Tanja Košutić-Gulija

Application of short monolithic columns for improved detection of viruses.

Monolithic chromatography media represent a novel generation of stationary phases introduced in the last 10-15 years providing a chromatography matrix with enhanced mass transfer and hydrodynamic properties. These features allow for an efficient and fast separation of especially large biomolecules like e.g., DNA and viruses. In this study, the enrichment of virus RNA on short monolithic columns prior to molecular detection of viruses is described. Measles and mumps viruses were chosen as model viruses. The results show that it is possible to bind viral RNA on monoliths and concentrate viral nucleic acids from a fairly dilute sample. Consequently, a potential application of short monolithic columns is the concentration of virus RNA to improve the sensitivity and selectivity of viral detection with the possibility of isolating viral RNA from cell-free biological fluids.

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.

Genetic heterogeneity of L-Zagreb mumps virus vaccine strain

BackgroundThe most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous.ResultsWe identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo.ConclusionL-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.

Incidence of hepatitis C virus RNA in anti-HCV negative plasma pools in Croatia

The risks of transmitting viral infection by blood and products derived from plasma have long been known and still remain an area of concern. Blood banks and transfusion centres are faced with the imminent introduction of nucleic acid amplification testing (NAT) of plasma pools as used by the plasma industry. In this paper, we show a part of our results of a validation study of an in-house method for routine polymerase chain reaction (PCR) screening for hepatitis C virus (HCV) RNA in plasma pools and the results of testing 2,718 anti-HCV negative plasma pools for the presence of HCV RNA. The European Committee for Proprietary Medical Products (CPMP) recommended that from 1 July 1999, only batches derived from plasma pools tested and found non-reactive for HCV RNA, using validated test methods of suitable sensitivity and specificity, should be batch released by authorities. The quality and efficiency of NAT detection of HCV RNA is among others influenced by the efficacy of RNA isolation, the primer selection and the use of control samples. Using modern molecular biology techniques (sensitive and specific in-house amplification methods for detection of HCV RNA and automated sequencing), we analysed samples of plasma pools from different Croatian transfusion centres. By detection of HCV RNA in an NIBSC working reagent (genotype 3) and a Pelispy HCV RNA run control (genotype 1) we determined a high reproducibility and sensitivity (below 100 International Units (IU)/ml) for our in-house method. By direct sequencing PCR cDNAs we proved the specificity of the test system and the possibility of determining the HCV genotype when the method was used for PCR screening of HCV RNA in single donations. Of 2,718 anti-HCV negative plasma pools we have found that 2.1

Genetic analysis of human parainfluenza virus type 3 obtained in Croatia, 2011–2015

were HCV RNA positive. Results of our investigation confirm the necessity of testing HCV RNA in plasma pools to further increase the safety of human plasma-derived drugs.

A study of genetic variability of human parainfluenza virus type 1 in Croatia, 2011-2014.

Purpose. This study investigated the HPIV3 circulating strains in Croatia and whether the other parts of HPIV3 genome (F gene and HN 582 nucleotides fragment) could be equally suitable for genetic and phylogenetic analysis. Methodology. Clinical materials were collected in period 2011‐2015 from children suffering from respiratory illnesses. In positive HPIV3 samples viral genome was partially amplified and sequenced for HN and F genes. Obtained sequences were analysed by phylogenetic analysis and genetic characterization was performed. Results. All samples from this study belonged to subcluster C and over a short period of time, genetic lineage C3a gained prevalence over the other C genetic lineages, from 39 % in 2011 to more than 90 % in 2013 and 2014. Phylogenetic classifications of HPIV3 based on the entire HN gene, HN 582 nt fragment and entire fusion (F) gene showed identical classification results for Croatian strains and the reference strains. Molecular analysis of the F and HN glycoproteins, showed their similar nucleotide diversity (Fcds P=0.0244 and HNcds P=0.0231) and similar Ka/Ks ratios (F Ka/Ks=0.0553 and HN Ka/Ks=0.0428). Potential N‐glycosylation sites, cysteine residues and antigenic sites are generally strongly conserved in HPIV3 glycoproteins from both our and the reference samples. Conclusion. The HPIV3 subclaster C3 (genetic lineage C3a) became the most detected circulating HPIV3 strain in Croatia. The results indicated that the HN 582 nt and the entire F gene sequences were as good for phylogenetic analysis as the entire HN gene sequence.

