Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe hemorrhagic disease in terrestrial poultry and are a threat to the poultry industry, wild life, and human health. HPAIVs arise from low pathogenic avian influenza viruses (LPAIVs), which circulate in wild aquatic birds. HPAIV emergence is thought to occur in poultry and not wild aquatic birds, but the reason for this species-restriction is not known. We hypothesized that, due to species-specific tropism and replication, intrahost HPAIV selection is favored in poultry and disfavored in wild aquatic birds. We tested this hypothesis by co-inoculating chickens, representative of poultry, and ducks, representative of wild aquatic birds, with a mixture of H7N7 HPAIV and LPAIV, mimicking HPAIV emergence in an experimental setting. Virus selection was monitored in swabs and tissues by RT-qPCR and immunostaining of differential N-terminal epitope tags that were added to the hemagglutinin protein. HPAIV was selected in four of six co-inoculated chickens, whereas LPAIV remained the major population in co-inoculated ducks on the long-term, despite detection of infectious HPAIV in tissues at early time points. Collectively, our data support the hypothesis that HPAIVs are more likely to be selected at the intrahost level in poultry than in wild aquatic birds and point towards species-specific differences in HPAIV and LPAIV tropism and replication levels as possible explanations.

Continued adaptation of A/H2N2 viruses during pandemic circulation in humans.

Influenza A viruses of the H2N2 subtype sparked a pandemic in 1957 and circulated in humans until 1968. Because A/H2N2 viruses still circulate in wild birds worldwide and human population immunity is low, the transmissibility of six avian A/H2N2 viruses was investigated in the ferret model. None of the avian A/H2N2 viruses was transmitted between ferrets, suggesting that their pandemic risk may be low. The transmissibility, receptor binding preference and haemagglutinin (HA) stability of human A/H2N2 viruses were also investigated. Human A/H2N2 viruses from 1957 and 1958 bound to human-type α2,6-linked sialic acid receptors, but the 1958 virus had a more stable HA, indicating adaptation to replication and spread in the new host. This increased stability was caused by a previously unknown stability substitution G205S in the 1958 H2N2 HA, which became fixed in A/H2N2 viruses after 1958. Although individual substitutions were identified that affected the HA receptor binding and stability properties, they were not found to have a substantial effect on transmissibility of A/H2N2 viruses via the air in the ferret model. Our data demonstrate that A/H2N2 viruses continued to adapt during the first year of pandemic circulation in humans, similar to what was previously shown for the A/H1N1pdm09 virus.

Substitutions near the HA receptor binding site explain the origin and major antigenic change of the B/Victoria and B/Yamagata lineages.

Influenza B virus primarily infects humans, causing seasonal epidemics globally. Two antigenic variants-Victoria-like and Yamagata-like-were detected in the 1980s, of which the molecular basis of emergence is still incompletely understood. Here, the antigenic properties of a unique collection of historical virus isolates, sampled from 1962 to 2000 and passaged exclusively in mammalian cells to preserve antigenic properties, were determined with the hemagglutination inhibition assay and an antigenic map was built to quantify and visualize the divergence of the lineages. The antigenic map revealed only three distinct antigenic clusters-Early, Victoria, and Yamagata-with relatively little antigenic diversity in each cluster until 2000. Viruses with Victoria-like antigenic properties emerged around 1972 and diversified subsequently into two genetic lineages. Viruses with Yamagata-like antigenic properties evolved from one lineage and became clearly antigenically distinct from the Victoria-like viruses around 1988. Recombinant mutant viruses were tested to show that insertions and deletions (indels), as observed frequently in influenza B virus hemagglutinin, had little effect on antigenic properties. In contrast, amino-acid substitutions at positions 148, 149, 150, and 203, adjacent to the hemagglutinin receptor binding site, determined the main antigenic differences between the Early, Victoria-like, and Yamagata-like viruses. Surprisingly, substitutions at two of the four positions reverted in recent viruses of the Victoria lineage, resulting in antigenic properties similar to viruses circulating ∼50 y earlier. These data shed light on the antigenic diversification of influenza viruses and suggest there may be limits to the antigenic evolution of influenza B virus.

Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct.

The emergence and rapid spread of SARS-CoV-2 variants may affect vaccine efficacy substantially. The Omicron variant termed BA.2, which differs substantially from BA.1 based on genetic sequence, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed. Here, we used antigenic cartography to quantify and visualize antigenic differences between early SARS-CoV-2 variants (614G, Alpha, Beta, Gamma, Zeta, Delta, and Mu) using hamster antisera obtained after primary infection. We first verified that the choice of the cell line for the neutralization assay did not affect the topology of the map substantially. Antigenic maps generated using pseudo-typed SARS-CoV-2 on the widely used VeroE6 cell line and the human airway cell line Calu-3 generated similar maps. Maps made using authentic SARS-CoV-2 on Calu-3 cells also closely resembled those generated with pseudo-typed viruses. The antigenic maps revealed a central cluster of SARS-CoV-2 variants, which grouped on the basis of mutual spike mutations. Whereas these early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape vaccine-induced antibody responses as a result of different antigenic characteristics. Thus, antigenic cartography could be used to assess antigenic properties of future SARS-CoV-2 variants of concern that emerge and to decide on the composition of novel spike-based (booster) vaccines.

