytial virus (HRSV) is a common etiological agent of acute lower respiratory tract disease in infants. The molecular epidemiology of HRSV in Croatia over four consecutive seasons (from 2006 to 2008) was investigated. A total of 72 HRSV samples were chosen from 696 screened cases in a pediatric clinic in Zagreb. Molecular characterization of HRSV revealed the predominance of HRSV group B viruses in the first two epidemic seasons and HRSV group A viruses in the next two seasons. According to the phylogenetic analysis, NA1 and BA9 were the predominant circulating HRSV genotypes detected during the study. Overall, 82.9% of all HRSV A strains belonged to the NA1 genotype. The HRSV B genotype BA9, detected in two consecutive seasons (2006 and 2007), was the predominant circulating HRSV B genotype, accounting for 80.6% of all HRSV B strains. This study provides data on the circulation pattern of HRSV genotypes in Croatia and their molecular characterization. J. Med. Virol. 84:1985–1992, 2012.

Variability of hemagglutinin-neuraminidase and nucleocapsid protein of vaccine and wild-type mumps virus strains.

The mumps virus (MuV) molecular evolution is characterized by the co-circulation of numerous distinct strains. Standardized phylogenetic analyses based on the nucleotide sequences of the SH gene are important for mumps surveillance, but lack the information regarding antigenic properties. So far, the location of antigenic epitopes has been determined for two MuV proteins, the hemagglutinin-neuraminidase (HN) and the nucleocapsid (N) protein. We performed multiple sequence comparisons of putative HN and N protein sequences in order to describe their diversity and plasticity, and to determine the level of similarity between vaccine and wild-type strains. The results of full-length HN or N protein phylogeny showed that MuV strains form a number of differing clades which are in concordance with grouping obtained by standard MuV genotyping. When vaccine strains are compared to all wild-type strains, the highest mean percentage of amino acid differences in both HN and N protein analysis was found for Jeryl Lynn 5 and Jeryl Lynn 2 strains while the lowest value was obtained for Leningrad-3 and L-Zagreb strains. When only 3 antigenic regions of the HN protein, comprising 45 amino acids in total, were investigated, the diversity is considerably diminished: 51.5% of all putative HN proteins show identical sequences (including those of vaccine strains L-Zagreb, Leningrad-3, Hoshino and Urabe). Another 26.5% proteins (including Miyahara vaccine strain) differ in only one amino acid, while the others differ in two to five amino acids from the most common sequence. Jeryl Lynn 2 and Jeryl Lynn 5 strains differ in four amino acids each. N protein antigenic sites have been mapped within its hypervariable C-terminus. Our results indicate that there might be genotype-specific amino acids residing in this antigenic region. The results of our study present the background information for investigations of MuV heterogeneity and antigenic diversity.

A molecular epidemiological study of human respiratory syncytial virus in Croatia, 2011-2014

Human respiratory syncytial virus (HRSV) causes common respiratory tract infections in infants, young children and the elderly. The diversity of HRSV strains circulating in Croatia was investigated throughout a period of four consecutive years from March 2011-March 2014. The analysis was based on sequences from the second hypervariable region of the G gene. A predominance of HRSV group A was observed in the first three years of the study, while group B became slightly predominant during the first few months of 2014. Overall, 76% of viruses belonged to group A including the genotypes NA1, ON1 and GA5. NA1 was by far the most common genotype within group A in 2011-2013; however, only ON1 and a few GA5 viruses were detected in the first three months of 2014. The majority of group B strains were of genotype BA9 (97%), and a few BA10 genotypes were detected. BA9 had the highest substitution rate of all the detected genotypes, followed by ON1. Multiple analyses showed that HRSV group A strains were more diverse than group B strains. Gly at residue 232 (previously described to be specific for ON1) was also detected in three NA1 strains, which were phylogenetically placed on separate branches within the NA1 genotype. For all genotypes, the diversity was higher at the amino acid level than at the nucleotide level, although positive selection of mutations was shown for only a few sites using four different methods of codon-based analysis of selective pressure. More codons were predicted to be negatively selected. The complexity of the HRSV pools present during each epidemic peak was determined and compared to previous epidemiological data. In addition to presenting genetic versatility of HRSV in this geographic region, the collected sequences provide data for further geographical and temporal comparative analyses of HRSV and its evolutionary pathways.

Isolation of cell-free DNA from plasma by chromatography on short monolithic columns and quantification of non-apoptotic fragments by real-time polymerase chain reaction.

Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.

Early Evolution of Human Respiratory Syncytial Virus ON1 Strains: Analysis of the Diversity in the C-Terminal Hypervariable Region of Glycoprotein Gene within the First 3.5 Years since Their Detection.

