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Immune escape mutants of highly pathogenic avian influenza H5N1 selected using polyclonal sera: Identification of key amino acids in the HA protein
Sitaras, I. ; Kalthof, D. ; Beer, M. ; Peeters, B.P.H. ; Jong, M.C.M. de - \ 2014
PLoS ONE 9 (2014)2. - ISSN 1932-6203 - 13 p.
virus hemagglutinin - antigenic drift - a viruses - monoclonal-antibodies - evolution - poultry - egypt - vaccination - molecule - neutralization
Evolution of Avian Influenza (AI) viruses – especially of the Highly Pathogenic Avian Influenza (HPAI) H5N1 subtype – is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks) is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1), using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA) protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.
Unraveling the Entry Mechanism of Baculoviruses and Its Evolutionary Implications
Wang, M. ; Wang, J. ; Yin, F. ; Tan, Y. ; Deng, F. ; Chen, X. ; Jehle, J.A. ; Vlak, J.M. ; Hu, Zhihong ; Wang, H. - \ 2014
Journal of Virology 88 (2014)4. - ISSN 0022-538X - p. 2301 - 2311.
envelope fusion protein - californica multicapsid nucleopolyhedrovirus - nuclear polyhedrosis-virus - mammalian-cells - f-proteins - gp64 - infectivity - insect - identification - neutralization
The entry of baculovirus budded virus into host cells is mediated by two distinct types of envelope fusion proteins (EFPs), GP64 and F protein. Phylogenetic analysis suggested that F proteins were ancestral baculovirus EFPs, whereas GP64 was acquired by progenitor group I alphabaculovirus more recently and may have stimulated the formation of the group I lineage. This study was designed to experimentally recapitulate a possible major step in the evolution of baculoviruses. We demonstrated that the infectivity of an F-null group II alphabaculovirus (Helicoverpa armigera nucleopolyhedrovirus [HearNPV]) can be functionally rescued by coinsertion of GP64 along with the nonfusogenic Fdef (furin site mutated HaF) from HearNPV. Interestingly, HearNPV enters cells by endocytosis and, less efficiently, by direct membrane fusion at low pH. However, this recombinant HearNPV coexpressing Fdef and GP64 mimicked group I virus not only in its EFP composition but also in its abilities to enter host cells via low-pH-triggered direct fusion pathway. Neutralization assays indicated that the nonfusogenic F proteins contribute mainly to binding to susceptible cells, while GP64 contributes to fusion. Coinsertion of GP64 with an F-like protein (Ac23) from group I virus led to efficient rescue of an F-null group II virus. In summary, these recombinant viruses and their entry modes are considered to resemble an evolutionary event of the acquisition of GP64 by an ancestral group I virus and subsequent adaptive inactivation of the original F protein. The study described here provides the first experimental evidence to support the hypothesis of the evolution of baculovirus EFPs.
Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of bovine viral diarrhea virus type 1 and 2
Jelsma, H. ; Loeffen, W.L.A. ; Beuningen, A.R. van; Rijn, P.A. van - \ 2013
Veterinary Microbiology 166 (2013)1-2. - ISSN 0378-1135 - p. 195 - 199.
swine-fever virus - random peptide library - hog-cholera virus - b-cell epitope - monoclonal-antibodies - strain brescia - vaccines - protein - identification - neutralization
Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed.
Passive immunization of guinea-pigs with llama single-domain antibody fragments against foot-and-mouth disease
Harmsen, M.M. ; Solt, C.B. van; Fijten, H.P.D. ; Keulen, L. van; Rosalia, R.A. ; Weerdmeester, K. ; Cornelissen, A.H.M. ; Bruin, M.G.M. de; Eble, P.L. ; Dekker, A. - \ 2007
Veterinary Microbiology 120 (2007)3-4. - ISSN 0378-1135 - p. 193 - 206.
4 antigenic sites - resistant mutants - escape mutants - virus - protection - sequence - neutralization - identification - immunoglobulin - involvement
Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody fragments (VHHs). Twenty-four VHHs binding serotype O FMDV in vitro were isolated from immunized llamas by phage display and expressed in bakers yeast for further characterization. They recognized four functionally independent antigenic sites. Six strongly FMDV neutralizing VHHs bound to a peptide representing the GH-loop of viral protein 1 known to be involved in binding to the cellular receptor of FMDV. Clone M8, recognizing this antigenic site, and clone M23, recognizing another antigenic site, showed synergistic in vitro virus neutralization. Three FMDV specific VHHs were PEGylated in order to decrease their rapid blood clearance and thus enable in vivo guinea pig protection experiments. Passive immunization with individual VHHs showed no protection, but a mixture of M8 and M23 showed partial transient protection. The protection afforded by these VHHs was however low as compared to the complete protection afforded by convalescent guinea pig serum. In contrast, these VHHs showed far more efficient in vitro FMDV neutralization than convalescent guinea pig serum. This lack of correlation between in vitro neutralization and in vivo protection lends further credence to the notion that opsonophagocytosis of FMDV is important for protection in vivo.