Contribution of Neuraminidase to the Efficacy of Seasonal Split Influenza Vaccines in the Ferret Model.

Seasonal influenza vaccination takes into account primarily hemagglutinin (HA)-specific neutralizing antibody responses. However, the accumulation of substitutions in the antigenic regions of HA (i.e., antigenic drift) occasionally results in a mismatch between the vaccine and circulating strains. To prevent poor vaccine performance, we investigated whether an antigenically matched neuraminidase (NA) may compensate for reduced vaccine efficacy due to a mismatched HA. Ferrets were vaccinated twice with adjuvanted split inactivated influenza vaccines containing homologous HA and NA (vacH3N2), only homologous HA (vacH3N1), only homologous NA (vacH1N2), heterologous HA and NA (vacH1N1), or phosphate-buffered saline (vacPBS), followed by challenge with H3N2 virus (A/Netherlands/16190/1968). Ferrets vaccinated with homologous HA (vacH3N2 and vacH3N1) displayed minimum fever and weight loss compared to vacH1N1 and vacPBS ferrets, while ferrets vaccinated with NA-matched vacH1N2 displayed intermediate fever and weight loss. Vaccination with vacH1N2 further led to a reduction in virus shedding from the nose and undetectable virus titers in the lower respiratory tract, similarly to when the homologous vacH3N2 was used. Some protection was observed upon vacH1N1 vaccination, but this was not comparable to that observed for vacH1N2, again highlighting the important role of NA in vaccine-induced protection. These results illustrate that NA antibodies can prevent severe disease caused by influenza virus infection and that an antigenically matched NA in seasonal vaccines might prevent lower respiratory tract complications. This underlines the importance of considering NA during the yearly vaccine strain selection process, which may be particularly beneficial in seasons when the HA component of the vaccine is mismatched. IMPORTANCE Despite the availability of vaccines, influenza virus infections continue to cause substantial morbidity and mortality in humans. Currently available influenza vaccines take primarily the hemagglutinin (HA) into account, but the highly variable nature of this protein as a result of antigenic drift has led to a recurrent decline in vaccine effectiveness. While the protective effect of neuraminidase (NA) antibodies has been highlighted by several studies, there are no requirements with regard to quantity or quality of NA in licensed vaccines, and NA immunity remains largely unexploited. Since antigenic changes in HA and NA are thought to occur asynchronously, NA immunity could compensate for reduced vaccine efficacy when drift in HA occurs. By matching and mismatching the HA and NA components of monovalent split inactivated vaccines, we demonstrated the potential of NA immunity to protect against disease, virus replication in the lower respiratory tract, and virus shedding in the ferret model.

Hemagglutinin Traits Determine Transmission of Avian A/H10N7 Influenza Virus between Mammals.

In 2014, an outbreak of avian A/H10N7 influenza virus occurred among seals along North-European coastal waters, significantly impacting seal populations. Here, we examine the cross-species transmission and mammalian adaptation of this influenza A virus, revealing changes in the hemagglutinin surface protein that increase stability and receptor binding. The seal A/H10N7 virus was aerosol or respiratory droplet transmissible between ferrets. Compared with avian H10 hemagglutinin, seal H10 hemagglutinin showed stronger binding to the human-type sialic acid receptor, with preferential binding to α2,6-linked sialic acids on long extended branches. In X-ray structures, changes in the 220-loop of the receptor-binding pocket caused similar interactions with human receptor as seen for pandemic strains. Two substitutions made seal H10 hemagglutinin more stable than avian H10 hemagglutinin and similar to human hemagglutinin. Consequently, identification of avian-origin influenza viruses across mammals appears critical to detect influenza A viruses posing a major threat to humans and other mammals.

Packaging of the Influenza Virus Genome Is Governed by a Plastic Network of RNA- and Nucleoprotein-Mediated Interactions.

