ABSTRACT
Acute respiratory infection (ARI) is the most common infection in children under 5â¯years of age and it is frequently caused by two pneumoviruses, human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). Epidemic seasons of these viruses overlap and disease manifestations are highly similar, including severe lower ARI such as bronchiolitis or pneumonia. Reinfections with pneumoviruses are frequent and limited prevention treatment is available. Genetic diversity of HRSV and HMPV strains circulating in Croatia was monitored during four consecutive years (2014-2017). Co-circulation of multiple lineages was observed for both viruses. Within HRSV group A, ON1 strains gained strong predominance during the 4-year period, while previously dominant genotype NA1 was detected only sporadically. Similarly, newly occurring HMPV genotype A2c gained predominance over genotype A2b during this period, resulting in all infection in 2017 being caused by A2c. Along with phylogenetic analysis based on the commonly used fragments for detection and genotyping of these viruses, full length G and SH genes were also analysed. Evolutionary dynamics showed that inferred substitution rates of HRSV and HMPV are between 2.51â¯×â¯10-3 and 3.61â¯×â¯10-3 substitutions/site/year. This study established presence of recently described HMPV strains containing large duplications in the G gene in Croatia. Viruses with either of the two duplications belong to a subcluster A2c, which has completely replaced all other group A subclusters in 2017.
Subject(s)
Metapneumovirus/classification , Paramyxoviridae Infections/epidemiology , Respiratory Syncytial Virus Infections/epidemiology , Respiratory Syncytial Virus, Human/classification , Respiratory Tract Infections/virology , Child , Child, Preschool , Croatia/epidemiology , Drug Substitution , Evolution, Molecular , Female , Humans , Infant , Male , Metapneumovirus/genetics , Metapneumovirus/isolation & purification , Molecular Epidemiology , Phylogeny , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/isolation & purificationABSTRACT
Immunogenicity testing in animals is a necessary preclinical assay for demonstration of vaccine efficacy the results of which are often the basis for the decision whether to proceed or withdraw the further development of the novel vaccine candidate. However, in vivo assays are rarely, if at all, optimized and validated. Here we clearly demonstrate the importance of in vivo assay (mumps virus immunogenicity testing in guinea pigs) optimization for gaining reliable results and the suitability of Fractional factorial design of experiments (DoE) for such a purpose. By the use of DoE with resolution IV (2IV((4-1))) we clearly revealed that the parameters significantly increasing assay sensitivity were interval between animal immunizations followed by the body weight of experimental animals. The quantity (0 versus 2%) of the stabilizer (fetal bovine serum, FBS) in the sample was shown as non-influencing parameter in DoE setup. However, the separate experiment investigating only the FBS influence, and performed under other parameters optimally set, showed that FBS also influences the results of immunogenicity assay. Such finding indicated that (a) factors with strong influence on the measured outcome can hide the effects of parameters with modest/low influence and (b) the matrix of mumps virus samples to be compared for immunogenicity must be identical for reliable virus immunogenicity comparison. Finally the 3 mumps vaccine strains widely used for decades in the licensed vaccines were for the first time compared in an animal model, and results obtained were in line with their reported immunogenicity in human population supporting the predictive power of the optimized in vivo assay.
Subject(s)
Animal Experimentation/standards , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/standards , Mumps Vaccine/immunology , Mumps virus/immunology , Animals , Body Weight , Female , Guinea Pigs , Immunization Schedule , Mumps Vaccine/administration & dosage , Research Design , Treatment OutcomeABSTRACT
Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. Also it has been reported as a strong suppressor of interferon (IFN) expression. IFN-α has opposite effect on fibroblast function and IFN induction. Here is our early report on the effect of low concentration of PDGF-AB alone or in combination with IFN-α on IFN mRNA production in MRC5 fibroblasts. MRC5 cells incubated with IFN-α or PDGF-AB, alone or in combination, produced significant induction of IFN-α, -ß and -γ mRNA in comparison with untreated cells. The induction was dose-dependent, with higher effect in cells treated with lower concentrations of PDGF-AB. Also, low concentration of PDGF-AB showed synergism with IFN-α in IFN-ß and -γ induction. Results presented here open new possibilities in multi-cytokine therapy and expand previous results on PDGF activity.
Subject(s)
Fibroblasts/metabolism , Interferon-alpha/metabolism , Interferon-beta/genetics , Interferon-gamma/genetics , Platelet-Derived Growth Factor/pharmacology , Cell Line , Fibroblasts/drug effects , Gene Expression Regulation/drug effects , Humans , Interferon-alpha/genetics , Interferon-alpha/pharmacology , Interferon-beta/metabolism , Interferon-gamma/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Native human interferon-α (nHuIFN-α) has a stronger reductive effect on procollagen type I mRNA expression than recombinant human interferon-α (rHuIFN-α). It is partially due to the additive activity of interleukin-1ß (IL-1ß), which is present in small concentrations in nHuIFN-α. Here, we show that the reductive effect is also the result of the endogenous cytokines induced by the activity of nHuIFN-α. In the culture of MRC5 fibroblasts, we have further found that nHuIFN-α induces endogenous interferons in higher amounts than rHuIFN-α, measured with PCR. That is more pronounced when interferon-γ (IFN-γ) is measured. This result puts a new light on IFN-γ activity in the nHuIFN-α treatment because its role was neglected due to the loss of its activity during the nHuIFN-α preparation process. The findings lead to the conclusion that endogenous cytokines play a significant role in the nHuIFN-α -mediated reduction of procollagen type I mRNA and are therefore an important factor in potential therapeutic usage.
