Molecular evolution of the hemagglutinin-neuraminidase (HN) gene in human respirovirus 3.

Human respirovirus 3 (HRV3) is a major causative agent of acute respiratory infections in humans. HRV3 can manifest as a recurrent infection, although exactly how is not known. In the present study, we conducted detailed molecular evolutionary analyses of the major antigen-coding hemagglutinin-neuraminidase (HN) gene of this virus detected/isolated in various countries. We performed analyses of time-scaled evolution, similarity, selective pressure, phylodynamics, and conformational epitope prediction by mapping to HN protein models. In this way, we estimated that a common ancestor of the HN gene of HRV3 and bovine respirovirus 3 diverged around 1815 and formed many lineages in the phylogenetic tree. The evolutionary rates of the HN gene were 1.1 × 10-3 substitutions/site/year, although the majority of these substitutions were synonymous. Some positive and many negative selection sites were predicted in the HN protein. Phylodynamic fluctuations of the gene were observed, and these were different in each lineage. Furthermore, most of the predicted B cell epitopes did not correspond to the neutralization-related mouse monoclonal antibody binding sites. The lack of a link between the conformational epitopes and neutralization sites may explain the naturally occurring HRV3 reinfection.

Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine.

Modeling the paramyxovirus hemagglutinin-neuraminidase protein.

The paramyxovirus hemagglutinin-neuraminidase (HN) protein exhibits neuraminidase activity and has an active site functionally similar to that in influenza neuraminidases. Earlier work identified conserved amino acids among HN sequences and proposed similarity between HN and influenza neuraminidase sequences. In this work we identify the three-dimensional fold and develop a more detailed model for the HN protein, in the process we examine a variety of protein structure prediction methods. We use the known structures of viral and bacterial neuraminidases as controls in testing the success of protein structure prediction and modeling methods, including knowledge-based threading, discrete three-dimensional environmental profiles, hidden Markov models, neural network secondary structure prediction, pattern matching, and hydropathy plots. The results from threading show that the HN protein sequence has a 6 beta-sheet propellor fold and enable us to assign the locations of the individual beta-strands. The three-dimensional environmental profile and hidden Markov model methods were not successful in this work. The model developed in this work helps to understand better the biological function of the HN protein and design inhibitors of the enzyme and serves as an assessment of some protein structure prediction methods, especially after the x-ray crystallographic solution of its structure.