Objective: Characterization of the phylogeny and diversity of human respiratory syncytial virus (HRSV) genotype ON1 that occurred during its early evolution (within the first 3.5 years since the detection of the first ON1 strains). ON1 strains have a 72-nucleotide-long in-frame duplication within the second hypervariable domain of the glycoprotein gene (HVR2). Methods: All available HVR2 sequences of strains belonging to the ON1 genotype published prior to June 20, 2014 were collected. Multiple sequence alignments, phylogeny, phylogeography, sequence clustering and putative protein analyses were performed. Results: The worldwide spread and diversification of ON1 strains are presented. Only in a minority of ON1 strains do the two replicas remain identical, and various ON1 strains possess common differences between the first and the second copy (segments A and B). Mutations of the progenitor sequence were more frequent in segment B, a higher overall diversity on the protein level and more putative glycosylation sites exist in segment B, and, unlike in segment A, positive selection acts on that protein region. Conclusions: The fast spread of the novel HRSV genotype ON1 has been accompanied by its rapid concurrent diversification. Differences in variability of the two replicas within HVR2 were detected, with C-terminal replica being more variable.

Recovery of infective virus particles in ion-exchange and hydrophobic interaction monolith chromatography is influenced by particle charge and total-to-infective particle ratio.

Viral particles are used in medical applications as vaccines or gene therapy vectors. In order to obtain product of high purity, potency and safety for medical use purification of virus particles is a prerequisite, and chromatography is gaining increased attention to meet this aim. Here, we report on the use of ion-exchange and hydrophobic interaction chromatography on monolithic columns for purification of mumps virus (MuV) and measles virus (MeV). Efficiency of the process was monitored by quantification of infective virus particles (by 50% cell culture infective dose assay) and total virus particles, and monitoring of their size (by Nanoparticle Tracking Analysis). Ion-exchange chromatography was shown to be inefficient for MuV and best results for MeV were obtained on QA column with recovery around 17%. Purification of MuV and MeV by hydrophobic interaction chromatography resulted in recoveries around 60%. Results showed that columns with small channels (d=1.4μm) are not suitable for MuV and MeV, although their size is below 400nm, whereas columns with large channels (6μm) showed to be efficient and recoveries independent on the flow rate up to 10mL/min. Heterogeneity of the virus suspension and its interday variability mostly regarding total-to-infective particle ratio was observed. Interestingly, a trend in recovery depending on the day of the harvest was also observed for both viruses, and it correlated with the total-to-infective particle ratio, indicating influence of the virus sample composition on the chromatography results.

Common position of indels that cause deviations from canonical genome organization in different measles virus strains

BackgroundThe canonical genome organization of measles virus (MV) is characterized by total size of 15 894 nucleotides (nts) and defined length of every genomic region, both coding and non-coding. Only rarely have reports of strains possessing non-canonical genomic properties (possessing indels, with or without the change of total genome length) been published. The observed mutations are mutually compensatory in a sense that the total genome length remains polyhexameric. Although programmed and highly precise pseudo-templated nucleotide additions during transcription are inherent to polymerases of all viruses belonging to family Paramyxoviridae, a similar mechanism that would serve to non-randomly correct genome length, if an indel has occurred during replication, has so far not been described in the context of a complete virus genome.MethodsWe compiled all complete MV genomic sequences (64 in total) available in open access sequence databases. Multiple sequence comparisons and phylogenetic analyses were performed with the aim of exploring whether non-recombinant and non-evolutionary linked measles strains that show deviations from canonical genome organization possess a common genetic characteristic.ResultsIn 11 MV sequences we detected deviations from canonical genome organization due to short indels located within homopolymeric stretches or next to them. In nine out of 11 identified non-canonical MV sequences, a common feature was observed: one mutation, either an insertion or a deletion, was located in a 28 nts long region in F gene 5′ untranslated region (positions 5051–5078 in genomic cDNA of canonical strains). This segment is composed of five tandemly linked homopolymeric stretches, its consensus sequence is G6-7C7-8A6-7G1-3C5-6. Although none of the mononucleotide repeats within this segment has fixed length, the total number of nts in canonical strains is always 28. These nine non-canonical strains, as well as the tenth (not mutated in 5051–5078 segment), can be grouped in three clusters, based on their passage histories/epidemiological data/genetic similarities. There are no indications that the 3 clusters are evolutionary linked, other than the fact that they all belong to clade D.ConclusionsA common narrow genomic region was found to be mutated in different, non-related, wild type strains suggesting that this region might have a function in non-random genome length corrections occurring during MV replication.

Influence of population diversity on neurovirulence potential of plaque purified L- Zagreb variants

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.