Subject(s)
Collagen Type I/biosynthesis , Cytokines/metabolism , Interferon-alpha/pharmacology , Interferons/biosynthesis , Procollagen/biosynthesis , Cycloheximide/pharmacology , Cytokines/biosynthesis , Gene Expression/drug effects , Humans , Interferon-alpha/antagonists & inhibitors , Interferons/drug effects , Interleukin-1beta/metabolism , Recombinant Proteins/pharmacologyABSTRACT
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.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , Mumps Vaccine/immunology , Mumps virus/immunology , Mumps/immunology , Animals , Epitopes/immunology , Genotype , Guinea Pigs/immunology , Humans , Mumps/prevention & control , Mumps/virology , PhylogenyABSTRACT
In order to assure the virological safety of blood products, in addition to serological testing of individual donations and virus inactivation steps undertaken during manufacture, routine PCR testing for HCV RNA of starting materials (plasma, cells), intermediates or final product is necessary. The aim of this study was to determine the rate of HCV RNA positive batches of human native leukocyte interferon during large-scale production. Our findings indicate the presence of HCV RNA in 6.1% batches despite acidification of intermediates in order to inactivate Sendai virus.
Subject(s)
Hepacivirus/isolation & purification , Interferon-alpha/chemistry , Leukocytes/chemistry , RNA, Viral/analysis , Base Sequence , Cells, Cultured , Drug Contamination , Genome, Viral , Hepacivirus/genetics , Humans , In Vitro Techniques , Interferon-alpha/isolation & purification , Molecular Sequence Data , Polymerase Chain Reaction , Reference Standards , Sequence Homology, Nucleic AcidABSTRACT
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$ 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.
Subject(s)
Hepacivirus/isolation & purification , Hepatitis C/epidemiology , RNA, Viral/blood , Viremia/epidemiology , Base Sequence , Croatia/epidemiology , Hepacivirus/classification , Hepacivirus/genetics , Hepatitis C/blood , Hepatitis C/prevention & control , Hepatitis C/transmission , Humans , Incidence , Mass Screening , Molecular Sequence Data , Polymerase Chain Reaction , Reference Standards , Risk , Safety , Sampling Studies , Viremia/bloodABSTRACT
Studied at the level of the individual cell, the pattern of [Ca2+]i mobilization of in vivo sensitized mouse lymphocytes by T-dependent antigen (KLH), challenged in vitro by Con A, PHA or anti-CD3epsilon mAb in different periods after immunization, was as follows. In the entire DLN lymphocyte population and in tested T cell subsets from immunized mice, baseline [Ca2+]i was significantly increased and cells were able to respond additionally to stimuli. In KLH-primed DLN lymphocytes, calcium mobilization in response to membrane receptor-dependent stimuli (anti-CD3epsilon, PHA, and ConA) was increased. Enhancement of Ca2+ mobilization is parallel with changed immunophenotype. These findings suggested that: (a) [Ca2+]i mobilization could correlate with lymphocyte behaviour during immunization and that mobilization clearly depended on kinetics of immune reaction; (b) the higher level of activity among sensitized lymphocytes was due to the increased number of specific B-cells (Ia(k+)) and gammadeltaTCR+ cells; (c) the quantitative measurement of [Ca2+]i could be an important biochemical parameter to study cellular reaction to a specific antigen.
Subject(s)
Calcium/metabolism , Animals , Female , Immunization , Immunophenotyping , Lymph Nodes/cytology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Lymphocyte Subsets/metabolism , Mice , Mice, Inbred CBA , PhenotypeABSTRACT
To assess the gamma delta TCR T cells in the control of the timing of the mucosal response to enteric parasitic infections, we used C57BL mice, orally infected with 200 viable T. spiralis larvae. The small intestine, spleens and Peyer's patches (PP) were excised on 1, 4, 7, 14, 21 and 29 postinfection days (p.i.) for immunophenotyping and histological studies. Uninfected mice served as control. Characterization of isolated lymphocytes of C57BL control mice, confirmed that T cell immunophenotype differs in spleen, PP and i-IEL. Practically all i-IEL were CD3+ cells (83%). In addition, most of the i-IEL expressed Ly-2 (65%). Among the i-IEL, the level of gamma delta TCR+ cells was significantly higher (29%) than that found in spleen (3%) and PP (3%). The expression was high on CD3+ and Ly-2+ (26 and 21%, respectively) and low on L3T4+ i-IEL (< 1%). During T. spiralis infection alpha beta TCR+ CD3+, gamma delta TCR+ CD3+ and gamma delta TCR+ Ly-2+ i-IEL increased on day 4 and 7. However, infected mice displayed a reduction in i-IEL number from 14 to 29 p.i. day. At the same time the proportion of gamma delta TCR on spleen Ly-2+ and on PP CD3+ and Ly-2+ cells increased on 14 and 21 p.i. day. Adult worms were expelled from the gut by day 14. Thus, the kinetics of gamma delta TCR+ i-IEL, but not spleen and PP gamma delta TCR, corresponded to the kinetics of worm expulsion in C57BL mice. Most murine i-IEL of the gamma delta T cell lineage tend to be cytolytic when activated. We speculated that gamma delta T cells of i-IEL during the early stages of infection recognize and eliminate damaged epithelial cells generated by parasite antigens, simultaneously accelerating the worm expulsion.