The genome of influenza A virus is organized into eight ribonucleoproteins, each composed of a distinct RNA segment bound by the viral polymerase and oligomeric viral nucleoprotein. Packaging sequences unique to each RNA segment together with specific nucleoprotein amino acids are thought to ensure the precise incorporation of these eight ribonucleoproteins into single virus particles, and yet the underlying interaction network remains largely unexplored. We show here that the genome packaging mechanism of an H7N7 subtype influenza A virus widely tolerates the mutation of individual packaging sequences in three different RNA segments. However, combinations of these modified RNA segments cause distinct genome packaging defects, marked by the absence of specific RNA segment subsets from the viral particles. Furthermore, we find that combining a single mutated packaging sequence with sets of specific nucleoprotein amino acid substitutions greatly impairs the viral genome packaging process. Along with previous reports, our data propose that influenza A virus uses a redundant and plastic network of RNA-RNA and potentially RNA-nucleoprotein interactions to coordinately incorporate its segmented genome into virions.IMPORTANCE The genome of influenza A virus is organized into eight viral ribonucleoproteins (vRNPs); this provides evolutionary advantages but complicates genome packaging. Although it has been shown that RNA packaging sequences and specific amino acids in the viral nucleoprotein (NP), both components of each vRNP, ensure selective packaging of one copy of each vRNP per virus particle, the required RNA-RNA and RNA-NP interactions remain largely elusive. We identified that the genome packaging mechanism tolerates the mutation of certain individual RNA packaging sequences, while their combined mutation provokes distinct genome packaging defects. Moreover, we found that seven specific amino acid substitutions in NP impair the function of RNA packaging sequences and that this defect is partially restored by another NP amino acid change. Collectively, our data indicate that packaging of the influenza A virus genome is controlled by a redundant and plastic network of RNA/protein interactions, which may facilitate natural reassortment processes.

Sequence diversity of the Rh blood group system in Basques.

Basques show specific cultural, demographic, and genetic characteristics that have placed them as an isolated and unique population within Europe, such as their non-Indo-European language, Euskara. They have historically lived along the Western Pyrenees, between Spain and France, in one of the most important European glacial refugia during the Last Glacial Maximum. The most striking genetic characteristic is their highest frequency of the RhD blood group negative allele, a variant related to the hemolytic disease of the newborn. Both demographic and adaptive processes have been suggested as possible causes of the high frequency of RhD negative in Basques, but neither hypothesis has been clearly demonstrated. While previous studies on the Rh system in Basques have been mostly focused on serological and genotyping diversity, in this work we analyze genotyping and next generation sequencing data in order to provide a general framework of the genetic scenario of the system in Basques. In particular, we genotyped the most relevant variants of the system (D/d, E/e, and C/c), and sequenced three ~6 kb flanking regions surrounding the Rh genes in Basques and also in other populations for comparison. Our results are in agreement with previous studies, with Basques presenting the highest frequency of the RHD deletion (47.2%). Haplotype analyses of D/d, E/e, and C/c variants confirmed an association between the RhC allele, previously suggested to be under positive selection, and the RhD positive variant in non-sub-Saharan populations, including Basques. We also found extreme differentiation for the C/c variant when comparing sub-Saharan to non-sub-Saharan populations.

Human Clade 2.3.4.4 A/H5N6 Influenza Virus Lacks Mammalian Adaptation Markers and Does Not Transmit via the Airborne Route between Ferrets.

Since their emergence in 1997, A/H5N1 influenza viruses of the A/goose/Guangdong/1/96 lineage have diversified in multiple genetic and antigenic clades upon continued circulation in poultry in several countries in Eurasia and Africa. Since 2009, reassortant viruses carrying clade 2.3.4.4 hemagglutinin (HA) and internal and neuraminidase (NA) genes of influenza A viruses of different avian origin have been detected, yielding various HA-NA combinations, such as A/H5N1, A/H5N2, A/H5N3, A/H5N5, A/H5N6, and A/H5N8. Previous studies reported on the low pathogenicity and lack of airborne transmission of A/H5N2 and A/H5N8 viruses in the ferret model. However, although A/H5N6 viruses are the only clade 2.3.4.4 viruses that crossed the species barrier and infected humans, the risk they pose for human health remains poorly characterized. Here, the characterization of A/H5N6 A/Guangzhou/39715/2014 virus in vitro and in ferrets is described. This A/H5N6 virus possessed high polymerase activity, mediated by the E627K substitution in the PB2 protein, which corresponds to only one biological trait out of the three that were previously shown to confer airborne transmissibility to A/H5N1 viruses between ferrets. This might explain its lack of airborne transmission between ferrets. After intranasal inoculation, A/H5N6 virus replicated to high titers in the respiratory tracts of ferrets and was excreted for at least 6 days. Moreover, A/H5N6 virus caused severe pneumonia in ferrets upon intratracheal inoculation. Thus, A/H5N6 virus causes a more severe disease in ferrets than previously investigated clade 2.3.4.4 viruses, but our results demonstrate that the risk from airborne spread is currently low. IMPORTANCE Avian influenza A viruses are a threat to human health, as they cross the species barrier and infect humans occasionally, often with severe outcome. The antigenic and genetic diversity of A/H5 viruses from the A/goose/Guangdong/1/96 lineage is increasing, due to continued circulation and reassortment in poultry, posing a constant risk for public health and requiring regular risk assessments. Here we performed an in-depth characterization of the properties of the newly emerged zoonotic A/H5N6 virus in vitro and in ferrets. The lack of airborne transmission in the ferret model indicates that A/H5N6 virus does not pose a direct public health threat, despite the fact that it can replicate to high titers throughout the respiratory tracts of ferrets and cause more severe disease than other clade 2.3.4.4 viruses.