Pseudotype reporter viruses are being used as safe, quantitative, and high-throughput tools for assessing antibody neutralization for many viruses, including influenza. However, characterization of pseudotypes containing influenza hemagglutinin (HA-pseudotypes) is needed before this system is widely adopted for evaluating neutralizing antibodies in sera following vaccination or infection. In this report HA-pseudotype stocks were analyzed for HA content, stability, and performance in neutralization assays under various conditions. HA-pseudotypes produced with HA genes of H5 strains representing clades 1, 2.2, and 2.3.4 consistently contain similar HA contents, and infectivity was not greatly affected by the purity of the HA-pseudotype preparations or variations in storage conditions. HA-pseudotype neutralization titers using a reference serum panel were also consistent across a wide range of dilutions of HA-pseudotype stocks and correlated well with results from microneutralization assays involving replicating influenza. Concentrated HA-pseudotypes were further shown to work well in hemagglutination inhibition assays. Finally, antisera elicited by genetically modified HA, with changes in the polybasic cleavage site that have been used in some H5 vaccines and reduce pathogenicity, gave identical neutralization titers against HA-pseudotypes with wild type or modified HA. These findings support continued development of HA-pseudotypes as a robust tool for analyzing sera in vaccine and serologic studies.
"The advantages of using pseudotyped viruses as alternatives to handling live virus have been utilised by many laboratories for over two decades. They have primarily been used as gene delivery or gene therapy vectors (Naldini et al., 1996; Zufferey et al., 1997) but more recently as antigen substitutes in PNAs evaluating antiviral drug potency (Aljofan et al., 2009; Parry et al., 2009; Su et al., 2008) and serum VNAb titres (Temperton et al., 2005, 2007; Wright et al., 2008), as vaccine immunogens (Szecsi et al., 2006, 2009; Yang et al., 2007) or target antigens in antibody binding assays (Wang et al., 2010). Also, PNAs have been shown to aid the identification of more potent and broader cross-neutralising MAbs than has been possible using other Fig. 1 "
[Show abstract][Hide abstract] ABSTRACT: It is likely that phylogroup 2 lyssaviruses circulate within bat reservoirs. We adapted a pseudotype (pt) neutralisation assay (PNA) to a multiplex format enabling serosurveillance for Lagos bat virus (LBV), Mokola virus (MOKV) and West Caucasian bat virus (WCBV) in a potential reservoir, the African straw-coloured fruit bat, Eidolon helvum. Highly correlated titres were observed between single and multiplex PNAs using ptLBV and ptMOKV (r=0.97, p<0.0001), validating its use for bat serosurveillance. Of the bat serum samples screened 56% neutralised ptLBV, 27% ptMOKV and 1% ptWCBV. Mean VNAb titres were 1:266, 1:35 and 1:7 against ptLBV, ptMOKV and ptWCBV respectively. The high seroprevalence estimates suggest that the infection rate of LBV in E. helvum remains high enough to persist in this species. This supports the hypothesis that LBV is endemic in Ghanaian E. helvum and we speculate that LBV may have co-evolved with African megachiroptera.
[Show abstract][Hide abstract] ABSTRACT: The 2009 H1N1 pandemic highlights the need to better understand influenza A infectivity and antigenicity. Relative to other recent seasonal H1N1 influenza strains, the 2009 H1N1 virus grew less efficiently in eggs, which hindered efforts to rapidly supply vaccine. Using lentiviral pseudotypes bearing influenza hemagglutinin (HA-pseudotypes) we evaluated a glutamine to arginine mutation at position 223 (Q223R) and glycosylation at residue 276 in HA for their effects on infectivity and neutralization. Q223R emerged during propagation in eggs and lies in the receptor binding site. We found that the Q223R mutation greatly enhanced infectivity of HA-pseudotypes in human cells, which was further augmented by inclusion of the viral neuraminidase (NA) and M2 proteins. Loss of glycosylation at residue 276 did not alter infectivity. None of these modifications affected neutralization. These findings provide information for increasing 2009 H1N1HA-pseudotype titers without altering antigenicity and offer insights into receptor use.
[Show abstract][Hide abstract] ABSTRACT: Pseudotyped viral particles are being used as safe surrogates to mimic the structure and surface of many viruses, including highly pathogenic viruses such as avian influenza H5N1, to investigate biological functions mediated by the envelope proteins derived from these viruses. The first part of this article evaluates and discusses the differences in the production and characterization of influenza pseudoparticles. The second part focuses on the applications that such a flexible tool can provide in modern influenza research, in particular in the fields of drug discovery, molecular biology and diagnosis.
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