A recent resurgence of mumps in doubly vaccinated cohorts has been observed, identifying genotype G as the current predominant genotype. In this study, the neutralization efficacy of guinea pig sera ...immunized with three vaccine viruses: L-Zagreb, Urabe AM9 and JL5, was tested against seven mumps viruses: three vaccine strains and four wild-type strains (two of genotype G, one of genotype C, one of genotype D) isolated during 1998–2011. All sera neutralized all viruses although at different levels. The neutralization efficiency of sera decreases several fold by temporal order of virus isolation. Therefore, we concluded that gradual evolution of mumps viruses, rather than belonging to a certain genotype, results in an antigenic divergence from the vaccine strains that decrease the neutralization capacity of vaccine-induced antibodies. Moreover, the amino-acid sequence alignment revealed three new potentially relevant regions for escape from neutralization, i.e. 113–130, 375–403 and 440–443.
To confirm the genetic stability of the Edmonston–Zagreb vaccine strain, we determined and compared the nucleotide sequences of genuine Edmonston–Zagreb master seed (EZ D22) and current working seed ...lot (EZ D24 2/99). Sequence analysis and comparison of the two sequences confirmed that these two sequences are the same at the molecular level. The obtained sequences were also compared to reference strains, i.e. Edmonston wild-type (Edmonston Wt) AF266288 and Edmonston–Zagreb (EZ) AF266290 vaccine strain. The sequence of EZ D22 differed from the Edmonston Wt in 32 nucleotides. EZ D22 differed from EZ AF266290 in six nucleotides. Coding substitution at position 441 and two silent substitutions at positions 11999 and 14612 in the
L gene are unique to EZ D22. The differences found between EZ from different sources can be a good reason for periodical sequence analysis of the same strain in the hands of different manufactures.
Eleven mumps vaccine strains, all containing live attenuated virus, have been used throughout the world. Although L-Zagreb mumps vaccine has been licensed since 1972, only its partial nucleotide ...sequence was previously determined (accession numbers
AJ272363,
AY623666 and
AY583323). Therefore, we sequenced the entire genome of L-Zagreb vaccine strain (Institute of Immunology Inc., Zagreb, Croatia). In order to investigate the genetic stability of the vaccine, sequences of both L-Zagreb master seed and currently produced vaccine batch were determined and no difference between them was observed.
A phylogenetic analysis based on SH gene sequence has shown that L-Zagreb strain does not belong to any of established mumps genotypes and that it is most similar to old, laboratory preserved European strains (1950s–1970s).
L-Zagreb nucleotide and deduced protein sequences were compared with other mumps virus sequences obtained from the GenBank. Emphasis was put on functionally important protein regions and known antigenic epitopes. The extensive comparisons of nucleotide and deduced protein sequences between L-Zagreb vaccine strain and other previously determined mumps virus sequences have shown that while the functional regions of HN, V, and L proteins are well conserved among various mumps strains, there can be a substantial amino acid difference in antigenic epitopes of all proteins and in functional regions of F protein. No molecular pattern was identified that can be used as a distinction marker between virulent and attenuated strains.
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 2718 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 2718 anti-HCV negative plasma pools we have found that 2.1% 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.
The aim of this study was the molecular characterization of a historical mumps isolate (an alleged individual sample). After RNA extraction and cDNA synthesis, selective nested PCR amplification with ...specific primers, automated DNA sequencing and RFLP analyses were performed. The relative ratios of the detected virus sequences were determined by GeneScan electrophoresis. Phylogenetic tree based on the 316 nucleotide region of the SH gene of the mumps virus was generated by the neighbor-joining method.
Results obtained by the described molecular approach show: (a) there are two mumps virus variants, A and B, detected in the fourth passage of wild type virus in the amniotic cavity of embryonated chicken eggs (ECE); (b) variants A and B belong to different genotypes; (c) variants A and B differ in the HN and NP genes which code for amino acid sequences comprising immunogenic epitopes; (d) variant B contains one or more minor variants.
We discuss whether the observed differences between the two variants are a consequence of natural heterogeneity or of laboratory contamination in the early passages.
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.
The viral safety of plasma-derived products with respect to hepatitis C virus (HCV) is assured by selection of donors, screening of individual donations for antibodies to HCV and the incorporation of ...effective viral inactivation-removal steps into manufacturing processes. As antibody screening of single donations is not sufficient to completely eliminate HCV RNA positive plasmas from plasma pools, testing for HCV RNA by gene amplification techniques may be necessary to identify positive donations. Using modern molecular biology techniques, we developed a specific, sensitive and reproducible method for routine PCR screening for HCV RNA in plasma pools.
Anion-exchange chromatography is one of the most important methods in downstream processing of plasmid DNA, both as a process and as an analytical technique. Separation of plasmid DNA on traditional ...particle-based anion-exchange supports is usually slow. Moreover, such supports have a low capacity for plasmid DNA due to the steric exclusion effects. In this work, the separation of plasmid DNA using short monolithic columns, Convective Interaction Media, will be presented. It will be demonstrated that plasmid DNA can be purified from bacterial cells using alkaline lysis followed by chromatography on a very short weak anion-exchange chromatographic columns—disks—with good purity and quality within a short time. Furthermore, the separation of plasmid DNA from cell RNA can be carried out without the need of adding RNAse. Fast and efficient method for
in-
process control of the purified plasmid will be described as well.
A residual risk of HCV infection by different blood products exists due to blood donations collected during the serological window period in the early stages of infection. The aim of this study is ...nucleic acid amplification technique (NAT)-based screening of the anti-HCV negative plasma pools obtained from various Croatian transfusion centres between 2001 and 2003 for HCV RNA. During this period 2647 anti-HCV negative plasma pools were tested by NAT and 12 (0.45%) HCV RNA positive pools were detected. In comparison to the results of our previous study Forčić D, Zgorelec R, Branović K, Košutić-Gulija T, Šantak M, Mažuran R. Incidence of hepatitis C virus RNA in anti-HCV negative plasma pools in Croatia. Transfus Apher Sci 2001;24:269–78, a remarkable decrease in the number of positive plasma pools (from 2.1% to 0.45%) was demonstrated.
