Substitutions near the hemagglutinin receptor-binding site determine the antigenic evolution of influenza A H3N2 viruses in U.S. swine

Swine influenza A virus is an endemic and economically important pathogen in pigs, with the potential to infect other host species. The hemagglutinin (HA) protein is the primary target of protective immune responses and the major component in swine influenza A vaccines. However, as a result of antigenic drift, vaccine strains must be regularly updated to reflect currently circulating strains. Characterizing the cross-reactivity between strains in pigs and seasonal influenza virus strains in humans is also important in assessing the relative risk of interspecies transmission of viruses from one host population to the other. Hemagglutination inhibition (HI) assay data for swine and human H3N2 viruses were used with antigenic cartography to quantify the antigenic differences among H3N2 viruses isolated from pigs in the United States from 1998 to 2013 and the relative cross-reactivity between these viruses and current human seasonal influenza A virus strains. Two primary antigenic clusters were found circulating in the pig population, but with enough diversity within and between the clusters to suggest updates in vaccine strains are needed. We identified single amino acid substitutions that are likely responsible for antigenic differences between the two primary antigenic clusters and between each antigenic cluster and outliers. The antigenic distance between current seasonal influenza virus H3 strains in humans and those endemic in swine suggests that population immunity may not prevent the introduction of human viruses into pigs, and possibly vice versa, reinforcing the need to monitor and prepare for potential incursions

Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion

H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the ‘Eastern’ G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages

The selection pressure on the neuraminidase gene of influenza viruses isolated in Ukraine from 2009 to 2015

A broad range of naturally occurring antigenic variants of the influenza virus is caused by its rapid evolutionary variability. The survival of viable influenza virus variants occurs through natural selection. The treatment of influenza infection with modern antiviral drugs – neuraminidase (NA) inhibitors – leads to the occurrence of mutations in the NA gene, which thereby result in the emergence of virus resistance to these drugs. The goal of this study was to determine the selection pressure on the NA protein of influenza viruses isolated in Ukraine from 2009 to 2015. The main method for assessing the selection pressure on proteins is to quantify the ratio of substitution rates at nonsynonymous (dN) and synonymous (dS) sites. With the help of this method, we showed that only a few codons in the NA gene were under positive selection resulting in mutations at the following sites: for influenza A viruses of the A(H1N1)pdm09 subtype – site 40, for viruses of the A(H3N2) subtype – sites 93 and 402, for Influenza B viruses of the B/Yamagata lineage – sites 74, 99, and 268, and for the viruses of the B/Victoria lineage – sites 358, 288, and 455. These sites are not associated with the NA active site, transmembrane domain, or the antigenic sites of this protein. We concluded that NA inhibitors are not a significant factor in the process of selection of the influenza viruses in Ukraine because the sites associated with the resistance of influenza viruses to NA inhibitors were not affected by positive selection. This finding could be explained by the limited use of NA inhibitors for the treatment of influenza infections in Ukraine. A broad range of naturally occurring antigenic variants of the influenza virus is caused by its rapid evolutionary variability. The survival of viable influenza virus variants occurs through natural selection. The treatment of influenza infection with modern antiviral drugs – neuraminidase (NA) inhibitors – leads to the occurrence of mutations in the NA gene, which thereby result in the emergence of virus resistance to these drugs. The goal of this study was to determine the selection pressure on the NA protein of influenza viruses isolated in Ukraine from 2009 to 2015. The main method for assessing the selection pressure on proteins is to quantify the ratio of substitution rates at nonsynonymous (dN) and synonymous (dS) sites. With the help of this method, we showed that only a few codons in the NA gene were under positive selection resulting in mutations at the following sites: for influenza A viruses of the A(H1N1)pdm09 subtype – site 40, for viruses of the A(H3N2) subtype – sites 93 and 402, for Influenza B viruses of the B/Yamagata lineage – sites 74, 99, and 268, and for the viruses of the B/Victoria lineage – sites 358, 288, and 455. These sites are not associated with the NA active site, transmembrane domain, or the antigenic sites of this protein. We concluded that NA inhibitors are not a significant factor in the process of selection of the influenza viruses in Ukraine because the sites associated with the resistance of influenza viruses to NA inhibitors were not affected by positive selection. This finding could be explained by the limited use of NA inhibitors for the treatment of influenza infections in Ukraine.

MISSEL: a method to identify a large number of small species-specific genomic subsequences and its application to viruses classification

Continuous improvements in next generation sequencing technologies led to ever-increasing collections of genomic sequences, which have not been easily characterized by biologists, and whose analysis requires huge computational effort. The classification of species emerged as one of the main applications of DNA analysis and has been addressed with several approaches, e.g., multiple alignments-, phylogenetic trees-, statistical- and character-based methods

Mapping of the Influenza-A Hemagglutinin Serotypes Evolution by the ISSCOR Method

Analyses and visualizations by the ISSCOR method of influenza virus hemagglutinin genes of different A-subtypes revealed some rather striking temporal relationships between groups of individual gene subsets. Based on these findings we consider application of the ISSCOR-PCA method for analyses of large sets of homologous genes to be a worthwhile addition to a toolbox of genomics - allowing for a rapid diagnostics of trends, and ultimately even aiding an early warning of newly emerging epidemiological threats.Comment: 26 pages with figures (Figs. 1-4 in the main text, and Figs. S1-S5 in supplementary materials