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

Molecular epidemiology of human parainfluenza viruses type 1 (HPIV1) was investigated. Samples were collected from patients hospitalized in Croatia during the three consecutive epidemic seasons (2011-2014). Results indicated co-circulation of two major genetic clusters of HPIV1. Samples from the current study refer to clades II and III in a phylogenetic tree of haemagglutinin-neuraminidase (HN) gene. Additional phylogenetic trees of fusion (F) and phosphoprotein (P) genes confirmed the topology. Analysis of nucleotide diversity of entire P, F and HN genes demonstrated similar values: 0.0255, 0.0236 and 0.0237, respectively. However, amino acid diversity showed F protein to be the most conserved, while P protein was the most tolerant to mutations. Potential N- and O-glycosylation sites suggested that HPIV1 HN protein is abundantly glycosylated, and a specific N-glycosylation pattern could distinguish between clades II and III. Analysis of potential O-glycosylation sites in F protein indicated that samples from this study have two potential O-glycosylation sites, while publicly available sequences have five potential sites. This study provides data on the molecular characterization and epidemic pattern of HPIV1 in Croatia.

Induction of IFN-α Subtypes and Their Antiviral Activity in Mumps Virus Infection

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.

Genetic Variability and Sequence Relatedness of Matrix Protein in Viruses of the Families Paramyxoviridae and Pneumoviridae

Human type I interferons (IFNs) comprise one IFN-β, -ω, -κ, and -ɛ and 12 different IFN-α subtypes, which play an important role in early host antiviral response. Despite their high structural homology and signaling through the same receptor, IFN-α subtypes exhibit different antiviral, antiproliferative, and immunomodulatory activities. Differences in the production of IFN-α subtypes therefore determine the quality of an antiviral response. In this study, we investigated the pattern of IFN-α subtypes induced in infection with different mumps virus (MuV) strains and examined the MuV sensitivity to the action of IFN-α subtypes. We found that all IFN-α subtypes are being expressed in response to MuV infection with a highly similar IFN-α subtype pattern between the virus strains. We assessed an antiviral activity of several IFN-α subtypes: IFN-α1, IFN-α2, IFN-α4, IFN-α6, IFN-α8, IFN-α14, IFN-α17, and IFN-α21. Although they were all effective in suppressing MuV replication, the intensity and pattern of their action varied between MuV strains. Our results indicate that the overall IFN antiviral activity as well as the activity of specific IFN-α subtypes against MuV depend on a virus strain.

Mumps virus strains isolated in Croatia in 1998 and 2005: Genotyping and putative antigenic relatedness to vaccine strains

Background: The families Paramyxoviridae and Pneumoviridae comprise a broad spectrum of viral pathogens that affect human health. The matrix (M) protein of these viruses has a central role in their life cycle. In line with this, molecular characteristics of the M proteins from variable viruses that circulated in Croatia were investigated. Methods: Sequences of the M proteins of human parainfluenza virus (HPIV) 1–3 within the family Paramyxoviridae, human metapneumovirus (HMPV), and human respiratory syncytial virus from the family Pneumoviridae were obtained and analyzed. Results: M proteins were very diverse among HPIVs, but highly conserved within each virus. More variability was seen in nucleotide sequences of M proteins from the Pneumoviridae family. An insertion of 8 nucleotides in the 3′ untranslated region in 1 HMPV M gene sequence was discovered (HR347-12). As there are no samples with such an insertion in the database, this insertion is of interest and requires further research. Conclusion: While we have confirmed that M proteins were conserved among individual viruses, any changes that are observed should be given attention and further researched. Of special interest is inclusion of HPIV2 M proteins in this analysis, as these proteins have not been studied to the same extent as other paramyxoviruses.