Mumps virus neurovirulence assessment-impact of viral doses, animal sex and age on results dispersion.

Mumps epidemics highlight the necessity for new live mumps vaccines. For vaccine candidate neurosafety assessment, the most commonly used test is the rat-based neurovirulence test (RNVT). Its main experimental and ethical deficiency is the results dispersion which requires large number of animals to obtain statistically relevant results.

Measles cases in Split-Dalmatia County (a Croatian tourist region), in May-July 2019: outbreak report and lessons learnt.

Background: Measles elimination was accomplished in Croatia in 2016. Split-Dalmatia County, with population of ca. 425 000 inhabitants, is among the most important Croatian tourist areas with numerous seasonal workers coming during summer months.

Is Better Standardization of Therapeutic Antibody Quality in Emerging Diseases Epidemics Possible?

During the ongoing COVID-19 epidemic many efforts have gone into the investigation of the SARS-CoV-2-specific antibodies as possible therapeutics. Currently, conclusions cannot be drawn due to the lack of standardization in antibody assessments. Here we describe an approach of establishing antibody characterisation in emergent times which would, if followed, enable comparison of results from different studies.

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.

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.

Measles outbreak in Dubrovnik-Neretva County, Croatia, May to June 2018.

In May 2018, measles was introduced in the Dubrovnik region by an adult who recently travelled to Kosovo*. Control measures and an outbreak investigation were implemented: 15 epidemiologically-linked cases met the outbreak case definition of a visitor/resident of Dubrovnik-Neretva County with laboratory-confirmed measles and symptom onset beginning on May 19. New cases were identified through hospitals and primary care physicians.

Variability analysis and inter-genotype comparison of human respiratory syncytial virus small hydrophobic gene.

Background: Small hydrophobic (SH) gene is one of the mostly diverse genomic regions of human respiratory syncytial virus (HRSV). Its coding region constitutes less than 50% of the complete gene length, enabling SH gene to be highly variable and the SH protein highly conserved. In standard HRSV molecular epidemiology studies, solely sequences of the second hypervariable region of the glycoprotein gene (HVR2) are analyzed.

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).

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

Background: The 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.

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.

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.

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).

The first genetic characterization of a D4 measles virus strain derived from a patient with subacute sclerosing panencephalitis.

Measles virus (MV) strains derived from patients with subacute sclerosing panencephalitis (SSPE), SSPE strains, possess numerous mutations when compared to viruses belonging to the same genotype and circulating in similar time period. Although many SSPE strains have been extensively characterized, none of them belongs to D4 genotype which currently predominates in Europe where it has caused a number of recent outbreaks/epidemics. We sequenced an MV derived from a patient with long-term SSPE; the virus was named MVs/Zagreb.

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.

Comparative analysis of CE-SSCP to standard RFLP-CE-FLA method in quantification of known viral variants within an RNA virus quasispecies.

RNA viruses display the highest replication error rate in our biosphere, leading to highly diverse viral populations termed quasispecies. The gold standard method for detection and quantification of variants in a quasispecies is cloning and sequencing, but it is expensive, laborious and time consuming. Therefore, other mutation detection approaches, including SSCP, are often used.

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.

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.

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.

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.

Purification of plant viral and satellite double-stranded RNAs on DEAE monoliths.

Replicative double-stranded RNA (dsRNA) is useful in preliminary identification of Cucumber mosaic virus and its satellite RNA (satRNA). This plant pathogen complex yields sufficient quantity of the replicative RNA form that can be isolated by chromatography on chemically unmodified graded cellulose powder (CF-11). In this work, much faster and more efficient procedure using DEAE monoliths was developed in which dsRNA was separated from other species in total nucleic acids extract originating from the infected plant tissue.

Restriction enzyme cleavage of fluorescently labeled DNA fragments--analysis of the method and its usage in examination of digestion completeness.

Restriction enzymes have proven to be among the most valuable tools in molecular biology. In this work, we demonstrate that the cleavage of fluorescently labeled, PCR-amplified DNA can be used as a simple and highly sensitive technique for detection of sequences present in a percentage as low as 0.6% in a DNA pool.