Significance of the oligosaccharides of the porcine reproductive and respiratory syndrome virus glycoproteins GP2a and GP5 for infectious virus production
Wissink, E.H.J. ; Kroese, M.V. ; Maneschijn-Bonsing, J.G. ; Meulenberg, J.J. ; Rijn, P.A. van; Rijsewijk, F.A.M. ; Rottier, P.J.M. - \ 2004
Journal of General Virology 85 (2004)12. - ISSN 0022-1317 - p. 3715 - 3723.
equine arteritis virus - dehydrogenase-elevating virus - n-linked glycans - lelystad-virus - influenza hemagglutinin - structural proteins - envelope proteins - neutralization - ectodomain - identification
The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains four glycoproteins, GP2a, GP3, GP4 and GP5, the functions of which are still largely unresolved. In this study, the significance of the N-glycosylation of the GP2a and GP5 proteins of PRRSV strain LV was investigated. Both glycoproteins contain two predicted N-glycosylation sites that are highly conserved between North American-type and European-type PRRSV. Using site-directed mutagenesis, single and double mutant full-length PRRSV cDNA clones were generated. After analysing the expression of the mutant proteins and the actual use of the four putative glycosylation sites in the wild-type proteins, the production of mutant virus particles and their infectivities were investigated. The results showed that the N-linked glycans normally present on the GP2a protein are not essential for particle formation, as is the oligosaccharide attached to N53 of the GP5 protein. In contrast, the oligosaccharide linked to N46 of the GP5 protein is strongly required for virus particle production. The specific infectivities of the mutant viruses were investigated by comparing their infectivity-per-particle ratios with that of wild-type virus. The results showed that the lack of either one or both of the N-linked oligosaccharides on GP2a or of the oligosaccharide attached to N53 of GP5 did not significantly affect the infectivities of the viruses. In contrast, the two recombinant viruses lacking the oligosaccharide bound to N46 exhibited a significantly reduced specific infectivity compared with the wild-type virus. The implications of the differential requirements of the modifications of GP2a and GP5 for PRRSV assembly and infectivity are discussed
Feldspar weathering as the key to understanding soil acidification monitoring data; a study of acid sandy soils in the Netherlands
Mol, G. ; Vriend, S.P. ; Gaans, P.F.M. van - \ 2003
Chemical Geology 202 (2003)39541. - ISSN 0009-2541 - p. 417 - 441.
atmospheric deposition - woodland soils - forest soil - compositional variation - geochemical record - aluminum - water - ecosystem - rates - neutralization
Monitoring activities pose special demands on the type of survey results needed. In the early 1990s a soil acidification monitoring methodology was adopted in the Netherlands that leaned heavily on methods developed in more fundamental research, most notably the use of proton budgets. Consequently, various controversies still not resolved in the scientific debate reflect on the current practice of soil acidity monitoring and complicate interpretation of the monitoring results. In a pilot study we address the most pressing issues: capacity versus intensity parameters, choice of monitoring objective, and natural variation in the compartment to be monitored. Focus is on the major source of buffering, the possible usefulness of the historic approach, and the regional patterns present in the sandy soils of the Netherlands. In a field campaign 92 locations in sandy regions all over the country were sampled at two depths. The solid phase, the displaced soil solution, and solid phase extractions with 0.01 M CaCl2 and 0.43 M HNO3, for the 184 samples were analyzed by a variety of methods. Aluminum release is the major source of buffering and is shown to contribute substantially to acid buffering already under natural conditions. The predominant Al bearing phases in Dutch sandy soils are feldspars and secondary Al minerals; feldspars are found to be the determinative phase in acid buffering. Application of the historic approach using the subsoil as a proxy for the initial composition of the topsoil proved feasible for this regional dataset. The average depletion of the ANC(s) of 230 mmolc kg¿1 in the topsoil matches well with estimates of the total proton load since the last ice age, with the anthropogenic contribution being between 20% and 50%. Fuzzy c-means cluster analyses of the solid phase and soil solution data show a distinct regionality that was also reflected in the parameters generally used to indicate the acidity status of soils, ¿ANC(s) and Al/BC ratios. A combined insight into both solid phase and soil solution, based on a comprehensive set of parameters, proves essential for interpreting soil acidity monitoring data.